Gene Ther Mol Biol Vol 8, 259-290, 2004
Institute of Neurosciences, Frenchay Hospital, Bristol BS16 1LE
*Correspondence: Nikunj K. Patel, Specialist Neurosurgery Registrar, Department of Neurosurgery, Frenchay Hospital, Bristol BS16
1LE, UK; Tel: (44)117 9701212; Fax: (44)117 9701161; Email: firstname.lastname@example.org Key words: Parkinson’s disease; Animal model; Neurotrophic factor; Glial cell-line derived neurotrophic factor; Cell transplantation; Cell encapsulation; Gene therapy; Viral vectors
Abbreviations: 6-hydroxydopamine, (6-OHDA); adeno-associated virus, (AAV); Alzheimer’s disease, (AD); amyotrophic lateral sclerosis, (ALS); brain derived neurotrophic factor, (BDNF); Core Program for Intracerebral Transplantations, (CAPIT); central nervous system, (CNS); ciliary neurotrophic factor, (CNTF); cerebrospinal fluid, (CSF); dopamine, dopaminergic, (DA); deoxyribonucleic acid, (DNA); 3,4-dihydroxyphenylacetic acid, (DOPAC); Escherichia coli, (E coli); epidermal growth factor, (EGF); fibroblast growth factor, (FGF); À-amino butyric acid, (GABA); glial cell line-derived neurotrophic factor, (GDNF); GDNF family receptors-alpha 1 to 4, (GFRα1-4); homovanillic acid, (HVA); intracerebroventricular, (ICV); immunoglobulin (insulin-like) growth factor, (IGF); interleukin, (IL); 3,4-dihydroxyphenylalanine (levodopa), (L-dopa); 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine, (MPTP); magnetic resonance imaging, (MRI); nerve growth factor, (NGF); N-methyl-D-aspartate, (NMDA); neurotrophin, (NT); polymerase chain reaction, (PCR); PD, (PD); platelet-derived growth factor, (PDGF); positron – emission tomography, (PET); receptor tyrosine kinase, (Ret); recombinant-methionyl human GDNF, (r-metHuGDNF); substantia nigra pars compacta, (SNc); substantia nigra pars reticulata, (SNr); transforming growth factor, (TGF); tyrosine hydroxylase, (TH); tyrosine kinase, (Trk); Unified Parkinson’s Disease Rating Scale, (UPDRS)
Summary Parkinson’s disease (PD) is a neurodegenerative disease characterised by the progressive loss of neural dopaminergic (DA) neurons. Although symptomatic therapies to substitute for the missing neurotransmitter dopamine (DA) are efficient at the early stages of the disease, the goal is to find alternative therapies, which could protect DA neurons from the degenerative process. GDNF promotes recovery of the injured nigrostriatal DA system and improves motor function in both rodent and nonhuman primate models of PD, although intracerebral administration is necessary because of the limited penetration into the brain tissue from either the blood or the CSF. The powerful neuroprotective and neurorestorative properties of GDNF seen in preclinical studies suggest that trophic factors may play an important role in treating PD, and intraparenchymal delivery of GDNF may represent a new treatment option. Intraparenchymal delivery in animal studies is effective whether by bolus injection, by chronic infusion using a pump, by implantation of genetically engineered cell line releasing GDNF (ex vivo gene therapy) or by infecting the brain with live replication-deficient viral particles engineered to deliver GDNF (in vivo gene therapy). We have studied the effects of direct striatal chronic infusion of GDNF in 5 PD patients and noted significant symptomatic improvements and positron emission tomography (PET) scans of 18FDA uptake showed a significant increase in putamen DA storage after 24 months, suggesting a direct effect of GDNF on DA function. This study warrants careful examination of GDNF as a treatment for PD, and the clinical application of other methods of delivery.
SNc DA neurons innervate the caudate nucleus and
putamen (referred to as the striatum) and form the
nigrostriatal DA (DA) system (German et al, 1992). InParkinson’s disease (PD) is a neurodegenerative addition to the nigro-striatal DA neurons, evidence fromdisorder characterised by impairment of motor function animal studies suggests possible involvement of the DAdue to the progressive death of selected populations of DA neurons in the striatum itself in PD patients (Betarbet et al,neurons, particularly within the pars compacta of the 1997; Perier et al, 2000; Porritt et al, 2000). While the losssubstantia nigra (SNc). There are approximately 500,000 of striatal DA correlates with the severity of clinicalspecialized DA cells in the SNc of young adults. These disability, clinical manifestations are not apparent until 80
– 85% of SNc neurons have degenerated and striatal DA levels are depleted by 60 – 80% (Marsden, 1982). The cardinal features of PD are resting tremor, muscle rigidity, bradykinesia, and loss of postural reflexes (Lang et al, 1998a, 1998b). Accompanying signs include a mask-like facial expression, flexed posture, festinating gait, handwriting changes and cognitive decline (Lang et al, 1998a, 1998b). The symptoms become progressively worse as the neurodegeneration continues until patients are virtually unable to move. The clinical presentation of PD varies even within a family and it can pose a major clinical difficulty in its diagnosis. Signs of parkinsonism is also common in Alzheimer’s disease (AD) and the cognitive decline in AD is found to be associated with the progression of parkinsonism (Wilson et al, 2000). In fact, degeneration in PD involves, not only DA neurons, but variably also non-DA (cholinergic, noradrenergic and serotoninergic) neurons.
PD is the second most common progressive neurodegenerative disorder and occurs at a rate of approximately 1 in 1000 people over the age of 55 in North America with about more than 1 million affected individuals currently affected (Lang et al, 1998a); and an estimated overall prevalence in Europe of 1.6 per 100 in the population 65 years of age and older (de Rijk et al, 1997). There is no gender preference. Mortality among affected individuals is 2 to 5 times greater than for their age-matched unaffected peers (Bennett et al, 1996; Morens et al, 1996; Louis et al, 1997), and life expectancy is markedly reduced (Morens et al, 1996). The causes of PD are unknown but considerable evidence suggests a multifactorial aetiology involving genetic and environmental factors. Mutations in the gene encoding asynuclein have bee n ide ntified re cently in some families w ith a utos oma l domina nt PD (Polymeropoulos et a l, 1997; K ruge r e t a l, 1998). Inte re stingly, a -synuclein is one of the major compone nts of Lew y-bodies, inclusions found in SN neurons of sporadic PD patients. In addition, deletions in the parkin gene, a ge ne e ncoding a ubiquitin liga s e , ha ve be en identifie d in autosomal re ce ssive juve nile forms of the dise ase (K itada e t a l, 1998, Shimura et al, 2000). A mong other poss ible c ause s of s elec tive degene ra tion of nigral DA neurons in PD are defects in mitochondria l metabolism, mitochondrial gene dele tions , constitutive metabolic deficiencies and c ellular da mage due to oxidative injury (for re views s ee re fs: (B ec k e t al, 1995, Jenne r, 1998)). The single most consistent risk factor for the disease is age, and given the changing demography of industrialized nations, its burden upon societies is likely to increase.
Early treatment of the disease was neurosurgical, involving the creation of small lesions within the thalamus (thalamotomy), which was found to relieve tremor and rigidity, although generally not bradykinesia (Hassler, 1954); and the globus pallidus internus (pallidotomy) and the subthalamic region (subthalamotomy), which were found to significantly improve the 3 cardinal features (Svennilson, 1960). Such invasive treatment was largely superseded by the introduction in the late 1960s of orally administered L-3/4-dihydoxyphenylalanine (L-dopa), the immediate precursor of DA that is absorbed through the small intestine and is able, unlike DA itself, to cross the blood-brain barrier. L-dopa, when combined with an aromatic amino acid decarboxylase inhibitor, remains the most effective treatment currently, widely available for PD (Koller, 2000; Jankovic, 2002).
During the early stages, the disease generally responds well to symptomatic treatment with L-dopa (Koller, 2000), or DA agonists (Lang et al, 1998a), however, current pharmacological treatments are not entirely adequate. Unfortunately, with prolonged use of DA therapy, a high proportion of patients develop motor complications (fluctuations and dyskinesiae) and psychiatric complications (Marsden, 1982; Marsden et al, 1982; Obeso et al, 1989). The inconsistencies of patient responsiveness, as manifest by motor fluctuations, take the form of distinct “wearing-off” and “on-off” phenomena. “Wearing-off”, also described as “end-of-dose deterioration”, is the term given to the relatively gradual and predictable decline in response to a dose of L-dopa that occurs over time, and this contrasts with “on-off” fluctuations in motor performance that are not clearly related to L-dopa dosing. “On” periods, during which patients exhibit a beneficial response to the drug, and which may be as brief as hours or as long as days, are generally unpredictably interrupted by “off” periods, during which there is an absence of response to the drug and a return of parkinsonian symptoms, and which may be as brief as hours or as long as days. With natural progression of the disease, “off” periods become more frequent.
In the early stages, motor fluctuations may be counteracted by approaches that prolong the actions of L-dopa (eg, using slow release formulations of the drug or the co-administration of a catechol-O-methyltranferase inhibitor) or by use of longer acting DA agonists; however, these interventions cannot prevent an eventual increased unpredictability and lessened control of motor fluctuations and an increased incidence of dyskinesias during “on” periods that eventually become severely disabling (Jankovic, 2002, Koller, 2000, Lang et al, 1998a). Additionally, some specific motor functions such as gait, balance, speech, deglutition, etc, become less responsive as disease and duration of treatment increase. It seems that both “non-physiological” DA stimulation and disease progression simultaneously interact to induce such complications (Marsden, 1982; Marsden et al, 1982; Obeso et al, 1989). While DA therapy provides considerable benefits, the validity of Parkinson’s original observation that “the unhappy sufferer has considered [the disease] as an evil, from the domination of which he has no prospect of escape” still holds. As a result, newer therapeutic avenues continue to be explored with three major aims: (1) to treat symptomatically the complications associated with chronic drug therapy (2) to stop or slow down progression of the disease and (3) and ultimately restore a normal system.
Consequently, there has been a resurgence of interest in surgical treatments. One initially promising approach was the implantation of embryonic DA neurons into the brains of patients with PD. Human fetal mesencephalic cell grafts survive for years and have produced long-lasting effects (Lindvall et al, 1994; Kordower et al, 1995; Piccini et al, 1999; Freed et al, 2001). Nevertheless, in a randomized, double-blind trial in which patients either received intraputaminal transplants of cultured embryonic mesencephalic tissue or were given sham surgery in which the dura mater was not penetrated, no clinical improvement was observed as a result of the transplants in patients over 60 years of age at 1 year after surgery, and only moderate improvement was apparent in those aged 60 years or less (Freed et al, 2001). During continued follow-up of 12 to 36 months in patients who had received transplants, dystonia and dyskinesias had developed in 5 patients, all of whom had been < 60 years of age at the time of surgery and each of whom had experienced clinical improvement during the first year after transplantation. Ethical and safety issues, the large amounts of embryonic mesencephalic tissue needed, as well as incomplete reversal of functional deficits in PD patients require further improvement of the method (Boer, 1994; Dunnett et al, 1999). One of the problems that prevent a more wide-spread application of human fetal transplantation is the poor survival of grafted tissue, less than 20% of the implanted cells surviving the transplantation procedure (Kordower et al, 1995; Lindvall, 1998). Different strategies such as graft treatment with neurotrophic factors, free radical scavengers, antioxidants, and anti-apoptotic molecules, have been shown to increase transplant survival in experimental models (Brundin et al, 2000a). However, despite improved survival of mesencephalic grafts after treatment with lazaroids, symptomatic relief in implanted parkinsonian patients was not ameliorated compared to non-treated grafts (Brundin et al, 2000b). The future of embryonic neural transplantation must therefore be considered uncertain.
Renewed interest in ablative neurosurgical techniques has arisen as a consequence of increased understanding of the pathophysiology of basal ganglia and refinements in neurosurgical operating procedures, and pallidotomy, thalamotomy and subthalamotomy are again widely accepted options for consideration once a patient’s condition has become increasingly difficult to manage using medication alone (Lang et al, 1998a; Jankovic, 2002). Regardless of the success or otherwise of such techniques to date, however, by their very nature, any side effects resulting from such interventions may very likely be irreversible. An alternative, less-irrevocable approach that simulates lesional effects, deep-brain stimulation, is currently the treatment of choice. In this procedure, electrical stimulation is provided on a long-term basis through implanted deep-brain electrodes and appears to result in improvements in motor function similar to those observed with ablative lesions. Bilateral electrical stimulation of the subthalamic nucle us grea tly improves akinesia, rigidity, tremor and reduces dyskinesias (Limousin et al, 1998). The mechanism by which this improvement occurs is not well understood but may involve the inhibition (Lang et al, 1998a) or distal stimulation to disrupt the abnormal neuronal activity.
Although symptomatic therapies are efficacious in the early stages of the disease, the goal would be to identify neuroprotective therapies, i.e. find factors that could arrest or slow down the degenerative process. While there are multiple causes of neurodegenerative diseases including environmental, genetic and age-associated factors, the treatments may be directed at similar underlying mechanisms via neuroprotective or reparative interventions. In a theoretical framework, one working model of neuronal damage and the prevention of cell death is the concept of “neuronal resilience”. Depending on the status of the cell with respect to pretraumatic events and gene-expression relevant to neuronal preservation, the neuron will exist far from or close to the threshold for irreversible neuronal damage. The neuron can thus be thought of as oscillating between protected and vulnerable conditions. This model of neuronal haemostasis suggests that a number of separate therapeutic measures, including delivery of neurotrophic factors, may reduce the overall probability in degeneration in those neuronal populations that approach their specific threshold for degeneration (Isacson et al, 1997).
Neurotrophic factors are naturally occurring proteins that have an impact on cell survival and proliferation, differentiation, biochemical function and morphological plasticity (Alexi et al, 2000). They not only promote the diffe re ntia tion a nd grow th of developing ne urons a nd phe notypic ma intenanc e and s urviva l of adult ma ture ne urons but also re pre se nt a potentia l mea ns of modifying neuronal dysfunc tion, a stroc ytic ac tivation a nd inflammatory rea ctions under pa thological conditions. A large body of evidence suggests that some ne urotrophic factors under ce rtain c onditions als o modulate neuronal plasticity that emerges during aging and under tra uma tic or de genera tive c ondition (Bles c h, 1998). Previously, it was thought that different neuronal populations were each responsive to only a single neurotrophic factor. However, evidence indicates that there is overlap and redundancy, whereby a single neurotrophic factor may affect more than one cell type, and a specific cell type may respond to several neurotrophic factors (Korsching, 1993).
Classically, a neurotrophic factor is produced and secreted by target cells, be they nerve cells or other cells, and then taken up by the innervating nerve terminals to exert local effects and, via retrograde axonal transport, trophic effects on the nerve cell body (Oppenheim, 1989; Olson, 1996). However, the actions of neurotrophic factors are associated not only with retrograde transport from the target tissue but also autocrine and paracrine mechanisms (Kokaia et al, 1993; Miranda et al, 1993).
Neurotrophic factors are expressed in different regions of the nervous system during different phases of development (Maisonpierre et al, 1990; Schecterson et al, 1992). It has be en propos ed that grow ing a xons compe te for limite d amounts of neurotrophic factors, which are produced by target tissues (Grimes et al, 1996; Yuen et al, 1996). Neurons which fail to obtain a sufficient quantity of the necessary neurotrophic factors die by a proce s s ca lle d progra mmed ce ll-de ath (Thoe nen e t a l, 1987; C onnor e t a l, 1998). Furthe r, in adulthood, neurotrophic factors are required to maintain neuronal functions and specific neuronal phenotype (Blesch, 1998); howe ver, it is unclear as to w ha t degree the ma ture neurons remain dependent upon target-derive d support. The site-specific neurotrophic factor expression in the adult brain suggests various mechanisms of action in relation to the observed selective neuronal trophism. In response to injury, trophic factors and their receptors increase in concentration, suggesting an endogenous regenerative response of these molecules (Hughes, 1999), and insufficiency of such trophic support due to decreased neurotrophic factor supply or impaired target cell response may account for some of the cell death in neurodegenerative diseases (Appel, 1981; Hefti, 1983). Re ce nt evide nc e s ugges ts that alte rations in the neurotrophic levels either due to age, genetic background or other factors might contribute to neurodegenera tion. It has be en propos e d tha t the los s of e ndoge nous target-derived trophic support for selective neuronal populations ma y lea d to the neuronal de ge neration c harac teristic of Alzheimer’s, Parkinson’s and other neurodegenera tive dis ea s es but direc t s upport for this hypothes is is currently lacking (Connor et al, 1998).
Neurotrophic factors serve the function of neuroprotectant molecules against cytotoxic cell damage. They can act as antiexcitotoxins and antioxidants and, as such, they have the capacity to enhance mitochondrial function. They have been shown to upregulate calcium buffering proteins, antioxidant enzymes and antiapoptotic factors (Mattson, 1998). Taken together with the widespread expression of the receptors for neurotrophic factors and the pleiotropic effects on different neuronal and glial cell types, these lines of evidence make it necessary to target trophic molecules to either a specific subpopulation of neurons or to the injury site itself and its immediate vicinity in order to provide neuronal protection, administered either prior to or following a neurotoxic insult. Based on their specificities, neurotrophic factors have become attractive drug candidates for the treatment of neurodegenerative diseases that affect specific populations of neurons.
Since the discovery of nerve growth factor (NGF) in the 1950s (Cohen, 1954; Levi-Montalcini, 1987) the prospect of applying neurotrophic factors to the treatment of neurological disorders has motivated investigators and excited clinicians. Over the past 2 decades in particular, great advances have been made in discovering new factors, characterizing and cloning them, and demonstrating their therapeutic potential in animal models of neurological disease (Apfel, 2001).
There are currently more than 20 trophic factors that have been identified, showing potential for use in a variety of neurodegenerative diseases, including PD (Collier et al, 1999; Thorne et al, 2001).
1. Candidate neurotrophic factors (Table 1)
Most neurotrophic factors belong to several families of structurally and functionally related molecules: (1) NGF-superfamily; (2) Glial cell line-derived neurotrophic factor (GDNF) family; (3) neurokine or neuropoietin superfamily; (4) non-neuronal growth factor-superfamily. All these neurotrophic factors signal via specific multicomponent receptor complexes. NGF-superfamily receptors include p75 and the receptor protein tyrosine kinases (Trk), TrkA, TrkB and TrkC. GDNF family receptors include a receptor complex of Ret and growth factor receptor (GFR) α1-4. The neurokine superfamily ligands mediate via the receptors gp130 and leukemia inhibitory factor receptor-β (LIFR-β).
NGF was the first neurotrophic factor to be discovered in the 1950s (Cohen, 1954; Levi-Montalcini, 1987). NGF is a member of a gene family of structurally related proteins called "neurotrophins". They include brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and NT-4, NT-5 and NT-6 (Maisonpierre et al, 1990; Schecterson and Bothwell, 1992). As few as six regions of amino acid non-identity confer specificity for each neurotrophin (Ip et al, 1993). From a clinical perspective, the most important neurons that respond to NGF include the central cholinergic basal forebrain neurons that consistently degenerate in AD, peripheral small fiber sensory neurons, which mediate pain and temperature sensation and post-ganglionic sympathetic neurons (Hefti et al, 1989). BDNF supports some sensory fibers as well, but also supports spinal motor neurons, and ventral DA neurons that degenerate in PD (Hyman et al, 1991; Sendtner et al, 1992; Ernfors et al, 1994a); and may also support the central cholinergic forebrain neurons that degenerate in AD. NT-3 is trophic for motor neurons and
T able 1. Ne urotrophic fa ctors a nd the ir c linic a lly importa nt re s po ns ive ne ur ona l popul a tion s
|Neurotrophic factor||Receptor||Responsive Neurons|
|NGF||Trk A, p75||Small fibre sensory|
|BDNF and NT-4/5||Trk B, p75||Medium fibre sensory|
|DA (Subst. Nigra)|
|NT-3||Trk C, p75||Large diameter sensory|
|CNTF||Gp 130, LIF R‚ and CNTFRα||Motor neurons|
|IGF-1||Type I & II IGF Receptors||Motor neurons|
|GDNF||Ret tyrosine kinase GDNFRα||Motor neurons|
|DA (Subst. Nigra)|
large diameter sensory neurons that mediate proprioceptive sensation (Hory-Lee et al, 1993).
The GDNF family, distantly related to the transforming growth factor-beta (TGFβ) superfamily, includes GDNF and three structurally related members called neurturin, persephin and artemin. GDNF was originally isolated as a factor from glial cell conditioned medium that promotes the survival and phenotype of ventral DA neurons (Lin et al, 1993). It was subsequently discovered to be trophic for a wide variety of peripheral neurons, most notably spinal motor neurons, for which it is particularly potent (Henderson et al, 1994; Buj-Bello et al, 1995). Most pre-clinical and clinical studies have focused on the possible application of GDNF for the treatment of motor neuron disease and PD. Detail on GDNF and its application for the treatment of PD is addressed in the next section.
The neurokine family includes ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), interleukin-6 (IL-6), cardiotrophin-1 (CT-1) and oncostatin-M. CNTF, a member of the alpha-helical cytokine superfamily (Ip et al, 1992; Lindsay, 1994) was originally identified as a protein that provided neurotrophic support for parasympathetic ciliary neurons (Adler et al, 1979). It was subsequently discovered to be trophic for many different types of peripheral and central neurons. Clinically, its most significant activity may be its support of motor neurons. It was the first growth factor discovered to promote the survival of lower motor neurons, and has demonstrated efficacy in rescuing a variety of animal models of motor neuron degeneration (Adler et al, 1979).
Non-neuronal growth factors present in significant concentrations in the nervous system include acidic fibroblast growth factor (aFGF), also called FGF-1; basic fibroblast growth factor (bFGF), also called FGF-2; epidermal growth factor (EGF), immunoglobulin growth factor (IGF) and bone morphogenetic protein (BMP). Most available information concerning neurotrophic effects is related to aFGF and bFGF, which are present in 500-fold and 50-fold greater concentrations than NGF. The immunoglobulin factors (IGFs) are a family of structurally and functionally related proteins that include insulin, IGF-1 and IGF-2. The IGFs are widely expressed throughout the nervous system and may play a number of important roles, particularly in stimulating nerve regeneration (Kanje et al, 1989; Sendtner et al, 1997). Several pre-clinical studies have suggested that IGF-1 in particular may be trophic for motor neurons, and consequently most of its clinical development in the nervous system has focused on the treatment of motor neuron disease (Lewis et al, 1993).
Neurotrophic factors of the NGF family and their receptors may be localized by immunocytochemistry in SNc of human brains. Melanin-positive neurons in SNc are immunoreactive for these neurotrophic factors and their receptors, indicating both autocrine and paracrine functions for these factors. The proportions of melanin-containing neurons immunoreactive for these factors or their receptors were: 66% for NGF and TrkA; 74% for BDNF, 71% for TrkB; 85% for NT-3, 86% for TrkC (Nishio et al, 1998). Thus, there is considerable overlap of these factors and receptors in the same cells in control brain.
BDNF measured by ELISA in portions of SN, striatum, cerebellum and frontal cortex in control and PD brains, is found significantly reduced in SN and striatum of PD brain. NGF, measured by ELISA, is also found reduced in PD SN (Mogi et al, 1999). However, studies of homogenates or extracts do not address the content of factor per neuron, which is the more critical value since there are large proportions of neuronal dropout with development of PD.
In SNc of PD brains compared to age-matched controls, the number of pigmented neurons containing BDNF was reduced to 9.6% of the control value while the number of pigmented neurons non-immunoreactive for BDNF was reduced only to 23.9% of the control value, thus suggesting that SNc pigmented neurons not expressing BDNF have a greater probability of surviving than BDNF-positive pigmented neurons. All the DA neurons observed to have Lewy bodies were immunoreactive for BDNF (Parain et al, 1999). These data suggest that BDNF either does not protect the neuron or actually confers a greater sensitivity to degeneration in PD. In a quantitative assessment of neurotrophic factor content within individual neurons based on density of staining with either immunoperoxidase or immunofluorescent labeling (Chauhan et al, 2001), there were observed small decreases in BDNF and CNTF (5 to 10%) but little or no significant changes in NGF, NT-3 or NT-4/5 in surviving neurons of SNc in PD.
Possibly, significant alterations in regulation of synthesis or transport of growth factors may result in changes in plasma levels in clinically diseased individuals. Using an immuno-enzymatic assay for plasma levels, Lorigados et al. (1998) found significant reductions in NGF levels in patients with PD and Huntington's disease. Such data require further surveys for substantiation, for a valid specific assay may lead to detection of early or preclinical disease.
With regard to receptors for the NGF family, an in situ hybridization study of TrkB mRNA did not show change in the mRNA content per surviving neuron in SN of PD brains despite the extensive reduction in the total number of neurons (Benisty et al, 1998). However, comparable data for all the other receptors are lacking. Since there is now evidence for both anterograde as well as retrograde transport of BDNF and NT-3, determination of critical pathogenic events requires in situ data for factors and their receptors in both SN and striatum in the same brains. The primary defect may be in one site and the observed outcome in a remote site. The critical pathogenic or compensatory process may be related to the regulation of synthesis or of axoplasmic transport. Moreover, in order to identify putative factors critical to neuronal loss, it is necessary to measure factor content per surviving neuron in situ before neuronal demise
Lesions of the nigrostriatal pathway or of the striatum in rodents lead to alterations in various peptides, including BDNF and NT-3, in the striatum. The effect on BDNF is consistent with evidence for its anterograde transport into striatum.
While comparable data for all pertinent neurotrophic factor receptors are not available, an in situ hybridization study of mRNAs for TrkB and TrkC in rat striatum after 6OHDA lesions shows increases in the hybridization density for TrkB but not for TrkC in the ipsilateral caudate-putamen. The upregulation of TrkB might be a compensatory response due to loss of DA input or loss of transported BDNF from the ventral midbrain (Numan et al, 1997).
The earliest study of GDNF mRNA expression by in situ hybridization in sections of mesencephalon and striatum from control and PD brain failed to show any labeling, suggesting that mRNA levels are very low in human adult brain (Hunot et al, 1996). However, utilizing PCR to produce amplification, Schaar et al. (1994) have found highest expression of GDNF mRNA in human caudate, with low levels in the putamen and no detectable mRNA in the SN. Springer et al. (1994) have observed GDNF transcripts in human striatum, hippocampus, cortex and spinal cord but not cerebellum. In adult rat tissues, GDNF transcripts are found in all regions of CNS and in a Schwann cell line that secretes a DA neuron trophic factor (Springer et al, 1994). In situ hybridization, in contrast to polymerase chain reaction (PCR), does not produce labeling in human brain (Hunot et al, 1996) or in rodent except in dorsal root ganglia of 1-day-old rats (Springer et al, 1994). Hence, analyses of PD brain for GDNF-mRNA expression require PCR amplification.
Tissue sections of SN from control and PD brain show Ret immunoreactivity localized in the DA neurons of SN, appearing as punctate deposits within cells. Expression of Ret is observed in many surviving neurons in all the cases of PD (Walker et al, 1998a). However, GFRα1, which is more specific for the GDNF, was not measured. This same group found that a human THP-1 macrophage cell line expressed immunoreactive Ret but not the mRNA for GFRa, thus demonstrating that Ret by itself is not a sufficient measure of the potential cell response to GDNF.
It has been found that GDNF treatment of cultured human fetal ventral mesencephalon (8 weeks postconception) nearly doubles the DA neuron survival while halving the rate of apoptosis from 6% to 3% (Clarkson et al, 1997) and increases the density of calbindin-positive neurons, which are proposed to be less vulnerable to degeneration in PD (Meyer et al, 1999). The data from immunoperoxidase and immunofluorescence methods (Chauhan et al, 2001) show significantly reduced levels of immunoreactive GDNF (about 20%) in all sampled surviving melanin-containing neurons in SNc of PD brain compared to age-matched controls. Since mRNA transcripts of GDNF have not been detected in SN (Schaar et al, 1994), the decreased GDNF per surviving neuron in SNc in PD may result from either reduced synthesis in or retrograde transport from the striatum. Decreased GDNF in neurons may be one of the early events anticipating neuronal loss in SNc in PD.
In one of the earliest studies of neurotrophic factors in human brain, Mogi et a l. (1994) found incre as es in a number of cytokines , including IL-6, in the striatum but not cerebral cortex of PD brains. These may be related to astrocytic activation. Immunore active bFG F is found depleted in midbra in D A ne urons , more s o tha n reduc tions in TH , in PD bra in (Tooya ma e t al, 1993). Both aFG F and bFGF a re increased in reactive astrocytes and bFGF is detected in plaque s , ne uritic ta ngle s a nd ne uropil threa ds in A D . O ther fa ctors w e re not te s te d in t h i s s tudy. It is possibly relevant that nicotine induces upregulation of bFGF in rat brain and that cigarette smoking is negatively correlated with the incidence of PD (Belluardo et al, 1998). An immunoc ytochemica l study of the re ceptor for FG F, FGFR-1, shows that immunoreactive product in the SN declines with age in control brain and is retained in many s urviving neurons of SN in PD bra in (Wa lke r et al, 1998b). It is of inte res t that IGF-1 was reduced in the mucus covering the nasal olfactory epithelium of patients with PD, late onset cereb e l l a r a ta xia a nd amyotropic latera l sc leros is (Fe de ric o e t al, 1999).
In s ummary, it w ould a ppe a r tha t bFG F a nd GD N F a re c a ndida tes for fa c tors whos e a lte re d regula tion is poss ibly critical in early pathogenetic steps in the degeneration in PD. However, the summary of available data in PD brain indicates the considerable gaps existing, particularly rega rding s triatum.
A v a i l a b l e d a t a r e g a r d i n g c h a n g e s i n e x p r e s s i o n o f n e u r o t r o p h i c f a c t o r s o r t h e i r r e c e p t o r s d u r i n g a gi ng in h um a n br a i n a re li mi t e d . A s m e nt i o n e d e a r l i e r , i m m u n o r e a c t i v e F G F R - 1 d e c l i n e s i n S N w i t h a g e i n h u m a n b r a i n ( W a l k e r e t a l , 1 9 9 8 b ) . I n n u c l e u s b a s a l i s o f M e y n e r t , S a l e h i e t a l. ( 19 96 ) ob s e r ve d n o r e l a t i on s h i p be t w e e n a g e o r s e x a nd the e xpre s s ion of p75 in c e ll b odie s but did f ind a p o s i t i v e r e l a t i o n s h i p o f a g e w i t h d e c r e a s e d s t a i n i n g i n ne rv e f ib e rs i n A D , w h il e W u e t a l . (1 99 7) a ls o f ou nd no s ig ni f ic a n t a ge - r e l a t e d c h a ng e s in p 7 5 or c h ol i ne a c e t yl a s e i m mu n o r e a c t i vi t y bu t d id f i nd l o s s o f c a l b in d i n -b i n d in g pr ot e in i mmu no re a c t ivi ty in c hol in e rg ic ne ur ons . In hu ma n h i p p o c a m p u s , B D N F i m m u n o s t a i n i n g w i d e n e d w i t h a g e w h il e t ha t o f N G F , N T - 3 a n d N T - 4 w a s
o b s e r v e d t o be g e n e r a l l y h i g h e r a t e a r l i e r a g e s ( Q u a r t u e t a l , 1 9 9 9 ) .
I n a s i m i l a r s t u d y D e l F i a c c o e t a l . (2002) s h o w e d G D N F i m m u n o r e a c t i v i t y mainly localized to the spinal trigeminal, cuneate, solitary, vestibular, and cochlear sensory nuclei, dorsal motor nucleus of the vagus nerve, ventral grey column, hypoglossal nucleus, dorsal and ventrolateral medullary reticular formation, pontine subventricular grey and locus coeruleus, lateral regions of the rostral pontine tegmentum, tectal plate, trochlear nucleus, dorsal and median raphe nuclei, caudal and rostral linear nuclei, cuneiform nucleus, and SN.
Comparison between pre- and full-term newborns and adult subjects revealed changes with age in density of positive innervation and frequency of immunoreactive perikarya. The results obtained provide detailed information on the occurrence of GDNF-like immunoreactive neurons in the human brain stem and suggest that the protein is present in a variety of neuronal systems, which subserve different functional activities, at developmental ages and in adult brains.
G o l d m a n ( G o l d m a n , 1 9 9 8 ) h a s r e v i e w e d n e u r o g e n e s i s i n a d u l t v e rt e b r a t e b ra i n . N e u r on a l p r e c u r s or s , w h i c h a r e lo c a t e d m a i n l y i n t h e s u b v e n t r i c u l a r z o n e , p e r s i s t i n a d u l t v e r t e b r a t e b r a i n . I n h u m a n b r a i n , t h e s u b v e n t r i c u l a r z o n e i n v i vo a p pe a r s t o c e a s e it s n e u r o n og e n e s i s d ur i n g d e v e lo p m e n t t o th e a du l t s t a te . H ow e v e r, i n v it r o s e r ia l a pp li c a t io n o f b F G F a n d B D N F h a s a l l o w e d t h e g e n e r a t i o n a n d m a i n t e n a n c e o f n e u r o n p r e c u r s o r c e l l s a r i s i n g f r o m t h e a d ul t h um a n s u b v e nt r i c ul a r z o n e . T h e s e d a t a s u gg e s t t h e f e a s i b i l i t y o f i n d u c i n g n e u r o g e n e s i s i n t h e a d u l t h u m a n b r a i n i n s i t u o r w i t h i m p l a n t e d p r o g e n i t o r c e l l s w i t h a p p r o p r i a t e l y s e l e c t e d a n d / o r r e g u l a t e d n e u r o t r o p h i c f a c t o r s ( G o l d m a n , 1 9 9 8 ) .
I n t h e f i r s t a n d o n l y a v a i l a b l e s t u d y o f i t s k i n d i n p r i m a t e s , S m i t h e t a l . ( 1 9 9 9 ) m e a s u r e d t h e n u m b e r a n d v o lu m e of c h ol i n e rg i c im m u n ol a b e le d n e u r o n s i n b a s a l f o r e b r a i n r e g i o n s o f R h e s u s m o n k e y s a n d f o u n d a n a g e - r e la t e d d e c l in e t ha t i s a l m os t c om p l e te l y r e ve r s e d b y a d m i n i s t r a t i o n o f h u m a n N G F . T h e s e a u - t h o r s s u g g e s t t h a t s u b c o r t i c a l s t r u c t u r e s a r e m o r e v u l n e r a b l e t o a g i n g e f f e c t s t h a n i s c o r t e x .
T he a v a i la b le o b s e rv a ti on s f ro m a gi n g hu m a n o r a ni ma l b ra i n ( G i bb s , 19 9 8; G o ld ma n , 19 9 8; S m it h e t a l , 1 99 9) a n d t he i n fo rm a ti on c o nc e rn in g n e u r on a l s u rv iva l in g e ne ra l, s u gge s t a t l e a s t thr e e br oa d p ri nc i pl e s r e g a r d i n g N e u r o t r o p h i c f a c t o r s a n d b r a i n r e p a i r :
I t i s c l e a r f r o m t h e q u e s t i o n s r a i s e d a n d t h e s c a r c i t y o f d a t a i n hu m a n t i s s ue s t ha t c on s id e r a bl e i nv e s t ig a t i on i s w a r r a n te d t o u nd e r s ta nd th e c ha ng e s in n e u ro t ro ph i c fa c to rs a n d i n th e ir r e s p e c t iv e r e c e p t or s b ro ug h t on by a g in g a nd t h e re g ul a t i on o f a g i n g i n th e a d ul t n e r v o u s s y s te m b y n e u ro t r o ph i c fa c t o rs a n d t h e ir re c e pto rs .
The slower progressive nature of DA neuronal loss in PD coupled to the relatively long run-in period for the condition (approximately 5 years; (Fearnley et al, 1991; Morrish et al, 1996; Marek et al, 2001)) makes this disorder an attractive candidate for neurotrophic factor “rescue”. In other words, the delivery of a neurotrophic factor at the time of clinical presentation could arrest and even reverse the parkinsonian symptoms and by doing so cure the patient. Of course, this assumes that solely rescuing the DA neurons in the nigrostriatal pathway will achieve a cure, eventhough there is pathology outside this system. Nevertheless, as a starting point, rescuing the DA neurons and their striatal projections is useful, and in this respect several factors have been shown to produce significant beneficial effects on DA neurons in culture and in animal models (Collier et al, 1999). Those producing effects on DA neurons in vitro include, but are not limited to, BDNF (Hyman et al, 1991, 1994); NT-3 (Hyman et al, 1994); NT-4/5 (Hynes et al, 1994); IGF-1 (Knusel et al, 1991); both aFGF and bFGF (Date et al, 1990; Engele et al, 1991); EGF (Casper et al, 1991); GDNF (Lin et al, 1993; Engele et al, 1996); CNTF; PDGF and TGF-α (Collier et al, 1999; Thorne et al, 2001). All these factors have been shown to support survival of embryonic DA neurons with varying degrees of potency and specificity.
In MPTP- and 6-OHDA-lesioned animal models of PD, BDNF (Frim et al, 1994) and GDNF (Tomac et al, 1995; Gash et al, 1998a) have been shown to promote the survival of mesencephalic DA neurons. In the initial rodent studies, low doses of BDNF failed to attenuate nigral DA loss following medial forebrain bundle transection (Lapchak et al, 1993). However, later studies with higher doses showed ameliorative effects. Supranigral implants of fibroblasts engineered to secrete human BDNF are shown to protect DA neurons from MPTP toxicity (Frim et al, 1994). Studies on BDNF and NT-3 in a 6-OHDA striatal perfusion neurodegeneration model show evidence of increased DA metabolism and turnover as determined by homovanillic acid (HVA) and DA ratios, and improvement in amphetamine-induced rotation despite no obvious effect on neuron survival or sprouting (Altar et al, 1994). The transplantation of BDNF-transduced astrocytes into the striatum of 6-OHDA lesioned animals did not enhance DA neuron survival, although it significantly reduced amphetamine-induced rotation indicating behavioral recovery in one study (Yoshimoto et al, 1995). Studies with the MPTP-primate model have shown potent and long-lasting effects on the SN, preservation of DA neurons and a significant amelioration of behavioral symptoms after the administration of GDNF (Gash et al, 1996).
Where there are comparative data, GDNF has been found to be among the most potent and specific neurotrophic factors for the nigrostriatal pathway (Bowers et al, 1997), and has consistently been shown to dramatically protect and enhance the function of DA neurons in animal models (Gash et al, 1998b; Grondin et al, 1998). GDNF is as potent as CNTF and NT-4 and five to 10 times more potent than BDNF in promoting survival of all axotomized nigrostriatal neurons in lesioned rats (Lu et al, 1997). Not only is GDNF found in the SN (Kawamoto et al, 2000; Del Fiacco et al, 2002) and the striatum (Nosrat et al, 1996; Kawamoto et al, 2000), but there is some evidence that PD patients may have reduced levels of GDNF in the SN (Chauhan et al, 2001). This suggests that loss of GDNF may contribute to the process of degeneration of DA cells in the SN, making it a frontrunner for therapeutic trials for PD.
The major difficulties e ncountered in the us e of ne urotro phic fa c to rs fo r the tre a tme nt of ne u rode g e ne ra tive dis ea s es a re the imposs ibility for the se molec ule s to cross th e blo od -br a i n-b a r rie r, th e i r i ns ta b il ity in a fl ui d e nv iro nm e nt , a nd t he i r s id e - effe cts a ssociate d with systemic administra tion and from binding to extra ta rget re ceptors with intra ce rebroventricula r (IC V) infus ion or inje ction (H aller e t a l, 1998; R idet et al, 2000; Ae bischer et a l, 2001). Local a dministration is the refore required to ac hieve therapeutic conc entra tions in the tissue. The efficie nc y of loc al distribution, and henc e effec tiveness of loca l the rapy, de pe nds on the ra te of protein migration through tiss ue .
Different strategies for delivery of GDNF in animal models of PD have been explored: ICV infusion (Hoane e t al, 1999); intraparenc hymatous injection (Rosenblad et al, 1998); the gra fting of genetically engineere d cells (Na kao e t al, 2000); in vivo gene transfer (Kordower, 2003); fibrin glue preparation (Cheng et al, 1998); and the use of biodegradable microspheres (Jollivet et al, 2004) and non-biodegradable polymeric devices (Tornqvist et al, 2000).
In re s pons e to the prec linic al studies , a ttempts to a pply neurotrophic fa c tors clinica lly, how ever, have s o far be en dis appointing be ca us e of the ir poor effic ac y and induction of troublesome side effects. In these clinical trials, the recombinant protein was delivered into the c e r e bros pina l flu id (C SF) ( intra ve ntr ic ula rly o r int ra the c a lly ) in pa tie nts suffering from amyotrophic lateral sclerosis (ALS), peripheral neuropathy, PD or A lz he ime r dis e a s e (A ebis c he r e t a l, 2001; Tus zyns ki, 2002). Re s ults from thes e s tudie s indic a te tha t the neurotrophic factors, whose receptors are widely distributed, are pr on e t o ind uc ing pr on oun c e d s id e e ff e c t s w he n de l iv e re d by th e s e route s . Excruciating pain was indeed described with the ICV administration of NGF in AD patients (Olson et al, 1992; Eriksdotter Jonhagen et al, 1998); weight loss, nausea and abnormal sexual behaviour was reported with the ICV administration of GDNF in PD patients (Kordower et al, 1999b). The poor pe netra tion a cros s the blood-bra in ba rrie r, a s we ll as the limi te d p a s s a g e of prote ins f rom t he C SF into the brain tissue, has made it necessary to administer the factors at doses that a re likely to induc e s ide e ffe c ts . The se e ffe c ts ma y not be s o evide n t in s ma ll -s iz e d e xp e rime nta l a nima ls . F or th is re a s on, the y ma y have gone unnoticed in the preclinical studies and may have become a pp a re nt in s ome c a s e s only a t th e pha s e II /III s ta ge of the c lini c a l trails when larger numbers of patients were included.
Intranasal administration offers a method for bypassing the blood-brain barrier and contributes to better central nervous system (CNS) penetration of neurotrophic factors (Thorne et al, 2001). However, the nasal route does not allow for the focal delivery of these potent molecules and high doses of trophic factors may be necessary to elicit therapeutic effects.
Th e the ra pe u ti c v a l ue of ne ur otr op hic -f a c t or de li ve r y, th e r e fo re , may not be possible to achieve unless the factors are specifically targeted and regionally restricted to the area of interest within the CNS to achieve significant results without widespread, unwanted adverse effects. Animal experiments have shown that direct parenchymal administration dramatically reduces the occurrence of side-effects reported with ICV application (Anderson et al, 1995, Morse et al, 1993). Int ra c e r e bra l de l ive ry of n e urot rophi c fa c tors ma y b e a c h ie ve d e ith e r by dire c t pro te in infusion or by gene transfer techniques, both ex vivo and in vivo, and the results of these delivery systems are discussed below.
Polymer-based, infusion, and cellular delivery systems provide prote in to the brain via drama tica lly diffe re nt me cha nisms : diffus ion from a polyme r matrix, flow through a s ynthe tic c ha nne l, a n d s e c re tio n a ft e r s y nt he s is by c e ll ul a r ma c hi ne ry. These approaches are similar in at le as t one importa nt re ga rd: a fte r re lea s e from the ma trix, ca nnula , or cell, protein molecules move to their cellular sites of action by migrating through the inte rstitial s pac e of tissue. With some nota ble e xc eptions (high-flow infus ion w he re migra tion re lie s on conve ction (Morrison e t al, 1994), diffus ion is the principal me chanism of prote in distribution through brain tis s ue . D iffus ion through the tis s ue inters titiu m is a s lo w pro c e s s (C l a us s e t a l, 1990 ); s ubs ta ntia l me ta bolis m or c le a r a nc e can occur during the period of migration. As a result, the volume of tiss ue expos ed to protein c an be re la tive ly s ma ll. Penetrability of a neurotrophic factor administered by interstitial drug delivery is dependant on the rate of dispersion versus elimination. If diffusion is the primary mechanism for dispersion, the extent of dispersion is dependant on the concentration gradient and molecular weight of the protein. Elimination is dependant on the stability of the protein within the interstitial space; and for proteins eliminated by receptor-mediated internalization and degradation, dependant on the number of receptors and the affinity to the receptor.
Typically for secreted proteins, neurotrophic factors are produced in the form of a precursor, preproneurotrophic factor. The signal sequence is cleaved on secretion, and activation of the pro-neurotrophic factor probably occurs by proteolytic cleavage. GDNF-family ligands seem to bind heparin-sulphate side chains of extracellular-matrix proteoglygans, which might restrict their diffusion and raise their local concentration (Hamilton et al, 2001). The specific pro-teases that cleave and activate GDNF precursors have not been identified. Interestingly, recent evidence indicates that secreted pro-neurotrophic factors are biologically active (Lee et al, 2001). Secreted proforms of NGF and BDNF are cleaved extracellularly by the serine protease plasmin and selective matrix metalloproteinases. Pro-NGF binds with high affinity to p75NTR, a receptor that induces apoptosis in cultured neurons, with minimal activation of the differentiation/survival pathway that is mediated by the TrkA. It is now of interest to determine whether the biological actions of GDNF are also regulated by proteolytic cleavage and whether proforms of GDNF have functions that are distinct from those of the mature ligands.
Interestingly, in one recent primate study in which aged rhesus monkeys received unilateral intra-striatal infusions of 22.5 µg of GDNF per day (Ai et al, 2003a), the authors reported a maximal diffusion distance of 11mm from the catheter tip, in addition to retrograde and anterograde transport mechanisms of this protein. GDNF had diffused up to 11 mm from the catheter openings in the putamen into the rostral putamen, internal capsule, external capsule, caudate nucleus, and globus pallidus. Anisotropic flow along the external capsule tracts carried GDNF into the anterior amygdaloid area. Backflow of GDNF along the catheter track from the frontal cortex infiltrated juxtaposed corpus callosal and cortical tissue. In addition, following interaction with the receptor and internalization, GDNF was carried by retrograde transport to DA neurons in the ipsilateral SN, stimulating an 18% increase in the number of TH-positive DA neurons and a 28% increase in DA neuron perikaryal size. Also, TH-positive fiber density was increased in the ipsilateral globus pallidus, caudate nucleus, and putamen. Anatomic effects from GDNF stimulation of the DA system were restricted to the ipsilateral hemisphere. Retrograde GDNF labeling was also present in a few TH-positive neurons in the locus coeruleus and a large cluster of TH-negative neurons in the ventral anterior thalamus. The authors also reported anterograde transport of GDNF in axons in the pyramidal tract from the cerebral peduncle to the caudal spinal cord.
GDNF, was first isolated from the conditioned medium of cultured rat glial cells from the B49 cell line (Lin et al, 1993)as a potent neurotrophic factor described as having relative specificity for DA neurons within dissociated rat embryonic midbrain cultures (Lachyankar et al, 1997, Li Duan et al, 2002, Lin et al, 1993, Lin et al, 1994). GDNF and related factors, neuturin, artemin and persephin, constitute a family of neurotrophic factors distantly related to the transforming growth factor-β (TGF-β) superfamily (Airaksinen et al, 2002, Baloh et al, 2000). Although there is only limited amino acid-sequence homology between GDNF and prototypic members of the TGF-β family, they do share marked conformational similarity (Eigenbrot et al, 1997).
After intracellular processing, GDNF is secreted as a glycosylated mature protein of 134 amino-acid residues. In its active form, GDNF is a disulphide-bonded homodimer of Mr 32 kDa to 42 kDa (Lin et al, 1993, 1994). The first 37 N-terminal amino acids constitute a high-affinity, heparin-binding domain that may limit diffusion of the molecule in vivo through its interactions with extracellular matrix heparan sulphate proteoglycans, thereby allowing local concentrations to increase. GDNF undergoes N-terminal cleavage both in vitro, in mammalian cell cultures, and in vivo, in rhesus monkeys, but the truncated form, des37-GDNF, retains full biologic activity in promoting DA neuronal survival despite lacking the heparin-binding domain. Monoclonal antibody inhibition data also indicate that the N-terminal region of GDNF is not critical for activity (Xu et al, 1998). The human GDNF gene has been cloned, and recombinant human GDNF displaying full biological activity has been expressed in E coli (Lin et al, 1993).
GDNF signals through a unique multicomponent receptor complex comprising the specific GFRα1 receptor, which is anchored to lipid rafts within the plasma membrane by glycosylphosphatidylinositol, and the transmembrane Ret receptor that incorporates an intracellular Trk domain (Takahashi, 2001; Airaksinen et al, 2002). The process involves the binding of GDNF to GFRα1 and subsequent recruitment of Ret to the lipid raft, thereby triggering its association with Src, which is required for downstream signalling. The binding of GDNF-GFRα1 to the extracellular domain of Ret leads to activation of the intracellular Trk domain and the subsequent activation of intracellular signalling pathways that lead to neuronal differentiation and survival (Tansey et al, 2000).
GDNF-GFRα1 has also been shown capable of signalling in a Ret-independent manner via direct activation of an Src-like kinase in an immortalized neuronal precursor cell line not expressing Ret (Trupp et al, 1999), although the physiologic significance of this alternative mechanism remains to be clarified (Airaksinen et al, 2002).
Analyses of tissue distribution of GDNF mRNA have provided insight into the potential roles of this neurotrophic factor and indicate that GDNF acts as a target-derived neurotrophic factor for both DA and motor neurons. GDNF mRNA was expressed in the striatum and skeletal muscle, the target fields for DA SN neurons and motor neurons, respectively. In situ hybridization and reverse transcription polymerase chain reaction studies have demonstrated GDNF mRNA expression in many regions of the developing and adult brain, as well as in peripheral tissues (e.g. kidney, gut), indicating that GDNF may have multiple actions (Choi-Lundberg et al, 1995; Pochon et al, 1997; Trupp et al, 1997). Indeed, GDNF has potent survival promoting actions on other CNS and PNS neurons, including sensory and autonomic ganglia (Zurn et a l, 1994; B uj-B e llo e t al, 1995; Trupp e t a l, 1995), Purkinje c ells of the ce rebellum (Mount e t a l, 1995), loc us coe rule us neurons (Arena s et al, 1995), tha lamic and hippocampal neurons (Martin et al, 1995), as w e ll a s ne urons in the c ingula te d c orte x a nd olfa c tory bulb (Trupp et al, 1997), with effects on noradrenergic, serotoninergic, and cholinergic cell populations (Lin et al, 1993; Arenas et al, 1995; Beck et al, 1996; Lapchak, 1996; Martin et al, 1996; Williams et al, 1996; Weis et al, 2001).
Analysis of GDNFR-α mRNA demonstrated its presence from embryonic day 15 and distribution in the adult rat ventral midbrain, spinal cord, subpopulations of the dorsal root ganglia, developing kidneys (nephrons), and smooth and striated muscle associated with the enteric nervous system. It was also found in the retina, thalamus, pons, medulla oblongata, pituitary gland, urogenital tract and pancreatic primordium (Treanor et al, 1996). Similarly, elevated c-ret mRNA expression was observed in the adult rat spinal cord, pons, medulla, hypothalamus, thalamus and cerebellum. Levels of c-ret mRNA increased progressively during the postnatal development in the ventral midbrain containing the SN, with a peak of expression between postnatal day 6 and 8, the period during which axons of DA neurons of the SN make functional contact with the striatum (Trupp et al, 1996).
Jing et al (1996) showed that GFR-α was expressed by cultured rat spinal cord motoneurons and that addition of GDNF to these cultures led to autophosphorylation of Ret. Similarly, Trupp et al (1996) reported that GDNF bound to Ret induced Ret autophosphorylation in a GDNF-responsive motoneuron cell line derived from embryonic mouse spinal cord motoneurons. High levels of Ret were reported in adult rat spinal cord, pons, medulla, hypothalamus, thalamus and cerebellum, but Ret was barely detectable in the striatum, hippocampus and cortex. Interestingly, a progressive increase in Ret level was observed postnatally in rat ventral mesencephalon, and a high level of Ret was detected in the adult rat SN suggesting DA neuronal soma expression of Ret.
Knockout mice lacking GDNF (Moore et al, 1996; Pichel et al, 1996; Sanchez et al, 1996), GFRαl (Cacalano et al, 1998), or Ret (Schuchardt et al, 1994) die shortly after birth and share a phenotype of kidney agenesis and an absence of many parasympathetic and enteric neurons. The similar phenotypes of ligand and receptor knockouts indicate a specific pairing of GDNF with GFRα1 and Ret in vivo. In all 3 knockouts, there is a significant loss of spinal and cranial motor neurons and a corresponding increase in dying cells (Airaksinen et al, 2002). In contrast, motor neuron survival is promoted by the muscle specific overexpression of GDNF or by GDNF treatment in utero (Oppenheim et al, 2000). This finding may have therapeutic relevance: In a transgenic ALS model, virus mediated intramuscular GDNF expression led to enhanced motor neuron survival, resulting in delayed disease onset and increased survival of the mice (Acsadi et al, 2002; Wang et al, 2002). GDNF promotes survival of a subgroup of developing sensory neurons (Buj-Bello et al, 1995), which show reduced soma size in knockout mice lacking GDNF or GFRal (Baudet et al, 2000). GDNF has potent and selective effects on a subset of dorsal root ganglia cells involved in nociception (Bennett et al, 1998), and it has been reported as a therapeutic treatment in rat models of neuropathic pain states (Boucher et al, 2000).
Finally, analysis of adult GDNF hemizygous mice has shown another function of GDNF outside the brain: GDNF regulates spermatogonial differentiation, and low GDNF levels lead to disturbed spermatogenesis (Meng et al, 2000).
The a bil ity o f G D N F to s timu la te nigro s tria ta l fun c tion in i nta c t and lesioned animals may, at least in part, reflect a direct action of GDNF on the function of DA neurons. These effects, as obse rve d in in v itr o studies , include increa ses in the spontaneous firing rate and the quantal s ize of terminal DA rele as e (Pothos e t a l, 1998), as we ll as a n inc re a se d e xc ita bility of the D A ne urons that is media ted by A-type K + channels (Ya ng et al, 2001) and high voltage-activated Ca2+ channels (Wa ng et al, 2003).
Furthermore, in addition to its in vitro effects on neuronal survival, results from multiple studies on the effects of r-metHuGDNF in chemically lesioned rodents and rhesus monkeys, reveal both neuroprotective and neurorestorative properties, supporting the scientific rationale for its therapeutic use as a neurotrophic factor in the treatment of PD, and ALS (Zurn et al, 1994; Beck et al, 1995; Tomac et al, 1995; Yan et al, 1995; Gash et al, 1998a).
Infus ions of the rec ombina nt protein a re adva nta ge ous in tha t the dos e c an be w e ll c on troll e d a n d tha t the infu s ion c a n be s toppe d in c a s e of un w a nte d s id e e ff e c ts . The dis a dva n ta ge is th a t th e pro te in is de live r e d fr om a point s ou rc e , w hic h c re a te s s te e p c onc e ntra t ion g ra die nts a nd re s tric t s the a c c e s s of the fa c to r to the t is s ue tha t is c los e to the i nfus i on c a nnula . Lon g-te r m pro te in de liv e ry, mor e ove r , ma y be c ompli c a te d by m a inte na nc e prob le ms a s s oc ia t e d w i th th e inf us ion de vi c e .
Evidence from a number of laboratories suggests that GDNF is capable of halting or reversing the progressive degeneration of the nigrostriatal DA system in animal models of PD. Intranigral, intrastriatal and/or ICV injections of recombinant GDNF have been shown to be protective in rodent models of PD (Grondin et al, 1998; Rosenblad et al, 1999; Kirik et al, 2000a); and similarly in rhesus monkeys previously lesioned with MPTP (Gash et al, 1996). In monkeys, intraparenchymal delivery has been shown to be effective whether delivered by bolus injection or chronic infusion using a pump (Gash et al, 1998b; Grondin et al, 2002).
1. Recombinant GDNF infusion in rodents models of PD
Most studies investigating the in vivo properties of GDNF have been performed in the rat. Although crucial and informative, studies involving GDNF treatment in rodent models are limited in their relevance to the human. R o de nts h a ve a mu c h s m a l le r n e rv ou s s ys te m , w hi c h di ff e rs s ign if ic a nt ly in nu me ro u s n e u r o a n a t o m i c a l a n d n e u r o c h e m i c a l p a r a m e t e r s f r o m t h e h u m a n . I n c on tr a s t , n on hu m a n p r im a t e s po s s e s s a C N S a nd be ha v i o u r a l r e p e r t o i r e m u c h c l o s e r t o t h e h u m a n t h a n t h e r o d e n t .
Early studies showed that direct nigral injection resulted in local increases in DA levels accompanied by neuritic sprouting of TH-positive neurons and up-regulation of DA activity within the striatum (Hudson et al, 1995; Hebert et al, 1996). In keeping with such findings in the intact rat, nigral administration of GDNF was also found to protect nigral DA neurons from retrograde cell death after axotomy (Beck et al, 1995; Lu et al, 1997).
Initial studies demonstrated, to varying extents, the neuroprotective and neurorestorative effects of GDNF in classical rodent models of PD, in which virtually total destruction of nigral DA neurons was brought about by the injection of the DA neurotoxin 6-hydroxyDA (6-OHDA) into the SN or medial forebrain bundle (Hoffer et al, 1994; Bowenkamp et al, 1995, 1996, 1997; Kearns et al, 1995; Lapchak et al, 1997b). The variability inherent in the effects observed is epitomized by the wide range of doses of GDNF (10 to 1000 µg) reported to be effective.
Subsequent studies have used the closer analogue of PD provided by the partial lesion model, in which the unilateral striatal injection of 6-OHDA leads initially to the local destruction of DA axons with the subsequent gradual and protracted degeneration of cell bodies (Bjorklund et al, 1997). This model allows for the investigation of both the neuroprotective properties of GDNF in the acute phase of injury and its neuroregenerative properties in the later chronic phase, when a proportion of neurons persist in an atrophic and dysfunctional state.
A series of studies using the partial lesion model has indicated that the protection afforded DA cell bodies by GDNF is, on its own, insufficient to achieve functional recovery, which is associated with terminal axonal sprouting and reinnervation of the striatum (Bjorklund et al, 1997; Rosenblad et al, 1998, 1999, 2000b). For example, while the continuous infusion of GDNF over the SN for 14 days immediately after an intrastriatal 6-OHDA insult resulted in the considerable protection of tyrosine hydroxylase (TH)-positive cell bodies, it failed to prevent the progressive death of axons from the striatum. Despite subsequent multiple intrastriatal injections of GDNF resulting in axon spreading in the globus pallidus and close to the injection site in the striatum, no improvement was seen in motor behaviour 56 days after insult and no increase was seen in the overall DA innervation of the striatum (Rosenblad et al, 2000b).
It thus became apparent that functional recovery may only be achievable if GDNF were continuously available within the striatum, ideally over a much longer period.
2. Recombinant GDNF infusion in nonhuman primate models of PD
The number of studies performed to date in nonhuman primates is necessarily somewhat limited, and all have been relatively small. Nevertheless, it has been possible to examine the effect of GDNF in a hemiparkinsonian monkey model of PD, induced by carotid arterial infusion of the neurotoxin MPTP (1methyl-4-phenyl-1,2,3,6-tetrahydropyridine). This model is closer clinically to the human condition than may be achieved with rodent lesion models, where less than 20% of nigral DA neurons remain and striatal DA amounts are reduced by more that 97% (Gash et al, 1996; Gerhardt et al, 1999; Grondin et al, 2002).
In this model, a single unilateral intranigral or intracaudate injection of GDNF was initially shown to bring about significant functional improvement in the 3 cardinal parkinsonian features (bradykinesia, rigidity, and postural instability) within 2 weeks and to result in increased TH immunoreactivity and increased perikaryal size of midbrain DA neurons (Gash et al, 1996). This contrasts with the situation described previously in the rat, in which functional recovery appeared to be consistently achievable only through the continuous provision of GDNF to the striatum. Interestingly, the same degree of functional improvement could be achieved with repeated ICV bolus injections of GDNF at 4-weekly intervals within the dose range 100 to 1000 µg GDNF per injection (Gash et al, 1996; Zhang et al, 1997) without any reversal of the MPTP-induced depletion of DA in the striatum, although increased DA levels were noted within the SN, ventral tegmental area, and globus pallidus (Gash et al, 1996).
Of particular relevance to the potential usefulness of GDNF in the treatment of PD, ICV injection of GDNF was found to reduce the incidence of adverse responses typically vomiting, dyskinesias, dystonias, and stereotypy to L-dopa treatment. Moreover, since the effects of GDNF and L-dopa appear to be additive, the combined use of the two anti-parkinsonian agents may allow a reduction in L-dopa dosing (Miyoshi et al, 1997).
Sim ila r func t iona l c ha n ge s , a t bo th th e ne u roc he mic a l a nd be ha v iora l leve l, ha ve be e n obs e rve d in M PTP-le sione d monke ys in w hic h GDNF was administered by c onti nuous IC V or by intr a puta mina l de livery at 7.5-22.5 µg/da y (G rondin et al, 2002). As a pilot study had shown the threshold dose of GDNF required to bring about behavioral improvements in parkinsonian monkeys to be >3.75 µg/day, infusions over the first 4 weeks delivered a dose calculated to provide at least 5 µg GDNF/day after taking into account absorbance to materials. Four of the animals (3 intraventricularly catheterized, 1 intraputaminally catheterized) responded to this dose, but in an attempt to achieve a similar level of behavioral improvements in the remaining animals, the quantity of GDNF infused for the remaining 8 weeks was increased to provide a daily dose of at least 15 µg after correction for absorption.
Statistical analysis revealed no significant effect of the route of infusion on weekly behavioral scores, DA and DA metabolite levels in the striatum and globus pallidus, nigral cell size and number, or TH-positive fiber density. Behavioral, neurochemical, and histological changes were similar among dose administration routes.
The chronic infusion of GDNF was well tolerated and did not induce any observable adverse effects. Body weight loss, previously observed with repeated injections of GDNF (Gash et al, 1996), was not significant. No significant behavioral changes were seen in either group of animals throughout the initial 4-week period during which vehicle was infused.
However, while parkinsonian features continued to be expressed at the same level in control animals for the remaining 3 months of the study, a steady improvement was seen in GDNF-treated animals. An average improvement of 2.5 points was noted on the rating scale after 1 month of GDNF infusion, which had increased to
3.5 points after 3 months, representing an overall 36% improvement in disability. Within this, consistent improvements of up to 60% were evident in bradykinesia, rigidity, balance, and posture, at peak effect.
DA and dihydroxyphenylacetic acid (DOPAC) were increased by 233% and 180%, respectively, in the lesioned periventricular (medial) striatum of GDNF-treated animals, the response being more variable in the intermediate and lateral portions of the striatum. In addition, HVA levels were increased by about 70% in each of the medial, intermediate, and lateral portions of the nonlesioned striatum. The levels of DA, DOPAC, and HVA were increased by 155%, 190%, and 47%, respectively, in the lesioned globus pallidus of GDNFtreated animals; and HVA levels were increased by 67% in the non-lesioned globus pallidus.
In the GDNF-treated animals, there was a mean 5fold increase in the density of TH-positive fibers in the lesioned periventricular (medial) striatal region, ranging from a moderate increase in some animals to the creation of a dense fiber network in others. While the striatal response was restricted to the lesioned side, the effects of GDNF on nigral DA neurons were bilateral, and the numbers of these expressing TH were increased by > 80% on the lesioned side and by about 20% on the intact left side compared with vehicle-treated animals. Similarly, their perikaryal size was increased by > 30% on both sides.
The beneficial effects of GDNF noted in this study were attributed primarily to its neuroregenerative rather than its neuroprotective properties, since it was not administered until 3 months after MPTP-induced nigrostriatal injury, by which time the expression of parkinsonian features is known to be stable (Bankiewicz et al, 1986; Smith et al, 1993). In achieving a mean 3.5-point improvement on the behavioral rating scale, the chronic infusion of daily doses of 7.5 – 15 µg GDNF/day proved superior to monthly injections of between 100 – 1000 µg, previously shown to bring about improvements of 1.5 to
2.5 points (Gash et al, 1996; Miyoshi et al, 1997; Zhang et al, 1997). As also noted in rodent studies (Rosenblad et al, 2000b; Kirik et al, 2001), the results indicate the importance of residual DA fibers in the striatum for GDNF-induced recovery. The effect of GDNF in the lesioned side of the striatum occurred in the periventricular (medial) region, the area in vehicle-treated animals that had the greatest number of DA fibers and the highest concentration of DA itself. The action of GDNF in restoring striatal DA innervation may thus have been one of the principal components of recovery.
The bilateral nature of the effect of GDNF on nigral DA neurons, previously noted after injection of GDNF in hemi-parkinsonian monkeys (Gash et al, 1996), indicates the wide distribution of GDNF within the brain parenchyma. Because the globus pallidus receives DA input from the SN and is involved in regulating motor functions by sending outputs to the motor cortex via the thalamus (Smith et al, 2000), the bilateral effect of GDNF on pallidal DA and DA metabolite levels is also of importance. Recombinant-GDNF has been shown to be transported in an anterograde manner from the striatum to the globus pallidus in rats (Kirik et al, 2000b), and the same would appear to be the case in monkeys.
3. Recombinant GDNF infusion in aged monkeys
Compared with young animals, middle-aged rhesus monkeys display motor deficits that correlate with changes in the functional properties of DA neurons (Gerhardt et al, 1995).
In a s e rie s of furthe r s tudie s in prima te s , Gerhardt, Gas h and colleague s (Ma swood et a l, 2002; A i et al, 2003b, Grondin et al, 2003) ha ve s tu die d the e ffe c t of G D N F t ha t w a s infu s e d ove r a 2 -mont h pe r iod b y pro gra mm a ble s ub c uta n e ous pumps . The fa c t or w a s de l ive r e d IC V or di re c tl y int o the p uta me n ( un ila te ra l ly ) a t a d os e o f 7.5
22.5 µg/day in aged monkeys that showed an age-dependent decline in motor function. Delivered in this way, GDNF induced an improvement in motor function that developed gradually ov e r 2- 6 w e e ks a n d w a s ma in ta i ne d d ur ing t he 2- mon th w a s h -ou t period w he n G DN F w as re plac e d with ve hic le . Inc rea se d bas e line and e v ok e d D A r e le a s e , a s mo n i t or e d by m i c r o d i a l y s i s , w a s obse rve d bilate ra lly in SN , puta me n a nd c a uda te nuc leus in the IC V group (Grondin e t a l, 2003). Inc re a se s in DA and D A me tabolite s in tis s u e s w e re ob s e rve d, mo s t pro mine n tly o n the inje c te d s ide , in p uta me n, c a uda te nuc le us a nd g lobus pa ll idus in th e int ra put a mina l group (SN was not included in this analysis)(Maswood et al, 2002), indicatin g tha t a s us ta i ne d, ge ne r a l up re gul a t ion o f D A n e ur on fun c tion ma y be in duc e d by e ithe r rout e of a dm inis t ra tio n. In comparison with vehicle-treated animals, GDNF-treated animals had DA levels that were greatly increased in the caudate nucleus and globus pallidus on the lesioned side (by 50% and 390%, respectively). DOPAC levels were increased bilaterally in the caudate nucleus (by 122% on the lesioned side and 76% on the non-lesioned side) despite GDNF infusion having been on the lesioned side only. The non-lesioned increase may have been a result of intraventricular diffusion of the growth factor. Levels of HVA were increased in the lesioned putamen, caudate nucleus, and globus pallidus (by 217%, 212%, and 171%, respectively).
B. Ex-vivo gene therapy
Transpla nts of c ell lines ge ne tic ally enginee red to c ontinuous ly r e l e a s e ne u ro tr o ph ic fa c t o rs , u s e d a s b io lo g ic a l minipumps, cons titute a us eful tool to de live r the ra pe utic molec ules continuous ly and at a relative ly low dose dire ctly within the brain. However, xenoge ne ic ce lls would be re je c te d u nle s s the y a re prote c te d from the imm une s yste m of the hos t. N ove l mate rials have bee n e ngine e re d to s e rve a s a ba rr ie r a nd to s e qu e s te r tra ns pla nte d c e lls . The y consist of se mipermeable membrane s tha t allow the diffusion of s mall molec ule s suc h as nutrie nts a nd trophic fa ctors into a nd out of the polyme r enve lope, but pre ve nt immune rejec tion of the c ells (Lys aght et a l, 1999). Ce ll encapsulation has s e v e ra l impor ta nt a dv a nta g e s : ( i) it a llo w s th e s us ta ine d a n d loc a l iz e d de li ve r y of s m a ll d os e s of th e ra pe uti c molecules directly within the brain; (ii) xenogeneic cells ca n be trans planted in the abs ence of immunos uppre ss ion; (iii) in case of capsule breakage or appearance of side-effects, the capsule can be retrieved. Th e lim ita ti on of this " e x v iv o " ge n e tra ns fe r te c hniqu e , a t its current state of development, is that transgene expression may not be stable over time. With encapsulated cells, stable protein secretion has, however, been obtained for several months from devices implanted in the brain parenchyma (Aebischer et al, 2001).
1. E x - v i v o g e n e t h e r a p y i n r o d e n t m o d e l s o f P D
E nca psula ted genetically engineere d Baby Hamster Kidne y (BH K) ce lls or murine C 2C 12 myoblas ts re lea sing the neurotrophic fa ctors CN TF or GDNF have been successfully used for ne ur oprot e c tio n s tu die s in e xp e rime nta l mode l s of A L S, PD a nd H u nti ng ton 's di s e a s e (Z ur n e t a l, 1 994 ; Sa g ot e t a l, 19 96; E me r ic h e t a l , 199 7) .
E n c a p s u l a t e d G D N F - pr o d u c i n g ba b y ha m s t e r k i dn e y c e l l s h a v e b e e n i mp la n te d i nt o t he s t ri a t u m or SN i n ra t mod e l s o f PD a n d s ho w n a l s o to in c r e a s e D A fiber outgrowth (Lindner et al, 1995; Date et al, 2001) and a me li o ra te s o me be ha v io ra l d e f i c i ts ( Sh in g o e t a l , 2 00 2, T s e ng e t a l , 19 9 7) . Imp la nta tion of ot he r c e ll t ype s s uc h a s G D N F- produ c ing f i b r o b l a s t s , P 1 9 c a r c i n o m a c e l l s , o r a s t r o c y t e s h a s s h o w n t ha t t he y a re a l l a b l e to pr e v e nt n ig ra l D A n e u ro na l de a t h fol low in g a l e s ion (A k e rud e t a l, 1999 ; N a k a o e t a l, 2000; C unn ingha m e t a l, 2 002). M o r e o v e r , in tr a s t ri a ta l imp la nta ti on of G D N F -de li ve r in g mi c r os p he re s o r n e u r a l s t e m c e l l s e n g i n e e r e d t o p r o d u c e G D N F , o r t r a n s p l a n t a t i o n o f b o n e m a r r o w c e l l s t r a n s d u c e d w i t h G D N F, c a n a ls o i mp ro v e b e h a v i or a n d pr e ve nt D A d e g e n e ra t i o n i n ro d e n t m o d e l s
o f P D ( A ke r u d e t a l, 2 0 01 , G ou h i e r e t a l , 2 00 2 , Pa r k e t a l , 2 0 0 1) . G D N F e x p r e s s i n g n e u r a l s t e m c e l l s s t i l l d i f f e r e n t i a t e d int o a tr oc yte s , ne urons , or oli gode n droc y te s ; a nd e ve n a fte r d i f f e r e n t i a t i n g , t h e y c o n t i n u e d t o e x p r e s s G D N F . N e u r a l s t e m c e l l s o v e r - e x p r e s s i n g p e r s e p h i n , a m e m b e r o f t h e G D N F f a m i l y , a l s o d e m o n s t r a t e d i n - v i v o f i n d i n g s s i m i l a r t o t h o s e w i t h G D N F w h e n i n j e c t e d i n t o m o u s e s t r i a t u m .
2. Ex-vivo gene therapy in non-human primate models of PD
The effects reported in non-human primates implanted with encapsulated cells are limited and variable. In one study, baboons chronically lesioned with MPTP w ere impla nte d ICV w ith tw o ca psule s rele as ing G DN F (100-200 ng/capsule per day) 2 years after MPTP treat-me nt (Zurn e t al, 2001). Hypokinesia signific antly decrea sed in thes e anima ls, w ith the GD NF-impla nted anima ls almost completely back to the pre-les ion values a fte r 4 months . Importa nt sprouting of rema ining TH-pos itive fibe rs in the SN a nd the puta me n oc c urre d in the pre sence of G DNF. This study re porte d no deleterious side effects and s howed tha t low dose s of GDNF continuous ly r e le a s e d i n t o t h e C S F w a s s u f f ic i e n t t o revers e be havioral s ymptoms in M PTP-les ioned ba boons .
A nothe r study e xamine d the long-term efficacy and late complications of a xenotransplant approach utilizing GDNF-expressing encapsulated baby hamster kidney (BHK) cells (Blanchet et al, 2003). Five MPTP-lesioned parkinsonian cynomolgus monkeys received five devices containing active or inert cells grafted bilaterally in the striatum in a two-stage procedure 9 months apart and animals were sacrificed 4 months later for analyses. They observed no definite motor benefit. DA levels were comparable between GDNF- and control cell-implanted striata, and TH immunoreactivity in the SN showed no consistent recovery. Cell viability and GDNF synthesis in the explanted devices was found to be negligible. The brain tissue surrounding all implants showed an intense immune reaction with prominent "foreign body" inflammatory infiltrates. Membrane biophysics, the cell type used, and the extended period of time the devices remained in situ may have contributed to the negative outcome.
C. In-vivo gene therapy
Ex-vivo gene therapy using encapsulated ge ne tic ally e n gi ne e re d c e l ls c a n r e a dil y be us e d to de li ve r n e ur o-trophic factors for se ve ral w eeks or months. H ow e ve r, the s e me t hods ha ve re la t ive ly limi te d n e urot r o p h i c d e l i v e r y i n t e r m s o f t h e e x t e n t o f t h e i r d i f f u s i o n w i th in th e C N S a n d a ls o ge n e r a te a ra ng e o f pra c t ic a l a nd s a f e t y i s s u e s t h a t a r i s e t h r o u g h t h e n e e d t o b e a b l e t o c h r o n i c a l l y d e l i v e r G D N F , a s c e l l d e a t h i n P D i s p r o g r e s s i v e a n d s u s t a i n e d . A s a r e s u l t , m o r e e f f i c i e n t d e l i v e r y s y s t e m s h a v e b e e n s o u g h t , i n c l u d i n g t h e u s e o f vi ra l v e c tor s .
T he re a r e m a n y d if fe r e n t t yp e s of vi ra l v e c tor t ha t c a n b e c on s id e r e d fo r d e l i ve ri n g G D N F, i n c l ud i ng a d e n ovi ru s , a d e no -a s s o c i a te d vir a l ve c t ors , a nd le nt ivi ru s e s . A de novir a l ve c tors c a n infe c t bot h di vi din g a n d non -d ivi di ng c e lls a nd in c or po ra t e la r ge pi e c e s of D N A ; ho w e ve r , the y ma y c a us e inf la mma ti on a n d r e c rui t a n im m un e r e s po n s e w h e n i n tr od u c e d i nt o t he h o s t . A d e n o-a s s oc i a te d vir a l (A A V ) v e c tor s a re pote ntia l ly s a f e r a s the ma jor ity o f t h e vi r a l g e n o me i s r e m o ve d , a n d t h e y a r c c a p a b l e of in fe c ti ng no n- div id ing c e ll s w it h hi gh e f fi c a c y (S a m uls ki , 1 99 9). Th e y a r e t hu s i de a l fo r t r a n s f e c t i n g n e u r on s . T h e p r i m a r y d i s a d v a n t a g e o f A A V s is t ha t h igh l e ve ls of t ra n s g e ne e xpr e s s io n a re n ot a p pa re n t u n t i l 2 - 3 w e e k s f o l l o w i n g a d m i n i s t r a t i o n . I n c o n t r a s t , HIV-derived le ntivirus es e x pr e s s t he tr a ns ge ne a l mo s t im me d ia te l y a nd a re capable of stably integrating transgenes into the c hromos ome of pos tmitotic c ells such as diffe rentia te d ne urons (B lomer e t a l, 1997; Trono, 2000). In addition, in c ontra st to adenoviruses a nd he rpe s simplex virus, le ntiviral vectors do not e ncode vira l pr ot e in s a nd th e r e fo re do n ot e l ic i t a n im mun e re s po ns e ; a re c a pa bl e of c lon in g l a r ge a mo un t s of D N A ; a n d c a n e x pr e s s h ig h l e v e l s o f t he t r a n s ge nc lo ng -te rm .
Th e us e o f v ir a l ve c tors in pa tie nt s , ho w e ve r , ra i s e s imp orta n t s a f e ty i s s ue s re la te d t o the ir po te nti a l imm unoge nic it y, th e ir r is k o f mut a ge ne s is b y ins e rtio n int o the ge no me of the hos t c e lls and the possible side effects induced by the expressed transgene. For safety reasons, therefore, it may be necessary to use vector constructs, such as the tetracycline-regulated promote r, that a llow the e xpre s s ion of the: trans ge ne to be e xte rnally re gula ted or s hut off.
1. In-vivo gene therapy in rodent models of PD
I nt ra - ni gr a l in je c ti on of a re pli c a tio n- de f e c tiv e a d e n ovi ra l v e c tor e nc oding huma n G D N F in a rod e nt mod e l of PD r e s ult e d in i n c r e a s e d d o pa m i n c r g i c n e ur o n a l s u r vi v a l ( C ho i - L un d b e rg e t a l , 19 9 7 ; C o n n or e t a l , 19 9 9 ) a n d i n s o m e c a s e s improved behavior (Lapchak et al, 1997a). However, as d e s c r i b e d a b ov e w it h d i re c t G D N F in j e c ti o n s , G D N F- e x p r e s s i n g a d e n o v i r a l v e c t o r s d e l i v e r e d i n t o t h e s t r i a t u m a r e c on s i s te nt ly mo re e f fe c ti ve in p re v e n tin g bot h D A d e g e n e ra t i o n a n d b e ha v i o ra l d e f i c i ts ( B il a n g -B l e u e l e t a l , 19 9 7 ; C h o i L u nd b e r g e t a l , 1 99 8 ; K o z l o w s k i e t a l , 2 0 0 0) .
S im il a rl y, ( M a nd e l e t a l , 1 99 9, M a nd e l e t a l , 1 99 7) t ra ns d uc ti o n of rat nigral cells by GDNF-expressing AAV vectors either 3 w e e ks be fore or ju s t a f te r a n int ra s tr ia ta l 6-h ydrox yD A ( 6-O H D A ) le s ion re s ul te d i n a n inc re a s e i n D A ne urona l numbe r. In the s ame les ion mode l, K ink e l a l . ( K i r i k e t a l , 2 0 0 0 a ) inj e c te d A A V e ithe r int o the s tr ia tum , nig ra , o r bot h 4 w e e ks before the lesion. Intranigral injections were most effective at in c r e a s in g D A ne ur ona l s ur vi va l , but o nly i ntr a s t ri a ta l inj e c tio ns e n ha nc e d D A fi be r i nne rv a ti on a nd impr ove d motor be h a viou r. Si mila r ly, i ntra s tria t a l a d mini s t ra t io n o f G D N F- e xp re s s i ng A A V v e c to r s 4- 5 w e e k s a f t e r t h e l e s i o n p r e v e n t e d D A n e u r o n a l d e g e n e r a t i o n a n d p r om o t e d s i g ni f i c a n t b e h a v i or a l re c o v e r y ( M c G r a t h e t a l , 2 0 0 2, W a ng e t a l , 20 0 2 ) .
Fu rt he r mo re , i nje c t ion o f G D N F-e xp rc s s i ng le nt ivi ra l v e c tor s int o the s tri a t um or SN b e fo re le s i oni ng in r ode nt PD m od e l s p ro t e c te d D A n e u ro n s ( D e gl o n e t a l, 2 0 00 , G e o rg i e v s k a e t a l, 2 0 00 a , 20 02 a , b) a nd p r ov i d e b e h a vi o r a l r e c ov e ry ( B e ns a do un e t a l , 20 0 0) , a lt ho u gh s o me s p on ta n e o us mo to r d e f i c i ts ma y r e m a i n i mp a i r e d ( G e or g ie vs k a e t a l , 2 00 2a ) .
These findings, together with those of comparative studies from other laboratories (Connor et al, 1999; Kirik et al, 2000b), emphasize the need for continuous overexpression of GDNF within the striatum for full functional recovery. Whereas nigral administration of vector protects DA cell bodies within the SN, only striatal delivery leads to proper striatal reinnervation with consequent behavioral recovery. Furthermore, rather longer-term studies than have generally been done would seem more appropriate for the analysis of cellular and behavioral activities in that, in the longest-term study done (Kirik et al, 2000b), reinnervation of the lesioned striatum occurred only gradually over 4 to 5 months.
3. In-vivo gene therapy in non-human primates (PD models and aged)
K or do w e r e t a l . ( 20 00 ) s t ud y i s p e r ha p s mo s t c on vi n c i ng w i th re ga r d to le nt i vi ra l v e c t or de l ive ry of G D N F. Pa rkins onia n R he s us mo nke ys re c e ive d i n tr a s t ri a t a l a n d i n t r a n i g r a l i n je c t i on s 1 w e e k a f t e r M P TP i n s u l t , resulting in the complete neuroprotection of nigral DA neurons and progressive functional improvement in a specific task that correlated with the degree of enhancement of striatal TH immunoreactivity. Si mi la r ly , t he simultaneous injection of the same amount of vector into multiple locations within the SN and striatum of aged monkeys resulted in an increase in a number of markers of DA function within both these regions of the brain. M o re o ve r, th e n um be r o f D A c e lls w it hin t he s t ria tu m, w h ic h n orma l ly in c re a s e s f ol lo w in g D A de p le ti o n, w a s e n h a n c e d i n b ot h a ge d a n d p a r k i n s o n i a n m o n k e y s f o l l o w i n g l e n t i v i r a l G D N F d e li v e r y ( P a l f i e t a l , 2 0 0 2) . Th e r e fo r e , t h e us e o f l e nt i v i r u s e s t o d e l i v e r G D N F i n P D p a t i e n t s a p p e a r s m o s t e nc ou r a g in g , gi v e n t h e s e p os it i ve r e s u lt s i n a n on hu m a n pr im a te m ode l a s w e ll a s de mo ns t ra tio n of c o nt inu e d lo ng -t e r m e xp re s s i on of G D N F 8 mo nt h s a f t e r i ni ti a l a d m in is t r a t i o n o f t h e l e n t i v i r u s ( K o r d o w e r , 2 0 0 3 ) . H o w e v e r , i n the s e s t ud ie s t w o a n ima ls di e d w i th in a w e e k fol lo w in g le n ti vir a l ve c t or de liv e r y. A l th o u g h t h e a u t h or s a tt r i b ut e d th e s e d e a t hs t o M P T P t o xi c i t y r a t he r t ha n vi ra l v e c tor a dmi ni s tr a t ion , c a u ti on is w a rr a nt e d re ga rdi ng th e s a f e t y o f thi s approach in treating neurodegenerative diseases such as PD (Bjorklund et al, 2000; Kordower et al, 2000).
D. C om bin ed s ymp tom at ic an d n eu rop rotective th erapy: improved mesencephalic graft survival with GDNF
In l a s t d e c a de s n e u ra l t ra n s p la n ta tio n of fe ta l D A t is s u e i nt o t he de ne r va te d s tr i a t um ha s b e e n c o n s i d e r e d a s a n a p p r o a c h t o r e p l e n i s h s t r i a t a l D A l e ve l a nd t o r e fo r m th e n ig r o s tr i a t a l p a th w a y ( B jo r k l un d , 19 9 2 ; M e nd e z e t a l , 1 9 9 2; N i kk h a h e t a l, 1 9 95 ) . H ow e ve r l i m i t s o f d o n o r f e t a l t i s s u e a t t h e t i m e o f t r a n s p l a n t a t i o n , p o o r c e l l s u r v i v a l ( ~ 5 - 1 0 % ) ( K o r d o w e r e t a l , 1 9 9 8 ) a n d l i m i t e d h o s t r e in n e r va t i o n a r e t h e m a j o r s h o rt c o m in g s of t h is a p pr o a c h. A l th o u g h t h e r e a s o n s fo r t he p o or s u rv i v a l o f t r a n s p l a n t a r e y e t t o b e e s t a b l i s h e d , i t i s l i k e l y t h a t p o o r c e l l s ur vi v a l o w e t o ma n y fa c to rs s u c h a s m i s m a t c h c o n di t io ns , l a c k o f t ro ph i c s u p po rt , f re e r a d i c a l m e d ia t e d t o xi c i t y ( J e nn e r, 1 99 4) a n d a p o p t o s i s ( Z a w a d a e t a l , 1 9 9 8 ) . A t t e m p t s h a v e b e e n m a d e t o i m p r o v e t h e s u r v i v a l o f g r a f t e d n i g ra l n e u ro n s us i ng n e ur ot r op hi c f a c t or s ( M a ye r e t a l, 1 99 3; J o ha n s s on e t a l , 19 9 5; T a ka ya m a e t a l , 1 99 5; R os e n b la d e t a l , 1 99 6 ; Si n c l a i r e t a l, 1 9 96 ; Y ur e k e t a l , 1 99 6) , a n t i o x i d a n t s ( N a k a o e t a l , 1 9 9 4 ; D u g a n e t a l , 2 0 0 1 ) a n d a n ti a po pt oti c a ge nt s (M yt ili ne ou e t a l , 199 7; Sc hi e rl e e t a l , 1 99 9).
Injection of recombinant GDNF close to fetal ni gr a l gr a f ts ha s b e e n s ho w n to in c r e a s e gra ft s u rv iva l, fiber outgrowth, and gra ft func tion, le ading to more rapid and more complete compe nsa tion of ampheta mine-induc ed turning be havior in rodent mode ls of PD (Rose nblad et a l, 1996, Sulliva n e t al, 1998). The s e prote c tive e ffe c ts ha ve be en obta ine d w ith re pea te d inje ctions of microgram quantitie s of GDN F (R os enbla d e t al, 1996).
In an ex vivo g e n e t h e r a p y s t r a t e g y t o c o n t i n u o u s l y d e l i v e r s m a l l a m ou nts o f G D N F c lo s e to fe ta l n ig ra l gr a f ts (S a u tte r e t a l , 1 99 8), e nc apsulated ge netic ally enginee re d B HK ce lls releas ing G DN F w ere implante d intrastriata lly in rats tha t had been unilaterally lesioned with 6-hydroxyDA 5 weeks earlier. Then 1 week later, the rats received intrastriatal grafts of ve ntra l me s e nc e phalic tis s ue. A vera ge in vitro G D N F r e l e a s e s f r om c a ps ul e s a s s e s s e d b y EL I SA w e re 3 9 ± 0 . 4 n g / d a y p r i o r t o i m p l a n t a t i o n a n d 1 0 9 ± 3 1 . 6 n g /d a y a t e x pl a n t a t i o n 7 w e e k s l a t e r . M e s e n c e ph a l i c grafts from ra ts with BH K -G DN F c aps ules c onta ined a 2.5-fold highe r number of TH immuno-re a c tive c e l ls c o mpa re d to gra ft s fro m c on trol ra ts . In a ddition, the re we re a signific antly larger number of fibe rs b e tw e e n t h e g r a ft a n d t h e c a p s ul e i n t h e B H K - G D N F group compare d to the B HK control group. Evaluation of druginduc e d rotation s how ed that there w as a ne ar complete re ve rsa l of turning 3 we eks post-grafting in ra ts with BH KGD NF ca ps ule s, while c ontrol rats showe d only little re c o ve r y a t 3 a nd 6 w e e k s (S a u tte r e t a l , 1 99 8). Th us , im pr ove d gra ft survival and increased sprouting of DA fibers le a ds to a c c e le ra t e d a n d mor e c om ple te be ha viora l re covery.
Sinc e impla nta tion of ca ps ule s 1 w e e k prior to grafting of e mbry onic tis s u e w ou ld no t be a va l id a pp roa c h for c li ni c a l t r ia ls , e ff e c t s o f G D N F on gr a f t s ur vi v a l a n d function w e re e valua ted in the ra t a fter s imultaneous co-transplantation of grafts and capsules. The type of lesion and the size of the mesencephalic grafts were the same as in the pre vious study, exc ept tha t genetica lly e ngine ere d C2C12 myoblasts releasing GDNF were used instead of B HK c e lls. C on ti n uo us de livery of GD NF via enc apsulated ge netic ally engineered cells thus constitutes a n efficient mea ns for improving gra ft s u rv i v a l a n d f u n c t i o n . I n a d d i t io n , it m a y r e d uc e t he amount of huma n e mbryonic donor tissue required for trans planta tion in PD patie nts .
III. Human studies
A. Ven tricu lar d elivery of recombinant GDN F
B a s e d on the promi s ing s tudi e s of the e ffe c ts of G D N F in a nima l mod e ls o f PD , a n in iti a l c l in ic a l tri a l te s t ing G D N F b y v e n tri c u la r d e li ve ry us ing a n indwelling reservoir was carried out in 50 parkinsonian patients for 8 months in a randomized, double-blind placebo-controlled trial (Nutt et al, 2003). While the doses of GDNF (25-4000 µg/month) were in excess of those e m p l o y e d f o r n o n h u m a n p r i m a t e s t u d i e s , l i t t l e t h e r a p e u t i c e f f i c a c y w a s obs e rve d in t he s e pa rki ns oni a n pa tie nt s a nd in f a c t w a s a s s o c ia te d w it h mul tiple s ide e ffe c ts i nc lud ing na u s e a , vomit ing, a nore xia , w e igh t los s , pa ra e s t he s ia s a nd hyp ona tr a e mia . Furthermore, a postm or te m r e p o rt o n o ne 6 5- ye a r- ol d p a t i e n t, w i th a 23 - ye a r hi s t o ry o f P D , th a t h a d r e c e iv e d mo n th ly in je c ti on s o f G D N F w it h no s y mp tom a t ic im pro ve me n t ha d n o a p p a r e n t D A r e g e n e r a t i o n o r G D N F d i f f u s i o n f r om t h e v e n tr i c l e i n to a p pr o p r ia t e br a i n r e g i on s a t p o s tm o r t e m ( K o r d o w e r e t a l , 1 9 9 9 b ) . Th e p ro bl e m ma y h a v e b e e n w it h t he s it e a nd me th od of de li ve ry; i.e . mont hly inj e c tio ns of the troph ic fa c tor into the la tera l ve ntric le . Suffic ient titre s of G DN F may not ha ve diffus e d through the ve nt ric ul a r w a ll a n d bra in pa re nc h yma t o the ta rg e te d D A ne uro ns in the SN a n d the ir a f fe re n t pro je c ti ons t o the put a me n.
B. C hron ic in t rapu t am en al GD N F d elivery u s in g p rogramm ab le pumps In our phase I safety study, five advanced PD patients (Table 2) with a previous history of g o od r e s p o n s e s t o L - d o pa u n de r w e nt s t e r e o t a c t i c u n i l a t e r a l ( P 1 ) o r bi l a t e r a l i n s e r t i on ( P 2 – 5 ) o f in - h o us e d ru g i nf us i on c a th e t e rs (F ig ur e 1 , 2 ) i nt o t he po s t e ro -d o rs a l pu ta m e n ( F ig ur e 3) . Infusion into the postero-dorsal putamen (i.e. its sensorimotor component) was chosen because in PD this is the most severely dopamine depleted region. We anticipated that if clinical benefits were shown, this would be due to local dopamine terminal sprouting in the putamen along with retrograde transport of GDNF down the surviving nigro-striatal axons, as previously reported in primate models (Figure 4) (Grondin et al, 2002).
H um a n re c o m bi na nt G D N F w a s c hr on ic a ll y i nf us e d v i a in dw e ll in g S yn c hr oM e d™ p um ps imp la nte d in the a bd omina l re g ion (Fig ur e 5). Pa tie nts w e re a s s e s s e d before and after surgery a c c ordi ng to the c ore a s s e s s me nt pro gra m for i ntra c e re br a l tr a n s pl a n ta t io ns (C A PI T) , i n or de r t o doc um e nt c ha n ge s i n dis e a s e s e ve r it y a nd m e d ic a ti on re qu i re me n ts ( La ng s to n e t a l , 1 99 2 ). T he p a t ie n ts a l s o u n de rw e nt q u a l it y o f l if e a nd n e u ro p s y c h o lo gy a s s e s s m e n t s ; a nd 18 F- d op a p o s i t r o n e m is s i o n t o m og r a p hy s c a n s a t b a s e l i n e a n d a t 6 -
Figure 3. Stereotactic MRI-directed targeting of postero-dorsal putamen for catheter implantation. Baseline 18F-dopa PET scan used for co-localization within the posterior putamen dopamine deficient areas.
Figure 4. Hypothesis: GDNF infusion into the postero-dorsal (sensorimotor) putamen is retrogradely transported to the substantia nigra down surviving dopaminergic neurons leading to upregulation, neuroprotection and neurorestoration through neurite branching
Figure 5. Intraparenchymal catheters connected to SynchroMed pumps implanted in the abdominal wall for GDNF infusion
m o nt h l y i n t e rv a l s a f t e r G D N F inf us ion to a s s e s s put a me n D A te rmina l fun c tion a nd c orre la te t hi s w it h a ny s ym pt o ma ti c b e n e fi ts .
After implantation, the SynchroMed pumps were primed with recombinant-methionyl human GDNF (rmetHuGDNF) (Amgen Inc., Thousand Oaks, California) and programmed to deliver a continuous infusion of 14.4µg of r-metHuGDNF per putamen per day at rate of 6µl per hour. The pumps were refilled monthly with fresh solution.The low concentration of r-metHuGDNF was maintained for a period of 8 weeks. At 2 months the pumps were refilled with fresh solution of higher concentration and programmed to deliver 43.2µg of rmetHuGDNF per putamen per day at a rate of 6µl per hour. Providing good tolerance and no side effects, this dose was to be maintained for the duration of the trial (12 months). However, due to the development of local reversible high-signal MRI changes of uncertain significance, the infusion parameters were altered to deliver lower doses (10.8 – 14.4µg of r-metHuGDNF) at lower rates (2-6µl per hour), in attempt to establish safe and clinically effective parameters, with repeat MRI monitoring at regular intervals up to 12 months. Between 12 and 18 months, all patients received a continuous infusion of 14.4µg of r-metHuGDNF per putamen per day at rate of 6µl per hour. At 18 months the dose of GDNF was increased to 28.8µg per putamen per day at rate of 6µl per hour, and remained so until 24 months except in P4 who reverted back to 14.4µg at 20 months.
I n t h i s p r e d o m i n a n t l y s a f e t y t r i a l d r u g - i n f u s i o n w a s t o l e r a t e d w e l l a n d s i d e e f f e c t s w e r e l i m i t e d ( T a b l e 2) a n d i n c l u d e d consistently L’hermitte’s phenomenon,
Table 2. Patient data and overall effects of GDNF which occurred in all patients and remained mild, non-distressing, and intermittent. Other side effects, which were inconsistent and intermittent, included non-specific headaches, vivid dreams, taste and smell abnormalities, apthous ulceration and hypersalivation. There was no nausea, anorexia, vomiting, weight loss or hyponatraemia reported as in the previous intraventricular trial (Kordower et al, 1999b; Nutt et al, 2003). In all patients, T2 MR images showed a region of high-signal intensity around the tips of the catheters with drug infusion. This response varied between patients, and even between the two hemispheres in bilaterally implanted cases. The signal change was most evident following the dose escalation of GDNF. The explanation for this signal change remains unclear as documented previously (Gill et al, 2003), however, with consistency and stability of findings, we are inclined to believe that these areas of high signal represent GDNF protein build up (Figure 6).
C h r o n i c G D N F i n f u s i o n r e s u l t e d i n i m p r o v e d m o t o r f u n c t i o n i n a l l p a t i e n t s , r e d u c t i o n i n " o f f ” - t i m e d u r a t i o n a n d s e v e r i t y , r e d u c t i o n i n d y s k i n e s i a s d u r a t i o n a n d s e v e r i t y , a n d a c o r r e s p o n d i n g i n c r e a s e i n g o o d " o n " - t i m e d u r a t i o n . A f t e r 1 2 m o n t h s , t h e r e w a s a 39% improvement in the off-medication motor sub-score of the Unified Parkinson’s Disease Rating Scale (UPDRS) and 61% improvement in the activities of daily living sub-score (Figure 7, Table 3). In all patients, the rate of symptomatic improvement was maximal in the first 3 months of GDNF infusion and, thereafter, there was slower but sustained improvement up to 24 months.
|Duration of PD||6||13||30||27||19|
|Unilateral/bilateral pump (U/B)||U||B||B||B||B|
|L-DOPA equivalents at 0 months||667||615||2154||680||762|
|Change in L-DOPA at 1 year||+10%||+6%||-51%||+10%||-44%|
|Procedural Adverse Events|
|Repositioning of catheter||*|
Other Clinical Effects
Recovery taste/smell * * * Revival sexual function * * * Improved bladder function * Reduction in tinnitus *
Figure 6. Peri-catheter high-signal changes at high – 43.2 µg/putamen/day (a), and after reduction to the low dose – 14.4 µg/putamen/day (b) in P1 (* with unilateral administration)
Table 3. Effects of GDNF on UPDRS and CAPIT clinical rating scores off and on medication
|Time post GDNF treatment|
|Meds||Baseline||3 Months||6 Months||12 Months|
|UPDRS I - Total||Off||66 ± 15||46 ± 8.9 (-30%)||44 ± 6.5 (-33%)||35 ± 11 (-48%)|
|On||28 ± 3.7||15 ± 3.2 (-48%)||20 ± 2.4 (-28%)||15 ± 3.4 (-45%)|
|UPDRS II - Activities of daily living||Off||21 ± 3.7||15 ± 3.0 (-30%)||13 ± 2.7 (-37%)||8.2 ± 3.3 (-61%)|
|On||5.2 ± 2.2||2.0 ± 1.6 (-62%)||3.4 ± 0.9 (-35%)||2.6 ± 2.3 (-50%)|
|UPDRS III - Motor examination||Off||33 ± 6.9||25 ± 4.8 (-24%)||23 ± 2.9 (-32%)||20 ± 7.5 (-39%)|
|On||10 ± 2.8||6.6 ± 3.6 (-39%)||9.0 ± 4.1 (-17%)||6.8 ± 4.2 (-37%)|
|UPDRS IVa - Dyskinesias||On||5.0 ± 2.6||1.8 ± 1.1 (-64%)||3.0 ± 1.5 (-40%)||1.8 ± 1.1 (-64%)|
|UPDRS IVb - Fluctuations||On||4.8 ± 2.6||3.2 ± 1.9 (-33%)||3.8 ± 1.6 (-21%)||3.4 ± 1.9 (-29%)|
|CAPIT - Pronation - supernation||Off||38.4 ± 23||16.8 ± 4.9 (-56%)||16.0 ± 4.0 (-58%)||14.1 ± 4.4 (-63%)|
|On||14.0 ± 3.2||11.6 ± 2.1 (-17%)||11.2 ± 1.9 (-20%)||10.9 ± 2.3 (-20%)|
|CAPIT - Hand-arm movements||Off||18.4 ± 5.6||10.4 ± 2.9 (-43%)||9.3 ± 1.9 (-50%)||8.6 ± 3.2 (-53%)|
|On||7.0 ± 1.8||5.6 ± 1.0 (-20%)||5.5 ± 1.1 (-22%)||5.6 ± 1.5 (-28%)|
|CAPIT - Finger dexterity||Off||64.8 ± 45||27.7 ± 8.8 (-57%)||28.2 ± 7.3 (-56%)||24.5 ± 5.1 (-62%)|
|On||27.4 ± 9.4||20.9 ± 5.3 (-24%)||21.6 ± 3.9 (-21%)||19.8 ± 3.5 (-28%)|
|CAPIT - Leg movements||Off||17.1 ± 6.7||8.4 ± 1.8 (-51%)||7.4 ± 2.1 (-56%)||7.5 ± 1.3 (-56%)|
|On||6.6 ± 0.5||5.7 ± 0.6 (-14%)||5.7 ± 0.4 (-14%)||5.5 ± 0.4 (-17%)|
|UPDRS - Numbers represent average scores for 5 patients ± SD|
|CAPIT - Numbers represent time (seconds) to complete task ± SD for the left limb|
Medication induced dyskinesias were reduced by about 70%. 18F-dopa PET showed a 16-28% increase in putamen and nigral DA storage at 18 months, with uptake in the putamen shown to be greatest in the region immediately surrounding the catheter tip, suggesting a direct effect of GDNF on DA function (Figure 8, Figure 9). Health-related quality of life measures (PDQ-39 and SF-36) showed general improvement over time, with the overall scores tending towards levels expected in a control population. Neuropsychological assessment results indicated no significant detrimental effects of GDNF infusion on cognition.
Three patients had long-standing loss of sensation of smell and taste, as is often the case in PD. These symptoms greatly improved or resolved completely between 3 and 6 weeks of GDNF infusion (Table 2). However, with this recovery, abnormal sensations of taste were intermittently experienced, with “metallic” or “soapy” tastes being reported. At the highest dose of GDNF, three patients reported recovery of normal sexual function, both in terms of interest and potency. This recovery subsided as the dose was reduced.
We conclude that GDNF delivered by intraparenchymal infusion is safe, causes significant symptomatic improvement, and represents a potential neuroprotective and restorative therapy for PD. The early changes in sense of smell and motor function suggest an initial pharmacological action of GDNF within the putamen, likely, in part, to involve a direct stimulatory effect on DA release as shown in rodent models (Hoffman et al, 1997). The reduction in dyskinesia duration and severity was not related to levodopa equivalent medication reduction in these patients, suggesting that GDNF might regulate DA production, release and metabolism in the striatum, thus improving the processing of motor output; quality of life when on medication, and as seen in primates and this may explain why our patients experienced better (Miyoshi et al, 1997).
Figure 7. UPDRS scores at 0, 3, 6 and 12 months following GDNF infusion in the “off” and “on” medication states. (A). UPDRS total scores, (B). UPDRS III (motor subscores), (C). UPDRS II (activities of daily living subscores)
|Pre-op||6 months||12 months|
|P1 (�) P2 (�) P3 ( P4 ( ) P5 (�)||0.0061 0.0045 0.0041 0.0051 0.0039||0.0091 (+49.2%) 0.0052 (+15.6%) 0.0054 (+31.7%) 0.0051 (0%) 0.0047 (+20.5%)||0.0068 (+11.5%) 0.0040 (-11.1%) 0.0051 (+24.4%) 0.0071 (+39.2%) 0.0051 (+30.8%)|
|Mean S.D. P values||0.00474 0.00089||0.0059(+24.5%) 0.0018 0.042||0.00562(+18.6%) 0.0013 0.048|
Figure 8. GDNF increases 18F-Dopa influx. A, PET image of P1 before GDNF infusion. B, same patient 12 months after unilateral GDNF infusion to the putamen. Circle represents region of interest around the catheter tip used in the analysis. c, Values of the 18FDopa influx constants (Ki) for the five patients preoperatively and at 6 and 12 months postoperatively. The values for P1 are for the right side only the remaining values are an average of both left and right sides. The percentage change from baseline is represented in brackets. P values based on one tailed student t test vs pre-op. * Patient moved in scanner
Figure 9. Statistical Parametric Maps demonstrating the spatial distribution of regions of increases in 18F-dopa uptake in the total patient group following 6 months of GDNF delivery (darker boxes indicate significantly increased regions: P<0.05, uncorrected at cluster level). a, coronal section and b, sagittal section. Put = putamen; SN = substantia nigra.
T hi s e xc it i ng i n it ia l t ri a l is be in g f ol l ow e d up i n t he P ha s e - II m u lt ic e nt e r ra nd o mi z e d p la c e b o- c on tr o ll e d tr ia l U ni te d States, at the University of Kentucky, with an s po ns o re d b y A m g e n I n c . u s in g b il a t e ra l c hr on i c FDA-approved Phase-I safety t r i a l o n t h e u s e o f a dm in i s t ra t io n i n pa t ie nt s w it h a dv a nc e d PD . u n i l a t e r a l c h r o n i c a l l y a d m i n i s t e r e d G D N F i n t e n p a t i e n t s w i t h a dv a n c e d P D ; a n d a n F D A -a p pr ov e d
I V . C on clu sions: Tow ards fu rth er clin ical trials
T h e q u e s t i o n r e m a i n s a s t o w h e t h e r t h e s e t h e r a p i e s w i ll t r a n s l a te t o t h e c l i n i c , e s pe c i a ll y g iv e n t h e r e c e n t c o n t r o v e r s i e s s t e m m i n g f r o m n e u r a l t r a n s p l a n t s ( F r e e d e t a l , 2 0 0 1 ; O l a n o w e t a l , 2 0 0 3 ) . These controvers ie s h a v e c on c e n tr a t e d on th e d e v e l o pm e n t o f d ys ki n e s ia s p os t -t ra n s p la n ta ti o n th a t in s om e c a s e s ha ve be e n di s a b li ng ( O l a n o w e t a l , 2 00 1, 2 00 3) . T he o r ig in of t h e s e m ot or a b no r m a li t i e s r e m a i n s o b s c u r e , b u t i t h a s b e e n a r gu e d th a t t he y a re r e la te d t o e it he r e xc e s s iv e D A r e l e a s e in t h e s t r ia t u m ( F re e d e t a l , 2 0 01 ) , un e v e n d i s tr i b u ti o n of D A re l e a s e a c ro s s th e s tr ia t um ( M a e t a l, 20 02 ) , o r e v e n no t e no ug h D A re l e a s e (O la now e t a l, 20 03 ). In a d di tio n, qu e s tio ns ha ve a r is e n a s t o w he the r the d ys k in e s i a s o n ly o c c u r i n s o m e t y p e s
As this was a Phase I safety trial with no control group, there is a danger of over interpreting the significant reductions in UPDRS and timed motor scores. However, the substantial changes in the clinical status of the patients in this trial warrant further discussion. It is unlikely that the effects could be related to lesioning or inflammatory changes from the catheter and GDNF infusion because: (a) a putamenal lesion is likely to make the patients worse (Bhatia et al, 1994) and (b) the PET data showed increased 18F-dopa uptake in a region around the catheter making tissue toxicity unlikely. It is important to note that placebo effects are known to occur in drug treatments for PD, but patients generally improve 30% at most and this is rarely sustained on repeated testing over 6 months (Goetz et al, 2000). Furthermore, no placebo effect has been seen in two double blind controlled neurosurgical interventions for PD (Freed et al, 2001). Our overall 57% reductions in “off” UPDRS scores are higher than might be expected from placebo. These improvements were progressive, with the off period scores at 24 months tending closely towards the baseline best on period scores. A decline in most CAPIT timed tests adds further evidence substantiating an overall subjective improvement. With caveats about lack of blinding, small patient numbers and the need for further double-blinded randomized placebo controlled trials, the investigators feel that GDNF is likely to be responsible for a substantial part of this improvement in clinical status.
T he p o te nt i a l o f G D N F a s a t he r a p e u t ic a g e n t i n P D , s t e m s f ro m i ts a b il it y n ot on ly to p r ov id e s ym pt o ma ti c r e l i e f , b ut a l s o t o m od i fy t h e di s e a s e s t a t e , d is ti n c t f r om o t he r c ur re n t th e ra pe u ti c s tr a t e gi e s fo r P D , s uc h a s de e p br a in s t im ul a ti on a n d D A re p la c e m e n t t he ra p y. Y e t, i t i s p os s i b le t h a t a c o mb i ne d a pp ro a c h u s i ng , f or e x a m p le , f e t a l D A c e ll g r a f ts a n d G D N F m a y p ro ve to b e e ve n m or e p ot e nt i n r e v e rs in g t he p a r ki n s o ni a n s y m pt om s i n pa t ie nt s . Ex p e r im e nt a l l y, G D N F i s bo t h ne u ro pr o te c t i ve a n d a b l e to in du c e a p ro mi n e n t f un c t i on a l up re g ul a t i on i n i nt a c t a n d le s io ne d n ig ra l D A n e u ro n s ; a n d in s o me c a s e s , it c a n a ls o i nd uc e a p ro n ou nc e d re g e n e r a ti ve re s p o ns e . Th e v ir a l ve c t o r e xp e r i me nt s , in pa rt i c u la r , in d ic a t e t ha t t he mo s t p ro no u nc e d fu nc t io na l e ff e c t s m a y r e s u lt fr om a c om bi n e d a c ti on in vo l vi ng a l l t hr e e me c h a ni s m s . Th e e xt e n t o f t he s e e f fe c ts i n o ur hu ma n p il o t s t u dy r e ma in s t o be fu ll y e lu c id a t e d. T h e e a r ly o n s e t o f s y m pt om a ti c i mp ro v e m e n t , a c c om pa n ie d b y a n in c r e a s e i n 18 F -d op a u pt a k e l im i te d t o th e a re a i mm e di a t e ly s u rr ou n di ng th e c a n nu l a ti p , s e e ms c o mp a t i bl e w it h a f un c ti on a l u pr e g u la ti o n in re s i d ua l D A ne u ro ns . T he pr og r e s s i v e a nd s u s t a i n e d i m pr ov e me nt in s y mp to m ol og y , a n d t he i nc re a s e d 1 8F -d o pa u p ta ke th ro u gh ou t t he w h ol e p ut a m e n a t 24 -m o nt hs , s ug g e s ts re du c e d p r og re s s i on of d is e a s e (n e ur op r ot e c t io n) , a nd a n u n qu a n t if ia b le r e g e n e ra ti v e re s po ns e b e y o nd f u nc ti o na l u pr e g u la ti o n.
T he h u ma n i nt ra p a r e n c hy ma l s tu d ie s make an important first step towards demonstrating both safety and efficacy of growth factors delivered directly into the brain parenchyma; and c ou ld no t o nl y h e l p d e s ig n b e t t e r t re a t m e n t for PD, but could also lay the foundation for further related studies in other neurodegenerative diseases such as AD, ALS and Huntington’s disease where various neurotrophic factors have also been shown to have beneficial effects in animal models (Kordower et al, 1999a; Beck et al, 2001; Sofroniew et al, 2001). H o w e v e r , w h i l e t h e d a t a f r o m o u r in tr a pa re nc h ym a l c l ini c a l t ri a l in hu ma ns lo ok e n c ou ra gin g (G il l e t a l, 2 003 ), re s ul ts fr om a c u rre nt ph a s e I I mul ti c e n tr e , ra ndo mi s e d , pla c e bo- c o nt rol le d s tu dy in 34 p a ti e n ts a n d f ur the r bli nd e d e f fi c a c y tri a l s w il l n e e d t o be c o ndu c t e d be f ore i t c a n be de t e rmin e d if chronic treatment with GDNF or other trophic molecules will prove useful i n tr e a t in g p a t i e n ts w it h P D .
D ue to t he pr ogre s s ive na tur e of PD , s us ta i ne d o r c on tinuo us de live ry o f t ro p hi c f a c to r s ma y b e n e c e s s a r y f or o p ti ma l , lo n g- te r m ne u ro na l effects. It is clear from animal studies that functional upregulation in intact or lesioned nigral DA neurons can be obtained by ICV, intranigral or intrastriatal routes, although the ICV route effects do not translate to humans. Only intrastriatal GDNF is capable of protecting degenerating or damaged nigrostriatal axons and terminals and inducing any substantial regenerative growth response, however, is effective in cases only when a significant portion of the nigrostiatal projection remains intact, and thus efficacy is diminished in animals with advanced parkinsonism. In advanced cases, as suggested by primate data, intranigral delivery, acting through increased transmission in downstream targets may provide symptomatic relief. Conversely, direct infusion into the SN in early disease may be protective to prevent disease progression. In PD patients, the optimal site of GDNF delivery may also depend on the site of the primary insult. Degeneration involving the lateral part of SNc (also called lateral area A9 of Dahlstrom and Fuxe), which projects to the posterior dorsal motor striatum and globus pallidus externus, is associated with rigidity and bradykinesia whereas degeneration involving the medial SNc (medial area A9), parts of area A8 and A10 which innervate the subthalamic nucleus/ zona incerta and globus pallidus internus has been reported to be associated with tremor predominant PD and “On/Off” fluctuations (Jellinger et al, 1980, 2002; Hassani et al, 1997; Kolmac et al, 1998; Francois et al, 1999, 2000; Smith et al, 2000), and may explain the limited effect on tremor in the tremor predominant PD cases. Therefore, whether the optimal choice involves GDNF acting on axon terminals in the striatum or the subthalamic region, or on cell bodies in the SN or a combination of targets remains to be elucidated.
With striatal delivery, it is not clear how far this protein will diffuse away or is transported from the delivery site, and it possible that more rostral portions of the putamen will continue to degenerate if the GDNF does not diffuse this far. In this study, chronic infusion of GDNF results in very localised increases in striatal 18Fdopa uptake. As shown in this study, increasing the dose, and therefore the concentration gradient can increase GDNF penetration. Future studies delivering the GDNF through multiple sites within the putamen may be necessary to optimise the therapeutic effect. Alternatively, penetration of GDNF may be increased using modified equally active forms of the drug with a lower molecular weight and with reduced affinity to the heparin-binding sites, increasing its diffusion through the interstitial spaces.
With delivery of GDNF to the subthalamic or nigral regions, diffusion of this protein within this more confined target would encompass not only the terminal axons in the subthalamic and zona incerta regions and the cell bodies in the nigra, but also involve degenerating non-DA fibre tracts and nuclei dispersed within the midbrain and pons. Additionally, a combination of neurotrophic factor delivery acting through different mechanisms and on a variety of neuronal populations may serve as the optimal therapy in attempt to modify the disease state. For example, infusion of a combination of GDNF and BDNF to the nigral region, would not only involve the local effects of GDNF as described, but also effects from BDNF taken up by local receptors and transported anterogradely to exert its effect on distant innervated targets for both DA and non-DA neuronal populations.
D e s ig n o f a d e l i ve ry s y s t e m th a t tr e a t s a p re - s pe c i f ie d v ol um e o f t is s u e r e q u ir e s ma ni p ul a t i on o f b a s ic me c h a ni s m s o f t is s u e p e n e tr a t i on . F or a s y s t e m t ha t r e l ie s o n d if fu s io n, a l te r a t io n o f t he d o s e d e li ve r e d a nd t h e r e f o re t h e c o n c e nt r a t io n g ra d ie nt , a nd re du c ti on
o f th e m ol e c u la r w e i g ht o f t he pr ot e in , f or e x a m pl e u s i ng a bi o lo gi c a l ly a c ti v e lo w e r m o le c u l a r w e ig ht pr o- G D N F, w o ul d i nc r e a s e pe ne t ra ti o n th r ou gh th e i nt e r s ti ti a l s p a c e . A dd it i on a l l y, p e ne tr a ti on w o ul d b e i nc re a s e d b y re d uc in g t he e l im i na ti o n or in c r e a s in g t he s ta bi l it y o f th e p ro t e i n, w h ic h f or G D N F w o ul d b e e nh a n c e d b y u ti l iz in g a p r o- G D N F w i t h a r e d uc e d a ff in i ty t o t he he pa r in -s u lp ha t e side chains of extracellular-matrix proteoglygans, increasing the availability of the free drug within the interstitial space and therefore its diffusion. With the appropriate modifications, it could be realistic that GDNF migration could be predicted and appropriately customized to treat a pre-specified volume of tissue based on the extent of degeneration, or customised to the size of the appropriate target chosen on the basis of predominant symptomology.
Continuous delivery of recombinant GDNF protein has potential problems, including complications associated with a chronically implanted infusion device. It w o u l d c l e a r l y b e a d v a n t a g e o u s f o r P D p a t i e n t s t o r e c e i v e a s i n g l e " o n e - o f f " i n j e c t i o n o f G D N F . Su c h a n a ppr oa c h us in g a v ira l ve c to r d e l ive ry s y s t e m do e s ge ne r a t e i s s ue s of s a fe ty , i n tha t if pr obl e m s a ri s e , i t i s e a s ie r t o s w i t c h
pr og re s s a nd p rob a b ly w i ll li mit t he s u p p l y o f a v a i l a b l e t i s s u e i f r e n e w a b l e s o u r c e s o f f un c t i on a l c e ll s a re no t f ou nd . S e v e ra l s c i e n t if ic hu rd l e s a l s o r e ma in . S te m c e l l s m a y n o t be im mu n ol og i c a ll y c om p a t ib l e w i t h mo s t p a t ie n ts , a nd t h e y c o ul d c a us e t e r a t o ma s i f t i g ht c o nt r o l
o v e r d i ff e r e nt i a t io n a nd c e l l p r o l i f e r a t i o n i s n o t a c h i e v e d , a n d m a y r e q u i r e t h e n e e d t o i m m u n o s u p p r e s s t h e h o s t s i n o r d e r t o p r e v e n t t h e r e j e c t i o n o f g r a f t s i n c e r t a i n t r a n s p l a n t a t i o n c o n d i t i o n s . I n t h e f u t u r e , i t w o u l d b e i m p o r t a n t t o d e v e l o p s t r a t e g i e s t o d e c r e a s e t h e r i s k o f g r a f t r e j e c t i o n , b y e i t h e r i d e n t i f y i n g a n d m a t c h i n g a n t i g e n s i n h o s t a n d d o n o r s , o r b y p e r f o r m i n g g r a f t s o f m u l t i p o t e n t s t e m c e l l s i s o l a t e d f r o m t h e s a m e i n d i v i d u a l , i n c l u d i n g b o n e m a r r o w s t e m c e l l s . O u r u n d e r s t a n d i n g o f t h e r u l e s a n d l i m it s t o i n t e g r a t io n o f d i ff e r e nt i a t e d c e ll s int o ne u ra l c irc ui ts is a ls o in i ts inf a nc y. A c o mple t e un de rs t a n din g w il l be ne c e s s a ry if w e a re to re a l iz e f ull y t h e p o t e n t i a l o f s t e m c e l l s . N o n e t h e l e s s , t h e a c t i v e i n t e r e s t a n d s u b s t a n t i a l r e c e n t p r o g r e s s i n t h i s a r e a s us ta i ns t h e e n t hu s i a s t ic h op e t ha t t hi s a pp ro a c h c o ul d e v e n t u a l l y a c h i e v e t h e u l t i m a t e g o a l o f r e p a i r i n g t h e d a m a g e d br a in , a nd m a y p ro ve to b e a m o re s a fe a n d a m e na bl e s tr a te gy th a n th e d ir e c t g e n e ti c m a n ip u la ti o n of th e h os t b ra in .
We would like to thank Amgen Inc. for providing the GDNF used in our phase I trial and a wealth of information on their previous GDNF infusion trial, and dedicate this manuscript in memory of the late Dr. Michael Traub (Amgen Inc.), whose support and enthusiasm was responsible for the phase II study currently in progress. We are also grateful to the Parkinson’s disease society (United Kingdom) for providing a support grant towards equipment and salary; and the Medical Research Council for providing support towards a salary for NKP. In our phase I study, the PET scans and analysis were performed at the Hammersmith hospital (London), and we are grateful to both Professor David Brooks and Dr. Gary Hotton.
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From left to right: Nikunj K. Patel and Steven S. Gill