GDNF synthesis, signaling, and retrograde transport in motor neurons.

Glial cell line-derived neurotrophic factor (GDNF) is a 134 amino acid protein belonging in the GDNF family ligands (GFLs). GDNF was originally isolated from rat glial cell lines and identified as a neurotrophic factor with the ability to promote dopamine uptake within midbrain dopaminergic neurons. Since its discovery, the potential neuroprotective effects of GDNF have been researched extensively, and the effect of GDNF on motor neurons will be discussed herein. Similar to other members of the TGF-ß superfamily, GDNF is first synthesized as a precursor protein (pro-GDNF). After a series of protein cleavage and processing, the 211 amino acid pro-GDNF is finally converted into the active and mature form of GDNF. GDNF has the ability to trigger receptor tyrosine kinase RET phosphorylation, whose downstream effects have been found to promote neuronal health and survival. The binding of GDNF to its receptors triggers several intracellular signaling pathways which play roles in promoting the development, survival, and maintenance of neuron-neuron and neuron-target tissue interactions. The synthesis and regulation of GDNF have been shown to be altered in many diseases, aging, exercise, and addiction. The neuroprotective effects of GDNF may be used to develop treatments and therapies to ameliorate neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). In this review, we provide a detailed discussion of the general roles of GDNF and its production, delivery, secretion, and neuroprotective effects on motor neurons within the mammalian neuromuscular system.

Glial-derived neurotrophic factor regulates enteric mast cells and ameliorates dextran sulfate sodium-induced experimental colitis.

BACKGROUND & AIMS: Although interactions between enteric glial cells (EGCs) and enteric mast cells have been demonstrated to play an important role in the pathogenesis of inflammatory bowel disease (IBD), the exact mechanisms by which EGCs regulate enteric mast cells are still unknown. The aims of this study were to investigate whether glial-derived neurotrophic factor (GDNF), which has been confirmed to be produced mostly by EGCs, might regulate enteric mast cells and ameliorate dextran sulfate sodium (DSS)-induced experimental colitis. METHODS: Recombinant adenoviral vectors encoding GDNF (Ad-GDNF) were administered intracolonically in experimental colitis induced by DSS. The disease activity index and histological score were measured. The expression of tumour necrosis factor-α (TNF-α), interleukin-6 and myeloperoxidase (MPO) activity were measured by ELISA assay. The expression of trypsin and ß-hexosaminidase were evaluated. GDNF specific receptor (GFR-α1/RET) was detected. The calcium reflux was tested by microplate reader. The expression p-JNK was analyzed by western blot assay. RESULTS: GDNF resulted in a significant inhibition of the activation of enteric mast cells by down-regulating JNK signal pathway, lessening intracellular calcium influx, and then reducing the degranulation as well as the expression of pro-inflammatory cytokines via combing with its receptor (GFR-α1/RET) in mast cells, and these inhibitory effects were abrogated by treatment with neutralizing antibody against GDNF. Moreover, the administration of GDNF led to an amelioration of experimental colitis. CONCLUSIONS: GDNF are able to regulate enteric mast cells and ameliorate experimental colitis. GDNF might be an important mediator of the cross-talk between EGCs and enteric mast cells, and GDNF might be a useful therapeutic drug for IBD.

Blockade of glial-derived neurotrophic factor in laryngeal muscles promotes appropriate reinnervation.

OBJECTIVES/HYPOTHESIS: Synkinetic reinnervation of the laryngeal muscles is one of the causes of the poor functional recovery after a recurrent laryngeal nerve (RLN) injury. Glial-derived neurotrophic factor (GDNF) is elevated in rat laryngeal muscles during RLN reinnervation. The specific aim of this investigation was to evaluate the effect of anti-GDNF on RLN reinnervation. METHODS: Anti-GDNF antibody was injected into the posterior cricoarytenoid (PCA) 3 days following RLN transection and anastomosis. Larynges were harvested at 7, 14, 28, 56, and 112 days post injury (DPI). Prior to sacrifice, the vocal fold mobility was assessed. Immunostaining to identify neuromuscular junctions was used to evaluate the extent of axonal reinnervation of the PCA, lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA). RESULTS: After anti-GDNF injection into PCA, RLN reinnervation in all muscles was altered when compared to the controls. PCA innervation was delayed. At 7 DPI, only a few axons made synapses in the PCA. In contrast, axons prematurely innervated the LTA and MTA when compared to controls. Innervation was similar to controls at 56 and 112 DPI. Vocal fold motion was enhanced in 10 of 24 animals studied. CONCLUSIONS: After injection of anti-GDNF into the PCA, early arriving axons bypass the PCA and enter the LTA. Later arriving axons innervate the PCA and MTA. Vocal fold function is improved as compared to controls. Anti-GDNF injection into the PCA influences the pattern of reinnervation and may result in less synkinetic, more functional innervation. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E337-E342, 2016.

Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones.

Immune protective mechanism of rMOMP protein ophthalmic vaccine regarding intraocular hypertension and retinal optic nerve injury in rats.

The aim of this study was to explore the immune protective mechanism of rMOMP protein vaccine in intraocular hypertension and retinal optic nerve injury in rats. The rMOMP protein ophthalmic vaccine was prepared and quality-controlled. Sixty normal adult SD rats were randomly divided into two groups to establish a chronic ocular hypertension model and an optic nerve injury model. The model rats were vaccinated with rMOMP-CS ophthalmic vaccine. Fluorogold retrograde tracing was used to observe retinal ganglion cells, and an immunofluorescence method to determine the expression of retinal GAP43, CD3, BDNF, and GDNF. rMOMP protein ophthalmic vaccine met the requirements for medicinal use. The number of retinal ganglion cells (RGCs) of the rMOMP-CS group in the chronic ocular hypertension model was significantly higher than that of the CS group (P < 0.05). The count of RGCs of the rMOMP-CS group in the optic nerve clamping injury model was significantly higher than that of the CS group (P < 0.01). Thus, rMOMP protein ophthalmic vaccine can induce an increase in the expression of retinal neurotrophic factors, thereby exerting a protective effect on damaged retinal optic nerve.

Estrogen Increases Striatal GDNF Immunoreactivity with no Effect on Striatal FGF-2 Immunoreactivity of MPTP-Treated Mice.

BACKGROUND: Glial derived neurotrophic factor (GDNF) and basic fibroblast growth factor (FGF-2) protect nigrostriatal dopaminergic (DA) neurons and their projections in animal models of Parkinson's disease (PD). Recent data indicate neuroprotective effects of estrogen in PD animal models through its anti-inflammatory and anti-oxidative effects, yet the hormonal effects on GDNF and FGF-2 expression in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice remain uninvestigated. OBJECTIVE: To determine the effects of 17 beta-estradiol (E2) on DA innervation and the expression ofGDNFandFGF-2 in the striatum of MPTP-treated mice. MATERIAL AND METHOD: Adult male mice were treated with E2 or vehicle for 11 days during which they were injected with MPTP or saline on the sixth day. The striatum was collected on day 11 and processedfor tyrosine hydroxylase (TH), GDNF and FGF-2 immunohistochemistry. Extent ofDA innervation and the expression of GDNF and FGF-2 in the striatum were assessed by measuring optical density of TH, GDNF and FGF-2 immunoreactivity, respectively. RESULTS: MPTP induced loss of DA axons and upregulation of FGF-2 expression, but did not alter GDNF level. E2 alleviated loss of DA axons, increased GDNF level, yet caused no change in FGF-2 level ofthe MPTP-intoxicated animals. CONCLUSION: One possible mechanism by which E2 protects nigrostriatal DA axons against MPTP is through upregulation ofstriatal GDNF.

Adenoviral-mediated GDNF protects bone marrow mesenchymal stem cells against apoptosis induced by hydrogen peroxide.

In this study, the effects of adenoviral-mediated glial cell line-derived neurotrophic factor (Ad-GDNF) on apoptosis of cultured bone marrow mesenchymal stem cells (BMSCs) induced by hydrogen peroxide (H2O2) were investigated. After BMSCs infected with Ad-GDNF were treated with 500 µM H2O2 at 37° C for 1 h, lactate dehydrogenase (LDH), MTT and TUNEL methods were used to detect cell viability and apoptosis. In addition, the levels of GDNF in the supernatants in BMSCs cultures were detected by ELISA, pro-inflammatory cytokines IL-6 and TNF-a in the supernatants and apoptosis-related protein Bax and Bcl-2 in cell pellets were investigated. The results showed that H2O2 treatment significantly induced apoptosis of BMSCs and decreased the viability of BMSCs. However, Ad-GDNF significantly reversed the effects of H2O2 on BMSCs. Furthermore, Ad-GDNF significantly decreased the levels of IL-6 and TNF-a and increased Bcl-2/Bax ratio in BMSCs treated by H2O2. In conclusion, Ad-GDNF inhibits apoptosis of BMSCs induced by H2O2, and the mechanisms may be related to down-regulating the expression of IL-6 and TNF-a and up-regulating the ratio of Bcl-2/Bax in BMSCs.

Naringin protects the nigrostriatal dopaminergic projection through induction of GDNF in a neurotoxin model of Parkinson's disease.

This study investigated the effect of naringin, a major flavonoid in grapefruit and citrus fruits, on the degeneration of the nigrostriatal dopaminergic (DA) projection in a neurotoxin model of Parkinson's disease (PD) in vivo and the potential underlying mechanisms focusing on the induction of glia-derived neurotrophic factor (GDNF), well known as an important neurotrophic factor involved in the survival of adult DA neurons. 1-Methyl-4-phenylpyridinium (MPP(+)) was unilaterally injected into the medial forebrain bundle of rat brains for a neurotoxin model of PD in the presence or absence of naringin by daily intraperitoneal injection. To ascertain whether naringin-induced GDNF contributes to neuroprotection, we further investigated the effects of intranigral injection of neutralizing antibodies against GDNF in the MPP(+) rat model of PD. Our observations demonstrate that naringin could increase the level of GDNF in DA neurons, contributing to neuroprotection in the MPP(+) rat model of PD, with activation of mammalian target of rapamycin complex 1. Moreover, naringin could attenuate the level of tumor necrosis factor-α in microglia increased by MPP(+)-induced neurotoxicity in the substantia nigra. These results indicate that naringin could impart to DA neurons the important ability to produce GDNF as a therapeutic agent against PD with anti-inflammatory effects, suggesting that naringin is a beneficial natural product for the prevention of DA degeneration in the adult brain.

GDNF fails to inhibit LPS-mediated activation of mouse microglia.

GDNF is a potent neuroprotective factor for midbrain dopaminergic (mDA) neurons. In LPS-mediated models for mDA degeneration GDNF increases neuron survival and further reduces microglia activation. To elucidate the effects of GDNF on LPS-induced activation, primary microglia from C57BL/6 and NMRI mice have been analysed. In this study we demonstrate that GDNF is not able to inhibit LPS-mediated upregulation and release of the proinflammatory factors IL6 and TNFα. Moreover, we provide evidence that mouse microglia, in contrast to rat microglia, lack expression of the GDNF signalling receptor c-Ret resulting in abrogated activation of downstream signalling kinases Akt and Erk1/2.

Pericyte-derived glial cell line-derived neurotrophic factor increase the expression of claudin-5 in the blood-brain barrier and the blood-nerve barrier.

The destruction of blood-brain barrier (BBB) and blood-nerve barrier (BNB) has been considered to be a key step in the disease process of a number of neurological disorders including cerebral ischemia, Alzheimer's disease, multiple sclerosis, and diabetic neuropathy. Although glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) facilitate neuronal or axonal regeneration in the brain or peripheral nerves, their action in the BBB and BNB remains unclear. The purpose of the present study was to elucidate whether these neurotrophic factors secreted from the brain or peripheral nerve pericytes increase the barrier function of the BBB or BNB, using our newly established human brain microvascular endothelial cell (BMEC) line or peripheral nerve microvascular endothelial cell (PnMEC) line. GDNF increased the expression of claudin-5 and the transendothelial electrical resistance (TEER) of BMECs and PnMECs, whereas BDNF did not have this effect. Furthermore, we herein demonstrate that the GDNF secreted from the brain and peripheral nerve pericytes was one of the key molecules responsible for the up-regulation of claudin-5 expression and the TEER value in the BBB and BNB. These results indicate that the regulation of GDNF secreted from pericytes may therefore be a novel therapeutic strategy to modify the BBB or BNB functions and promote brain or peripheral nerve regeneration.

Harpagoside attenuates MPTP/MPP⁺ induced dopaminergic neurodegeneration and movement disorder via elevating glial cell line-derived neurotrophic factor.

Parkinson's disease is a chronic neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. New therapeutic approaches aiming at delaying or reversing the neurodegenerative process are under active investigations. In this work, we found that harpagoside, an iridoid purified from the Chinese medicinal herb Scrophularia ningpoensis, could not only prevent but also rescue the dopaminergic neurodegeneration in MPTP/MPP(+) intoxication with promising efficacy. Firstly, in cultured mesencephalic neurons, harpagoside significantly attenuated the loss of TH-positive neuron numbers and the shortening of axonal length. Secondly, in a chronic MPTP mouse model, harpagoside dose-dependently improved the loco-motor ability (rotarod test), increased the TH-positive neuron numbers in the substantia nigra pars compacta (unbiased stereological counting) and increased the striatal DAT density ((125) I-FP-CIT autoradiography). Thirdly, harpagoside markedly elevated the GDNF mRNA and GDNF protein levels in MPTP/MPP(+) lesioned models. However, the protecting effect of harpagoside on the dopaminergic degeneration disappeared when the intrinsic GDNF action was blocked by either the Ret inhibitor PP1 or the neutralizing anti-GDNF antibody. Taken together, we conclude that harpagoside attenuates the dopaminergic neurodegeneration and movement disorder mainly through elevating glial cell line-derived neurotrophic factor.

MET signaling in GABAergic neuronal precursors of the medial ganglionic eminence restricts GDNF activity in cells that express GFRα1 and a new transmembrane receptor partner.

GDNF (glial cell line-derived neurotrophic factor) promotes the differentiation and migration of GABAergic neuronal precursors of the medial ganglionic eminence (MGE). These functions are dependent on the GPI-anchored receptor GFRα1, but independent of its two known transmembrane receptor partners RET and NCAM. Here we show that soluble GFRα1 is also able to promote differentiation and migration of GABAergic MGE neurons. These activities require endogenous production of GDNF. Although GDNF responsiveness is abolished in Gfra1(-/-) neurons, it can be restored upon addition of soluble GFRα1, a result that is only compatible with the existence of a previously unknown transmembrane signaling partner for the GDNF-GFRα1 complex in GABAergic neurons. The roles of two candidate transmembrane receptors previously implicated in GABAergic interneuron development--MET, a receptor for hepatocyte growth factor (HGF), and ErbB4, the neuregulin receptor--were examined. GDNF did not induce the activation of either receptor, nor did inhibition of MET or ErbB4 impair GDNF activity in GABAergic MGE neurons. Unexpectedly, however, inhibition of MET or HGF per se promoted neuronal differentiation and migration and enhanced the activity of GDNF on MGE neurons. These effects were dependent on endogenous GDNF and GFRα1, suggesting that MET signaling negatively regulates GDNF activity in the MGE. In agreement with this, Met mutant MGE neurons showed enhanced responses to GDNF and inhibition of MET or HGF increased Gfra1 mRNA expression in MGE cells. In vivo, expression of MET and GFRα1 overlapped in the MGE, and a loss-of-function mutation in Met increased Gfra1 expression in this region. Together, these observations demonstrate the existence of a novel transmembrane receptor partner for the GDNF-GFRα1 complex and uncover an unexpected interplay between GDNF-GFRα1 and HGF-MET signaling in the early diversification of cortical GABAergic interneuron subtypes.

Chronic dosing of mice with a transferrin receptor monoclonal antibody-glial-derived neurotrophic factor fusion protein.

Glial-derived neurotrophic factor (GDNF) is a potential neurotrophic factor treatment of brain disorders, including Parkinson's disease. However, GDNF does not cross the blood-brain barrier (BBB). A brain-penetrating form of GDNF, which is a fusion protein of human GDNF and a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), has been engineered for the mouse and is designated the cTfRMAb-GDNF fusion protein. The present study examined the potential toxic side effects and immune response after treatment of mice with twice-weekly cTfRMAb-GDNF fusion protein at a dose of 2 mg/kg i.v. for 12 consecutive weeks. Chronic treatment with the fusion protein caused no change in body weight, no change in 23 serum chemistry measurements, and no histologic changes in brain and cerebellum, kidney, liver, spleen, heart, or pancreas. Chronic treatment caused a low-titer immune response against the fusion protein, which was directed against the variable region of the antibody part of the fusion protein, with no immune response directed against either the constant region of the antibody or against GDNF. A pharmacokinetics and brain uptake study was performed at the end of the 12 weeks of treatment. There was no change in clearance of the fusion protein mediated by the TfR in peripheral organs, and there was no change in BBB permeability to the fusion protein mediated by the TfR at the BBB. The study shows no toxic side effects from chronic cTfRMAb-GDNF systemic treatment and the absence of neutralizing antibodies in vivo.

Prenatal LPS increases inflammation in the substantia nigra of Gdnf heterozygous mice.

Prenatal systemic inflammation has been implicated in neurological diseases, but optimal animal models have not been developed. We investigated whether a partial genetic deletion of glial cell line-derived neurotrophic factor (Gdnf(+/-)) increased vulnerability of dopamine (DA) neurons to prenatal lipopolysaccharide (LPS). LPS [0.01 mg/kg intraperitoneal (i.p.)] or saline was administered to wild-type (WT) or Gdnf(+/-) pregnant mice on gestational day 9.5. Male offspring were examined at 3 weeks, 3 and 12 months of age. There was a progressive degeneration of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN) with age in Gdnf(+/-) but not in WT mice, with no observed effects on locus coeruleus (LC) noradrenergic neurons or DA neurons of the ventral tegmental area. Inflammatory markers were elevated in SN of LPS treated offspring, with exacerbation in Gdnf(+/-) mice. Intracellular accumulation of α-synuclein (α-syn) immunoreactivity in DA neurons of SN was observed in all groups of Gdnf(+/-) and in WT mice with prenatal LPS, with altered distribution between pars reticulata (pr) and pars compacta (pc). The findings suggest that prenatal LPS leads to accelerated neuropathology in the SN with age, and that a partial loss of GDNF exacerbates these effects, providing a novel model for age-related neuropathology of the nigrostriatal DA system.

Neuroprotection from diazinon-induced toxicity in differentiating murine N2a neuroblastoma cells.

In previous work, the outgrowth of axon-like processes by differentiating mouse N2a neuroblastoma cells was shown to be inhibited by exposure to 10 microM diazinon. In the present work, N2a cells were induced to differentiate for 24 h in the presence and absence of 10 microM diazinon and 20% (v/v) conditioned medium derived from differentiating rat C6 glioma cells. Cells were then stained or lysed for morphological and biochemical analyses, respectively. The data showed that co-treatment with conditioned medium prevented the neurite inhibitory effect of diazinon. Furthermore, a significant recovery was also observed in the reduced levels of neurofilament heavy chain (NFH), heat shock protein-70 (HSP-70) and growth-associated protein-43 (GAP-43) observed as a result of diazinon treatment in the absence of conditioned medium, as seen by densitometric analysis of Western blots of cell lysates probed with monoclonal antibodies N52, BRM-22 and GAP-7B10. By contrast, no significant change was noted in the reactivity of cell lysates with antibodies against alpha- and beta-tubulin under any condition tested. After pre-incubation with a polyclonal anti-glial cell line-derived neurotrophic factor (GDNF) antibody, conditioned medium derived from rat C6 glioma cells lost its ability to protect N2a cells against the neurite inhibitory effects of diazinon. In conclusion, these data demonstrate that C6 conditioned medium protects N2a cells from the neurite inhibitory effects of diazinon by blocking molecular events leading to axon damage and that GDNF is implicated in these effects.

Function of neurotrophic factors beyond the nervous system: inflammation and autoimmune demyelination.

In the nervous system, neurotrophic factors play a role during development, especially for the differentiation of neuronal and glial cells. Moreover, they promote cell survival of neurons, axons, and oligodendrocytes, as well as their precursors, in vitro and in lesional paradigms. In recent years, several functions of neurotrophic factors outside the nervous system have been described, with a special focus on the immune system as well as on models of autoimmune demyelination, such as experimental autoimmune encephalomyelitis (EAE). In the family of neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were investigated. NGF may influence B-cell as well as T-cell function and particularly plays a role in macrophage migration into inflamed lesions. BDNF is produced by several immune-cell subtypes in vitro and also in multiple sclerosis (MS) plaques. This observation gave rise to the concept of neuroprotective autoimmunity, implying that immune-cell infiltration in the nervous system may not only be detrimental but may also play a beneficial role, for example, through the production of neurotrophic factors. In the family of neurotrophic cytokines, ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) share some common protective roles in axons and oligodendrocytes. In EAE, endogenous CNTF targets myelin, oligodendroglial cells, and axons. In contrast, LIF exerts protective functions on oligodendrocytes in some models but is also able to interact with the immune response and may modulate T-cell, monocyte and neutrophil functions. In summary, neurotrophic factors have distinct roles in the immune system during autoimmunity and may modulate immune responses as well as the susceptibility of the target tissue.

Monocyte chemoattractant protein-1 immunoreactivity in sensory ganglia and hindpaw after adjuvant injection.

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 is a member of the CC chemokine family that exhibits potent chemotactic activity for monocytes/macrophages. In the current study, the proportion of monocyte chemoattractant protein-1-immunoreactive (IR) neurons in the dorsal root ganglion (DRG) of rats was shown to increase markedly following adjuvant injection into the hindpaw. MCP-1-IR axon terminals were not found in the spinal cord or hindpaw of control or adjuvant-treated rats. Instead, the inflamed hindpaw dermis was infiltrated by numerous MCP-1-IR inflammatory cells. Following adjuvant injection, the majority of MCP-1-IR neurons in the DRG colocalized with IB4 binding. Our findings suggest that peripheral tissue inflammation induces increased MCP-1 expression in DRG neurons and this may be dependent upon glial cell line-derived neurotrophic factor.

Six-month continuous intraputamenal infusion toxicity study of recombinant methionyl human glial cell line-derived neurotrophic factor (r-metHuGDNF in rhesus monkeys.

Recombinant human glial cell line-derived neurotrophic factor (r-metHuGDNF) is a potent neuronal growth and survival factor that has been considered for clinical use in the treatment of Parkinson's disease (PD). Here we present results of a 6-month toxicology study in rhesus monkeys conducted to support clinical evaluation of chronic intraputamenal infusion of r-metHuGDNF for PD. Monkeys (6-9/sex/group) were treated with 0 (vehicle), 15, 30, or 100 microg/day r-metHuGDNF by continuous unilateral intraputamenal infusion (150 microl/day flow rate) for 6 months; a subset of animals (2-3/sex/group) underwent a subsequent 3-month treatment-free recovery period. Notable observations included reduced food consumption and body weight at 100 microg/day and meningeal thickening underlying the medulla oblongata and/or overlying various spinal cord segments at 30 and 100 microg/day. In addition, multifocal cerebellar Purkinje cell loss (with associated atrophy of the molecular layer and, in some cases, granule cell loss) was observed in 4 monkeys in the 100-microg/day group. This cerebellar finding has not been observed in previous nonclinical studies evaluating r-metHuGDNF. The small number of affected animals precludes definitive conclusions regarding the pathogenesis of the cerebellar lesion, but the data support an association with r-metHuGDNF treatment.

Six-month continuous intraputamenal infusion toxicity study of recombinant methionyl human glial cell line-derived neurotrophic factor (r-metHuGDNF) in rhesus monkeys.

Recombinant human glial cell line-derived neurotrophic factor (r-metHuGDNF) is a potent neuronal growth and survival factor that has been considered for clinical use in the treatment of Parkinson's disease (PD). Here we present results of a 6-month toxicology study in rhesus monkeys conducted to support clinical evaluation of chronic intraputamenal infusion of r-metHuGDNF for PD. Monkeys (6-9/sex/group) were treated with 0 (vehicle), 15, 30, or 100 micro g/day r-metHuGDNF by continuous unilateral intraputamenal infusion (150 micro l/day flow rate) for 6 months; a subset of animals (2-3/sex/group) underwent a subsequent 3-month treatment-free recovery period. Notable observations included reduced food consumption and body weight at 100 micro g/day and meningeal thickening underlying the medulla oblongata and/or overlying various spinal cord segments at 30 and 100 micro g/day. In addition, multifocal cerebellar Purkinje cell loss (with associated atrophy of the molecular layer and, in some cases, granule cell loss) was observed in 4 monkeys in the 100-micro g/day group. This cerebellar finding has not been observed in previous nonclinical studies evaluating r-metHuGDNF. The small number of affected animals precludes definitive conclusions regarding the pathogenesis of the cerebellar lesion, but the data support an association with r-metHuGDNF treatment.

Development of a maturing T-cell-mediated immune response in patients with idiopathic Parkinson's disease receiving r-metHuGDNF via continuous intraputaminal infusion.

The development of a maturing T-cell-mediated immune response was characterized in Parkinson's disease subjects receiving recombinant human glial-derived neurotrophic factor (r-metHuGDNF) via continuous bilateral intraputaminal infusion. Eighteen of 34 subjects tested positive for anti-r-metHuGDNF-binding antibodies. Four subjects developed neutralizing activity, three of which demonstrated classic immunoglobulin class switching from IgM to IgG. An increase of anti-r-metHuGDNF IgG-binding antibodies correlated with the development of neutralizing activity. All serum samples from two subjects with neutralizing activity were characterized for IgG subclasses. These data revealed an initial anti-r-metHuGDNF IgG population where IgG1 >> IgG2 >> IgG4, and IgG3 concentrations were negligible. However, continued antigenic stimulation resulted in concentration changes where IgG4 > IgG1> IgG2, indicating a mature immune response. In addition, using in silico techniques, two immunodominant MHC class II T-cell epitopes were predicted for the native GDNF sequence. These data demonstrate development of a mature T-cell-mediated immune response in these subjects.