Richard Grondin

Caloric restriction increases neurotrophic factor levels and attenuates neurochemical and behavioral deficits in a primate model of Parkinson's disease

We report that a low-calorie diet can lessen the severity of neurochemical deficits and motor dysfunction in a primate model of Parkinsons disease. Adult male rhesus monkeys were maintained for 6 months on a reduced-calorie diet [30% caloric restriction (CR)] or an ad libitum control diet after which they were subjected to treatment with a neurotoxin to produce a hemiparkinson condition. After neurotoxin treatment, CR monkeys exhibited significantly higher levels of locomotor activity compared with control monkeys as well as higher levels of dopamine (DA) and DA metabolites in the striatal region. Increased survival of DA neurons in the substantia nigra and improved manual dexterity were noted but did not reach statistical significance. Levels of glial cell line-derived neurotrophic factor, which is known to promote the survival of DA neurons, were increased significantly in the caudate nucleus of CR monkeys, suggesting a role for glial cell line-derived neurotrophic factor in the anti-Parkinsons disease effect of the low-calorie diet.

Antiparkinsonian effect of a new selective adenosine A2A receptor antagonist in MPTP-treated monkeys

Background: Chronic treatment with l-3,4-dihydroxyphenylalanine (l-dopa) is often associated with motor side effects in PD patients. The search for new therapeutic approaches has led to study the role of other neuromodulators including adenosine. Among the four adenosine receptors characterized so far, the A2A subtype is distinctively present on striatopallidal output neurons containing enkephalin and mainly bearing dopamine (DA) D2 receptors (indirect pathway). Studies in DA-denervated rats suggest that blockade of adenosine A2A receptors might be used in PD. Objective: To evaluate the antiparkinsonian effect of a new selective adenosine A2A receptor antagonist, KW-6002, in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. Methods: In the present study, we used six MPTP-exposed cynomolgus monkeys already primed and exhibiting l-dopa–induced dyskinesias to evaluate both the antiparkinsonian and dyskinetic effect upon challenge with two oral doses (60 and 90 mg/kg) of KW-6002 administered alone or in combination with l-dopa/benserazide (50/12.5 mg). Results: KW-6002 administered alone produced a dose-dependent antiparkinsonian response that reached the level of efficacy of l-dopa/benserazide but was less likely to reproduce dyskinesias in these animals. When co-administered, KW-6002 potentiated the effects of l-dopa/benserazide on motor activity (up to 30%) without affecting the dyskinetic response. Conclusion: Adenosine A2A receptor antagonists have antiparkinsonian effects of their own with a reduced propensity to elicit dyskinesias. They might therefore be useful agents in the treatment of PD.

Point source concentration of GDNF may explain failure of phase II clinical trial.

Significant differences have been reported in results from three clinical trials evaluating intraputamenal infusion of glial cell line-derived neurotrophic factor (GDNF) for the treatment of Parkinsons disease. To determine if problems in drug bioavailability could have contributed to the discrepancies between studies, we have analyzed the distribution of intraputamenally infused GDNF in the rhesus monkey brain using the delivery system and infusion protocol followed in a phase 2 clinical trial that failed to achieve its primary endpoint. I125-GDNF was unilaterally infused into the putamen of three adult rhesus monkeys for 7 days. Three age- and sex-matched animals received vehicle infusions following identical procedures. GDNF levels in the brain, peripheral organs, blood and CSF were quantified and mapped by GDNF immunocytochemistry, GDNF ELISAs and I125 measurements. Infused GDNF was found to be unevenly concentrated around the catheter, with tissue levels dropping exponentially with increasing distance from the point source of the single opening in the catheter tip. The volume of distribution of GDNF around the catheter, as determined by immunocytochemistry, varied over four-fold between animals ranging from 87 to 369 mm3. The concentration of GDNF around the catheter tip and limited diffusion into surrounding brain parenchyma support the hypothesis that drug bioavailability was limited to a small portion (2-9%) of the human putamen in the clinical trial using this catheter and infusion protocol.

Intraputamenal infusion of GDNF in aged rhesus monkeys: Distribution and dopaminergic effects

Site‐specific delivery of trophic factors in the brain may be important for achieving therapeutic efficacy without unwanted side effects. This study evaluated the site‐specific infusion of glial cell line–derived neurotrophic factor (GDNF) into the right putamen of aged rhesus monkeys. After 4 weeks of continuous infusion at a rate of 22.5 μg/day, 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. GDNF was carried by retrograde transport to dopamine neurons in the ipsilateral substantia nigra, stimulating an 18% increase in the number of tyrosine hydroxylase (TH)–positive dopamine neurons and a 28% increase in dopamine 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 dopaminergic 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. Anterograde transport of GDNF was evident in axons in the pyramidal tract from the cerebral peduncle to the caudal spinal cord. Tissue injury from the intraparenchymal catheter and continuous infusion was confined primarily to a narrow zone surrounding the track and was mild to moderate in severity. J. Comp. Neurol. 461:250–261, 2003.

Glial cell line-derived neurotrophic factor (GDNF): a drug candidate for the treatment of Parkinson’s disease

Abstract Considerable effort has been devoted to the search for molecules that might exert trophic influences on midbrain dopamine neurons, and potentially be of therapeutic value in the treatment of Parkinson’s disease. One such candidate is glial cell line-derived neurotrophic factor (GDNF). GNDF is distantly related to the transforming growth factor-β superfamily and is widely expressed in many neuronal and non-neuronal tissues. GDNF uses a multisubunit receptor system in which GFRα-1 and Ret function as the ligand-binding and signalling components, respectively. In addition to its effects on cultured fetal midbrain dopamine neurons, GDNF promotes recovery of the injured nigrostriatal dopamine system and improves motor functions in rodent and nonhuman primate models of Parkinson’s disease. Intraventricular, intrastriatal and intranigral routes of administration are efficacious in both models. In parkinsonian nonhuman primates, GDNF treatment improves bradykinesia, rigidity and postural instability. In this model, adult midbrain dopamine neurons stimulated by GDNF show increased cell size, neuritic extent, and expression of phenotypic markers. The neurorestorative effects of a single administration of GDNF last for at least a month and can be maintained in rhesus monkeys by monthly injections. GDNF also induces neuroprotective changes in dopamine neurons, which are active within hours following trophic factor administration in rodents. The powerful neuroprotective and neurorestorative properties of GDNF seen in preclinical studies suggest that trophic factors may play an important role in treating Parkinson’s disease.

Glial Cell Line-Derived Neurotrophic Factor Increases Stimulus-Evoked Dopamine Release and Motor Speed in Aged Rhesus Monkeys

Changes in the functional dynamics of dopamine release and regulation in the basal ganglia have been posited to contribute to age-related slowing of motor functions. Here, we report the effects of glial cell line-derived neurotrophic factor (GDNF) on the stimulus-evoked release of dopamine and motor speed in aged monkeys (21–27 years of age; n = 10). Although no changes were observed in the vehicle controls (n = 5), chronic infusions of 7.5 μg of GDNF per day for 2 months into the right lateral ventricle initially increased hand movement speed up to 40% on an automated hand-reach task. These effects were maintained for at least 2 months after replacing GDNF with vehicle, and increased up to another 10% after the reinstatement of GDNF treatment for 1 month. In addition, upper-limb motor performance times of the aged GDNF-treated animals (n = 5) recorded at the end of the study were similar to those of five young adult monkeys (8–12 years of age). The stimulus-evoked release of dopamine was significantly increased, up to 130% in the right caudate nucleus and putamen and up to 116% in both the right and left substantia nigra of the aged GDNF recipients compared with vehicle controls. Also, basal extracellular levels of dopamine were bilaterally increased, up to 163% in the substantia nigra of the aged GDNF-treated animals. The data suggest that the effects of GDNF on the release of dopamine in the basal ganglia may be responsible for the improvements in motor functions and support the hypothesis that functional changes in dopamine release may contribute to motor dysfunctions characterizing senescence.

Differential Regulation of Striatal Preproenkephalin and Preprotachykinin mRNA Levels in MPTP-Lesioned Monkeys Chronically Treated with Dopamine D1 or D2 Receptor Agonists

Abstract : Studies in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐lesioned monkeys and in parkinsonian patients show elevated preproenkephalin (PPE) mRNA levels, unaltered by chronic l‐DOPA therapy, whereas preprotachykinin (PPT) mRNA levels are decreased by the lesion and corrected by l‐DOPA. The relative contributions of the dopamine D1 and D2 receptors for PPE mRNA regulation were investigated in the present study and compared with those for PPT mRNA. In situ hybridization was used to measure peptide mRNA levels in the striatum of MPTP cynomolgus monkeys after chronic 1‐month treatment with the D1 agonist SKF‐82958, administered subcutaneously in pulsatile or continuous mode, compared with the long‐acting D2 agonist cabergoline. Normal as well as untreated MPTP animals were also studied. PPE mRNA levels were elevated in the caudate nucleus and putamen of untreated MPTP monkeys compared with control animals with a more pronounced increase in the lateral as compared with the medial part of both structures. PPT mRNA levels showed a rostrocaudal gradient, with higher values in the middle of the caudate‐putamen and more so in the medial versus the lateral parts. PPT mRNA levels were decreased in the caudate and putamen of untreated MPTP monkeys compared with control animals, and this was observed in the middle and posterior parts of these brain areas. Elevated PPE and decreased PPT mRNA levels observed after MPTP exposure were corrected after treatment with cabergoline (0.25 mg/kg, every other day), a dose that had antiparkinsonian effects and did not give sustained dyskinesia. In contrast, elevated PPE mRNA levels observed in untreated MPTP monkeys were markedly increased by pulsatile administration of SKF‐82958 (1 mg/kg, three times daily) in two monkeys in which the parkinsonian symptoms were improved and dyskinesias developed, whereas it remained close to control values in a third one that did not display dyskinesias despite a sustained improvement in disability ; a shorter duration of motor benefit (wearing off) over time was observed in these three animals. By contrast, pulsatile administration of SKF‐82958 corrected the decreased PPT level observed in untreated MPTP monkeys. Continuous treatment with SKF‐82958 (equivalent daily dose) produced no clear antiparkinsonian and dyskinetic responses and did not alter the denervation‐induced elevation of PPE or decrease of PPT mRNA levels. The present data suggest an opposite contribution of the dopamine D1 receptors (stimulatory) as compared with the dopamine D2 receptors (inhibitory) on PPE mRNA, whereas a similar stimulatory contribution of D1 or D2 receptors is observed for PPT mRNA. An increase in PPE expression could be involved in the induction of dyskinesias and wearing off, whereas our data do not support this link for PPT. The antiparkinsonian response was associated with a correction of the lesion‐induced decrease of PPT.

Dopamine-receptor stimulation: biobehavioral and biochemical consequences

The MPTP monkey is a well-characterized animal model of parkinsonism and provides an exceptional tool for the study of dyskinesias induced by dopamine-like agents. Several such agents have been tested during the past 15 years, and it has been found that the duration of action of these compounds is the most reliable variable with which to predict their dyskinesiogenic profile. It is proposed that L-dopa-induced dyskinesias represent a form of pathological learning caused by chronic pulsatile (nonphysiological) stimulation of dopamine receptors, which activates a cascade of molecular and biochemical events. These events include defective regulation of Fos proteins that belong to the deltaFosB family, increased expression of neuropeptides, and defective GABA- and glutamate-mediated neurotransmission in the output structures of the basal ganglia.

Six-month partial suppression of Huntingtin is well tolerated in the adult rhesus striatum

Huntingtons disease is caused by expression of a mutant form of Huntingtin protein containing an expanded polyglutamine repeat. One possible treatment for Huntingtons disease may be to reduce expression of mutant Huntingtin in the brain via RNA interference. Unless the therapeutic molecule is designed to be allele-specific, both wild-type and mutant protein will be suppressed by an RNA interference treatment. A key question is whether suppression of wild-type as well as mutant Huntingtin in targeted brain regions can be tolerated and result in a net benefit to patients with Huntingtons disease. Whether Huntingtin performs essential functions in the adult brain is unclear. Here, we tested the hypothesis that the adult primate brain can tolerate moderately reduced levels of wild-type Huntingtin protein for an extended period of time. A serotype 2 adeno-associated viral vector encoding for a short hairpin RNA targeting rhesus huntingtin messenger RNA (active vector) was bilaterally injected into the striatum of four adult rhesus monkeys. Four additional animals received a comparable vector encoding a scrambled control short hairpin RNA (control vector). General health and motor behaviour were monitored for 6 months. Upon termination, brain tissues were sampled and assessed blindly for (i) huntingtin messenger RNA knockdown; (ii) Huntingtin protein expression; and (iii) neuropathological changes. Reduction in wild-type huntingtin messenger RNA levels averaging ∼30% was measured in the striatum of active vector recipients 6 months post-injection. A widespread reduction in Huntingtin protein levels was also observed by immunohistochemistry in these animals, with an average protein reduction of ∼45% relative to controls measured by western blot analysis in the putamen of active vector recipients. As with control vector recipients, no adverse effects were observed behaviourally, and no neurodegeneration was found on histological examination of active vector recipients. Our results suggest that long-term partial suppression of wild-type Huntingtin may be safe, and thus if a comparable level of suppression of mutant Huntingtin is beneficial, then partial suppression of both wild-type and mutant Huntingtin may result in a net benefit in patients with heterozygous Huntingtons disease.

Alteration of glutamate receptors in the striatum of dyskinetic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys following dopamine agonist treatment.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal lesion and dopaminomimetic treatment on parameters of glutamatergic activity within the basal ganglia of monkeys were studied in relation with the development of dyskinesias. Drug-naive controls, saline-treated MPTP monkeys, as well as MPTP monkeys treated with either a long-acting D2 agonist (cabergoline) or a D1 agonist (SKF-82958) given by intermittent injections or continuous infusion, were included in this study. 3H-L-glutamate, 3H-alpha-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA), 3H-glycine, 3H-CGP39653 (an N-methyl-D-aspartate, NMDA, antagonist selective for NR1/NR2A assembly) and 3H-Ro 25-6981 (an NMDA antagonist selective for NR1/NR2B assembly), specific binding to glutamate receptors, the expression of the NR1 subunit of NMDA receptors and glutamate, glutamine and glycine concentrations were studied by autoradiography, in situ hybridization and high-performance liquid chromatography (HPLC), respectively. Pulsatile SKF-82958 and cabergoline treatment relieved parkinsonian symptoms, whereas animals continuously treated with SKF-82958 remained akinetic. Pulsatile SKF-82958 induced dyskinesias in two of the three animals tested, whereas cabergoline did not. MPTP induced no significant changes of striatal specific binding of the radioligands used, NR1 mRNA expression and amino acid concentrations. In the putamen, pulsatile SKF-82958 treatment was associated with decreased content of glycine and glutamate, whereas only glycine was decreased in cabergoline-treated monkeys. Cabergoline and continuous administration of SKF-82958 led to lower levels of NR1 mRNA in the caudate in comparison to pulsatile SKF-82958 administration. The development of dyskinesias following a D1 agonist treatment was associated with an upregulation of 3H-glutamate [+49%], 3H-AMPA [+38%], 3H-CGP39653 [+ 111%], 3H-glycine [+ 26%, nonsignificant] and 3H-Ro 25-6981 [+ 33%] specific binding in the striatum in comparison to nondyskinetic MPTP monkeys. Our data suggest that supersensitivity to glutamatergic input in the striatum might play a role in the pathogenesis of dopaminomimetic-induced dyskinesias and further support the therapeutic potential of glutamate antagonists in Parkinsons disease.