AAV2-mediated delivery of human neurturin to the rat nigrostriatal system: long-term efficacy and tolerability of CERE-120 for Parkinson's disease

Neurturin (NTN) is a neurotrophic factor with known potential to protect and restore the function of dopaminergic substantia nigra neurons whose degeneration has been most closely linked to the major motor deficits in Parkinson's disease (PD). CERE-120, an adeno-associated virus serotype 2 (AAV2)-based gene delivery vector encoding human NTN, is being developed as a potential therapeutic for PD. In a series of preclinical studies reported herein, CERE-120 delivery to the striatum produced a dose-related neuroprotection of nigrostriatal neurons in the rat 6-hydroxydopamine (6-OHDA) lesion model. Long-lasting efficacy of CERE-120 was evidenced by substantia nigra cell protection, preserved fiber innervation of the striatum, and behavioral recovery for at least 6 months. In addition, striatal infusion of CERE-120 was found to have a safety and tolerability profile devoid of side effects or toxicological responses, for at least 12 months post-treatment, even at dose multiples 125 times that of the lowest efficacious dose tested. These results support the ongoing CERE-120 clinical program in PD patients.     

Predictive value of the smell identification test for nigrostriatal dopaminergic depletion in Korean tremor patients

BackgroundThe predictive value of Cross-Cultural Smell Identification Test for nigrostriatal dopaminergic depletion in Korean tremor patients has yet to be assessed.MethodsThree hundred nineteen drug-naive patients who visited our clinic for the diagnosis of their tremor, and took both Cross-Cultural Smell Identification Test and dopamine transporter PET were included in the data analysis. Visual grading of each PET image was performed by two independent neurologists.ResultsSmell test scores were significantly correlated to the striatal dopaminergic activity (Kendall's τb = ?0.291, p < 0.001). However, smell test score alone appeared to have relatively weak power for predicting dopaminergic depletion (area under the curve = 0.693). Multivariate logistic regression model with inclusion of the patient's age and symptom duration as independent variables enhanced predictive power for dopaminergic depletion (area under the curve = 0.812).ConclusionsThese results demonstrated that Cross-Cultural Smell Identification Test measurements alone may be insufficient to predict striatal dopaminergic depletion in Korean tremor patients.     

Stereotaxic injection of GD1a ganglioside induces limited recovery of striatal dopaminergic system in MPTP-treated aging mice.

The systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to young (2 months old) and aging (12 months old) C57BL/6 mice (4 x 20 mg/kg i.p. given 12 hr apart) reduced tyrosine hydroxylase (TH)-immunoreactive (IR) fibers in the striatum and reduced dopamine (DA) concentration to 35% of controls in young and 22% of controls in aging mouse brain 5 weeks after administration. Stereotaxic injection of GD1a ganglioside (3 x 100 micrograms, 5 days apart) into the striatum of MPTP-treated young mice restored striatal DA concentration to 52% of the control concentration 5 weeks after MPTP injection. Similar injections of GD1a ganglioside restored striatal DA concentration of MPTP-treated aging mice to only 31% of the control concentration. Immunocytochemical analysis showed significant recovery of TH-IR fibers in the striatum of MPTP-depleted young mice treated with GD1a ganglioside, while TH-IR fibers in the striatum of MPTP-depleted aging mice treated with GD1a ganglioside showed less recovery. We conclude that treatment of MPTP-depleted aging mice with GD1a ganglioside results in more limited recovery in the nigrostriatal DA system than in young mice.     

AAV2-mediated gene transfer of GDNF to the striatum of MPTP monkeys enhances the survival and outgrowth of co-implanted fetal dopamine neurons

Neural transplantation offers the potential of treating Parkinson's disease by grafting fetal dopamine neurons to depleted regions of the brain. However, clinical studies of neural grafting in Parkinson's disease have produced only modest improvements. One of the main reasons for this is the low survival rate of transplanted neurons. The inadequate supply of critical neurotrophic factors in the adult brain is likely to be a major cause of early cell death and restricted outgrowth of fetal grafts placed into the mature striatum. Glial derived neurotrophic factor (GDNF) is a potent neurotrophic factor that is crucial to the survival, outgrowth and maintenance of dopamine neurons, and so is a candidate for protecting grafted fetal dopamine neurons in the adult brain. We found that implantation of adeno-associated virus type 2 encoding GDNF (AAV2-GDNF) in the normal monkey caudate nucleus induced overexpression of GDNF that persisted for at least 6 months after injection. In a 6-month within-animal controlled study, AAV2-GDNF enhanced the survival of fetal dopamine neurons by 4-fold, and increased the outgrowth of grafted fetal dopamine neurons by almost 3-fold in the caudate nucleus of MPTP-treated monkeys, compared with control grafts in the other caudate nucleus. Thus, the addition of GDNF gene therapy to neural transplantation may be a useful strategy to improve treatment for Parkinson's disease.     

A novel LRRK2 mutation in an Austrian cohort of patients with Parkinson's disease.

To investigate the frequency of mutations in the Leucine-Rich Repeat Kinase 2 gene (LRRK2) in a sample of Austrian Parkinson's disease (PD) patients, we sequenced the complete coding region in 16 patients with autosomal dominant PD. Furthermore, we sequenced exons 31, 35, and 41 additionally in 146 patients with idiopathic PD and 30 patients with dementia with Lewy bodies. Furthermore, all 192 patients were screened for 21 putative LRRK2 mutations. While the most common mutation G2019S and the risk variant G2385R were not found in our samples, we detected a novel missense mutation (S973N) in a patient with familial, late-onset and dopa-responsive PD.     

GFRalpha-1 mRNA in dopaminergic and nondopaminergic neurons in the substantia nigra and ventral tegmental area.

Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for several types of neurons, including dopaminergic (DAergic) neurons. GDNF binds with high affinity to the GDNF family receptor alpha-1 (GFRalpha-1), which is highly expressed in the midbrain. Using anatomical and lesion techniques, we demonstrated that GFRalpha-1 was expressed in DAergic and non-DAergic neurons in the rat midbrain. Immunohistochemical characterization of GFRalpha-1-expressing neurons indicated that most of the neurons that were immunopositive for the DAergic marker tyrosine hydroxylase (TH) expressed GFRalpha-1 in the substantia nigra pars compacta (SNC). In contrast, fewer TH-containing neurons expressed GFRalpha-1 in the substantia nigra pars reticulata (SNR) and the ventral tegmental area (VTA). Depletion of GFRalpha-1/TH neurons was observed in the SNC following treatment with the neurotoxin 6-hydroxydopamine (6-OHDA); however, GFRalpha-1 expression remained in some neurons located in the SNR. The gamma-aminobutyric acid (GABA)ergic nature of GFRalpha-1-expressing neurons located in the SNR, which were resistant to (6-hydroxydopamine) 6-OHDA, was established by their expression of glutamic acid decarboxylase (GAD; the synthesizing enzyme for GABA). Further analysis indicated that coexpression of GFRalpha-1 and GAD varied in a rostrocaudal gradient in the SNR, substantia nigra pars lateralis (SNL), and VTA. Midbrain DAergic and GABAergic neurons have been previously classified according to their Ca(2+) binding protein (CaBP) content; thus, we also sought to investigate the proportion of midbrain GFRalpha-1-expressing neurons containing parvalbumin (PV), calbindin (CB), and calretinin (CR) in the midbrain. Although GFRalpha-1 expression was found mainly in CB- and CR-immunoreactive neurons, it was rarely observed in PV-immunolabeled neurons. Analysis of the proportion of GFRalpha-1-expressing neurons for each CaBP subpopulation indicated the coexistence of GFRalpha-1 with CR in the VTA and all subdivisions of the SN; double-labeled GFRalpha-1/CR neurons were distributed in the SNC, SNR, SNL, and VTA. GFRalpha-1/CB neurons were also detected in the SNC, SNL, and VTA. Expression of GFRalpha-1 in DAergic and non-DAergic neurons in the rat SN and VTA suggests that GDNF, via GFRalpha-1, might modulate DAergic and GABAergic functions in the nigrostriatal, mesolimbic, and nigrothalamic circuits of the adult rat.     

N-0923, A novel soluble dopamine D2 agonist in the treatment of parkinsonism

N-0923, a novel aminotetralin dopamine D₂ agonist, was shown to effectively reverse parkinsonian symptoms in nine dopa/agonist-responsive Parkinson's disease patients. The drug was given up to 4.5 hours by continuous intravenous (IV) infusion using an IV pump. The onset of anti-parkinsonian effect was seen within minutes of the initiation of the infusion and was absent within 90 minutes of cessation of the infusion. The short elimination half-life of N-0923 (90 min) would allow for the rapid initiation of drug effect when necessary and at the same time permit the effect to be terminated quickly if necessary. The drug would be useful in situations where oral medication is not feasible or is associated with erratic absorption. The patients tolerated the drug well. Dose escalation load was limited by nausea and vomiting. It should be noted that the doses were increased until these symptoms occurred, but therapeutic effects were noted well before the side effects occurred. Using a modified Columbia scale, maximum improvement consisted of a 27–95% drop in score. Maximum response was obtained at infusion rates varying from 2–16 μg/kg per hour and at blood levels of 0.11–1.49 μg/mL.     

Effects of naringin,a flavanone glycoside in grapefruits and citrus fruits,on the nigrostriatal dopaminergic projection in the adult brain

Recently, we have demonstrated the ability of naringin, a well-known flavanone glycoside of grapefruits and citrus fruits, to prevent neurodegeneration in a neurotoxin model of Parkinson＇s disease. Intraperitoneal injection of naringin protected the nigrostriatal dopaminergic projection by increasing glial cell line-derived neurotrophic factor expression and decreasing the level of tumor necrosis factor-alpha in dopaminergic neurons and microglia, respectively. These results suggest that naringin can impart to the adult dopaminergic neurons the ability to produce glial cell line-derived neurotrophic factor against Parkinson＇s disease with anti-inflammatory effects. Based on these results, we would like to describe an important perspective on its possibility as a therapeutic agent for Parkinson＇s disease.     

Response of the GABAergic and dopaminergic mesostriatal projections to the lesion of the contralateral dopaminergic mesostriatal pathway in the rat.

Although dopamine is the main neurotransmitter in the mesostriatal system, recent studies indicate the existence of two nigrostriatal GABAergic projections: one arising from neurons immunoreactive for GABA, glutamic acid decarboxylase (GAD67), and parvalbumin (PV) lying in the substantia nigra pars reticulata (nigrostriatal GABA cells) and the other arising from a subpopulation of dopaminergic neurons lying in the substantia nigra pars compacta and ventral tegmental area, which under normal conditions, contains mRNA for GAD65 (one of the two isoforms of glutamic acid decarboxylase), but which is not immunoreactive for GABA and GAD65 (nigrostriatal dopaminergic [DA]/GABA cells). With the aim of improving our knowledge about the interaction between the nigrostriatal system of both brain hemispheres, we have studied the response of these three components of the mesostriatal system (GABA, DA/GABA, and DA) to the lesion of the contralateral mesostriatal DA pathway, by using morphological and neurophysiological techniques. Our findings show that, in the side contralateral to the lesion, (1) the number of nigrostriatal GABA cells increases from 6% to 17% with respect to the total number of nigrostriatal cells, (2) the soma of DA/GABA cells becomes immunoreactive for GABA and GAD65, and (3) there is an increase in the firing rate and burst activity of DA-neurons, except in those projecting to the striatum, which may be under the action of the GABA hyperactivity. Taken together, our results suggest that the GABAergic components of the mesostriatal projection play a regulatory role on the DA component, activated or upregulated after contralateral DA lesion and are probably addressed to restoring the functional symmetry in basal ganglia and to slowing down the contralateral progression of DA-cell degeneration in Parkinson's disease.     

The number of dopaminergic cells is increased in the olfactory bulb of monkeys chronically exposed to MPTP

We investigated the impact of the nigrostriatal lesion on the olfactory tyrosine hydroxylase-immunoreactive (TH-ir) cells in monkeys. The majority of these TH-ir cells appeared in the glomerular layer of the olfactory bulb and many were immature but functional dopaminergic neurons. In parkinsonian monkeys the number of olfactory dopaminergic neurons increased up to 100% as compared to controls, but their phenotype did not change. This increased TH-ir cell population might be a direct consequence of the nigral cell loss and contribute to the hyposmia reported by Parkinson's disease patients.     

Analysis of 14 LRRK2 mutations in Parkinson's plus syndromes and late-onset Parkinson's disease.

The pleomorphic pathology of postmortem LRRK2-positive patients and the frequent association with late-onset Parkinson's disease (LOPD) symptoms suggest that LRRK2 mutations may play a role in Parkinson's Plus disorders and LOPD. Published studies primarily focus on the common G2019S mutation. Analysis of a spectrum of LRRK2 mutations in Parkinson's Plus disorders has yet to be reported. We investigated 14 leucine-rich repeat kinase 2 (LRRK2) mutations in a cohort of Parkinson's Plus disorders and LOPD. A total of 458 patients with progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal ganglionic degeneration (CBGD), atypical Parkinsonism (AP), and LOPD were screened for 14 mutations that span exons 19 to 41 of the LRRK2 gene. Among the LOPD cases, 1 patient was found to harbor the R1441C mutation. He presented with typical features of PD at age of 58 years old and responded well to levodopa. We did not detect any of the 14 mutations in PSP, MSA, CBGD, and AP patients. We highlight the first case of LRRK2 R1441C mutation in late onset sporadic PD of non-European ancestry. Furthermore, extensive mutational screen found LRRK2 mutations to be rare among patients who presented with PSP, MSA, CBGD, and AP.     

Chronic treatment with small doses of cabergoline prevents dopa-induced dyskinesias in parkinsonian monkeys.

Levodopa continues to be the most effective agent for the symptomatic treatment of Parkinson's disease (PD). But over time, initial benefits decline in efficacy because of a rise in adverse effects such as dyskinesias. The pathophysiology of levodopa-induced dyskinesias (LID) is not completely understood, but it appears to result from deficient regulation by dopamine of corticostriatal glutamatergic inputs leading to a cascade of neurochemical changes in the striatum and the output pathways. In the present study, we examined if the addition of small doses of cabergoline (a long-acting D(2) receptor agonist) to levodopa could prevent LID. The major hypothesis is that sustained activation of postsynaptic D(2) receptors on medium spiny neurons even by small doses of cabergoline could prevent or reduce LID. The minor hypothesis, and the more controversial of the two, is that the long-acting stimulation by small doses of cabergoline could diminish the release of glutamate by the corticostriatal pathway and prevent LID. Eight MPTP-treated monkeys with a long-standing and stable parkinsonian syndrome and having never received dopaminergic agents were used. Two groups of four were treated for 1 month with levodopa/benserazide administered orally (100 mg/25 mg). The second group received in addition a threshold dose of cabergoline (dose ranging from 0.015 to 0.035 mg/kg, SC). During the treatment, we observed LID in the levodopa group but not in the group receiving levodopa+cabergoline. Furthermore, the combination produced a comparable antiparkinsonian effect in terms of quality but prolonged the duration (by 1 to 2 hours) and increased the locomotion (mean for 2 weeks congruent with 104%). Our data suggest that a small dose of a long-acting D(2) agonist combined with high doses of levodopa could be preventive of LID in patients with PD and could be an alternative to using antiglutamatergic agents for this purpose.     

Comprehensive sequencing of the LRRK2 gene in patients with familial Parkinson's disease from North Africa

The LRRK2 gene is a key player in Parkinson's disease (PD), however prevalence and pathogenicity of LRRK2 variants remain to be investigated in ethnically diverse populations. Herein, we performed comprehensive sequencing of the LRRK2 gene in 92 Tunisian probands with familial PD. We then performed an association study using all identified variants in a series of 167 Lrrk2 p.G2019S‐negative patients with sporadic PD and 365 Lrrk2 p.G2019S‐negative healthy control subjects, all from the same Arab‐Berber ethnicity. We identified one novel coding substitution (p.M2408I) and 24 known coding changes. Only the Lrrk2 p.G2019S mutation segregated with disease within families and was found in 39% of familial probands. None of the variants displayed significant association with risk for sporadic PD, however a trend was observed for Lrrk2 p.Y2189C. The present study underscores the importance of the LRRK2 gene in the Tunisian PD population. © 2010 Movement Disorder Society     

1-Benzyl-1,2,3,4-tetrahydroisoquinoline, a Parkinsonism-inducing endogenous toxin, increases alpha-synuclein expression and causes nuclear damage in human dopaminergic cells

1-Benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ), an endogenous neurotoxin, is known to cause parkinsonism in rodents and nonhuman primates. The levels of 1BnTIQ in cerebrospinal fluid of patients with Parkinson's disease (PD) were reported to be three times higher than those in control subjects. In the present study, we have evaluated the effects of 1BnTIQ on alpha-synuclein (alpha-syn) expression together with biochemical and morphological changes in human dopaminergic SH-SY5Y cells in culture. 1BnTIQ at lower concentrations (1-50 microM) increased alpha-syn protein expression in a time- and dose-dependent manner in these cells. There was also up-regulation of alpha-syn mRNA by 1BnTIQ. Inhibition of complex I by rotenone and depletion of glutathione by L-buthionine sulfoxamine also correlated with an increase in alpha-syn expression, suggesting that oxidative stress may cause an increase in alpha-syn levels in dopaminergic cells. Furthermore, 1BnTIQ significantly depleted glutathione levels. 1BnTIQ at higher concentrations (500 microM) increased reactive oxygen species levels, decreased ATP levels, and caused nuclear damage in the cells. The 1BnTIQ-induced alpha-syn up-regulation was inhibited by cotreatment with the antioxidants selegiline, coenzyme Q(10), and N-acetylcystein and the caspase inhibitor DEVD-CHO. Taken together, these results suggest that alpha-syn up-regulation and oxidative stress are contributing factors in 1BnTIQ-induced neurotoxicity in dopaminergic neurons in PD.     

Generation of dopamine neurons from embryonic stem cells in the presence of the neuralizing activity of bone marrow stromal cells derived from adult mice

Stromal cell lines such as PA6 and MS5 have been employed for generating dopamine (DA) neurons from embryonic stem (ES) cells. The present study was designed to test whether bone marrow stromal cells (BMSC) derived from adult mice might be available as a feeder layer to produce DA cells efficiently from ES cells. When ES cells were grown on BMSC in the presence of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), about 40% of TuJ1-positive neurons expressed tyrosine hydroxylase (TH). Because these cells labeled with TH were negative for dopamine-beta-hydroxylasae (DBH), the marker for noradrenergic and adrenergic neurons, the TH-positive cells were most likely DA neurons. They indeed expressed midbrain DA neuron markers such as Nurr 1, Ptx-3, and c-ret and were capable of synthesizing and releasing DA in vitro. Furthermore, DA neurons differentiated from ES cells in this differentiation protocol survived transplantation in rats with 6-hydroxydopamine lesions and reversed the lesion-induced circling behavior. The data indicate that BMSC can facilitate an efficient induction of DA neurons from ES cells and that the generated DA neurons are biologically functional both in vitro and in vivo. Insofar as BMSC have recently been employed in autologous cell therapy for ischemic heart and arteriosclerotic limb diseases, the present study raises the possibility that autologous BMSC can be applied in future cell transplantation therapy in Parkinson's disease.