Hemraj B. Dodiya

Disease duration and the integrity of the nigrostriatal system in Parkinson’s disease

The pace of nigrostriatal degeneration, both with regards to striatal denervation and loss of melanin and tyrosine hydroxylase-positive neurons, is poorly understood especially early in the Parkinsons disease process. This study investigated the extent of nigrostriatal degeneration in patients with Parkinsons disease at different disease durations from time of diagnosis. Brains of patients with Parkinsons disease (n=28) with post-diagnostic intervals of 1-27 years and normal elderly control subjects (n=9) were examined. Sections of the post-commissural putamen and substantia nigra pars compacta were processed for tyrosine hydroxylase and dopamine transporter immunohistochemistry. The post-commissural putamen was selected due to tissue availability and the fact that dopamine loss in this region is associated with motor disability in Parkinsons disease. Quantitative assessments of putaminal dopaminergic fibre density and stereological estimates of the number of melanin-containing and tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta (both in total and in subregions) were performed by blinded investigators in cases where suitable material was available (n=17). Dopaminergic markers in the dorsal putamen showed a modest loss at 1 year after diagnosis in the single case available for study. There was variable (moderate to marked) loss, at 3 years. At 4 years post-diagnosis and thereafter, there was virtually complete loss of staining in the dorsal putamen with only an occasional abnormal dopaminergic fibre detected. In the substantia nigra pars compacta, there was a 50-90% loss of tyrosine hydroxylase-positive neurons from the earliest time points studied with only marginal additional loss thereafter. There was only a ∼10% loss of melanized neurons in the one case evaluated 1 year post-diagnosis, and variable (30 to 60%) loss during the first several years post-diagnosis with more gradual and subtle loss in the second decade. At all time points, there were more melanin-containing than tyrosine hydroxylase-positive cells. Loss of dopaminergic markers in the dorsal putamen occurs rapidly and is virtually complete by 4 years post-diagnosis. Loss of melanized nigral neurons lags behind the loss of dopamine markers. These findings have important implications for understanding the nature of Parkinsons disease neurodegeneration and for studies of putative neuroprotective/restorative therapies.

Alterations in lysosomal and proteasomal markers in Parkinson's disease: Relationship to alpha-synuclein inclusions

We explored the relationship between ubiquitin proteasome system (UPS) and lysosomal markers and the formation of alpha-synuclein (alpha-syn) inclusions in nigral neurons in Parkinson disease (PD). Lysosome Associated Membrane Protein 1(LAMP1), Cathepsin D (CatD), and Heat Shock Protein73 (HSP73) immunoreactivity were significantly decreased within PD nigral neurons when compared to age-matched controls. This decrease was significantly greater in nigral neurons that contained alpha-syn inclusions. Immunoreactivity for 20S proteasome was similarly reduced in PD nigral neurons, but only in cells that contained inclusions. In aged control brains, there is staining for alpha-syn protein, but it is non-aggregated and there is no difference in LAMP1, CatD, HSP73 or 20S proteasome immunoreactivity between alpha-syn positive or negative neuromelanin-laden nigral neurons. Targeting over-expression of mutant human alpha-syn in the rat substantia nigra using viral vectors revealed that lysosomal and proteasomal markers were significantly decreased in the neurons that displayed alpha-syn-ir inclusions. These findings suggest that alpha-syn aggregation is a key feature associated with decline of proteasome and lysosome and support the hypothesis that cell degeneration in PD involves proteosomal and lysosomal dysfunction, impaired protein clearance, and protein accumulation and aggregation leading to cell death.

Alpha-synuclein in colonic submucosa in early untreated Parkinson's disease.

The diagnosis of Parkinsons disease rests on motor signs of advanced central dopamine deficiency. There is an urgent need for disease biomarkers. Clinicopathological evidence suggests that α‐synuclein aggregation, the pathological signature of Parkinsons disease, can be detected in gastrointestinal tract neurons in Parkinsons disease. We studied whether we could demonstrate α‐synuclein pathology in specimens from unprepped flexible sigmoidoscopy of the distal sigmoid colon in early subjects with Parkinsons disease. We also looked for 3‐nitrotyrosine, a marker of oxidative stress. Ten subjects with early Parkinsons disease not treated with dopaminergic agents (7 men; median age, 58.5 years; median disease duration, 1.5 years) underwent unprepped flexible sigmoidoscopy with biopsy of the distal sigmoid colon. Immunohistochemistry studies for α‐synuclein and 3‐nitrotyrosine were performed on biopsy specimens and control specimens from a tissue repository (23 healthy subjects and 23 subjects with inflammatory bowel disease). Nine of 10 Parkinsons disease samples were adequate for study. All showed staining for α‐synuclein in nerve fibers in colonic submucosa. No control sample showed this pattern. A few showed light α‐synuclein staining in round cells. 3‐Nitrotyrosine staining was seen in 87% of Parkinsons disease cases but was not specific for Parkinsons disease. This study suggests a pattern of α‐synuclein staining in Parkinsons disease that was distinct from healthy subjects and those with inflammatory bowel disease. The absence of this pattern in subjects with inflammatory bowel disease suggests it is not a sequel of inflammation or oxidative stress. 3‐Nitrotyrosine immunostaining was common in all groups studied, suggesting oxidative stress in the colonic submucosa.

Increased intestinal permeability correlates with sigmoid mucosa alpha-synuclein staining and endotoxin exposure markers in early Parkinson's disease.

Parkinsons disease (PD) is the second most common neurodegenerative disorder of aging. The pathological hallmark of PD is neuronal inclusions termed Lewy bodies whose main component is alpha-synuclein protein. The finding of these Lewy bodies in the intestinal enteric nerves led to the hypothesis that the intestine might be an early site of PD disease in response to an environmental toxin or pathogen. One potential mechanism for environmental toxin(s) and proinflammatory luminal products to gain access to mucosal neuronal tissue and promote oxidative stress is compromised intestinal barrier integrity. However, the role of intestinal permeability in PD has never been tested. We hypothesized that PD subjects might exhibit increased intestinal permeability to proinflammatory bacterial products in the intestine. To test our hypothesis we evaluated intestinal permeability in subjects newly diagnosed with PD and compared their values to healthy subjects. In addition, we obtained intestinal biopsies from both groups and used immunohistochemistry to assess bacterial translocation, nitrotyrosine (oxidative stress), and alpha-synuclein. We also evaluated serum markers of endotoxin exposure including LPS binding protein (LBP). Our data show that our PD subjects exhibit significantly greater intestinal permeability (gut leakiness) than controls. In addition, this intestinal hyperpermeability significantly correlated with increased intestinal mucosa staining for E. coli bacteria, nitrotyrosine, and alpha-synuclein as well as serum LBP levels in PD subjects. These data represent not only the first demonstration of abnormal intestinal permeability in PD subjects but also the first correlation of increased intestinal permeability in PD with intestinal alpha–synuclein (the hallmark of PD), as well as staining for gram negative bacteria and tissue oxidative stress. Our study may thus shed new light on PD pathogenesis as well as provide a new method for earlier diagnosis of PD and suggests potential therapeutic targets in PD subjects. Trial Registration Clinicaltrials.gov NCT01155492

Is alpha‐synuclein in the colon a biomarker for premotor Parkinson's Disease? Evidence from 3 cases

Background: Despite clinicopathological evidence that Parkinsons disease (PD) may begin in peripheral tissues, identification of premotor Parkinsons disease is not yet possible. Alpha‐synuclein aggregation underlies Parkinsons disease pathology, and its presence in peripheral tissues may be a reliable disease biomarker. Objective: We sought evidence of alpha‐synuclein pathology in colonic tissues before the development of characteristic Parkinsons disease motor symptoms. Methods: Old colon biopsy samples were available for three subjects with PD. Biopsies were obtained 2‐5 years before PD onset. We performed immunohistochemistry studies for the presence of alpha‐synuclein and Substance P in these samples. Results: All subjects showed immunostaining for alpha‐synuclein (two, five and two years before first motor Parkinsons disease symptom). No similar alpha‐synuclein immunostaining was seen in 23 healthy controls. Staining of samples for substance P suggested colocalization of alpha‐synuclein and substance P in perikarya and neurites. Conclusions: This is the first demonstration of alpha‐synuclein in colon tissue prior to onset of PD. Additional study is required to determine whether colonic mucosal biopsy may be a biomarker of premotor PD.

TRANSFER OF HOST-DERIVED ALPHA SYNUCLEIN TO GRAFTED DOPAMINERGIC NEURONS IN RAT

Multiple laboratories have recently demonstrated that long-term dopaminergic transplants form Lewy bodies in patients with Parkinsons disease. Debate has arisen as to whether these Lewy bodies form from the transfer of α synuclein from the host to the graft or whether they form from intrinsic responses of the graft from being placed into what was, or became, an inflammatory focus. To test whether the former hypothesis was possible, we grafted fetal rat ventral mesencephalon into the dopamine depleted striatum of rats that had previously received 6-hydroxydopamine lesions. One month after the transplant, rats received viral over expression of human α synuclein (AAV2/6-α synuclein) or green fluorescent protein (AAV2/6-GFP) into the striatum rostral to the grafts. Care was taken to make sure that the AAV injections were sufficiently distal to the graft so no cells would be directly transfected. All rats were sacrificed five weeks after the virus injections. Double label immunohistochemistry combined with confocal microscopy revealed that a small number of grafted tyrosine hydroxylase (TH) neurons (5.7% ± 1.5% (mean ± SEM) of grafted dopamine cells) expressed host derived α synuclein but none of the grafted cells expressed host-derived GFP. The α synuclein in a few of these cells was misfolded and failed to be digested with proteinase K. These data indicate that it is possible for host derived α synuclein to transfer to grafted neurons supporting the concept that this is one possible mechanism by which grafted dopamine neurons form Lewy bodies in Parkinsons disease patients.

Differential Transduction Following Basal Ganglia Administration of Distinct Pseudotyped AAV Capsid Serotypes in Nonhuman Primates

We examined the transduction efficiency of different adeno-associated virus (AAV) capsid serotypes encoding for green fluorescent protein (GFP) flanked by AAV2 inverted terminal repeats in the nonhuman primate basal ganglia as a prelude to translational studies, as well as clinical trials in patients with Parkinsons disease (PD). Six intact young adult cynomolgus monkeys received a single 10 microl injection of AAV2/1-GFP, AAV2/5-GFP, or AAV2/8-GFP pseudotyped vectors into the caudate nucleus and putamen bilaterally in a pattern that resulted in each capsid serotype being injected into at least four striatal sites. GFP immunohistochemistry revealed excellent transduction rates for each AAV pseudotype. Stereological estimates of GFP+ cells within the striatum revealed that AAV2/5-GFP transduces significantly higher number of cells than AAV2/8-GFP (P < 0.05) and there was no significant difference between AAV2/5-GFP and AAV2/1-GFP (P = 0.348). Consistent with this result, Cavalieri estimates revealed that AAV2/5-GFP resulted in a significantly larger transduction volume than AAV2/8-GFP (P < 0.05). Each pseudotype transduced striatal neurons effectively [>95% GFP+ cells colocalized neuron-specific nuclear protein (NeuN)]. The current data suggest that AAV2/5 and AAV2/1 are superior to AAV2/8 for gene delivery to the nonhuman primate striatum and therefore better candidates for therapeutic applications targeting this structure.

Cell Transplantation and Gene Therapy in Parkinson's Disease

Parkinsons disease is a progressive neurodegenerative disorder affecting, in part, dopaminergic motor neurons of the ventral midbrain and their terminal projections that course to the striatum. Symptomatic strategies focused on dopamine replacement have proven effective at remediating some motor symptoms during the course of disease but ultimately fail to deliver long-term disease modification and lose effectiveness due to the emergence of side effects. Several strategies have been experimentally tested as alternatives for Parkinsons disease, including direct cell replacement and gene transfer through viral vectors. Cellular transplantation of dopamine-secreting cells was hypothesized as a substitute for pharmacotherapy to directly provide dopamine, whereas gene therapy has primarily focused on restoration of dopamine synthesis or neuroprotection and restoration of spared host dopaminergic circuitry through trophic factors as a means to enhance sustained controlled dopamine transmission. This seems now to have been verified in numerous studies in rodents and nonhuman primates, which have shown that grafts of fetal dopamine neurons or gene transfer through viral vector delivery can lead to improvements in biochemical and behavioral indices of dopamine deficiency. However, in clinical studies, the improvements in parkinsonism have been rather modest and variable and have been plagued by graft-induced dyskinesias. New developments in stem-cell transplantation and induced patient-derived cells have opened the doors for the advancement of cell-based therapeutics. In addition, viral-vector-derived therapies have been developed preclinically with excellent safety and efficacy profiles, showing promise in clinical trials thus far. Further progress and optimization of these therapies will be necessary to ensure safety and efficacy before widespread clinical use is deemed appropriate.

Human Neural Stem Cells Survive Long Term in the Midbrain of Dopamine-Depleted Monkeys After GDNF Overexpression and Project Neurites Toward an Appropriate Target

Transplanted multipotent human fetal neural stem cells (hfNSCs) significantly improved the function of parkinsonian monkeys in a prior study primarily by neuroprotection, with only 3%–5% of cells expressing a dopamine (DA) phenotype. In this paper, we sought to determine whether further manipulation of the neural microenvironment by overexpression of a developmentally critical molecule, glial cell‐derived neurotrophic factor (GDNF), in the host striatum could enhance DA differentiation of hfNSCs injected into the substantia nigra and elicit growth of their axons to the GDNF‐expressing target. hfNSCs were transplanted into the midbrain of 10 green monkeys exposed to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydro‐pyridine. GDNF was delivered concomitantly to the striatum via an adeno‐associated virus serotype 5 vector, and the fate of grafted cells was assessed after 11 months. Donor cells remained predominantly within the midbrain at the injection site and sprouted numerous neurofilament‐immunoreactive fibers that appeared to course rostrally toward the striatum in parallel with tyrosine hydroxylase‐immunoreactive fibers from the host substantia nigra but did not mature into DA neurons. This work suggests that hfNSCs can generate neurons that project long fibers in the adult primate brain. However, in the absence of region‐specific signals and despite GDNF overexpression, hfNSCs did not differentiate into mature DA neurons in large numbers. It is encouraging, however, that the adult primate brain appeared to retain axonal guidance cues. We believe that transplantation of stem cells, specifically instructed ex vivo to yield DA neurons, could lead to reconstruction of some portion of the nigrostriatal pathway and prove beneficial for the parkinsonian condition.

Neonatal immune-tolerance in mice does not prevent xenograft rejection.

Assessing the efficacy of human stem cell transplantation in rodent models is complicated by the significant immune rejection that occurs. Two recent reports have shown conflicting results using neonatal tolerance to xenografts in rats. Here we extend this approach to mice and assess whether neonatal tolerance can prevent the rapid rejection of xenografts. In three strains of neonatal immune-intact mice, using two different brain transplant regimes and three independent stem cell types, we conclusively show that there is rapid rejection of the implanted cells. We also address specific challenges associated with the generation of humanized mouse models of disease.