Yaping Chu

Lewy body–like pathology in long-term embryonic nigral transplants in Parkinson's disease

Fourteen years after transplantation into the striatum of an individual with Parkinsons disease, grafted nigral neurons were found to have Lewy body–like inclusions that stained positively for α-synuclein and ubiquitin and to have reduced immunostaining for dopamine transporter. These pathological changes suggest that Parkinsons disease is an ongoing process that can affect grafted cells in the striatum in a manner similar to host dopamine neurons in the substantia nigra. These findings have implications for cell-based therapies and for understanding the cause of Parkinsons disease.

Loss and atrophy of layer II entorhinal cortex neurons in elderly people with mild cognitive impairment.

Layer II of the entorhinal cortex contains the cells of origin for the perforant path, plays a critical role in memory processing, and consistently degenerates in end‐stage Alzheimers disease. The extent to which neuron loss in layer II of entorhinal cortex is related to mild cognitive impairment without dementia has not been extensively investigated. We analyzed 29 participants who came to autopsy from our ongoing longitudinal study of aging and dementia composed of religious clergy (Religious Orders Study). All individuals underwent detailed clinical evaluation within 12 months of death and were categorized as having no cognitive impairment (n = 8), mild cognitive impairment (n = 10), or mild or moderate Alzheimers disease (n = 11). Sections through the entorhinal cortex were immunoreacted with an antibody directed against a neuron‐specific nuclear protein (NeuN). Stereological counts of NeuN‐immunoreactive stellate cells, their volume, and the volume of layer II entorhinal cortex were estimated. Cases exhibiting no cognitive impairment averaged 639,625 ± 184,600 layer II stellate neurons in the right entorhinal cortex. Individuals with mild cognitive impairment (63.5%; p < 0.0003) and mild or moderate Alzheimers disease (46.06%; p < 0.0017) displayed significant losses of layer II entorhinal cortex neurons relative to those with no cognitive impairment but not relative to each other (p > 0.33). There was also significant atrophy of layer II entorhinal cortex neurons in individuals with mild cognitive impairment (24.1%) and Alzheimers disease (25.1%). The volume of layer II was also reduced in individuals with mild cognitive impairment (26.5%), with a further reduction in those with Alzheimers disease (46.4%). The loss and atrophy of layer II entorhinal cortex neurons significantly correlated with performance on clinical tests of declarative memory. Atrophy of layer II entorhinal cortex and the neurons within this layer significantly correlated with performance on the Mini Mental Status Examination. These data indicate that atrophy and loss of layer II entorhinal cortex neurons occur in elderly subjects with mild cognitive impairment prior to the onset of dementia and suggests that these changes are not exacerbated in early Alzheimers disease. Ann Neurol 2001;49:202–213

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.

Transplanted dopaminergic neurons develop PD pathologic changes: A second case report

This report describes pathological changes within the grafted neurons of another patient with Parkinsons disease (PD) who died 14 years posttransplantation. Although numerous healthy appearing grafted neurons were present at this long‐term time point, some displayed Lewy bodies as evidenced by alpha‐synuclein, ubiquitin, and thioflavin‐S staining. Additionally, there was a general loss of dopamine transporter‐immunoreactivity in grafted neurons. Some grafted cell displayed a loss of tyrosine hydroxylase. These data support the emerging concept that PD‐like pathology is seen in young grafted neurons when they survive long term.

Lentiviral Gene Transfer to the Nonhuman Primate Brain

Lentiviral vectors infect quiescent cells and allow for the delivery of genes to discrete brain regions. The present study assessed whether stable lentiviral gene transduction can be achieved in the monkey nigrostriatal system. Three young adult Rhesus monkeys received injections of a lentiviral vector encoding for the marker gene beta galatosidase (beta Gal). On one side of the brain, each monkey received multiple lentivirus injections into the caudate and putamen. On the opposite side, each animal received a single injection aimed at the substantia nigra. The first two monkeys were sacrificed 1 month postinjection, while the third monkey was sacrificed 3 months postinjection. Robust incorporation of the beta Gal gene was seen in the striatum of all three monkeys. Stereological counts revealed that 930,218; 1,192,359; and 1,501,217 cells in the striatum were beta Gal positive in monkeys 1 (n = 2) and 3 (n = 1) months later, respectively. Only the third monkey had an injection placed directly into the substantia nigra and 187,308 beta Gal-positive cells were identified in this animal. The injections induced only minor perivascular cuffing and there was no apparent inflammatory response resulting from the lentivirus injections. Double label experiments revealed that between 80 and 87% of the beta Gal-positive cells were neurons. These data indicate that robust transduction of striatal and nigral cells can occur in the nonhuman primate brain for up to 3 months. Studies are now ongoing testing the ability of lentivirus encoding for dopaminergic trophic factors to augment the nigrostriatal system in nonhuman primate models of Parkinsons disease.

Age-related decreases in Nurr1 immunoreactivity in the human substantia nigra

Nuclear receptor‐related factor 1 (Nurr1), a member of the nuclear receptor superfamily, is associated with the induction of dopaminergic (DA) phenotypes in developing and mature midbrain neurons. It is well established that dopaminergic nigrostriatal function decreases with age. Whether age‐related deficits in DA phenotypic markers are associated with alterations in Nurr1 expression is unknown. The present study found that virtually all of tyrosine hydroxylase‐immunoreactive (TH‐ir) neurons within the young adult human substantia nigra were Nurr1‐immunoreactive (Nurr1‐ir) positive. Stereologic counts revealed a significant reduction in the number of Nurr1‐ir nigral neurons in middle‐aged (23.13%) and aged (46.33%) individuals relative to young subjects. The loss of Nurr1‐ir neurons was associated with a similar decline in TH‐ir neuron number. In this regard, TH‐ir neuronal number was decreased in middle‐aged (11.10%) and in aged (45.97%) subjects, and this loss of TH‐ir neurons was highly correlated (r = 0.92) with the loss of Nurr1‐ir neurons. In contrast, the number of melanin‐containing nigral neuron number was generally stable across age groups, indicating that changes in Nurr1 and TH reflect phenotypic age‐related changes and not frank neuronal degeneration. In support of this concept, confocal microscopic analyses of Nurr1‐ir and TH‐ir fluorescence intensity revealed parallel decreases in Nurr1‐ and TH‐immunofluorescence as a function of age. These data demonstrate that age‐related decline of DA phenotypic markers is associated with down‐regulation of Nurr1 expression in the SN. J. Comp. Neurol. 450:203–214, 2002.

Failure of proteasome inhibitor administration to provide a model of Parkinson's disease in rats and monkeys

McNaught and colleagues 1 reported recently that systemic administration of proteasome inhibitors PSI (Z‐Ileu‐Glu(OtBu)‐Ala‐Leu‐CHO) or epoxomicin recapitulated many of the degenerative changes seen in Parkinsons disease including loss of striatal dopamine and cell loss in the substantia nigra, locus ceruleus, dorsal motor nucleus of the X cranial nerve, and nucleus basalis of Meynert. Intracytoplasmic inclusions resembling Lewy bodies were also described. All experiments administering PSI to rats using identical procedures and multiple attempts failed to induce any of the previously described changes. Furthermore, administration of PSI or epoxomicin to monkeys in an attempt to extend the model to a primate species failed. Currently, systemic proteasome inhibition is not a reliable model for Parkinsons disease. Ann Neurol 2006;60:264–268

Proteasome inhibition and Parkinson's disease modeling.

Impaired proteasome function is a potential mechanism for dopaminergic neuron degeneration. To model this molecular defect, we administered systemically the reversible lipophilic proteasome inhibitor, carbobenzoxy‐L‐isoleucyl‐γ‐t‐butyl‐L‐glutamyl‐L‐alanyl‐L‐leucinal (PSI), to rodents. In contrast to a previous report, this approach failed to cause any detectable behavioral or neuropathological abnormality in either rats or mice. Although theoretically appealing, this specific model of Parkinsons disease appears to exhibit poor reproducibility. Ann Neurol 2006;60:260–264

Nurr1 in Parkinson's disease and related disorders

In mammals, the transcription factor Nurr1 is expressed early in development and continues to be detectable throughout the organisms lifetime. Nurr1 is involved in the establishment and maintenance of the dopaminergic phenotype within specific central nervous system neuronal subpopulations including the nigrostriatal dopamine system. This protein is reduced over the course of normal aging, which is a major risk factor for Parkinsons disease (PD). However, whether Nurr1 expression is affected by PD has not been documented. The present study examined the role of Nurr1 in the maintenance of the dopaminergic phenotype within neurons in substantia nigra in PD compared with patients with diagnoses of progressive supranuclear palsy (PSP) or Alzheimers disease (AD) or age‐matched‐matched controls. In PD, the optical density (OD) of Nurr1 immunofluorescence was significantly decreased in nigral neurons containing α‐synuclein‐immunoreactive inclusions. Similarly, the OD of Nurr1 immunofluorescence intensity in the nigra of AD cases was decreased in neurons with neurofibrillary tangles (NFTs). In contrast to PD and AD, the OD of Nurr1 immunofluorescence intensity was severely decreased in the neurons with or without NFTs in PSP cases. Decline of Nurr1‐ir neuronal number and OD was observed within substantia nigra (SN) neurons in PD but not within hippocampal neurons. The decline in Nurr1‐ir expression was correlated with loss of tyrosine hydroxylase immunofluorescence across the four groups. These data demonstrate that Nurr1 deficiency in dopaminergic neurons is associated with the intracellular pathology in both synucleinopathies and tauopathies. J. Comp. Neurol. 494:495–514, 2006.