Ippolita Cantuti-Castelvetri

PGC-1α, A Potential Therapeutic Target for Early Intervention in Parkinson’s Disease

Abnormal expression of genes for energy regulation in Parkinson’s disease patients identifies a master regulator as a possible therapeutic target for early intervention. Getting to the Root of Parkinson’s Disease Parkinson’s disease (PD) is a debilitating neurodegenerative disorder that results in the loss of dopamine neurons in the substantia nigra of the brain. Degeneration of these movement-related neurons predictably causes rigidity, slowness of movement, and resting tremor, but patients also show cognitive changes. Although gene mutations have been identified in several families with PD, the cause of the more common sporadic form is not known. Certain environmental factors, such as exposure to the pesticide rotenone, combined with a genetic susceptibility, are thought to confer risk for developing PD. A key pathological feature seen in postmortem brain tissue from PD patients is Lewy bodies, neuronal inclusions containing clumps of the α-synuclein protein (which is mutated in familial PD), as well as damaged mitochondria. Taking a systems biology approach to pinpoint the root cause of PD, Zheng et al. now implicate altered activity of the master transcription factor PGC-1α and the genes it regulates in the early stages of PD pathogenesis. To detect new sets of genes that may be associated with PD, the investigators did a meta-analysis of 17 independent genome-wide gene expression microarray studies that had been performed on a total of 322 postmortem brain tissue samples and 88 blood samples. The samples came from presymptomatic and symptomatic PD patients, as well as from control individuals who did not show any neurological deficits at autopsy. Nine genome-wide expression studies were conducted either on dopaminergic neurons obtained by laser capture from substantia nigra (three studies) or on substantia nigra homogenates (six studies). The authors then used a powerful tool called Gene Set Enrichment Analysis to sift through 522 gene sets (a gene set is a group of genes involved in one biological pathway or process). At the end of this tour-de-force analysis, they identified 10 gene sets that were all associated with PD. The gene sets with the strongest association contained nuclear genes encoding subunits of the electron transport chain proteins found in mitochondria. These genes all showed decreased expression in substantia nigra dopaminergic neurons (obtained by laser capture) even in the earliest stages of PD. Furthermore, a second gene set associated with PD and also underexpressed in the earliest stages of PD encodes enzymes involved in glucose metabolism. These results are compelling because many studies have already implicated dysfunctional mitochondria and altered energy metabolism as well as defective glucose metabolism in PD. The authors realized that these gene sets had in common the master transcriptional regulator, PGC-1α, and surmised that disruption of PGC-1α expression might be a root cause of PD. They tested this hypothesis in cultured dopaminergic neurons from embryonic rat midbrain forced to express a mutant form of α-synuclein. Overexpression of PGC-1α in these neurons resulted in activation of electron transport genes and protection against neuronal damage induced by mutant α-synuclein. In other cultured neurons treated with rotenone, overexpression of PGC-1α also was protective, blocking pesticide-induced neuronal cell death. These exciting findings identify altered expression of PGC-1α and the genes it regulates as key players during early PD pathogenesis. This potential new target could be exploited therapeutically to interfere with the pathological process during the earliest stages before permanent damage and neuronal loss occurs. Parkinson’s disease affects 5 million people worldwide, but the molecular mechanisms underlying its pathogenesis are still unclear. Here, we report a genome-wide meta-analysis of gene sets (groups of genes that encode the same biological pathway or process) in 410 samples from patients with symptomatic Parkinson’s and subclinical disease and healthy controls. We analyzed 6.8 million raw data points from nine genome-wide expression studies, and 185 laser-captured human dopaminergic neuron and substantia nigra transcriptomes, followed by two-stage replication on three platforms. We found 10 gene sets with previously unknown associations with Parkinson’s disease. These gene sets pinpoint defects in mitochondrial electron transport, glucose utilization, and glucose sensing and reveal that they occur early in disease pathogenesis. Genes controlling cellular bioenergetics that are expressed in response to peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) are underexpressed in Parkinson’s disease patients. Activation of PGC-1α results in increased expression of nuclear-encoded subunits of the mitochondrial respiratory chain and blocks the dopaminergic neuron loss induced by mutant α-synuclein or the pesticide rotenone in cellular disease models. Our systems biology analysis of Parkinson’s disease identifies PGC-1α as a potential therapeutic target for early intervention.

Neurobehavioral aspects of antioxidants in aging.

Both aging and age‐associated neurodegenerative diseases are associated with various degrees of behavioral impairments, and among the prime candidates responsible for producing the neuronal changes mediating these behavioral deficits appear to be free radicals and the oxidative stress they generate. Therefore, there have been a number of studies which have examined the putative positive benefits of antioxidants in altering, reversing, or forestalling these neuronal/behavioral decrements, with varying degrees of success. Additional experiments have examined the effects of diets rich in fruits and vegetables or herbal extracts in reducing certain types of cancer and cardiovascular diseases, and evidence emerging from such experiments suggests that these kinds of dietary modifications may be beneficial in altering neuronal/behavioral deficits in aging, as well. These kinds of diets are particularly rich in antioxidants such as vitamins A, C, E, and bioflavonoids (such as flavones, tannins, and anthocyanins), and thus, there may be synergistic effects among them. The present paper will review studies concerning the influence of dietary and synthetic antioxidants on normal, pathological age‐related, and reactive oxygen species‐induced behavioral changes in human and animal subjects. The antioxidants reviewed are vitamin E, α‐lipoic acid, and the phytochemicals contained in herbals, fruits and vegetables.

Distinct Roles In Vivo for the Ubiquitin–Proteasome System and the Autophagy–Lysosomal Pathway in the Degradation of α-Synuclein

Increased intracellular levels of α-synuclein are implicated in Parkinsons disease and related disorders and may be caused by alterations in the ubiquitin–proteasome system (UPS) or the autophagy–lysosomal pathway (ALP). A critical question remains how α-synuclein is degraded by neurons in vivo. To address this, our study uses α-synuclein transgenic mice, expressing human α-synuclein or α-synuclein-eGFP under the (h)PDGF-β promoter, in combination with in vivo pharmacologic and multiphoton imaging strategies to systematically test degradation pathways in the living mouse brain. We demonstrate that the UPS is the main degradation pathway for α-synuclein under normal conditions in vivo while with increased α-synuclein burden the ALP is recruited. Moreover, we report alterations of the UPS in α-synuclein transgenic mice and age dependence to the role of the UPS in α-synuclein degradation. In addition, we provide evidence that the UPS and ALP might be functionally connected such that impairment of one can upregulate the other. These results provide a novel link between the UPS, the ALP, and α-synuclein pathology and may have important implications for future therapeutics targeting degradation pathways.

Effects of Gender on Nigral Gene Expression and Parkinson Disease

To identify gene expression patterns in human dopamine (DA) neurons in the substantia nigra pars compacta (SNc) of male and female control and Parkinson disease (PD) patients, we harvested DA neurons from frozen SNc from 16 subjects (4 male PDs, 4 female PDs, 4 male and 4 female controls) using Laser Capture microdissection and microarrays. We assessed for enrichment of functional categories with a hypergeometric distribution. The data were validated with QPCR. We observed that gender has a pervasive effect on gene expression in DA neurons. Genes upregulated in females relative to males are mainly involved in signal transduction and neuronal maturation, while in males some of the upregulated genes (alpha-synuclein and PINK1) were previously implicated in the pathogenesis of PD. In females with PD we found alterations in genes with protein kinase activity, genes involved in proteolysis and WNT signaling pathway, while in males with PD there were alterations in protein-binding proteins and copper-binding proteins. Our data reveal broad gender-based differences in gene expression in human dopaminergic neurons of SNc that may underlie the predisposition of males to PD. Moreover, we show that gender influences the response to PD, suggesting that the nature of the disease and the response to treatment may be gender-dependent.

Dopaminergic neuron loss and up-regulation of chaperone protein mRNA induced by targeted over-expression of alpha-synuclein in mouse substantia nigra

Several transgenic mouse lines with altered α‐synuclein expression have been developed that show a variety of Parkinson’s disease‐like symptoms without specific loss of dopaminergic neurons. Targeted over‐expression of human α‐synuclein using viral‐vector mediated gene delivery into the substantia nigra of rats and non‐human primates leads to dopaminergic cell loss and the formation of α‐synuclein aggregates reminiscent of Lewy bodies. In the context of these recent findings, we used adeno‐associated virus (AAV) to over‐express wild type human α‐synuclein in the substantia nigra of mice. We hypothesized that this over‐expression would recapitulate pathological hallmarks of Parkinson’s disease, creating a mouse model to further characterize the disease pathogenesis. Recombinant AAV expressing α‐synuclein was stereotaxically injected into the substantia nigra of mice, leading to a 25% reduction of dopaminergic neurons after 24 weeks of transduction. Furthermore, examination of mRNA levels of stress‐related proteins using laser capture microdissection and quantitative PCR revealed a positive correlation of Hsp27 expression with the extent of viral transduction at 4 weeks and a positive correlation of Hsp40, Hsp70 and caspase 9 with the extent of viral transduction at 24 weeks. Taken together, our findings suggest that targeted over‐expression of α‐synuclein can induce pathology at the gross anatomical and molecular level in the substantia nigra, providing a mouse model in which upstream changes in Parkinson’s disease pathogenesis can be further elucidated.

Somatic mitochondrial DNA mutations in single neurons and glia

Somatic mitochondrial DNA (mtDNA) point mutations reach high levels in the brain. However, the cell types that accumulate mutations and the patterns of mutations within individual cells are not known. We have quantified somatic mtDNA mutations in 28 single neurons and in 18 single glia from post-mortem human substantia nigra of six control subjects. Both neurons and glia contain mtDNA with somatic mutations. Single neurons harbor a geometric mean (95% CI) of 200.3 (152.9-262.4) somatic mtDNA point mutations per million base pairs, compared to 133.8 (97.5-184.9) for single glia (p=0.0251). If mutations detected multiple times in the same cell are counted only once, the mean mutation level per million base pairs remains elevated in single neurons (146.9; 124.0-174.2) compared to single glia (100.5; 81.5-126.5; p=0.009). Multiple distinct somatic point mutations are present in different cells from the same subject. Most of these mutations are individually present at low levels (less than 10-20% of mtDNA molecules), but with high aggregate mutation levels, particularly in neurons. These mutations may contribute to changes in brain function during normal aging and neurodegenerative disorders.

Transcriptional dysregulation in a transgenic model of Parkinson disease

Alpha-synuclein has been implicated in Parkinson disease, yet the mechanism by which alpha-synuclein causes cell injury is not understood. Using a transgenic mouse model, we evaluated the effect of alpha-synuclein overexpression on gene expression in the substantia nigra. Nigral mRNA from wild type and alpha-synuclein transgenic mice was analyzed using Affymetrix gene arrays. At 3 months, before pathological changes are apparent, we observed modest alterations in gene expression. However, nearly 200 genes were altered in expression at 9 months, when degenerative changes are more apparent. Functional genomic analysis revealed that the genes altered at 9 months were predominantly involved in gene transcription. As in human Parkinson disease, gene expression changes in the transgenic model were also modulated by gender. These data demonstrate that alterations of gene expression are widespread in this animal model, and suggest that transcriptional dysregulation may be a disease mechanism that can be targeted therapeutically.

Cognitive deficits induced by 56Fe radiation exposure.

Exposing rats to particles of high energy and charge (e.g., 56Fe) disrupts neuronal systems and the behaviors mediated by them; these adverse behavioral and neuronal effects are similar to those seen in aged animals. Because cognition declines with age, and our previous study showed that radiation disrupted Morris water maze spatial learning and memory performance, the present study used an 8-arm radial maze (RAM) to further test the cognitive behavioral consequences of radiation exposure. Control rats or rats exposed to whole-body irradiation with 1.0 Gy of 1 GeV/n high-energy 56Fe particles (delivered at the alternating gradient synchrotron at Brookhaven National Laboratory) were tested nine months following exposure. Radiation adversely affected RAM performance, and the changes seen parallel those of aging. Irradiated animals entered baited arms during the first 4 choices significantly less than did controls, produced their first error sooner, and also tended to make more errors as measured by re-entries into non-baited arms. These results show that irradiation with high-energy particles produces age-like decrements in cognitive behavior that may impair the ability of astronauts to perform critical tasks during long-term space travel beyond the magnetosphere.

Somatic mitochondrial DNA mutations in early parkinson and incidental lewy body disease

Somatic mutations in mitochondrial DNA (mtDNA) are hypothesized to play a role in Parkinson disease (PD), but large increases in mtDNA mutations have not previously been found in PD, potentially because neurons with high mutation levels degenerate and thus are absent in late stage tissue. To address this issue, we studied early stage PD cases and cases of incidental Lewy body disease (ILBD), which is thought to represent presymptomatic PD. We show for the first time that mtDNA mutation levels in substantia nigra neurons are significantly elevated in this group of early PD and ILBD cases. Ann Neurol 2012;71:850–854

Dysregulation of CalDAG-GEFI and CalDAG-GEFII predicts the severity of motor side-effects induced by anti-parkinsonian therapy

Voluntary movement difficulties in Parkinsons disease are initially relieved by l-DOPA therapy, but with disease progression, the repeated l-DOPA treatments can produce debilitating motor abnormalities known as l-DOPA-induced dyskinesias. We show here that 2 striatum-enriched regulators of the Ras/Rap/ERK MAP kinase signal transduction cascade, matrix-enriched CalDAG-GEFI and striosome-enriched CalDAG-GEFII (also known as RasGRP), are strongly and inversely dysregulated in proportion to the severity of abnormal movements induced by l-DOPA in a rat model of parkinsonism. In the dopamine-depleted striatum, the l-DOPA treatments produce down-regulation of CalDAG-GEFI and up-regulation of CalDAG-GEFII mRNAs and proteins, and quantification of the mRNA levels shows that these changes are closely correlated with the severity of the dyskinesias. As these CalDAG-GEFs control ERK cascades, which are implicated in l-DOPA-induced dyskinesias, and have differential compartmental expression patterns in the striatum, we suggest that they may be key molecules involved in the expression of the dyskinesias. They thus represent promising new therapeutic targets for limiting the motor complications induced by l-DOPA therapy.