Linda B. Moran

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.

Whole genome expression profiling of the medial and lateral substantia nigra in Parkinson’s disease

We have used brain tissue from clinically well-documented and neuropathologically confirmed cases of sporadic Parkinson’s disease to establish the transcriptomic expression profile of the medial and lateral substantia nigra. In addition, the superior frontal cortex was analyzed in a subset of the same cases. DNA oligonucleotide microarrays were employed, which provide whole human genome coverage. A total of 570 genes were found to be differentially regulated at a high level of significance. A large number of differentially regulated expressed sequence tags were also identified. Levels of mRNA sequences encoded by genes of key interest were validated by means of quantitative real-time polymerase chain reaction (PCR). Comparing three different normalization procedures, results based on the recently published GeneChip Robust Multi Array algorithm were found to be the most accurate predictor of real-time PCR results. Several new candidate genes which map to PARK loci are reported. In addition, the DNAJ family of chaperones is discussed in the context of Parkinson’s disease pathogenesis.

Mechanisms of cell death in neurodegenerative diseases: Fashion, fiction, and facts

Apoptosis has become a most popular concept of cell death. However, the term is now so widely used and employed in such general terms in relation to neurological diseases that its application is very problematic. In addition, with the exception of developmental conditions, there is essentially no evidence of apoptosis fulfilling the criteria of its classical definition in any of the important human neurodegenerative diseases, including Alzheimers, Parkinsons, Huntingtons, Amyotrophic Lateral Sclerosis, and Creutzfeldt‐Jakob disease. Importantly, a number of new cell death forms have been described in the literature and there is good reason to pay attention to these emerging concepts as they may provide a rationale for the development of disease‐specific therapies.

Transcriptome analysis reveals link between proteasomal and mitochondrial pathways in Parkinson’s disease

There is growing evidence that dysfunction of the mitochondrial respiratory chain and failure of the cellular protein degradation machinery, specifically the ubiquitin–proteasome system, play an important role in the pathogenesis of Parkinson’s disease. We now show that the corresponding pathways of these two systems are linked at the transcriptomic level in Parkinsonian substantia nigra. We examined gene expression in medial and lateral substantia nigra (SN) as well as in frontal cortex using whole genome DNA oligonucleotide microarrays. In this study, we use a hypothesis-driven approach in analysing microarray data to describe the expression of mitochondrial and ubiquitin–proteasomal system (UPS) genes in Parkinson’s disease (PD). Although a number of genes showed up-regulation, we found an overall decrease in expression affecting the majority of mitochondrial and UPS sequences. The down-regulated genes include genes that encode subunits of complex I and the Parkinson’s-disease-linked UCHL1. The observed changes in expression were very similar for both medial and lateral SN and also affected the PD cerebral cortex. As revealed by “gene shaving” clustering analysis, there was a very significant correlation between the transcriptomic profiles of both systems including in control brains. Therefore, the mitochondria and the proteasome form a higher-order gene regulatory network that is severely perturbed in Parkinson’s disease. Our quantitative results also suggest that Parkinson’s disease is a disease of more than one cell class, i.e. that it goes beyond the catecholaminergic neuron and involves glia as well.

Towards a pathway definition of Parkinson’s disease: a complex disorder with links to cancer, diabetes and inflammation

We have previously established a first whole genome transcriptomic profile of sporadic Parkinson’s disease (PD). After extensive brain tissue-based validation combined with cycles of iterative data analysis and by focusing on the most comparable cases of the cohort, we have refined our analysis and established a list of 892 highly dysregulated priority genes that are considered to form the core of the diseased Parkinsonian metabolic network. The substantia nigra pathways, now under scrutiny, contain more than 100 genes whose association with PD is known from the literature. Of those, more than 40 genes belong to the highly significantly dysregulated group identified in our dataset. Apart from the complete list of 892 priority genes, we present pathways revealing PD ‘hub’ as well as ‘peripheral’ network genes. The latter include Lewy body components or interact with known PD genes. Biological associations of PD with cancer, diabetes and inflammation are discussed and interactions of the priority genes with several drugs are provided. Our study illustrates the value of rigorous clinico-pathological correlation when analysing high-throughput data to make optimal use of the histopathological phenome, or morphonome which currently serves as the key diagnostic reference for most human diseases. The need for systematic human tissue banking, following the highest possible professional and ethical standard to enable sustainability, becomes evident.

Towards a transcriptome definition of microglial cells

Abstract.This study provides an expression signature of interferon-gamma (IFN-γ)-activated microglia. Microglia are macrophage precursor cells residing in the brain and spinal cord. The microglial phenotype is highly plastic and changes in response to numerous pathological stimuli. IFN-γ has been established as a strong immunological activator of microglial cells both in vitro and in vivo. Affymetrix RG_U34A microarrays were used to determine the effect of IFN-γ stimulation on migroglia cells isolated from newborn Lewis rat brains. More than 8,000 gene sequences were examined, i.e., 7,000 known genes and 1,000 expressed sequence tag (EST) clusters. Under baseline conditions, microglia expressed 326 of 8,000 genes examined (approximately 4% of all genes, 182 known and 144 ESTs). Transcription of only 34 of 7,000 known genes and 8 of 1,000 ESTs was induced by IFN-γ stimulation. The majority of the newly expressed genes encode pro-inflammatory cytokines and components of the MHC-mediated antigen presentation pathway. The expression of 60 of 182 identified genes and of 9 of 144 ESTs was increased by IFN-γ, whereas 29 of 182 known genes and 7 of 144 ESTs were down-regulated or undetectable in IFN-γ-stimulated cultures. Overall, the activating effect of IFN-γ on the microglial transcriptome showed restriction to pathways involved in antigen presentation, protein degradation, actin binding, cell adhesion, apoptosis, and cell signaling. In comparison, down-regulatory effects of IFN-γ stimulation appeared to be confined to pathways of growth regulation, remodeling of the extracellular matrix, lipid metabolism, and lysosomal processing. In addition, transcriptomic profiling revealed previously unknown microglial genes that were de novo expressed, such as calponin 3, or indicated differential regulatory responses, such as down-regulation of cathepsins that are up-regulated in response to other microglia stimulators.

DnaJB6 Is Present in the Core of Lewy Bodies and Is Highly Up-Regulated in Parkinsonian Astrocytes

DnaJ/Hsp40 chaperones determine the activity of Hsp70s by stabilizing their interaction with substrate proteins. We have predicted, based on the in silico analysis of a brain‐derived whole‐genome transcriptome data set, an increased expression of DnaJ/Hsp40 homologue, subfamily B, member 6 (DnaJB6) in Parkinsons disease (PD; Moran et al. [ 2006 ] Neurogenetics 7:1–11). We now show that DnaJB6 is a novel component of Lewy bodies (LBs) in both PD substantia nigra and PD cortex and that it is strongly up‐regulated in parkinsonian astrocytes. The presence of DnaJB6 in the center of LBs suggests an early and direct involvement of this chaperone in the neuronal disease process associated with PD. The strong concomitant expression of DnaJB6 in astrocytes emphasizes the involvement of glial cells in PD and could indicate a route for therapeutic intervention. Extracellular alpha‐synuclein originating from intravesicular alpha‐synuclein is prone to aggregation and the potential source of extracellular aggregates (Lee [ 2008 ] J. Mol. Neurosci. 34:17–22). The observed strong expression of DnaJB6 by astrocytes could reflect a protective reaction, so reducing the neuronal release of toxic alpha‐synuclein and supporting the astrocyte response in PD might limit the progression of the disease process.

Increased incidence of gap junctional coupling between spinal motoneurones following transient blockade of NMDA receptors in neonatal rats.

Neonatal rat motoneurones are electrically coupled via gap junctions and the incidence of this coupling declines during postnatal development. The mechanisms involved in this developmental regulation of gap junctional communication are largely unknown. Here we have studied the role of NMDA receptor‐mediated glutamatergic synaptic activity in the regulation of motoneurone coupling. Gap junctional coupling was demonstrated by the presence of graded, short latency depolarising potentials following ventral root stimulation, and by the transfer of the low molecular weight tracer Neurobiotin to neighbouring motoneurones. Sites of close apposition between the somata and/or dendrites of the dye‐coupled motoneurones were identified as potential sites of gap junctional coupling. Early postnatal blockade of the NMDA subtype of glutamate receptors using the non‐competitive antagonist dizocilpine maleate (MK801) arrested the developmental decrease in electrotonic and dye coupling during the first postnatal week. These results suggest that the postnatal increase in glutamatergic synaptic activity associated with the onset of locomotion promote the loss of gap junctional connections between developing motoneurones.

Analysis of alpha-synuclein, dopamine and parkin pathways in neuropathologically confirmed parkinsonian nigra

The identification of mutations that cause familial Parkinson’s disease (PD) provides a framework for studies into pathways that may be perturbed also in the far more common, non-familial form of the disorder. Following this hypothesis, we have examined the gene regulatory network that links alpha-synuclein and parkin pathways with dopamine metabolism in neuropathologically verified cases of sporadic PD. By means of an in silico approach using a database of eukaryotic molecular interactions and a whole genome transcriptome dataset validated by qRT-PCR and histological methods, we found parkin and functionally associated genes to be up-regulated in the lateral substantia nigra (SN). In contrast, alpha-synuclein and ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene expression levels were significantly reduced in both the lateral and medial SN in PD. Gene expression for Septin 4, a member of the GTP-binding protein family involved in alpha-synuclein metabolism was elevated in the lateral parkinsonian SN. Additionally, catalase and mitogen-activated protein kinase 8 and poly(ADP-ribose) polymerase family member 1 (PARP1) known to function in DNA repair and cell death induction, all members of the dopamine synthesis pathway, were up-regulated in the lateral SN. In contrast, two additional PD-linked genes, glucocerebrosidase and nuclear receptor subfamily 4, group A, member 2 (NR4A2) showed reduced expression. We show that in sporadic PD, parkin, alpha-synuclein and dopamine pathways are co-deregulated. Alpha-synuclein is a member of all three gene regulatory networks. Our analysis results support the view that alpha-synuclein has a central role in the familial as well as the non-familial form of the disease and provide steps towards a pathway definition of PD.

Neuronal pentraxin II is highly upregulated in Parkinson’s disease and a novel component of Lewy bodies

Neuronal pentraxin II (NPTX2) is the most highly upregulated gene in the Parkinsonian substantia nigra based on our whole genome expression profiling results. We show here that it is a novel component of Lewy bodies and Lewy neurites in sporadic Parkinson’s disease (PD). NPTX2 is also known as the neuronal activity-regulated protein (Narp), which is secreted and involved in long-term neuronal plasticity. Narp further regulates AMPA receptors which have been found to mediate highly selective non-apoptotic cell death of dopaminergic neurons. NPTX2/Narp is found in close association with alpha-synuclein aggregates in both substantia nigra and cerebral cortex in PD but unlike alpha-synuclein gene expression, which is down-regulated in the Parkinsonian nigra, NPTX2 could represent a driver of the disease process. In view of its profound (>800%) upregulation and its established role in synaptic plasticity as well as dopaminergic nerve cell death, NPTX2 is a very interesting novel player which is likely to be involved in the pathway dysregulation which underlies PD.