Regulation and function of novel brain-specific PGC-1alpha isoforms

Perturbations of PGC-1alpha expression and function have been implicated in the pathogenesis of several neurodegenerative disorders. Our recent investigation to clarify the function of an SNP (rs7665116) in intron 2 of the reference PPARGC1A gene, that showed associations with Huntington disease (HD) age-of-onset, led to the discovery of multiple transcripts that initiated from a common novel promoter located 587 kb upstream of exon 2. Using real-time PCR, RNase protection assays and Northern blotting, we showed that the majority of these transcripts were brain-specific and perhaps more abundant than the reference sequence PPARGC1A transcripts in whole brain. Two main transcripts containing independent methionine start codons were shown to encode full length brain-specific PGC-1alpha proteins that differed only at their N-termini from reference PGC-1alpha. Additional truncated isoforms containing these N-termini exist that are similar to N-terminal (NT)-PGC-1alpha. Other transcripts that lack the second LXXLL motif that serves as an interaction site for several nuclear receptors encode dominant negative forms. Furthermore, we showed that the new promoter is active in neuronal cell lines and described haplotypes encompassing this region that are associated with HD disease age-of-onset. The discovery of such a large PPARGC1A locus and multiple isoforms in brain warrant further studies on their basic biology. The primary objective this proposal is to compare the function and regulation of the new brain-specific isoforms with PGC-1alpha, encoded by the reference gene, in mouse and human brain and neuronal cell models. A secondary objective is to determine how potential differences in their regulation and function may affect the pathogenesis of HD and Parkinson disease (PD). These objectives will be approached by the following specific aims: i) to quantify the levels of the brain-specific and reference gene transcripts and their respective proteins in control and HD-transgenic mouse models subjected to neuroprotective regimens, in post-mortem human brain specimens and in cellular models of HD and PD; ii) to characterize the function of the brain-specific isoforms in relation to PGC-1alpha in control and HD and PD cell culture models with respect to their coactivation properties, their bioenergetic effects and their role in the defense against reactive oxygen species (ROS); iii) To further characterize the new brain-specific PPARGC1A promoter in comparison to the reference gene promoter and delineate the signaling pathways that converge at the two distinct promoter regions. These studies should provide essential data on the recently discovered brain-specific isoforms and their potential role in neurodegenerative disorders. Hence, results of this project may identify new tissue-specific targets for treating neurodegenerative diseases.

Parkinson Disease (PD) and Alzheimer Disease (AD) are the most common neurodegenerative disorders (ND) worldwide. While the clinical and neuropathological hallmarks differ between PD and AD, experimental and genetic studies strongly support defects in protein disposal via the proteasome and/or lysosome-autophagy system, mitochondrial function and damage by reactive oxygen species (ROS) in both disorders. PGC-1?, encoded by PPARGC1A, is a transcriptional co-activator that integrates transcriptional programs implicated in the pathogenesis of ND including mitochondrial biogenesis and function, the defense against ROS and auto- and mitophagy. We recently discovered multiple new PPARGC1A transcripts that initiate from a novel promoter located 587 kb upstream of exon 2. We showed that these new transcripts are specific for the central nervous system (CNS), are more abundant than the reference gene (RG) transcripts in whole brain and are, for the most part, conserved in rodents. Two main transcripts with independent methionine start codons encode full-length (FL) CNS-isoforms that differ only at their N-termini from the RG encoded PGC-1?. Several truncated isoforms of CNS-specific isoforms also exist. The main objective of the proposal was to gain basic information on CNS-specific PGC-1? isoforms. We studied the expression levels of CNS and RG PPARGC1A transcripts and isoforms in post-mortem tissues of cases with PD and controls and observed a moderate reduction of CNS-specific transcripts in PD in the substantia nigra that was attributed to loss of dopaminergic neurons. However, we observed in PD an upregulation of transcripts encoding a CNS-specific 17 kDa protein that inhibited the co-activation of several transcription factors by FL proteins. Furthermore, we identified haplotypes in the CNS-specific region of PPARGC1A that were protective for PD in clinical and post-mortem samples. Thus, functional and genetic studies implicate the CNS-specific PPARGC1A locus in PD. In two established animal models of PD, the PPARGC1A expression profiles differed from post-mortem human tissues, as several brain regions were affected. In AD, CNS-specific transcripts were also reduced in post-mortem brain tissue. Our additional studies suggest that PGC-1? induces the transcription of TOMM40, which was associated with AD according to our genetic studies. Finally, we identified several signalling pathways that activate the CNS or RG or both promoters. Importantly, hypoxia activated the CNS-specific promoter via HIF1A and HIF2 in cell culture experiments. The latter results were substantiated in an in vivo model animal model of brain hypoxia. Thus, the CNS-specific PGC-1a isoforms are likely to play role in disorders resulting from hypoxia such as ischemic stroke. In conclusion, our studies provide new mechanistic insight into the pathogenesis of common ND disorders and allow the formulation of new hypotheses to be tested in future studies.