Molecular Cloning of a Novel Brain‐specific RGS6 Isoform Preferentially Expressed in the Substantia Nigra of Humans with Parkinson's Disease

Katelin E Ahlers-Dannen; Adele Stewart; Jianqi Yang; John G Koland; Rory A Fisher

doi:10.1096/fasebj.2018.32.1_supplement.557.4

RGS proteins modulate G protein‐coupled receptor (GPCR) signaling by facilitating heterotrimeric G protein inactivation through their GTPase‐activating (GAP) activity toward Gα subunits, a function bestowed by their RGS domain. RGS6 (Gαi/o‐ GAP), a member of the R7 subfamily, is critically involved in several CNS disorders for which it may be a novel therapeutic target. Remarkably, RGS6−/− mice have reduced anxiety/depression, exhibit diminished alcohol seeking/reward behaviors, and develop late‐onset Parkinson's disease (PD). The role of RGS6 in these disorders depends on its ability to inhibit various GPCRs, including: cortical and hippocampal 5‐HT1ARs (anxiety/depression), mesolimbic GABABRs and D2Rs (alcoholism), and D2Rs in the substantia nigra (SNc, PD). Potentially key to RGS6's ability to regulate numerous GPCRs are unidentified domains arising via alternative mRNA splicing. Our initial cloning effort identified 36 RGS6 mRNAs in human brain encoding proteins ≤56kDa. Recently, we identified, in mouse and human, additional brain‐specific RGS6 protein isoforms that are larger (~61, 69kDa) than the ubiquitously expressed ~56kDa RGS6L isoforms. The function of these RGS6 isoforms is unknown, but they may be critical for normal CNS function and pathology as both are expressed in brain regions affected by the disorders described above. Here we report the cloning of a novel RGS6 transcript arising via novel exon (Alternative 3, A3) inclusion. This transcript was named RGS6LA3α1 to indicate it resembles the RGS6Lα1 transcript identified in our initial cloning effort. RGS6LA3α1 exhibits near exclusive CNS expression, encodes a protein that co‐migrates with the 69kDa brain‐specific RGS6, and has a C‐terminal extension near the RGS domain that may be a novel protein interaction site or regulatory domain. Of particular interest, is the role of the 69kDa RGS6 in PD pathogenesis as its expression, as well as its transcript, is upregulated in the SNc of PD patients relative to other RGS6L proteins which are down‐regulated. This latter finding is consistent with our evidence that RGS6 is required for SNc dopamine (DA) neuron survival. However, these data also raise the intriguing possibility that while RGS6L isoforms promote SNc DA neuron survival, RGS6LA3α1/the 69kDa brain‐specific RGS6 protein isoform may fail to do so or even contribute to SNc DA neuron death. We hypothesize that dysregulated splicing of primary RGS6 transcripts, and A3 exon inclusion, could lead to both a physical loss of RGS6L protein isoforms as well as a functional loss of RGS6‐mediated GAP activity due to RGS domain interference caused by C‐terminal extension, a prediction supported by molecular modeling studies. We predict that decreased expression of RGS6L isoforms and increased expression of the 69kDa protein disinhibits SNc D2R signaling culminating in accumulation of cytotoxic DA byproducts as well as decreased mitochondrial number and size ultimately inducing SNc DA neuron death. Together, this research begins to elucidate the functional significance of RGS6 alternative mRNA splicing in brain function and pathology. Support or Funding Information NIH CA161882 and MJ Fox 11551 This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.

Molecular Cloning of a Novel Brain‐specific RGS6 Isoform Preferentially Expressed in the Substantia Nigra of Humans with Parkinson's Disease

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