Identification of RBMX as a splicing regulator in Parkinsonian mimetic induced alternative splicing of α-synuclein.

α-Synuclein (α-syn) plays a precipitating role in Parkinson's disease (PD) due to its tendency to form oligomers and fibrils. The presence of smaller isoforms of α-syn was widely noticed in the affected brain regions of PD patients. 112-synuclein (112-syn) which lacks exon-5, possess enhanced aggregation propensity and forms intracellular inclusions. However, the factors responsible for the skipping of exon-5 are not completely understood. In this context, we aimed to identity the cis & trans-acting elements governing alternative splicing (AS) events by the Parkinsonian agent (MPP+) using minigene constructs. Minigene-I and -II were constructed by pruning the intron-4 and -5 regions respectively without altering the branch point adenosine to preserve splicing machinery. Also, chimeric minigenes were engineered by replacing either 5' (Mini-III) or 3' (Mini-IV) flanking intronic regions of exon-5 with other intronic regions (intron-3 and -2) that are not responsive to MPP+ induced splicing. While all the above minigenes exhibited MPP+-induced skipping of exon-5, Minigene-III did not generate the spliced product indicating that the 5' flanking intronic region (316 bp) of exon-5 possess cis-acting elements responsible for oxidant-induced alternative splicing. RNA-Binding Protein Database (RBDP) analysis revealed the presence of four putative RNA binding proteins (RBPs), namely, RBMX, MBNL1, KHDRBS3 and SFRS1 that may bind to the 316 bp region of intron-4and their expression was substantially reduced following MPP+ treatment. Further, overexpression of RBMX mitigated MPP+-induced generation of 112-syn and also reduced intracellular α-syn aggregates. Overall, our study identified the pivotal role of the splicing regulator, RBMX, in the pathophysiology of PD.

Identification of an Alternatively Spliced α-Synuclein Isoform That Generates a 41-Amino Acid N-Terminal Truncated Peptide, 41-syn: Role in Dopamine Homeostasis.

The presynaptic protein, α-synuclein (α-syn), has been shown to play a crucial role in multiple neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), and dementia with Lewy bodies (DLB). The three major domains of α-syn protein were shown to govern its membrane interaction, protein fibrillation, and chaperone activity. So far, four different alternatively spliced isoforms of α-syn, which lack either exon 3 (syn-126) or exon 5 (syn-112) or both (syn-98) resulting in altered function of the proteins, have been identified. In the present study, we have identified the smallest isoform of α-syn due to the skipping of exons 3 and 4 generating a 238 bp transcript. Due to the presence of a premature stop codon, the 238 bp transcript generated a 41 aa N-terminal peptide instead of the 78 aa protein, which is secreted into the extracellular medium when overexpressed in cells. The presence of 41-syn was initially noticed in the substantia nigra of PD autopsy tissues, as well as in cells undergoing oxidative stress. In vitro studies inferred that 41-syn neither aggregates nor alters the aggregation propensity of either WT or 112-syn. Overexpression of 41-syn or treatment of cells with 41-syn peptide did not affect cell viability. However, PC-12 cells treated with 41-syn exhibited a time and dose dependent enhancement in the cellular uptake of dopamine. Based on the physiological role of the N-terminal region of α-syn in modulating membrane trafficking events, we believe that the identification of 41-syn may provide novel impetus in unraveling the physiological basis of alternative splicing events in governing PD pathophysiology.

The unintended mitochondrial uncoupling effects of the FDA-approved anti-helminth drug nitazoxanide mitigates experimental parkinsonism in mice.

Mitochondria play a primary role in the pathophysiology of Parkinson's disease (PD), and small molecules that counteract the initial stages of disease may offer therapeutic benefit. In this regard, we have examined whether the off-target effects of the Food and Drug Administration (FDA)-approved anti-helminth drug nitazoxanide (NTZ) on mitochondrial respiration could possess any therapeutic potential for PD. Results indicate that MPP+-induced loss in oxygen consumption rate (OCR) and ATP production by mitochondria were ameliorated by NTZ in real time by virtue of its mild uncoupling effect. Pretreatment of cells with NTZ mitigated MPP+-induced loss in mitochondrial OCR and reactive oxygen species (ROS). Similarly, addition of NTZ to cells pretreated with MPP+ could reverse block in mitochondrial OCR and reactive oxygen species induced by MPP+ in real time. The observed effects of NTZ were found to be transient and reversible as removal of NTZ from incubation medium restored the mitochondrial respiration to that of controls. Apoptosis induced by MPP+ was ameliorated by NTZ in a dose-dependent manner. In vivo results demonstrated that oral administration of NTZ (50 mg/kg) in an acute MPTP mouse model of PD conferred significant protection against the loss of tyrosine hydroxylase (TH)-positive neurons of substantia nigra. Based on the above observations we believe that repurposing of NTZ for PD may offer therapeutic benefit.