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.

Extracellular-Signal-Regulated Kinase Inhibition Switches APP Processing from ß- to α-Secretase under Oxidative Stress: Modulation of ADAM10 by SIRT1/NF-κB Signaling.

The sequential cleavage of full-length amyloid precursor protein (APP) by secretases has been at the center of efforts for understanding the onset of Alzheimer's disease (AD). A decrease in α-secretase activity was observed during the progression of AD; however, the precise molecular mechanism involved in the downregulation of α-secretase under oxidative stress is not fully understood. In the present study, we have demonstrated that pharmacological inhibition of mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) by mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor (PD98059) restored the expression of a disintegrin and metalloproteinase 10 (ADAM10) with a concomitant decrease in ß-site APP cleavage enzyme 1 (BACE1) under oxidative stress. Silent mating-type information regulation 2 homologue 1 (SIRT1) activation by resveratrol also mitigated alterations in secretase levels through MAPK/ERK signaling. Intracerebroventricular (ICV) administration of streptozotocin in rats showed amyloidogenic processing of APP and altered the SIRT1/ERK axis in the hippocampus. We also observed that the ADAM10 expression is controlled at the transcriptional level by oxidative stress. Using the luciferase reporter activity of ADAM10 promoter deletion constructs, we have identified the region 290 bp upstream of the transcription start site (TSS) possessing regulatory elements responsible for ADAM10 downregulation with hydrogen peroxide (H2O2) treatment. Further, bioinformatics analysis revealed the presence of putative nuclear factor kappa B (NF-κB) binding sites in the ADAM10 promoter region. Treatment of cortical neurons with the NF-κB inhibitor (Bay 11-7082) mitigated the transcriptional upregulation of ADAM10 by PD98059. Overall, our findings suggest that SIRT1/ERK/NF-κB axis contributes to the downregulation of ADAM10, resulting in the shift from nonamyloidogenic to amyloidogenic processing of APP under oxidative stress.

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.