Functional Assessment of Single Nucleotide Polymorphisms in Nir2 in the Context of Neurodegeneration

Abstract

Nir2 is a lipid transfer protein that operates at the endoplasmic reticulum (ER) - plasma membrane (PM) contact sites, facilitating the transfer of phosphatidylinositol (PI) from ER to PM and phosphatidic acid (PA) in the opposite direction. This process plays a crucial role in maintaining phospholipase C (PLC)-dependent Phosphatidylinositol 4,5-bisphosphate (PIP2) signalling. Whole exome sequencing data from a cohort of patients with clinical features like cerebellar dysfunction, suggestive of Spinocerebellar Ataxia (SCA), revealed Single Nucleotide Polymorphisms (SNPs) in the Nir2-encoding PITPNM1 gene in six patients. Two of these SNPs (K191E and Q135P) were found in the PITP domain of Nir2. We hypothesise that SNPs in this domain might cause aberrant PI-transfer function of the protein, consequently disrupting PIP2 signalling and thereby perturbing the downstream Ca2+ homeostasis. Previous studies show evidence that dysregulated Ca2+ homeostasis could contribute to the onset and progression of several neurodegenerative disorders, including SCA. Molecular dynamics simulation predicted that only one of the SNPs (K191E) adversely affected the structure, stability, and function of Nir2. Experimental validation revealed no alterations in the expression and localisation of Nir2 containing either of the SNPs (K191E and Q135P). However, assessment of receptor-activated intracellular Ca2+ release as a readout of Nir2 function demonstrated significant functional impairment.