Differential Expression of Piccolo Splice Isoforms During Cerebellar Development

Piccolo is a multi‐domain protein with restricted expression to the presynaptic active zone, the site of synaptic vesicle fusion and neurotransmitter release. Alternative splicing of the Piccolo gene ( PCLO) results in two primary splice isoforms: isoform‐1 (I‐1) with two C2 domains (C2A and C2B) and isoform‐2 (I‐2) with only a single C2A motif. In addition, an exon‐skipping event generates Piccolo isoforms that alter the C2A domain resulting in exclusion of a 27‐nucleotide exon located in the C2A domain (e.g. short variant). The result is a lack of 9 amino acids in the domain resulting in increased calcium and phospholipid binding affinity versus the longer C2A domain (e.g. long variant) that includes the 9‐peptide region. Consistent with the importance of the Ca 2+ ‐binding property of C2 domain‐containing proteins in synaptic function, genome‐wide association studies have identified variations in or near the C2A domain of human PCLO that predispose individuals to affective disorders. The most compelling evidence for the importance of the C2A domain comes from recent identification of the genetic cause of pontocerebellar hypoplasia type III (PCH3): a neurological disorder that results in severe underdevelopment of the cerebellum. Analysis of genomic DNA from two affected individuals with PCH3 identified a homozygous nonsense variant in PCLO that introduces a STOP codon in the 3′ coding region including the C2A domain. The purpose of this study was to examine the temporal expression of Piccolo splice variants during critical periods of mouse cerebellar and cortical development. We performed real‐time quantitative RT‐PCR gene expression analysis of Piccolo splice isoforms (e.g. I‐1, I‐2, long and short) from RNA isolated from cerebellum at key stages of cerebellar granule cell differentiation (P0, P5, P15, adult) and also from the cortex at the same ages. Our analysis demonstrated that expression levels of the I‐1 and I‐2 splice variants were relatively consistent between cerebellum and cortex at all examined developmental time points. In contrast, we showed that during initial stages of cerebellar development (P0 and P5), the more calcium‐sensitive short variant had significantly increased expression levels relative to the long variant. Our results indicate that increased expression of the short variant may play a role in neuronal proliferation during initial cerebellar development when considering the augmented calcium permeability of immature neurons during this time period. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .