Identification of causative and susceptibility variants in the neurexin-neuroligin pathway in patients with Alzheimer's disease. The role of a truncating mutation in Neuroligin 1

Abstract

Neurexins (NRXN) and neuroligins (NLGN) are synaptic cell-adhesion proteins involved in neurodevelopmental disorders, as autism and intellectual disability. Previously, we have postulated a role of NRNX-NLGN in Alzheimer's disease (AD). Here, we present genetic and functional approaches to identify variants of the NRNX-NLGN pathway in AD patients. Using next-generation sequencing, we have studied a panel of 36 genes of the NRNX-NLGN synaptic pathway in 192 AD patients. Potential causal variants were studied by in silico analysis and validated by Sanger sequencing. Susceptibility variants were analyzed by manual inspection in IGV (Integrative Genomics Viewer). Functional studies of selected variants were performed in N2A and COS cells and cultured hipocampal neurons. To identify susceptibility alleles, we selected SNPs whose allelic frequency differed among the genotyped AD patients and the control databases. These SNPs were then genotyped in a geographically-matched control population. On the other hand, potential causative mutations were selected among novel variants with a predicted pathogenic effect. We present a two base-pair insertion in NLGN1 (p.Thr271fs) in a patient with familial history of AD. The frameshift mutation in NLGN1 predicts a premature STOP codon that truncates the extracellular domain. We generated expression vectors for the p.Thr271fs mutation. In western blot experiments we detected a band of ~130 kD corresponding to wild type NLGN1. In contrast, the p.Thr271fs mutation resulted in truncated proteins of ~30 kDa. Localization studies in COS cells showed that p.Thr271fs NLGN1 failed to reach the plasma membrane and accumulated in the ER. In neurons, NLGN1 induces the formation of glutamatergic synapses. We showed that p.Thr271fs NLGN1 failed to induce the formation of glutamatergic synapses and accumulated in the soma of transfected neurons. Thus, the synaptic activity of NLGN1 was abolished by the AD-associated p.Thr271fs mutation. Our data report the first inactivating mutation in the NLGN1 gene in AD patients and support a role for the NRXN-NLGN pathway in the etiology of AD.