Neurexin (NRXN) proteins are presynaptic cell adhesion molecules that regulate neuronal communication. Deletions in the NRXN1 gene (chromosomal location 2p16.3) are associated with an increased risk of developing neurodevelopmental disorders (NDDs), including autism and schizophrenia, in which serotonergic dysfunction is evident. Recent brain imaging data, reporting altered dorsal raphé metabolism, also support the potential serotonergic dysfunction as a consequence of NRXN1a deletion. Understanding the impact of NRXN1a deficiency on the serotonin system is essential for evaluating the potential of serotonergic drug therapies for 2p16.3 deletion syndrome, among other NDDs. In Alzheimer's disease (AD), NRXN1a is also depleted from synapses by amyloid-b, disrupting synaptic function. Given the potential involvement of synaptic NRXN1a deficiency in both NDDs and AD, direct modulation of cell surface NRXN1a protein expression levels is also of interest. The mechanisms regulating the synaptic availability of NRXN1a are yet to be elucidated.
In the current study, a Nrxn1a heterozygous (HZ) mouse model was used to characterise serotonin receptor subtype gene expression in the prefrontal cortex (PFC) using RT-qPCR. Guided by observations from these studies, the 5-HT2A receptor antagonist/inverse agonist, volinanserin (MDL100,907), was administered to Nrxn1a HZ and wild-type (WT) mice, prior to behavioural assessment. Additionally, the impact on cerebral metabolism was then analysed using [14C]-2-Deoxyglucose brain imaging. Furthermore, a range of plasmid based NRXN1a expression systems and stable cell lines were developed to characterize NRXN1a expression, the mechanisms of NRXN1a proteolytic processing, and the impact on cell proliferation and survival.
Nrxn1a HZ mice displayed selective overexpression of Htr2a in the PFC, with no changes to other targets measured. Behaviourally, HZ mice showed improved fine motor skills, but displayed similar locomotor activity, hedonic response, memory, and repetitive behaviours to WT mice. Behaviourally volinanserin treatment reduced rotarod performance and locomotion for both genotypes. This might suggest that volinanserin may have use to correct abnormal locomotor phenotypes seen in Nrxn1a mice, the lack of genotype x treatment interaction precluded any firm conclusion, requiring further study. Genotype-mediated regions of hypometabolism were observed within regions of the basal ganglia, thalamus, and hippocampus of Nrxn1a HZ mice. Volinanserin treatment resulted in hyperactivation of the serotonergic raphé but decreased cerebral metabolism in the primary motor cortex potentially explaining the treatment-related behavioural motor deficits. However, drug treatment did not correct genotype induced hypometabolism deficits.
In the cell models generated, full-length NRXN1a (~160 kDa) was found to undergo cleavage at two ectodomain sites, generating soluble fragments (~150 kDa and ~45 kDa), and resulting in the production of a C-terminal fragment (CTF) (~15 kDa). A putative NRXN1a A Disintegrin and Metalloproteinase (ADAM) cleavage sequence was predicted in the juxtamembrane region. Mutation of this site influenced NRXN1a cleavage and expression in SH-SY5Y neuroblastoma cells but not in non-neuronal Human Embryonic Kidney (HEK) cells. NRXN1a shedding was reduced by Batimastat but not b-secretase Inhibitor IV. Additionally, the phorbol ester, phorbol myristate acetate (PMA), a protein kinase C (PKC) activator increased both protein expression and shedding, suggesting that NRXN1a expression is dynamically regulated by PKC signalling. Mutations at the predicted juxtamembrane cleavage site (E1400-V1401) reduced CTF generation relative to the full-length protein expression in SH-SY5Y cells but had no effect on the efficiency of Batimastat mediated inhibition of NRXN1a cleavage. NRXN1a overexpression did not influence amyloid precursor protein (APP) expression or proteolysis. Based on published observations that APP can bind directly to NRXN1a to decrease synaptic levels of NRXN1a, suggests that the regulatory interactions between these proteins are uni-directional.
The presented findings support PFC serotonergic dysfunction in Nrxn1a HZ mice, which may contribute to the deficits in PFC function reported in these animals. However, the potential utility of serotonergic drugs for the treatment of 2p16.3-related disorders requires further validation. Novel insights were provided into the proteolytic processing of NRXN1a with the data suggesting this could be ADAM-mediated. Thus, regulating the proteolytic cleavage of cell surface NRXN1a by ADAMs may be a novel interventional strategy by which to regulate NRXN1a in NDDs and AD.