Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function

Abstract

Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function.

Diagnosis of traumatic brain injury using miRNA signatures in nanomagnetically isolated brain-derived extracellular vesicles

“…Lysates from mouse EVs were analyzed using Western blot analysis as we described. 49–51 Briefly, proteins were resolved on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to a polyvinylidene difluoride membrane using transfer buffer (5.8% tris-base, 29% glycine, 20% methanol, 45.2% distilled water) for 1 hour. Blots were blocked for 1 hour at room temperature in 5% bovine serum albumin (BSA) dissolved in TBST (500mM tris-base, 60mM KCl and 2.8M NaCl, pH 7.4 with 0.1% Tween).…”

d-Serine administration affects nitric oxide synthase 1 adaptor protein and DISC1 expression in sex-specific manner

“…Cortical neuron cultures were prepared from embryonic rats at gestation day 18 as previously described (Hernandez et al, 2016; Kutzing et al, 2011, 2012). Cells were plated at a density of 2 × 10 5 /well on 12- mm glass coverslips (Thermofisher) coated with poly-D-lysine (0.1mg/ml) for dendrite branching analysis or 35mm dishes for protein analysis.…”

“…Lysates from cultures and drug-treated rats were analyzed using Western blot analysis as previously described (Carrel et al, 2009; Hadzimichalis et al, 2010; Hernandez et al, 2016). Proteins were resolved on 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to polyvinylidene difluoride membrane without SDS.…”

“…This disrupts nitric oxide (NO) signaling, and thus, NMDAR signaling (Jaffrey et al, 2002; Jaffrey et al, 1998). We previously reported that NOS1AP long (NOS1AP-L), short (NOS1AP-S), and short’ (NOS1AP-S’) isoforms are upregulated in Brodmann’s area 46 in postmortem tissue of patients with SCZ (Hadzimichalis et al, 2010) and that overexpression of NOS1AP-L and NOS1AP-S reduces dendrite branching (Carrel et al, 2009) and alters dendritic spines in vitro (Hernandez et al, 2016), reproducing neuronal and synaptic abnormalities associated with SCZ (Black et al, 2004; Kolomeets et al, 2005; Kulkarni and Firestein, 2012). Chronic antipsychotic treatment correlates with increased NOS1AP-L mRNA expression in the DLPFC of SCZ patients (Xu et al, 2005).…”

“…Here, we investigate the effects of haloperidol and D-serine administration in vivo on expression of NOS1AP, D2 receptor, and DISC1. Using primary neuronal culture as a model for the contribution of NOS1AP to SCZ phenotypes (Carrel et al, 2009; Carrel et al, 2015; Hernandez et al, 2016), we examine how treatment with antipsychotic medications or D-serine affects NOS1AP protein expression and dendrite branching in vitro . Our data indicate that antipsychotic medications and D-serine exert distinct sex-specific effects on protein expression and neuronal development.…”