N-methyl-D-aspartate receptors (NMDARs) are important postsynaptic receptors that contribute to normal synaptic function and cell survival; however, when overactivated, as in Huntington’s disease (HD), NMDARs cause excitotoxicity. HD-affected striatal neurons show altered NMDAR currents and augmented ratio of surface to internal GluN2B-containing NMDARs, with augmented accumulation at extrasynaptic sites. Fyn protein is a member of the Src kinase family (SKF) with an important role in NMDARs phosphorylation and synaptic localization and function; recently, we demonstrated that Fyn is reduced in several HD models. Thus, in this study, we aimed to explore the impact of HD-mediated altered Fyn levels at post-synaptic density (PSD), and their role in distorted NMDARs function and localization, and intracellular neuroprotective pathways in YAC128 mouse primary striatal neurons. We show that reduced synaptic Fyn levels and activity in HD mouse striatal neurons is related to decreased phosphorylation of synaptic GluN2B-composed NMDARs; this occurs concomitantly with augmented extrasynaptic NMDARs activity and currents and reduced cAMP response element-binding protein (CREB) activation, along with induction of cell death pathways. Importantly, expression of a constitutive active form of SKF reestablishes NMDARs localization, phosphorylation, and function at PSD in YAC128 mouse neurons. Enhanced SKF levels and activity also promotes CREB activation and reduces caspase-3 activation in YAC128 mouse striatal neurons. This work supports, for the first time, a relevant role for Fyn protein in PSD modulation, controlling NMDARs synaptic function in HD, and favoring neuroprotective pathways and cell survival. In this respect, Fyn Tyr kinase constitutes an important potential HD therapeutic target directly acting at PSD.

Soluble Aβ_1–42 oligomers (AβO) are formed in the early stages of Alzheimer’s disease (AD) and were previously shown to trigger enhanced Ca²⁺ levels and mitochondrial dysfunction via the activation of N-methyl-D-aspartate receptors (NMDAR). Src kinase is a ubiquitous redox-sensitive non-receptor tyrosine kinase involved in the regulation of several cellular processes, which was demonstrated to have a reciprocal interaction towards NMDAR activation. However, little is known about the early-stage mechanisms associated with AβO-induced neurodysfunction involving Src. Thus, in this work, we analysed the influence of brief exposure to oligomeric Aβ_1–42 on Src activation and related mechanisms involving mitochondria and redox changes in mature primary rat hippocampal neurons. Data show that brief exposure to AβO induce H₂O₂-dependent Src activation involving different cellular events, including NMDAR activation and mediated intracellular Ca²⁺ rise, enhanced cytosolic and subsequent mitochondrial H₂O₂ levels, accompanied by mild mitochondrial fragmentation. Interestingly, these effects were prevented by Src inhibition, suggesting a feedforward modulation. The current study supports a relevant role for Src kinase activation in promoting the loss of postsynaptic glutamatergic synapse homeostasis involving cytosolic and mitochondrial ROS generation after brief exposure to AβO. Therefore, restoring Src activity can constitute a protective strategy for mitochondria and related hippocampal glutamatergic synapses.

Huntington's disease (HD) is a neurodegenerative disorder characterized by psychiatric disturbances, such as obsessive-compulsive disorder (OCD), as well as motor and cognitive impairment, being caused by expression of mutant huntingtin protein (mHTT). Synaptic disturbances have been described in HD, affecting mainly the cortico-striatal synapse. Striatal GABAergic neurons, the main component of the striatum, are enriched in NMDA receptors (NMDARs), rendering it susceptible to neuronal damage due to excitotoxicity. A dense network of scaffolding proteins reside at post-synaptic densities (PSDs), namely PSD-95, which interacts with SAPAP and Shank proteins, and possibly HTT. Previous studies have proven that SAPAP family member SAPAP3 is involved in cortico-striatal synaptic defects observed in OCD; however, interaction of mHTT with the PSD-SAPAP3 complex in HD has not been established yet. Using PSDs isolated from both HD caudate human post-mortem samples and 3-month old YAC128 HD mouse striatum and cortex (early phenotype), along with immunocytochemistry of primary striatal cultures (E16), we investigated whether altered SAPAP3 protein is involved in the dysfunction of cortico-striatal glutamatergic synapses. PSD isolates showed a consistent decrease in striatal SAPAP3, both in human HD caudate (mean 55 years) and YAC128 striatum, but not in YAC128 cortex. We also found a stable decrease in NMDAR GluN1, GluN2A and GluN2B subunits in YAC128 striatal PSDs and an increase in GluN2A subunits in the cortex, suggesting a differential excitotoxic effect in early stages of HD involving NMDARs in these brain regions. While Homer 1b/c scaffold protein remained unchanged in the two brain areas analysed, both PSD-95 and β-actin were specifically downregulated at YAC128 striatal synapses. Furthermore, SAPAP3 protein levels observed in primary cultures of striatal neurons showed a tendency for an enrichment in the soma and a decrease in the distal dendrites of YAC128 vs WT mice. These data suggest a possible defect in the post-synaptic complex involving SAPAP3, PSD-95, β-actin and NMDAR subunits that may underlie disease-related psychiatric disturbances at early stages in HD.