NCK1 Modulates Neuronal Actin Dynamics and Promotes Dendritic Spine, Synapse, and Memory Formation.

Memory formation and maintenance is a dynamic process involving the modulation of the actin cytoskeleton at synapses. Understanding the signaling pathways that contribute to actin modulation is important for our understanding of synapse formation and function, as well as learning and memory. Here, we focused on the importance of the actin regulator, noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1), in hippocampal dependent behaviors and development. We report that male mice lacking NCK1 have impairments in both short-term and working memory, as well as spatial learning. Additionally, we report sex differences in memory impairment showing that female mice deficient in NCK1 fail at reversal learning in a spatial learning task. We find that NCK1 is expressed in postmitotic neurons but is dispensable for neuronal proliferation and migration in the developing hippocampus. Morphologically, NCK1 is not necessary for overall neuronal dendrite development. However, neurons lacking NCK1 have lower dendritic spine and synapse densities in vitro and in vivo EM analysis reveal increased postsynaptic density (PSD) thickness in the hippocampal CA1 region of NCK1-deficient mice. Mechanistically, we find the turnover of actin-filaments in dendritic spines is accelerated in neurons that lack NCK1. Together, these findings suggest that NCK1 contributes to hippocampal-dependent memory by stabilizing actin dynamics and dendritic spine formation.SIGNIFICANCE STATEMENT Understanding the molecular signaling pathways that contribute to memory formation, maintenance, and elimination will lead to a better understanding of the genetic influences on cognition and cognitive disorders and will direct future therapeutics. Here, we report that the noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) adaptor protein modulates actin-filament turnover in hippocampal dendritic spines. Mice lacking NCK1 show sex-dependent deficits in hippocampal memory formation tasks, have altered postsynaptic densities, and reduced synaptic density. Together, our work implicates NCK1 in the regulation of actin cytoskeleton dynamics and normal synapse development which is essential for memory formation.

The Stability of Glutamatergic Synapses Is Independent of Activity Level, but Predicted by Synapse Size.

Neuronal activity is thought to drive the remodeling of circuits in the mammalian cerebral cortex. However, its precise function in the underlying formation and elimination of glutamatergic synapses has remained controversial. To clarify the role of activity in synapse turnover, we have assessed the effects of inhibition of glutamate release from a sparse subset of cultured hippocampal neurons on synapse turnover. Sustained chemogenetic attenuation of release through presynaptic expression of a designer receptor exclusively activated by designer drugs (DREADD) had no effect on the formation or elimination of glutamatergic synapses. Sparse expression of tetanus neurotoxin light chain (TeNT-LC), a synaptobrevin-cleaving protease that completely abolishes neurotransmitter release, likewise did not lead to changes in the rate of synapse elimination, although it reduced the rate of synapse formation. The stability of active and silenced synapses correlated with measures of synapse size. While not excluding a modulatory role in synapse elimination, our findings show that synaptic activity is neither required for the removal nor the maintenance of glutamatergic synapses between hippocampal neurons. Our results also demonstrate that the stability of glutamatergic synapses scales with their size irrespective of their activity.

Pan-neurexin perturbation results in compromised synapse stability and a reduction in readily releasable synaptic vesicle pool size.

Neurexins are a diverse family of cell adhesion molecules that localize to presynaptic specializations of CNS neurons. Heterologous expression of neurexins in non-neuronal cells leads to the recruitment of postsynaptic proteins in contacting dendrites of co-cultured neurons, implicating neurexins in synapse formation. However, isoform-specific knockouts of either all α- or all ß-neurexins show defects in synaptic transmission but an unaltered density of glutamatergic synapses, a finding that argues against an essential function of neurexins in synaptogenesis. To address the role of neurexin in synapse formation and function, we disrupted the function of all α- and ß-neurexins in cultured hippocampal neurons by shRNA knockdown or by overexpressing a neurexin mutant that is unable to bind to postsynaptic neurexin ligands. We show that neurexin perturbation results in an attenuation of neurotransmitter release that is in large part due to a reduction in the number of readily releasable synaptic vesicles. We also find that neurexin perturbation fails to alter the ability of neurons to form synapses, but rather leads to more frequent synapse elimination. These experiments suggest that neurexins are dispensable for the formation of initial synaptic contacts, but play an essential role in the stabilization and functional maturation of synapses.