Structural plasticity of dendritic spines is a key component of the refinement of synaptic connections during learning. Recent studies highlight a novel role for the NMDA receptor (NMDAR), independent of ion flow, in driving spine shrinkage and LTD. Yet little is known about the molecular mechanisms that link conformational changes in the NMDAR to changes in spine size and synaptic strength. Here, using two-photon glutamate uncaging to induce plasticity at individual dendritic spines on hippocampal CA1 neurons from mice and rats of both sexes, we demonstrate that p38 MAPK is generally required downstream of non-ionotropic NMDAR signaling to drive both spine shrinkage and LTD. In a series of pharmacological and molecular genetic experiments, we identify key components of the non-ionotropic NMDAR signaling pathway driving dendritic spine shrinkage, including the interaction between NOS1AP (nitric oxide synthase 1 adaptor protein) and neuronal nitric oxide synthase (nNOS), nNOS enzymatic activity, activation of MK2 (MAPK-activated protein kinase 2) and cofilin, and signaling through CaMKII. Our results represent a large step forward in delineating the molecular mechanisms of non-ionotropic NMDAR signaling that can drive shrinkage and elimination of dendritic spines during synaptic plasticity.

Dystroglycan (Dag1) is a cell adhesion molecule that links the extracellular matrix to the actin cytoskeleton, and is critical for normal muscle and brain development. Mutations in Dag1 or the genes required for its functional glycosylation result in dystroglycanopathy, which is characterized by a wide range of phenotypes including muscle weakness, brain defects, and cognitive impairments. Whereas Dystroglycans role in muscle and early brain development are well defined, much less is known about its role at later stages of neural circuit development including synapse formation and refinement. Recent work has found that selective deletion of Dag1 from pyramidal neurons leads to a loss of presynaptic CCK+ inhibitory neurons (INs) early in development. In this study, we investigated how IN development is affected in multiple mouse models of dystroglycanopathy. Widespread forebrain deletion of Dag1 or Pomt2, which is required for Dystroglycan glycosylation, recapitulates brain phenotypes seen in severe forms of dystroglycanopathy. CCK+ INs were present in Dag1 and Pomt2 mutant mice, but their axons failed to properly target the somatodendritic compartment of pyramidal neurons in the hippocampus. In contrast, CCK+ IN axon targeting was largely normal in mouse models of mild dystroglycanopathy with partially reduced Dystroglycan glycosylation (B4Gat1,Fkrp). Furthermore, the intracellular domain of Dystroglycan appears to be dispensable for CCK+ IN axon targeting. Collectively, these data show that synaptic defects are a hallmark of severe dystroglycanopathy.

The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience-dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity-dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF-dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome-dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5-GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity-dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site-specific formation of new spines.

Predicted loss-of-function and missense heterozygous de novo mutations of TBR1 are strongly associated with intellectual disability and autism. The functional effects of these heterogeneous mutations on cortical development and genotype-phenotype relationships have yet to be explored. We characterized mouse models carrying patient mutations A136PfsX80 and K228E, finding convergent and discordant phenotypes. The A136PfsX80 mutation is loss-of-function and allelic to the Tbr1 knockout. In contrast, K228E causes significant upregulation of TBR1. Heterozygosity of either mutation produces axon defects, including reduction of the anterior commissure, and CTIP2 downregulation in adult cortex. While mice lacking TBR1 show extensive cortical apoptosis and inverted layering, K228E homozygotes show normal apoptosis levels and a complex layering phenotype-suggesting partial, yet abnormal, function of the allele. The construct and face validity of these Tbr1 patient mutation mice suggests they will be valuable translational models for studying the function of this essential brain transcription factor.