SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Small microdeletions and point mutations in SHANK3 have been identified in a small subgroup of individuals with autism spectrum disorder (ASD) and intellectual disability. SHANK3 also plays a key role in the chromosome 22q13.3 microdeletion syndrome (Phelan-McDermid syndrome), which includes ASD and cognitive dysfunction as major clinical features. To evaluate the role of Shank3 in vivo, we disrupted major isoforms of the gene in mice by deleting exons 4-9. Isoform-specific Shank3(e4-9) homozygous mutant mice display abnormal social behaviors, communication patterns, repetitive behaviors and learning and memory. Shank3(e4-9) male mice display more severe impairments than females in motor coordination. Shank3(e4-9) mice have reduced levels of Homer1b/c, GKAP and GluA1 at the PSD, and show attenuated activity-dependent redistribution of GluA1-containing AMPA receptors. Subtle morphological alterations in dendritic spines are also observed. Although synaptic transmission is normal in CA1 hippocampus, long-term potentiation is deficient in Shank3(e4-9) mice. We conclude that loss of major Shank3 species produces biochemical, cellular and morphological changes, leading to behavioral abnormalities in mice that bear similarities to human ASD patients with SHANK3 mutations.

Whole-cell recordings from optic tectal neurons in Xenopus tadpoles were used to study the maturation of a glutamatergic synapse. The first glutamatergic transmission is mediated only by N-methyl-D-aspartate (NMDA) receptors and is silent at resting potentials. More mature synapses acquire transmission by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. This maturational program is mimicked by postsynaptic expression of constitutively active calcium-calmodulin-dependent protein kinase II (CaMKII). Newly formed synapses may be silent unless sufficient depolarization is provided by coincident activity that could activate postsynaptic CaMKII, resulting in the appearance of AMPA responses.

The Rho family of small GTPases controls a wide range of cellular processes in eukaryotic cells, such as normal cell growth, proliferation, differentiation, gene regulation, actin cytoskeletal organization, cell fate determination, and neurite outgrowth. The activation of RhoGTPases requires the exchange of GDP for GTP, a process catalyzed by the Dbl family of guanine nucleotide exchange factors. We demonstrate that a newly identified guanine nucleotide exchange factor, GEFT, is widely expressed in the brain and highly concentrated in the hippocampus, and the Purkinje and granular cells of the cerebellum. Exogenous expression of GEFT promotes dendrite outgrowth in hippocampal neurons, resulting in spines with larger size as compared with control spines. In neuroblastoma cells, GEFT promotes the active GTPbound state of Rac1, Cdc42, and RhoA and increases neurite outgrowth primarily via Rac1. Furthermore, we demonstrated that PAK1 and PAK5, both downstream effectors of Rac1/Cdc42, are necessary for GEFT-induced neurite outgrowth. AP-1 and NF-B, two transcriptional factors involved in neurite outgrowth and survival, were up-regulated in GEFT-expressing cells. Together, our data suggest that GEFT enhances dendritic spine formation and neurite outgrowth in primary neurons and neuroblastoma cells, respectively, through the activation of Rac/Cdc42-PAK signaling pathways.

The immunophilin FKBP12 binds the skeletal muscle Ca2+ release channel or ryanodine receptor (RyR1), but the functional consequences of this interaction are not known. In this study, we have generated skeletal muscle specific FKBP12-deficient mice to investigate the role of FKBP12 in skeletal muscle. Primary myotubes from these mice show no obvious change in either Ca2+ stores or resting Ca2+ levels but display decreased voltage-gated intracellular Ca2+ release and increased L-type Ca2+ currents. Consistent with the decreased voltage-gated Ca2+ release, maximal tetanic force production is decreased and the force frequency curves are shifted to the right in extensor digitorum longus (EDL) muscles of the mutant mice. In contrast, there is no decrease in maximal tetanic force production in the mutant diaphragm or soleus muscle. The force frequency curve is shifted to the left in the FKBP12-deficient diaphragm muscle compared with controls. No changes in myosin heavy chain (MHC) phenotype are observed in EDL or soleus muscle of the FKBP12-deficient mice, but diaphragm muscle displays an increased ratio of slow to fast MHC isoforms. Also, calcineurin levels are increased in the diaphragm of the mutant mice but not in the soleus or EDL. In summary, FKBP12 deficiency alters both orthograde and retrograde coupling between the L-type Ca2+ channel and RyR1 and the consequences of these changes depend on muscle type and activity. In highly used muscles such as the diaphragm, adaptation to the loss of FKBP12 occurs, possibly due to the increased Ca2+ influx.

The Rho-family of small GTPases consists of essential regulators of neurite outgrowth, axonal pathfinding, and dendritic arborization. Previous work has demonstrated in nonneuronal cell types that Smurf1, an E3 ubiquitin ligase, regulates cell polarity and protrusive activity via PKCf-dependent recruitment to cellular protrusion sites, and subsequent ubiquitination and proteasomal degradation of RhoA. In this study, we show that Smurf1 enhances neurite outgrowth in Neuro2a neuroblastoma cells. We demonstrate that RhoA is ubiquitinated, and that Smurf1 and RhoA physically interact in vivo. Interestingly, Smurf1 overexpression in Neuro2a cells dramatically reduces RhoA protein levels during dibutyric cyclic AMP, but not retinoic acid induced neurite outgrowth. This Smurf1-dependent reduction in RhoA protein levels was abrogated using the general proteasome inhibitor MG132, suggesting that RhoA is targeted for ubiquitination and degradation via Smurf1. Together, our data suggest that localized regulation of different subsets of Rho GTPases by specific guidance signals results in an intracellular asymmetry of RhoA activity, which could regulate neurite outgrowth and guidance.