Rho GTPases activated by GDP/GTP exchange factors (GEFs) play key roles in the developing and adult nervous system. Kalirin-7 (Kal7), the predominant adult splice form of the multifunctional Kalirin RhoGEF, includes a PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] binding domain and localizes to the postsynaptic side of excitatory synapses.In vitrostudies demonstrated that overexpression of Kal7 increased dendritic spine density, whereas reduced expression of endogenous Kal7 decreased spine density. To evaluate the role of Kal7in vivo, mice lacking the terminal exon unique to Kal7 were created. Mice lacking both copies of the Kal7 exon (Kal7^KO) grew and reproduced normally. Golgi impregnation and electron microscopy revealed decreased hippocampal spine density in Kal7^KOmice. Behaviorally, Kal7^KOmice showed decreased anxiety-like behavior in the elevated zero maze and impaired acquisition of a passive avoidance task, but normal behavior in open field, object recognition, and radial arm maze tasks. Kal7^KOmice were deficient in hippocampal long-term potentiation. Western blot analysis confirmed the absence of Kal7 and revealed compensatory increases in larger Kalirin isoforms. PSDs purified from the cortices of Kal7^KOmice showed a deficit in Cdk5, a kinase known to phosphorylate Kal7 and play an essential role in synaptic function. The early stages of excitatory synaptic development proceeded normally in cortical neurons prepared from Kal7^KOmice, with decreased excitatory synapses apparent only after 21 d invitro. Expression of exogenous Kal7 in Kal7^KOneurons rescued this deficit. Kal7 plays an essential role in synaptic structure and function, affecting a subset of cognitive processes.

The structures of dendritic spines and the dendritic tree, key determinants of neuronal function, are regulated by diverse inputs that affect many scaffolding and signaling molecules. Nevertheless, here we show that reduced expression of a single gene results in loss of dendritic spines and a decrease in dendritic complexity. Kalirin, a dual Rho GDP-GTP exchange factor, causes spine formation when overexpressed. Reduced expression of Kalirin in CA1 hippocampal neurons resulted in a reduction in linear spine density, with dispersion of postsynaptic density markers and elimination of presynaptic endings. Simplification of the apical dendritic tree preceded simplification of basal dendrites. Pyramidal cell axons were not dramatically altered. Although many factors determine dendrite shape and spine formation, expression of Kalirin is necessary for the normal function of these many regulatory elements.

A critical role for peptide C-terminal amidation was apparent when the first bioactive peptides were identified. The conversion of POMC into adrenocorticotropic hormone and then into α-melanocyte-stimulating hormone, an amidated peptide, provided a model system for identifying the amidating enzyme. Peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that catalyzes this modification, is essential; mice lacking PAM survive only until mid-gestation. Purification and cloning led to the discovery that the amidation of peptidylglycine substrates proceeds in two steps: peptidylglycine α-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate; peptidyl-α-hydroxyglycine α-amidating lyase cleaves the N–C bond, producing amidated product and glyoxylate. Both enzymes are contained in the luminal domain of PAM, a type 1 integral membrane protein. The structures of both catalytic cores have been determined, revealing how they interact with metals, molecular oxygen, and substrate to catalyze both reactions. Although not essential for activity, the intrinsically disordered cytosolic domain is essential for PAM trafficking. A phylogenetic survey led to the identification of bifunctional membrane PAM inChlamydomonas, a unicellular eukaryote. Accumulating evidence points to a role for PAM in copper homeostasis and in retrograde signaling from the lumen of the secretory pathway to the nucleus. The discovery of PAM in cilia, cellular antennae that sense and respond to environmental stimuli, suggests that much remains to be learned about this ancient protein.

Bipolar disorder, schizophrenia, autism and intellectual disability are complex neurodevelopmental disorders, debilitating millions of people. Therapeutic progress is limited by poor understanding of underlying molecular pathways. Using a targeted search, we identified an enrichment of de novo mutations in the gene encoding the 330-kDa triple functional domain (TRIO) protein associated with neurodevelopmental disorders. By generating multiple TRIO antibodies, we show that the smaller TRIO9 isoform is the major brain protein product, and its levels decrease after birth. TRIO9 contains two guanine nucleotide exchange factor (GEF) domains with distinct specificities: GEF1 activates both Rac1 and RhoG; GEF2 activates RhoA. To understand the impact of disease-associated de novo mutations and other rare sequence variants on TRIO function, we utilized two FRET-based biosensors: a Rac1 biosensor to study mutations in TRIO (T)GEF1, and a RhoA biosensor to study mutations in TGEF2. We discovered that one autism-associated de novo mutation in TGEF1 (K1431M), at the TGEF1/Rac1 interface, markedly decreased its overall activity toward Rac1. A schizophrenia-associated rare sequence variant in TGEF1 (F1538Intron) was substantially less active, normalized to protein level and expressed poorly. Overall, mutations in TGEF1 decreased GEF1 activity toward Rac1. One bipolar disorder-associated rare variant (M2145T) in TGEF2 impaired inhibition by the TGEF2 pleckstrin-homology domain, resulting in dramatically increased TGEF2 activity. Overall, genetic damage to both TGEF domains altered TRIO catalytic activity, decreasing TGEF1 activity and increasing TGEF2 activity. Importantly, both GEF changes are expected to decrease neurite outgrowth, perhaps consistent with their association with neurodevelopmental disorders.

The vacuolar H⁺-ATPase (V-ATPase) establishes pH gradients along secretory and endocytic pathways. Progressive acidification is essential for proteolytic processing of prohormones and aggregation of soluble content proteins. The V-ATPase V₀ subunit is thought to have a separate role in budding and fusion events. Prolonged treatment of professional secretory cells with selective V-ATPase inhibitors (bafilomycin A1, concanamycin A) was used to investigate its role in secretory-granule biogenesis. As expected, these inhibitors eliminated regulated secretion and blocked prohormone processing. Drug treatment caused the formation of large, mixed organelles, with components of immature granules and lysosomes and some markers of autophagy. Markers of the trans-Golgi network and earlier secretory pathway were unaffected. Ammonium chloride and methylamine treatment blocked acidification to a similar extent as the V-ATPase inhibitors without producing mixed organelles. Newly synthesized granule content proteins appeared in mixed organelles, whereas mature secretory granules were spared. Following concanamycin treatment, selected membrane proteins enter tubulovesicular structures budding into the interior of mixed organelles. shRNA-mediated knockdown of the proteolipid subunit of V₀ also caused vesiculation of immature granules. Thus, V-ATPase has a role in protein sorting in immature granules that is distinct from its role in acidification.

In the mammalian brain, the majority of excitatory synapses are housed in micron-sized dendritic protrusions called spines, which can undergo rapid changes in shape and number in response to increased or decreased synaptic activity. These dynamic alterations in dendritic spines require precise control of the actin cytoskeleton. Within spines, multidomain Rho guanine nucleotide exchange factors (Rho GEFs) coordinate activation of their target Rho GTPases by a variety of pathways. In this review, we focus on the handful of disease-related Rho GEFs (Kalirin; Trio; Tiam1; P-Rex1,2; RasGRF1,2; Collybistin) localized at synapses and known to affect electrophysiology, spine morphology, and animal behavior. The goal is to integrate structure/function studies with measurements of synaptic function and behavioral phenotypes in animal models.