Rac-maninoff and Rho-vel: The symphony of Rho-GTPase signaling at excitatory synapses.

Synaptic connections between neurons are essential for every facet of human cognition and are thus regulated with extreme precision. Rho-family GTPases, molecular switches that cycle between an active GTP-bound state and an inactive GDP-bound state, comprise a critical feature of synaptic regulation. Rho-GTPases are exquisitely controlled by an extensive suite of activators (GEFs) and inhibitors (GAPs and GDIs) and interact with many different signalling pathways to fulfill their roles in orchestrating the development, maintenance, and plasticity of excitatory synapses of the central nervous system. Among the mechanisms that control Rho-GTPase activity and signalling are cell surface receptors, GEF/GAP complexes that tightly regulate single Rho-GTPase dynamics, GEF/GAP and GEF/GEF functional complexes that coordinate multiple Rho-family GTPase activities, effector positive feedback loops, and mutual antagonism of opposing Rho-GTPase pathways. These complex regulatory mechanisms are employed by the cells of the nervous system in almost every step of development, and prominently figure into the processes of synaptic plasticity that underlie learning and memory. Finally, misregulation of Rho-GTPases plays critical roles in responses to neuronal injury, such as traumatic brain injury and neuropathic pain, and in neurodevelopmental and neurodegenerative disorders, including intellectual disability, autism spectrum disorder, schizophrenia, and Alzheimer's Disease. Thus, decoding the mechanisms of Rho-GTPase regulation and function at excitatory synapses has great potential for combatting many of the biggest current challenges in mental health.

The Rac-GEF Tiam1 Promotes Dendrite and Synapse Stabilization of Dentate Granule Cells and Restricts Hippocampal-Dependent Memory Functions.

The dentate gyrus (DG) controls information flow into the hippocampus and is critical for learning, memory, pattern separation, and spatial coding, while DG dysfunction is associated with neuropsychiatric disorders. Despite its importance, the molecular mechanisms regulating DG neural circuit assembly and function remain unclear. Here, we identify the Rac-GEF Tiam1 as an important regulator of DG development and associated memory processes. In the hippocampus, Tiam1 is predominantly expressed in the DG throughout life. Global deletion of Tiam1 in male mice results in DG granule cells with simplified dendritic arbors, reduced dendritic spine density, and diminished excitatory synaptic transmission. Notably, DG granule cell dendrites and synapses develop normally in Tiam1 KO mice, resembling WT mice at postnatal day 21 (P21), but fail to stabilize, leading to dendrite and synapse loss by P42. These results indicate that Tiam1 promotes DG granule cell dendrite and synapse stabilization late in development. Tiam1 loss also increases the survival, but not the production, of adult-born DG granule cells, possibly because of greater circuit integration as a result of decreased competition with mature granule cells for synaptic inputs. Strikingly, both male and female mice lacking Tiam1 exhibit enhanced contextual fear memory and context discrimination. Together, these results suggest that Tiam1 is a key regulator of DG granule cell stabilization and function within hippocampal circuits. Moreover, based on the enhanced memory phenotype of Tiam1 KO mice, Tiam1 may be a potential target for the treatment of disorders involving memory impairments.SIGNIFICANCE STATEMENT The dentate gyrus (DG) is important for learning, memory, pattern separation, and spatial navigation, and its dysfunction is associated with neuropsychiatric disorders. However, the molecular mechanisms controlling DG formation and function remain elusive. By characterizing mice lacking the Rac-GEF Tiam1, we demonstrate that Tiam1 promotes the stabilization of DG granule cell dendritic arbors, spines, and synapses, whereas it restricts the survival of adult-born DG granule cells, which compete with mature granule cells for synaptic integration. Notably, mice lacking Tiam1 also exhibit enhanced contextual fear memory and context discrimination. These findings establish Tiam1 as an essential regulator of DG granule cell development, and identify it as a possible therapeutic target for memory enhancement.

Cranial irradiation induces axon initial segment dysfunction and neuronal injury in the prefrontal cortex and impairs hippocampal coupling.

BACKGROUND: Radiation therapy for brain tumors commonly induces cognitive dysfunction. The prefrontal cortex (PFC) is crucial for a diverse array of cognitive processes, however, its role in radiation-induced cognitive dysfunction is unknown. We previously found that cranial irradiation impairs neuroplasticity along the hippocampal-PFC pathway. Herein, we hypothesized that brain irradiation directly affects the firing properties of PFC neurons, contributing to deficits in neuronal functions. METHODS: In vivo recordings were used to monitor the firing activities of PFC neurons and local field potentials in both PFC and hippocampal CA1/subicular regions after cranial irradiation of Sprague Dawley rats. We further assessed the impacts of irradiation on axon initial segments (AISs) with immunofluorescence assays of PFC slices. RESULTS: We found that PFC neurons exhibited increased excitation 3 days after radiation and the timing of increased excitation coincided with elongation of the AIS. At 2 weeks, excitation levels returned to nearly normal levels however the population of spontaneously firing neurons decreased. While the number of NeuN-positive neurons in the PFC was not different, persistent neuronal injury, manifested as ATF-3 staining, was present at 2 weeks. Radiation also disrupted communication along the hippocampal-PFC pathway, with elongation of the phase lag between regions. Analysis of paired-pulse ratios suggested that this was secondary to presynaptic dysfunction. CONCLUSIONS: Cranial irradiation excited and injured surviving PFC neurons and was associated with a partial block of PFC's functional coupling to the hippocampus. These deficits in the PFC may contribute to radiation-induced cognitive dysfunction.

An autism-linked missense mutation in SHANK3 reveals the modularity of Shank3 function.

Genome sequencing has revealed an increasing number of genetic variations that are associated with neuropsychiatric disorders. Frequently, studies limit their focus to likely gene-disrupting mutations because they are relatively easy to interpret. Missense variants, instead, have often been undervalued. However, some missense variants can be informative for developing a more profound understanding of disease pathogenesis and ultimately targeted therapies. Here we present an example of this by studying a missense variant in a well-known autism spectrum disorder (ASD) causing gene SHANK3. We analyzed Shank3's in vivo phosphorylation profile and identified S685 as one phosphorylation site where one ASD-linked variant has been reported. Detailed analysis of this variant revealed a novel function of Shank3 in recruiting Abelson interactor 1 (ABI1) and the WAVE complex to the post-synaptic density (PSD), which is critical for synapse and dendritic spine development. This function was found to be independent of Shank3's other functions such as binding to GKAP and Homer. Introduction of this human ASD mutation into mice resulted in a small subset of phenotypes seen previously in constitutive Shank3 knockout mice, including increased allogrooming, increased social dominance, and reduced pup USV. Together, these findings demonstrate the modularity of Shank3 function in vivo. This modularity further indicates that there is more than one independent pathogenic pathway downstream of Shank3 and correcting a single downstream pathway is unlikely to be sufficient for clear clinical improvement. In addition, this study illustrates the value of deep biological analysis of select missense mutations in elucidating the pathogenesis of neuropsychiatric phenotypes.

The adhesion-GPCR BAI1 shapes dendritic arbors via Bcr-mediated RhoA activation causing late growth arrest.

Dendritic arbor architecture profoundly impacts neuronal connectivity and function, and aberrant dendritic morphology characterizes neuropsychiatric disorders. Here, we identify the adhesion-GPCR BAI1 as an important regulator of dendritic arborization. BAI1 loss from mouse or rat hippocampal neurons causes dendritic hypertrophy, whereas BAI1 overexpression precipitates dendrite retraction. These defects specifically manifest as dendrites transition from growth to stability. BAI1-mediated growth arrest is independent of its Rac1-dependent synaptogenic function. Instead, BAI1 couples to the small GTPase RhoA, driving late RhoA activation in dendrites coincident with growth arrest. BAI1 loss lowers RhoA activation and uncouples it from dendrite dynamics, causing overgrowth. None of BAI1's known downstream effectors mediates BAI1-dependent growth arrest. Rather, BAI1 associates with the Rho-GTPase regulatory protein Bcr late in development and stimulates its cryptic RhoA-GEF activity, which functions together with its Rac1-GAP activity to terminate arborization. Our results reveal a late-acting signaling pathway mediating a key transition in dendrite development.

The Adhesion-GPCR BAI1 Promotes Excitatory Synaptogenesis by Coordinating Bidirectional Trans-synaptic Signaling.

Excitatory synapses are specialized cell-cell contacts located on actin-rich dendritic spines that mediate information flow and storage in the brain. The postsynaptic adhesion-G protein-coupled receptor (A-GPCR) BAI1 is a critical regulator of excitatory synaptogenesis, which functions in part by recruiting the Par3-Tiam1 polarity complex to spines, inducing local Rac1 GTPase activation and actin cytoskeletal remodeling. However, a detailed mechanistic understanding of how BAI1 controls synapse and spine development remains elusive. Here, we confirm that BAI1 is required in vivo for hippocampal spine development, and we identify three distinct signaling mechanisms mediating BAI1's prosynaptogenic functions. Using in utero electroporation to sparsely knock down BAI1 expression in hippocampal pyramidal neurons, we show that BAI1 cell-autonomously promotes spinogenesis in the developing mouse brain. BAI1 appears to function as a receptor at synapses, as its extracellular N-terminal segment is required for both its prospinogenic and prosynaptogenic functions. Moreover, BAI1 activation with a Stachel-derived peptide, which mimics a tethered agonist motif found in A-GPCRs, drives synaptic Rac1 activation and subsequent spine and synapse development. We also reveal, for the first time, a trans-synaptic function for BAI1, demonstrating in a mixed-culture assay that BAI1 induces the clustering of presynaptic vesicular glutamate transporter 1 (vGluT1) in contacting axons, indicative of presynaptic differentiation. Finally, we show that BAI1 forms a receptor complex with the synaptogenic cell-adhesion molecule Neuroligin-1 (NRLN1) and mediates NRLN1-dependent spine growth and synapse development. Together, these findings establish BAI1 as an essential postsynaptic A-GPCR that regulates excitatory synaptogenesis by coordinating bidirectional trans-synaptic signaling in cooperation with NRLN1.SIGNIFICANCE STATEMENT Adhesion-G protein-coupled receptors are cell-adhesion receptors with important roles in nervous system development, function, and neuropsychiatric disorders. The postsynaptic adhesion-G protein-coupled receptor BAI1 is a critical regulator of dendritic spine and excitatory synapse development. However, the mechanism by which BAI1 controls these functions remains unclear. Our study identifies three distinct signaling paradigms for BAI1, demonstrating that it mediates forward, reverse, and lateral signaling in spines. Activation of BAI1 by a Stachel-dependent mechanism induces local Rac1 activation and subsequent spinogenesis/synaptogenesis. BAI1 also signals trans-synaptically to promote presynaptic differentiation. Furthermore, BAI1 interacts with the postsynaptic cell-adhesion molecule Neuroligin-1 (NRLN1) and facilitates NRLN1-dependent spine growth and excitatory synaptogenesis. Thus, our findings establish BAI1 as a functional synaptogenic receptor that promotes presynaptic and postsynaptic development in cooperation with synaptic organizer NRLN1.

Radiation induces age-dependent deficits in cortical synaptic plasticity.

Background: Radiation-induced cognitive dysfunction is a significant side effect of cranial irradiation for brain tumors. Clinically, pediatric patients are more vulnerable than adults. However, the underlying mechanisms of dysfunction, including reasons for age dependence, are still largely unknown. Previous studies have focused on the loss of hippocampal neuronal precursor cells and deficits in memory. However, survivors may also experience deficits in attention, executive function, or other non-hippocampal-dependent cognitive domains. We hypothesized that brain irradiation induces age-dependent deficits in cortical synaptic plasticity. Methods: In vivo recordings were used to test neuronal plasticity along the direct pathway from the cornu ammonis 1 (CA1)/subicular region to the prefrontal cortex (PFC). Specifically, long-term potentiation (LTP) in the CA1/subicular-PFC pathway was assessed after cranial irradiation of juvenile and adult Sprague Dawley rats. We further assessed a potential role for glutamate toxicity by evaluating the potential neuroprotective effects of memantine. Results: LTP was greatly inhibited in both adult and juvenile animals at 3 days after radiation but returned to near-normal levels by 8 weeks-only in adult rats. Memantine given before, but not after, irradiation partially prevented LTP inhibition in juvenile and adult rats. Conclusion: Cranial radiation impairs neuroplasticity along the hippocampal-PFC pathway; however, its effects vary by age. Pretreatment with memantine offered protection to both juvenile and adult animals. Deficits in cortical plasticity may contribute to radiation-induced cognitive dysfunction, including deficits in attention and age-dependent sensitivity of such pathways, which may underlie differences in clinical outcomes between juveniles and adults after cranial irradiation.

Particle Radiation Induced Neurotoxicity in the Central Nervous System.

For patients with primary or metastatic brain tumors, radiation therapy plays a central role in treatment. However, despite its efficacy, cranial radiation is associated with a range of side effects ranging from mild cognitive impairment to overt brain necrosis. Given the negative effects on patient quality of life, radiation-induced neurotoxicities have been the subject of intense study for decades. Photon-based therapy has been and largely remains the standard of care for the treatment of brain tumors. This is particularly true for patients with metastatic tumors who may need treatment to the whole brain or those with very aggressive tumors and a limited life expectancy. Particle therapy is now becoming more widely available for clinical use with the two most common particles used being protons and carbon ions. For patients with favorable prognoses, particularly childhood brain tumors, proton therapy is increasingly used for treatment. This is, in part, driven by the desire to reduce the potential for radiation-induced side effects, including lasting cognitive impairment, which may potentially be achieved by reducing dose to normal tissues using the unique physical properties of particle therapy. There is also interest in using carbon ion therapy for the treatment of aggressive brain tumors, as this form of particle therapy not only spares normal tissues but may also improve tumor control. The biological effects of particle therapy, both proton and carbon, may differ substantially from those of photon radiation. In this review, we briefly describe the unique physical properties of particle therapy that produce differential biological effects. Focusing on the effects of various radiation types on brain parenchyma, we then describe biological effects and potential mechanisms underlying these, comparing to photon studies and highlighting potential clinical implications.

Memantine prevents acute radiation-induced toxicities at hippocampal excitatory synapses.

Background: Memantine has shown clinical utility in preventing radiation-induced cognitive impairment, but the mechanisms underlying its protective effects remain unknown. We hypothesized that abnormal glutamate signaling causes radiation-induced abnormalities in neuronal structure and that memantine prevents synaptic toxicity. Methods: Hippocampal cultures expressing enhanced green fluorescent protein were irradiated or sham-treated and their dendritic spine morphology assessed at acute (minutes) and later (days) times using high-resolution confocal microscopy. Excitatory synapses, defined by co-localization of the pre- and postsynaptic markers vesicular glutamate transporter 1 and postsynaptic density protein 95, were also analyzed. Neurons were pretreated with vehicle, the N-methyl-d-aspartate-type glutamate receptor antagonist memantine, or the glutamate scavenger glutamate pyruvate transaminase to assess glutamate signaling. For animal studies, Thy-1-YFP mice were treated with whole-brain radiotherapy or sham with or without memantine. Results: Unlike previously reported long-term losses of dendritic spines, we found that the acute response to radiation is an initial increase in spines and excitatory synapses followed by a decrease in spine/synapse density with altered spine dynamics. Memantine pre-administration prevented this radiation-induced synaptic remodeling. Conclusion: These results demonstrate that radiation causes rapid, dynamic changes in synaptic structural plasticity, implicate abnormal glutamate signaling in cognitive dysfunction following brain irradiation, and describe a protective mechanism of memantine.

RhoGTPases Spread the Word for Synaptic Crosstalk.

Excitatory synaptic strengthening and the corresponding enlargement of dendritic spines are thought to be essential for learning and memory. In two recent Nature papers, Harward et al. (2016) and Hedrick et al. (2016) provide insight into the mechanisms that regulate these processes and illuminate the molecular basis of crosstalk between synapses.

Emerging Roles of BAI Adhesion-GPCRs in Synapse Development and Plasticity.

Synapses mediate communication between neurons and enable the brain to change in response to experience, which is essential for learning and memory. The sites of most excitatory synapses in the brain, dendritic spines, undergo rapid remodeling that is important for neural circuit formation and synaptic plasticity. Abnormalities in synapse and spine formation and plasticity are associated with a broad range of brain disorders, including intellectual disabilities, autism spectrum disorders (ASD), and schizophrenia. Thus, elucidating the mechanisms that regulate these neuronal processes is critical for understanding brain function and disease. The brain-specific angiogenesis inhibitor (BAI) subfamily of adhesion G-protein-coupled receptors (adhesion-GPCRs) has recently emerged as central regulators of synapse development and plasticity. In this review, we will summarize the current knowledge regarding the roles of BAIs at synapses, highlighting their regulation, downstream signaling, and physiological functions, while noting the roles of other adhesion-GPCRs at synapses. We will also discuss the relevance of BAIs in various neurological and psychiatric disorders and consider their potential importance as pharmacological targets in the treatment of these diseases.

Mechanisms for spatiotemporal regulation of Rho-GTPase signaling at synapses.

Synapses mediate information flow between neurons and undergo plastic changes in response to experience, which is critical for learning and memory. Conversely, synaptic defects impair information processing and underlie many brain pathologies. Rho-family GTPases control synaptogenesis by transducing signals from extracellular stimuli to the cytoskeleton and nucleus. The Rho-GTPases Rac1 and Cdc42 promote synapse development and the growth of axons and dendrites, while RhoA antagonizes these processes. Despite its importance, many aspects of Rho-GTPase signaling remain relatively unknown. Rho-GTPases are activated by guanine nucleotide exchange factors (GEFs) and inhibited by GTPase-activating proteins (GAPs). Though the number of both GEFs and GAPs greatly exceeds that of Rho-GTPases, loss of even a single GEF or GAP often has profound effects on cognition and behavior. Here, we explore how the actions of specific GEFs and GAPs give rise to the precise spatiotemporal activation patterns of Rho-GTPases in neurons. We consider the effects of coupling GEFs and GAPs targeting the same Rho-GTPase and the modular pathways that connect specific cellular stimuli with a given Rho-GTPase via different GEFs. We discuss how the creation of sharp borders between Rho-GTPase activation zones is achieved by pairing a GEF for one Rho-GTPase with a GAP for another and the extensive crosstalk between different Rho-GTPases. Given the importance of synapses for cognition and the fundamental roles that Rho-GTPases play in regulating them, a detailed understanding of Rho-GTPase signaling is essential to the progress of neuroscience.

New functions and signaling mechanisms for the class of adhesion G protein-coupled receptors.

The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region. In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF are associated noncovalently as a heterodimer at the plasma membrane. While the biological function of the GAIN domain-mediated autocleavage is not fully understood, mounting evidence suggests that the NTF and CTF possess distinct biological activities in addition to their function as a receptor unit. We discuss recent advances in understanding the biological functions, signaling mechanisms, and disease associations of the aGPCRs.

Dynamic control of excitatory synapse development by a Rac1 GEF/GAP regulatory complex.

The small GTPase Rac1 orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. Here, we identify a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases.

The adhesion-GPCR BAI1 regulates synaptogenesis by controlling the recruitment of the Par3/Tiam1 polarity complex to synaptic sites.

Excitatory synapses are polarized structures that primarily reside on dendritic spines in the brain. The small GTPase Rac1 regulates the development and plasticity of synapses and spines by modulating actin dynamics. By restricting the Rac1-guanine nucleotide exchange factor Tiam1 to spines, the polarity protein Par3 promotes synapse development by spatially controlling Rac1 activation. However, the mechanism for recruiting Par3 to spines is unknown. Here, we identify brain-specific angiogenesis inhibitor 1 (BAI1) as a synaptic adhesion GPCR that is required for spinogenesis and synaptogenesis in mice and rats. We show that BAI1 interacts with Par3/Tiam1 and recruits these proteins to synaptic sites. BAI1 knockdown results in Par3/Tiam1 mislocalization and loss of activated Rac1 and filamentous actin from spines. Interestingly, BAI1 also mediates Rac-dependent engulfment in professional phagocytes through its interaction with a different Rac1-guanine nucleotide exchange factor module, ELMO/DOCK180. However, this interaction is dispensable for BAI1's role in synapse development because a BAI1 mutant that cannot interact with ELMO/DOCK180 rescues spine defects in BAI1-knockdown neurons, whereas a mutant that cannot interact with Par3/Tiam1 rescues neither spine defects nor Par3 localization. Further, overexpression of Tiam1 rescues BAI1 knockdown spine phenotypes. These results indicate that BAI1 plays an important role in synaptogenesis that is mechanistically distinct from its role in phagocytosis. Furthermore, our results provide the first example of a cell surface receptor that targets members of the PAR polarity complex to synapses.

Orai-STIM-mediated Ca2+ release from secretory granules revealed by a targeted Ca2+ and pH probe.

Secretory granules (SGs) sequester significant calcium. Understanding roles for this calcium and potential mechanisms of release is hampered by the difficulty of measuring SG calcium directly in living cells. We adapted the Förster resonance energy transfer-based D1-endoplasmic reticulum (ER) probe to develop a unique probe (D1-SG) to measure calcium and pH in secretory granules. It significantly localizes to SGs and reports resting free Ca(2+) of 69 ± 15 µM and a pH of 5.8. Application of extracellular ATP to activate P2Y receptors resulted in a slow monotonic decrease in SG Ca(2+) temporally correlated with the occurrence of store-operated calcium entry (SOCE). Further investigation revealed a unique receptor-mediated mechanism of calcium release from SGs that involves SG store-operated Orai channels activated by their regulator stromal interaction molecule 1 (STIM1) on the ER. SG Ca(2+) release is completely antagonized by a SOCE antagonist, by switching to Ca(2+)-free medium, and by overexpression of a dominant-negative Orai1(E106A). Overexpression of the CRAC activation domain (CAD) of STIM1 resulted in a decrease of resting SG Ca(2+) by ∼75% and completely abolished the ATP-mediated release of Ca(2+) from SGs. Overexpression of a dominant-negative CAD construct(CAD-A376K) induced no significant changes in SG Ca(2+). Colocalization analysis suggests that, like the plasma membrane, SG membranes also possess Orai1 channels and that during SG Ca(2+) release, colocalization between SGs and STIM1 increases. We propose Orai channel opening on SG membranes as a potential mode of calcium release from SGs that may serve to raise local cytoplasmic calcium concentrations and aid in refilling intracellular calcium stores of the ER and exocytosis.

Control of synapse development and plasticity by Rho GTPase regulatory proteins.

Synapses are specialized cell-cell contacts that mediate communication between neurons. Most excitatory synapses in the brain are housed on dendritic spines, small actin-rich protrusions extending from dendrites. During development and in response to environmental stimuli, spines undergo marked changes in shape and number thought to underlie processes like learning and memory. Improper spine development, in contrast, likely impedes information processing in the brain, since spine abnormalities are associated with numerous brain disorders. Elucidating the mechanisms that regulate the formation and plasticity of spines and their resident synapses is therefore crucial to our understanding of cognition and disease. Rho-family GTPases, key regulators of the actin cytoskeleton, play essential roles in orchestrating the development and remodeling of spines and synapses. Precise spatio-temporal regulation of Rho GTPase activity is critical for their function, since aberrant Rho GTPase signaling can cause spine and synapse defects as well as cognitive impairments. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and inhibited by GTPase-activating proteins (GAPs). We propose that Rho-family GEFs and GAPs provide the spatiotemporal regulation and signaling specificity necessary for proper Rho GTPase function based on the following features they possess: (i) existence of multiple GEFs and GAPs per Rho GTPase, (ii) developmentally regulated expression, (iii) discrete localization, (iv) ability to bind to and organize specific signaling networks, and (v) tightly regulated activity, perhaps involving GEF/GAP interactions. Recent studies describe several Rho-family GEFs and GAPs that uniquely contribute to spinogenesis and synaptogenesis. Here, we highlight several of these proteins and discuss how they occupy distinct biochemical niches critical for synaptic development.

Calcium transport mechanisms of PC12 cells.

Many studies of Ca2+ signaling use PC12 cells, yet the balance of Ca2+ clearance mechanisms in these cells is unknown. We used pharmacological inhibition of Ca2+ transporters to characterize Ca2+ clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), plasma membrane Ca2+ ATPase (PMCA), and Na+/Ca2+ exchanger (NCX) account for almost all Ca2+ clearance in both cell states, with NCX and PMCA making the greatest contributions. Any contribution of mitochondrial uniporters is small. The ATP pool in differentiated cells was much more labile than that of undifferentiated cells in the presence of agents that dissipated mitochondrial proton gradients. Differentiated PC12 cells have a small component of Ca2+ clearance possessing pharmacological characteristics consistent with secretory pathway Ca2+ ATPase (SPCA), potentially residing on Golgi and/or secretory granules. Undifferentiated and differentiated cells are similar in overall Ca2+ transport and in the small transport due to SERCA, but they differ in the fraction of transport by PMCA and NCX. Transport in neurites of differentiated PC12 cells was qualitatively similar to that in the somata, except that the ER stores in neurites sometimes released Ca2+ instead of clearing it after depolarization. We formulated a mathematical model to simulate the observed Ca2+ clearance and to describe the differences between these undifferentiated and NGF-differentiated states quantitatively. The model required a value for the endogenous Ca2+ binding ratio of PC12 cell cytoplasm, which we measured to be 268 +/- 85. Our results indicate that Ca2+ transport in undifferentiated PC12 cells is quite unlike transport in adrenal chromaffin cells, for which they often are considered models. Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models. Comparison with other cell types shows that different cells emphasize different Ca2+ transport mechanisms.

Contributions of intracellular compartments to calcium dynamics: implicating an acidic store.

Many cells show a plateau of elevated cytosolic Ca(2+) after a long depolarization, suggesting delayed Ca(2+) release from intracellular compartments such as mitochondria and endoplasmic reticulum (ER). Mouse pancreatic beta-cells show a thapsigargin-sensitive plateau ('hump') of Ca(2+) after a 30 s depolarization but not after a 10 s depolarization. Surprisingly, this hump depends primarily on compartments other than the mitochondria or ER. It is reduced by only 22% upon blocking mitochondrial Na(+)-Ca(2+) exchange and by only 18% upon blocking ryanodine or IP(3) receptors together. Further, the time course of ER Ca(2+) measured by a targeted cameleon does not depend on the duration of depolarizations. Instead, the hump is reduced 35% by treatments with the dipeptide glycylphenylalanine beta-napthylamide, a tool often used to lyse lysosomes. We show that this dipeptide does not disturb ER functions, but it lyses acidic compartments and releases Ca(2+) into the cytosol. Moreover, it induces leaks in and possibly lyses insulin granules and stops mobilization of secretory granules to the readily releasable pool in beta-cells. We conclude that the dipeptide compromises dense-core secretory granules and that these granules comprise an acidic calcium store in beta-cells whose loading and/or release is sensitive to thapsigargin and which releases Ca(2+) after cytosolic Ca(2+) elevation.

What is the role of SNARE proteins in membrane fusion?

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins have been at the fore-front of research on biological membrane fusion for some time. The subcellular localization of SNAREs and their ability to form the so-called SNARE complex may be integral to determining the specificity of intracellular fusion (the SNARE hypothesis) and/or serving as the minimal fusion machinery. Both the SNARE hypothesis and the idea of the minimal fusion machinery have been challenged by a number of experimental observations in various model systems, suggesting that SNAREs may have other functions. Considering recent advances in the SNARE literature, it appears that SNAREs may actually function as part of a complex fusion "machine." Their role in the machinery could be any one or a combination of roles, including establishing tight membrane contact, formation of a scaffolding on which to build the machine, binding of lipid surfaces, and many others. It is also possible that complexations other than the classic SNARE complex participate in membrane fusion.