Loss of EPAC2 results in altered synaptic composition of dendritic spines and excitatory and inhibitory balance

EPAC2 is a guanine nucleotide exchange factor that regulates GTPase activity of the small GTPase Rap and Ras) and is highly enriched at synapses. Activation of EPAC2 has been shown to induce dendritic spine shrinkage and increase spine motility, effects that are necessary for synaptic plasticity. These morphological effects are dysregulated by rare mutations of EPAC2 associated with autism spectrum disorders. In addition, EPAC2 destabilizes synapses through the removal of synaptic GluA2/3-containing AMPA receptors. Previous work has shown that Epac2 knockout mice (Epac2-/-) display abnormal social interactions, as well as gross disorganization of the frontal cortex and abnormal spine motility in vivo. In this study we sought to further understand the cellular consequences of knocking out Epac2 on the development of neuronal and synaptic structure and organization of cortical neurons. Using primary cortical neurons generated from Epac2+/+ or Epac2-/- mice, we confirm that EPAC2 is required for cAMP-dependent spine shrinkage. Neurons from Epac2-/- mice also displayed increased synaptic expression of GluA2/3-containing AMPA receptors, as well as of the adhesion protein N-cadherin. Intriguingly, analysis of excitatory and inhibitory synaptic proteins revealed that loss of EPAC2 resulted in altered of expression of vesicular glutamate transporter 1 (VGluT1) and vesicular GABA transporter (VGAT), indicating a potential imbalance in excitatory/inhibitory inputs onto neurons. Finally, examination of cortical neurons located within the anterior cingulate cortex further revealed subtle deficits in the establishment of dendritic arborization in vivo. These data provide evidence that EPAC2 is required for the correct composition of synapses and that loss of this protein could result in an imbalance of excitatory and inhibitory synapses.

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Loss of Depalmitoylation Exaggerates Synaptic Upscaling and Leads to Neuroinflammation in a Lysosomal Storage Disease

10.1101/2021.12.16.473002 ◽

2021 ◽

Author(s):

Kevin P Koster ◽

Eden Flores-Barrera ◽

Emilce Artur de la Villarmois ◽

Thu T.A. Nguyen ◽

Amanda Niqula ◽

...

Keyword(s):

Ampa Receptors ◽

Protein Trafficking ◽

Cortical Neurons ◽

Microglial Activation ◽

Storage Disease ◽

Homeostatic Plasticity ◽

Synaptic Proteins ◽

Lysosomal Storage ◽

Palmitoyl Protein Thioesterase

Palmitoylation and depalmitoylation are the dichotomic processes of lipid modification regulating protein trafficking, recycling, and degradation, thereby controlling proteostasis. Despite our understanding of palmitoylation, depalmitoylation is far less studied. Here, we study a lysosomal depalmitoylating enzyme, palmitoyl-protein thioesterase 1 (PPT1), associated with the devastating neurodegenerative condition CLN1 disease and show that dark-rearing Ppt1-/- mice, which induces synaptic upscaling in vivo, worsen the symptoms. In Ppt1-/- cortical neurons, upscaling induction triggers exaggerated responses of synaptic calcium-permeable AMPA receptors composed of palmitoylated GluA1 subunits. Consequently, Ppt1-/- visual cortex exhibits hypersynchrony in vivo. Remarkably, we also find an overload of palmitoylated A-kinase anchor protein 5 (Akap5) in Ppt1-/- mouse brains, leading to microglial activation through NFAT. These findings indicate Ppt1 acts as a gatekeeper of homeostatic plasticity by regulating the proteostasis of palmitoylated synaptic proteins. Moreover, our results suggest that perturbed depalmitoylation results in neuroinflammation, which is common to neurodegenerative diseases.

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Glutathione S-transferase Pi (Gstp) Proteins Regulate Neuritogenesis in the Developing Cerebral Cortex

10.1101/2020.05.21.103036 ◽

2020 ◽

Author(s):

Xiaonan Liu ◽

Sara M. Blazejewski ◽

Sarah A. Bennison ◽

Kazuhito Toyo-oka

Keyword(s):

Cortical Neurons ◽

Cortical Development ◽

Apical Dendrite ◽

Autism Spectrum ◽

Neuronal Morphology ◽

Jnk Signaling ◽

Neurite Elongation ◽

Neurite Initiation ◽

Neurite Formation

AbstractGSTP proteins are metabolic enzymes involved in removal of oxidative stress and intracellular signaling and also have inhibitory effects on JNK activity. However, the functions of Gstp proteins in the developing brain are unknown. In mice, there are three Gstp proteins, Gstp1, 2 and 3, while there is only one GSTP in humans. By RT-PCR analysis, we found that Gstp1 was expressed beginning at E15.5 in the cortex, but Gstp2 and 3 started expressing at E18.5. Gstp 1 and 2 knockdown caused decreased neurite number in cortical neurons, implicating them in neurite initiation. Using in utero electroporation to knockdown Gstp1 and 2 in layer 2/3 pyramidal neurons in vivo, we found abnormal swelling of the apical dendrite at P3 and reduced neurite number at P15. Using time-lapse live imaging, we found that the apical dendrite orientation was skewed compared to the control, but these defects were ameliorated. Overexpression of Gstp 1 or 2 resulted in changes in neurite length, suggesting a role in neurite elongation. We explored the molecular mechanism and found that JNK inhibition rescued reduced neurite number caused by Gstp knockdown, indicating that Gstp regulates neurite formation through JNK signaling. Thus, we found novel functions of Gstp proteins in neurite initiation during cortical development. Furthermore, the overexpression experiments suggest different functions of Gstp1 and 2 in neurite elongation. Since previous studies have shown the potential implication of Gstp in Autism Spectrum Disorder, our findings will attract more clinical interests in Gstp proteins in neurodevelopmental disorders.SignificanceNeurite formation, including neurite initiation and elongation, is the first step of generating polarized neuronal morphology in developing neurons, and thus is essential for establishing a neuronal network. Therefore, it is crucial to understand the mechanisms of neurite formation. Limited studies have been performed to clarify the mechanisms of neurite formation, especially neurite initiation. In this present study, we report a novel, essential role of Gstp in neurite initiation in mouse cortical neurons in vitro and in vivo. We also found that Gstp regulates neurite formation via JNK signaling pathways. These findings not only provide novel functions of Gstp proteins in neuritogenesis during cortical development but also help us to understand the complexity of neurite formation.

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Ras-like Gem GTPase induced by Npas4 promotes activity-dependent neuronal tolerance for ischemic stroke

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2018850118 ◽

2021 ◽

Vol 118 (32) ◽

pp. e2018850118

Author(s):

Hiroo Takahashi ◽

Ryo Asahina ◽

Masayuki Fujioka ◽

Takeshi K. Matsui ◽

Shigeki Kato ◽

...

Keyword(s):

Ischemic Stroke ◽

Cortical Neurons ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Small Gtpase ◽

Neuroprotective Effects ◽

Necessary And Sufficient ◽

Activity Dependent

Ischemic stroke, which results in loss of neurological function, initiates a complex cascade of pathological events in the brain, largely driven by excitotoxic Ca2+ influx in neurons. This leads to cortical spreading depolarization, which induces expression of genes involved in both neuronal death and survival; yet, the functions of these genes remain poorly understood. Here, we profiled gene expression changes that are common to ischemia (modeled by middle cerebral artery occlusion [MCAO]) and to experience-dependent activation (modeled by exposure to an enriched environment [EE]), which also induces Ca2+ transients that trigger transcriptional programs. We found that the activity-dependent transcription factor Npas4 was up-regulated under MCAO and EE conditions and that transient activation of cortical neurons in the healthy brain by the EE decreased cell death after stroke. Furthermore, both MCAO in vivo and oxygen-glucose deprivation in vitro revealed that Npas4 is necessary and sufficient for neuroprotection. We also found that this protection involves the inhibition of L-type voltage-gated Ca2+ channels (VGCCs). Next, our systematic search for Npas4-downstream genes identified Gem, which encodes a Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of L-type VGCCs to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death after in vitro and in vivo ischemia. Collectively, our findings indicate that Gem expression via Npas4 is necessary and sufficient to promote neuroprotection in the injured brain. Importantly, Gem is also induced in human cerebral organoids cultured under an ischemic condition, revealing Gem as a new target for drug discovery.

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The small GTPase KlRho5 responds to oxidative stress and affects cytokinesis

Journal of Cell Science ◽

10.1242/jcs.258301 ◽

2021 ◽

Vol 134 (18) ◽

Author(s):

Marius Musielak ◽

Carolin C. Sterk ◽

Felix Schubert ◽

Christian Meyer ◽

Achim Paululat ◽

...

Keyword(s):

Oxidative Stress ◽

Mitochondrial Dynamics ◽

Kluyveromyces Lactis ◽

Neck Region ◽

Small Gtpase ◽

Nutrient Sensing ◽

Deletion Mutants ◽

Nucleotide Exchange ◽

Morphological Defects

ABSTRACT Rho5 is the yeast homolog of the human small GTPase Rac1. We characterized the genes encoding Rho5 and the subunits of its dimeric activating guanine-nucleotide-exchange factor (GEF), Dck1 and Lmo1, in the yeast Kluyveromyces lactis. Rapid translocation of the three GFP-tagged components to mitochondria upon oxidative stress and carbon starvation indicate a similar function of KlRho5 in energy metabolism and mitochondrial dynamics as described for its Saccharomyces cerevisiae homolog. Accordingly, Klrho5 deletion mutants are hyper-resistant towards hydrogen peroxide. Moreover, synthetic lethalities of rho5 deletions with key components in nutrient sensing, such as sch9 and gpr1, are not conserved in K. lactis. Instead, Klrho5 deletion mutants display morphological defects with strengthened lateral cell walls and protruding bud scars. The latter result from aberrant cytokinesis, as observed by following the budding process in vivo and by transmission electron microscopy of the bud neck region. This phenotype can be suppressed by KlCDC42G12V, which encodes a hyper-active variant. Data from live-cell fluorescence microscopy support the notion that KlRho5 interferes with the actin moiety of the contractile actomyosin ring, with consequences different from those previously reported for mutants lacking myosin.

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Regulation of AMPA receptor subunit GluA1 surface expression by PAK3 phosphorylation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518382112 ◽

2015 ◽

Vol 112 (43) ◽

pp. E5883-E5890 ◽

Cited By ~ 22

Author(s):

Natasha K. Hussain ◽

Gareth M. Thomas ◽

Junjie Luo ◽

Richard L. Huganir

Keyword(s):

Signal Transduction ◽

Synaptic Plasticity ◽

Ampa Receptors ◽

Cortical Neurons ◽

Molecular Mechanisms ◽

Signal Transduction Pathway ◽

Cognitive Disorders ◽

Surface Expression ◽

Nucleotide Exchange

AMPA receptors (AMPARs) are the major excitatory receptors of the brain and are fundamental to synaptic plasticity, memory, and cognition. Dynamic recycling of AMPARs in neurons is regulated through several types of posttranslational modification, including phosphorylation. Here, we identify a previously unidentified signal transduction cascade that modulates phosphorylation of serine residue 863 (S863) in the GluA1 AMPAR subunit and controls surface trafficking of GluA1 in neurons. Activation of the EphR–Ephrin signal transduction pathway enhances S863 phosphorylation. Further, EphB2 can interact with Zizimin1, a guanine–nucleotide exchange factor that activates Cdc42 and stimulates S863 phosphorylation in neurons. Among the numerous targets downstream of Cdc42, we determined that the p21-activated kinase-3 (PAK3) phosphorylates S863 in vitro. Moreover, specific loss of PAK3 expression and pharmacological inhibition of PAK both disrupt activity-dependent phosphorylation of S863 in cortical neurons. EphB2, Cdc42, and PAKs are broadly capable of controlling dendritic spine formation and synaptic plasticity and are implicated in multiple cognitive disorders. Collectively, these data delineate a novel signal cascade regulating AMPAR trafficking that may contribute to the molecular mechanisms that govern learning and cognition.

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Nrg1 Intracellular Signaling Is Neuroprotective upon Stroke

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/3930186 ◽

2019 ◽

Vol 2019 ◽

pp. 1-15

Author(s):

Carmen Navarro-González ◽

Alba Huerga-Gómez ◽

Pietro Fazzari

Keyword(s):

Cortical Neurons ◽

Intracellular Signaling ◽

Neuronal Survival ◽

Neuroprotective Effect ◽

Inhibitory Synapses ◽

Ischemic Lesion ◽

Stroke Treatment

The schizophrenia risk gene NRG1 controls the formation of excitatory and inhibitory synapses in cortical circuits. While the expression of different NRG1 isoforms occurs during development, adult neurons primarily express the CRD-NRG1 isoform characterized by a highly conserved intracellular domain (NRG1-ICD). We and others have demonstrated that Nrg1 intracellular signaling promotes dendrite elongation and excitatory connections during neuronal development. However, the role of Nrg1 intracellular signaling in adult neurons and pathological conditions remains largely unaddressed. Here, we investigated the role of Nrg1 intracellular signaling in neuroprotection and stroke. Our bioinformatic analysis revealed the evolutionary conservation of the NRG1-ICD and a decrease in NRG1 expression with age in the human frontal cortex. Hence, we first evaluated whether Nrg1 signaling may affect pathological hallmarks in an in vitro model of neuronal senescence; however, our data failed to reveal a role for Nrg1 in the activation of the stress-related pathway p38 MAPK and DNA damage. Previous studies demonstrated that the soluble EGF domain of Nrg1 alleviated brain ischemia, a pathological process involving the generation of free radicals, reactive oxygen species (ROS), and excitotoxicity. Hence, we tested the hypothesis that Nrg1 intracellular signaling could be neuroprotective in stroke. We discovered that Nrg1 expression significantly increased neuronal survival upon oxygen-glucose deprivation (OGD), an established in vitro model for stroke. Notably, the specific activation of Nrg1 intracellular signaling by expression of the Nrg1-ICD protected neurons from OGD. Additionally, time-lapse experiments confirmed that Nrg1 intracellular signaling increased the survival of neurons exposed to OGD. Finally, we investigated the relevance of Nrg1 intracellular signaling in stroke in vivo. Using viral vectors, we expressed the Nrg1-ICD in cortical neurons and subsequently challenged them by a focal hemorrhagic stroke; our data indicated that Nrg1 intracellular signaling improved neuronal survival in the infarcted area. Altogether, these data highlight Nrg1 intracellular signaling as neuroprotective upon ischemic lesion both in vitro and in vivo. Given the complexity of the neurotoxic effects of stroke and the involvement of various mechanisms, such as the generation of ROS, excitotoxicity, and inflammation, further studies are required to determine the molecular bases of the neuroprotective effect of Nrg1 intracellular signaling. In conclusion, our research highlights the stimulation of Nrg1 intracellular signaling as a promising target for cortical stroke treatment.

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In Vivo Dynamics of Rac-Membrane Interactions

Molecular Biology of the Cell ◽

10.1091/mbc.e06-01-0005 ◽

2006 ◽

Vol 17 (6) ◽

pp. 2770-2779 ◽

Cited By ~ 67

Author(s):

Konstadinos Moissoglu ◽

Boris M. Slepchenko ◽

Nahum Meller ◽

Alan F. Horwitz ◽

Martin A. Schwartz

Keyword(s):

Dissociation Rate ◽

Small Gtpase ◽

Nucleotide Exchange ◽

Hairpin Rna ◽

Nucleotide Exchange Factors ◽

Gtpase Activating Proteins ◽

Detectable Rate ◽

Major Route ◽

Membrane Bound

The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (koff) of membrane-bound GFP-Rac. We find that koff is 0.048 s−1 for wtRac and ∼10-fold less (0.004 s−1) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased koff for wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on koff. These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.

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CalDAG-GEFI deficiency protects mice in a novel model of FcγRIIA-mediated thrombosis and thrombocytopenia

Blood ◽

10.1182/blood-2011-03-342352 ◽

2011 ◽

Vol 118 (4) ◽

pp. 1113-1120 ◽

Cited By ~ 45

Author(s):

Moritz Stolla ◽

Lucia Stefanini ◽

Pierrette André ◽

Timothy D. Ouellette ◽

Michael P. Reilly ◽

...

Keyword(s):

Platelet Activation ◽

Near Infrared ◽

Infrared Imaging ◽

Small Gtpase ◽

In Vivo Studies ◽

Critical Event ◽

Nucleotide Exchange ◽

P2y12 Inhibitor ◽

Novel Model

AbstractPlatelet activation via Fcγ receptor IIA (FcγRIIA) is a critical event in immune-mediated thrombocytopenia and thrombosis syndromes (ITT). We recently identified signaling by the guanine nucleotide exchange factor CalDAG-GEFI and the adenosine diphosphate receptor P2Y12 as independent pathways leading to Rap1 small GTPase activation and platelet aggregation. Here, we evaluated the contribution of CalDAG-GEFI and P2Y12 signaling to platelet activation in ITT. Mice transgenic for the human FcγRIIA (hFcR) and deficient in CalDAG-GEFI−/− (hFcR/CDGI−/−) were generated. Compared with controls, aggregation of hFcR/CDGI−/− platelets or P2Y12 inhibitor-treated hFcR platelets required more than 5-fold and approximately 2-fold higher concentrations of a FcγRIIA stimulating antibody against CD9, respectively. Aggregation and Rap1 activation were abolished in P2Y12 inhibitor-treated hFcR/CDGI−/− platelets. For in vivo studies, a novel model for antibody-induced thrombocytopenia and thrombosis was established. FcγRIIA-dependent platelet thrombosis was induced by infusion of Alexa750-labeled antibodies to glycoprotein IX (CD42a), and pulmonary thrombi were detected by near-infrared imaging technology. Anti-GPIX antibodies dose-dependently caused thrombocytopenia and pulmonary thrombosis in hFcR-transgenic but not wild-type mice. CalDAG-GEFI-deficient but not clopidogrel-treated hFcR-transgenic mice were completely protected from ITT. In summary, we established a novel mouse model for ITT, which was used to identify CalDAG-GEFI as a potential new target in the treatment of ITT.

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COPI Recruitment Is Modulated by a Rab1b-dependent Mechanism

Molecular Biology of the Cell ◽

10.1091/mbc.e02-09-0625 ◽

2003 ◽

Vol 14 (5) ◽

pp. 2116-2127 ◽

Cited By ~ 86

Author(s):

Cecilia Alvarez ◽

Rafael Garcia-Mata ◽

Elizabeth Brandon ◽

Elizabeth Sztul

Keyword(s):

Active Form ◽

Brefeldin A ◽

Small Gtpase ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Inactive Form ◽

Exact Function ◽

Guanine Nucleotide Binding

The small GTPase Rab1b is essential for endoplasmic reticulum (ER) to Golgi transport, but its exact function remains unclear. We have examined the effects of wild-type and three mutant forms of Rab1b in vivo. We show that the inactive form of Rab1b (the N121I mutant with impaired guanine nucleotide binding) blocks forward transport of cargo and induces Golgi disruption. The phenotype is analogous to that induced by brefeldin A (BFA): it causes resident Golgi proteins to relocate to the ER and induces redistribution of ER-Golgi intermediate compartment proteins to punctate structures. The COPII exit machinery seems to be functional in cells expressing the N121I mutant, but COPI is compromised, as shown by the release of β-COP into the cytosol. Our results suggest that Rab1b function influences COPI recruitment. In support of this, we show that the disruptive effects of N121I can be reversed by expressing known mediators of COPI recruitment, the GTPase ARF1 and its guanine nucleotide exchange factor GBF1. Further evidence is provided by the finding that cells expressing the active form of Rab1b (the Q67L mutant with impaired GTPase activity) are resistant to BFA. Our data suggest a novel role for Rab1b in ARF1- and GBF1-mediated COPI recruitment pathway.

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Ras recruits mitotic exit regulator Lte1 to the bud cortex in budding yeast

The Journal of Cell Biology ◽

10.1083/jcb.200301128 ◽

2003 ◽

Vol 161 (5) ◽

pp. 889-897 ◽

Cited By ~ 46

Author(s):

Satoshi Yoshida ◽

Ryuji Ichihashi ◽

Akio Toh-e

Keyword(s):

Small Gtpase ◽

Mitotic Exit ◽

Effector Protein ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Downstream Effector ◽

Nucleotide Exchange Factor ◽

Family Protein

ACdc25 family protein Lte1 (low temperature essential) is essential for mitotic exit at a lowered temperature and has been presumed to be a guanine nucleotide exchange factor (GEF) for a small GTPase Tem1, which is a key regulator of mitotic exit. We found that Lte1 physically associates with Ras2-GTP both in vivo and in vitro and that the Cdc25 homology domain (CHD) of Lte1 is essential for the interaction with Ras2. Furthermore, we found that the proper localization of Lte1 to the bud cortex is dependent on active Ras and that the overexpression of a derivative of Lte1 without the CHD suppresses defects in mitotic exit of a Δlte1 mutant and a Δras1 Δras2 mutant. These results suggest that Lte1 is a downstream effector protein of Ras in mitotic exit and that the Ras GEF domain of Lte1 is not essential for mitotic exit but required for its localization.

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