74 research outputs found
Identification of a Novel Formin-GAP Complex and Its Role in Macrophage Migration and Phagocytosis
No full textEssential and diverse biological processes such as cell division, morphogenesis and migration are regulated by a family of molecular switches called Rho GTPases. These proteins cycle between active, GTP-bound states and inactive, GDP-bound state and this cycle is regulated by families of proteins called Rho GEFs and GAPs. GAPs are proteins that stimulate the intrinsic GTPase activity of Rho-family proteins, potentiating the active to inactive transition. GAPs target specific spatiotemporal pools of GTPases by responding to cellular cues and utilizing protein-protein interactions. By dissecting these interactions and pathways, we can infer and then decipher the biological functions of these GAPs.This work focuses on the characterization of a novel Rho-family GAP called srGAP2. In this study, we identify that srGAP2 is a Rac-specific GAP that binds a Formin-family member, Formin-like 1 (FMNL1). FMNL1 is activated by Rac and polymerizes, bundles and severs actin filaments. srGAP2 specifically inhibits the actin severing of active FMNL1, and the assembly of an srGAP2-FMNL1 complex is regulated by Rac. Work on FMNL1 shows that it plays important roles in regulating phagocytosis and adhesion in macrophages. To learn more about srGAP2 and its role in regulating FMNL1, we studied macrophages isolated from an srGAP2 KO mouse we have recently generated. This has proven quite fruitful: loss of srGAP2 decreases the ability for macrophages to invade through extracellular matrix but increases phagocytosis. These results suggest that these two processes might be coordinated in vivo by srGAP2 and that srGAP2 might be a critical regulator of the innate immune system.</p
Novel Regulators of Actin Signaling During the Developmental Stage of Spine Formation and Maturation
No full textExcitatory synapse formation during development involves the complex orchestration of both structural and functional alterations at the postsynaptic side, beginning with the formation of transient dendritic filopodia. Abnormalities in synapse development are linked to developmental brain disorders such as autism spectrum disorders, schizophrenia, and intellectual disability. However, the molecular mechanisms that underlie excitatory synaptogenesis remain elusive, in part because the internal machinery of developing synapses is largely unknown. Unlike mature excitatory synapses, there is currently no way to biochemically isolate the dendritic filopodia of nascent synapses. This lack of understanding is a critical barrier to our grasp of synapse development as well as the etiology of many neurodevelopmental disorders. This dissertation work focuses on the detection and analysis of proteins which localize to and are critical for spinogenesis and synaptogenesis. Using state-of-the-art in vivo proteomics, we identified a network of proteins which localize to the receiving end of the developing excitatory synapse, the dendritic filopodia. We then used the CRISPR/Cas9 system to identify candidates which drive the formation and maturation of dendritic filopodia. We finally did careful functional analysis of CARMIL3 and the Arp2/3 complex to identify their critical and diverse roles in synaptogenesis. In our analysis, we found that CARMIL3 is expressed in the brain predominately during synaptogenesis, localizes to developing dendritic protrusions, and is important for the morphological and functional maturation of synapses, likely through its role in recruiting capping protein to maturing synapses. Loss of CARMIL3 leads to structurally and functionally immature synapses that are capping protein deficient. Further, we found that the Arp2/3 complex, a critical regulator of the actin cytoskeleton which creates branched actin networks, is required for both the functional and morphological maturation of dendritic spines. In the absence of the Arp2/3 complex, dendritic protrusions make presynaptic contact, recruit key proteins such as MAGUKs, and recruit certain receptors such as NMDA receptors, but lack AMPA receptors which are required for synapse unsilencing.Together, this work demonstrates that the actin cytoskeleton controls the functional maturation of synapses by altering the cytoskeletal dynamics towards the creation of a branched actin network. CARMIL3 contributes to this process by providing capping protein, which biases actin nucleation towards branched actin networks. Arp2/3 creates the branched actin network. Without this network, there is not a sufficient framework to dock AMPA receptors in the post-synaptic density, and without AMPA receptors, dendritic protrusions remain functionally silent. Together, this work shows that the dynamics of the actin cytoskeleton drive synapse unsilencing.</p
Understanding the role of Cnksr2 in Epilepsy-Aphasia Syndrome
No full textEpilepsy-Aphasia Syndromes (EAS) are debilitating childhood focal epilepsies and epileptic encephalopathies with severe cognitive and language dysfunction. Patients with EAS experience loss of previously normal language, seizures, sleep-related EEG abnormalities, and cognitive deficits. Seizures are often refractory to treatments, and the prognosis of EAS is generally poor. Even when epilepsy is resolved, most patients have life-long language and cognitive disturbances. The link between EEG abnormalities, seizures, and cognito-behavioral comorbidities seen in this spectrum is poorly understood, making it more challenging to develop therapies. While the clinical manifestations of EAS have been long studied, the etiology of EAS has been unknown. Recently, loss of function mutations in X-linked gene CNKSR2(Connector Enhancer Of Kinase Suppressor Of Ras 2) have been implicated in EAS. A small number of in vitro studies suggest that CNKSR2 encodes for a putative scaffold protein that localizes to the synapse. Yet, there have been no functional studies of Cnksr2 to examine how its absence may lead to EAS. This dissertation focuses on uncovering the role of Cnksr2 in the brain and investigating the neuropathological effects of Cnksr2 loss in the context of EAS.
Here, I first present that Cnksr2 is expressed in cortical, striatal, and cerebellar regions and is localized at excitatory and inhibitory postsynapses in the mouse brain. Next, we generate a novel transgenic Cnksr2 knockout mouse model and confirm the loss of Cnksr2. Then, using proteomics analysis, I demonstrate that Cnksr2 anchors key binding partners in synapses, and its loss results in significant alterations of synaptic proteins implicated in neurological disorders. I also find that loss of Cnksr2 leads to increased spontaneous activity of neurons, and Cnksr2 KO mice exhibit electrographic seizures and epileptiform discharges.
Notably, Cnksr2 KO mice show significantly increased anxiety, impaired learning, and a progressive and dramatic loss of ultrasonic vocalizations. Next, I investigate the cell-specific contributions to the core Cnksr2-loss phenotypes in mice. Particularly, I seek to determine the effects of Cnksr2 deletion in excitatory vs. inhibitory cells using Cre/Lox system. Surprisingly, I discover that cell-specific deletion of Cnksr2 results in distinct phenotypic outcomes: glutamatergic neuron-specific deletion of Cnksr2 leads to behavioral deficits, and the GABAergic neuron-specific deletion leads to electrographic seizures.
Taken together, my doctoral dissertation research validates that loss of CNKSR2 leads to EAS, as well as highlights the fundamental roles of Cnksr2 in synaptic protein organization and excitation of neuronal networks. Furthermore, I show that Cnksr2 KO mice exhibit electrophysiological and behavioral phenotypes similar to those of patients, providing an exciting new model for future therapeutics studies of EAS. Lastly, my research provides novel insights into the cellular mechanisms leading to distinct EAS-related pathology.
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Uncovering the Role of the WASH Complex in Neurological Disorders
No full textMultiple neurodevelopmental and neurodegenerative disorders are driven by disruptions to intracellular trafficking between organelles in neurons. One branch of this trafficking network, the endo-lysosomal pathway, is thought to tightly regulate many neuronal processes, including the dynamic recycling of synaptic receptors and the homeostatic regulation of membrane lipid composition. This idea is supported by numerous accounts of human neurological diseases that develop from the disruption of endo-lysosomal processes. In particular, mutations in subunits of the endosomal WASH complex, Strumpellin and SWIP, are implicated in motor and cognitive disorders, but the cellular etiologies of these associations are unknown (Assoum et al., 2020; De Bot et al., 2013; Elliott et al., 2013; Ropers et al., 2011). This dissertation focuses on dissecting the molecular function of the WASH complex in mouse brain and determining how mutation of the WASH subunit, SWIP, drives neurological dysfunction.To explore the role of the WASH complex in neurons, we first sought to identify the molecular interactors of WASH in mouse brain. We utilized in vivo BioID (iBioID) to delineate the neuronal WASH complex proteome in wild-type mouse brain, revealing a rich network of endosomal proteins. Comparison of this network to previously reported WASH interactors in other cell types demonstrated the conservation of WASH function in endosomal trafficking within the mouse nervous system. Then, to uncover how dysfunction of endosomal SWIP leads to disease, we generated a novel mouse model of a human WASHC4c.3056C>G mutation. We performed quantitative spatial proteomics analysis of SWIPP1019R mouse brain, which demonstrated that this mutation destabilizes the WASH complex and causes significant perturbations in both endosomal and lysosomal pathways. We then utilized cellular and histological analyses to confirm that SWIPP1019R results in an endo-lysosomal disruption in vitro and in vivo. We also found indications of neurodegeneration in SWIPP1019R mice that appeared selective for the motor cortex. To determine if these structural changes produced functional effects in SWIPP1019R brain, we conducted behavioral assays in wild-type and mutant SWIPP1019R littermates at adolescence and adulthood. These studies revealed that SWIPP1019R not only impacts cognition, but also causes significant progressive motor deficits in mice. By analyzing human SWIPP1019R patient data, we found that this WASHC4 mutation produces similar movement deficits in humans. Combined, our research provides the first molecular analysis of WASH complex function in the mammalian nervous system, and supports the model that WASH complex destabilization, resulting from SWIPP1019R, drives cognitive and motor impairments via endo-lysosomal dysfunction in the brain.</p
Statistical Inference and Community Detection in Proximity and Spatial Proteomics: Resolving the Organization of the Neuronal Proteome
No full textTechnological advances in protein mass spectrometry (MS), aka proteomics, haveenabled high-throughput quantification of spatially-resolved,
subcellular-specific proteomes. Biological insight in these experiments depends
upon sound statistical analysis. Despite the myriad of existing proprietary and
open-source software solutions for statistical analysis of proteomics data,
these tools suffer a drawback inherent in any general solution: a loss of
specificity. These tools often fail to be easily adapted to analyze
experiment-specific designs. I present a flexible, linear mixed-effects model
framework for assessing differential abundance in protein mass spectrometry
experiments. Combined with methods to identify communities of proteins in
biological networks, I extend this framework to perform inference at the level
of protein groups or modules. Using these software tools, I demonstrate how
module-level insight in proximity and spatial proteomics
generates hypotheses that identify foci of biological function and dysfunction
which may underlie the neuropathology of disease.</p
Linking WRP/srGAP3 to the Cognitive Deficits in 3p- Syndrome and Its Role in the Regulation of Dendritic Filopodia Formation
No full textRho GTPase signaling regulates a wide variety of cellular functions in the developing and adult central nervous system. These molecular switches are in turn spatially and temporally regulated by an over abundance of positive and negative regulatory proteins: the activating guanine nucleotide exchange factors (GEFs) and the deactivating GTPase activating proteins (GAPs). The WAVE-associated Rac GAP (WRP) is thought to regulate key aspects of synapse development and function, and has been implicated in a form of mental retardation in humans called 3p- Syndrome. WRP is a member of the srGAP family of Rho GAP domain containing proteins, which share a characteristic domain organization and are expressed throughout the brain. Recently, one of the members of this family was found to contain a newly described inverse F-BAR (IF-BAR) domain of unknown function and to regulate cortical migration in developing neurons. This study focuses on the regulatory capacity of WRP during the development of neuronal connections in the central nervous system, and what role its loss may have on cognitive functions.To assess these roles, biochemical studies were performed to characterize the way in which WRP's novel IF-BAR domain interacted with lipid membranes. Additionally, WRP's role in regulating neuronal function was assessed both in vitro and in vivo through the use of mouse model systems for critical genes in the WAVE complex pathway, including a conditional WRP KO mouse developed in our lab. Finally, because WRP is implicated in mental retardation, behaviors of WRP heterozygous and null mice have been evaluated.This study shows that WRP's IF-BAR domain senses, or facilitates, outward membrane protrusions through a convex lipid-binding surface of the dimerized WRP IF-BAR domain. WRP localizes to the membranes of dendritic shafts via its IF-BAR domain where it is enriched in filopodia like projections. During dendritic filopodia formation, WRP functions to regulate the WAVE-1 complex and its downstream effectors, including the Arp2/3 complex. Loss of WRP in vivo and in vitro results in a reduction of dendritic spines, and that this is a function of WRP's role in the initiation of dendritic filopodia, not during the maturation of dendritic filopodia into mature dendritic spines. Finally, this study demonstrates that the loss of WRP results in deficits in learning and memory, linking WRP to the cognitive deficits seen in 3p- syndrome.</p
Spatiotemporal Dynamics of CaMKI During Structural Plasticity of Single Dendritic Spines
No full textMultifunctional calcium/calmodulin dependent protein kinases (CaMKs) are key regulators of spine structural plasticity and long-term potentiation (LTP) in neurons. CaMKs have promiscuous and overlapping substrate recognition motifs, and are distinguished in their regulatory role based on differences in the spatiotemporal dynamics of activity. While the function and activity of CaMKII in synaptic plasticity has been extensively studied, that of CaMKI, another major class of CaMK required for LTP, still remain elusive. Here, we develop a Förster’s Resonance Energy Transfer (FRET) based sensor to measure the spatiotemporal activity dynamics of CaMK1. We monitored CaMKI activity using 2-photon fluorescence lifetime imaging, while inducing LTP in single dendritic spines of rat (Rattus Norvegicus, strain Sprague Dawley) hippocampal CA1 pyramidal neurons using 2-photon glutamate uncaging. Using RNA-interference and pharmacological means, we also characterize the role of CaMKI during spine structural plasticity. We found that CaMKI was rapidly and transiently activated with a rise time of ~0.3 s and decay time of ~1 s in response to each uncaging pulse. Activity of CaMKI spread out of the spine. Phosphorylation of CaMKI by CaMKK was required for this spreading and for the initial phase of structural LTP. Combined with previous data showing that CaMKII is restricted to the stimulated spine and required for long-term maintenance of structural LTP, these results suggest that CaMK diversity allows the same incoming signal – calcium – to independently regulate distinct phases of LTP by activating different CaMKs with distinct spatiotemporal dynamics.</p
Mechanisms of Cellular Protrusions Branch Out
No full textF-BAR domains bind curved membranes and induce membrane invagination. In a recent Cell paper, Guerrier et al. describe an “inverse” F-BAR family member that induces outward curvature and filopodia in migrating neurons. These findings suggest that F-BAR domains are functionally diverse and regulate different types of membrane morphology
Review of: © 2014, Kim et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
No full textCdc42 is a signaling protein important for reorganization of actin cytoskeleton and morphogenesis of cells. However, the functional role of Cdc42 in synaptic plasticity and in behaviors such as learning and memory are not well understood. Here we report that postnatal forebrain deletion of Cdc42 leads to deficits in synaptic plasticity and in remote memory recall using conditional knockout of Cdc42. We found that deletion of Cdc42 impaired LTP in the Schaffer collateral synapses and postsynaptic structural plasticity of dendritic spines in CA1 pyramidal neurons in the hippocampus. Additionally, loss of Cdc42 did not affect memory acquisition, but instead significantly impaired remote memory recall. Together these results indicate that the postnatal functions of Cdc42 may be crucial for the synaptic plasticity in hippocampal neurons, which contribute to the capacity for remote memory recall.DOI: http://dx.doi.org/10.7554/eLife.02839.001View Full TextTo Top Cdc42 is a signaling protein important for reorganization of actin cytoskeleton and morphogenesis of cells. However, the functional role of Cdc42 in synaptic plasticity and in behaviors such as learning and memory are not well understood. Here we report that postnatal forebrain deletion of Cdc42 leads to deficits in synaptic plasticity and in remote memory recall using conditional knockout of Cdc42. We found that deletion of Cdc42 impaired LTP in the Schaffer collateral synapses and postsynaptic structural plasticity of dendritic spines in CA1 pyramidal neurons in the hippocampus. Additionally, loss of Cdc42 did not affect memory acquisition, but instead significantly impaired remote memory recall. Together these results indicate that the postnatal functions of Cdc42 may be crucial for the synaptic plasticity in hippocampal neurons, which contribute to the capacity for remote memory recall. DOI: http://dx.doi.org/10.7554/eLife.02839.001 Neurons communicate with one another at junctions called synapses, which are typically formed between the dendrite of one neuron and the axon terminus of another. The dendrites are protrusions coming out of the cell body that receive inputs from other cells; the axon is a cable-like structure that enables neurons to contact other cells. In excitatory neurons in part of the brain called the hippocampus, the dendrites are themselves covered in structures called spines, so most synapses are formed between an axon terminus (belonging to the presynaptic cell) and a dendritic spine (on the postsynaptic cell). The hippocampus is necessary for the formation of long-term memories. The strength of a synapse can increase or decrease over time—a property that is called synaptic plasticity. Changes in the strength of synapses are thought to underlie learning and memory, and long-lasting changes in synaptic strength involve increases or decreases in the number and size of dendritic spines. Such changes are possible because spines have an internal skeleton that can be assembled and disassembled in a matter of minutes. This ‘remodeling’ process is regulated by a family of enzymes called small GTPases. One of these, known as Cdc42, has been shown to promote the formation and maintenance of spines in cell culture, but its role in synaptic plasticity, learning and memory remains unknown. Now, Kim, Wang et al. have used genetically modified mice who have had Cdc42 deleted from excitatory neurons in their forebrain to examine the functions of this enzyme in living animals. These ‘knockout’ mice showed a small but statistically significant reduction in the number of dendritic spines in the hippocampus. They also showed smaller changes in spine volume and impaired long-term synaptic plasticity in the hippocampus. When the mice performed long-term memory tests where they learnt to associate a specific set of visual cues with an impending electric shock, the knockout mice performed well for up to a few days. However, when tested again on the same task 45 days later, the knockout mice did not perform as well as normal mice. This is surprising, given the presumed role of long-term synaptic plasticity in learning and memory, and indicates that Cdc42 is required for ‘remote memory’, the form of memory lasting for many days. Similar results were obtained with another memory test using a water maze, where the animals have to remember the location of a hidden platform. Normal mice remember the location for more than 30 days. In contrast, the knockout mice could only remember the location for a few days. As well as providing the first demonstration of the role of Cdc42 in synaptic plasticity in live animals, the work of Kim, Wang et al. has provided new insights into the functions of this enzyme in memory. Further work is required to determine how Cdc42 interacts with other proteins present at synapses. DOI: http://dx.doi.org/10.7554/eLife.02839.00
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