MARK1 regulates dendritic spine morphogenesis and cognitive functions in vivo.

Dendritic spines play a pivotal role in synaptic communication and are crucial for learning and memory processes. Abnormalities in spine morphology and plasticity are observed in neurodevelopmental and neuropsychiatric disorders, yet the underlying signaling mechanisms remain poorly understood. The microtubule affinity regulating kinase 1 (MARK1) has been implicated in neurodevelopmental disorders, and the MARK1 gene shows accelerated evolution in the human lineage suggesting a role in cognition. However, the in vivo role of MARK1 in synaptogenesis and cognitive functions remains unknown. Here we show that forebrain-specific conditional knockout (cKO) of Mark1 in mice causes defects in dendritic spine morphogenesis in hippocampal CA1 pyramidal neurons with a significant reduction in spine density. In addition, we found loss of MARK1 causes synaptic accumulation of GKAP and GluA2. Furthermore, we found that MARK1 cKO mice show defects in spatial learning in the Morris water maze and reduced anxiety-like behaviors in the elevated plus maze. Taken together, our data show a novel role for MARK1 in regulating dendritic spine morphogenesis and cognitive functions in vivo.

Neuronal connectivity, behavioral, and transcriptional alterations associated with the loss of MARK2.

Neuronal connectivity is essential for adaptive brain responses and can be modulated by dendritic spine plasticity and the intrinsic excitability of individual neurons. Dysregulation of these processes can lead to aberrant neuronal activity, which has been associated with numerous neurological disorders including autism, epilepsy, and Alzheimer's disease. Nonetheless, the molecular mechanisms underlying aberrant neuronal connectivity remains unclear. We previously found that the serine/threonine kinase Microtubule Affinity Regulating Kinase 2 (MARK2), also known as Partitioning Defective 1b (Par1b), is important for the formation of dendritic spines in vitro. However, despite its genetic association with several neurological disorders, the in vivo impact of MARK2 on neuronal connectivity and cognitive functions remains unclear. Here, we demonstrate that loss of MARK2 in vivo results in changes to dendritic spine morphology, which in turn leads to a decrease in excitatory synaptic transmission. Additionally, loss of MARK2 produces substantial impairments in learning and memory, anxiety, and social behavior. Notably, MARK2 deficiency results in heightened seizure susceptibility. Consistent with this observation, RNAseq analysis reveals transcriptional changes in genes regulating synaptic transmission and ion homeostasis. These findings underscore the in vivo role of MARK2 in governing synaptic connectivity, cognitive functions, and seizure susceptibility.

MARK1 regulates dendritic spine morphogenesis and cognitive functions in vivo.

Dendritic spines play a pivotal role in synaptic communication and are crucial for learning and memory processes. Abnormalities in spine morphology and plasticity are observed in neurodevelopmental and neuropsychiatric disorders, yet the underlying signaling mechanisms remain poorly understood. The microtubule affinity regulating kinase 1 (MARK1) has been implicated in neurodevelopmental disorders, and the MARK1 gene shows accelerated evolution in the human lineage suggesting a role in cognition. However, the in vivo role of MARK1 in synaptogenesis and cognitive functions remains unknown. Here we show that forebrain-specific conditional knockout (cKO) of Mark1 causes defects in dendritic spine morphogenesis in hippocampal CA1 pyramidal neurons with a significant reduction in spine density. In addition, we found that MARK1 cKO mice show defects in spatial learning in the Morris Water Maze and reduced anxiety-like behaviors in the Elevated Plus Maze. Furthermore, we found loss of MARK1 causes synaptic accumulation of GKAP and GluR2. Taken together, our data show a novel role for MARK1 in regulating dendritic spine morphogenesis and cognitive functions in vivo .

Loss of the polarity protein Par3 promotes dendritic spine neoteny and enhances learning and memory.

The Par3 polarity protein is critical for subcellular compartmentalization in different developmental processes. Variants of PARD3 , which encodes PAR3, are associated with intelligence and neurodevelopmental disorders. However, the role of Par3 in glutamatergic synapse formation and cognitive functions in vivo remains unknown. Here, we show that forebrain conditional knockout of Par3 leads to an increase in long, thin dendritic spines without significantly impacting mushroom spines in vivo . In addition, we observed a decrease in the amplitude of miniature excitatory postsynaptic currents. Surprisingly, loss of Par3 in vivo enhances hippocampal- dependent spatial learning. Phosphoproteomic analysis revealed proteins regulating cytoskeletal dynamics are significantly dysregulated downstream of Par3. Mechanistically, we found Par3 deletion causes increased activation of the Rac1 pathway. Together, our data reveal an unexpected role for Par3 as a molecular gatekeeper in regulating the pool of immature dendritic spines, a rate-limiting step of learning and memory, through modulating Rac1 activation in vivo .

SHANK3 Mutations Associated with Autism and Schizophrenia Lead to Shared and Distinct Changes in Dendritic Spine Dynamics in the Developing Mouse Brain.

Autism Spectrum Disorders (ASD) and schizophrenia are distinct neurodevelopmental disorders that share certain symptoms and genetic components. Both disorders show abnormalities in dendritic spines, which are the main sites of excitatory synaptic inputs. Recent studies have identified the synaptic scaffolding protein Shank3 as a leading candidate gene for both disorders. Mutations in the SHANK3 gene have been linked to both ASD and schizophrenia; however, how patient-derived mutations affect the structural plasticity of dendritic spines during brain development is unknown. Here we use live two photon in vivo imaging to examine dendritic spine structural plasticity in mice with SHANK3 mutations associated with ASD and schizophrenia. We identified shared and distinct phenotypes in dendritic spine morphogenesis and plasticity in the ASD-associated InsG3680 mutant mice and the schizophrenia-associated R1117X mutant mice. No significant changes in dendritic arborization were observed in either mutant, raising the possibility that synaptic dysregulation may be a key contributor to the behavioral defects previously reported in these mice. These findings shed light on how patient-linked mutations in SHANK3 affect dendritic spine dynamics in the developing brain, which provides insight into the synaptic basis for the distinct phenotypes observed in ASD and schizophrenia.

Polarity proteins: Shaping dendritic spines and memory.

The morphogenesis and plasticity of dendritic spines are associated with synaptic strength, learning, and memory. Dendritic spines are highly compartmentalized structures, which makes proteins involved in cellular polarization and membrane compartmentalization likely candidates regulating their formation and maintenance. Indeed, recent studies suggest polarity proteins help form and maintain dendritic spines by compartmentalizing the spine neck and head. Here, we review emerging evidence that polarity proteins regulate dendritic spine plasticity and stability through the cytoskeleton, scaffolding molecules, and signaling molecules. We specifically analyze various polarity complexes known to contribute to different forms of cell polarization processes and examine the essential conceptual context linking these groups of polarity proteins to dendritic spine morphogenesis, plasticity, and cognitive functions.

Mitochondrial Fission Factor Is a Novel Interacting Protein of the Critical B Cell Survival Regulator TRAF3 in B Lymphocytes.

Proteins controlling mitochondrial fission have been recognized as essential regulators of mitochondrial functions, mitochondrial quality control and cell apoptosis. In the present study, we identified the critical B cell survival regulator TRAF3 as a novel binding partner of the key mitochondrial fission factor, MFF, in B lymphocytes. Elicited by our unexpected finding that the majority of cytoplasmic TRAF3 proteins were localized at the mitochondria in resting splenic B cells after ex vivo culture for 2 days, we found that TRAF3 specifically interacted with MFF as demonstrated by co-immunoprecipitation and GST pull-down assays. We further found that in the absence of stimulation, increased protein levels of mitochondrial TRAF3 were associated with altered mitochondrial morphology, decreased mitochondrial respiration, increased mitochondrial ROS production and membrane permeabilization, which eventually culminated in mitochondria-dependent apoptosis in resting B cells. Loss of TRAF3 had the opposite effects on the morphology and function of mitochondria as well as mitochondria-dependent apoptosis in resting B cells. Interestingly, co-expression of TRAF3 and MFF resulted in decreased phosphorylation and ubiquitination of MFF as well as decreased ubiquitination of TRAF3. Moreover, lentivirus-mediated overexpression of MFF restored mitochondria-dependent apoptosis in TRAF3-deficient malignant B cells. Taken together, our findings provide novel insights into the apoptosis-inducing mechanisms of TRAF3 in B cells: as a result of survival factor deprivation or under other types of stress, TRAF3 is mobilized to the mitochondria through its interaction with MFF, where it triggers mitochondria-dependent apoptosis. This new role of TRAF3 in controlling mitochondrial homeostasis might have key implications in TRAF3-mediated regulation of B cell transformation in different cellular contexts. Our findings also suggest that mitochondrial fission is an actionable therapeutic target in human B cell malignancies, including those with TRAF3 deletion or relevant mutations.

Metformin reduces neuroinflammation and improves cognitive functions after traumatic brain injury.

Within the brain, traumatic brain injury (TBI) alters synaptic plasticity and increases neuroinflammation and neuronal death. Yet, there lacks effective TBI treatments providing pleiotropic beneficial effects on these diverse cellular processes necessary for functional recovery. Here, we show the diabetes drug, metformin, significantly improves cognitive functions after controlled cortical impact (CCI) injury in mice, showing improved spatial learning and nest building. Furthermore, injured animals treated with metformin exhibit increased ramification of microglia processes, indicating reduced neuroinflammation. Finally, metformin treatment in vitro increased neuronal activation of partitioning defective 1 (Par1), a family of Ser/Thr kinases playing a key role in synaptic plasticity and neuroinflammation. These results suggest metformin is a promising therapeutic agent for targeting multiple cellular processes necessary for functional TBI recovery.

Translational derepression of Elavl4 isoforms at their alternative 5' UTRs determines neuronal development.

Neurodevelopment requires precise regulation of gene expression, including post-transcriptional regulatory events such as alternative splicing and mRNA translation. However, translational regulation of specific isoforms during neurodevelopment and the mechanisms behind it remain unknown. Using RNA-seq analysis of mouse neocortical polysomes, here we report translationally repressed and derepressed mRNA isoforms during neocortical neurogenesis whose orthologs include risk genes for neurodevelopmental disorders. We demonstrate that the translation of distinct mRNA isoforms of the RNA binding protein (RBP), Elavl4, in radial glia progenitors and early neurons depends on its alternative 5' UTRs. Furthermore, 5' UTR-driven Elavl4 isoform-specific translation depends on upstream control by another RBP, Celf1. Celf1 regulation of Elavl4 translation dictates development of glutamatergic neurons. Our findings reveal a dynamic interplay between distinct RBPs and alternative 5' UTRs in neuronal development and underscore the risk of post-transcriptional dysregulation in co-occurring neurodevelopmental disorders.

Long-lasting Behavioral and Neuroanatomical Effects of Postnatal Valproic Acid Treatment.

Valproic acid (VPA) administered to mice during the early postnatal period causes social, cognitive, and motor deficits similar to those observed in humans with autism spectrum disorder (ASD). However, previous studies on the effects of early exposure to VPA have largely focused on behavioral deficits occurring before or during the juvenile period of life. Given that ASD is a life-long condition, the present study ought to extend our understanding of the behavioral profile following early postnatal VPA into adulthood. Male mice treated with VPA on postnatal day 14 (P14) displayed increased aggression, decreased avoidance of the open arms in the elevated plus maze, and impaired reversal learning in the Y maze. This may indicate a disinhibited or impulsive phenotype in male, but not female, mice treated with VPA during the second week of postnatal life. Decreased dendritic spine density and dendritic spine morphological abnormalities in the mPFC of VPA-treated mice may be indicative of PFC hypofunction, consistent with the observed behavioral differences. Since these types of long-lasting deficits are not exclusively found in ASD, early life exposure to VPA may reflect dysfunction of a neurobiological domain common to several developmental disorders, including ASD, ADHD, and conduct disorder.

Oxidation of KCNB1 potassium channels in the murine brain during aging is associated with cognitive impairment.

Voltage-gated potassium (K+) channel sub-family B member 1 (KCNB1, Kv2.1) is known to undergo oxidation-induced oligomerization during aging but whether this process affects brain's physiology was not known. Here, we used 10, 16 and 22 month-old transgenic mice overexpressing a KCNB1 variant that does not oligomerize (Tg-C73A) and as control, mice overexpressing the wild type (Tg-WT) channel and non-transgenic (non-Tg) mice to elucidate the effects of channel's oxidation on cognitive function. Aging mice in which KCNB1 oligomerization is negligible (Tg-C73A), performed significantly better in the Morris Water Maze (MWM) test of working memory compared to non-Tg or Tg-WT mice. KCNB1 and synapsin-1 co-immunoprecipitated and the cognitive impairment in the MWM was associated with moderate loss of synapsin-1 in pre-synaptic structures of the hippocampus, whereas neurodegeneration and neuronal loss were not significantly different in the various genotypes. We conclude that moderate oxidation of the KCNB1 channel during aging can influence neuronal networks by affecting synaptic function.

Par3 regulates polarized convergence between APP and BACE1 in hippocampal neurons.

The convergence between amyloid precursor protein (APP) and its ß-secretase ß-site APP cleaving enzyme 1 (BACE1) is a prerequisite for the generation of ß-amyloid peptide, a key pathogenic agent for Alzheimer's disease. Yet the underlying molecular mechanisms regulating their convergence remain unclear. Here, we show that the polarity protein partitioning-defective 3 (Par3) regulates the polarized convergence between APP and BACE1 in hippocampal neurons. Par3 forms a complex with BACE1 through its first PDZ domain, which is important for regulating BACE1 endosome-to-TGN trafficking. In the absence of Par3, there is an increase in the convergence between internalized APP and BACE1. In hippocampal neurons, loss of Par3 leads to increased APP and BACE1 convergence in axons but not in dendrites. This polarized convergence mainly occurs in retrograde or stalled axonal late endocytic organelles and is likely due to compartment-specific regulation of APP trafficking by Par3. Together, our data show a novel function for Par3 in regulating polarized convergence between APP and BACE1 in hippocampal neurons.

Loss of Par1b/MARK2 primes microglia during brain development and enhances their sensitivity to injury.

BACKGROUND: Microglia, the resident immune cells of the brain, exhibit various morphologies that correlate with their functions under physiological and pathological conditions. In conditions such as aging and stress, microglia priming occurs, which leads to altered morphology and lower threshold for activation upon further insult. However, the molecular mechanisms that lead to microglia priming are unclear. METHODS: To understand the role of Par1b/MARK2 in microglia, we first expressed shRNA targeting luciferase or Par1b/MARK2 in primary microglial cells and imaged the cells using fluorescent microscopy to analyze for morphological changes. A phagocytosis assay was then used to assess functional changes. We then moved in vivo and used a Par1b/MARK2 knockout mouse model to assess for changes in microglia density, morphology, and phagocytosis using immunohistochemistry, confocal imaging, and 3D image reconstruction. Next, we used two-photon in vivo imaging in live Par1b/MARK2 deficient mice to examine microglia dynamics. In addition, a controlled-cortical impact injury was performed on wild-type and Par1b/MARK2-deficient mice and microglial response was determined by confocal imaging. Finally, to help rule out non-cell autonomous effects, we analyzed apoptosis by confocal imaging, cytokine levels by multiplex ELISA, and blood-brain barrier permeability using Evans Blue assay. RESULTS: Here, we show that loss of the cell polarity protein Par1b/MARK2 facilitates the activation of primary microglia in culture. We next found that microglia in Par1b/MARK2 deficient mice show increased density and a hypertrophic morphology. These morphological changes are accompanied with alterations in microglia functional responses including increased phagocytosis of neuronal particles early in development and decreased surveillance of the brain parenchyma, all reminiscent of a primed phenotype. Consistent with this, we found that microglia in Par1b/MARK2 deficient mice have a significantly lower threshold for activation upon injury. CONCLUSIONS: Together, our studies show that loss of Par1b/MARK2 switches microglia from a surveillant to a primed state during development, resulting in an increased neuroinflammatory response to insults.

The Endolysosomal System and Proteostasis: From Development to Degeneration.

How do neurons adapt their endolysosomal system to address the particular challenge of membrane transport across their elaborate cellular landscape and to maintain proteostasis for the lifetime of the organism? Here we review recent findings that address this central question. We discuss the cellular and molecular mechanisms of endolysosomal trafficking and the autophagy pathway in neurons, as well as their role in neuronal development and degeneration. These studies highlight the importance of understanding the basic cell biology of endolysosomal trafficking and autophagy and their roles in the maintenance of proteostasis within the context of neurons, which will be critical for developing effective therapies for various neurodevelopmental and neurodegenerative disorders.

Ras and Rap Signal Bidirectional Synaptic Plasticity via Distinct Subcellular Microdomains.

How signaling molecules achieve signal diversity and specificity is a long-standing cell biology question. Here we report the development of a targeted delivery method that permits specific expression of homologous Ras-family small GTPases (i.e., Ras, Rap2, and Rap1) in different subcellular microdomains, including the endoplasmic reticulum, lipid rafts, bulk membrane, lysosomes, and Golgi complex, in rodent hippocampal CA1 neurons. The microdomain-targeted delivery, combined with multicolor fluorescence protein tagging and high-resolution dual-quintuple simultaneous patch-clamp recordings, allows systematic analysis of microdomain-specific signaling. The analysis shows that Ras signals long-term potentiation via endoplasmic reticulum PI3K and lipid raft ERK, whereas Rap2 and Rap1 signal depotentiation and long-term depression via bulk membrane JNK and lysosome p38MAPK, respectively. These results establish an effective subcellular microdomain-specific targeted delivery method and unveil subcellular microdomain-specific signaling as the mechanism for homologous Ras and Rap to achieve signal diversity and specificity to control multiple forms of synaptic plasticity.

The GluN2A Subunit Regulates Neuronal NMDA receptor-Induced Microglia-Neuron Physical Interactions.

Microglia are known to engage in physical interactions with neurons. However, our understanding of the detailed mechanistic regulation of microglia-neuron interactions is incomplete. Here, using high resolution two photon imaging, we investigated the regulation of NMDA receptor-induced microglia-neuron physical interactions. We found that the GluN2A inhibitor NVPAAM007, but not the GluN2B inhibitor ifenprodil, blocked the occurrence of these interactions. Consistent with the well-known developmental regulation of the GluN2A subunit, these interactions are absent in neonatal tissues. Furthermore, consistent with a preferential synaptic localization of GluN2A subunits, there is a differential sensitivity of their occurrence between denser (stratum radiatum) and less dense (stratum pyramidale) synaptic sub-regions of the CA1. Finally, consistent with differentially expressed GluN2A subunits in the CA1 and DG areas of the hippocampus, these interactions could not be elicited in the DG despite robust microglial chemotactic capabilities. Together, these results enhance our understanding of the mechanistic regulation of NMDA receptor-dependent microglia-neuronal physical interactions phenomena by the GluN2A subunit that may be relevant in the mammalian brain during heightened glutamatergic neurotransmission such as epilepsy and ischemic stroke.

Par3 and aPKC regulate BACE1 endosome-to-TGN trafficking through PACS1.

The cleavage of amyloid precursor protein (APP) by ß-site APP cleaving enzyme 1 (BACE1) is the rate-limiting step in beta amyloid generation during Alzheimer's disease (AD) pathogenesis. In AD brains, BACE1 is abnormally accumulated in endocytic compartments, where the acidic pH is optimal for its activity. However, mechanisms regulating the endosome-to-trans-Golgi network (TGN) retrieval of BACE1 remain unclear. Here, we show that partitioning defective 3 (Par3) facilitates BACE1 retrograde trafficking from endosomes to the TGN. Par3 functions through aPKC-mediated phosphorylation of BACE1 on Ser498, which in turn promotes the interaction between BACE1 and phosphofurin acidic cluster sorting protein 1 and facilitates the retrograde trafficking of BACE1 to the TGN. In human AD brains, there is a significant decrease in Ser498 phosphorylation of BACE1 suggesting that defective phosphorylation-dependent retrograde transport of BACE1 is important in AD pathogenesis. Together, our studies provide mechanistic insight into a novel role for Par3 and aPKC in regulating the retrograde endosome-to-TGN trafficking of BACE1 and shed light on the mechanisms of AD pathogenesis.