SIK1 Downregulates Synaptic AMPA Receptors and Contributes to Cognitive Defects in Alzheimer's Disease.

A reduction in AMPA receptor (AMPAR) expression and weakened synaptic activity is early cellular phenotypes in Alzheimer's disease (AD). However, the molecular processes leading to AMPAR downregulation are complex and remain less clear. Here, we report that the salt inducible kinase SIK1 interacts with AMPARs, leading to a reduced accumulation of AMPARs at synapses. SIK1 protein level is sensitive to amyloid beta (Aß) and shows a marked increase in the presence of Aß and in AD brains. In neurons, Aß incubation causes redistribution of SIK1 to synaptic sites and enhances SIK1-GluA1 association. SIK1 function is required for Aß-induced AMPAR reduction. Importantly, in 3xTG AD mice, knockdown of SIK1 in the brain leads to restoration of AMPAR expression and a rescue of the cognitive deficits. These findings indicate an important role for SIK1 in meditating the cellular and functional pathology in AD.

Sexually dimorphic phenotypes and the role of androgen receptors in UBE3A-dependent autism spectrum disorder.

Autism spectrum disorders (ASDs) are characterized by social, communication, and behavioral challenges. UBE3A is one of the most common ASD genes. ASDs display a remarkable sex difference with a 4:1 male to female prevalence ratio; however, the underlying mechanism remains largely unknown. Using the UBE3A-overexpressing mouse model for ASD, we studied sex differences at behavioral, genetic, and molecular levels. We found that male mice with extra copies of Ube3A exhibited greater impairments in social interaction, repetitive self-grooming behavior, memory, and pain sensitivity, whereas female mice with UBE3A overexpression displayed greater olfactory defects. Social communication was impaired in both sexes, with males making more calls and females preferring complex syllables. At the molecular level, androgen receptor (AR) levels were reduced in both sexes due to enhanced degradation mediated by UBE3A. However, AR reduction significantly dysregulated AR target genes only in male, not female, UBE3A-overexpressing mice. Importantly, restoring AR levels in the brain effectively normalized the expression of AR target genes, and rescued the deficits in social preference, grooming behavior, and memory in male UBE3A-overexpressing mice, without affecting females. These findings suggest that AR and its signaling cascade play an essential role in mediating the sexually dimorphic changes in UBE3A-dependent ASD.

Increased gene dosage of RFWD2 causes autistic-like behaviors and aberrant synaptic formation and function in mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interactions, communication deficits and repetitive behaviors. A study of autistic human subjects has identified RFWD2 as a susceptibility gene for autism, and autistic patients have 3 copies of the RFWD2 gene. The role of RFWD2 as an E3 ligase in neuronal functions, and its contribution to the pathophysiology of ASD, remain unknown. We generated RFWD2 knockin mice to model the human autistic condition of high gene dosage of RFWD2. We found that heterozygous knockin (Rfwd2+/-) male mice exhibited the core symptoms of autism. Rfwd2+/- male mice showed deficits in social interaction and communication, increased repetitive and anxiety-like behavior, and spatial memory deficits, whereas Rfwd2+/- female mice showed subtle deficits in social communication and spatial memory but were normal in anxiety-like, repetitive, and social behaviors. These autistic-like behaviors in males were accompanied by a reduction in dendritic spine density and abnormal synaptic function on layer II/III pyramidal neurons in the prelimbic area of the medial prefrontal cortex (mPFC), as well as decreased expression of synaptic proteins. Impaired social behaviors in Rfwd2+/- male mice were rescued by the expression of ETV5, one of the major substrates of RFWD2, in the mPFC. These findings indicate an important role of RFWD2 in the pathogenesis of autism.

The role of glia in the dysregulation of neuronal spinogenesis in Ube3a-dependent ASD.

Overexpression of the Ube3a gene and the resulting increase in Ube3a protein are linked to autism spectrum disorder (ASD). However, the cellular and molecular processes underlying Ube3a-dependent ASD remain unclear. Using both male and female mice, we find that neurons in the somatosensory cortex of the Ube3a 2× Tg ASD mouse model display reduced dendritic spine density and increased immature filopodia density. Importantly, the increased gene dosage of Ube3a in astrocytes alone is sufficient to confer alterations in neurons as immature dendritic protrusions, as observed in primary hippocampal neuron cultures. We show that Ube3a overexpression in astrocytes leads to a loss of astrocyte-derived spinogenic protein, thrombospondin-2 (TSP2), due to a suppression of TSP2 gene transcription. By neonatal intraventricular injection of astrocyte-specific virus, we demonstrate that Ube3a overexpression in astrocytes in vivo results in a reduction in dendritic spine maturation in prelimbic cortical neurons, accompanied with autistic-like behaviors in mice. These findings reveal an astrocytic dominance in initiating ASD pathobiology at the neuronal and behavior levels. SIGNIFICANCE STATEMENT: Increased gene dosage of Ube3a is tied to autism spectrum disorders (ASDs), yet cellular and molecular alterations underlying autistic phenotypes remain unclear. We show that Ube3a overexpression leads to impaired dendritic spine maturation, resulting in reduced spine density and increased filopodia density. We find that dysregulation of spine development is not neuron autonomous, rather, it is mediated by an astrocytic mechanism. Increased gene dosage of Ube3a in astrocytes leads to reduced production of the spinogenic glycoprotein thrombospondin-2 (TSP2), leading to abnormalities in spines. Astrocyte-specific Ube3a overexpression in the brain in vivo confers dysregulated spine maturation concomitant with autistic-like behaviors in mice. These findings indicate the importance of astrocytes in aberrant neurodevelopment and brain function in Ube3a-depdendent ASD.

mRNA nuclear retention reduces AMPAR expression and promotes autistic behavior in UBE3A-overexpressing mice.

UBE3A is a common genetic factor in ASD etiology, and transgenic mice overexpressing UBE3A exhibit typical autistic-like behaviors. Because AMPA receptors (AMPARs) mediate most of the excitatory synaptic transmission in the brain, and synaptic dysregulation is considered one of the primary cellular mechanisms in ASD pathology, we investigate here the involvement of AMPARs in UBE3A-dependent ASD. We show that expression of the AMPAR GluA1 subunit is decreased in UBE3A-overexpressing mice, and that AMPAR-mediated neuronal activity is reduced. GluA1 mRNA is trapped in the nucleus of UBE3A-overexpressing neurons, suppressing GluA1 protein synthesis. Also, SARNP, an mRNA nuclear export protein, is downregulated in UBE3A-overexpressing neurons, causing GluA1 mRNA nuclear retention. Restoring SARNP levels not only rescues GluA1 mRNA localization and protein expression, but also normalizes neuronal activity and autistic behaviors in mice overexpressing UBE3A. These findings indicate that SARNP plays a crucial role in the cellular and behavioral phenotypes of UBE3A-induced ASD by regulating nuclear mRNA trafficking and protein translation of a key AMPAR subunit.

Heterozygous Nexmif female mice demonstrate mosaic NEXMIF expression, autism-like behaviors, and abnormalities in dendritic arborization and synaptogenesis.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a strong genetic basis. ASDs are commonly characterized by impairments in language, restrictive and repetitive behaviors, and deficits in social interactions. Although ASD is a highly heterogeneous disease with many different genes implicated in its etiology, many ASD-associated genes converge on common cellular defects, such as aberrant neuronal morphology and synapse dysregulation. Our previous work revealed that, in mice, complete loss of the ASD-associated X-linked gene NEXMIF results in a reduction in dendritic complexity, a decrease in spine and synapse density, altered synaptic transmission, and ASD-like behaviors. Interestingly, human females of NEXMIF haploinsufficiency have recently been reported to demonstrate autistic features; however, the cellular and molecular basis for this haploinsufficiency-caused ASD remains unclear. Here we report that in the brains of Nexmif± female mice, NEXMIF shows a mosaic pattern in its expression in neurons. Heterozygous female mice demonstrate behavioral impairments similar to those of knockout male mice. In the mosaic mixture of neurons from Nexmif± mice, cells that lack NEXMIF have impairments in dendritic arborization and spine development. Remarkably, the NEXMIF-expressing neurons from Nexmif± mice also demonstrate similar defects in dendritic growth and spine formation. These findings establish a novel mouse model of NEXMIF haploinsufficiency and provide new insights into the pathogenesis of NEXMIF-dependent ASD.

The interaction of Synapsin 2a and Synaptogyrin-3 regulates fear extinction in mice.

The mechanisms behind a lack of efficient fear extinction in some individuals are unclear. Here, by employing a principal components analysis-based approach, we differentiated the mice into extinction-resistant and susceptible groups. We determined that elevated synapsin 2a (Syn2a) in the infralimbic cortex (IL) to basolateral amygdala (BLA) circuit disrupted presynaptic orchestration, leading to an excitatory/inhibitory imbalance in the BLA region and causing extinction resistance. Overexpression or silencing of Syn2a levels in IL neurons replicated or alleviated behavioral, electrophysiological, and biochemical phenotypes in resistant mice. We further identified that the proline-rich domain H in the C-terminus of Syn2a was indispensable for the interaction with synaptogyrin-3 (Syngr3) and demonstrated that disrupting this interaction restored extinction impairments. Molecular docking revealed that ritonavir, an FDA-approved HIV drug, could disrupt Syn2a-Syngr3 binding and rescue fear extinction behavior in Syn2a-elevated mice. In summary, the aberrant elevation of Syn2a expression and its interaction with Syngr3 at the presynaptic site were crucial in fear extinction resistance, suggesting a potential therapeutic avenue for related disorders.

Senktide blocks aberrant RTN3 interactome to retard memory decline and tau pathology in social isolated Alzheimer's disease mice.

Sporadic or late-onset Alzheimer's disease (LOAD) accounts for more than 95% of Alzheimer's disease (AD) cases without any family history. Although genome-wide association studies have identified associated risk genes and loci for LOAD, numerous studies suggest that many adverse environmental factors, such as social isolation, are associated with an increased risk of dementia. However, the underlying mechanisms of social isolation in AD progression remain elusive. In the current study, we found that 7 days of social isolation could trigger pattern separation impairments and presynaptic abnormalities of the mossy fibre-CA3 circuit in AD mice. We also revealed that social isolation disrupted histone acetylation and resulted in the downregulation of 2 dentate gyrus (DG)-enriched miRNAs, which simultaneously target reticulon 3 (RTN3), an endoplasmic reticulum protein that aggregates in presynaptic regions to disturb the formation of functional mossy fibre boutons (MFBs) by recruiting multiple mitochondrial and vesicle-related proteins. Interestingly, the aggregation of RTN3 also recruits the PP2A B subunits to suppress PP2A activity and induce tau hyperphosphorylation, which, in turn, further elevates RTN3 and forms a vicious cycle. Finally, using an artificial intelligence-assisted molecular docking approach, we determined that senktide, a selective agonist of neurokinin3 receptors (NK3R), could reduce the binding of RTN3 with its partners. Moreover, application of senktide in vivo effectively restored DG circuit disorders in socially isolated AD mice. Taken together, our findings not only demonstrate the epigenetic regulatory mechanism underlying mossy fibre synaptic disorders orchestrated by social isolation and tau pathology but also reveal a novel potential therapeutic strategy for AD.

The autism spectrum disorder risk gene NEXMIF over-synchronizes hippocampal CA1 network and alters neuronal coding.

Mutations in autism spectrum disorder (ASD) risk genes disrupt neural network dynamics that ultimately lead to abnormal behavior. To understand how ASD-risk genes influence neural circuit computation during behavior, we analyzed the hippocampal network by performing large-scale cellular calcium imaging from hundreds of individual CA1 neurons simultaneously in transgenic mice with total knockout of the X-linked ASD-risk gene NEXMIF (neurite extension and migration factor). As NEXMIF knockout in mice led to profound learning and memory deficits, we examined the CA1 network during voluntary locomotion, a fundamental component of spatial memory. We found that NEXMIF knockout does not alter the overall excitability of individual neurons but exaggerates movement-related neuronal responses. To quantify network functional connectivity changes, we applied closeness centrality analysis from graph theory to our large-scale calcium imaging datasets, in addition to using the conventional pairwise correlation analysis. Closeness centrality analysis considers both the number of connections and the connection strength between neurons within a network. We found that in wild-type mice the CA1 network desynchronizes during locomotion, consistent with increased network information coding during active behavior. Upon NEXMIF knockout, CA1 network is over-synchronized regardless of behavioral state and fails to desynchronize during locomotion, highlighting how perturbations in ASD-implicated genes create abnormal network synchronization that could contribute to ASD-related behaviors.

Tau pathology epigenetically remodels the neuron-glial cross-talk in Alzheimer's disease.

The neuron-glia cross-talk is critical to brain homeostasis and is particularly affected by neurodegenerative diseases. How neurons manipulate the neuron-astrocyte interaction under pathological conditions, such as hyperphosphorylated tau, a pathological hallmark in Alzheimer's disease (AD), remains elusive. In this study, we identified excessively elevated neuronal expression of adenosine receptor 1 (Adora1 or A1R) in 3×Tg mice, MAPT P301L (rTg4510) mice, patients with AD, and patient-derived neurons. The up-regulation of A1R was found to be tau pathology dependent and posttranscriptionally regulated by Mef2c via miR-133a-3p. Rebuilding the miR-133a-3p/A1R signal effectively rescued synaptic and memory impairments in AD mice. Furthermore, neuronal A1R promoted the release of lipocalin 2 (Lcn2) and resulted in astrocyte activation. Last, silencing neuronal Lcn2 in AD mice ameliorated astrocyte activation and restored synaptic plasticity and learning/memory. Our findings reveal that the tau pathology remodels neuron-glial cross-talk and promotes neurodegenerative progression. Approaches targeting A1R and modulating this signaling pathway might be a potential therapeutic strategy for AD.

CIP2A deficiency promotes depression-like behaviors in mice through inhibition of dendritic arborization.

Major depressive disorder (MDD) is a severe mental illness. Decreased brain plasticity and dendritic fields have been consistently found in MDD patients and animal models; however, the underlying molecular mechanisms remain to be clarified. Here, we demonstrate that the deletion of cancerous inhibitor of PP2A (CIP2A), an endogenous inhibitor of protein phosphatase 2A (PP2A), leads to depression-like behaviors in mice. Hippocampal RNA sequencing analysis of CIP2A knockout mice shows alterations in the PI3K-AKT pathway and central nervous system development. In primary neurons, CIP2A stimulates AKT activity and promotes dendritic development. Further analysis reveals that the effect of CIP2A in promoting dendritic development is dependent on PP2A-AKT signaling. In vivo, CIP2A deficiency-induced depression-like behaviors and impaired dendritic arborization are rescued by AKT activation. Decreased CIP2A expression and impaired dendrite branching are observed in a mouse model of chronic unpredictable mild stress (CUMS). Indicative of clinical relevance to humans, CIP2A expression is found decreased in transcriptomes from MDD patients. In conclusion, we discover a novel mechanism that CIP2A deficiency promotes depression through the regulation of PP2A-AKT signaling and dendritic arborization.

Regulatory Effects of Gradient Microtopographies on Synapse Formation and Neurite Growth in Hippocampal Neurons.

Among approaches aiming toward functional nervous system restoration, those implementing microfabrication techniques allow the manufacture of platforms with distinct geometry where neurons can develop and be guided to form patterned connections in vitro. The interplay between neuronal development and the microenvironment, shaped by the physical limitations, remains largely unknown. Therefore, it is crucial to have an efficient way to quantify neuronal morphological changes induced by physical or contact guidance of the microenvironment. In this study, we first devise and assess a method to prepare anisotropic, gradient poly(dimethylsiloxane) micro-ridge/groove arrays featuring variable local pattern width. We then demonstrate the ability of this single substrate to simultaneously profile the morphologcial and synaptic connectivity changes of primary cultured hippocampal neurons reacting to variable physical conditons, throughout neurodevelopment, in vitro. The gradient microtopography enhanced adhesion within microgrooves, increasing soma density with decreasing pattern width. Decreasing pattern width also reduced dendritic arborization and increased preferential axon growth. Finally, decreasing pattern geometry inhibited presynaptic puncta architecture. Collectively, a method to examine structural development and connectivity in response to physical stimuli is established, and potentially provides insight into microfabricated geometries which promote neural regeneration and repair.

Role of the DUB enzyme USP7 in dendritic arborization, neuronal migration, and autistic-like behaviors in mice.

Duplication and haploinsufficiency of the USP7 gene are implicated in autism spectrum disorders (ASD), but the role for USP7 in neurodevelopment and contribution to ASD pathogenesis remain unknown. We find that in primary neurons, overexpression of USP7 increases dendritic branch number and total dendritic length, whereas knockdown leads to opposite alterations. Besides, USP7 deubiquitinates the X-linked inhibitor of apoptosis protein (XIAP). The USP7-induced increase in XIAP suppresses caspase 3 activity, leading to a reduction in tubulin cleavage and suppression of dendritic pruning. When USP7 is introduced into the brains of prenatal mice via in utero electroporation (IUE), it results in abnormal migration of newborn neurons and increased dendritic arborization. Importantly, intraventricular brain injection of AAV-USP7 in P0 mice leads to autistic-like phenotypes including aberrant social interactions, repetitive behaviors, as well as changes in somatosensory sensitivity. These findings provide new insights in USP7-related neurobiological functions and its implication in ASD.

Prkn knockout mice show autistic-like behaviors and aberrant synapse formation.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with high genetic heterogeneity, affecting one in 44 children in the United States. Recent genomic sequencing studies from autistic human individuals indicate that PARK2, a gene that has long been considered in the pathogenesis of Parkinson's disease, is involved in ASD. Here, we report that Prkn knockout (KO) mice demonstrate autistic-like behaviors including impaired social interaction, elevated repetitive behaviors, and deficits in communication. In addition, Prkn KO mice show reduced neuronal activity in the context of sociability in the prelimbic cortex. Cell morphological examination of layer 5 prelimbic cortical neurons shows a reduction in dendritic arborization and spine number. Furthermore, biochemistry and immunocytochemistry analyses reveal alterations in synapse density and the molecular composition of synapses. These findings indicate that Prkn is implicated in brain development and suggest the potential use of the Prkn KO mouse as a model for autism research.

Social isolation reinforces aging-related behavioral inflexibility by promoting neuronal necroptosis in basolateral amygdala.

Aging is characterized with a progressive decline in many cognitive functions, including behavioral flexibility, an important ability to respond appropriately to changing environmental contingencies. However, the underlying mechanisms of impaired behavioral flexibility in aging are not clear. In this study, we reported that necroptosis-induced reduction of neuronal activity in the basolateral amygdala (BLA) plays an important role in behavioral inflexibility in 5-month-old mice of the senescence-accelerated mice prone-8 (SAMP8) line, a well-established model with age-related phenotypes. Application of Nec-1s, a specific inhibitor of necroptosis, reversed the impairment of behavioral flexibility in SAMP8 mice. We further observed that the loss of glycogen synthase kinase 3α (GSK-3α) was strongly correlated with necroptosis in the BLA of aged mice and the amygdala of aged cynomolgus monkeys (Macaca fascicularis). Moreover, genetic deletion or knockdown of GSK-3α led to the activation of necroptosis and impaired behavioral flexibility in wild-type mice, while the restoration of GSK-3α expression in the BLA arrested necroptosis and behavioral inflexibility in aged mice. We further observed that GSK-3α loss resulted in the activation of mTORC1 signaling to promote RIPK3-dependent necroptosis. Importantly, we discovered that social isolation, a prevalent phenomenon in aged people, facilitated necroptosis and behavioral inflexibility in 4-month-old SAMP8 mice. Overall, our study not only revealed the molecular mechanisms of the dysfunction of behavioral flexibility in aged people but also identified a critical lifestyle risk factor and a possible intervention strategy.

Large-scale voltage imaging in behaving mice using targeted illumination.

Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold transmembrane voltage in vivo. To perform high-speed voltage imaging of many neurons simultaneously over a large anatomical area, widefield microscopy remains an essential tool. However, the lack of optical sectioning makes widefield microscopy prone to background cross-contamination. We implemented a digital-micromirror-device-based targeted illumination strategy to restrict illumination to the cells of interest and quantified the resulting improvement both theoretically and experimentally with SomArchon expressing neurons. We found that targeted illumination increased SomArchon signal contrast, decreased photobleaching, and reduced background cross-contamination. With the use of a high-speed, large-area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in behaving mice. Thus, the targeted illumination strategy described here offers a simple solution for widefield voltage imaging of many neurons over a large field of view in behaving animals.

Non-genetic photoacoustic stimulation of single neurons by a tapered fiber optoacoustic emitter.

Neuromodulation at high spatial resolution poses great significance in advancing fundamental knowledge in the field of neuroscience and offering novel clinical treatments. Here, we developed a tapered fiber optoacoustic emitter (TFOE) generating an ultrasound field with a high spatial precision of 39.6 µm, enabling optoacoustic activation of single neurons or subcellular structures, such as axons and dendrites. Temporally, a single acoustic pulse of sub-microsecond converted by the TFOE from a single laser pulse of 3 ns is shown as the shortest acoustic stimuli so far for successful neuron activation. The precise ultrasound generated by the TFOE enabled the integration of the optoacoustic stimulation with highly stable patch-clamp recording on single neurons. Direct measurements of the electrical response of single neurons to acoustic stimulation, which is difficult for conventional ultrasound stimulation, have been demonstrated. By coupling TFOE with ex vivo brain slice electrophysiology, we unveil cell-type-specific responses of excitatory and inhibitory neurons to acoustic stimulation. These results demonstrate that TFOE is a non-genetic single-cell and sub-cellular modulation technology, which could shed new insights into the mechanism of ultrasound neurostimulation.

RTP801 regulates motor cortex synaptic transmission and learning.

BACKGROUND: RTP801/REDD1 is a stress-regulated protein whose upregulation is necessary and sufficient to trigger neuronal death in in vitro and in vivo models of Parkinson's and Huntington's diseases and is up regulated in compromised neurons in human postmortem brains of both neurodegenerative disorders. Indeed, in both Parkinson's and Huntington's disease mouse models, RTP801 knockdown alleviates motor-learning deficits. RESULTS: We investigated the physiological role of RTP801 in neuronal plasticity and we found RTP801 in rat, mouse and human synapses. The absence of RTP801 enhanced excitatory synaptic transmission in both neuronal cultures and brain slices from RTP801 knock-out (KO) mice. Indeed, RTP801 KO mice showed improved motor learning, which correlated with lower spine density but increased basal filopodia and mushroom spines in the motor cortex layer V. This paralleled with higher levels of synaptosomal GluA1 and TrkB receptors in homogenates derived from KO mice motor cortex, proteins that are associated with synaptic strengthening. CONCLUSIONS: Altogether, these results indicate that RTP801 has an important role modulating neuronal plasticity and motor learning. They will help to understand its role in neurodegenerative disorders where RTP801 levels are detrimentally upregulated.