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Autism spectrum disorders (ASDs) are considered neural dysconnectivity syndromes. To better understand ASD and uncover potential treatments, it is imperative to know and dissect the connectivity deficits under conditions of autism. Here, we apply a whole-brain immunostaining and quantification platform to demonstrate impaired structural and functional connectivity and aberrant whole-brain synchronization in a Tbr1+/- autism mouse model. We express a channelrhodopsin variant oChIEF fused with Citrine at the basolateral amygdala (BLA) to outline the axonal projections of BLA neurons. By activating the BLA under blue light theta-burst stimulation (TBS), we then evaluate the effect of BLA activation on C-FOS expression at a whole brain level to represent neural activity. We show that Tbr1 haploinsufficiency almost completely disrupts contralateral BLA axonal projections and results in mistargeting in both ipsilateral and contralateral hemispheres, thereby globally altering BLA functional connectivity. Based on correlated C-FOS expression among brain regions, we further show that Tbr1 deficiency severely disrupts whole-brain synchronization in the absence of salient stimulation. Tbr1+/- and wild-type (WT) mice exhibit opposing responses to TBS-induced amygdalar activation, reducing synchronization in WT mice but enhancing it in Tbr1+/- mice. Whole-brain modular organization and intermodule connectivity are also affected by Tbr1 deficiency and amygdalar activation. Following BLA activation by TBS, the synchronizations of the whole brain and the default mode network, a specific subnetwork highly relevant to ASD, are enhanced in Tbr1+/- mice, implying a potential ameliorating effect of amygdalar stimulation on brain function. Indeed, TBS-mediated BLA activation increases nose-to-nose social interactions of Tbr1+/- mice, strengthening evidence for the role of amygdalar connectivity in social behaviors. Our high-resolution analytical platform reveals the inter- and intrahemispheric connectopathies arising from ASD. Our study emphasizes the defective synchronization at a whole-brain scale caused by Tbr1 deficiency and implies a potential beneficial effect of deep brain stimulation at the amygdala for TBR1-linked autism.

Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.

Dendritic spines, the tiny and actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain that control synaptic activity and plasticity. Dendritic spines contain a specialized form of endoplasmic reticulum (ER), i.e., the spine apparatus, required for local calcium signaling and that is involved in regulating dendritic spine enlargement and synaptic plasticity. Many autism-linked genes have been shown to play critical roles in synaptic formation and plasticity. Among them, KLHL17 is known to control dendritic spine enlargement during development. As a brain-specific disease-associated gene, KLHL17 is expected to play a critical role in the brain, but it has not yet been well characterized. In this study, we report that KLHL17 expression in mice is strongly regulated by neuronal activity and KLHL17 modulates the synaptic distribution of synaptopodin (SYNPO), a marker of the spine apparatus. Both KLHL17 and SYNPO are F-actin-binding proteins linked to autism. SYNPO is known to maintain the structure of the spine apparatus in mature spines and contributes to synaptic plasticity. Our super-resolution imaging using expansion microscopy demonstrates that SYNPO is indeed embedded into the ER network of dendritic spines and that KLHL17 is closely adjacent to the ER/SYNPO complex. Using mouse genetic models, we further show that Klhl17 haploinsufficiency and knockout result in fewer dendritic spines containing ER clusters and an alteration of calcium events at dendritic spines. Accordingly, activity-dependent dendritic spine enlargement and neuronal activation (reflected by extracellular signal-regulated kinase (ERK) phosphorylation and C-FOS expression) are impaired. In addition, we show that the effect of disrupting the KLHL17 and SYNPO association is similar to the results of Klhl17 haploinsufficiency and knockout, further strengthening the evidence that KLHL17 and SYNPO act together to regulate synaptic plasticity. In conclusion, our findings unravel a role for KLHL17 in controlling synaptic plasticity via its regulation of SYNPO and synaptic ER clustering and imply that impaired synaptic plasticity contributes to the etiology of KLHL17-related disorders.

Summary: Synaptopathy, which encompasses morphological deficits and any abnormal protein distribution of synapses, is a critical feature of many neurological diseases. We here provide a protocol using mice stably expressing a Thy1-YFP transgene to assess synaptic features in vivo. We describe steps for recording the entire morphology of projection neurons using confocal microscopy based on YFP signals. We detail assessment of the density and size of dendritic spines and the distributions of synaptic proteins using ImageJ and statistical analysis using Prism.For complete details on the use and execution of this protocol, please refer to Shih et al. (2020).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Since SARM1 mutations have been identified in human neurological disease, SARM1 inhibition has become an attractive therapeutic strategy to preserve axons in a variety of disorders of the peripheral (PNS) and central nervous system (CNS). While SARM1 has been extensively studied in neurons, it remains unknown whether SARM1 is present and functional in myelinating glia? This is an important question to address. Firstly, to identify whether SARM1 dysfunction in other cell types in the nervous system may contribute to neuropathology in SARM1 dependent diseases? Secondly, to ascertain whether therapies altering SARM1 function may have unintended deleterious impacts on PNS or CNS myelination? Surprisingly, we find that oligodendrocytes express sarm1 mRNA in the zebrafish spinal cord and that SARM1 protein is readily detectable in rodent oligodendrocytes in vitro and in vivo. Furthermore, activation of endogenous SARM1 in cultured oligodendrocytes induces rapid cell death. In contrast, in peripheral glia, SARM1 protein is not detectable in Schwann cells and satellite glia in vivo and sarm1/Sarm1 mRNA is detected at very low levels in Schwann cells, in vivo, in zebrafish and mouse. Application of specific SARM1 activators to cultured mouse Schwann cells does not induce cell death and nicotinamide adenine dinucleotide (NAD) levels remain unaltered suggesting Schwann cells likely contain no functionally relevant levels of SARM1. Finally, we address the question of whether SARM1 is required for myelination or myelin maintenance. In the zebrafish and mouse PNS and CNS, we show that SARM1 is not required for initiation of myelination and myelin sheath maintenance is unaffected in the adult mouse nervous system. Thus, strategies to inhibit SARM1 function to treat neurological disease are unlikely to perturb myelination in humans.

Autism impacts synapses. This study reports that autism-linked mutations of CTTNBP2 regulate phase separation to control synaptic enrichment of that protein. A zinc-induced liquid-to-gel transition improves synaptic retention of CTTNBP2 and SHANK3.

Abstract Background Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by a dyad of behavioural symptoms—social and communication deficits and repetitive behaviours. Multiple aetiological genetic and environmental factors have been identified as causing or increasing the likelihood of ASD, including serum zinc deficiency. Our previous studies revealed that dietary zinc supplementation can normalise impaired social behaviours, excessive grooming, and heightened anxiety in a Shank3 mouse model of ASD, as well as the amelioration of synapse dysfunction. Here, we have examined the efficacy and breadth of dietary zinc supplementation as an effective therapeutic strategy utilising a non-Shank-related mouse model of ASD—mice with Tbr1 haploinsufficiency. Methods We performed behavioural assays, amygdalar slice whole-cell patch-clamp electrophysiology, and immunohistochemistry to characterise the synaptic mechanisms underlying the ASD-associated behavioural deficits observed in Tbr1 +/− mice and the therapeutic potential of dietary zinc supplementation. Two-way analysis of variance (ANOVA) with Šídák's post hoc test and one-way ANOVA with Tukey’s post hoc multiple comparisons were performed for statistical analysis. Results Our data show that dietary zinc supplementation prevents impairments in auditory fear memory and social interaction, but not social novelty, in the Tbr1 +/− mice. Tbr1 haploinsufficiency did not induce excessive grooming nor elevate anxiety in mice. At the synaptic level, dietary zinc supplementation reversed α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-d-aspartate receptor (NMDAR) hypofunction and normalised presynaptic function at thalamic-lateral amygdala (LA) synapses that are crucial for auditory fear memory. In addition, the zinc supplemented diet significantly restored the synaptic puncta density of the GluN1 subunit essential for functional NMDARs as well as SHANK3 expression in both the basal and lateral amygdala (BLA) of Tbr1 +/− mice. Limitations The therapeutic effect of dietary zinc supplementation observed in rodent models may not reproduce the same effects in human patients. The effect of dietary zinc supplementation on synaptic function in other brain structures affected by Tbr1 haploinsufficiency including olfactory bulb and anterior commissure will also need to be examined. Conclusions Our data further the understanding of the molecular mechanisms underlying the effect of dietary zinc supplementation and verify the efficacy and breadth of its application as a potential treatment strategy for ASD.

Abstract Background Dendritic spines, the actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain. Many actin-regulating molecules modulate dendritic spine morphology. Since dendritic spines are neuron-specific structures, it is reasonable to speculate that neuron-specific or -predominant factors are involved in dendritic spine formation. KLHL17 (Kelch-like 17, also known as Actinfilin), an actin-binding protein, is predominantly expressed in brain. Human genetic study has indicated an association of KLHL17/Actinfilin with infantile spasms, a rare form of childhood epilepsy also resulting in autism and mental retardation, indicating that KLHL17/Actinfilin plays a role in neuronal function. However, it remains elusive if and how KLHL17/Actinfilin regulates neuronal development and brain function. Methods Fluorescent immunostaining and electrophysiological recording were performed to evaluate dendritic spine formation and activity in cultured hippocampal neurons. Knockdown and knockout of KLHL17/Actinfilin and expression of truncated fragments of KLHL17/Actinfilin were conducted to investigate the function of KLHL17/Actinfilin in neurons. Mouse behavioral assays were used to evaluate the role of KLHL17/Actinfilin in brain function. Results We found that KLHL17/Actinfilin tends to form circular puncta in dendritic spines and are surrounded by or adjacent to F-actin. Klhl17 deficiency impairs F-actin enrichment at dendritic spines. Knockdown and knockout of KLHL17/Actinfilin specifically impair dendritic spine enlargement, but not the density or length of dendritic spines. Both N-terminal Broad-Complex, Tramtrack and Bric-a-brac (BTB) domain and C-terminal Kelch domains of KLHL17/Actinfilin are required for F-actin remodeling and enrichment at dendritic spines, as well as dendritic spine enlargement. A reduction of postsynaptic and presynsptic markers at dendritic spines and altered mEPSC profiles due to Klhl17 deficiency evidence impaired synaptic activity in Klhl17-deficient neurons. Our behavioral assays further indicate that Klhl17 deficiency results in hyperactivity and reduced social interaction, strengthening evidence for the physiological role of KLHL17/Actinfilin. Conclusion Our findings provide evidence that KLHL17/Actinfilin modulates F-actin remodeling and contributes to regulation of neuronal morphogenesis, maturation and activity, which is likely relevant to behavioral impairment in Klhl17-deficient mice. Trial registration Non-applicable.

Abstract Abnormal synaptic formation and signaling is one of the key molecular features of autism spectrum disorders (ASD). Cortactin binding protein 2 (CTTNBP2), an ASD-linked gene, is known to regulate the subcellular distribution of synaptic proteins, such as cortactin, thereby controlling dendritic spine formation and maintenance. However, it remains unclear how ASD-linked mutations of CTTNBP2 influence its function. Here, using cultured hippocampal neurons and knockin mouse models, we screen seven ASD-linked mutations in the short form of the Cttnbp2 gene and identify that M120I, R533* and D570Y mutations impair CTTNBP2 protein–protein interactions via divergent mechanisms to reduce dendritic spine density in neurons. R533* mutation impairs CTTNBP2 interaction with cortactin due to lack of the C-terminal proline-rich domain. Through an N–C terminal interaction, M120I mutation at the N-terminal region of CTTNBP2 also negatively influences cortactin interaction. D570Y mutation increases the association of CTTNBP2 with microtubule, resulting in a dendritic localization of CTTNBP2, consequently reducing the distribution of CTTNBP2 in dendritic spines and impairing the synaptic function of CTTNBP2. Finally, we generated heterozygous M120I knockin mice to mimic the genetic variation of patients and found they exhibit reduced social interaction. Our study elucidates that different ASD-linked mutations of CTTNBP2 result in diverse molecular deficits, but all have the similar consequence of synaptic impairment.

Toll-like receptor (TLR) signaling is critical for defense against pathogenic infection, as well as for modulating tissue development. Activation of different TLRs triggers common inflammatory responses such as cytokine induction. Here, we reveal differential impacts of TLR3 and TLR7 signaling on transcriptomic profiles in bone marrow-derived macrophages (BMDMs). Apart from self-regulation, TLR3, but not TLR7, induced expression of other TLRs, suggesting that TLR3 activation globally enhances innate immunity. Moreover, we observed diverse influences of TLR3 and TLR7 signaling on genes involved in methylation, caspase and autophagy pathways. We compared endogenous TLR3 and TLR7 by using CRISPR/Cas9 technology to knock in a dual Myc-HA tag at the 3’ ends of mouse Tlr3 and Tlr7. Using anti-HA antibodies to detect endogenous tagged TLR3 and TLR7, we found that both TLRs display differential tissue expression and posttranslational modifications. C-terminal tagging did not impair TLR3 activity. However, it disrupted the interaction between TLR7 and myeloid differentiation primary response 88 (MYD88), the Tir domain-containing adaptor of TLR7, which blocked its downstream signaling necessary to trigger cytokine and chemokine expression. Our study demonstrates different properties for TLR3 and TLR7, and also provides useful mouse models for further investigation of these two RNA-sensing TLRs.

Abstract Toll-like receptors (TLRs) are well known as critical pattern recognition receptors that trigger innate immune responses. In addition, TLRs are expressed in neurons and may act as the gears in the neuronal detection/alarm system for making good connections. As neuronal differentiation and circuit formation take place along with programmed cell death, neurons face the challenge of connecting with appropriate targets while avoiding dying or dead neurons. Activation of neuronal TLR3, TLR7 and TLR8 with nucleic acids negatively modulates neurite outgrowth and alters synapse formation in a cell-autonomous manner. It consequently influences neural connectivity and brain function and leads to deficits related to neuropsychiatric disorders. Importantly, neuronal TLR activation does not simply duplicate the downstream signal pathways and effectors of classical innate immune responses. The differences in spatial and temporal expression of TLRs and their ligands likely account for the diverse signaling pathways of neuronal TLRs. In conclusion, the accumulated evidence strengthens the idea that the innate immune system of neurons serves as an alarm system that responds to exogenous pathogens as well as intrinsic danger signals and fine-tune developmental processes of neurons.

Abstract Background Autism spectrum disorders (ASD) exhibit two clusters of core symptoms, i.e., social and communication impairment, and repetitive behaviors and sensory abnormalities. Our previous study demonstrated that TBR1, a causative gene of ASD, controls axonal projection and neuronal activation of amygdala and regulates social interaction and vocal communication in a mouse model. Behavioral defects caused by Tbr1 haploinsufficiency can be ameliorated by increasing neural activity via D-cycloserine treatment, an N-methyl-D-aspartate receptor (NMDAR) coagonist. In this report, we investigate the role of TBR1 in regulating olfaction and test whether D-cycloserine can also improve olfactory defects in Tbr1 mutant mice. Methods We used Tbr1 +/− mice as a model to investigate the function of TBR1 in olfactory sensation and discrimination of non-social odors. We employed a behavioral assay to characterize the olfactory defects of Tbr1 +/− mice. Magnetic resonance imaging (MRI) and histological analysis were applied to characterize anatomical features. Immunostaining was performed to further analyze differences in expression of TBR1 subfamily members (namely TBR1, TBR2, and TBX21), interneuron populations, and dendritic abnormalities in olfactory bulbs. Finally, C-FOS staining was used to monitor neuronal activation of the olfactory system upon odor stimulation. Results Tbr1 +/− mice exhibited smaller olfactory bulbs and anterior commissures, reduced interneuron populations, and an abnormal dendritic morphology of mitral cells in the olfactory bulbs. Tbr1 haploinsufficiency specifically impaired olfactory discrimination but not olfactory sensation. Neuronal activation upon odorant stimulation was reduced in the glomerular layer of Tbr1 +/− olfactory bulbs. Furthermore, although the sizes of piriform and perirhinal cortices were not affected by Tbr1 deficiency, neuronal activation was reduced in these two cortical regions in response to odorant stimulation. These results suggest an impairment of neuronal activation in olfactory bulbs and defective connectivity from olfactory bulbs to the upper olfactory system in Tbr1 +/− mice. Systemic administration of D-cycloserine, an NMDAR co-agonist, ameliorated olfactory discrimination in Tbr1 +/− mice, suggesting that increased neuronal activity has a beneficial effect on Tbr1 deficiency. Conclusions Tbr1 regulates neural circuits and activity in the olfactory system to control olfaction. Tbr1 +/− mice can serve as a suitable model for revealing how an autism causative gene controls neuronal circuits, neural activity, and autism-related behaviors.

Summary: Voluntary exercise is well known to benefit brain performance. In contrast, forced exercise induces inflammation-related stress responses and may cause psychiatric disorders. Here, we unexpectedly found that rotarod testing, a frequently applied assay for evaluating rodent motor coordination, induces anxiety and alters spatial learning/memory performance of mice. Rotarod testing upregulated genes involved in the unfolded protein response and stress responses and downregulated genes associated with neurogenesis and neuronal differentiation. It impacts two downstream pathways. The first is the IL-6-dependent pathway, which mediates rotarod-induced anxiety. The second is the Toll-like receptor 7 (TLR7)-dependent pathway, which is involved in the effect of rotarod exercise on gene expression and its impact on contextual learning and memory of mice. Thus, although rotarod exercise does not induce systemic inflammation, it influences innate immunity-related responses in the brain, controls gene expression and, consequently, regulates anxiety and contextual learning and memory.

Enterovirus 71 (EV71) is a positive single-stranded RNA (ssRNA) virus from the enterovirus genus of Picornaviridae family and causes diseases ranged from the mild disease of hand, foot and mouth disease (HFMD) to the severe disease of neurological involvement in young children. TLR7 is an intracellular pattern recognition receptor (PRR) recognizing viral ssRNA. In this study, we investigated the role of TLR7 in EV71 infection in mouse pups (10-12 days old) and found that wild-type (WT) and TLR7 knock-out (TLR7KO) mice infected with EV71 showed similar limb paralysis at the onset and peak of the disease, comparable loss of motor neurons, and similar levels of antiviral molecules in the spinal cord. These results suggest that TLR7 is not the absolute PRR for EV71 in the spinal cord. Interestingly, TLR7KO mice infected with EV71 exhibited significantly delayed recovery from limb paralysis compared with WT mice. TLR7KO mice infected with EV71 showed significantly decreased levels of IgM and IgG2, important antibodies for antiviral humoral immunity. Furthermore, TLR7KO mice infected with EV71 showed a decrease of germinal center B cells in the spleen compared with WT mice. Altogether, our study suggests that TLR7 plays a critical role in anti-viral humoral immunity rather than in being a PRR in the spinal cord during EV71 infection in young mice.

Summary: Both genetic variations and nutritional deficiency are associated with autism spectrum disorders and other neurological disorders. However, it is less clear whether or how nutritional deficiency and genetic variations influence each other under pathogenic conditions. “Valosin-containing protein” (VCP, also known as p97) is associated with multiple neurological disorders and regulates dendritic spine formation by controlling endoplasmic reticulum formation and protein synthesis efficiency. Increased protein synthesis ameliorates the dendritic spine defects of Vcp-deficient neurons. Therefore, we investigated if Vcp-deficient mice are sensitive to nutritional conditions. Here, we show that social interaction and contextual memory of Vcp-deficient mice are indeed influenced by different dietary protein levels. Moreover, leucine supplementation ameliorates the behavioral deficits and dendritic spine density of Vcp-deficient mice, strengthening evidence for the role of protein synthesis in VCP function. Our study illustrates that genetic variation and nutrient factors cross-talk to influence neuronal and behavioral phenotypes.

Summary: Synaptic dysregulation is a critical feature of autism spectrum disorders (ASDs). Among various autism-associated genes, cortactin binding protein 2 (CTTNBP2) is a cytoskeleton regulator predominantly expressed in neurons and highly enriched at dendritic spines. Here, using Cttnbp2 knockout and ASD-linked mutant mice, we demonstrate that Cttnbp2 deficiency reduces zinc levels in the brain, alters synaptic protein targeting, impairs dendritic spine formation and ultrastructure of postsynaptic density, and influences neuronal activation and autism-like behaviors. A link to autism, the NMDAR-SHANK pathway, and zinc-related regulation are three features shared by CTTNBP2-regulated synaptic proteins. Zinc supplementation rescues the synaptic expression of CTTNBP2-regulated proteins. Moreover, zinc supplementation and administration of D-cycloserine, an NMDAR coagonist, improve the social behaviors of Cttnbp2-deficient mice. We suggest that CTTNBP2 controls the synaptic expression of a set of zinc-regulated autism-associated genes and influences NMDAR function and signaling, providing an example of how genetic and environmental factor crosstalk controls social behaviors.

Summary: Neurofibromatosis type 1 (NF1) is a dominant genetic disorder manifesting, in part, as cognitive defects. Previous study indicated that neurofibromin (NF1 protein) interacts with valosin-containing protein (VCP)/P97 to control dendritic spine formation, but the mechanism is unknown. Here, using Nf1+/– mice and transgenic mice overexpressing wild-type Vcp/p97, we demonstrate that neurofibromin acts with VCP to control endoplasmic reticulum (ER) formation and consequent protein synthesis and regulates dendritic spine formation, thereby modulating contextual fear memory and social interaction. To validate the role of protein synthesis, we perform leucine supplementation in vitro and in vivo. Our results suggest that leucine can effectively enter the brain and increase protein synthesis and dendritic spine density of Nf1+/– neurons. Contextual memory and social behavior of Nf1+/– mice are also restored by leucine supplementation. Our study suggests that the “ER-protein synthesis” pathway downstream of neurofibromin and VCP is a critical regulator of dendritic spinogenesis and brain function.

The two hemispheres of the vertebrate brain are connected through several commissures. Although the anterior commissure (AC) is the most conserved white matter structure in the brains of different vertebrates, its complete physiological functionality remains elusive. Since the AC is involved in the connection between two amygdalae and because amygdalae are critical for emotional behaviors and social interaction, we assessed amygdalar activity and function to investigate the physiological role of the AC. We first performed ex vivo electrophysiological recording on mouse brains to demonstrate that the AC delivers a positive signal to facilitate synaptic responses and to recruit basolateral amygdalar neurons via glutamatergic synapses. Transection was then undertaken to investigate the role of the AC in vivo. Results from in vivo optogenetic stimulation suggest that AC transection impairs mutual activation between two basolateral amygdalae. Behavioral analyses were then used to assess if AC surgical lesioning results in hyperactivity, anxiety, social reduction or learning/memory impairment, which are behavioral features associated with neuropsychiatric disorders, such as autism spectrum disorders. We found that AC transection results in higher locomotor activity, aberrant social interaction and reduced associative memory, but not anxiety. Moreover, systemic administration of D-cycloserine, a coagonist of N-methyl-D-aspartate receptor, ameliorated auditory fear memory in AC-transected mice, reinforcing our evidence that the AC potentiates the activity of basolateral amygdalae. Our study suggests that the AC regulates basolateral amygdalar activity and influences neuropsychiatry-related behaviors in mice.

Abstract The endoplasmic reticulum (ER) is the biggest organelle in cells and is involved in versatile cellular processes. Formation and maintenance of ER morphology are regulated by a series of proteins controlling membrane fusion and curvature. At least six different ER morphology regulators have been demonstrated to be involved in neurological disorders—including Valosin-containing protein (VCP), Atlastin-1 (ATL1), Spastin (SPAST), Reticulon 2 (RTN2), Receptor expression enhancing protein 1 (REEP1) and RAB10—suggesting a critical role of ER formation in neuronal activity and function. Among these genes, mutations in VCP gene involve in inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD), familial amyotrophic lateral sclerosis (ALS), autism spectrum disorders (ASD), and hereditary spastic paraplegia (HSP). ATL1 is also one of causative genes of HSP. RAB10 is associated with Parkinson’s disease (PD). A recent study showed that VCP and ATL1 work together to regulate dendritic spine formation by controlling ER formation and consequent protein synthesis efficiency. RAB10 shares the same function with VCP and ATL1 to control ER formation and protein synthesis efficiency but acts independently. Increased protein synthesis by adding extra leucine to cultured neurons ameliorated dendritic spine deficits caused by VCP and ATL1 deficiencies, strengthening the significance of protein synthesis in VCP- and ATL1-regulated dendritic spine formation. These findings provide new insight into the roles of ER and protein synthesis in controlling dendritic spine formation and suggest a potential etiology of neurodegenerative disorders caused by mutations in VCP, ATL1 and other genes encoding proteins regulating ER formation and morphogenesis.

Summary: Impaired interhemispheric connectivity is commonly found in various psychiatric disorders, although how interhemispheric connectivity regulates brain function remains elusive. Here, we use the mouse amygdala, a brain region that is critical for social interaction and fear memory, as a model to demonstrate that contralateral connectivity intensifies the synaptic response of basolateral amygdalae (BLA) and regulates amygdala-dependent behaviors. Retrograde tracing and c-FOS expression indicate that contralateral afferents widely innervate BLA non-randomly and that some BLA neurons innervate both contralateral BLA and the ipsilateral central amygdala (CeA). Our optogenetic and electrophysiological studies further suggest that contralateral BLA input results in the synaptic facilitation of BLA neurons, thereby intensifying the responses to cortical and thalamic stimulations. Finally, pharmacological inhibition and chemogenetic disconnection demonstrate that BLA contralateral facilitation is required for social interaction and memory. Our study suggests that interhemispheric connectivity potentiates the synaptic dynamics of BLA neurons and is critical for the full activation and functionality of amygdalae. : Huang et al. show that contralateral innervation between two basolateral amygdalae (BLA) in the two brain hemispheres evokes the synaptic activity of BLA and intensifies the BLA response to ipsilateral afferents, including cortical and thalamic inputs. BLA contralateral connectivity is required for amygdala-dependent behaviors, including social interaction and memory. Keywords: amygdala, anterior commissure, associative memory, contralateral innervation, social interaction, synaptic facilitation

Protein homeostasis is crucial for maintaining a variety of cellular functions. Here the authors show that valosin-containing protein and its cofactors regulate tubular ER formation and protein synthesis efficiency, thereby control dendritic spine formation in neurons.

Members of the ribonuclease A (RNase A) superfamily regulate various physiological processes. RNase A, the best-studied member of the RNase A superfamily, is widely expressed in different tissues, including brains. We unexpectedly found that RNase A can trigger proliferation of neuronal progenitor cells (NPC) both in vitro and in vivo. RNase A treatment induced cell proliferation in dissociated neuronal cultures and increased cell mass in neurosphere cultures. BrdU (5-Bromo-2'-Deoxyuridine) labeling confirmed the effect of RNase A on cell proliferation. Those dividing cells were Nestin- and SOX2-positive, suggesting that RNase A triggers NPC proliferation. The proliferation inhibitor Ara-C completely suppressed the effect of RNase A on NPC counts, further supporting that RNase A increases NPC number mainly by promoting proliferation. Moreover, we found that RNase A treatment increased ERK phosphorylation and blockade of the ERK pathway inhibited the effect of RNase A on NPC proliferation. Intracerebroventricular injection of RNase A into mouse brain increased the population of 5-ethynyl-2'-deoxyuridine (EdU) or BrdU-labeled cells in the subventricular zone. Those RNase A-induced NPCs were able to migrate into other brain areas, including hippocampus, amygdala, cortex, striatum, and thalamus. In conclusion, our study shows that RNase A promotes proliferation of NPCs via an ERK-dependent pathway and further diversifies the physiological functions of the RNase A family.

Plasmacytoid dendritic cells (pDCs) are a specialized subset of DCs capable of rapidly producing copious amounts of type I IFN (IFN-I) in response to viral infections. The mechanism regulating rapid production of IFN-I after pDCs are exposed to viral nucleic acids remains elusive. Here, we show that the transcription factor Blimp-1 is promptly induced in pDCs after exposure to TLR7 and TLR9 ligands via a unique Ras-related C3 botulinum toxin substrate (Rac)-mediated pathway. Deletion of the Prdm1 gene encoding Blimp-1 impaired production of IFN-I, but not other cytokines, upon viral infection or treatment with CpG DNA in pDCs. Accordingly, mice lacking Blimp-1 in DCs failed to produce IFN-I after CpG stimulation and did not mount proper antiviral responses following flavivirus infection. The development of pDCs in bone marrow as well as the induction of several activation markers, such as CD86, CD69, and MHCII, by CpG stimulation was generally not affected by the absence of Blimp-1. Mechanistically, we found that Blimp-1 controls the activation of IKKα and IRF7 by directly suppressing interleukin-1 receptor-associated kinase 3 (Irak3), a negative regulator of TLR signaling, in pDCs. Together, we identify a Blimp-1-dependent pathway that rapidly facilitates IFN-I production by relieving interleukin-1 receptor-associated kinase M, encoded by Irak3, in pDCs.

Dendritic spines are the location of excitatory synapses in the mammalian nervous system and are neuron-specific subcellular structures essential for neural circuitry and function. Dendritic spine morphology is determined by the F-actin cytoskeleton. F-actin remodeling must coordinate with different stages of dendritic spinogenesis, starting from dendritic filopodia formation to the filopodia-spines transition and dendritic spine maturation and maintenance. Hundreds of genes, including F-actin cytoskeleton regulators, membrane proteins, adaptor proteins, and signaling molecules, are known to be involved in regulating synapse formation. Many of these genes are not neuron-specific, but how they specifically control dendritic spine formation in neurons is an intriguing question. Here, we summarize how ubiquitously expressed genes, including syndecan-2, NF1 (encoding neurofibromin protein), VCP, and CASK, and the neuron-specific gene CTTNBP2 coordinate with neurotransmission, transsynaptic signaling, and cytoskeleton rearrangement to control dendritic filopodia formation, filopodia-spines transition, and dendritic spine maturation and maintenance. The aforementioned genes have been associated with neurological disorders, such as autism spectrum disorders (ASDs), mental retardation, learning difficulty, and frontotemporal dementia. We also summarize the corresponding disorders in this report.

Memory tests are important indexes of the brain functions for rodents behavior assay. Many memory tasks require external forces (e.g. electric shocks) or intrinsic forces (e.g. hunger and thirsty) to trigger the responses. Under those conditions, rodents are under stresses, such as pain, tired, malnutrition or dehydration, which potentially affect the natural neural responses. Novel object recognition is a non-force driving and spontaneous memory test. It is derived from curiosity but easy to be interfered with manipulation factors. In addition to stepwise procedure, the protocol described here emphasizes how to reduce the noise in the novel object recognition.

Cognitive flexibility, the higher-order cognition involving reversal learning, has been defined as having the ability to shift one’s previous thoughts or actions to new situations depending on situational demands. Studies of neuropsychiatric disorders such as autism spectrum disorder (ASD) showed that restricted and repetitive patterns of activities are associated with the impairments of cognitive flexibility. Some behavioral tasks including attentional set-shifting task are used to assess cognitive flexibility in mouse models for psychiatric disorders (Birrell and Brown, 2000; Colacicco et al., 2002). Here we present a two-choice digging test, which is simplified and modified from set-shifting task, for using mice to study the reversal learning (Huang et al., 2014).

Since SARM1 mutations have been identified in human neurological disease, SARM1 inhibition has become an attractive therapeutic strategy to preserve axons in a variety of disorders of the peripheral (PNS) and central nervous system (CNS). While SARM1 has been extensively studied in neurons, it remains unknown whether SARM1 is present and functional in myelinating glia? This is an important question to address. Firstly, to identify whether SARM1 dysfunction in other cell types in the nervous system may contribute to neuropathology in SARM1 dependent diseases? Secondly, to ascertain whether therapies altering SARM1 function may have unintended deleterious impacts on PNS or CNS myelination? Surprisingly, we find that oligodendrocytes express sarm1 mRNA in the zebrafish spinal cord and that SARM1 protein is readily detectable in rodent oligodendrocytes in vitro and in vivo. Furthermore, activation of endogenous SARM1 in cultured oligodendrocytes induces rapid cell death. In contrast, in peripheral glia, SARM1 protein is not detectable in Schwann cells and satellite glia in vivo and sarm1/Sarm1 mRNA is detected at very low levels in Schwann cells, in vivo, in zebrafish and mouse. Application of specific SARM1 activators to cultured mouse Schwann cells does not induce cell death and nicotinamide adenine dinucleotide (NAD) levels remain unaltered suggesting Schwann cells likely contain no functionally relevant levels of SARM1. Finally, we address the question of whether SARM1 is required for myelination or myelin maintenance. In the zebrafish and mouse PNS and CNS, we show that SARM1 is not required for initiation of myelination and myelin sheath maintenance is unaffected in the adult mouse nervous system. Thus, strategies to inhibit SARM1 function to treat neurological disease are unlikely to perturb myelination in humans. [ABSTRACT FROM AUTHOR]