DNA cytosine methyltransferases differentially regulate genome-wide hypermutation and interhomolog recombination in Trichoderma reesei meiosis.

Trichoderma reesei is an economically important enzyme producer with several unique meiotic features. spo11, the initiator of meiotic double-strand breaks (DSBs) in most sexual eukaryotes, is dispensable for T. reesei meiosis. T. reesei lacks the meiosis-specific recombinase Dmc1. Rad51 and Sae2, the activator of the Mre11 endonuclease complex, promote DSB repair and chromosome synapsis in wild-type and spo11Δ meiosis. DNA methyltransferases (DNMTs) perform multiple tasks in meiosis. Three DNMT genes (rid1, dim2 and dimX) differentially regulate genome-wide cytosine methylation and C:G-to-T:A hypermutations in different chromosomal regions. We have identified two types of DSBs: type I DSBs require spo11 or rid1 for initiation, whereas type II DSBs do not rely on spo11 and rid1 for initiation. rid1 (but not dim2) is essential for Rad51-mediated DSB repair and normal meiosis. rid1 and rad51 exhibit a locus heterogeneity (LH) relationship, in which LH-associated proteins often regulate interconnectivity in protein interaction networks. This LH relationship can be suppressed by deleting dim2 in a haploid rid1Δ (but not rad51Δ) parental strain, indicating that dim2 and rid1 share a redundant function that acts earlier than rad51 during early meiosis. In conclusion, our studies provide the first evidence of the involvement of DNMTs during meiotic initiation and recombination.

Deep brain stimulation of the Tbr1-deficient mouse model of autism spectrum disorder at the basolateral amygdala alters amygdalar connectivity, whole-brain synchronization, and social behaviors.

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.

KLHL17 differentially controls the expression of AMPA- and KA-type glutamate receptors to regulate dendritic spine enlargement.

Kelch-like family member 17 (KLHL17), an actin-associated adaptor protein, is linked to neurological disorders, including infantile spasms and autism spectrum disorders. The key morphological feature of Klhl17-deficient neurons is impaired dendritic spine enlargement, resulting in the amplitude of calcium events being increased. Our previous studies have indicated an involvement of F-actin and the spine apparatus in KLHL17-mediated dendritic spine enlargement. Here, we show that KLHL17 further employs different mechanisms to control the expression of two types of glutamate receptors, that is, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainate receptors (KARs), to regulate dendritic spine enlargement and calcium influx. We deployed proteomics to reveal that KLHL17 interacts with N-ethylmaleimide-sensitive fusion protein (NSF) in neurons, with this interaction of KLHL17 and NSF enhancing NSF protein levels. Consistent with the function of NSF in regulating the surface expression of AMPAR, Klhl17 deficiency limits the surface expression of AMPAR, but not its total protein levels. The NSF pathway also contributes to synaptic F-actin distribution and the dendritic spine enlargement mediated by KLHL17. KLHL17 is known to act as an adaptor mediating degradation of the KAR subunit GluK2 by the CUL3 ubiquitin ligase complex, and Klhl17 deficiency impairs activity-dependent degradation of GluK2. Herein, we further demonstrate that GluK2 is critical to the increased amplitude of calcium influx in Klhl17-deficient neurons. Moreover, GluK2 is also involved in KLHL17-regulated dendritic spine enlargement. Thus, our study reveals that KLHL17 controls AMPAR and KAR expression via at least two mechanisms, consequently regulating dendritic spine enlargement. The regulatory effects of KLHL17 on these two glutamate receptors likely contribute to neuronal features in patients suffering from certain neurological disorders.

Noncanonical usage of stop codons in ciliates expands proteins with structurally flexible Q-rich motifs.

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.

Vcp overexpression and leucine supplementation extend lifespan and ameliorate neuromuscular junction phenotypes of a SOD1G93A-ALS mouse model.

Many genes with distinct molecular functions have been linked to genetically heterogeneous amyotrophic lateral sclerosis (ALS), including SuperOxide Dismutase 1 (SOD1) and Valosin-Containing Protein (VCP). SOD1 converts superoxide to oxygen and hydrogen peroxide. VCP acts as a chaperon to regulate protein degradation and synthesis and various other cellular responses. Although the functions of these two genes differ, in the current report we show that overexpression of wild-type VCP in mice enhances lifespan and maintains the size of neuromuscular junctions (NMJs) of both male and female SOD1G93A mice, a well-known ALS mouse model. Although VCP exerts multiple functions, its regulation of ER formation and consequent protein synthesis has been shown to play the most important role in controlling dendritic spine formation and social and memory behaviors. Given that SOD1 mutation results in protein accumulation and aggregation, it may direct VCP to the protein degradation pathway, thereby impairing protein synthesis. Since we previously showed that the protein synthesis defects caused by Vcp deficiency can be improved by leucine supplementation, to confirm the role of the VCP-protein synthesis pathway in SOD1-linked ALS, we applied leucine supplementation to SOD1G93A mice and, similar to Vcp overexpression, we found that it extends SOD1G93A mouse lifespan. In addition, the phenotypes of reduced muscle strength and fewer NMJs of SOD1G93A mice are also improved by leucine supplementation. These results support the existence of crosstalk between SOD1 and VCP and suggest a critical role for protein synthesis in ASL. Our study also implies a potential therapeutic treatment for ALS.

Improved CaP Nanoparticles for Nucleic Acid and Protein Delivery to Neural Primary Cultures and Stem Cells.

Efficiently delivering exogenous materials into primary neurons and neural stem cells (NSCs) has long been a challenge in neurobiology. Existing methods have struggled with complex protocols, unreliable reproducibility, high immunogenicity, and cytotoxicity, causing a huge conundrum and hindering in-depth analyses. Here, we establish a cutting-edge method for transfecting primary neurons and NSCs, named teleofection, by a two-step process to enhance the formation of biocompatible calcium phosphate (CaP) nanoparticles. Teleofection enables both nucleic acid and protein transfection into primary neurons and NSCs, eliminating the need for specialized skills and equipment. It can easily fine-tune transfection efficiency by adjusting the incubation time and nanoparticle quantity, catering to various experimental requirements. Teleofection's versatility allows for the delivery of different cargos into the same cell culture, whether simultaneously or sequentially. This flexibility proves invaluable for long-term studies, enabling the monitoring of neural development and synapse plasticity. Moreover, teleofection ensures the consistent and robust expression of delivered genes, facilitating molecular and biochemical investigations. Teleofection represents a significant advancement in neurobiology, which has promise to transcend the limitations of current gene delivery methods. It offers a user-friendly, cost-effective, and reproducible approach for researchers, potentially revolutionizing our understanding of brain function and development.

Autism-related KLHL17 and SYNPO act in concert to control activity-dependent dendritic spine enlargement and the spine apparatus.

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.

The Src-ZNRF1 axis controls TLR3 trafficking and interferon responses to limit lung barrier damage.

Type I interferons are important antiviral cytokines, but prolonged interferon production is detrimental to the host. The TLR3-driven immune response is crucial for mammalian antiviral immunity, and its intracellular localization determines induction of type I interferons; however, the mechanism terminating TLR3 signaling remains obscure. Here, we show that the E3 ubiquitin ligase ZNRF1 controls TLR3 sorting into multivesicular bodies/lysosomes to terminate signaling and type I interferon production. Mechanistically, c-Src kinase activated by TLR3 engagement phosphorylates ZNRF1 at tyrosine 103, which mediates K63-linked ubiquitination of TLR3 at lysine 813 and promotes TLR3 lysosomal trafficking and degradation. ZNRF1-deficient mice and cells are resistant to infection by encephalomyocarditis virus and SARS-CoV-2 because of enhanced type I interferon production. However, Znrf1-/- mice have exacerbated lung barrier damage triggered by antiviral immunity, leading to enhanced susceptibility to respiratory bacterial superinfections. Our study highlights the c-Src-ZNRF1 axis as a negative feedback mechanism controlling TLR3 trafficking and the termination of TLR3 signaling.

Quantification of the density and morphology of dendritic spines and synaptic protein distribution using Thy1-YFP transgenic mice.

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.

SARM1 detection in myelinating glia: sarm1/Sarm1 is dispensable for PNS and CNS myelination in zebrafish and mice.

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.

Sex bias in social deficits, neural circuits and nutrient demand in Cttnbp2 autism models.

Autism spectrum disorders caused by both genetic and environmental factors are strongly male-biased neuropsychiatric conditions. However, the mechanism underlying the sex bias of autism spectrum disorders remains elusive. Here, we use a mouse model in which the autism-linked gene Cttnbp2 is mutated to explore the potential mechanism underlying the autism sex bias. Autism-like features of Cttnbp2 mutant mice were assessed via behavioural assays. C-FOS staining identified sex-biased brain regions critical to social interaction, with their roles and connectivity then validated by chemogenetic manipulation. Proteomic and bioinformatic analyses established sex-biased molecular deficits at synapses, prompting our hypothesis that male-biased nutrient demand magnifies Cttnbp2 deficiency. Accordingly, intakes of branched-chain amino acids (BCAA) and zinc were experimentally altered to assess their effect on autism-like behaviours. Both deletion and autism-linked mutation of Cttnbp2 result in male-biased social deficits. Seven brain regions, including the infralimbic area of the medial prefrontal cortex (ILA), exhibit reduced neural activity in male mutant mice but not in females upon social stimulation. ILA activation by chemogenetic manipulation is sufficient to activate four of those brain regions susceptible to Cttnbp2 deficiency and consequently to ameliorate social deficits in male mice, implying an ILA-regulated neural circuit is critical to male-biased social deficits. Proteomics analysis reveals male-specific downregulated proteins (including SHANK2 and PSD-95, two synaptic zinc-binding proteins) and female-specific upregulated proteins (including RRAGC) linked to neuropsychiatric disorders, which are likely relevant to male-biased deficits and a female protective effect observed in Cttnbp2 mutant mice. Notably, RRAGC is an upstream regulator of mTOR that senses BCAA, suggesting that mTOR exerts a beneficial effect on females. Indeed, increased BCAA intake activates the mTOR pathway and rescues neuronal responses and social behaviours of male Cttnbp2 mutant mice. Moreover, mutant males exhibit greatly increased zinc demand to display normal social behaviours. Mice carrying an autism-linked Cttnbp2 mutation exhibit male-biased social deficits linked to specific brain regions, differential synaptic proteomes and higher demand for BCAA and zinc. We postulate that lower demand for zinc and BCAA are relevant to the female protective effect. Our study reveals a mechanism underlying sex-biased social defects and also suggests a potential therapeutic approach for autism spectrum disorders.

Phase separation and zinc-induced transition modulate synaptic distribution and association of autism-linked CTTNBP2 and SHANK3.

Many synaptic proteins form biological condensates via liquid-liquid phase separation (LLPS). Synaptopathy, a key feature of autism spectrum disorders (ASD), is likely relevant to the impaired phase separation and/or transition of ASD-linked synaptic proteins. Here, we report that LLPS and zinc-induced liquid-to-gel phase transition regulate the synaptic distribution and protein-protein interaction of cortactin-binding protein 2 (CTTNBP2), an ASD-linked protein. CTTNBP2 forms self-assembled condensates through its C-terminal intrinsically disordered region and facilitates SHANK3 co-condensation at dendritic spines. Zinc binds the N-terminal coiled-coil region of CTTNBP2, promoting higher-order assemblies. Consequently, it leads to reduce CTTNBP2 mobility and enhance the stability and synaptic retention of CTTNBP2 condensates. Moreover, ASD-linked mutations alter condensate formation and synaptic retention of CTTNBP2 and impair mouse social behaviors, which are all ameliorated by zinc supplementation. Our study suggests the relevance of condensate formation and zinc-induced phase transition to the synaptic distribution and function of ASD-linked proteins.

Dietary zinc supplementation rescues fear-based learning and synaptic function in the Tbr1+/- mouse model of autism spectrum disorders.

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 Sídák'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.

Vaccination with SARS-CoV-2 spike protein lacking glycan shields elicits enhanced protective responses in animal models.

A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc-decorated state (SMG) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from SMG-immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.

Sarm1 activation produces cADPR to increase intra-axonal Ca++ and promote axon degeneration in PIPN.

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a "dying-back" axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN).

Protein synthesis as a modifiable target for autism-related dendritic spine pathophysiologies.

Autism spectrum disorder (ASD) is increasingly recognized as a condition of altered brain connectivity. As synapses are fundamental subcellular structures for neuronal connectivity, synaptic pathophysiology has become one of central themes in autism research. Reports disagree upon whether the density of dendritic spines, namely excitatory synapses, is increased or decreased in ASD and whether the protein synthesis that is critical for dendritic spine formation and function is upregulated or downregulated. Here, we review recent evidence supporting a subgroup of ASD models with decreased dendritic spine density (hereafter ASD-DSD), including Nf1 and Vcp mutant mice. We discuss the relevance of branched-chain amino acid (BCAA) insufficiency in relation to unmet protein synthesis demand in ASD-DSD. In contrast to ASD-DSD, ASD models with hyperactive mammalian target of rapamycin (mTOR) may represent the opposite end of the disease spectrum, often characterized by increases in protein synthesis and dendritic spine density (denoted ASD-ISD). Finally, we propose personalized dietary leucine as a strategy tailored to balancing protein synthesis demand, thereby ameliorating dendritic spine pathophysiologies and autism-related phenotypes in susceptible patients, especially those with ASD-DSD.

Macro Photography with Lightsheet Illumination Enables Whole Expanded Brain Imaging with Single-cell Resolution.

Macro photography allows direct visualization of the enlarged whole mouse brain by a combination of lightsheet illumination and expansion microscopy with single-cell resolution.  Taking advantage of the long working distance of a camera lens, we imaged a 3.7 cm thick, transparent, fluorescently-labeled expanded brain. In order to improve 3D sectioning capability, we used lightsheet excitation confined as the depth of field of the camera lens. Using 4x sample expansion and 5x optical magnification, macro photography enables imaging of expanded whole mouse brain with an effective resolution of 300 nm, which provides the subcellular structural information at the organ level.

Transcriptomic Analysis and C-Terminal Epitope Tagging Reveal Differential Processing and Signaling of Endogenous TLR3 and TLR7.

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.

TLR7 and IL-6 differentially regulate the effects of rotarod exercise on the transcriptomic profile and neurogenesis to influence anxiety and memory.

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.