Three-dimensional surface motion capture of multiple freely moving pigs using MAMMAL.

Understandings of the three-dimensional social behaviors of freely moving large-size mammals are valuable for both agriculture and life science, yet challenging due to occlusions in close interactions. Although existing animal pose estimation methods captured keypoint trajectories, they ignored deformable surfaces which contained geometric information essential for social interaction prediction and for dealing with the occlusions. In this study, we develop a Multi-Animal Mesh Model Alignment (MAMMAL) system based on an articulated surface mesh model. Our self-designed MAMMAL algorithms automatically enable us to align multi-view images into our mesh model and to capture 3D surface motions of multiple animals, which display better performance upon severe occlusions compared to traditional triangulation and allow complex social analysis. By utilizing MAMMAL, we are able to quantitatively analyze the locomotion, postures, animal-scene interactions, social interactions, as well as detailed tail motions of pigs. Furthermore, experiments on mouse and Beagle dogs demonstrate the generalizability of MAMMAL across different environments and mammal species.

Disruption of ER ion homeostasis maintained by an ER anion channel CLCC1 contributes to ALS-like pathologies.

Although anion channel activities have been demonstrated in sarcoplasmic reticulum/endoplasmic reticulum (SR/ER), their molecular identities and functions remain unclear. Here, we link rare variants of Chloride Channel CLIC Like 1 (CLCC1) to amyotrophic lateral sclerosis (ALS)-like pathologies. We demonstrate that CLCC1 is a pore-forming component of an ER anion channel and that ALS-associated mutations impair channel conductance. CLCC1 forms homomultimers and its channel activity is inhibited by luminal Ca2+ but facilitated by phosphatidylinositol 4,5-bisphosphate (PIP2). We identified conserved residues D25 and D181 in CLCC1 N-terminus responsible for Ca2+ binding and luminal Ca2+-mediated inhibition on channel open probability and K298 in CLCC1 intraluminal loop as the critical PIP2-sensing residue. CLCC1 maintains steady-state [Cl-]ER and [K+]ER and ER morphology and regulates ER Ca2+ homeostasis, including internal Ca2+ release and steady-state [Ca2+]ER. ALS-associated mutant forms of CLCC1 increase steady-state [Cl-]ER and impair ER Ca2+ homeostasis, and animals with the ALS-associated mutations are sensitized to stress challenge-induced protein misfolding. Phenotypic comparisons of multiple Clcc1 loss-of-function alleles, including ALS-associated mutations, reveal a CLCC1 dosage dependence in the severity of disease phenotypes in vivo. Similar to CLCC1 rare variations dominant in ALS, 10% of K298A heterozygous mice developed ALS-like symptoms, pointing to a mechanism of channelopathy dominant-negatively induced by a loss-of-function mutation. Conditional knockout of Clcc1 cell-autonomously causes motor neuron loss and ER stress, misfolded protein accumulation, and characteristic ALS pathologies in the spinal cord. Thus, our findings support that disruption of ER ion homeostasis maintained by CLCC1 contributes to ALS-like pathologies.

A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration.

Emerging evidence suggests that intron-detaining transcripts (IDTs) are a nucleus-detained and polyadenylated mRNA pool for cell to quickly and effectively respond to environmental stimuli and stress. However, the underlying mechanisms of detained intron (DI) splicing are still largely unknown. Here, we suggest that post-transcriptional DI splicing is paused at the Bact state, an active spliceosome but not catalytically primed, which depends on Smad Nuclear Interacting Protein 1 (SNIP1) and RNPS1 (a serine-rich RNA binding protein) interaction. RNPS1 and Bact components preferentially dock at DIs and the RNPS1 docking is sufficient to trigger spliceosome pausing. Haploinsufficiency of Snip1 attenuates neurodegeneration and globally rescues IDT accumulation caused by a previously reported mutant U2 snRNA, a basal spliceosomal component. Snip1 conditional knockout in the cerebellum decreases DI splicing efficiency and causes neurodegeneration. Therefore, we suggest that SNIP1 and RNPS1 form a molecular brake to promote spliceosome pausing, and that its misregulation contributes to neurodegeneration.

TCF7L2 acts as a molecular switch in midbrain to control mammal vocalization through its DNA binding domain but not transcription activation domain.

Vocalization is an essential medium for social signaling in birds and mammals. Periaqueductal gray (PAG) a conserved midbrain structure is believed to be responsible for innate vocalizations, but its molecular regulation remains largely unknown. Here, through a mouse forward genetic screening we identified one of the key Wnt/ß-catenin effectors TCF7L2/TCF4 controls ultrasonic vocalization (USV) production and syllable complexity during maternal deprivation and sexual encounter. Early developmental expression of TCF7L2 in PAG excitatory neurons is necessary for the complex trait, while TCF7L2 loss reduces neuronal gene expressions and synaptic transmission in PAG. TCF7L2-mediated vocal control is independent of its ß-catenin-binding domain but dependent of its DNA binding ability. Patient mutations associated with developmental disorders, including autism spectrum disorders, disrupt the transcriptional repression effect of TCF7L2, while mice carrying those mutations display severe USV impairments. Therefore, we conclude that TCF7L2 orchestrates gene expression in midbrain to control vocal production through its DNA binding but not transcription activation domain.

Dual-gRNA approach with limited off-target effect corrects C9ORF72 repeat expansion in vivo.

C9ORF72 GGGGCC repeat expansion is the most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia, which generates abnormal DNA and RNA structures and produces toxic proteins. Recently, efficacy of CRISPR/Cas9-mediated editing has been proven in treatment of disease. However, DNA low complexity surrounding C9ORF72 expansion increases the off-target risks. Here we provide a dual-gRNA design outside of the low complexity region which enables us to remove the repeat DNA in a 'cutting-deletion-fusion' manner with a high fusion efficiency (50%). Our dual-gRNA design limits off-target effect and does not significantly affect C9ORF72 expression. In neurons carrying patient C9ORF72 expansion, our approach removes the repeat DNA and corrects the RNA foci in vitro and in vivo. Therefore, we conclude that our proof-of-concept design correct C9ORF72 repeat expansion, which may have potential therapeutic value for the patients.

An L1 retrotransposon insertion-induced deafness mouse model for studying the development and function of the cochlear stria vascularis.

Dysregulation of ion and potential homeostasis in the scala media is the most prevalent cause of hearing loss in mammals. However, it is not well understood how the development and function of the stria vascularis regulates this fluid homeostasis in the scala media. From a mouse genetic screen, we characterize a mouse line, named 299, that displays profound hearing impairment. Histology suggests that 299 mutant mice carry a severe, congenital structural defect of the stria vascularis. The in vivo recording of 299 mice using double-barreled electrodes shows that endocochlear potential is abolished and potassium concentration is reduced to ∼20 mM in the scala media, a stark contrast to the +80 mV endocochlear potential and the 150 mM potassium concentration present in healthy control mice. Genomic analysis revealed a roughly 7-kb-long, interspersed nuclear element (LINE-1 or L1) retrotransposon insertion on chromosome 11. Strikingly, the deletion of this L1 retrotransposon insertion from chromosome 11 restored the hearing of 299 mutant mice. In summary, we characterize a mouse model that enables the study of stria vascularis development and fluid homeostasis in the scala media.

In vivo stress granule misprocessing evidenced in a FUS knock-in ALS mouse model.

Many RNA-binding proteins, including TDP-43, FUS, and TIA1, are stress granule components, dysfunction of which causes amyotrophic lateral sclerosis (ALS). However, whether a mutant RNA-binding protein disrupts stress granule processing in vivo in pathogenesis is unknown. Here we establish a FUS ALS mutation, p.R521C, knock-in mouse model that carries impaired motor ability and late-onset motor neuron loss. In disease-susceptible neurons, stress induces mislocalization of mutant FUS into stress granules and upregulation of ubiquitin, two hallmarks of disease pathology. Additionally, stress aggravates motor performance decline in the mutant mouse. By using two-photon imaging in TIA1-EGFP transduced animals, we document more intensely TIA1-EGFP-positive granules formed hours but cleared weeks after stress challenge in neurons in the mutant cortex. Moreover, neurons with severe granule misprocessing die days after stress challenge. Therefore, we argue that stress granule misprocessing is pathogenic in ALS, and the model we provide here is sound for further disease mechanistic study.

An ENU-induced mutation in Twist1 transactivation domain causes hindlimb polydactyly with complete penetrance and dominant-negatively impairs E2A-dependent transcription.

Twist1 encodes a basic helix-loop-helix transcription factor (TF), which forms homodimer or heterodimer with other TFs, like E2A, to regulate target genes' expression. Mutations in TWIST1 are associated with Saethre-Chotzen syndrome (SCS), a rare congenital disorder characterized with osteogenesis abnormalities. However, how dysfunction of TWIST1 leads to SCS is still largely unknown. Here, using an unbiased ENU-induced mutagenesis screening, we identified a novel Twist1 mutation and the mutant mouse phenocopies some features of SCS in a dominant manner. Physically, our mutation p.F191S lies at the edge of a predicted α-helix in Twist1 transactivation (TA) domain. Adjacent to F191, a consecutive three-residue (AFS) has been hit by 3 human and 2 mouse disease-associated mutations, including ours. Unlike previously reported mouse null and p.S192P alleles that lead to hindlimb polydactyly with incomplete penetrance but a severe craniofacial malformation, our p.F191S causes the polydactyly (84.2% bilateral and 15.8% unilateral) with complete penetrance but a mild craniofacial malformation. Consistent with the higher penetrance, p.F191S has stronger impairment on E2A-dependent transcription than p.S192P. Although human p.A186T and mouse p.S192P disease mutations are adjacent to ours, these three mutations function differently to impair the E2A-dependent transcription. Unlike p.A186T and p.S192S that disturb local protein conformation and unstabilize the mutant proteins, p.F191S keeps the mutant protein stable and its interaction with E2A entire. Therefore, we argue that p.F191S we identified acts in a dominant-negative manner to impair E2A-dependent transcription and to cause the biological consequences. In addition, the mutant mouse we provided here could be an additional and valuable model for better understanding the disease mechanisms underlying SCS caused by TWIST1 dysfunction.

TRPC Channels and Programmed Cell Death.

Neurotrophins, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), bind to their high-affinity receptors to promote neuronal survival during brain development. One of the key downstream pathways is the phospholipase C (PLC) pathway, which not only plays a central role in calcium release from internal store but also in activation of TRPC channels coupled with neurotrophin receptors. TRPC channels are required for the neurotrophin-mediated neuronal protective effects. In addition, activation of TRPC channels is able to protect neurons in the absence of neurotrophin. In some circumstances, TRPC channels coupled with metabotropic glutamate receptor may mediate the excitotoxicity by calcium overload. One of the key questions in the field is the channel gating mechanisms; understanding of which would help design compounds to modulate the channel properties. The development and identification of TRPC channel agonists or blockers are promising and may unveil new therapeutic drugs for the treatment of neurodegenerative diseases and epilepsy.

TRPC Channels and Neuron Development, Plasticity, and Activities.

In this chapter, we mainly focus on the functions of TRPC channels in brain development, including neural progenitor proliferation, neurogenesis, neuron survival, axon guidance, dendritic morphology, synaptogenesis, and neural plasticity. We also notice emerging advances in understanding the functions of TRPC channels in periphery, especially their functions in sensation and nociception in dorsal root ganglion (DRG). Because TRPC channels are expressed in all major types of glial cells, which account for at least half of total cells in the brain, TRPC channels may act as modulators for glial functions as well. The future challenges for studying these channels could be (1) the detailed protein structures of these channels, (2) their cell type-specific functions, (3) requirement for their specific blockers or activators, and (4) change in the channel conformation in the brain.

Activity-induced histone modifications govern Neurexin-1 mRNA splicing and memory preservation.

Epigenetic mechanisms regulate the formation, consolidation and reconsolidation of memories. However, the signaling path from neuronal activation to epigenetic modifications within the memory-related brain circuit remains unknown. We report that learning induces long-lasting histone modifications in hippocampal memory-activated neurons to regulate memory stability. Neuronal activity triggers a late-onset shift in Nrxn1 splice isoform choice at splicing site 4 by accumulating a repressive histone marker, H3K9me3, to modulate the splicing process. Activity-dependent phosphorylation of p66α via AMP-activated protein kinase recruits HDAC2 and Suv39h1 to establish repressive histone markers and changes the connectivity of the activated neurons. Removal of Suv39h1 abolished the activity-dependent shift in Nrxn1 splice isoform choice and reduced the stability of established memories. We uncover a cell-autonomous process for memory preservation in which memory-related neurons initiate a late-onset reduction of their rewiring capacities through activity-induced histone modifications.

Identification of a Novel ENU-Induced Mutation in Mouse Tbx1 Linked to Human DiGeorge Syndrome.

The patients with DiGeorge syndrome (DGS), caused by deletion containing dozens of genes in chromosome 22, often carry cardiovascular problem and hearing loss associated with chronic otitis media. Inside the deletion region, a transcription factor TBX1 was highly suspected. Furthermore, similar DGS phenotypes were found in the Tbx1 heterozygous knockout mice. Using ENU-induced mutagenesis and G1 dominant screening strategy, here we identified a nonsynonymous mutation p.W118R in T-box of TBX1, the DNA binding domain for transcription activity. The mutant mice showed deficiency of inner ear functions, including head tossing and circling, plus increased hearing threshold determined by audiometry. Therefore, our result further confirms the pathogenic basis of Tbx1 in DGS, points out the crucial role of DNA binding activity of TBX1 for the ear function, and provides additional animal model for studying the DGS disease mechanisms.

Loss of Clcc1 results in ER stress, misfolded protein accumulation, and neurodegeneration.

Folding of transmembrane and secretory proteins occurs in the lumen of the endoplasmic reticulum (ER) before transportation to the cell surface and is monitored by the unfolded protein response (UPR) signaling pathway. The accumulation of unfolded proteins in the ER activates the UPR that restores ER homeostasis by regulating gene expression that leads to an increase in the protein-folding capacity of the ER and a decrease in the ER protein-folding load. However, prolonged UPR activity has been associated with cell death in multiple pathological conditions, including neurodegeneration. Here, we report a spontaneous recessive mouse mutation that causes progressive cerebellar granule cell death and peripheral motor axon degeneration. By positional cloning, we identify the mutation in this strain as a retrotransposon insertion in the Clcc1 gene, which encodes a putative chloride channel localized to the ER. Furthermore, we demonstrate that the C3H/HeSnJ inbred strain has late onset cerebellar degeneration due to this mutation. Interestingly, acute knockdown of Clcc1 expression in cultured cells increases sensitivity to ER stress. In agreement, GRP78, the major HSP70 family chaperone in the ER, is upregulated in Clcc1-deficient granule cells in vivo, and ubiquitinated proteins accumulate in these neurons before their degeneration. These data suggest that disruption of chloride homeostasis in the ER disrupts the protein-folding capacity of the ER, leading to eventual neuron death.

Mutation of a U2 snRNA gene causes global disruption of alternative splicing and neurodegeneration.

Although uridine-rich small nuclear RNAs (U-snRNAs) are essential for pre-mRNA splicing, little is known regarding their function in the regulation of alternative splicing or of the biological consequences of their dysfunction in mammals. Here, we demonstrate that mutation of Rnu2-8, one of the mouse multicopy U2 snRNA genes, causes ataxia and neurodegeneration. Coincident with the observed pathology, the level of mutant U2 RNAs was highest in the cerebellum and increased after granule neuron maturation. Furthermore, neuron loss was strongly dependent on the dosage of mutant and wild-type snRNA genes. Comprehensive transcriptome analysis identified a group of alternative splicing events, including the splicing of small introns, which were disrupted in the mutant cerebellum. Our results suggest that the expression of mammalian U2 snRNA genes, previously presumed to be ubiquitous, is spatially and temporally regulated, and dysfunction of a single U2 snRNA causes neuron degeneration through distortion of pre-mRNA splicing.

Critical role of TRPC6 channels in the formation of excitatory synapses.

The transient receptor potential canonical (TRPC) channels are Ca2+-permeable, nonselective cation channels with different biological functions, but their roles in brain are largely unknown. Here we report that TRPC6 was localized to excitatory synapses and promoted their formation via a CaMKIV-CREB-dependent pathway. TRPC6 transgenic mice showed enhancement in spine formation, and spatial learning and memory in Morris water maze. These results reveal a previously unknown role of TRPC6 in synaptic and behavioral plasticity.

Anti-tumor effect of beta-elemene in glioblastoma cells depends on p38 MAPK activation.

beta-Elemene, a natural plant drug extracted from Curcuma wenyujin, has been used as an antitumor drug for different tumors, including glioblastoma. However, the mechanism of its anti-tumor effect is largely unknown. Here we report that anti-proliferation of glioblastoma cells induced by beta-elemene was dependent on p38 MAPK activation. Treatment of glioblastoma cell lines with beta-elemene, led to phosphorylation of p38 MAPK, cell-cycle arrest in G0/G1 phase and inhibition of proliferation of these cells. Inhibition of p38 MAPK reversed beta-elemene-mediated anti-proliferation effect. Furthermore, the growth of glioblastoma cell-transplanted tumors in nude mice was inhibited by intraperitoneal injection of beta-elemene. Taken together, our findings indicate that activation of p38 MAPK is critical for the anti-proliferation effect of beta-elemene and that p38 MAPK might be a putative pharmacological target for glioblastoma therapy.

TRPC channels promote cerebellar granule neuron survival.

Channels formed by the transient receptor potential (TRP) family of proteins have a variety of physiological functions. Here we report that two members of the TRP cation channel (TRPC) subfamily, TRPC3 and 6, protected cerebellar granule neurons (CGNs) against serum deprivation-induced cell death in cultures and promoted CGN survival in rat brain. In CGN cultures, blocking TRPC channels or downregulating TRPC3 or 6 suppressed brain-derived neurotrophic factor (BDNF)-mediated protection, BDNF-triggered intracellular Ca2+ elevation and BDNF-induced CREB activation. By contrast, overexpressing TRPC3 or 6 increased CREB-dependent reporter gene transcription and prevented apoptosis in the neurons deprived of serum, and this protection was blocked by the dominant negative form of CREB. Furthermore, downregulating TRPC3 or 6 induced CGN apoptosis in neonatal rat cerebellum, and this effect was rescued by overexpressing either TRPC3 or 6. Thus, our findings provide in vitro and in vivo evidence that TRPC channels are important in promoting neuronal survival.

Characterization of ZNF23, a KRAB-containing protein that is downregulated in human cancers and inhibits cell cycle progression.

The Krupple-associated box-containing zinc-finger proteins (KRAB-ZFPs) make up one of the largest family of transcription factors. Several members of the KRAB-ZFPs modulate cell growth, survival and are implicated in malignant disorders. However, most members are not well characterized and their functions are largely unknown. Here we report that ZNF23, a member of KRAB-ZFPs, inhibits cell cycle progression. ZNF23 protein localized to the nucleus and was ubiquitously expressed in all tested normal tissues. However, the expression levels of ZNF23 protein were lost or greatly reduced in human cancer. Ectopic expression of ZNF23 led to enhancement of p27(kip-1) expression, growth inhibition and cell cycle arrest in G(1) phase. Downregulation of p27(kip-1) by siRNA against p27(kip-1) reversed growth inhibition induced by ZNF23. Furthermore, the growth-inhibitory effect of ZNF23 was p53-independent. Deletion analysis revealed that the effect of ZNF23 did not rely on its KRAB domain, but on the C-terminal zinc fingers. Thus, we have identified a new member of KRAB-ZNF superfamily with growth-inhibitory ability and its downregulation may contribute to carcinogenesis.