Mutant Mice Research Articles

Detailed phenotyping of Tbr1-2A-CreER knock-in mice demonstrates significant impacts on TBR1 protein levels and axon development.

Cre recombinase knock-in mouse lines have served as invaluable genetic tools for understanding key developmental processes altered in autism. However, insertion of exogenous DNA into the genome can have unintended effects on local gene regulation or protein function that must be carefully considered. Here, we analyze a recently generated Tbr1-2A-CreER knock-in mouse line, where a 2A-CreER cassette was inserted in-frame before the stop codon of the transcription factor gene Tbr1. Heterozygous TBR1 mutations in humans and mice are known to cause autism or autism-like behavioral phenotypes accompanied by structural brain malformations, most frequently a reduction of the anterior commissure (AC). Thus, it is critical for modified versions of Tbr1 to exhibit true wild-type-like activity. We evaluated the Tbr1-2A-CreER allele for its potential impact on Tbr1 function and complementation to Tbr1 loss-of-function alleles. In mice with one copy of the Tbr1-2A-CreER allele, we identified reduction of TBR1 protein in early postnatal cortex along with thinning of the AC, suggesting hypersensitivity of this structure to TBR1 dosage. Comparing Tbr1-2A-CreER and Tbr1-null mice to Tbr1-null complementation crosses showed reductions of TBR1 dosage ranging from 20% to 100%. Using six combinatorial genotypes, we found that moderate to severe TBR1 reductions (≥44%) were associated with cortical layer 5 expansion, while only the complete absence of TBR1 was associated with reeler-like "inverted" cortical layering. In total, these results strongly support the conclusion that Tbr1-2A-CreER is a hypomorphic allele. We advise caution when interpreting experiments using this allele, considering the sensitivity of various corticogenic processes to TBR1 dosage and the association of heterozygous TBR1 mutations with complex neurodevelopmental disorders.

ATP citrate lyase is an essential player in the metabolic rewiring induced by PTEN loss during T-ALL development.

Alterations inactivating the tumor suppressor gene PTEN drive the development of solid and hematological cancers, such as T-cell acute lymphoblastic leukemia (T-ALL), whereby PTEN loss defines poor-prognosis patients. We investigated the metabolic rewiring induced by PTEN loss in T-ALL, aiming at identifying novel metabolic vulnerabilities. We showed that the enzyme ATP citrate lyase (ACLY) is strictly required for the transformation of thymic immature progenitors and for the growth of human T-ALL, which remain dependent on ACLY activity even upon transformation. Whereas PTEN mutant mice all died within 17 weeks, the concomitant ACLY deletion prevented disease initiation in 70% of the animals. In these animals, ACLY promoted BCL-2 epigenetic upregulation and prevented the apoptosis of pre-malignant DP thymocytes. Transcriptomic and metabolic analysis of primary T-ALL cells next translated our findings to the human pathology, showing that PTEN-altered T-ALL cells activate ACLY and are sensitive to its genetic targeting. ACLY activation thus represents a metabolic vulnerability with therapeutic potential for high-risk T-ALL patients.

A pilot study to directly estimate radiation-induced mutation in large Japanese field mouse duo sample, mother and offspring, excluding unknown father, using ddRAD sequencing.

DNA mutations are one of the effects of radiation exposure. A large amount of radioactive materials was released into the environment from the Fukushima Daiichi Nuclear Power Plant after a major earthquake and tsunami. Wild animals and plants living in highly radiation-contaminated areas are constantly exposed to high doses of radiation, and concerns occur about its effects on their health and the next generations. As a pilot study, double-digest restriction site-associated DNA (ddRAD) sequencing was conducted to assess the incidence of mutations in wild large Japanese field mice collected from the evacuation area. The optimal combination of restriction enzymes, encompassing the functionally important coding regions, was selected using in silico analysis. These enzymes were used for ddRAD sequence analysis of females and their fetuses to evaluate mutation rates. The results indicated that no significant differences were observed in mutation rates between mothers and fetuses in the study areas.

ATP1A3 dysfunction causes motor hyperexcitability and afterhyperpolarization loss in a dystonia model.

Mutations in the gene encoding the alpha3 Na+/K+-ATPase isoform (ATP1A3) lead to movement disorders that manifest with dystonia, a common neurological symptom with many different origins, but for which the underlying molecular mechanisms remain poorly understood. We have generated an ATP1A3 mutant mouse that displays motor impairments and a hyperexcitable motor phenotype compatible with dystonia. We show that neurons harboring this mutation are compromised in their ability to extrude raised levels of intracellular sodium, highlighting a profound deficit in neuronal sodium homeostasis. We show that the spinal motor network in ATP1A3 mutant mice has a reduced responsiveness to activity-dependent rises in intracellular sodium and that this is accompanied by loss of the Na+/K+-ATPase-mediated afterhyperpolarization in motor neurons. Taken together, our data support that the alpha3 Na+/K+-ATPase is important for cellular and spinal motor network homeostasis. These insights suggest that it may be useful to consider ways to compensate for this loss of a critical afterhyperpolarization-dependent control of neuronal excitability when developing future therapies for dystonia.

Abstract 4136847: Administration of the Recombinant Activated Protein C Rescues the Cardiac Vulnerability to Ischemic Insults in Aging through Modulating Inflammatory Response during Ischemia and Reperfusion

Introduction: Activated protein C (APC) is an endogenous vitamin-K-dependent serine protease and exhibits therapeutic potential for ischemic heart disease. The APC derivatives with anticoagulant and/or cytoprotective properties were produced through chemical engineering to characterize the functional domain of APC for limiting ischemia/reperfusion (I/R) injury. Hypothesis: The thromboinflammatory regulation by APC ameliorates ischemic insults caused by I/R through the receptor EPCR/PAR1. Methods: Young (3 months)/aged (24 months) wild type C57BL/6J mice and PAR1 R46Q/R46Q (block cleavage by APC) knock in mutant (3 months, C57BL/6J) mice were subjected to ligation of left anterior descending coronary artery with 45 min of ischemia. 5 min prior to reperfusion, wild type APC, cytoprotective selective APC-2Cys, or anticoagulant selective APC-E170A (0.2 μg/kg i.v.) was administered followed by 24 hr of reperfusion. The left ventricles of hearts were used for immunoblotting and flow cytometry analysis. Results: APC and APC-2Cys but not APC-E170A reduced I/R-induced myocardial infarction. Echocardiography showed that APC and APC-2Cys but not APC-E170A can rescue systolic dysfunction by I/R in young/aged WT but not in PAR1 R46Q/R46Q mice. Biochemical analysis showed that administration of APC and APC-2Cys inhibited activation of inflammation signaling c-Jun N-terminal protein kinase (JNK) and NF-κB by I/R and reduced mRNA levels of the proinflammatory cytokines TNFα and IL-6 in young/aged WT but not in PAR1 R46Q/R46Q hearts. The plasma cytokine array demonstrated that APC and APC2-Cys reduced potency of pro-inflammatory cytokines and apoptotic signaling cytokines during I/R. Moreover, I/R-triggered inflammasome NLRP3 was reduced by APC and APC-2Cys in young/aged WT but not in PAR1 R46Q/R46Q hearts. The flow cytometry showed that APC and APC-2Cys increased lymphocyte and decreased myeloid cell abundance and increased the recruitment of inflammation-mediating CD8a + T-cells during I/R in young/aged WT but not PAR1 R46Q/R46Q hearts, indicating that the APC/EPCR/PAR1 axis mediates APC's immune regulation under I/R. Moreover, APC and APC-2Cys reduced I/R-triggered macrophage abundance in young/aged WT hearts, although the proportion of M1 and M2 macrophages was unchanged. Conclusions: The cytoprotective domain of APC is critical for its cardioprotection against ischemic insults. EPCR/PAR1 receptor complex mediates APC's immune regulation in aging during I/R conditions.

Contributions of mirror-image hair cell orientation to mouse otolith organ and zebrafish neuromast function.

Otolith organs in the inner ear and neuromasts in the fish lateral-line harbor two populations of hair cells oriented to detect stimuli in opposing directions. The underlying mechanism is highly conserved: the transcription factor EMX2 is regionally expressed in just one hair cell population and acts through the receptor GPR156 to reverse cell orientation relative to the other population. In mouse and zebrafish, loss of Emx2 results in sensory organs that harbor only one hair cell orientation and are not innervated properly. In zebrafish, Emx2 also confers hair cells with reduced mechanosensory properties. Here, we leverage mouse and zebrafish models lacking GPR156 to determine how detecting stimuli of opposing directions serves vestibular function, and whether GPR156 has other roles besides orienting hair cells. We find that otolith organs in Gpr156 mouse mutants have normal zonal organization and normal type I-II hair cell distribution and mechano-electrical transduction properties. In contrast, gpr156 zebrafish mutants lack the smaller mechanically evoked signals that characterize Emx2-positive hair cells. Loss of GPR156 does not affect orientation-selectivity of afferents in mouse utricle or zebrafish neuromasts. Consistent with normal otolith organ anatomy and afferent selectivity, Gpr156 mutant mice do not show overt vestibular dysfunction. Instead, performance on two tests that engage otolith organs is significantly altered - swimming and off-vertical-axis rotation. We conclude that GPR156 relays hair cell orientation and transduction information downstream of EMX2, but not selectivity for direction-specific afferents. These results clarify how molecular mechanisms that confer bi-directionality to sensory organs contribute to function, from single hair cell physiology to animal behavior.

Contributions of mirror-image hair cell orientation to mouse otolith organ and zebrafish neuromast function

Otolith organs in the inner ear and neuromasts in the fish lateral-line harbor two populations of hair cells oriented to detect stimuli in opposing directions. The underlying mechanism is highly conserved: the transcription factor EMX2 is regionally expressed in just one hair cell population and acts through the receptor GPR156 to reverse cell orientation relative to the other population. In mouse and zebrafish, loss of Emx2 results in sensory organs that harbor only one hair cell orientation and are not innervated properly. In zebrafish, Emx2 also confers hair cells with reduced mechanosensory properties. Here, we leverage mouse and zebrafish models lacking GPR156 to determine how detecting stimuli of opposing directions serves vestibular function, and whether GPR156 has other roles besides orienting hair cells. We find that otolith organs in Gpr156 mouse mutants have normal zonal organization and normal type I-II hair cell distribution and mechano-electrical transduction properties. In contrast, gpr156 zebrafish mutants lack the smaller mechanically evoked signals that characterize Emx2-positive hair cells. Loss of GPR156 does not affect orientation-selectivity of afferents in mouse utricle or zebrafish neuromasts. Consistent with normal otolith organ anatomy and afferent selectivity, Gpr156 mutant mice do not show overt vestibular dysfunction. Instead, performance on two tests that engage otolith organs is significantly altered – swimming and off-vertical-axis rotation. We conclude that GPR156 relays hair cell orientation and transduction information downstream of EMX2, but not selectivity for direction-specific afferents. These results clarify how molecular mechanisms that confer bi-directionality to sensory organs contribute to function, from single hair cell physiology to animal behavior.

TMOD-12. DEFINING THE ROLE OF THE DAAM2 R414W GERMLINE VARIANT IN FAMILIAL GLIOMA

Abstract Malignant glioma is the most common primary brain tumor in adults, leading to high morbidity and mortality due to its aggressive nature and limited efficacious treatment options. To improve this prognosis, research has focused on genetic factors that may contribute to glioma. While most glioma cases arise from sporadic somatic mutations, approximately 5-10% are classified as familial glioma, stemming from inherited genetic variations. Although several studies have explored mechanisms behind somatic mutations in glioma, less is known about hereditary variants that promote glioma susceptibility. We recently discovered five germline mutations in Daam2 (Dishevelled associated activator of morphogenesis 2) from familial glioma patients. To examine the role of these mutations in familial glioma, we overexpressed these mutations in patient-derived glioma stem-like cells, which were subsequently orthotopically xenografted into immunodeficient mice. We identified that one of these mutations, Daam2-R414W, promotes tumor cell proliferation both in vitro and in vivo. To further understand the role of the Daam2-R414W mutation in familial glioma, we generated a knock-in immunocompetent mouse model with the Daam2-R414W mutation. In these mutant mice, we found an increased number of proliferating glial progenitor cells in the subventricular zone compared to Daam2 wildtype mice, suggesting the potential of the Daam2-R414W mutation to influence glial progenitor expansion to stimulate glioma initiation. Furthermore, to establish a potential mechanism, we performed proteomic profiling of Daam2 wildtype and Daam2-R414W overexpression tumors and found enrichment of Git1, a GTPase-activating protein involved in cytoskeletal remodeling, suggesting that the Daam2-R414W variant may interact with Git1 to promote tumor invasion. Our findings thus far identify a hereditary genetic variant with the potential to increase the risk of glioma, shedding light on the repercussions of a human developmental gene variant and its potential to disrupt normal cellular machinery to promote malignancy and glioma development.

DDDR-26. IDENTIFICATION OF MODULATORS OF IDH INHIBITOR SENSITIVITY INIDH-MUTATED DIFFUSE GLIOMAS

Abstract Driver mutations in IDH1 and IDH2 characterize a substantial proportion of lower-grade diffuse gliomas in adults. Mutated IDH molecules cause the accumulation of D-2-hydroxyglutarate (D-2-HG), which perturbs many cellular functions, including metabolism, epigenetic regulation, and the ability to differentiate. This is notably associated with DNA and histone hyper-methylation. Recently, the newly developed IDH inhibitor, vorasidenib, was shown to delay tumor progression and the need for additional treatment in the groundbreaking INDIGO clinical trial. Nevertheless, the responses to vorasidenib were variable between patients, and so far have been established only for less aggressive disease. In addition, it is unclear to what extent vorasidenib can cause tumor regression. This highlights the need to identify molecules and pathways that control IDH inhibitor sensitivity. Towards this goal, we generated a new mouse model that combines Idh1R132H and Trp53 loss-of-function, targeted to oligodendrocyte progenitors. All the mutant mice develop diffuse gliomas that recapitulate the cardinal features of the corresponding human disease. We established multiple cell lines from these tumors, which retained expression of mutant IDH. The cells robustly produced D-2-HG, which was concentration-dependently suppressed by vorasidenib. Accordingly, vorasidenib could lower DNA methylation, and induce the expression of mature glial cell markers. To identify determinants of vorasidenib sensitivity, we performed genome-wide loss-of-function CRISPR/Cas9 functional genomics screens. In those experiments, targeting genes associated with astrocyte differentiation or Notch signaling enhanced cell fitness in the presence of vorasidenib, consistent with the anticipated effects of the drug on inducing cell differentiation. Conversely, vorasidenib-treated cells were more sensitive to the loss of regulators of cell growth and metabolism, and of some epigenetic regulators. These studies point to strategies that may enhance the efficacy of IDH inhibitors for the treatment of IDH mutated gliomas.

Host genetics and gut microbiota influence lipid metabolism and inflammation: potential implications for ALS pathophysiology in SOD1G93A mice

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the progressive loss of motor neurons, with genetic and environmental factors contributing to its complex pathogenesis. Dysregulated immune responses and altered energetic metabolism are key features, with emerging evidence implicating the gut microbiota (GM) in disease progression. We investigated the interplay among genetic background, GM composition, metabolism, and immune response in two distinct ALS mouse models: 129Sv_G93A and C57Ola_G93A, representing rapid and slow disease progression, respectively.Using 16 S rRNA sequencing and fecal metabolite analysis, we characterized the GM composition and metabolite profiles in non-transgenic (Ntg) and SOD1G93A mutant mice of both strains. Our results revealed strain-specific differences in GM composition and functions, particularly in the abundance of taxa belonging to Erysipelotrichaceae and the levels of short and medium-chain fatty acids in fecal samples. The SOD1 mutation induces significant shifts in GM colonization in both strains, with C57Ola_G93A mice showing changes resembling those in 129 Sv mice, potentially affecting disease pathogenesis. ALS symptom progression does not significantly alter microbiota composition, suggesting stability.Additionally, we assessed systemic immunity and inflammatory responses revealing strain-specific differences in immune cell populations and cytokine levels.Our findings underscore the substantial influence of genetic background on GM composition, metabolism, and immune response in ALS mouse models. These strain-specific variations may contribute to differences in disease susceptibility and progression rates. Further elucidating the mechanisms underlying these interactions could offer novel insights into ALS pathogenesis and potential therapeutic targets.

Oligodendrocyte Slc48a1 (Hrg1) encodes a functional heme transporter required for myelin integrity.

Oligodendrocytes (OLs) of the central nervous system require iron for proteolipid biosynthesis during the myelination process. Although most heme is found complexed to hemoglobin in red blood cells, surprisingly, we found that Slc48a1, encoding the heme transporter Hrg1, is expressed at higher levels in OLs than any other cell type in rodent and humans. We confirmed in situ that Hrg1 is expressed in OLs but not their precursors (OPCs) and found that Hrg1 proteins in CNS white matter co-localized within myelin sheaths. In older Hrg1 null mutant mice we observed reduced expression of myelin associated glycoprotein (Mag) and ultrastructural myelin defects reminiscent of Mag-null animals, suggesting myelin adhesion deficiency. Further, we confirmed reduced myelin iron levels in Hrg1 null animals in vivo, and show that OLs in vitro can directly import both the fluorescent heme analogue ZnMP and heme itself, which rescued iron deficiency induced inhibition of OL differentiation in a heme-oxidase-dependent manner. Together these findings indicate OL Hrg1 encodes a functional heme transporter required for myelin integrity.

Tufm lactylation regulates neuronal apoptosis by modulating mitophagy in traumatic brain injury.

Lactates accumulation following traumatic brain injury (TBI) is detrimental. However, whether lactylation is triggered and involved in the deterioration of TBI remains unknown. Here, we first report that Tufm lactylation pathway induces neuronal apoptosis in TBI. Lactylation is found significantly increased in brain tissues from patients with TBI and mice with controlled cortical impact (CCI), and in neuronal injury cell models. Tufm, a key factor in mitophagy, is screened and identified to be mostly lactylated. Tufm is detected to be lactylated at K286 and the lactylation inhibits the interaction of Tufm and Tomm40 on mitochondria. The mitochondrial distribution of Tufm is then inhibited. Consequently, Tufm-mediated mitophagy is suppressed while mitochondria-induced neuronal apoptosis is increased. In contrast, the knockin of a lactylation-deficient TufmK286R mutant in mice rescues the mitochondrial distribution of Tufm and Tufm-mediated mitophagy, and improves functional outcome after CCI. Likewise, mild hypothermia, as a critical therapeutic method in neuroprotection, helps in downregulating Tufm lactylation, increasing Tufm-mediated mitophagy, mitigating neuronal apoptosis, and eventually ameliorating the outcome of TBI. A novel molecular mechanism in neuronal apoptosis, TBI-initiated Tufm lactylation suppressing mitophagy, is thus revealed.

An EED/PRC2-H19 Loop Regulates Cerebellar Development.

EED (embryonic ectoderm development) is a core subunit of the polycomb repressive complex 2 (PRC2), which senses the trimethylation of histone H3 lysine 27 (H3K27). However, its biological function in cerebellar development remains unknown. Here, we show that EED deletion from neural stem cells (NSCs) or cerebellar granule cell progenitors (GCPs) leads to reduced GCPs proliferation, cell death, cerebellar hypoplasia, and motor deficits in mice. Joint profiling of transcripts and ChIP-seq analysis in cerebellar granule cells reveals that EED regulates bunches of genes involved in cerebellar development. EED ablation exhibits overactivation of a developmental repressor long non-coding RNA H19. Importantly, an obvious H3K27ac enrichment is found at Ctcf, a trans-activator of H19, and H3K27me3 enrichment at the H19 imprinting control region (ICR), suggesting that EED regulates H19 in an H3K27me3-dependent manner. Intriguingly, H19 deletion reduces EED expression and the reprogramming of EED-mediated H3K27me3 profiles, resulting in increased proliferation, differentiation, and decreased apoptosis of GCPs. Finally, molecular and genetic evidence provides that increased H19 expression is responsible for cerebellar hypoplasia and motor defects in EED mutant mice. Thus, this study demonstrates that EED, H19 forms a negative feedback loop, which plays a crucial role in cerebellar morphogenesis and controls cerebellar development.

Progressive Optic Neuropathy in Hydrocephalic Ccdc13 Mutant Mice Caused by Impaired Axoplasmic Transport at the Optic Nerve Head.

Optic nerve head (ONH) atrophy is frequently associated with hydrocephalic conditions. Cerebrospinal fluid (CSF)-containing meninges form a subarachnoid space that terminates at the ONH, which physically impacts it. This study aims to characterize optic neuropathy in congenital hydrocephalic mice with genetic disruption of the Ccdc13 gene. The ccdc13 germline knockout mice were generated. The hydrocephalus phenotype and subarachnoid space surrounding the optic nerve were evaluated using routine histology and Evans blue stain. Optic neuropathy was examined with immunohistochemistry and transmission electron microscopy (TEM). Axon transport was indicated by cholera toxin subunit B (CTB) fluorescence conjugate. Retinal function was evaluated by electroretinography (ERG), and Ccdc13 expression was revealed by a knock-in Gfp reporter. Ccdc13 mutant mice manifested hydrocephalus at birth. ONH displacement, or negative cupping, and enlarged subarachnoid space at the optic terminus occurred as early as 1 month after birth. Intraocular pressure (IOP) was normal. Optic neuropathy was first observed at the ONH, followed by a distal-to-proximal progression of optic nerve pathology indicated by alteration of axonal ultrastructure and deposition of unphosphorylated neurofilament heavy chain. Anterograde axonal transport was also hampered. Retinal ganglion cell (RGC) function was compromised as early as postnatal day 21 (P21), along with reduced neurofilament heavy chain expression. Optic neuropathy caused by disruption of Ccd13 was non-cell autonomous, stemming from hydrocephalus with presumed high intracranial pressure (ICP), which physically impacts the ONH by increasing the translaminar pressure gradient. We provided knowledge of optic neuropathy from a congenital mouse model for hydrocephalus. The hydrocephalus in mice could damage the ONH by increasing the translaminar pressure gradient and negative cupping, leading to impairment in axoplasmic transport and RGC pathology. Our findings highlight the importance of the interplay between IOP and ICP in the development of glaucoma.

Molecular basis of neurodegeneration in a mouse model of Polr3-related disease

Pathogenic variants in subunits of RNA polymerase (Pol) III cause a spectrum of Polr3-related neurodegenerative diseases including 4H leukodystrophy. Disease onset occurs from infancy to early adulthood and is associated with a variable range and severity of neurological and non-neurological features. The molecular basis of Polr3-related disease pathogenesis is unknown. We developed a postnatal whole-body mouse model expressing pathogenic Polr3a mutations to examine the molecular mechanisms by which reduced Pol III transcription results primarily in central nervous system phenotypes. Polr3a mutant mice exhibit behavioral deficits, cerebral pathology and exocrine pancreatic atrophy. Transcriptome and immunohistochemistry analyses of cerebra during disease progression show a reduction in most Pol III transcripts, induction of innate immune and integrated stress responses and cell-type-specific gene expression changes reflecting neuron and oligodendrocyte loss and microglial activation. Earlier in the disease when integrated stress and innate immune responses are minimally induced, mature tRNA sequencing revealed a global reduction in tRNA levels and an altered tRNA profile but no changes in other Pol III transcripts. Thus, changes in the size and/or composition of the tRNA pool have a causal role in disease initiation. Our findings reveal different tissue- and brain region-specific sensitivities to a defect in Pol III transcription.

Molecular basis of neurodegeneration in a mouse model of Polr3-related disease.

Pathogenic variants in subunits of RNA polymerase (Pol) III cause a spectrum of Polr3-related neurodegenerative diseases including 4H leukodystrophy. Disease onset occurs from infancy to early adulthood and is associated with a variable range and severity of neurological and non-neurological features. The molecular basis of Polr3-related disease pathogenesis is unknown. We developed a postnatal whole-body mouse model expressing pathogenic Polr3a mutations to examine the molecular mechanisms by which reduced Pol III transcription results primarily in central nervous system phenotypes. Polr3a mutant mice exhibit behavioral deficits, cerebral pathology and exocrine pancreatic atrophy. Transcriptome and immunohistochemistry analyses of cerebra during disease progression show a reduction in most Pol III transcripts, induction of innate immune and integrated stress responses and cell-type-specific gene expression changes reflecting neuron and oligodendrocyte loss and microglial activation. Earlier in the disease when integrated stress and innate immune responses are minimally induced, mature tRNA sequencing revealed a global reduction in tRNA levels and an altered tRNA profile but no changes in other Pol III transcripts. Thus, changes in the size and/or composition of the tRNA pool have a causal role in disease initiation. Our findings reveal different tissue- and brain region-specific sensitivities to a defect in Pol III transcription.

Novel voltage-dependent Cl− channels in striatal medium spiny neurons are unrelated to ClC-1 or other known Ca2+-induced Cl− channel/transporter types

Intracellular chloride (Cl−) homeostasis is a critical regulator of neuronal excitability. Voltage-dependent neuronal Cl− channels remain the least understood in terms of their role as a source of Cl− entry controlling excitability. We have shown recently that striatal medium spiny neurons (MSNs) express a functional Cl− conducting ClC-1-like channel with properties similar but not identical to native ClC-1 channels (Yarotskyy, V., Lark, A.R.S., Nass S.R., Hahn, Y.K., Marone, M.G., McQuiston, A.R., Knapp, P.E., Hauser, K.F. (2022) Am. J. Physiol. Cell. Physiol. 322 (2022) C395-C409). Using a myotonic SWR/J-Clcn1adr-mto/J mouse model with a premature stop codon for the ClC-1 channel rendering it non-functional, we demonstrate that striatal MSNs isolated from wild type (wt) and homozygous mutant (adr) mouse embryos have identical voltage-dependent outwardly rectifying Cl− currents. In contrast and as expected, homozygous adr skeletal muscle flexor digitorum brevis (FDB) fibers display nominal macroscopic Cl− currents compared to heterozygous wild-type adr FDB fibers. Together, our findings demonstrate that the novel ClC-1-like channels in MSNs are unrelated to skeletal muscle-specific ClC-1 channels, and therefore represent a unique voltage-dependent neuronal Cl− channel of unknown identity.

Comprehensive phenotypic assessment of nonsense mutations in mitochondrial ND5 in mice.

Mitochondrial dysfunction induced by mitochondrial DNA (mtDNA) mutations has been implicated in various human diseases. A comprehensive analysis of mitochondrial genetic disorders requires suitable animal models for human disease studies. While gene knockout via premature stop codons is a powerful method for investigating the unique functions of target genes, achieving knockout of mtDNA has been rare. Here, we report the genotypes and phenotypes of heteroplasmic MT-ND5 gene-knockout mice. These mutant mice presented damaged mitochondrial cristae in the cerebral cortex, hippocampal atrophy, and asymmetry, leading to learning and memory abnormalities. Moreover, mutant mice are susceptible to obesity and thermogenetic disorders. We propose that these mtDNA gene-knockdown mice could serve as valuable animal models for studying the MT-ND5 gene and developing therapies for human mitochondrial disorders in the future.

BDNF and Cerebellar Ataxia.

Brain-derived neurotrophic factor (BDNF) has been proposed as a treatment for neurodegeneration, including diseases of the cerebellum, where BDNF levels or those of its main receptor, TrkB, are often diminished relative to controls, thereby serving as replacement therapy. Experimental evidence indicates that BDNF signaling countered cerebellar degeneration, sensorimotor deficits, or both, in transgenic ATXN1 mice mutated for ataxin-1, Cacna1a knock-in mice mutated for ataxin-6, mice injected with lentivectors encoding RNA sequences against human FXN into the cerebellar cortex, Kcnj6Wv (Weaver) mutant mice with granule cell degeneration, and rats with olivocerebellar transaction, similar to a BDNF-overexpressing transgenic line interbred with Cacng2stg mutant mice. In this regard, this study discusses whether BDNF is effective in cerebellar pathologies where BDNF levels are normal and whether it is effective in cases with combined cerebellar and basal ganglia damage.

Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.

Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel invivo model of mitochondrial myopathy. The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model invivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, exvivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes. Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls. SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in exvivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.