Knockout Mouse Line Research Articles

Mouse models of Slc35a2 brain mosaicism reveal mechanisms of mild malformations of cortical development with oligodendroglial hyperplasia in epilepsy.

Brain somatic variants in SLC35A2 were recently identified as a genetic marker for mild malformations of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). The role of SLC35A2 in cortical development and the contributions of abnormal neurons and oligodendrocytes to seizure activity in MOGHE remain largely unexplored. Here, we generated a novel Slc35a2 floxed allele, which we used to develop two Slc35a2 conditional knockout mouse lines targeting (1) the Emx1 dorsal telencephalic lineage (excitatory neurons and glia) and (2) the Olig2 lineage (oligodendrocytes). We examined brain structure, behavior, and seizure activity. Knockout of Slc35a2 from the Emx1 lineage, which targets both cortical neurons and oligodendrocytes, resulted in early lethality and caused abnormal cortical development, increased oligodendroglial cell density, early onset seizures, and developmental delays akin to what is observed in patients with MOGHE. By tracing neuronal development with 5-Ethynyl-2'-deoxyuridine (EdU) birthdating experiments, we found that Slc35a2 deficiency disrupts corticogenesis by delaying radial migration of neurons from the subventricular zone. To discern the contributions of oligodendrocytes to these phenotypes, we knocked out Slc35a2 from the Olig2 lineage. This recapitulated the increased oligodendroglial cell density and resulted in abnormal electroencephalographic activity, but without a clear seizure phenotype, suggesting Slc35a2 deficiency in neurons is required for epileptogenesis. This study presents two novel Slc35a2 conditional knockout mouse models and characterizes the effects on brain development, behavior, and epileptogenesis. Together, these results demonstrate a direct causal role for SLC35A2 in MOGHE-like phenotypes, including a critical role in neuronal migration during brain development, and identify neurons as key contributors to SLC35A2-related epileptogenesis.

The Intestinal Transporter SLC30A1 Plays a Critical Role in Regulating Systemic Zinc Homeostasis.

The essential trace element, zinc, regulates virtually all aspects of cellular physiology, particularly cell proliferation and survival. Diverse families of metal transporters, metallothioneins, and metal-responsive transcriptional regulators are linked to zinc homeostasis. However, the mechanism underlying the regulation of systemic zinc homeostasis remains largely unknown. Here, it is reported that the intestinal transporter SLC30A1 plays an essential role in maintaining systemic zinc homeostasis. Using several lines of tissue-specific knockout mice, it is found that intestinal Slc30a1 plays a critical role in survival. Furthermore, lineage tracing reveals that Slc30a1 is localized to the basolateral membrane of intestinal epithelial cells (IECs). It is also found that Slc30a1 safeguards both intestinal barrier integrity and systemic zinc homeostasis. Finally, an integrative analysis of the cryo-EM structure and site-specific mutagenesis of human SLC30A1 are performed and a zinc transport mechanism of SLC30A1 unique within the SLC30A family, with His43 serving as a critical residue for zinc selectivity, is identified.

TRPC5 controls the adrenaline-mediated counter regulation of hypoglycemia.

Hypoglycemia triggers autonomic and endocrine counter-regulatory responses to restore glucose homeostasis, a response that is impaired in patients with diabetes and its long-term complication hypoglycemia-associated autonomic failure (HAAF). We show that insulin-evoked hypoglycemia is severely aggravated in mice lacking the cation channel proteins TRPC1, TRPC4, TRPC5, and TRPC6, which cannot be explained by alterations in glucagon or glucocorticoid action. By using various TRPC compound knockout mouse lines, we pinpointed the failure in sympathetic counter-regulation to the lack of the TRPC5 channel subtype in adrenal chromaffin cells, which prevents proper adrenaline rise in blood plasma. Using electrophysiological analyses, we delineate a previously unknown signaling pathway in which stimulation of PAC1 or muscarinic receptors activates TRPC5 channels in a phospholipase-C-dependent manner to induce sustained adrenaline secretion as a crucial step in the sympathetic counter response to insulin-induced hypoglycemia. By comparing metabolites in the plasma, we identified reduced taurine levels after hypoglycemia induction as a commonality in TRPC5-deficient mice and HAAF patients.

Impact of essential genes on the success of genome editing experiments generating 3313 new genetically engineered mouse lines

The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps – generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.

Impaired hippocampal plasticity associated with loss of recycling endosomal SLC9A6/NHE6 is ameliorated by the TrkB agonist 7,8-dihydroxyflavone

Proper maintenance of intracellular vesicular pH is essential for cargo trafficking during synaptic function and plasticity. Mutations in the SLC9A6 gene encoding the recycling endosomal pH regulator (Na+, K+)/H+ exchanger isoform 6 (NHE6) are causal for Christianson syndrome (CS), a severe form of X-linked intellectual disability. NHE6 expression is also downregulated in other neurodevelopmental and neurodegenerative disorders, such as autism spectrum disorder and Alzheimer's disease, suggesting its dysfunction could contribute more broadly to the pathophysiology of other neurological conditions. To understand how ablation of NHE6 function leads to severe learning impairments, we assessed synaptic structure, function, and cellular mechanisms of learning in a novel line of Nhe6 knockout (KO) mice expressing a plasma membrane-tethered green fluorescent protein within hippocampal neurons. We uncovered significant reductions in dendritic spines density, AMPA receptor (AMPAR) expression, and AMPAR-mediated neurotransmission in CA1 pyramidal neurons. The neurons also failed to undergo functional and structural enhancement during long-term potentiation (LTP). Significantly, the selective TrkB agonist 7,8-dihydroxyflavone restored spine density as well as functional and structural LTP in KO neurons. TrkB activation thus may act as a potential clinical intervention to ameliorate cognitive deficits in CS and other neurodegenerative disorders.

Loss of CCDC188 causes male infertility with defects in the sperm head-neck connection in mice.

Acephalic spermatozoa syndrome (ASS) represents a rare genetic and reproductive disease, which is defined as semen composed of mostly headless spermatozoa. The connecting piece in the neck region, also known as the head-to-tail coupling apparatus (HTCA), plays a crucial role in the tight linkage between the sperm head and tail. Dysfunction of this structure can lead to separation of sperm heads and tails, and male infertility. Using the mouse as an experimental model, several proteins have been identified as associated with the HTCA and disruption of these proteins causes acephalic spermatozoa. However, the molecular mechanism underlying this morphologic anomaly and HTCA remains elusive. In this study, we focused on coiled-coil domain containing 188 (Ccdc188), which shows testis-enriched expression. To elucidate the physiological role of CCDC188, we generated a knockout (KO) mouse line using the CRISPR/Cas9 system. Ccdc188 KO male mice were sterile, indicating that CCDC188 is indispensable for male fertility. Most Ccdc188-null spermatozoa were acephalic. Transmission electron microscopy revealed that while the sperm HTCA could assemble properly without CCDC188, the HTCA failed to attach to the nucleus during spermiogenesis, leading to sperm head and neck separation. In addition, we found almost all of the spermatozoa in the cauda epididymis lacked a mitochondrial sheath. Taken together, we demonstrated that CCDC188 plays a crucial role in forming a tight sperm head-neck junction.

Identification of novel genes regulating the development of the palate.

The International Mouse Phenotyping Consortium (IMPC) has generated thousands of knockout mouse lines, many of which exhibit embryonic or perinatal lethality. Using micro-computed tomography (micro-CT), the IMPC has created and publicly released 3D image datasets of embryos from these lethal and subviable lines. In this study, we leveraged this dataset to screen homozygous null mutants for anomalies in secondary palate development. We analyzed optical sections from 2,987 embryos at embryonic days E15.5 and E18.5, representing 484 homozygous mutant lines. Our analysis identified 45 novel genes implicated in palatogenesis. Gene set enrichment analysis highlighted biological processes and pathways relevant to palate development and uncovered 18 genes jointly regulating the development of the eye and the palate. These findings present a valuable resource for further research, offer novel insights into the molecular mechanisms underlying palatogenesis, and provide important context for understanding the etiology of rare human congenital disorders involving simultaneous malformations of the palate and other organs, including the eyes, ears, kidneys, and lungs.

Determining the requirements of proper N-linked glycosylation during placental morphogenesis via examination of Alg3 and Stt3b knockout mouse lines.

Determining the requirements of proper N-linked glycosylation during placental morphogenesis via examination of Alg3 and Stt3b knockout mouse lines.

Generation of Astrocyte-specific BEST1 Conditional Knockout Mouse with Reduced Tonic GABA Inhibition in the Brain.

Bestrophin-1 (BEST1) is a Ca2+-activated anion channel known for its role in astrocytes. Best1 is permeable to gliotransmitters, including GABA, to contribute to tonic GABA inhibition and modulate synaptic transmission in neighboring neurons. Despite the crucial functions of astrocytic BEST1, there is an absence of genetically engineered cell-type specific conditional mouse models addressing these roles. In this study, we developed an astrocyte-specific BEST1 conditional knock-out (BEST1 aKO) mouse line. Using the embryonic stem cell (ES cell) targeting method, we developed Best1 floxed mice (C57BL/6JCya-Best1em1flox/Cya), which have exon 3, 4, 5, and 6 of Best1 flanked by two loxP sites. By crossing with hGFAP-CreERT2 mice, we generated Best1 floxed/hGFAP-CreERT2 mice, which allowed for the tamoxifen-inducible deletion of Best1 under the human GFAP promoter. We characterized its features across various brain regions, including the striatum, hippocampal dentate gyrus (HpDG), and Parafascicular thalamic nucleus (Pf). Compared to the Cre-negative control, we observed significantly reduced BEST1 protein expression in immunohistochemistry (IHC) and tonic GABA inhibition in patch clamp recordings. The reduction in tonic GABA inhibition was 66.7% in the striatum, 46.4% in the HpDG, and 49.6% in the Pf. Our findings demonstrate that the BEST1 channel in astrocytes significantly contributes to tonic inhibition in the local brain areas. These mice will be valuable for future studies not only on tonic GABA release but also on tonic release of gliotransmitters mediated by astrocytic BEST1.

ZG16 impacts gut microbiota-associated intestinal inflammation and pulmonary mucosal function through bacterial metabolites

Zymogen granule 16 (ZG16) is a secretory glycoprotein found in zymogen granules, which also plays an important role in colorectal inflammation and cancer. Herein, a ZG16 gene knock-out (ZG16−/−) mouse line was established and we found that ZG16 deletion damaged the intestinal mucosal barrier and gut microbiota, which resulted in low-level inflammation and further promoted the development of ulcerative colitis and inflammation-related colorectal cancer. Meanwhile, a metabolomics analysis on mouse feces showed that the metabolites significantly differed between ZG16−/− and WT mice, which were important mediators of host-microbiota communication and may impact the pulmonary inflammation of mice. Indeed, ZG16−/− mice showed more severe inflammation in a bronchial asthma model. Taken together, the results demonstrate that ZG16 plays a pivotal role in inhibiting inflammation and regulating immune responses in colorectum and lung of experimental animals, which may provide a better understanding of the underlying mechanism of human inflammatory diseases associated with ZG16.

Mll4 regulates tooth enamel development.

Amelogenesis, or enamel development, is a highly regulated process that leads to the formation of tooth enamel, which is critical for protecting teeth from decay and wear. Disruptions in the amelogenesis process can result in amelogenesis imperfecta, a group of genetic conditions characterized by inadequately formed enamel. This condition can include enamel hypoplasia, marked by thinning or underdevelopment of the enamel layer. Mutations in the MLL4 ( KMT2D ) gene, which encodes a histone H3-lysine 4-methyltransferase, are associated with Kabuki syndrome, a developmental disorder that can involve dental anomalies such as enamel hypoplasia. However, the specific role of MLL4 in amelogenesis and its underlying mechanisms remain poorly understood. To investigate the role of Mll4 in amelogenesis, we generated a conditional knockout mouse line with an ectoderm-specific deletion of Mll4 ( Krt14-Cre;Mll4 fl/fl , or Mll4 -cKO) and examined the gross, radiographic, histological, cellular, and molecular features in these mice. Micro-computed tomography and scanning electron microscopy analyses revealed that adult Mll4 -cKO mice exhibited 100% penetrant amelogenesis imperfecta, characterized by hypoplastic and hypomineralized enamel, partially phenocopying human Kabuki syndrome. Additionally, Mll4 -cKO neonates developed molar tooth germs with minor cusp shape alterations and mild delays in ameloblast differentiation at birth. RNA-seq analysis of the first molar tooth germ at birth revealed that approximately 33.7% of known amelogenesis-related genes were significantly downregulated in the Mll4 -cKO teeth. Intersection with Mll4 CUT&RUN-seq results identified 8 overlapping genes directly targeted by Mll4. Re-analysis of a single-cell RNA-seq dataset in the developing mouse incisor teeth revealed distinct roles for these genes in Mll4-regulated differentiation across various cell subtypes within the dental epithelium. Among these genes, Satb1 and Sp6 are likely directly targeted by Mll4 during the differentiation of pre-ameloblasts into ameloblasts. Taken together, we propose that Mll4 plays a crucial role in amelogenesis by directly activating key genes involved in ameloblast differentiation.

Mice lacking Astn2 have ASD-like behaviors and altered cerebellar circuit properties

Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. Individuals with ASTN2 mutations exhibit neurodevelopmental disorders, including autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), learning difficulties, and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity, repetitive behaviors, altered behavior in the three-chamber test, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors are also prominent in Astn2 cKO animals, but they do not show altered behavior in the three-chamber test. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrate a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.

Zinc-Finger Protein ZFP488 Regulates the Timing of Oligodendrocyte Myelination and Remyelination.

Oligodendrocyte myelination and remyelination after injury are intricately regulated by various intrinsic and extrinsic factors, including transcriptional regulators. Among these, the zinc-finger protein ZFP488 is an oligodendrocyte-enriched transcriptional regulator that promotes oligodendrocyte differentiation in the developing neural tube and in oligodendroglial cell lines. However, the specific in vivo genetic requirements for ZFP488 during oligodendrocyte development and remyelination have not been defined. To address this gap, we generated a lineage-traceable ZFP488 knock-out mouse line, wherein an H2b-GFP reporter replaces the ZFP488-coding region. Using these mice of either sex, we examined the dynamics of ZFP488 expression from the endogenous promoter in the developing central nervous system (CNS). We observed a unique expression pattern in the oligodendrocyte lineage, with ZFP488 expression particularly enriched in differentiated oligodendrocytes. ZFP488 loss resulted in delayed myelination in the developing CNS and impaired remyelination after demyelinating injury in the brain. Integrated transcriptomic and genomic profiling further revealed that ZFP488 loss decreased the expression of myelination-associated genes but not oligodendrocyte progenitor-associated genes, suggesting that ZFP488 serves as a positive regulator of myelination by regulating maturation programs. Thus, our genetic loss-of-function study revealed that ZFP488 regulates a stage-dependent differentiation program that controls the timing of CNS myelination and remyelination.

Abstract We146: CHD8 is Essential for Postnatal Heart Function through Preserving Mitochondrial Integrity

Objective: Epigenetic regulation plays a crucial role in heart development and disease, although its mechanisms remain elusive. Known mutations in epigenetic regulators have been linked to cardiac disease. For instance, the mutation in the chromodomain helicase DNA-binding (CHD) protein 7 gene, which leads to CHARGE syndrome, is known to results in heart defects. However, the role of CHD8, an interacting partner of CHD7, in the heart has not been investigated. This study aims to explore the function of CHD8 in the heart. Methods and Results: We generated a cardiac-specific Chd8 conditional knockout mouse line (Chd8 cKO) by breeding Chd8 fl/fl mice with aMHC Cre mice. Over 60% of Chd8 cKO mice died between 7 and 9 months of age, with increased heart weight to bodyweight ratio and reduced cardiac function, increased fibrosis, and signs of dilated cardiomyopathy. Electron microscopy analysis revealed disordered sarcomeres and escalated mitophagy in CHD8-deficient hearts. Using Genome-wide transcriptome analysis though RNA sequencing, we discovered that genes involved in mitochondrial oxidative phosphorylation and muscle contraction were down-regulated in Chd8 cKO hearts. Proteins in the electron transport chain complexes were down regulated and mitophagy marker LC3 increased in the Chd8 cKO hearts compared with the control mice. Upregulated pathways include inflammatory response suggesting that the innate immune response may contribute to dilated cardiomyopathy. Conclusions: Our findings underscore CHD8 as a key regulator of cardiac function and regeneration by influencing chromatin accessibility of genes involved in mitochondrial oxidative phosphorylation and contractility. Overall, our study provides new insights into the epigenetic regulation of cardiac function and the CHD8’s role in heart development and disease. It implies that activating CHD8 may be a promising therapeutic strategy for treating dilated cardiomyopathy and other heart-related diseases.

Generation of an Armcx1 Conditional Knockout Mouse.

Armadillo repeat-containing X-linked protein-1 (Armcx1) is a poorly characterized transmembrane protein that regulates mitochondrial transport in neurons. Its overexpression has been shown to induce neurite outgrowth in embryonic neurons and to promote retinal ganglion cell (RGC) survival and axonal regrowth in a mouse optic nerve crush model. In order to evaluate the functions of endogenous Armcx1 in vivo, we have created a conditional Armcx1 knockout mouse line in which the entire coding region of the Armcx1 gene is flanked by loxP sites. This Armcx1fl line was crossed with mouse strains in which Cre recombinase expression is driven by the promoters for β-actin and Six3, in order to achieve deletion of Armcx1 globally and in retinal neurons, respectively. Having confirmed deletion of the gene, we proceeded to characterize the abundance and morphology of RGCs in Armcx1 knockout mice aged to 15 months. Under normal physiological conditions, no evidence of aberrant retinal or optic nerve development or RGC degeneration was observed in these mice. The Armcx1fl mouse should be valuable for future studies investigating mitochondrial morphology and transport in the absence of Armcx1 and in determining the susceptibility of Armcx1-deficient neurons to degeneration in the setting of additional heritable or environmental stressors.

USP28 protects development of inflammation in mouse intestine by regulating STAT5 phosphorylation and IL22 production in T lymphocytes.

Ubiquitin-specific proteases (USPs), a large subset of more than 50 deubiquitinase proteins, have recently emerged as promising targets in cancer. However, their role in immune cell regulation, particularly in T cell activation, differentiation, and effector functions, remains largely unexplored. We utilized a USP28 knockout mouse line to study the effect of USP28 on T cell activation and function, and its role in intestinal inflammation using the dextran sulfate sodium (DSS)-induced colitis model and a series of in vitro assays. Our results show that USP28 exerts protective effects in acute intestinal inflammation. Mechanistically, USP28 knockout mice (USP28-/-) exhibited an increase in total T cells mainly due to an increased CD8+ T cell content. Additionally, USP28 deficiency resulted in early defects in T cell activation and functional changes. Specifically, we observed a reduced expression of IL17 and an increase in inducible regulatory T (iTreg) suppressive functions. Importantly, activated T cells lacking USP28 showed increased STAT5 phosphorylation. Consistent with these findings, these mice exhibited increased susceptibility to acute DSS-induced intestinal inflammation, accompanied by elevated IL22 cytokine levels. Our findings demonstrate that USP28 is essential for T cell functionality and protects mice from acute DSS-induced colitis by regulating STAT5 signaling and IL22 production. As a T cell regulator, USP28 plays a crucial role in immune responses and intestinal health.

The interferon-induced protein, IFIT2, requires RNA-binding activity and neuronal expression to protect mice from intranasal vesicular stomatitis virus infection.

The interferon (IFN) system protects mammals from diseases caused by virus infections. IFN synthesis is induced by pattern recognition receptor signaling pathways activated by virus infection. IFN is secreted from the infected cells and acts upon neighboring cells by binding cell surface receptors and triggering induction of hundreds of IFN-stimulated genes and proteins, many of which block different steps of virus replication. The IFN-induced tetratricopeptide repeat proteins (IFIT) are a family of RNA-binding proteins. We and others have previously reported that IFIT2 protects mice from many neurotropic RNA viruses; indeed, Ifit2-/- mice are very susceptible to intranasal or subcutaneous infections with vesicular stomatitis virus (VSV). Here, using a newly generated conditional knockout mouse, we report that ablation of Ifit2 expression only in neuronal cells was sufficient to render mice susceptible to neuropathogenesis caused by intranasal, but not subcutaneous, infection of VSV. Another genetically modified mouse line, expressing a mutant IFIT2 that cannot bind RNA, was as susceptible to VSV infection as Ifit2-/- mice. These results demonstrated that IFIT2 RNA-binding activity is essential for protecting mice against neurological diseases caused by intranasal infection of VSV.IMPORTANCEInterferon's (IFN's) antiviral effects are mediated by the proteins encoded by the interferon-stimulated genes. IFN-stimulated genes (IFIT2) is one such protein, which inhibits replication of many RNA viruses in the mouse brain and the resultant neuropathology. Our study sheds light on how IFIT2 works. By ablating Ifit2 expression only in neuronal cells, using a newly generated conditional knockout mouse line, we showed that Ifit2 induction in the neurons of the infected mouse was necessary for antiviral function of interferon. IFIT2 has no known enzyme activity; instead, it functions by binding to cellular or viral proteins or RNAs. We engineered a new mouse line that expressed a mutant IFIT2 that cannot bind RNA. These mice were very susceptible to infection with vesicular stomatitis virus indicating that the RNA-binding property of IFIT2 was essential for its antiviral function in vivo.

Loss of DIS3L in the initial segment is dispensable for sperm maturation in the epididymis and male fertility

DIS3L, a catalytic exoribonuclease associated with the cytoplasmic exosome complex, degrades cytoplasmic RNAs and is implicated in cancers and certain other diseases in humans. Epididymis plays a pivotal role in the transport, maturation, and storage of sperm required for male fertility. However, it remains unclear whether DIS3L-mediated cytoplasmic RNA degradation plays a role in epididymis biology and functioning. Herein, we fabricated a Dis3l conditional knockout (Dis3l cKO) mouse line in which DIS3L was ablated from the principal cells of the initial segment (IS). Morphological analyses showed that spermatogenesis and IS differentiation occurred normally in Dis3l cKO mice. Additionally, the absence of DIS3L had no dramatic influence on the transcriptome of IS. Moreover, the sperm count, morphology, motility, and acrosome reaction frequency in Dis3l cKO mice were comparable to that of the control, indicating that the Dis3l cKO males had normal fertility. Collectively, our genetic model demonstrates that DIS3L inactivation in the IS is nonessential for sperm maturation and male fertility.

Mitochondrial DNA Instability Supersedes Parkin Mutations in Driving Mitochondrial Proteomic Alterations and Functional Deficits in Polg Mutator Mice.

Mitochondrial quality control is essential in mitochondrial function. To examine the importance of Parkin-dependent mechanisms in mitochondrial quality control, we assessed the impact of modulating Parkin on proteome flux and mitochondrial function in a context of reduced mtDNA fidelity. To accomplish this, we crossed either the Parkin knockout mouse or ParkinW402A knock-in mouse lines to the Polg mitochondrial mutator line to generate homozygous double mutants. In vivo longitudinal isotopic metabolic labeling was followed by isolation of liver mitochondria and synaptic terminals from the brain, which are rich in mitochondria. Mass spectrometry and bioenergetics analysis were assessed. We demonstrate that slower mitochondrial protein turnover is associated with loss of mtDNA fidelity in liver mitochondria but not synaptic terminals, and bioenergetic function in both tissues is impaired. Pathway analysis revealed loss of mtDNA fidelity is associated with disturbances of key metabolic pathways, consistent with its association with metabolic disorders and neurodegeneration. Furthermore, we find that loss of Parkin leads to exacerbation of Polg-driven proteomic consequences, though it may be bioenergetically protective in tissues exhibiting rapid mitochondrial turnover. Finally, we provide evidence that, surprisingly, dis-autoinhibition of Parkin (ParkinW402A) functionally resembles Parkin knockout and fails to rescue deleterious Polg-driven effects. Our study accomplishes three main outcomes: (1) it supports recent studies suggesting that Parkin dependence is low in response to an increased mtDNA mutational load, (2) it provides evidence of a potential protective role of Parkin insufficiency, and (3) it draws into question the therapeutic attractiveness of enhancing Parkin function.

Knockout mice with pituitary malformations help identify human cases of hypopituitarism

BackgroundCongenital hypopituitarism (CH) and its associated syndromes, septo-optic dysplasia (SOD) and holoprosencephaly (HPE), are midline defects that cause significant morbidity for affected people. Variants in 67 genes are associated with CH, but a vast majority of CH cases lack a genetic diagnosis. Whole exome and whole genome sequencing of CH patients identifies sequence variants in genes known to cause CH, and in new candidate genes, but many of these are variants of uncertain significance (VUS).MethodsThe International Mouse Phenotyping Consortium (IMPC) is an effort to establish gene function by knocking-out all genes in the mouse genome and generating corresponding phenotype data. We used mouse embryonic imaging data generated by the Deciphering Mechanisms of Developmental Disorders (DMDD) project to screen 209 embryonic lethal and sub-viable knockout mouse lines for pituitary malformations.ResultsOf the 209 knockout mouse lines, we identified 51 that have embryonic pituitary malformations. These genes not only represent new candidates for CH, but also reveal new molecular pathways not previously associated with pituitary organogenesis. We used this list of candidate genes to mine whole exome sequencing data of a cohort of patients with CH, and we identified variants in two unrelated cases for two genes, MORC2 and SETD5, with CH and other syndromic features.ConclusionsThe screening and analysis of IMPC phenotyping data provide proof-of-principle that recessive lethal mouse mutants generated by the knockout mouse project are an excellent source of candidate genes for congenital hypopituitarism in children.