Generation and characterization of a knock-in mouse model for spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM).

Solute carrier family 1 member 4 (SLC1A4), also referred to as Alanine/Serine/Cysteine/Threonine-preferring Transporter 1 (ASCT1), is a sodium-dependent neutral amino acid transporter. It is expressed in many tissues, including the brain, where it is expressed primarily on astrocytes and plays key roles in neuronal differentiation and development, maintaining neurotransmitter homeostasis, and N-methyl-D-aspartate neurotransmission, through regulation of L- and D-serine. Mutations in SLC1A4 are associated with the rare autosomal recessive neurodevelopmental disorder spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM, OMIM 616657). Psychomotor development and speech are significantly impaired in these patients, and many develop seizures. We generated and characterized a knock-in mouse model for the most common mutant allele, which results in a single amino acid change (p.Glu256Lys, or E256K). Homozygous mutants had increased D-serine uptake in the brain, microcephaly, and thin corpus callosum and cortex layer 1. While p.E256K homozygotes showed some significant differences in exploratory behavior relative to wildtype mice, their performance in assays for motor coordination, endurance, learning, and memory was normal, and they showed no significant differences in long-term potentiation. Taken together, these results indicate that the impact of the p.E256K mutation on cognition and motor function is minimal in mice, but other aspects of SLC1A4 function in the brain are conserved. Mice homozygous for p.E256K may be a good model for understanding the developmental basis of the corpus callosum and microcephaly phenotypes observed in SPATCCM patients and assessing whether they are rescued by serine supplementation.

Generation and characterization of a knock-in mouse model for Spastic Tetraplegia, Thin Corpus Callosum, and Progressive Microcephaly (SPATCCM).

SLC1A4 (solute carrier family 1 member 4, also referred to as ASCT1, Alanine/Serine/Cysteine/Threonine-preferring Transporter 1) is a sodium-dependent neutral amino acid transporter. It is highly expressed in many tissues, including the brain, where it is expressed primarily on astrocytes and plays key roles in neuronal differentiation and development, maintaining neurotransmitter homeostasis, and N-methyl-D-aspartate (NMDA) neurotransmission, through regulation of L- and D-serine. Mutations in SLC1A4 are associated with the rare autosomal recessive neurodevelopmental disorder spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM, OMIM 616657). Psychomotor development and speech are significantly impaired in these patients, and many develop seizures. We generated and characterized a knock-in mouse model for the most common mutant allele, which results in a single amino acid change (p.Glu256Lys, or E256K). Homozygous mutants had increased D-serine uptake in the brain, microcephaly, and thin corpus callosum and cortex layer 1. While p.E256K homozygotes showed some significant differences in exploratory behavior relative to wildtype mice, their performance in assays for motor coordination, endurance, learning, and memory was normal, and they showed no significant differences in long-term potentiation. Taken together, these results indicate that some aspects of SLC1A4 function in brain development are conserved between mice and humans, but the impact of the p.E256K mutation on cognition and motor function is minimal in mice.

35th International Mammalian Genome Conference: meeting overview.

The 35th International Mammalian Genome Conference (IMGC) was held on July 17-20, 2022 in Vancouver, British Columbia; this conference marked the first time the International Mammalian Genome Society (IMGS) hosted a meeting in Canada. Scientists from around the world participated to share advances in genetics and genomics research across mammalian species. A diverse attendance of pre-doctoral and post-doctoral trainees, young investigators, established researchers, clinicians, bioinformaticians, and computational biologists enjoyed a rich scientific program selected from 88 abstracts in the fields of cancer, conservation genetics, developmental biology, epigenetics, human disease modeling, immunology, infectious diseases, systems genetics, translational biology, and technological advances.

Gene-teratogen interactions influence the penetrance of birth defects by altering Hedgehog signaling strength.

Birth defects result from interactions between genetic and environmental factors, but the mechanisms remain poorly understood. We find that mutations and teratogens interact in predictable ways to cause birth defects by changing target cell sensitivity to Hedgehog (Hh) ligands. These interactions converge on a membrane protein complex, the MMM complex, that promotes degradation of the Hh transducer Smoothened (SMO). Deficiency of the MMM component MOSMO results in elevated SMO and increased Hh signaling, causing multiple birth defects. In utero exposure to a teratogen that directly inhibits SMO reduces the penetrance and expressivity of birth defects in Mosmo-/- embryos. Additionally, tissues that develop normally in Mosmo-/- embryos are refractory to the teratogen. Thus, changes in the abundance of the protein target of a teratogen can change birth defect outcomes by quantitative shifts in Hh signaling. Consequently, small molecules that re-calibrate signaling strength could be harnessed to rescue structural birth defects.

A Membrane-Tethered Ubiquitination Pathway Regulates Hedgehog Signaling and Heart Development.

The etiology of congenital heart defects (CHDs), which are among the most common human birth defects, is poorly understood because of its complex genetic architecture. Here, we show that two genes implicated in CHDs, Megf8 and Mgrn1, interact genetically and biochemically to regulate the strength of Hedgehog signaling in target cells. MEGF8, a transmembrane protein, and MGRN1, a RING superfamily E3 ligase, assemble to form a receptor-like ubiquitin ligase complex that catalyzes the ubiquitination and degradation of the Hedgehog pathway transducer Smoothened. Homozygous Megf8 and Mgrn1 mutations increased Smoothened abundance and elevated sensitivity to Hedgehog ligands. While mice heterozygous for loss-of-function Megf8 or Mgrn1 mutations were normal, double heterozygous embryos exhibited an incompletely penetrant syndrome of CHDs with heterotaxy. Thus, genetic interactions can arise from biochemical mechanisms that calibrate morphogen signaling strength, a conclusion broadly relevant for the many human diseases in which oligogenic inheritance is emerging as a mechanism for heritability.

Chronic and age-dependent effects of the spongiform neurodegeneration-associated MGRN1 E3 ubiquitin ligase on mitochondrial homeostasis.

Spongiform encephalopathy is an intriguing yet poorly understood neuropathology characterized by vacuoles, demyelination, and gliosis. It is observed in patients with prion disease, primary mitochondrial disease, HIV-1 infection of the brain, and some inherited disorders, but the underlying mechanism of disease remains unclear. The brains of mice lacking the MGRN1 E3 ubiquitin ligase develop vacuoles by 9 months of age. MGRN1-dependent ubiquitination has been reported to regulate mitofusin 1 and GP78, suggesting MGRN1 may have a direct effect on mitochondrial homeostasis. Here, we demonstrate that some MGRN1 localizes to mitochondria, most likely due to N-myristoylation, and mitochondria in cells from Mgrn1 null mutant mice display fragmentation and depolarization without recruitment of the parkin E3 ubiquitin ligase. The late onset of pathology in the brains of Mgrn1 null mutant mice suggests that a further, age-dependent effect on mitochondrial homeostasis may be required to trigger vacuolation. Parkin protein and mRNA levels showed a significant decline in the brains of Mgrn1 null mutant mice by 12 months of age. To test whether loss of parkin triggers vacuolation through a synergistic effect, we generated Mgrn1; parkin double mutant mice. By 1 month of age, their brains demonstrated more severe mitochondrial dysfunction than Mgrn1 null mutants, but there was no effect on the age-of-onset of spongiform neurodegeneration. Expression of the ATF4 transcription factor, a key regulator of the mitochondrial stress response, also declined in the brains of aged Mgrn1 null mutant mice. Together, the data presented here indicate that loss of MGRN1 has early, direct effects on mitochondrial homeostasis and late, indirect effects on the ability of cells to respond to mitochondrial stress.

Meeting report: 31st International Mammalian Genome Conference, Mammalian Genetics and Genomics: From Molecular Mechanisms to Translational Applications.

High on the Heidelberg hills, inside the Advanced Training Centre of the European Molecular Biology Laboratory (EMBL) campus with its unique double-helix staircase, scientists gathered for the EMBL conference "Mammalian Genetics and Genomics: From Molecular Mechanisms to Translational Applications," organized in cooperation with the International Mammalian Genome Society (IMGS) and the Mouse Molecular Genetics (MMG) group. The conference attracted 205 participants from 30 countries, representing 6 of the 7 continents-all except Antarctica. It was a richly diverse group of geneticists, clinicians, and bioinformaticians, with presentations by established and junior investigators, including many trainees. From the 24th-27th of October 2017, they shared exciting advances in mammalian genetics and genomics research, from the introduction of cutting-edge technologies to descriptions of translational studies involving highly relevant models of human disease.

Oligodendroglial deletion of ESCRT-I component TSG101 causes spongiform encephalopathy.

BACKGROUND INFORMATION: Vacuolation of the central nervous system (CNS) is observed in patients with transmissible spongiform encephalopathy, HIV-related encephalopathy and some inherited diseases, but the underlying cellular mechanisms remain poorly understood. Mice lacking the mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase develop progressive, widespread spongiform degeneration of the CNS. MGRN1 ubiquitinates and regulates tumour susceptibility gene 101 (TSG101), a central component of the endosomal trafficking machinery. As loss of MGRN1 is predicted to cause partial TSG101 loss-of-function, we hypothesised that CNS vacuolation in Mgrn1 null mice may be caused by the accumulation of multi-cisternal endosome-like 'class E' vacuolar protein sorting (vps) compartments similar to those observed in Tsg101-depleted cells in culture. RESULTS: To test this hypothesis, Tsg101 was deleted from mature oligodendroglia in vivo. This resulted in severe spongiform encephalopathy, histopathologically similar to that observed in Mgrn1 null mutant mice but with a more rapid onset. Vacuoles in the brains of Tsg101-deleted and Mgrn1 mutant mice labelled with endosomal markers, consistent with an endosomal origin. Vacuoles in the brains of mice inoculated with Rocky Mountain Laboratory (RML) prions did not label with these markers, indicating a different origin, consistent with previously published studies that indicate RML prions have a primary effect on neurons and cause vacuolation in an MGRN1-independent manner. Oligodendroglial deletion of Rab7, which mediates late endosome-to-lysosome trafficking and autophagosome-lysosome fusion, did not cause spongiform change. CONCLUSIONS: Our data suggest that the formation of multi-cisternal 'class E' vps endosomal structures in oligodendroglia leads to vacuolation. SIGNIFICANCE: This work provides the first evidence that disrupting multi-vesicular body formation in oligodendroglia can cause white matter vacuolation and demyelination. HIV is known to hijack the endosomal sorting machinery, suggesting that HIV infection of the CNS may also act through this pathway to cause encephalopathy.

Disrupted SOX10 function causes spongiform neurodegeneration in gray tremor mice.

Mice homozygous for the gray tremor (gt) mutation have a pleiotropic phenotype that includes pigmentation defects, megacolon, whole body tremors, sporadic seizures, hypo- and dys-myelination of the central nervous system (CNS) and peripheral nervous system, vacuolation of the CNS, and early death. Vacuolation similar to that caused by prions was originally reported to be transmissible, but subsequent studies showed the inherited disease was not infectious. The gt mutation mapped to distal mouse chromosome 15, to the same region as Sox10, which encodes a transcription factor with essential roles in neural crest survival and differentiation. As dominant mutations in mouse or human SOX10 cause white spotting and intestinal aganglionosis, we screened the Sox10 coding region for mutations in gt/gt DNA. An adenosine to guanine transversion was identified in exon 2 that changes a highly conserved glutamic acid residue in the SOX10 DNA binding domain to glycine. This mutant allele was not seen in wildtype mice, including the related GT/Le strain, and failed to complement a Sox10 null allele. Gene expression analysis revealed significant down-regulation of genes involved in myelin lipid biosynthesis pathways in gt/gt brains. Knockout mice for some of these genes develop CNS vacuolation and/or myelination defects, suggesting that their down-regulation may contribute to these phenotypes in gt mutants and could underlie the neurological phenotypes associated with peripheral demyelinating neuropathy-central dysmyelinating leukodystrophy-Waardenburg syndrome-Hirschsprung disease, caused by mutations in human SOX10.

Lack of prolidase causes a bone phenotype both in human and in mouse.

The degradation of the main fibrillar collagens, collagens I and II, is a crucial process for skeletal development. The most abundant dipeptides generated from the catabolism of collagens contain proline and hydroxyproline. In humans, prolidase is the only enzyme able to hydrolyze dipeptides containing these amino acids at their C-terminal end, thus being a key player in collagen synthesis and turnover. Mutations in the prolidase gene cause prolidase deficiency (PD), a rare recessive disorder. Here we describe 12 PD patients, 9 of whom were molecularly characterized in this study. Following a retrospective analysis of all of them a skeletal phenotype associated with short stature, hypertelorism, nose abnormalities, microcephaly, osteopenia and genu valgum, independent of both the type of mutation and the presence of the mutant protein was identified. In order to understand the molecular basis of the bone phenotype associated with PD, we analyzed a recently identified mouse model for the disease, the dark-like (dal) mutant. The dal/dal mice showed a short snout, they were smaller than controls, their femurs were significantly shorter and pQCT and µCT analyses of long bones revealed compromised bone properties at the cortical and at the trabecular level in both male and female animals. The differences were more pronounce at 1 month being the most parameters normalized by 2 months of age. A delay in the formation of the second ossification center was evident at postnatal day 10. Our work reveals that reduced bone growth was due to impaired chondrocyte proliferation and increased apoptosis rate in the proliferative zone associated with reduced hyperthrophic zone height. These data suggest that lack of prolidase, a cytosolic enzyme involved in the final stage of protein catabolism, is required for normal skeletogenesis especially at early age when the requirement for collagen synthesis and degradation is the highest.

RML prions act through Mahogunin and Attractin-independent pathways.

While the conversion of the normal form of prion protein to a conformationally distinct pathogenic form is recognized to be the primary cause of prion disease, it is not clear how this leads to spongiform change, neuronal dysfunction and death. Mahogunin ring finger-1 (Mgrn1) and Attractin (Atrn) null mutant mice accumulate vacuoles throughout the brain that appear very similar to those associated with prion disease, but they do not accumulate the protease-resistant scrapie form of the prion protein or become sick. A study demonstrating an interaction between cytosolically-exposed prion protein and MGRN1 suggested that disruption of MGRN1 function may contribute to prion disease pathogenesis, but we recently showed that neither loss of MGRN1 nor MGRN1 overexpression influences the onset or progression of prion disease following intracerebral inoculation with Rocky Mountain Laboratory prions. Here, we show that loss of ATRN also has no effect on prion disease onset or progression and discuss possible mechanisms that could cause vacuolation of the central nervous system in Mgrn1 and Atrn null mutant mice and whether the same pathways might contribute to this intriguing phenotype in prion disease.

Levels of the Mahogunin Ring Finger 1 E3 ubiquitin ligase do not influence prion disease.

Prion diseases are rare but invariably fatal neurodegenerative disorders. They are associated with spongiform encephalopathy, a histopathology characterized by the presence of large, membrane-bound vacuolar structures in the neuropil of the brain. While the primary cause is recognized as conversion of the normal form of prion protein (PrP(C)) to a conformationally distinct, pathogenic form (PrP(Sc)), the cellular pathways and mechanisms that lead to spongiform change, neuronal dysfunction and death are not known. Mice lacking the Mahogunin Ring Finger 1 (MGRN1) E3 ubiquitin ligase develop spongiform encephalopathy by 9 months of age but do not become ill. In cell culture, PrP aberrantly present in the cytosol was reported to interact with and sequester MGRN1. This caused endo-lysosomal trafficking defects similar to those observed when Mgrn1 expression is knocked down, implicating disrupted MGRN1-dependent trafficking in the pathogenesis of prion disease. As these defects were rescued by over-expression of MGRN1, we investigated whether reduced or elevated Mgrn1 expression influences the onset, progression or pathology of disease in mice inoculated with PrP(Sc). No differences were observed, indicating that disruption of MGRN1-dependent pathways does not play a significant role in the pathogenesis of transmissible spongiform encephalopathy.

Cardiomyocyte calcium cycling in a naturally occurring German shepherd dog model of inherited ventricular arrhythmia and sudden cardiac death.

OBJECTIVE: To further characterize arrhythmic mechanisms in German shepherd dogs (GSDs) affected with inherited ventricular arrhythmias by evaluating intracellular calcium cycling and expression of calcium handling genes. ANIMALS: Twenty five GSDs, 9 backcross dogs, and 6 normal mongrel dogs (controls) were studied. The GSDs and backcross dogs were from a research colony of inherited ventricular arrhythmias. The control research dogs were purchased. METHODS: Action potentials (APs) and pseudo-electrocardiograms (ECG) were recorded from left ventricular (LV) wedge preparations of GSDs and normal dogs. Midmyocardial (Mid) LV cells from GSDs and normal mongrels were isolated by enzymatic digestion. Cells were either field stimulated or voltage clamped and calcium transients were measured by confocal microscopy using the indicator Fluo-3AM. Expression of calcium handling genes was measured by quantitative RT-PCR. RESULTS: Mean calcium transient decay (tau) was not different between affected GSDs and control dogs, but striking cell-to-cell variability for tau was observed within affected GSDs and between affected GSDs and controls (P < 0.0001 each); within-dog variability accounted for 75% of total variability. Calcium sparks and afterdepolarizations occurred in GSD but not control cells. ATP2A2/SERCA2a expression was significantly reduced (P = 0.0063) in affected GSDs and inversely correlated (P = 0.0006) with severity of ventricular arrhythmias. CONCLUSIONS: German shepherd dogs with inherited ventricular arrhythmias have electrophysiologic abnormalities in calcium cycling associated with reduced ATP2A2/SERCA2a expression. These animals provide a unique opportunity to study calcium remodeling at the genetic and molecular level in familial ventricular arrhythmias.

MGRN1-dependent pigment-type switching requires its ubiquitination activity but not its interaction with TSG101 or NEDD4.

Mice lacking the E3 ubiquitin ligase mahogunin ring finger-1 (MGRN1) have a pleiotropic phenotype that includes spongiform neurodegeneration, embryonic patterning defects, and dark fur due to a defect in pigment-type switching. The only MGRN1 ubiquitination target identified to date is tumor susceptibility gene 101 (TSG101), a component of the endosomal trafficking machinery. Here, we show that MGRN1 also interacts with but does not ubiquitinate NEDD4, a HECT-domain ubiquitin ligase involved in endosomal trafficking. Using transgenesis in mice, we demonstrate that pigment-type switching likely requires MGRN1's ubiquitin ligase activity but not its ability to bind TSG101 or NEDD4. This indicates that MGRN1-dependent ubiquitination of an as-yet unidentified target protein is required for agouti-mediated melanocortin signaling.

Functional annotation and ENU.

Functional annotation of every gene in the mouse genome is a herculean task that requires a multifaceted approach. Many large-scale initiatives are contributing to this undertaking. The International Knockout Mouse Consortium (IKMC) plans to mutate every protein-coding gene, using a combination of gene trapping and gene targeting in embryonic stem cells. Many other groups are performing using the chemical mutagen ethylnitrosourea (ENU) or transpon-based systems to induce mutations, screening offspring for phenovariants and identifying the causative mutations. A recent paper in BMC Research Notes by Arnold et al. presents data from an ENU-based mutagenesis project that provides not only some of the first phenotype-genotype information for a large number of genes, but also a trove of information, all publicly available, that demonstrates the specificity and efficiency of ENU mutagenesis.

Evaluation of a fibrillin 2 gene haplotype associated with hip dysplasia and incipient osteoarthritis in dogs.

OBJECTIVE: To determine whether a mutation in the fibrillin 2 gene (FBN2) is associated with canine hip dysplasia (CHD) and osteoarthritis in dogs. ANIMALS: 1,551 dogs. Procedures-Hip conformation was measured radiographically. The FBN2 was sequenced from genomic DNA of 21 Labrador Retrievers and 2 Greyhounds, and a haplotype in intron 30 of FBN2 was sequenced in 90 additional Labrador Retrievers and 143 dogs of 6 other breeds. Steady-state values of FBN2 mRNA and control genes were measured in hip joint tissues of fourteen 8-month-old Labrador Retriever-Greyhound crossbreeds. RESULTS: The Labrador Retrievers homozygous for a 10-bp deletion haplotype in intron 30 of FBN2 had significantly worse CHD as measured via higher distraction index and extended-hip joint radiograph score and a lower Norberg angle and dorsolateral subluxation score. Among 143 dogs of 6 other breeds, those homozygous for the same deletion haplotype also had significantly worse radiographic CHD. Among the 14 crossbred dogs, as the dorsolateral subluxation score decreased, the capsular FBN2 mRNA increased significantly. Those dogs with incipient hip joint osteoarthritis had significantly increased capsular FBN2 mRNA, compared with those dogs without osteoarthritis. Dogs homozygous for the FBN2 deletion haplotype had significantly less FBN2 mRNA in their femoral head articular cartilage. CONCLUSIONS AND CLINICAL RELEVANCE: The FBN2 deletion haplotype was associated with CHD. Capsular gene expression of FBN2 was confounded by incipient secondary osteoarthritis in dysplastic hip joints. Genes influencing complex traits in dogs can be identified by genome-wide screening, fine mapping, and candidate gene screening.

Developmental cardiac hypertrophy in a mouse model of prolidase deficiency.

BACKGROUND: Hypertrophic cardiomyopathy, characterized by thickened ventricular walls and reduced ventricular chamber volume, is a common cause of sudden cardiac death in young people. Most inherited forms result from mutations in genes encoding sarcomeric proteins. METHODS: Histologic analysis identified embryonic cardiac hypertrophy in dark-like mutant mice. BrdU analysis was performed to measure proliferation and cardiomyocytes were isolated to measure cell size. The dark-like mutation was identified by positional cloning. RESULTS: The dark-like mutation causes cardiomyocyte hypertrophy due to loss-of-function of peptidase d (Pepd), which encodes prolidase, a cytosolic enzyme that recycles proline for collagen re-synthesis. Prolidase deficiency is a rare autosomal recessive disease in humans with a broad phenotypic spectrum not reported to include heart defects, but a conserved role for prolidase in heart development was confirmed by morpholino knockdown in zebrafish. We tested the hypothesis that loss of prolidase function disrupts collagen-mediated integrin signaling and determined that the levels of several key integrin transducers were reduced in the hearts of dark-like mutant embryos. CONCLUSIONS: This work identifies dark-like mice as a model of prolidase deficiency that will be valuable for studying the role of proline metabolism in normal physiology and disease processes, and suggests that integrin signaling may regulate the onset of hypertrophic cardiac growth.

Shades of meaning: the pigment-type switching system as a tool for discovery.

The pigment-type switching system, which controls whether melanocytes produce black/brown eumelanin or yellow/red pheomelanin, is responsible for many familiar coat coloration patterns in both domestic and wild mammals. In conjunction with the accessory proteins attractin and mahogunin ring finger 1, endogenous agonists and antagonists modulate signaling by the melanocortin 1 receptor to determine pigment type. Mutations in pigment-type switching genes can cause a variety of pleiotropic phenotypes, and these are often similar between mutants at different loci because the proteins encoded by these genes act together as part of conserved molecular pathways that are deployed in multiple biological contexts. When this is the case, pigment-type switching provides a powerful model system for elucidating the shared molecular mechanisms underlying the pigmentary and non-pigmentary phenotypes. This review outlines the current understanding of the pigment-type switching pathway and discusses the opportunities that exist for exploring the molecular basis of pleiotropic phenotypes using this model system.