Human ACE2 Gene Replacement Mice Support SARS-CoV-2 Viral Replication and Nonlethal Disease Progression.

Many mouse models of SARS-CoV-2 infection involve expression of the human ACE2 protein, the entry receptor for SARS-CoV-2 Spike protein, in mouse tissues. However, most of these models suffer from nonphysiological regulation of ACE2 expression, which can lead to atypically severe infections and aberrant sites of viral replication. In this report, we developed and characterized an ACE2 gene replacement (ACE2-GR) mouse strain in which the mouse Ace2 genomic locus was replaced by the entire human ACE2 gene locus, and we investigated the ability of these animals to respond to SARS-CoV-2 infection. We show that ACE2-GR mice support SARS-CoV-2 viral replication, but, in stark contrast to the widely used K18-hACE2 transgenic model, this infection leads to a mild disease with no detectable involvement of the CNS. Thus, ACE2-GR mice provide a novel, to our knowledge, model to explore immune responses and long-term consequences of SARS-CoV-2 infection.

A common Alu insertion in the 3'UTR of TMEM106B is associated with risk of dementia.

INTRODUCTION: Sequence variants in TMEM106B have been associated with an increased risk of developing dementia. METHODS: As part of our efforts to generate a set of mouse lines in which we replaced the mouse Tmem106b gene with a human TMEM106B gene comprised of either a risk or protective haplotype, we conducted an in-depth sequence analysis of these alleles. We also analyzed transcribed TMEM106B sequences using RNA-seq data (AD Knowledge portal) and full genome sequences (1000 Genomes). RESULTS: We identified an AluYb8 insertion in the 3' untranslated region (3'UTR) of the TMEM106B risk haplotype. We found this AluYb8 insertion in every risk haplotype analyzed, but not in either protective haplotypes or in non-human primates. DISCUSSION: We conclude that this risk haplotype arose early in human development with a single Alu-insertion event within a unique haplotype context. This AluYb8 element may act as a functional variant in conferring an increased risk of developing dementia. HIGHLIGHTS: We conducted an in-depth sequence analysis of (1) a risk and (2) a protective haplotype of the human TMEM106B gene. We also analyzed transcribed TMEM106B sequences using RNA-seq data (AD Knowledge Portal) and full genome sequences (1000 Genomes). We identified an AluYb8 insertion in the 3' untranslated region (3'UTR) of the TMEM106B risk haplotype. We found this AluYb8 insertion in every risk haplotype analyzed, but not in either protective haplotypes or in non-human primates. This AluYb8 element may act as a functional variant in conferring an increased risk of developing dementia.

Mapping SCA1 regional vulnerabilities reveals neural and skeletal muscle contributions to disease.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q;Nestin-Cre mice, which showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks of age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q;Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks of age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q;ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.

Gene replacement-Alzheimer's disease (GR-AD): Modeling the genetics of human dementias in mice.

INTRODUCTION: Genetic studies conducted over the past four decades have provided us with a detailed catalog of genes that play critical roles in the etiology of Alzheimer's disease (AD) and related dementias (ADRDs). Despite this progress, as a field we have had only limited success in incorporating this rich complexity of human AD/ADRD genetics findings into our animal models of these diseases. Our primary goal for the gene replacement (GR)-AD project is to develop mouse lines that model the genetics of AD/ADRD as closely as possible. METHODS: To do this, we are generating mouse lines in which the genes of interest are precisely and completely replaced in the mouse genome by their full human orthologs. RESULTS: Each model set consists of a control line with a wild-type human allele and variant lines that precisely match the human genomic sequence in the control line except for a high-impact pathogenic mutation or risk variant.

Delineating regional vulnerability in the neurodegenerative disease SCA1 using a conditional mutant ATXN1 mouse.

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ATXN1 protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockout mouse model ( f-ATXN1 146Q/2Q ) having mouse Atxn1 coding exons replaced by human exons encoding 146 glutamines. F-ATXN1 146Q/2Q mice manifest SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. CNS contributions to disease were revealed using ATXN1 146Q/2Q ; Nestin-Cre mice, that showed improved rotarod, open field and Barnes maze performances. Striatal contributions to motor deficits were examined using f-ATXN1 146Q/2Q ; Rgs9-Cre mice. Mice lacking striatal ATXN1 146Q/2Q had improved rotarod performance late in disease. Muscle contributions to disease were revealed in f-ATXN1 146Q/2Q ; ACTA1-Cre mice which lacked muscle pathology and kyphosis seen in f-ATXN1 146Q/2Q mice. Kyphosis was not improved in f-ATXN1 146Q/2Q ;Nestin - Cre mice. Thus, optimal SCA1 therapeutics will require targeting mutant ATXN1 toxic actions in multiple brain regions and muscle.

The Transcription Factor, α1ACT, Acts Through a MicroRNA Network to Regulate Neurogenesis and Cell Death During Neonatal Cerebellar Development.

MicroRNAs, a class of small RNA regulators, function throughout neurodevelopment, from neural stem cell neurogenesis to neuronal maturation, synaptic formation, and plasticity. α1ACT, a transcription factor (TF), plays a critical role in neonatal cerebellar development by regulating an ensemble of genes. Of these, ChIP-seq analysis matched near 50% genes directly regulated by α1ACT. Yet, more than half the regulated transcripts lacked direct interaction with α1ACT. To investigate whether α1ACT acts through a microRNA network, we studied α1ACT-associated simultaneous miRNA:mRNA transcriptome profiles, using miRNA-seq paired with RNA-seq. Thirty-one differentially expressed miRNAs (DEMs) associated with α1ACT-regulated differentially expressed genes (DEGs) were profiled in α1ACT-overexpressing PC12 cells and were further validated in neonatal transgenic mouse cerebellum overexpressing α1ACT in a context-dependent manner. Here, we also demonstrated that α1ACT facilitates neurogenesis and development of dendritic synapses and is partially a result of the downregulation of the miR-99 cluster, miR-143, miR-23, miR-146, miR-363, and miR-484. On the other hand, the miR-181, miR-125, and miR-708 clusters were upregulated by α1ACT, which inhibit MAPK signaling and cell death pathways by targeting Ask1, Odc1, Atf4, and Nuf2 for decreased expression. MiR-181a-5p was verified as the most abundant DEM in neonatal cerebellum, which was further induced by α1ACT. Overall, under α1ACT modulation, up-/downregulated miRNA clusters with their paired target genes may form a regulatory network controlling the balance between the neuronal proliferation, differentiation, and cell death in the cerebellum to promote neonatal development. Our findings concerning the α1ACT-related miRNA/mRNA expression profiles in neonatal cerebellum may inform future investigations for cerebellar development.

Genetic Deletion of KLHL1 Leads to Hyperexcitability in Hypothalamic POMC Neurons and Lack of Electrical Responses to Leptin.

Kelch-like 1 (KLHL1) is a neuronal actin-binding protein that modulates voltage-gated calcium channels. The KLHL1 knockout (KO) model displays altered calcium channel expression in various brain regions. We analyzed the electrical behavior of hypothalamic POMC (proopiomelanocortin) neurons and their response to leptin. Leptin's effects on POMC neurons include enhanced gene expression, activation of the ERK1/2 pathway and increased electrical excitability. The latter is initiated by activation of the Jak2-PI3K-PLC pathway, which activates TRPC1/5 (Transient Receptor Potential Cation) channels that in turn recruit T-type channel activity resulting in increased excitability. Here we report over-expression of CaV3.1 T-type channels in the hypothalamus of KLHL1 KO mice increased T-type current density and enhanced POMC neuron basal excitability, rendering them electrically unresponsive to leptin. Electrical sensitivity to leptin was restored by partial blockade of T-type channels. The overexpression of hypothalamic T-type channels in POMC neurons may partially contribute to the obese and abnormal feeding phenotypes observed in KLHL1 KO mice.

Developmental Pathogenicity of 4-Repeat Human Tau Is Lost with the P301L Mutation in Genetically Matched Tau-Transgenic Mice.

Tau is a microtubule-associated protein that becomes dysregulated in a group of neurodegenerative diseases called tauopathies. Differential tau isoforms, expression levels, promoters, and disruption of endogenous genes in transgenic mouse models of tauopathy make it difficult to draw definitive conclusions about the biological role of tau in these models. We addressed this shortcoming by characterizing the molecular and cognitive phenotypes associated with the pathogenic P301L tau mutation (rT2 mice) in relation to a genetically matched transgenic mouse overexpressing nonmutant (NM) 4-repeat (4R) human tau (rT1 mice). Both male and female mice were included in this study. Unexpectedly, we found that 4R NM human tau (hTau) exhibited abnormal dynamics in young mice that were lost with the P301L mutation, including elevated protein stability and hyperphosphorylation, which were associated with cognitive impairment in 5-month-old rT1 mice. Hyperphosphorylation of NM hTau was observed as early as 4 weeks of age, and transgene suppression for the first 4 or 12 weeks of life prevented abnormal molecular and cognitive phenotypes in rT1, demonstrating that NM hTau pathogenicity is specific to postnatal development. We also show that NM hTau exhibits stronger binding to microtubules than P301L hTau, and is associated with mitochondrial abnormalities. Overall, our genetically matched mice have revealed that 4R NM hTau overexpression is pathogenic in a manner distinct from classical aging-related tauopathy, underlining the importance of assaying the effects of transgenic disease-related proteins at appropriate stages in life.SIGNIFICANCE STATEMENT Due to differences in creation of transgenic lines, the pathological properties of the P301L mutation confers to the tau protein in vivo have remained elusive, perhaps contributing to the lack of disease-modifying therapies for tauopathies. In an attempt to characterize P301L-specific effects on tau biology and cognition in novel genetically matched transgenic mouse models, we surprisingly found that nonmutant human tau has development-specific pathogenic properties of its own. Our findings indicate that overexpression of 4-repeat human tau during postnatal development is associated with excessive microtubule binding, which may disrupt important cellular processes, such as mitochondrial dynamics, leading to elevated stability and hyperphosphorylation of tau, and eventual cognitive impairments.

Case Report: Early-Onset Behavioral Variant Frontotemporal Dementia in Patient With Retrotransposed Full-Length Transcript of Matrin-3 Variant 5.

Frontotemporal dementia (FTD) rarely occurs in individuals under the age of 30, and genetic causes of early-onset FTD are largely unknown. The current report follows a 27 year-old patient with no significant past medical history presenting with two years of progressive changes in behavior, rushed speech, verbal aggression, and social withdrawal. MRI and FDG-PET imaging of the brain revealed changes maximally in the frontal and temporal lobes, which along with the clinical features, are consistent with behavioral variant FTD. Next generation sequencing of a panel of 28 genes associated with dementia and amyotrophic lateral sclerosis (ALS) initially revealed a duplication of exon 15 in Matrin-3 (MATR3). Whole genome sequencing determined that this genetic anomaly was, in fact, a sequence corresponding with full-length MATR3 variant 5 inserted into chromosome 12, indicating retrotransposition from a messenger RNA intermediate. To our knowledge, this is a novel mutation of MATR3, as the majority of mutations in MATR3 linked to FTD-ALS are point mutations. Genomic DNA analysis revealed that this mutation is also present in one unaffected first-degree relative and one unaffected second-degree relative. This suggests that the mutation is either a disease-causing mutation with incomplete penetrance, which has been observed in heritable FTD, or a benign variant. Retrotransposons are not often implicated in neurodegenerative diseases; thus, it is crucial to clarify the potential role of this MATR3 variant 5 retrotransposition in early-onset FTD.