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.

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.

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.

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.

Transferring isolated mitochondria into tissue culture cells.

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a 'mitocytoplast' cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.

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.

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.

Nonreplicating intracellular bacterial vector for conjugative DNA transfer into mitochondria.

PURPOSE: We have previously shown that DNA constructs can be introduced into isolated mitochondria through the process of conjugative transfer from an E. coli host. We set out to generate a conjugative E. coli strain that would be able to introduce itself into the cytoplasm of a mammalian cell for the purpose of transferring DNA into the mitochondria in the cell. METHODS: We have now developed a method for making E. coli strains from which nonreplicating populations of daughter cells can be generated. We used this approach to modify a facultative intracellular enteroinvasive E. coli (EIEC) and introduced conjugative functions to this new strain. RESULTS: We demonstrate that this new strain can generate large populations of nonreplicating cells that are capable of conjugative transfer to other cells and can readily invade mammalian tissue culture cells, live in the cytoplasm of the cell for several days, and that do not kill the invaded mammalian cell. CONCLUSIONS: We successfully constructed an E. coli host suitable for intracellular conjugative transfer but, due to the lack of suitable mitochondrial screening or selectable markers, we have not yet been able to determine if these bacterial vectors can in fact transfer DNA into intracelluar mitochondria.

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.

Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli.

Now that many genomes have been sequenced and the products of newly identified genes have been annotated, the next goal is to engineer the desired phenotypes in organisms of interest. For the phenotypic engineering of microorganisms, we have developed novel artificial transcription factors (ATFs) capable of reprogramming innate gene expression circuits in Escherichia coli. These ATFs are composed of zinc finger (ZF) DNA-binding proteins, with distinct specificities, fused to an E. coli cyclic AMP receptor protein (CRP). By randomly assembling 40 different types of ZFs, we have constructed more than 6.4 x 10(4) ATFs that consist of 3 ZF DNA-binding domains and a CRP effector domain. Using these ATFs, we induced various phenotypic changes in E. coli and selected for industrially important traits, such as resistance to heat shock, osmotic pressure and cold shock. Genes associated with the heat-shock resistance phenotype were then characterized. These results and the general applicability of this platform clearly indicate that novel ATFs are powerful tools for the phenotypic engineering of microorganisms and can facilitate microbial functional genomic studies.

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.

PCR-based cloning of the complete mouse mitochondrial genome and stable engineering in Escherichia coli.

We have devised a method for cloning an entire mammalian mitochondrial genome (mtDNA) in Escherichia coli using PCR-based amplification and sequential ligation. Here we test this approach by cloning the complete mouse mtDNA. The mtDNA was divided into four to five fragments based on unique restriction enzyme sites and amplified by high-fidelity long-range DNA polymerase. The synthesized fragments were cloned individually to test their toxicity in the E. coli host and then combined sequentially into a vector containing the E. coli R6K origin of DNA replication. The synthetic complete mouse mtDNA clones were replicated stably and faithfully in E. coli when maintained at moderately low copy numbers per cell. The sequence integrity of the synthetic mouse mtDNA clones was confirmed by nucleotide sequencing; no mutations or rearrangements in the genome were found. This approach can facilitate the cloning of entire mammalian mitochondrial genomes in E. coli and assist in the introduction of desired modifications into the mitochondrial genome.

Intramitochondrial transfer and engineering of mammalian mitochondrial genomes in yeast.

Mitochondrial genomes (mtDNA) depend on the nuclear genome with which they have evolved to provide essential replication functions and have been known to replicate as xenotransplants only in the cells of closely related species. We now report that complete mouse mitochondrial genomes can be stably transplanted into the mitochondrial network in yeast devoid of their own mtDNA. Our analyses of these xenomitochondrial yeast cells show that they are accurately replicating intact mouse mtDNA genomes without rearrangement and that these mtDNA genomes have the same overall topology as the mtDNA present in the mouse mitochondrial network (i.e., circular monomers). Moreover, non-mtDNA replication and selection sequences required for maintaining the mitochondrial genomes in bacterial hosts are dispensable in these yeast mitochondria and could be efficiently and seamlessly removed by targeted homologous recombination within the mitochondria. These findings demonstrate that the yeast mtDNA replication system is capable of accurately replicating intact mammalian mtDNA genomes without sequence loss or rearrangement and that yeast mitochondria are a highly versatile host system for engineering complete mammalian mitochondrial genomes.

Factors other than hTau overexpression that contribute to tauopathy-like phenotype in rTg4510 mice.

The tauopathy-like phenotype observed in the rTg4510 mouse line, in which human tauP301L expression specifically within the forebrain can be temporally controlled, has largely been attributed to high overexpression of mutant human tau in the forebrain region. Unexpectedly, we found that in a different mouse line with a targeted-insertion of the same transgene driven by the same tetracycline-TransActivator (tTA) allele, but with even higher overexpression of tauP301L than rTg4510, atrophy and tau histopathology are delayed, and a different behavioral profile is observed. This suggests that it is not overexpression of mutant human tau alone that contributes to the phenotype in rTg4510 mice. Furthermore we show that the tauopathy-like phenotype seen in rTg4510 requires a ~70-copy tau-transgene insertion in a 244 kb deletion in Fgf14, a ~7-copy tTA-transgene insertion in a 508 kb deletion that disrupts another five genes, in addition to high transgene overexpression. We propose that these additional effects need to be accounted for in any studies using rTg4510.

Selection by drug resistance proteins located in the mitochondria of mammalian cells.

Transformation of mitochondria in mammalian cells is now a technical challenge. In this report, we demonstrate that the standard drug resistant genes encoding neomycin and hygromycin phosphotransferases can potentially be used as selectable markers for mammalian mitochondrial transformation. We re-engineered the drug resistance genes to express proteins targeted to the mitochondrial matrix and confirmed the location of the proteins in the cells by fusing them with GFP and by Western blot and mitochondrial content mixing analyses. We found that the mitochondrially targeted-drug resistance proteins confer resistance to high levels of G418 and hygromycin without affecting the viability of cells.

Targeted deletion of a single Sca8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits.

Spinocerebellar ataxia type 8 (SCA8) patients typically have a slowly progressive, adult-onset ataxia. SCA8 is dominantly inherited and is caused by large CTG repeat expansions in the untranslated antisense RNA of the Kelch-like 1 gene (KLHL1), but the molecular mechanism through which this expansion leads to disease is still unknown. To more fully characterize the underlying molecular mechanisms involved in SCA8, we developed a mouse model in which Klhl1 is deleted in either all tissues or is deleted specifically in Purkinje cells only. We found that mice that are either homozygous or heterozygous for the Klhl1 deletion have significant gait abnormalities at an early age and develop a significant loss of motor coordination by 24 weeks of age. This loss progresses more rapidly in homozygous knock-outs. Mice with Klhl1 specifically deleted in only Purkinje cells had a loss of motor coordination that was almost identical to the total-tissue deletion mice. Finally, we found significant Purkinje cell dendritic deficits, as measured by the thickness of the molecular layer, in all mice in which Klhl1 was deleted (both total and Purkinje cell-specific deletions) and an intermediate reduction in molecular layer thickness in mice with reduced levels of Klhl1 expression (heterozygous deletions). The results from this mouse model show that even a partial loss of Klhl1 function leads to degeneration of Purkinje cell function and indicates that loss of KLHL1 activity is likely to play a significant part in the underlying pathophysiology of SCA8.

The spinocerebellar ataxia 8 noncoding RNA causes neurodegeneration and associates with staufen in Drosophila.

Spinocerebellar Ataxia 8 (SCA8) appears unique among triplet repeat expansion-induced neurodegenerative diseases because the predicted gene product is a noncoding RNA. Little is currently known about the normal function of SCA8 in neuronal survival or how repeat expansion contributes to neurodegeneration. To investigate the molecular context in which SCA8 operates, we have expressed the human SCA8 noncoding RNA in Drosophila. SCA8 induces late-onset, progressive neurodegeneration in the Drosophila retina. Using this neurodegenerative phenotype as a sensitized background for a genetic modifier screen, we have identified mutations in four genes: staufen, muscle-blind, split ends, and CG3249. All four encode neuronally expressed RNA binding proteins conserved in Drosophila and humans. Although expression of both wild-type and repeat-expanded SCA8 induce neurodegeneration, the strength of interaction with certain modifiers differs between the two SCA8 backgrounds, suggesting that CUG expansions alter associations with specific RNA binding proteins. Our demonstration that SCA8 can recruit Staufen and that the interaction domain maps to the portion of the SCA8 RNA that undergoes repeat expansion in the human disease suggests a specific mechanism for SCA8 function and disease. Genetic modifiers identified in our SCA8-based screens may provide candidates for designing therapeutic interventions to treat this disease.

Interspecies mitochondrial fusion between mouse and human mitochondria is rapid and efficient.

A detailed molecular understanding of mitochondrial fusion and fission in mammalian cells is rapidly emerging. In this report, we demonstrate for the first time cross-species mitochondrial fusion between distantly related species using green and red fluorescent proteins targeted to the mitochondrial matrix. We found that mouse mitochondria were able to efficiently fuse to unmodified mitochondria of human cells and that the contents of the mitochondrial matrix were completely mixed in less than 4h. We also observed that mitochondria from the mtDNA-less (rho(0)) mouse cells can homogeneously fuse to the mitochondria of human cells. We were, however, unable to maintain human mitochondrial DNA in the mouse cells. These results indicate that mitochondrial fusion proteins in mouse and human cells have enough functional homology to mediate efficient cross-species mitochondrial fusion, but mouse nuclear and human mitochondrial genomes have not retained functional compatibility with one another.

Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system.

An increasing number of microbial genomes have been completely sequenced, and functional analyses of these genomic sequences are under way. To facilitate these analyses, we have developed a genome-engineering tool for determining essential genes and minimizing bacterial genomes. We made two large pools of independent transposon mutants in Escherichia coli using modified Tn5 transposons with two different selection markers and precisely mapped the chromosomal location of 800 of these transposons. By combining a mapped transposon mutation from each of the mutant pools into the same chromosome using phage P1 transduction and then excising the flanked genomic segment by Cre-mediated loxP recombination, we obtained E. coli strains in which large genomic fragments (59-117 kilobases) were deleted. Some of these individual deletions were then combined into a single "cumulative deletion strain" that lacked 287 open reading frames (313.1 kilobases) but that nevertheless exhibited normal growth under standard laboratory conditions.