Adult Mouse Model Research Articles

The Regulation of ZIP8 by Dietary Manganese in Mice.

ZIP8 is a newly identified manganese transporter. A lack of functional ZIP8 results in severe manganese deficiency in both humans and mice, indicating that ZIP8 plays a crucial role in maintaining body manganese homeostasis. Despite a well-acknowledged connection between ZIP8 and manganese metabolism, how ZIP8 is regulated under high-manganese conditions remains unclear. The primary goal of this study was to examine the regulation of ZIP8 by high-manganese intake. We used both neonatal and adult mouse models in which mice were supplied with dietary sources containing either a normal or a high level of manganese. We discovered that high-manganese intake caused a reduction in liver ZIP8 protein in young mice. Since a decrease in hepatic ZIP8 leads to reduced manganese reabsorption from the bile, our study identified a novel mechanism for the regulation of manganese homeostasis under high-manganese conditions: high dietary manganese intake results in a decrease in ZIP8 in the liver, which in turn decreases the reabsorption of manganese from the bile to prevent manganese overload in the liver. Interestingly, we found that a high-manganese diet did not cause a decrease in hepatic ZIP8 in adult animals. To determine the potential reason for this age-dependent variation, we compared the expressions of liver ZIP8 in 3-week-old and 12-week-old mice. We found that liver ZIP8 protein content in 12-week-old mice decreases when compared with that of 3-week-old mice under normal conditions. Overall, results from this study provide novel insights to facilitate the understanding of ZIP8's function in regulating manganese metabolism.

Comparative Efficacy of Mayaro Virus-Like Particle Vaccines Produced in Insect or Mammalian Cells.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes often debilitating rheumatic disease in tropical Central and South America. There are currently no licensed vaccines or antiviral drugs available for MAYV disease. Here, we generated Mayaro virus-like particles (VLPs) using the scalable baculovirus-insect cell expression system. High-level secretion of MAYV VLPs in the culture fluid of Sf9 insect cells was achieved, and particles with a diameter of 64 to 70 nm were obtained after purification. We characterize a C57BL/6J adult wild-type mouse model of MAYV infection and disease and used this model to compare the immunogenicity of VLPs from insect cells with that of VLPs produced in mammalian cells. Mice received two intramuscular immunizations with 1 μg of nonadjuvanted MAYV VLPs. Potent neutralizing antibody responses were generated against the vaccine strain, BeH407, with comparable activity seen against a contemporary 2018 isolate from Brazil (BR-18), whereas neutralizing activity against chikungunya virus was marginal. Sequencing of BR-18 illustrated that this virus segregates with genotype D isolates, whereas MAYV BeH407 belongs to genotype L. The mammalian cell-derived VLPs induced higher mean neutralizing antibody titers than those produced in insect cells. Both VLP vaccines completely protected adult wild-type mice against viremia, myositis, tendonitis, and joint inflammation after MAYV challenge. IMPORTANCE Mayaro virus (MAYV) is associated with acute rheumatic disease that can be debilitating and can evolve into months of chronic arthralgia. MAYV is believed to have the potential to emerge as a tropical public health threat, especially if it develops the ability to be efficiently transmitted by urban mosquito vectors, such as Aedes aegypti and/or Aedes albopictus. Here, we describe a scalable virus-like particle vaccine against MAYV that induced neutralizing antibodies against a historical and a contemporary isolate of MAYV and protected mice against infection and disease, providing a potential new intervention for MAYV epidemic preparedness.

The human skin organ culture model as an optimal complementary tool for murine pemphigus models.

Pemphigus is a severe autoimmune bullous disease of the skin and/or mucous membranes caused by autoantibodies that mainly target the adhesion proteins desmoglein (Dsg) 3 and/or Dsg1. Clinically, pemphigus is characterized by flaccid blistering, leading to severe water and electrolyte loss. Before the introduction of corticosteroid treatment, the disease turned out to be fatal in many cases. Despite recent therapeutic improvements, treatment of pemphigus patients is centred on prolonged systemic immunosuppression and remains challenging. Current drug development for pemphigus has a strong focus on disease-causing B cells and autoantibodies and, more recently, also on modulating autoantibody-induced tissue pathology and keratinocyte signalling. This drug development requires reliable pre-clinical model systems replicating the pathogenesis of the human disease. Among those are neonatal and adult mouse models based on the transfer of Dsg3, Dsg1/3 or Dsg1-specific autoantibodies. To reduce the number of animal experiments, we recently established a standardized human skin organ culture (HSOC) model for pemphigus. This model reproduces the clinical phenotype of autoantibody-induced tissue pathology in pemphigus vulgaris. For induction of blistering, a recombinant single-chain variable fragment (scFv) targeting both Dsg1 and 3 is injected into pieces of human skin (obtained from plastic surgeries). Further characterization of the HSOC model demonstrated that key morphologic, molecular and immunologic features of pemphigus are being replicated. Thus, the pemphigus HSOC model is an excellent alternative to pemphigus animal model systems that are based on the transfer of (auto)antibodies.

Transcriptional reprogramming restores UBE3A brain-wide and rescues behavioral phenotypes in an Angelman syndrome mouse model

Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. The UBE3A gene is paternally imprinted in brain neurons. Clinical features of AS are primarily due to the loss of maternally expressed UBE3A in the brain. A healthy copy of paternal UBE3A is present in the brain but is silenced by a long non-coding antisense transcript (UBE3A-ATS). Here, we demonstrate that an artificial transcription factor (ATF-S1K) can silence Ube3a-ATS in an adult mouse model of Angelman syndrome (AS) and restore endogenous physiological expression of paternal Ube3a. A single injection of adeno-associated virus (AAV) expressing ATF-S1K (AAV-S1K) into the tail vein enabled whole-brain transduction and restored UBE3A protein in neurons to ∼25% of wild-type protein. The ATF-S1K treatment was highly specific to the target site with no detectable inflammatory response 5weeks after AAV-S1K administration. AAV-S1K treatment of AS mice showed behavioral rescue in exploratory locomotion, a task involving gross and fine motor abilities, similar to low ambulation and velocity in AS patients. The specificity and tolerability of a single injection of AAV-S1K therapy for AS demonstrate the use of ATFs as a promising translational approach for AS.

Antisense oligonucleotide therapy rescues disturbed brain rhythms and sleep in juvenile and adult mouse models of Angelman syndrome.

UBE3A encodes ubiquitin protein ligase E3A, and in neurons its expression from the paternal allele is repressed by the UBE3A antisense transcript (UBE3A-ATS). This leaves neurons susceptible to loss-of-function of maternal UBE3A. Indeed, Angelman syndrome, a severe neurodevelopmental disorder, is caused by maternal UBE3A deficiency. A promising therapeutic approach to treating Angelman syndrome is to reactivate the intact paternal UBE3A by suppressing UBE3A-ATS. Prior studies show that many neurological phenotypes of maternal Ube3a knockout mice can only be rescued by reinstating Ube3a expression in early development, indicating a restricted therapeutic window for Angelman syndrome. Here, we report that reducing Ube3a-ATS by antisense oligonucleotides in juvenile or adult maternal Ube3a knockout mice rescues the abnormal electroencephalogram (EEG) rhythms and sleep disturbance, two prominent clinical features of Angelman syndrome. Importantly, the degree of phenotypic improvement correlates with the increase of Ube3a protein levels. These results indicate that the therapeutic window of genetic therapies for Angelman syndrome is broader than previously thought, and EEG power spectrum and sleep architecture should be used to evaluate the clinical efficacy of therapies.

Developmental exposure to thyroid disruptors: misprogramming of the brain's stem cells in later life?

Developmental exposure to thyroid disruptors: misprogramming of the brain's stem cells in later life?

Approaches and Vectors for Efficient Cochlear Gene Transfer in Adult Mouse Models

Inner ear gene therapy using adeno-associated viral vectors (AAVs) in neonatal mice can alleviate hearing loss in mouse models of deafness. However, efficient and safe transgene delivery to the adult mouse cochlea is critical for the effectiveness of AAV-mediated therapy. Here, we examined three gene delivery approaches including posterior semicircular canal (PSCC) canalostomy, round window membrane (RWM) injection, and tubing-RWM+PSCC (t-RP) in adult mice. Transduction rates and survival rates of cochlear hair cells were analyzed, hearing function was recorded, AAV distribution in the sagittal brain sections was evaluated, and cochlear histopathologic images were appraised. We found that an injection volume of 1 μL AAV through the PSCC is safe and highly efficient and does not impair hearing function in adult mice, but local injection allows AAV vectors to spread slightly into the brain. We then tested five AAV serotypes (PHP.eB, IE, Anc80L65, AAV2, and PHP.s) in parallel and observed the most robust eGFP expression in inner hair cells, outer hair cells, and spiral ganglion neurons throughout the cochlea after AAV-Anc80L65 injection. Thus, PSCC-injected Anc80L65 provides a foundation for gene therapy in the adult cochlea and will facilitate the development of inner ear gene therapy.

Neuroprotective Epigenetic Changes Induced by Maternal Treatment with an Inhibitor of Soluble Epoxide Hydrolase Prevents Early Alzheimer′s Disease Neurodegeneration

Modulation of Alzheimer's disease (AD) risk begins early in life. During embryo development and postnatal maturation, the brain receives maternal physiological influences and establishes epigenetic patterns that build its level of resilience to late-life diseases. The soluble epoxide hydrolase inhibitor N-[1-(1-oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl] urea (TPPU), reported as ant-inflammatory and neuroprotective against AD pathology in the adult 5XFAD mouse model of AD, was administered to wild-type (WT) female mice mated to heterozygous 5XFAD males during gestation and lactation. Two-month-old 5XFAD male and female offspring of vehicle-treated dams showed memory loss as expected. Remarkably, maternal treatment with TPPU fully prevented memory loss in 5XFAD. TPPU-induced brain epigenetic changes in both WT and 5XFAD mice, modulating global DNA methylation (5-mC) and hydroxymethylation (5-hmC) and reducing the gene expression of some histone deacetylase enzymes (Hdac1 and Hdac2), might be on the basis of the long-term neuroprotection against cognitive impairment and neurodegeneration. In the neuropathological analysis, both WT and 5XFAD offspring of TPPU-treated dams showed lower levels of AD biomarkers of tau hyperphosphorylation and microglia activation (Trem2) than the offspring of vehicle-treated dams. Regarding sex differences, males and females were similarly protected by maternal TPPU, but females showed higher levels of AD risk markers of gliosis and neurodegeneration. Taken together, our results reveal that maternal treatment with TPPU impacts in preventing or delaying memory loss and AD pathology by inducing long-term modifications in the epigenetic machinery and its marks.

Pathogenicity and Structural Basis of Zika Variants with Glycan Loop Deletions in the Envelope Protein.

The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.

Renal Injury Response in an Adult Pkd2 Mouse Model

Renal Injury Response in an Adult Pkd2 Mouse Model

Loss of E-cadherin is causal to pathologic changes in chronic lung disease

Epithelial cells line the lung mucosal surface and are the first line of defense against toxic exposures to environmental insults, and their integrity is critical to lung health. An early finding in the lung epithelium of patients with chronic obstructive pulmonary disease (COPD) is the loss of a key component of the adherens junction protein called E-cadherin. The cause of this decrease is not known and could be due to luminal insults or structural changes in the small airways. Irrespective, it is unknown whether the loss of E-cadherin is a marker or a driver of disease. Here we report that loss of E-cadherin is causal to the development of chronic lung disease. Using cell-type-specific promoters, we find that knockout of E-cadherin in alveolar epithelial type II but not type 1 cells in adult mouse models results in airspace enlargement. Furthermore, the knockout of E-cadherin in airway ciliated cells, but not club cells, increase airway hyperreactivity. We demonstrate that strategies to upregulate E-cadherin rescue monolayer integrity and serve as a potential therapeutic target.

Effects of Psychotropic Medication on Somatic Sterol Biosynthesis of Adult Mice.

Polypharmacy is commonly used to treat psychiatric disorders. These combinations often include drugs with sterol biosynthesis inhibiting side effects, including the antipsychotic aripiprazole (ARI), and antidepressant trazodone (TRZ). As the effects of psychotropic medications are poorly understood across the various tissue types to date, we investigated the effects of ARI, TRZ, and ARI + TRZ polypharmacy on the post-lanosterol biosynthesis in three cell lines (Neuro2a, HepG2, and human dermal fibroblasts) and seven peripheral tissues of an adult mouse model. We found that both ARI and TRZ strongly interfere with the function of 7-dehydrocholesterol reductase enzyme (DHCR7) and lead to robust elevation in 7-dehydrocholesterol levels (7-DHC) and reduction in desmosterol (DES) across all cell lines and somatic tissues. ARI + TRZ co-administration resulted in summative or synergistic effects across the utilized in vitro and in vivo models. These findings suggest that at least some of the side effects of ARI and TRZ are not receptor mediated but arise from inhibiting DHCR7 enzyme activity. We propose that interference with sterol biosynthesis, particularly in the case of simultaneous utilization of medications with such side effects, can potentially interfere with functioning or development of multiple organ systems, warranting further investigation.

Vitamin D3 supplementation may attenuate morphological and molecular abnormalities of the olfactory bulb in a mouse model of Down syndrome

Individuals with Down syndrome (DS) exhibit impaired olfactory function and are at a higher risk of developing Alzheimer's disease (AD). Olfactory dysfunction may be an early clinical symptom of AD. Recent studies have demonstrated that vitamin D3 (VD3) exerts neuroprotective effects in mouse models of AD. In this study, we investigated the effects of VD3 on the morphology, immunolocalization, and markers involved in neuropathogenic processes, apoptosis, proliferation, cell survival, and clearance of amyloid peptides, along with neuronal markers in the olfactory bulb (OB) of an adult female mouse model of DS. Morphological and molecular analyses revealed that trisomic mice exhibited a volume reduction in the external plexiform layer, a decrease in the number of mitral and granule cells, and an increase in the expression of amyloid-β 42, caspase-3 p12, and P-glycoprotein. VD3 reversed certain morphological abnormalities in the OB of control trisomic mice (Ts(CO)) and decreased the levels of caspase-3 p12 and methylenetetrahydrofolate reductase in the treated groups. The results demonstrated that trisomy factor causes morphofunctional abnormalities in the OB of Ts(CO) mice. Moreover, VD3 could represent a therapeutic target to attenuate morphological and molecular alterations in OB.

Turnover Kinetics of Pancreatic Macrophages in Lean and Obese Diabetic Mice.

Pancreatic resident macrophages, a heterogeneous family of cells with distinct origins and phenotypes, are the main myeloid cells in exocrine and endocrine tissues. Adult exocrine F4/80hi macrophages consist of three different subsets based on the embryonic marker Tim-4 and MHC II expression. Their frequencies shift during aging and obesity with the Tim-4-MHCII+ fraction becoming the predominant subpopulation in the inter acinar stroma. Endocrine resident F4/80hi macrophages are more homogenous and represent the prevalent leukocyte fraction residing within the islets in both lean and obese mice. We used an adult fate mapping mouse model to characterize turnover kinetics within the pancreatic resident macrophages under normal homeostasis and obese diabetic conditions. We demonstrate that islet resident macrophages show unique replenishment kinetics, with embryonic macrophages being gradually replaced by bone marrow-derived monocytes with increasing age. Their replenishment was independent of the CCL2/CCR2 axis. Furthermore, we confirmed that both exocrine Tim-4+MHCIIlow and Tim-4+MHCII+ fractions are long-lived and primarily independent from bone marrow-derived monocytes. In contrast, exocrine Tim-4-MHCII+ macrophages are gradually replaced through a CCR2-dependent influx of bone marrow-derived monocytes in aging. Moreover, we show that obesity and type 2 diabetes do not affect the turnover kinetics of any macrophage subpopulation residing in the pancreas. Our study uncovers new insights on pancreatic macrophage biology in aging and obesity.

A Non-Obese Hyperglycemic Mouse Model that Develops after Birth with Low Birthweight.

The number of low birthweight (LBW) infants weighing below 2500 g has not decreased in Japan. This study aimed to develop an adult non-obese hyperglycemic mouse model born with LBW to study the pathogenesis. At 16.5 days of gestation, transient intrauterine ischemia (blocked blood flow in both uterine arteries for 15 min) was performed in a subgroup of pregnant mice (group I). Non-occluded dams were used as sham controls (group C). After birth, female pups in each group were weaned at 4 weeks of age and reared on the normal diet until 8 weeks of age (n = 7). Fasting blood glucose levels, serum immunoreactive insulin (IRI), and body composition were then measured. Metabolite analyses was performed on the liver tissues. Birthweight was significantly lower in group I compared with group C. Pups from group I remained underweight with low fat-free mass and showed hyperglycemia with high serum IRI and homeostasis model assessment of insulin resistance levels, indicating insulin resistance. Metabolite analyses showed significantly reduced adenosine triphosphate and nicotinamide adenine dinucleotide production and increased lactic acid in group I. The pathogenesis of our non-obese hyperglycemic mouse model may be due to increased myogenic insulin resistance based on mitochondrial dysfunction and reduced lean body mass.

P1521: A SEVERE MOUSE MODEL OF ALPHA-THALASSEMIA SHOWS ABNORMAL IRON METABOLISM, ERYTHROPOIESIS AND COAGULATION, AND CAN BE RESCUED BY A NOVEL GENE THERAPY APPROACH

Background: Clinical presentation of deletional a-thal varies from an asymptomatic condition (one inactivated a-globin gene) to a complete knockout (Hb Bart’s Hydrops Fetalis). In patients with severe a-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation. Aims: The aims of this study are to develop a novel adult mouse model of a-thal and a gene therapy approach for this disease. Methods: We generated adult animals that do not produce a-globin chains (a-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. To generate a gene therapy tool, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human a-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human a-globin genes. Results: The a-KO animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only b-globin chains (HbH). RBC show aberrant morphology and aggregation of b-globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events, associated with neutrophil infiltration, fibrinogen staining, von Willebrand factor (vWF) released, platelet recruitment and activation. These animals also show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney. We identified ALS20a, a vector where a-globin is under control of the b-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous a-globin at a level comparable to that produced by one endogenous a-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Use of ALS20a resulted in correction of the disease phenotype in a dose-dependent manner in a-KO mice. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels. We tested ALS20a in CD34 cells isolated from four patients with both deletional and non-deletional HbH disease. We measured the change of b/a-globin mRNA ratio (b/aR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial b/aR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a b/aR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced a-thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2). Image:Summary/Conclusion: We generated an adult mouse model of lethal a-thal and, in preliminary experiments, we rescue it with ALS20a. Furthermore, ALS20a successfully improves a-globin levels in patient cells.

Impaired regulation of MMP2/16-MLCK3 by miR-146a-5p increased susceptibility to myocardial ischaemic injury in aging mice.

Aging impairs cardiac function and increases susceptibility to myocardial ischaemic injury. Cardiac myosin light chain kinase (MLCK3) phosphorylates cardiac myosin regulatory light chain (MLC2), controlling sarcomere organization and cardiomyocyte contraction. Dysregulation of MLCK3 and phosphorylated MLC2 (p-MLC2) contributes to heart failure after myocardial infarction (MI). We aimed at exploring how the MLCK3-p-MLC2 axis changes in aging hearts post MI and at investigating the underlying regulatory mechanisms. We generated adult (3 months) and aged (30 months) MI mouse models to compare their cardiac performance, and then detected MLCK3 expression and MLC2 activity. Aging increased the size of MI-induced infarctions and promoted cardiac contractile dysfunction. Furthermore, MLCK3 expression and MLC2 activity increased in adult hearts after MI, but not in aged hearts. miR-146a was found consistently increased in adult and aged hearts post MI. Mechanistic analyses performed in vitro demonstrated that miR-146a-5p down-regulated matrix metalloprotease (MMP)2/16 expression in cardiomyocytes. This down-regulation in turn increased MLCK3 expression and MLC2 activity. However, miR-146a-5p failed to regulate the MMP2/16-MLCK3-p-MLC2 axis in senescent cardiomyocytes or in cardiac miR-146a conditional knockout mice, with the latter experiencing an exacerbated deterioration of cardiac function post MI. These results suggest that an increase of MLCK3 and p-MLC2 contents through decreasing MMP2/16 by miR-146a-5p represents a compensatory mechanism that can protect cardiac contractile function after MI. Aging impairs this miR-146a-5p-regulated MMP2/16-MLCK3-p-MLC2 contractile axis, leading to compromised contractile function and increased susceptibility to heart failure.

Adenine Base Editing In Vivo with a Single Adeno-Associated Virus Vector.

Base editors (BEs) have opened new avenues for the treatment of genetic diseases. However, advances in delivery approaches are needed to enable disease targeting of a broad range of tissues and cell types. Adeno-associated virus (AAV) vectors remain one of the most promising delivery vehicles for gene therapies. Currently, most BE/guide combinations and their promoters exceed the packaging limit (∼5 kb) of AAVs. Dual-AAV delivery strategies often require high viral doses that impose safety concerns. In this study, we engineered an adenine base editor (ABE) using a compact Cas9 from Neisseria meningitidis (Nme2Cas9). Compared with the well-characterized Streptococcus pyogenes Cas9-containing ABEs, ABEs using Nme2Cas9 (Nme2-ABE) possess a distinct protospacer adjacent motif (N4CC) and editing window, exhibit fewer off-target effects, and can efficiently install therapeutically relevant mutations in both human and mouse genomes. Importantly, we show that in vivo delivery of Nme2-ABE and its guide RNA by a single AAV vector can efficiently edit mouse genomic loci and revert the disease mutation and phenotype in an adult mouse model of tyrosinemia. We anticipate that Nme2-ABE, by virtue of its compact size and broad targeting range, will enable a range of therapeutic applications with improved safety and efficacy due in part to packaging in a single-vector system.

Ttc21b deficiency attenuates autosomal dominant polycystic kidney disease in a kidney tubular- and maturation-dependent manner

Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multiprotein complexes. Deletion of several IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult autosomal dominant polycystic kidney disease (ADPKD) mouse models. However, deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, we investigate the role of global deletion of an IFT-A gene, Ttc21b, in juvenile and adult mouse models of ADPKD. Both juvenile (postnatal day 21) and adult (six months of age) ADPKD mice exhibited kidney cysts, increased kidney weight/body weight ratios, lengthened kidney cilia, inflammation, and increased levels of the nutrient sensor, O-linked β-N-acetylglucosamine (O-GlcNAc). Deletion of Ttc21b in juvenile ADPKD mice reduced cortical collecting duct cystogenesis and kidney weight/body weight ratios, increased proximal tubular and glomerular dilations, but did not reduce cilia length, inflammation, nor O-GlcNAc levels. In contrast, Ttc21b deletion in adult ADPKD mice markedly attenuated kidney cystogenesis and reduced cilia length, inflammation, and O-GlcNAc levels. Thus, unlike IFT-B, the effect of Ttc21b deletion in mouse models of ADPKD is development-specific. Unlike an IFT-A adaptor, deleting Ttc21b in juvenile ADPKD mice is partially ameliorative. Thus, our studies suggest that different microenvironmental factors, found in distinct nephron segments and in developing versus mature stages, modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a pathological feature of ADPKD.

Size anomaly and alteration of GABAergic enzymes expressions in cerebellum of a valproic acid mouse model of autism

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by social communication deficit and repetitive behaviour. In the past few years, increasing clinical evidence has shown that the cerebellum may contribute to the neuropathology of ASD. However, studies in the mechanism for the involvement of the cerebellum in autism remained speculative. Although some have suggested the possibility of a change of glutamate decarboxylases in the cerebellum of autistic patients, this remains controversial and is limited to the alteration in transcriptional level. This study aimed to investigate the cerebellar structure and determine the expression of rate-limiting GABAergic enzymes in GABA signalling of the autism cerebellum. Pregnant C57BL/6 J mice were intraperitoneally injected with a dosage of 500 mg/kg valproic acid (VPA) on embryonic day 10.5 for autistic behavioural induction. This study found that early prenatal exposure to VPA led to tail deformation and decreased cerebellar weight and size. Early adult mouse models with autistic behaviour showed reduced expression of both isoforms of glutamate decarboxylases (GAD) 65 and 67 in the cerebellum. Also, protein expressions of cerebellar type 1 GABA transporter (GAT-1) and GABA transaminase (GABAT) were reduced in VPA mice. It indicated that abnormal GABA production, recycling, and metabolism could alter the excitatory-inhibitory balance in the autistic cerebellum. Thus, our findings provide supporting evidence that cerebellum impairment could be an etiology of environmentally induced autism. Changes in cerebellar structure and the altered GABAergic enzymes in the cerebellum provide targets for future therapeutic studies in idiopathic autism.