CRISPR-mediated megabase-scale transgene de-duplication to generate a functional single-copy full-length humanized DMD mouse model.

BACKGROUND: The development of sequence-specific precision treatments like CRISPR gene editing therapies for Duchenne muscular dystrophy (DMD) requires sequence humanized animal models to enable the direct clinical translation of tested strategies. The current available integrated transgenic mouse model containing the full-length human DMD gene, Tg(DMD)72Thoen/J (hDMDTg), has been found to have two copies of the transgene per locus in a tail-to-tail orientation, which does not accurately simulate the true (single) copy number of the DMD gene. This duplication also complicates analysis when testing CRISPR therapy editing outcomes, as large genetic alterations and rearrangements can occur between the cut sites on the two transgenes. RESULTS: To address this, we performed long read nanopore sequencing on hDMDTg mice to better understand the structure of the duplicated transgenes. Following that, we performed a megabase-scale deletion of one of the transgenes by CRISPR zygotic microinjection to generate a single-copy, full-length, humanized DMD transgenic mouse model (hDMDTgSc). Functional, molecular, and histological characterisation shows that the single remaining human transgene retains its function and rescues the dystrophic phenotype caused by endogenous murine Dmd knockout. CONCLUSIONS: Our unique hDMDTgSc mouse model simulates the true copy number of the DMD gene, and can potentially be used for the further generation of DMD disease models that would be better suited for the pre-clinical assessment and development of sequence specific CRISPR therapies.

A longitudinal MRI analysis reveals altered brain connectivity and microstructural changes in a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by progressive cognitive decline and neuropathological changes, yet the underlying neurobiological mechanisms remain elusive. Here, we employed a multimodal longitudinal neuroimaging approach, using anatomical and functional sequences on a high field magnetic resonance imaging (MRI) preclinical scanner, to investigate alterations in brain connectivity and white matter microstructure in a transgenic mouse model of AD (J20) when compared to wild-type (WT) littermates. Functional connectivity analysis revealed distinct network disruptions in J20 mice, primarily involving connections between posterior and anterior brain regions; importantly, a significant interaction between group and age highlighted an exacerbation of these connectivity changes with advancing age in J20 mice. In addition, significant reductions in fractional anisotropy (FA) were observed in the corpus callosum of J20 mice compared to WT, indicative of microstructural alterations consistent with white matter pathology. The observed alterations in brain connectivity and microstructure provide valuable insights into the spatiotemporal processes underlying AD-related decline and underscore the utility of multimodal neuroimaging in elucidating the neurobiological substrates of AD pathology in animal models.

A novel mouse model reproducing frontal alterations related to the prodromal stage of dementia with LEWY bodies.

BACKGROUND: Dementia with Lewy bodies (DLB) is the second most common age-related neurocognitive pathology after Alzheimer's disease. Animal models characterizing this disease are lacking and their development would ameliorate both the understanding of neuropathological mechanisms underlying DLB as well as the efficacy of pre-clinical studies tackling this disease. METHODS: We performed extensive phenotypic characterization of a transgenic mouse model overexpressing, most prominently in the dorsal hippocampus (DH) and frontal cortex (FC), wild-type form of the human α-synuclein gene (mThy1-hSNCA, 12 to 14-month-old males). Moreover, we drew a comparison of our mouse model results to DH- and FC- dependent neuropsychological and neuropathological deficits observed in a cohort of patients including 34 healthy control subjects and 55 prodromal-DLB patients (males and females). RESULTS: Our study revealed an increase of pathological form of soluble α-synuclein, mainly in the FC and DH of the mThy1-hSNCA model. However, functional impairment as well as increase in transcripts of inflammatory markers and decrease in plasticity-relevant protein level were exclusive to the FC. Furthermore, we did not observe pathophysiological or Tyrosine Hydroxylase alterations in the striatum or substantia nigra, nor motor deficits in our model. Interestingly, the results stemming from the cohort of prodromal DLB patients also demonstrated functional deficits emanating from FC alterations, along with preservation of those usually related to DH dysfunctions. CONCLUSIONS: This study demonstrates that pathophysiological impairment of the FC with concomitant DH preservation is observed at an early stage of DLB, and that the mThy1-hSNCA mouse model parallels some markers of this pathology.

A patient-based murine model recapitulates human STAT3 gain-of-function syndrome.

STAT3 gain-of-function (GOF) variants results in a heterogeneous clinical syndrome characterized by early onset immunodeficiency, multi-organ autoimmunity, and lymphoproliferation. While 191 documented cases with STAT3 GOF variants have been reported, the impact of individual variants on immune regulation and the broad clinical spectrum remains unclear. We developed a Stat3p.L387R mouse model, mirroring a variant identified in a family exhibiting common STAT3 GOF symptoms, and rare phenotypes including pulmonary hypertension and retinal vasculitis. In vitro experiments revealed increased STAT3 phosphorylation, nuclear migration, and DNA binding of the variant. Our Stat3p.L387R model displayed similar traits from previous Stat3GOF strains, such as splenomegaly and lymphadenopathy. Notably, Stat3p.L387R/+ mice exhibited heightened embryonic lethality compared to prior Stat3GOF/+ models and ocular abnormalities were observed. This research underscores the variant-specific pathology in Stat3p.L387R/+ mice, highlighting the ability to recapitulate human STAT3 GOF syndrome in patient-specific transgenic murine models. Additionally, such models could facilitate tailored treatment development.

UFMylation is involved in serum inflammatory cytokines generation and splenic T cell activation induced by lipopolysaccharide.

UFMylation, a novel ubiquitin-like protein modification system, has been recently found to be activated in inflammation. However, the effects of UFMylation activation on inflammation in vivo remains unclear. In the present study, we generated a UFMylation activated mice using transgenic (TG) techniques. Lipopolysaccharide (LPS) was used to induce systemic inflammation in both TG and non-transgenic (NTG) mice. Serum cytokines were detected using a Mouse Cytokine Array, and the proportions of splenic NK, B and T cells were determined by using flow cytometry. We found that TG mice showed increased serum G-CSF, TNF RII and decreased serum TCA-3, CD30L, bFGF, IL-15 and MIG compared with NTG mice at baseline. Furthermore, serum cytokines in TG mice exhibited different responses to LPS compared to NTG mice. LPS up-regulated serum TNF RII, G-CSF, MCP-5, RANTES, KC, BLC, MIG and down-regulated IL-1b, IL-2, IL-3, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-15, IL-17, IFN-γ, TCA-3, Eotaxin-2, LIX, MCP-1, TNFα, GM-CSF in NTG mice, whereas LPS up-regulated G-CSF, MCP-5, RANTES, KC, BLC, MIG, ICAM-1, PF4, Eotaxin, CD30L, MIP-1a, TNFRI and down-regulated IL-1b, IL-3, LIX, MCP-1, TNFα, GM-CSF in TG mice. Data from flow cytometry indicated that LPS significantly reduced the percentages of NK and NKT cells in NTG mice, whereas UFMylation activation inhibited LPS-induced NKT cell decrease. The proportions of B cells, total CD4+ and total CD8+ T cells were comparable between TG and NTG mice in response to LPS treatment, whereas the percentages of CD4+CD69+ and CD8+CD69+T cells were lower in TG mice. These findings suggest that UFMylation may alter LPS-induced serum cytokine profile and participate in splenic T cell activation in vivo.

A rapidly progressive multiple system atrophy-cerebellar variant model presenting marked glial reactions with inflammation and spreading of α-synuclein oligomers and phosphorylated α-synuclein aggregates.

Multiple system atrophy (MSA) is a severe α-synucleinopathy facilitated by glial reactions; the cerebellar variant (MSA-C) preferentially involves olivopontocerebellar fibres with conspicuous demyelination. A lack of aggressive models that preferentially involve olivopontocerebellar tracts in adulthood has hindered our understanding of the mechanisms of demyelination and neuroaxonal loss, and thus the development of effective treatments for MSA. We therefore aimed to develop a rapidly progressive mouse model that recaptures MSA-C pathology. We crossed Plp1-tTA and tetO-SNCA*A53T mice to generate Plp1-tTA::tetO-SNCA*A53T bi-transgenic mice, in which human A53T α-synuclein-a mutant protein with enhanced aggregability-was specifically produced in the oligodendrocytes of adult mice using Tet-Off regulation. These bi-transgenic mice expressed mutant α-synuclein from 8 weeks of age, when doxycycline was removed from the diet. All bi-transgenic mice presented rapidly progressive motor deterioration, with wide-based ataxic gait around 22 weeks of age and death around 30 weeks of age. They also had prominent demyelination in the brainstem/cerebellum. Double immunostaining demonstrated that myelin basic protein was markedly decreased in areas in which SM132, an axonal marker, was relatively preserved. Demyelinating lesions exhibited marked ionised calcium-binding adaptor molecule 1-, arginase-1-, and toll-like receptor 2-positive microglial reactivity and glial fibrillary acidic protein-positive astrocytic reactivity. Microarray analysis revealed a strong inflammatory response and cytokine/chemokine production in bi-transgenic mice. Neuronal nuclei-positive neuronal loss and patchy microtubule-associated protein 2-positive dendritic loss became prominent at 30 weeks of age. However, a perceived decrease in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta in bi-transgenic mice compared with wild-type mice was not significant, even at 30 weeks of age. Wild-type, Plp1-tTA, and tetO-SNCA*A53T mice developed neither motor deficits nor demyelination. In bi-transgenic mice, double immunostaining revealed human α-synuclein accumulation in neurite outgrowth inhibitor A (Nogo-A)-positive oligodendrocytes beginning at 9 weeks of age; its expression was further increased at 10 to 12 weeks, and these increased levels were maintained at 12, 24, and 30 weeks. In an α-synuclein-proximity ligation assay, α-synuclein oligomers first appeared in brainstem oligodendrocytes as early as 9 weeks of age; they then spread to astrocytes, neuropil, and neurons at 12 and 16 weeks of age. α-Synuclein oligomers in the brainstem neuropil were most abundant at 16 weeks of age and decreased thereafter; however, those in Purkinje cells successively increased until 30 weeks of age. Double immunostaining revealed the presence of phosphorylated α-synuclein in Nogo-A-positive oligodendrocytes in the brainstem/cerebellum as early as 9 weeks of age. In quantitative assessments, phosphorylated α-synuclein gradually and successively accumulated at 12, 24, and 30 weeks in bi-transgenic mice. By contrast, no phosphorylated α-synuclein was detected in wild-type, tetO-SNCA*A53T, or Plp1-tTA mice at any age examined. Pronounced demyelination and tubulin polymerisation, promoting protein-positive oligodendrocytic loss, was closely associated with phosphorylated α-synuclein aggregates at 24 and 30 weeks of age. Early inhibition of mutant α-synuclein expression by doxycycline diet at 23 weeks led to fully recovered demyelination; inhibition at 27 weeks led to persistent demyelination with glial reactions, despite resolving phosphorylated α-synuclein aggregates. In conclusion, our bi-transgenic mice exhibited progressively increasing demyelination and neuroaxonal loss in the brainstem/cerebellum, with rapidly progressive motor deterioration in adulthood. These mice showed marked microglial and astrocytic reactions with inflammation that was closely associated with phosphorylated α-synuclein aggregates. These features closely mimic human MSA-C pathology. Notably, our model is the first to suggest that α-synuclein oligomers may spread from oligodendrocytes to neurons in transgenic mice with human α-synuclein expression in oligodendrocytes. This model of MSA is therefore particularly useful for elucidating the in vivo mechanisms of α-synuclein spreading from glia to neurons, and for developing therapies that target glial reactions and/or α-synuclein oligomer spreading and aggregate formation in MSA.

Elucidating the Mechanisms Underlying Interindividual Differences in the Onset of Adverse Drug Reactions.

Idiosyncratic drug toxicities (IDTs) pose a significant challenge; they are marked by life-threatening adverse reactions that emerge aftermarket release and are influenced by intricate genetic and environmental variations. Recent genome-wide association studies have highlighted a strong correlation between specific human leukocyte antigen (HLA) polymorphisms and IDT onset. This review provides an overview of current research on HLA-mediated drug toxicities. In the last six years, HLA-transgenic (Tg) mice have been instrumental in advancing our understanding of these underlying mechanisms, uncovering systemic immune reactions that replicate human drug-induced immune stimulation. Additionally, the potential role of immune tolerance in shaping individual differences in adverse effects highlights its relevance to the interplay between HLA polymorphisms and IDTs. Although HLA-Tg mice offer valuable insights into systemic immune reactions, further exploration is essential to decipher the intricate interactions that lead to organ-specific adverse effects, especially in organs such as the skin or liver. Navigating the intricate interplay of HLA, which may potentially trigger intracellular immune responses, this review emphasizes the need for a holistic approach that integrates findings from both animal models and molecular/cellular investigations. The overarching goal is to enhance our comprehensive understanding of HLA-mediated IDTs and identify factors shaping individual variations in drug reactions. This review aims to facilitate the development of strategies to prevent severe adverse effects, address existing knowledge gaps, and provide guidance for future research initiatives in the field of HLA-mediated IDTs.

APP mediates tau uptake and its overexpression leads to the exacerbated tau pathology.

Alzheimer's disease (AD), as the most common type of dementia, has two pathological hallmarks, extracellular senile plaques composed of ß-amyloid peptides and intracellular neurofibrillary tangles containing phosphorylated-tau protein. Amyloid precursor protein (APP) and tau each play central roles in AD, although how APP and tau interact and synergize in the disease process is largely unknown. Here, we showed that soluble tau interacts with the N-terminal of APP in vitro in cell-free and cell culture systems, which can be further confirmed in vivo in the brain of 3XTg-AD mouse. In addition, APP is involved in the cellular uptake of tau through endocytosis. APP knockdown or N-terminal APP-specific antagonist 6KApoEp can prevent tau uptake in vitro, resulting in an extracellular tau accumulation in cultured neuronal cells. Interestingly, in APP/PS1 transgenic mouse brain, the overexpression of APP exacerbated tau propagation. Moreover, in the human tau transgenic mouse brain, overexpression of APP promotes tau phosphorylation, which is significantly remediated by 6KapoEp. All these results demonstrate the important role of APP in the tauopathy of AD. Targeting the pathological interaction of N-terminal APP with tau may provide an important therapeutic strategy for AD.

One or two injections of MVA-vectored vaccine shields hACE2 transgenic mice from SARS-CoV-2 upper and lower respiratory tract infection.

Modified vaccinia virus Ankara (MVA) is a replication-restricted smallpox vaccine, and numerous clinical studies of recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases, including COVID-19, are in progress. Here, we characterize rMVAs expressing the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Modifications of full-length S individually or in combination included two proline substitutions, mutations of the furin recognition site, and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) is flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected cells and was recognized by anti-RBD antibody and soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced antibodies, which neutralized a pseudovirus in vitro and, upon passive transfer, protected hACE2 transgenic mice from lethal infection with SARS-CoV-2, as well as S-specific CD3+CD8+IFNγ+ T cells. Antibody boosting occurred following a second rMVA or adjuvanted purified RBD protein. Immunity conferred by a single vaccination of hACE2 mice prevented morbidity and weight loss upon intranasal infection with SARS-CoV-2 3 wk or 7 wk later. One or two rMVA vaccinations also prevented detection of infectious SARS-CoV-2 and subgenomic viral mRNAs in the lungs and greatly reduced induction of cytokine and chemokine mRNAs. A low amount of virus was found in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of low levels of subgenomic mRNAs in turbinates indicated that replication was aborted in immunized animals.

Overexpression of Human sFLT1 in the Spongiotrophoblast Is Sufficient to Induce Placental Dysfunction and Fetal Growth Restriction in Transgenic Mice.

Preeclampsia (PE) is characterized by maternal hypertension and placental dysfunction, often leading to fetal growth restriction (FGR). It is associated with an overexpression of the anti-angiogenic sFLT1 protein, which originates from the placenta and serves as a clinical biomarker to predict PE. To analyze the impact of sFLT1 on placental function and fetal growth, we generated transgenic mice with placenta-specific human sFLT1 (hsFLT1) overexpression. Immunohistochemical, morphometrical, and molecular analyses of the placentas on 14.5 dpc and 18.5 dpc were performed with a focus on angiogenesis, nutrient transport, and inflammation. Additionally, fetal development upon placental hsFLT1 overexpression was investigated. Dams exhibited a mild increase in serum hsFLT1 levels upon placental hsFLT1 expression and revealed growth restriction of the fetuses in a sex-specific manner. Male FGR fetuses expressed higher amounts of placental hsFLT1 mRNA compared to females. FGR placentas displayed an altered morphology, hallmarked by an increase in the spongiotrophoblast layer and changes in labyrinthine vascularization. Further, FGR placentas showed a significant reduction in placental glycogen storage and nutrient transporter expression. Moreover, signs of hypoxia and inflammation were observed in FGR placentas. The transgenic spongiotrophoblast-specific hsFLT1 mouse line demonstrates that low hsFLT1 serum levels are sufficient to induce significant alterations in fetal and placental development in a sex-specific manner.

Amyloid ß Proteoforms Elucidated by Quantitative LC/MS in the 5xFAD Mouse Model of Alzheimer's Disease.

Numerous Aß proteoforms, identified in the human brain, possess differential neurotoxic and aggregation propensities. These proteoforms contribute in unknown ways to the conformations and resultant pathogenicity of oligomers, protofibrils, and fibrils in Alzheimer's disease (AD) manifestation owing to the lack of molecular-level specificity to the exact chemical composition of underlying protein products with widespread interrogating techniques, like immunoassays. We evaluated Aß proteoform flux using quantitative top-down mass spectrometry (TDMS) in a well-studied 5xFAD mouse model of age-dependent Aß-amyloidosis. Though the brain-derived Aß proteoform landscape is largely occupied by Aß1-42, 25 different forms of Aß with differential solubility were identified. These proteoforms fall into three natural groups defined by hierarchical clustering of expression levels in the context of mouse age and proteoform solubility, with each group sharing physiochemical properties associated with either N/C-terminal truncations or both. Overall, the TDMS workflow outlined may hold tremendous potential for investigating proteoform-level relationships between insoluble fibrils and soluble Aß, including low-molecular-weight oligomers hypothesized to serve as the key drivers of neurotoxicity. Similarly, the workflow may also help to validate the utility of AD-relevant animal models to recapitulate amyloidosis mechanisms or possibly explain disconnects observed in therapeutic efficacy in animal models vs humans.

6-Mercaptopurine potently inhibits recruitment of SHP2 by phosphorylated PD-1 to inhibit PD-1 signalling and enhance T cell function.

Antibody inhibitors that block PD-1/PD-L1 interaction have been approved for oncological clinics, yielding impressive treatment effects. Small molecules inhibiting PD-1 signalling are at various stages of development, given that small molecular drugs are expected to outperform protein drugs in several ways. Currently, a significant portion of these small molecular inhibitors achieve this purpose by binding to a limited region of the PD-L1 protein, thereby limiting the choice of chemical structures. Alternative strategies for developing small-molecular PD-1 inhibitors are urgently needed to broaden the choice of chemical structures. Here, we report that 6-mercaptopurine (6-MP) inhibits PD-1 signalling, activates T cell function in vitro and in vivo and shrinks tumours by activating cytotoxic T cells. Mechanistically, 6-MP potently inhibited PD-1 signalling by blocking the recruitment of SHP2 by PD-1. Considering that 6-MP is a chemotherapeutic agent already approved by the FDA for childhood leukaemia, our work revealed a novel anti-tumour mechanism for this drug and suggests that 6-MP warrants further clinical evaluation for other tumour types.

IGF2BP1 induces neuroblastoma via a druggable feedforward loop with MYCN promoting 17q oncogene expression.

BACKGROUND: Neuroblastoma is the most common solid tumor in infants accounting for approximately 15% of all cancer-related deaths. Over 50% of high-risk neuroblastoma relapse, emphasizing the need of novel drug targets and therapeutic strategies. In neuroblastoma, chromosomal gains at chromosome 17q, including IGF2BP1, and MYCN amplification at chromosome 2p are associated with adverse outcome. Recent, pre-clinical evidence indicates the feasibility of direct and indirect targeting of IGF2BP1 and MYCN in cancer treatment. METHODS: Candidate oncogenes on 17q were identified by profiling the transcriptomic/genomic landscape of 100 human neuroblastoma samples and public gene essentiality data. Molecular mechanisms and gene expression profiles underlying the oncogenic and therapeutic target potential of the 17q oncogene IGF2BP1 and its cross-talk with MYCN were characterized and validated in human neuroblastoma cells, xenografts and PDX as well as novel IGF2BP1/MYCN transgene mouse models. RESULTS: We reveal a novel, druggable feedforward loop of IGF2BP1 (17q) and MYCN (2p) in high-risk neuroblastoma. This promotes 2p/17q chromosomal gains and unleashes an oncogene storm resulting in fostered expression of 17q oncogenes like BIRC5 (survivin). Conditional, sympatho-adrenal transgene expression of IGF2BP1 induces neuroblastoma at a 100% incidence. IGF2BP1-driven malignancies are reminiscent to human high-risk neuroblastoma, including 2p/17q-syntenic chromosomal gains and upregulation of Mycn, Birc5, as well as key neuroblastoma circuit factors like Phox2b. Co-expression of IGF2BP1/MYCN reduces disease latency and survival probability by fostering oncogene expression. Combined inhibition of IGF2BP1 by BTYNB, MYCN by BRD inhibitors or BIRC5 by YM-155 is beneficial in vitro and, for BTYNB, also. CONCLUSION: We reveal a novel, druggable neuroblastoma oncogene circuit settling on strong, transcriptional/post-transcriptional synergy of MYCN and IGF2BP1. MYCN/IGF2BP1 feedforward regulation promotes an oncogene storm harboring high therapeutic potential for combined, targeted inhibition of IGF2BP1, MYCN expression and MYCN/IGF2BP1-effectors like BIRC5.

A novel humanized tri-receptor transgenic mouse model of HAdV infection and pathogenesis.

Human adenovirus (HAdV) is a highly virulent respiratory pathogen that poses clinical challenges in terms of diagnostics and treatment. Currently, no effective therapeutic drugs or prophylactic vaccines are available for HAdV infections. One factor contributing to this deficiency is that existing animal models, including wild-type and single-receptor transgenic mice, are unsuitable for HAdV proliferation and pathology testing. In this study, a tri-receptor transgenic mouse model expressing the three best-characterized human cellular receptors for HAdV (hCAR, hCD46, and hDSG2) was generated and validated via analysis of transgene insertion, receptor mRNA expression, and protein abundance distribution. Following HAdV-7 infection, the tri-receptor mice exhibited high transcription levels at the early and late stages of the HAdV gene, as well as viral protein expression. Furthermore, the tri-receptor mice infected with HAdV exhibited dysregulated cytokine responses and multiple tissue lesions. This transgenic mouse model represents human HAdV infection and pathogenesis with more accuracy than any other reported animal model. As such, this model facilitates the comprehensive investigation of HAdV pathogenesis as well as the evaluation of potential vaccines and therapeutic modalities for HAdV.

Transgenic HPV11-E2 protein modulates URR activity in vivo.

In vitro experiments have shown that the E2 protein of human papillomaviruses (HPV) binds to the upstream regulatory region (URR) of the viral genome and modulates transcription. Additionally, it seems to be a necessary component for viral DNA replication together with E1. We have developed a transgenic mouse model containing the URR region of the low-risk virus HPV11 that regulates the expression of the lacZ reporter gene. Most interestingly, in these mice, the transgene was exclusively expressed in the bulge region of the hair follicle but not in any other tissues. Further experimental data indicate that in double transgenic mice that also express the HPV11-E2 protein under the control of the Ubiquitin C-promoter, the transcription of the reporter gene is modulated. When E2 is present, the expression of the reporter gene also occurs exclusively in the bulge region of the hair follicles as it does in the single transgenic mice, but the expression of the lacZ driven by the URR is increased and the statistical spread is greater. Even if the expression of the reporter gene occurs in the hair follicles of the dorsal skin of an animal uniform, E2 obviously has the capacity for both to induce and to repress the URR activity in vivo.

µCT to quantify muco-obstructive lung disease and effects of neutrophil elastase knockout in mice.

Muco-obstructive lung diseases are characterized by airway obstruction and hyperinflation, which can be quantified by imaging. Our aim was to evaluate µCT for longitudinal quantification of muco-obstructive lung disease in ß-epithelial Na+ channel overexpressing (Scnn1b-TG) mice and of the effects of neutrophil elastase (NE) knockout on its progression. Lungs from wild-type (WT), NE-/-, Scnn1b-TG, and Scnn1b-TG/NE-/- mice were scanned with 9-µm resolution at 0, 5, 14, and 60 days of age, and airway and parenchymal disease was quantified. Mucus adhesion lesions (MAL) were persistently increased in Scnn1b-TG compared with WT mice from 0 days (20.25 ± 6.50 vs. 9.60 ± 2.07, P < 0.05), and this effect was attenuated in Scnn1b-TG/NE-/- mice (5.33 ± 3.67, P < 0.001). Airway wall area percentage (WA%) was increased in Scnn1b-TG mice compared with WT from 14 days onward (59.2 ± 6.3% vs. 49.8 ± 9.0%, P < 0.001) but was similar in Scnn1b-TG/NE-/- compared with WT at 60 days (46.4 ± 9.2% vs. 45.4 ± 11.5%, P = 0.97). Air proportion (Air%) and mean linear intercept (Lm) were persistently increased in Scnn1b-TG compared with WT from 5 days on (53.9 ± 4.5% vs. 30.0 ± 5.5% and 78.82 ± 8.44 µm vs. 65.66 ± 4.15 µm, respectively, P < 0.001), whereas in Scnn1b-TG/NE-/-, Air% and Lm were similar to WT from birth (27.7 ± 5.5% vs. 27.2 ± 5.9%, P = 0.92 and 61.48 ± 9.20 µm vs. 61.70 ± 6.73 µm, P = 0.93, respectively). Our results suggest that µCT is sensitive to detect the onset and progression of muco-obstructive lung disease and effects of genetic deletion of NE on morphology of airways and lung parenchyma in Scnn1b-TG mice, and that it may serve as a sensitive endpoint for preclinical studies of novel therapeutic interventions for muco-obstructive lung diseases.

Alzheimer's disease amyloid-ß pathology in the lens of the eye.

Neuropathological hallmarks of Alzheimer's disease (AD) include pathogenic accumulation of amyloid-ß (Aß) peptides and age-dependent formation of amyloid plaques in the brain. AD-associated Aß neuropathology begins decades before onset of cognitive symptoms and slowly progresses over the course of the disease. We previously reported discovery of Aß deposition, ß-amyloidopathy, and co-localizing supranuclear cataracts (SNC) in lenses from people with AD, but not other neurodegenerative disorders or normal aging. We confirmed AD-associated Aß molecular pathology in the lens by immunohistopathology, amyloid histochemistry, immunoblot analysis, epitope mapping, immunogold electron microscopy, quantitative immunoassays, and tryptic digest mass spectrometry peptide sequencing. Ultrastructural analysis revealed that AD-associated Aß deposits in AD lenses localize as electron-dense microaggregates in the cytoplasm of supranuclear (deep cortex) fiber cells. These Aß microaggregates also contain αB-crystallin and scatter light, thus linking Aß pathology and SNC phenotype expression in the lenses of people with AD. Subsequent research identified Aß lens pathology as the molecular origin of the distinctive cataracts associated with Down syndrome (DS, trisomy 21), a chromosomal disorder invariantly associated with early-onset Aß accumulation and Aß amyloidopathy in the brain. Investigation of 1249 participants in the Framingham Eye Study found that AD-associated quantitative traits in brain and lens are co-heritable. Moreover, AD-associated lens traits preceded MRI brain traits and cognitive deficits by a decade or more and predicted future AD. A genome-wide association study of bivariate outcomes in the same subjects identified a new AD risk factor locus in the CTNND2 gene encoding δ-catenin, a protein that modulates Aß production in brain and lens. Here we report identification of AD-related human Aß (hAß) lens pathology and age-dependent SNC phenotype expression in the Tg2576 transgenic mouse model of AD. Tg2576 mice express Swedish mutant human amyloid precursor protein (APP-Swe), accumulate hAß peptides and amyloid pathology in the brain, and exhibit cognitive deficits that slowly progress with increasing age. We found that Tg2576 trangenic (Tg+) mice, but not non-transgenic (Tg-) control mice, also express human APP, accumulate hAß peptides, and develop hAß molecular and ultrastructural pathologies in the lens. Tg2576 Tg+ mice exhibit age-dependent Aß supranuclear lens opacification that recapitulates lens pathology and SNC phenotype expression in human AD. In addition, we detected hAß in conditioned medium from lens explant cultures prepared from Tg+ mice, but not Tg- control mice, a finding consistent with constitutive hAß generation in the lens. In vitro studies showed that hAß promoted mouse lens protein aggregation detected by quasi-elastic light scattering (QLS) spectroscopy. These results support mechanistic (genotype-phenotype) linkage between Aß pathology and AD-related phenotypes in lens and brain. Collectively, our findings identify Aß pathology as the shared molecular etiology of two age-dependent AD-related cataracts associated with two human diseases (AD, DS) and homologous murine cataracts in the Tg2576 transgenic mouse model of AD. These results represent the first evidence of AD-related Aß pathology outside the brain and point to lens Aß as an optically-accessible AD biomarker for early detection and longitudinal monitoring of this devastating neurodegenerative disease.

Hypoxic and Hypercapnic Responses in Transgenic Murine Model of Alzheimer's Disease Overexpressing Human AßPP: The Effects of Pretreatment with Memantine and Rivastigmine.

Despite the severe respiratory problems reducing the quality of life for Alzheimer's disease (AD) patients, their causes are poorly understood. We aimed to investigate hypoxic and hypercapnic respiratory responses in a transgenic mouse model of AD (AßPP V717I) overexpressing AßPP and mimicking early-onset AD. The cholinesterase inhibitor rivastigmine and the NMDA receptor antagonist memantine were used to investigate the effects of drugs, used to treat AD cognitive dysfunction, on breathing in hypoxia and hypercapnia. We found a significant increase in the respiratory response to hypercapnia and no difference in the hypoxic response in APP+ mice, compared with the control group (APP-). Memantine had no effect on respiration in either group, including responses to hypoxia and hypercapnia. Rivastigmine depressed resting ventilation and response to hypercapnia irrespective of the mice genotype. Reduction in hypoxia-augmented ventilation by rivastigmine was observed only in APP+ mice, which exhibited lower acetylcholinesterase activity in the hippocampus. Treatment with rivastigmine reduced the enzyme activity in both groups equally in the hippocampus and brainstem. The increased ventilatory response to hypercapnia in transgenic mice may indicate alterations in chemoreceptive respiratory nuclei, resulting in increased CO2 sensitivity. Rivastigmine is a potent reductant of normoxic and hypercapnic respiration in APP+ and APP- mice.

Targeted Disruption of Lats1 and Lats2 in Mice Impairs Testis Development and Alters Somatic Cell Fate.

Hippo signaling plays an essential role in the development of numerous tissues. Although it was previously shown that the transcriptional effectors of Hippo signaling Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) can fine-tune the regulation of sex differentiation genes in the testes, the role of Hippo signaling in testis development remains largely unknown. To further explore the role of Hippo signaling in the testes, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in the somatic cells of the testes using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that early stages of testis somatic cell differentiation were not affected in this model but progressive testis cord dysgenesis was observed starting at gestational day e14.5. Testis cord dysgenesis was further associated with the loss of polarity of the Sertoli cells and the loss of SOX9 expression but not WT1. In parallel with testis cord dysgenesis, a loss of steroidogenic gene expression associated with the appearance of myofibroblast-like cells in the interstitial space was also observed in mutant animals. Furthermore, the loss of YAP phosphorylation, the accumulation of nuclear TAZ (and YAP) in both the Sertoli and interstitial cell populations, and an increase in their transcriptional co-regulatory activity in the testes suggest that the observed phenotype could be attributed at least in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper differentiation of testis somatic cells.

DM1 Transgenic Mice Exhibit Abnormal Neurotransmitter Homeostasis and Synaptic Plasticity in Association with RNA Foci and Mis-Splicing in the Hippocampus.

Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disease mediated by a toxic gain of function of mutant RNAs. The neuropsychological manifestations affect multiple domains of cognition and behavior, but their etiology remains elusive. Transgenic DMSXL mice carry the DM1 mutation, show behavioral abnormalities, and express low levels of GLT1, a critical regulator of glutamate concentration in the synaptic cleft. However, the impact of glutamate homeostasis on neurotransmission in DM1 remains unknown. We confirmed reduced glutamate uptake in the DMSXL hippocampus. Patch clamp recordings in hippocampal slices revealed increased amplitude of tonic glutamate currents in DMSXL CA1 pyramidal neurons and DG granule cells, likely mediated by higher levels of ambient glutamate. Unexpectedly, extracellular GABA levels and tonic current were also elevated in DMSXL mice. Finally, we found evidence of synaptic dysfunction in DMSXL mice, suggestive of abnormal short-term plasticity, illustrated by an altered LTP time course in DG and in CA1. Synaptic dysfunction was accompanied by RNA foci accumulation in localized areas of the hippocampus and by the mis-splicing of candidate genes with relevant functions in neurotransmission. Molecular and functional changes triggered by toxic RNA may induce synaptic abnormalities in restricted brain areas that favor neuronal dysfunction.