Dt Mice Research Articles

Sensory-motor circuit is a therapeutic target for dystonia musculorum mice, a model of hereditary sensory and autonomic neuropathy 6.

Mutations in Dystonin (DST), which encodes cytoskeletal linker proteins, cause hereditary sensory and autonomic neuropathy 6 (HSAN-VI) in humans and the dystonia musculorum (dt) phenotype in mice; however, the neuronal circuit underlying the HSAN-VI and dt phenotype is unresolved. dt mice exhibit dystonic movements accompanied by the simultaneous contraction of agonist and antagonist muscles and postnatal lethality. Here, we identified the sensory-motor circuit as a major causative neural circuit using a gene trap system that enables neural circuit-selective inactivation and restoration of Dst by Cre-mediated recombination. Sensory neuron-selective Dst deletion led to motor impairment, degeneration of proprioceptive sensory neurons, and disruption of the sensory-motor circuit. Restoration of Dst expression in sensory neurons using Cre driver mice or a single postnatal injection of Cre-expressing adeno-associated virus ameliorated sensory degeneration and improved abnormal movements. These findings demonstrate that the sensory-motor circuit is involved in the movement disorders in dt mice and that the sensory circuit is a therapeutic target for HSAN-VI.

MEF2C ameliorates learning, memory, and molecular pathological changes in Alzheimer’s disease in vivo and in vitro

Myocyte enhancer factor 2C (MEF2C) is highly expressed in the nervous system, and regulates neuro-development, synaptic plasticity, and inflammation. However, its mechanism in Alzheimer's disease (AD) is underestimated. In this study, the role and mechanism of MEF2C were investigated in the brain tissue specimens from patients with AD, APPswe/PSEN1dE9 double transgenic (APP/PS1_DT) mice, and SH-SY5Y cells treated with β-amyloid peptide (Aβ). The results indicated that the expression of MEF2C is significantly reduced, and the expression of MEF2C/Aβ in different parts of brain is negatively correlated in patients with AD. Knockdown of MEF2C promotes cell apoptosis and the level of β-amyloid precursor protein cleaving enzyme 1 (BACE) but reduces BACE2 expression. In addition, knockdown of enhances the generation and aggregation of Aβ in the cortex of APP/PS1_DT mice, reduces the expression of synaptic proteins, exacerbates the ability of learning and memory of APP/PS1_DT mice, damages the structure of mitochondria, increases the oxidative stress (OS) level, and inhibits the expression levels of members of the Nrf2-ARE signal pathway. In summary, inhibition of MEF2C exacerbates the toxic effect of Aβ and , damages synaptic plasticity, reduces the ability of learning and memory of APP/PS1 mice, and increases the level of OS via the Nrf2-ARE signal pathway.

Mechanisms of Systolic Cardiac Dysfunction in PP2A, PP5 and PP2AxPP5 Double Transgenic Mice.

As part of our ongoing studies on the potential pathophysiological role of serine/threonine phosphatases (PP) in the mammalian heart, we have generated transgenic mice with cardiac muscle cell-specific overexpression of PP2Acα (PP2A) and PP5 (PP5). For further studies we crossbred PP2A and PP5 mice to obtain PP2AxPP5 double transgenic mice (PP2AxPP5, DT) and compared them with littermate wild-type mice (WT) serving as a control. The mortality of DT mice was greatly enhanced vs. other genotypes. Cardiac fibrosis was noted histologically and mRNA levels of collagen 1α, collagen 3α and fibronectin 1 were augmented in DT. DT and PP2A mice exhibited an increase in relative heart weight. The ejection fraction (EF) was reduced in PP2A and DT but while the EF of PP2A was nearly normalized after β-adrenergic stimulation by isoproterenol, it was almost unchanged in DT. Moreover, left atrial preparations from DT were less sensitive to isoproterenol treatment both under normoxic conditions and after hypoxia. In addition, levels of the hypertrophy markers atrial natriuretic peptide and B-type natriuretic peptide as well as the inflammation markers interleukin 6 and nuclear factor kappa B were increased in DT. PP2A enzyme activity was enhanced in PP2A vs. WT but similar to DT. This was accompanied by a reduced phosphorylation state of phospholamban at serine-16. Fittingly, the relaxation times in left atria from DT were prolonged. In summary, cardiac co-overexpression of PP2A and PP5 were detrimental to animal survival and cardiac function, and the mechanism may involve dephosphorylation of important regulatory proteins but also fibrosis and inflammation.

Over-Expression of Activation-Induced Cytidine Deaminase in TCL1 Mice Leads to the Development of IGHV-Mutated CLL Clones That Associate with More Aggressive Disease

Introduction. Activation-induced cytidine deaminase (AID) mediates somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes (IG s), both of which are necessary for an effective immune response in normal B cells. An unwanted consequence of AID's role is its contribution to cellular transformation and tumor progression by aberrantly mutagenizing genes outside the IG locus (off-target mutations). In chronic lymphocytic leukemia (CLL), higher levels of IGHV clonal mutations in IGHV-mutated CLL (M-CLL) correlate strongly with better clinical outcomes, while, IGHV -unmutated (U-CLL) cases are associated with worse patient outcome. Paradoxically, U-CLL cells express significantly higher levels of AID than M- CLL cells.Eμ-T-cell leukemia-1 (TCL1) transgenic (Tg) mice are a valuable model of CLL. However, because SHM and CSR occur rarely in the leukemic clones of these animals, TCL1 Tg mice mimic only U-CLL and hence may not provide an understanding of the roles of SHM and CSR in disease evolution. To address this issue, we developed a TCL1 strain by interbreeding mice over-expressing AID in B lymphocytes (Eμ-TCL1xVκ-AID).Methods. B-cell clonal expansions were identified in spleen cells from 14 TCL1 and 23 TCL1xAID (DT) mice by amplifying cDNAs by PCR using consensus IGHV FR and isotype-specific primers for IgH chains and Vκ FR and Cκ for IgL chain genes. Ig sequences were compared to murine germline IGHV s and IGKV s, and IGHV-D-JH and IGKV-JK rearrangements by IMGT V-Quest. Six DT and 2 TCL1 tumors were transferred to SCID mice (5 mice/donor)Results.Clonal expansions: Monoclonal/oligoclonal expansions were detected in all TCL1 mice; these used only µ H and κ L chains. Similar expansions were detected in 21 of 23 DT mice; each animal bore an IgMκ+ clone and 5 also exhibited a concomitant IgGκ+ clone. Approximately 50% used VH1-55, VH11-2, or VH12-3.SHM: The IgM+ TCL1 clones had a mutation frequency of 0.05% for IGHV and 0.09% for IGKV . In contrast, the IGHV and IGKV mutation frequencies were considerably higher in DT mice: IGHV 0.47% in IgM+ and 3.0% in IgG+ clones, and IGKV 0.9% for IgM+ and IgG+ combined. These mutations localized more frequently in AID hotspots than coldspots at a ratio of ≥7:1.However, SHM did not affect all clones equally. Although the mutation frequency in VH12-3 and VH11-2 clones was higher (0.38%) than the TCL1 level (0.05%), it was considerably less than that found in the DT clones using other IGHV s (0.80%). In addition, no mutations were detected in VH1-55 clones from DT mice.IGHV gene use and SHM in clones that underwent CSR: Notably, in 2 of 5 instances the same IGHV-D-J rearrangement was found in IgMκ+ and IgGκ+ clones from the same mice. For the remaining 3, only the IgG+ version was detected. Also, within the IgG-only group, IGHV1-47 was used by 2 different clones and these were highly mutated (8.9%).Stereotyped IGHV-D-J and IGkV-Jk rearrangements: Among 32 IgM clones from DT mice, 4 expressed VH11-2 and Vκ14-126 and 8 expressed VH12-3 and Vκ4-91; these rearrangements were very similar to canonical anti-phosphatidylcholine-producing clones.DT mice develop more aggressive disease: we observed shorter median survival in DT mice comparing to TCL1 mice (279 days vs 356 days, P =0.0034)Transfer tumor development: Spleen cells from 6 CLL-bearing mice were transferred into SCID recipients, leading to a selected outgrowth of VH12-3/Vκ4-91 and VH11-2/Vκ14-126 clones. Survival in these mice inversely correlated with IGHV mutations (Figure).Summary and conclusions.Over-expression of AID in TCL1 mice leads to markedly increased SHM and CSR. However, SHM is not equivalent for all IGHV genes: certain IGHV s and IGK Vs are less susceptible to having major increases in the extent of SHM and the occurrence of CSR. This property resembles some human CLL IGHV s that rarely develop SHMs or undergo CSR despite the B-cell's ability to synthesize AID (e.g., many IGHV1-69+ clones in U-CLL patients). AID overexpression also led to IgG+ clones for which an IgM precursor was not found. This resembles those human stereotyped CLL clones that are only found as IgGs (e.g., stereotyped subsets 4 and 8). Finally, transfer of DT clones to SCID mice led to more IGHV and IGKV mutations and shorter survival opposite to that in M-CLL patients. Possibly this adverse effect might be a consequence of AID's aberrant action outside the IG loci which is facilitated in DT mice. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Disruption of dystonin in Schwann cells results in late-onset neuropathy and sensory ataxia.

Dystonin (Dst) is a causative gene for Dystonia musculorum (dt) mice, which is an inherited disorder exhibiting dystonia-like movement and ataxia with sensory degeneration. Dst is expressed in a variety of tissues, including the central nervous system and the peripheral nervous system (PNS), muscles, and skin. However, the Dst-expressing cell type(s) for dt phenotypes have not been well characterized. To address the questions whether the disruption of Dst in Schwann cells induces movement disorders and how much impact does it have on dt phenotypes, we generated Dst conditional knockout (cKO) mice using P0-Cre transgenic mice and Dst gene trap mice. First, we assessed the P0-Cre transgene-dependent Cre recombination using tdTomato reporter mice and then confirmed the preferential tdTomato expression in Schwann cells. In the Dst cKO mice, Dst mRNA expression was significantly decreased in Schwann cells, but it was intact in most of the sensory neurons in the dorsal root ganglion. Next, we analyzed the phenotype of Dst cKO mice. They exhibited a normal motor phenotype during juvenile periods, and thereafter, started exhibiting an ataxia. Behavioral tests and electrophysiological analyses demonstrated impaired motor abilities and slowed motor nerve conduction velocity in Dst cKO mice, but these mice did not manifest dystonic movements. Electron microscopic observation of the PNS of Dst cKO mice revealed significant numbers of hypomyelinated axons and numerous infiltrating macrophages engulfing myelin debris. These results indicate that Dst is important for normal PNS myelin organization and Dst disruption in Schwann cells induces late-onset neuropathy and sensory ataxia. MAIN POINTS: Dystonin (Dst) disruption in Schwann cells results in late-onset neuropathy and sensory ataxia. Dst in Schwann cells is important for normal myelin organization in the peripheral nervous system.

Diverse dystonin gene mutations cause distinct patterns of Dst isoform deficiency and phenotypic heterogeneity in Dystonia musculorum mice.

ABSTRACTLoss-of-function mutations in dystonin (DST) can cause hereditary sensory and autonomic neuropathy type 6 (HSAN-VI) or epidermolysis bullosa simplex (EBS). Recently, DST-related diseases were recognized to be more complex than previously thought because a patient exhibited both neurological and skin manifestations, whereas others display only one or the other. A single DST locus produces at least three major DST isoforms: DST-a (neuronal isoform), DST-b (muscular isoform) and DST-e (epithelial isoform). Dystonia musculorum (dt) mice, which have mutations in Dst, were originally identified as spontaneous mutants displaying neurological phenotypes. To reveal the mechanisms underlying the phenotypic heterogeneity of DST-related diseases, we investigated two mutant strains with different mutations: a spontaneous Dst mutant (Dstdt-23Rbrc mice) and a gene-trap mutant (DstGt mice). The Dstdt-23Rbrc allele possesses a nonsense mutation in an exon shared by all Dst isoforms. The DstGt allele is predicted to inactivate Dst-a and Dst-b isoforms but not Dst-e. There was a decrease in the levels of Dst-a mRNA in the neural tissue of both Dstdt-23Rbrc and DstGt homozygotes. Loss of sensory and autonomic nerve ends in the skin was observed in both Dstdt-23Rbrc and DstGt mice at postnatal stages. In contrast, Dst-e mRNA expression was reduced in the skin of Dstdt-23Rbrc mice but not in DstGt mice. Expression levels of Dst proteins in neural and cutaneous tissues correlated with Dst mRNAs. Because Dst-e encodes a structural protein in hemidesmosomes (HDs), we performed transmission electron microscopy. Lack of inner plaques and loss of keratin filament invasions underneath the HDs were observed in the basal keratinocytes of Dstdt-23Rbrc mice but not in those of DstGt mice; thus, the distinct phenotype of the skin of Dstdt-23Rbrc mice could be because of failure of Dst-e expression. These results indicate that distinct mutations within the Dst locus can cause different loss-of-function patterns among Dst isoforms, which accounts for the heterogeneous neural and skin phenotypes in dt mice and DST-related diseases.

Activation of α7 nAChR by PNU-282987 improves synaptic and cognitive functions through restoring the expression of synaptic-associated proteins and the CaM-CaMKII-CREB signaling pathway.

Ligands of nicotinic acetylcholine receptors (nAChRs) are widely considered as potential therapeutic agents. The present study used primary hippocampus cells and APPswe/PSEN1dE9 double-transgenic mice models to study the possible therapeutic effect and underlying mechanism of the specific activation of α7 nAChR by PNU-282987 in the pathogenesis of Alzheimer’s disease. The results indicated that activation of α7 nAChR attenuated the Aβ-induced cell apoptosis, decreased the deposition of Aβ, increased the expression of synaptic-associated proteins, and maintained synaptic morphology. Furthermore, in the APP/PS1_DT mice model, activation of α7 nAChR attenuated Aβ-induced synaptic loss, reduced the deposition of Aβ in the hippocampus, maintained the integral structure of hippocampus-derived synapse, and activated the calmodulin (CaM)-calmodulin-dependent protein kinase II (CaMKII)-cAMP response element-binding protein signaling pathway by upregulation of its key signaling proteins. In addition, activation of α7 nAChR improved the learning and memory abilities of the APP/PS1_DT mice. Collectively, the activation of α7 nAChR by PNU-282987 attenuated the toxic effect of Aβ in vivo and in vitro, which including reduced deposition of Aβ in the hippocampus, maintained synaptic morphology by partially reversing the expression levels of synaptic-associated proteins, activation of the Ca2+ signaling pathway, and improvement of the cognitive abilities of APP/PS1_DT mice.

Essential Oil of Mentha aquatica var. Kenting Water Mint Suppresses Two-Stage Skin Carcinogenesis Accelerated by BRAF Inhibitor Vemurafenib.

The v-raf murine sarcoma viral homolog B1 (BRAF) inhibitor drug vemurafenib (PLX4032) is used to treat melanoma; however, epidemiological evidence reveals that it could cause cutaneous keratoacanthomas and squamous cell carcinoma in cancer patients with the most prevalent HRASQ61L mutation. In a two-stage skin carcinogenesis mouse model, the skin papillomas induced by 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) (DT) resemble the lesions in BRAF inhibitor-treated patients. In this study, we investigated the bioactivity of Mentha aquatica var. Kenting Water Mint essential oil (KWM-EO) against PDV cells, mouse keratinocytes bearing HRASQ61L mutation, and its effect on inhibiting papilloma formation in a two-stage skin carcinogenesis mouse model with or without PLX4032 co-treatment. Our results revealed that KWM-EO effectively attenuated cell viability, colony formation, and the invasive and migratory abilities of PDV cells. Induction of G2/M cell-cycle arrest and apoptosis in PDV cells was also observed. KWM-EO treatment significantly decreased the formation of cutaneous papilloma further induced by PLX4032 in DT mice (DTP). Immunohistochemistry analyses showed overexpression of keratin14 and COX-2 in DT and DTP skin were profoundly suppressed by KWM-EO treatment. This study demonstrates that KWM-EO has chemopreventive effects against PLX4032-induced cutaneous side-effects in a DMBA/TPA-induced two-stage carcinogenesis model and will be worth further exploration for possible application in melanoma patients.

Reduced Proliferation of Oligodendrocyte Progenitor Cells in the Postnatal Brain of Dystonia Musculorum Mice.

Dystonia musculorum (dt) mice show sensory neurodegeneration and movement disorder, such as dystonia and cerebellar ataxia. The causative gene Dystonin (Dst) encodes a cytoskeleton linker protein. Although sensory neurodegeneration has been well studied, glial cell responses in the central nervous system (CNS) are poorly understood. Here, we investigated cell proliferation in the CNS of Dst Gt homozygous mice using newly generated in situ hybridization (ISH) probes-Ki-67 and proliferating cell nuclear antigen (PCNA) probes-both of which effectively detect proliferating cells. We found that Ki-67-positive cells were significantly decreased in the corpus callosum and thalamus of dt brain at postnatal day 21 (P21). There is a similar but not significant tendency at postnatal day 14 (P14) in the dt brain. We also confirmed the reduced proliferation by PCNA ISH and Ki-67 immunohistochemistry. Double staining with cell-type-specific markers revealed that proliferating cells are oligodendrocyte progenitor cells (OPCs) in both wild-type and dt brain. We also observed a reduced number of Olig2-positive cells in the corpus callosum of Dst Gt homozygous mice at P21, indicating that reduced proliferation resulted in a reduced number of OPCs. Our data indicate that OPCs proliferation is reduced in the dt mouse brain at the postnatal stage and that it subsequently results in the reduced number of OPCs.

Motoneuron degeneration in the trigeminal motor nucleus innervating the masseter muscle in Dystonia musculorum mice

Dystonia musculorum (dt) mice, which have a mutation in the Dystonin (Dst) gene, are used as animal models to investigate the human disease known as hereditary sensory and autonomic neuropathy type VI. Massive neuronal cell death is observed, mainly in the peripheral nervous system (PNS) of dt mice. We and others have recently reported a histopathological feature of these mice that neurofilament (NF) accumulates in various areas of the central nervous system (CNS), including motor pathways. Although dt mice show motor disorder and growth retardation, the causes for these are still unknown. Here we performed histopathological analyses on motor units of the trigeminal motor nucleus (Mo5 nucleus), because they are a good system to understand neuronal responses in the mutant CNS, and abnormalities in this system may lead to problems in mastication, with subsequent growth retardation. We report that motoneurons with NF accumulation in the Mo5 nuclei of DstGt homozygous mice express the stress-induced genes CHOP, ATF3, and lipocalin 2 (Lcn2). We also show a reduced number of Mo5 motoneurons and a reduced size of Mo5 nuclei in DstGt homozygous mice, possibly due to apoptosis, given the presence of cleaved caspase 3-positive Mo5 motoneurons. In the mandibular (V3) branches of the trigeminal nerve, which contains axons of Mo5 motoneurons and trigeminal sensory neurons, there was infiltration of Iba1-positive macrophages. Finally, we report atrophy of the masseter muscles in DstGt homozygous mice, which showed abnormal nuclear localization of myofibrils and increased expression of atrogin-1 mRNA, a muscle atrophy-related gene and weaker masseter muscle strength with uncontrolled muscle activity by electromyography (EMG). Taken together, our findings strongly suggest that mastication in dt mice is affected due to abnormalities of Mo5 motoneurons and masseter muscles, leading to growth retardation at the post-weaning stages.

Osteocyte Apoptosis Attracts Myeloma Cells to Bone and Supports Progression through Regulation of the Bone Marrow Microenvironment

Multiple Myeloma (MM) pathogenesis and progression is intricately tied to the bone marrow (BM) microenvironment, and understanding this microenvironment is of upmost importance. Osteocytes are the most abundant cell type in bone, comprising over 90% of the cellular population. These cells were long thought to be primarily inert mechanical sensors, however osteocyte regulation of the BM is increasingly appreciated. Still, very little is known about the role of osteocytes in the context of cancer. We initially evaluated MM patient BM biopsies and determined that osteocyte apoptosis is increased compared to MGUS and healthy BM. We further validated this finding in the SCID-hu mouse model - increased osteocyte death was seen in the primary human bone grafts that were injected with aggressive MM cells compared to those injected with less aggressive MM cells. Surprisingly, and more interestingly, increased osteocyte apoptosis/death was also found in the contralateral, un-injected human bone grafts from mice with aggressive MM. Importantly, these changes in the secondary bone grafts occurred prior to detection of MM cells. In the present study, we investigated if and how osteocyte apoptosis at distant bone sites, before MM metastasis, feeds back to MM cells to support dissemination and progression.We developed a unique, syngeneic mouse model of MM in which osteocytes can be specifically ablated in vivo. Mice expressing the Diphtheria toxin receptor (DTR) in mature osteocytes (RIKEN BioResource Center, Japan) were crossed with C57Bl/KaLwRij mice, a strain that when injected with syngeneic 5TGM1 MM cells develops many characteristics of human MM. The resulting 8-week old progeny expressing the DTR gene and on the C57Bl/KaLwRij background were intraperitoneally (i.p.) injected with Diphtheria toxin (DT,12.5 μg/kg) or PBS as control (n=7 per group). After 7 days, mice were injected intravenously (i.v.) with 2x106 5TGM1 MM cells expressing firefly Luciferase. Weekly bioluminescent imaging and ELISA for IgG2bκ (secreted by 5TGM1 cells) tracked tumor location and growth over time. Remarkably, compared to control mice (DTR+PBS), osteocyte ablated (DTR+DT) mice showed significantly enhanced tumor homing to bone by bioluminescent imaging and significantly increased total tumor burden by both bioluminescent imaging and sera IgG2bκ ELISA.To understand mechanistically how osteocyte apoptosis enhances MM-bone homing and tumor progression, we evaluated BM microenvironmental changes after osteocyte apoptosis was induced. Syngeneic DTR mice were injected at 8 weeks with DT or PBS (n=3-4 per group). Mouse legs were collected after 7 days: one leg for sectioning and one leg for isolating BM cells and BM extracellular fluid. Flow cytometry was performed on BM cells and BM extracellular fluid was evaluated using a cytokine/chemokine array. Leg sections were stained via immunohistochemistry (IHC). Cytokine arrays of BM extracellular fluid revealed substantial increases of multiple cytokines/chemokines important for MM dissemination and progression (i.e. IL-6, HGF) in DTR+DT mice. Additionally, bone destruction-associated molecules DKK1, RANKL, and M-CSF were also increased in DTR + DT mice compared to DTR+PBS control mice. IHC showed increased numbers of osteocytes positive for sclerostin, HIF-1α and RANKL. Finally, flow cytometric analysis suggested heightened immune suppression in the BM as multiple immune-suppressive cell populations were enhanced in DTR+DT mice compared to DTR+PBS mice. These include myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and a regulatory B cell subset (Breg).In conclusion, we have successfully generated a syngeneic model of MM in which osteocytes are selectively ablated in vivo after a single injection of DT. Using this model, our data demonstrate that osteocyte apoptosis supports MM homing and growth in bone, likely as a result of an altered BM microenvironment. Interestingly, these changes include enhanced MM supportive/attractive soluble molecules, support of bone destruction, and enhanced immune suppression. Combined, our data suggests that osteocyte apoptosis is important for developing a pre-metastatic niche and contributes to MM progression in bone. Therefore, preventing osteocyte apoptosis may help stall and prevent dissemination of MM, and possibly other cancers, to bone. DisclosuresNo relevant conflicts of interest to declare.

Characterization of novel dystonia musculorum mutant mice: Implications for central nervous system abnormality

We identified a novel spontaneous mutant mouse showing motor symptoms that are similar to those of the dystonia musculorum (dt) mouse. The observations suggested that the mutant mice inherited the mild dt phenotype as an autosomal recessive trait. Linkage analysis showed that the causative gene was located near D1Mit373 and D1Mit410 microsatellite markers on chromosome 1, which are close to the dystonin (Dst) gene locus. To investigate whether Dst is the causative gene of the novel mutant phenotype, we crossed the mutant with Dst gene trap (DstGt) mice. Compound heterozygotes showed a typical dt phenotype with sensory degeneration and progressive motor symptoms. DNA sequencing analysis identified a nonsense mutation within the spectrin repeats of the plakin domain. The novel mutant allele was named dt23Rbrc. Motor abnormalities in homozygous dt23Rbrc/dt23Rbrc mice are not as severe as homozygous DstGt/DstGt mice. Histological analyses showed abnormal neurofilament (NF) accumulation in the nervous system of homozygous dt23Rbrc/dt23Rbrc mice, which is characteristic of the dt phenotype. We mapped the distribution of abnormal NF-accumulated neurons in the brain and found that they were located specifically in the brainstem, spinal cord, and in regions such as the vestibular nucleus, reticular nucleus, and red nucleus, which are implicated in posture and motor coordination pathways. The quantification of abnormal NF accumulation in the cytoplasm and spheroids (axons) of neurons showed that abnormal NF immunoreactivity was lower in homozygous dt23Rbrc/dt23Rbrc mice than in homozygous DstGt/DstGt mice. Therefore, we have identified a novel hypomorphic allele of dt, which causes histological abnormalities in the central nervous system that may account for the abnormal motor phenotype. This novel spontaneously occurring mutant may become a good model of hereditary sensory and autonomic neuropathy type 6, which is caused by mutations in the human DST gene.

β-Hydroxypyruvate: a new diabetogenic factor?

The incidence of type 2 diabetes mellitus (T2DM) is increasing at an alarming rate. Insulin secretory dysfunction and insulin resistance are characteristic features of T2DM. However, their relative contributions to the progression from normal glucose tolerance to impaired glucose tolerance (IGT) to T2DM remain unknown. This limits the capacity of researchers to establish therapeutic interventions for T2DM. Glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 are the two primary incretin hormones secreted by intestinal K and L cells, respectively. Secretion of these incretins increases postprandially and they potentiate glucose-stimulated insulin secretion (GSIS). Despite blunted incretin responses in patients with T2DM, insulinotropic responses to exogenously administered GLP-1 remain active in these patients (1). Although controversy exists, a recent study demonstrated that insulin secretory responses to GIP were retained in patients with T2DM (1,2). Additionally, incretin response to GIP is improved by reducing blood glucose levels in patients with T2DM (3). These findings support the rationale for use of incretin-based pharmacotherapies in the treatment and management of T2DM. However, identification of the mechanism by which the function of K cells contributes to the development of T2DM is still elusive and requires a good model of study. To determine the role of K cells in the regulation of metabolism, K cells were eliminated from mice by expressing the diphtheria toxin A-chain gene in GIP-producing cells (DT mice). Despite a severely impaired …

Global biochemical profiling identifies β-hydroxypyruvate as a potential mediator of type 2 diabetes in mice and humans.

Glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 are incretins secreted by respective K and L enteroendocrine cells after eating and amplify glucose-stimulated insulin secretion (GSIS). This amplification has been termed the “incretin response.” To determine the role(s) of K cells for the incretin response and type 2 diabetes mellitus (T2DM), diphtheria toxin–expressing (DT) mice that specifically lack GIP-producing cells were backcrossed five to eight times onto the diabetogenic NONcNZO10/Ltj background. As in humans with T2DM, DT mice lacked an incretin response, although GLP-1 release was maintained. With high-fat (HF) feeding, DT mice remained lean but developed T2DM, whereas wild-type mice developed obesity but not diabetes. Metabolomics identified biochemicals reflecting impaired glucose handling, insulin resistance, and diabetes complications in prediabetic DT/HF mice. β-Hydroxypyruvate and benzoate levels were increased and decreased, respectively, suggesting β-hydroxypyruvate production from d-serine. In vitro, β-hydroxypyruvate altered excitatory properties of myenteric neurons and reduced islet insulin content but not GSIS. β-Hydroxypyruvate–to–d-serine ratios were lower in humans with impaired glucose tolerance compared with normal glucose tolerance and T2DM. Earlier human studies unmasked a neural relay that amplifies GIP-mediated insulin secretion in a pattern reciprocal to β-hydroxypyruvate–to–d-serine ratios in all groups. Thus, K cells may maintain long-term function of neurons and β-cells by regulating β-hydroxypyruvate levels.

0346: Aldosterone promotes cardiac endothelial cells proliferation and angiogenesis

The mineralocorticoid receptor (MR) and its ligand, aldosterone (aldo), are involved in cardiac diseases. However, the cardiomyocyte-MR regulated processes leading to these injuries are still unclear. To identify new aldosterone-regulated functions involved in cardiac diseases. Double transgenic mice overexpressing MR in cardiomyocytes (DT) and their controls (CT) were treated for 7 days with aldosterone (60 μg/kg/day; n = 6-9 / group) and a genome microarray analysis was performed. In CT mice, aldo regulated 70 genes. In DT mice, aldo induced much more genes (343). To identify the biological processes modulated by aldo, a gene ontology analysis was performed. The majority of aldosteroneregulated genes were involved in cell division, as shown by the relative expression of Cycline B1 and Cdk1 (Cycline; CT untreated 1 ± 0.02, CT aldotreated 2.1 ± 0.5; Cdk1; CT untreated 1 ± 0.05, CT aldo-treated 2.2 ± 0.4). Moreover, the Ki-67 index of aldo-treated mice (CT and DT) was higher than in non-treated ones (% of positive Ki-67 nuclei vs total nuclei; CT untreated 15±4, CT aldo-treated 51±12%; DT untreated 29 ± 5, DT aldo-treated 61 ± 19%). Co-staining of Ki-67 with caveolin 1 revealed that the cycling cells were endothelial cells. Ki-67 index was increased in an experimental model of heart failure (transverse aortic constriction: TAC) in rats. Eplerenone (eple), a MR blocker, prevented this increase (% of positive Ki-67 nuclei vs total nuclei; sham 26 ± 3, TAC 42 ± 5, TAC+eple 23 ± 6%). Aldosterone induces cardiac endothelial cells proliferation in vivo and underlines the role of cardiomyocyte-MR activation in this process. Favre and collaborators (AJP 2011) reported that cardiomyocyte- MR activation is involved in coronary endothelial dysfunction, via pathways of oxidative stress. It appears interesting to study if the cardiac endothelial cells proliferation induced by aldo is oxidative stress dependent.

Loss of the TNFα function inhibits Wnt/β-catenin signaling, exacerbates obesity development in adolescent spontaneous obese mice

Tumor necrosis factor alpha (TNFα) is an adipokine involved in the regulation of cell differentiation and lipid metabolism, but its specific role has not been clearly understood. We validated a hypothesis that loss of TNFα function would inhibit Wnt/β-catenin signaling and accelerate adipogenesis in adolescent genetic obese mice. Epididymal white adipose tissues (eWAT) from TNFα deficient (TNFα−/−), leptin receptor deficient (db/db) and double gene mutant (db/db/TNFα−/−, DT) male mice were used for comparative analysis of key molecules in Wnt/β-catenin signaling and adipogenic markers by qRT-PCR and western blot techniques. Compared with TNFα−/− and WT mice of 28 days old, an obese trait was observed in both db/db and DT mice, while the latter showed more significant body weight gain and eWAT hypertrophy. The mRNA level of key molecules in Wnt/β-catenin pathway was reduced in both obese groups, while the DT group was the lowest. Expression of adipocyte-specific genes was up-regulated during obese development in the two obese groups, while the DT group revealed more correlation than that of db/db group. At the protein level, a down regulation of Wnt10b and β-catenin in obese eWAT showed similar tendency with that of mRNA level. Compared with the lean groups, the levels of adiponectin and PPARγ2 for the obese groups were down-regulated at 21-day-old age, while they were elevated at older age. Our results suggested that deficiency in TNFα inhibited Wnt/β-catenin signaling of the obese eWAT and up-regulated expression of adipokines, and accelerated adipogenesis in genetic obese mice on a chow diet.Electronic supplementary materialThe online version of this article (doi:10.1007/s11010-014-1987-5) contains supplementary material, which is available to authorized users.

Transgenic expression of neuronal dystonin isoform 2 partially rescues the disease phenotype of the dystonia musculorum mouse model of hereditary sensory autonomic neuropathy VI.

A newly identified lethal form of hereditary sensory and autonomic neuropathy (HSAN), designated HSAN-VI, is caused by a homozygous mutation in the bullous pemphigoid antigen 1 (BPAG1)/dystonin gene (DST). The HSAN-VI mutation impacts all major neuronal BPAG1/dystonin protein isoforms: dystonin-a1, -a2 and -a3. Homozygous mutations in the murine Dst gene cause a severe sensory neuropathy termed dystonia musculorum (dt). Phenotypically, dt mice are similar to HSAN-VI patients, manifesting progressive limb contractures, dystonia, dysautonomia and early postnatal death. To obtain a better molecular understanding of disease pathogenesis in HSAN-VI patients and the dt disorder, we generated transgenic mice expressing a myc-tagged dystonin-a2 protein under the regulation of the neuronal prion protein promoter on the dt(Tg4/Tg4) background, which is devoid of endogenous dystonin-a1 and -a2, but does express dystonin-a3. Restoring dystonin-a2 expression in the nervous system, particularly within sensory neurons, prevented the disorganization of organelle membranes and microtubule networks, attenuated the degeneration of sensory neuron subtypes and ameliorated the phenotype and increased life span in these mice. Despite these improvements, complete rescue was not observed likely because of inadequate expression of the transgene. Taken together, this study provides needed insight into the molecular basis of the dt disorder and other peripheral neuropathies including HSAN-VI.

Abstract 138: Altered Vascular Reactivity in Mice Overexpressing Adipocyte Mineralocorticoid Receptors - Role of Rho Kinase.

Aldosterone (aldo) plays a role in cardiovascular diseases, including hypertension and obesity. We previously demonstrated that adipocyte-derived factors regulate vascular function and cell signaling in cultured vascular smooth muscle cells. Moreover, adipocytes are able to produce aldo, which influences vascular reactivity. Plasma levels of aldo are positively correlated with obesity and hypertension. However, the pathophysiological role of aldo and mineralocorticoid receptors (MR) in adipose tissue and its interactions with the vasculature remains elusive. In our study, we investigated molecular mechanisms whereby activation of MR, in adipocytes, leads to release of vascular reactive factors and regulation of vascular tone, using a conditional transgenic mouse model that overexpresses MR only in the adipocytes. Vascular reactivity of resistance mesenteric arteries to acetylcholine (Ach), sodium nitroprusside and phenylephrine (Phe), in the absence or presence of fat conditioned medium (Fcm) from control and adipocyte overexpressing MR (DT) mice, was performed by myography. In basal conditions, endothelial dysfunction was not observed in DT or control mice. However, in the presence of Fcm from DT mice, relaxation to Ach was impaired in control mice (Ach 10 -6 M: 77.5±9.6% no Fcm vs. 49.8±7.5% Fcm, p<0.05), an effect blocked by N-acetyl-cysteine (anti-oxidant) (Ach 10 -6 M: 82.2±6.6%). Resistance arteries from DT mice had decreased Phe-induced contraction, compared to control mice (Phe 10 -5 M: 2.7±0.2 mN/mm CT vs. 1.7±0.2 mN/mm DT, p<0.05). Phosphorylation of ezrin, a marker of Rho kinase activation, measured by immunoblotting, was decreased in white and brown adipose tissues of DT (CT: 3.1±0.7 vs. DT: 0.6±0.1, arbitrary units, p<0.05). In conclusion, MR in adipocytes may play an important role in the regulation of vascular function, and may be involved in vascular oxidative stress. MR in adipocytes is also important to the anti-contractile properties of the adipose tissue through downregulation of Rho kinase signaling. Our study identiy novel mechanisms linking vascular and adipose biology through adipocyte MRs.

Microtubule stability, Golgi organization, and transport flux require dystonin-a2–MAP1B interaction

Loss of function of dystonin cytoskeletal linker proteins causes neurodegeneration in dystonia musculorum (dt) mutant mice. Although much investigation has focused on understanding dt pathology, the diverse cellular functions of dystonin isoforms remain poorly characterized. In this paper, we highlight novel functions of the dystonin-a2 isoform in mediating microtubule (MT) stability, Golgi organization, and flux through the secretory pathway. Using dystonin mutant mice combined with isoform-specific loss-of-function analysis, we found dystonin-a2 bound to MT-associated protein 1B (MAP1B) in the centrosomal region, where it maintained MT acetylation. In dt neurons, absence of the MAP1B-dystonin-a2 interaction resulted in altered MAP1B perikaryal localization, leading to MT deacetylation and instability. Deacetylated MT accumulation resulted in Golgi fragmentation and prevented anterograde trafficking via motor proteins. Maintenance of MT acetylation through trichostatin A administration or MAP1B overexpression mitigated the observed defect. These cellular aberrations are apparent in prephenotype dorsal root ganglia and primary sensory neurons from dt mice, suggesting they are causal in the disorder.

Cardiac FKBP12.6 overexpression protects against triggered ventricular tachycardia in pressure overloaded mouse hearts

Alterations in RyR2 function have been proposed as a major pathophysiological mechanism of arrhythmias and heart failure (HF). Cardiac FKBP12.6 overexpression protects against myocardial infarction-induced HF and catecholamine-promoted ventricular arrhythmias. We tested the hypothesis that FKBP12.6 overexpression protects against maladaptive LVH and triggered ventricular arrhythmias following transverse aorta constriction (TAC) in the mouse. The TAC-associated mortality rate was significantly lower in male transgenic (DT) than in Ctr mice (p<0.05). TAC-associated maladaptive hypertrophy was blunted in DT mice especially 1month post-TAC and their SERCA2a/PLB ratio remained unchanged 1 and 2months post-TAC. Two months after TAC, trains of 30 stimuli (burst pacing) performed following isoproterenol injection (0.2mg/kg, ip), induced VT in 50% of the TAC-Ctr and in none of the TAC-DT mice (p=0.022). The increase in myocyte shortening and Ca(2+) spark frequency observed in sham-operated Ctr mice in response to 50 nM isoproterenol was reduced in DT mice, and abolished in TAC-DT mice. NCX1 function was reduced in Sham-DT and TAC-DT compared with Sham-Ctr and TAC-Ctr mice, respectively (p<0.05 for the 2 comparisons). In mice killed after isoproterenol injection and burst pacing, RyR2 S2814 phosphorylation was decreased by 50% in TAC-DT versus TAC-Ctr mice (p<0.05), with no change in RyR2 S2808 and PLB S16 and T17 phosphorylation. Cardiac FKBP12.6 overexpression in the mouse blunts pressure overload-induced maladaptive LV remodelling and protects against catecholamine-promoted burst pacing-induced ventricular tachycardia by decreasing cardiac sensitivity to adrenergic stress and RyR2 S2814 phosphorylation, and decreasing NCX1 activity.