Dystonin-A3 upregulation is responsible for maintenance of tubulin acetylation in a less severe dystonia musculorum mouse model for hereditary sensory and autonomic neuropathy type VI.

Hereditary sensory and autonomic neuropathy type VI (HSAN-VI) is a recessive human disease that arises from mutations in the dystonin gene (DST; also known as Bullous pemphigoid antigen 1 gene). A milder form of HSAN-VI was recently described, resulting from loss of a single dystonin isoform (DST-A2). Similarly, mutations in the mouse dystonin gene (Dst) result in severe sensory neuropathy, dystonia musculorum (Dstdt). Two Dstdt alleles, Dstdt-Tg4 and Dstdt-27J, differ in the severity of disease. The less severe Dstdt-Tg4 mice have disrupted expression of Dst-A1 and -A2 isoforms, while the more severe Dstdt-27J allele affects Dst-A1, -A2 and -A3 isoforms. As dystonin is a cytoskeletal-linker protein, we evaluated microtubule network integrity within sensory neurons from Dstdt-Tg4 and Dstdt-27J mice. There is a significant reduction in tubulin acetylation in Dstdt-27J indicative of microtubule instability and severe microtubule disorganization within sensory axons. However, Dstdt-Tg4 mice have no change in tubulin acetylation, and microtubule organization was only mildly impaired. Thus, microtubule instability is not central to initiation of Dstdt pathogenesis, though it may contribute to disease severity. Maintenance of microtubule stability in Dstdt-Tg4 dorsal root ganglia could be attributed to an upregulation in Dst-A3 expression as a compensation for the absence of Dst-A1 and -A2 in Dstdt-Tg4 sensory neurons. Indeed, knockdown of Dst-A3 in these neurons resulted in a decrease in tubulin acetylation. These findings shed light on the possible compensatory role of dystonin isoforms within HSAN-VI, which might explain the heterogeneity in symptoms within the reported forms of the disease.

Pathologic Alterations in the Proteome of Synaptosomes from a Mouse Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a human genetic disorder characterized by muscle weakness, muscle atrophy, and death of motor neurons. SMA is caused by mutations or deletions in a gene called survival motor neuron 1 (SMN1). SMN1 is a housekeeping gene, but the most prominent pathologies in SMA are atrophy of myofibers and death of motor neurons. Further, degeneration of neuromuscular junctions, of synapses, and of axonal regions are features of SMA disease. Here, we have investigated the proteome dynamics of central synapses in P14 Smn2B/- mice, a model of SMA. Label-free quantitative proteomics on isolated synaptosomes from spinal cords of these animals identified 2030 protein groups. Statistical data analysis revealed 65 specific alterations in the proteome of the central synapses at the early onset stage of disease. Functional analysis of the dysregulated proteins indicated a significant enrichment of proteins associated with mitochondrial dynamics, cholesterol biogenesis, and protein clearance. These pathways represent potential targets for therapy development with the goal of providing stability to the central synapses, thereby preserving neuronal integrity in the context of SMA disease. Data are available via ProteomeXchange with identifier PXD012850.

Immune dysregulation may contribute to disease pathogenesis in spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) has long been solely considered a neurodegenerative disorder. However, recent work has highlighted defects in many other cell types that could contribute to disease aetiology. Interestingly, the immune system has never been extensively studied in SMA. Defects in lymphoid organs could exacerbate disease progression by neuroinflammation or immunodeficiency. Smn depletion led to severe alterations in the thymus and spleen of two different mouse models of SMA. The spleen from Smn depleted mice was dramatically smaller at a very young age and its histological architecture was marked by mislocalization of immune cells in the Smn2B/- model mice. In comparison, the thymus was relatively spared in gross morphology but showed many histological alterations including cortex thinning in both mouse models at symptomatic ages. Thymocyte development was also impaired as evidenced by abnormal population frequencies in the Smn2B/- thymus. Cytokine profiling revealed major changes in different tissues of both mouse models. Consistent with our observations, we found that survival motor neuron (Smn) protein levels were relatively high in lymphoid organs compared to skeletal muscle and spinal cord during postnatal development in wild type mice. Genetic introduction of one copy of the human SMN2 transgene was enough to rescue splenic and thymic defects in Smn2B/- mice. Thus, Smn is required for the normal development of lymphoid organs, and altered immune function may contribute to SMA disease pathogenesis.

Oligodendrocyte development and CNS myelination are unaffected in a mouse model of severe spinal muscular atrophy.

The childhood neurodegenerative disease spinal muscular atrophy (SMA) is caused by loss-of-function mutations or deletions in the Survival Motor Neuron 1 (SMN1) gene resulting in insufficient levels of survival motor neuron (SMN) protein. Classically considered a motor neuron disease, increasing evidence now supports SMA as a multi-system disorder with phenotypes discovered in cortical neuron, astrocyte, and Schwann cell function within the nervous system. In this study, we sought to determine whether Smn was critical for oligodendrocyte (OL) development and central nervous system myelination. A mouse model of severe SMA was used to assess OL growth, migration, differentiation and myelination. All aspects of OL development and function studied were unaffected by Smn depletion. The tremendous impact of Smn depletion on a wide variety of other cell types renders the OL response unique. Further investigation of the OLs derived from SMA models may reveal disease modifiers or a compensatory mechanism allowing these cells to flourish despite the reduced levels of this multifunctional protein.

Myogenic program dysregulation is contributory to disease pathogenesis in spinal muscular atrophy.

Mutations in the survival motor neuron (SMN1) gene lead to the neuromuscular disease spinal muscular atrophy (SMA). Although SMA is primarily considered as a motor neuron disease, the importance of muscle defects in its pathogenesis has not been fully examined. We use both primary cell culture and two different SMA model mice to demonstrate that reduced levels of Smn lead to a profound disruption in the expression of myogenic genes. This disruption was associated with a decrease in myofiber size and an increase in immature myofibers, suggesting that Smn is crucial for myogenic gene regulation and early muscle development. Histone deacetylase inhibitor trichostatin A treatment of SMA model mice increased myofiber size, myofiber maturity and attenuated the disruption of the myogenic program in these mice. Taken together, our work highlights the important contribution of myogenic program dysregulation to the muscle weakness observed in SMA.

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.

Isolation of small extracellular vesicles from regenerating muscle tissue using tangential flow filtration and size exclusion chromatography.

We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

Loss of miR-145 promotes remyelination and functional recovery in a model of chronic central demyelination.

Strategies for treating progressive multiple sclerosis (MS) remain limited. Here, we found that miR-145-5p is overabundant uniquely in chronic lesion tissues from secondary progressive MS patients. We induced both acute and chronic demyelination in miR-145 knockout mice to determine its contributions to remyelination failure. Following acute demyelination, no advantage to miR-145 loss could be detected. However, after chronic demyelination, animals with miR-145 loss demonstrated increased remyelination and functional recovery, coincident with altered presence of astrocytes and microglia within the corpus callosum relative to wild-type animals. This improved response in miR-145 knockout animals coincided with a pathological upregulation of miR-145-5p in wild-type animals with chronic cuprizone exposure, paralleling human chronic lesions. Furthermore, miR-145 overexpression specifically in oligodendrocytes (OLs) severely stunted differentiation and negatively impacted survival. RNAseq analysis showed altered transcriptome in these cells with downregulated major pathways involved in myelination. Our data suggest that pathological accumulation of miR-145-5p is a distinctive feature of chronic demyelination and is strongly implicated in the failure of remyelination, possibly due to the inhibition of OL differentiation together with alterations in other glial cells. This is mirrored in chronic MS lesions, and thus miR-145-5p serves as a potential relevant therapeutic target in progressive forms of MS.

The Rb/E2F pathway modulates neurogenesis through direct regulation of the Dlx1/Dlx2 bigene cluster.

During brain morphogenesis, the mechanisms through which the cell cycle machinery integrates with differentiation signals remain elusive. Here we show that the Rb/E2F pathway regulates key aspects of differentiation and migration through direct control of the Dlx1 and Dlx2 homeodomain proteins, required for interneuron specification. Rb deficiency results in a dramatic reduction of Dlx1 and Dlx2 gene expression manifested by loss of interneuron subtypes and severe migration defects in the mouse brain. The Rb/E2F pathway modulates Dlx1/Dlx2 regulation through direct interaction with a Dlx forebrain-specific enhancer, I12b, and the Dlx1/Dlx2 proximal promoter regions, through repressor E2F sites both in vitro and in vivo. In the absence of Rb, we demonstrate that repressor E2Fs inhibit Dlx transcription at the Dlx1/Dlx2 promoters and Dlx1/2-I12b enhancer to suppress differentiation. Our findings support a model whereby the cell cycle machinery not only controls cell division but also modulates neuronal differentiation and migration through direct regulation of the Dlx1/Dlx2 bigene cluster during embryonic development.

Wnt binding to Coatomer proteins directs secretion on exosomes independently of palmitoylation.

Wnt proteins are secreted hydrophobic glycoproteins that act over long distances through poorly understood mechanisms. We discovered that Wnt7a is secreted on extracellular vesicles (EVs) following muscle injury. Structural analysis identified the motif responsible for Wnt7a secretion on EVs that we term the Exosome Binding Peptide (EBP). Addition of the EBP to an unrelated protein directed secretion on EVs. Disruption of palmitoylation, knockdown of WLS, or deletion of the N-terminal signal peptide did not affect Wnt7a secretion on purified EVs. Bio-ID analysis identified Coatomer proteins as candidates responsible for loading Wnt7a onto EVs. The crystal structure of EBP bound to the COPB2 coatomer subunit, the binding thermodynamics, and mutagenesis experiments, together demonstrate that a dilysine motif in the EBP mediates binding to COPB2. Other Wnts contain functionally analogous structural motifs. Mutation of the EBP results in a significant impairment in the ability of Wnt7a to stimulate regeneration, indicating that secretion of Wnt7a on exosomes is critical for normal regeneration in vivo . Our studies have defined the structural mechanism that mediates binding of Wnt7a to exosomes and elucidated the singularity of long-range Wnt signalling.

Cdx2 regulation of posterior development through non-Hox targets.

The homeodomain transcription factors Cdx1, Cdx2 and Cdx4 play essential roles in anteroposterior vertebral patterning through regulation of Hox gene expression. Cdx2 is also expressed in the trophectoderm commencing at E3.5 and plays an essential role in implantation, thus precluding assessment of the cognate-null phenotype at later stages. Cdx2 homozygous null embryos generated by tetraploid aggregation exhibit an axial truncation indicative of a role for Cdx2 in elaborating the posterior embryo through unknown mechanisms. To better understand such roles, we developed a conditional Cdx2 floxed allele in mice and effected temporal inactivation at post-implantation stages using a tamoxifen-inducible Cre. This approach yielded embryos that were devoid of detectable Cdx2 protein and exhibited the axial truncation phenotype predicted from previous studies. This phenotype was associated with attenuated expression of genes encoding several key players in axial elongation, including Fgf8, T, Wnt3a and Cyp26a1, and we present data suggesting that T, Wnt3a and Cyp26a1 are direct Cdx2 targets. We propose a model wherein Cdx2 functions as an integrator of caudalizing information by coordinating axial elongation and somite patterning through Hox-independent and -dependent pathways, respectively.

Suppression of the necroptotic cell death pathways improves survival in Smn 2B/- mice.

Spinal muscular atrophy (SMA) is a monogenic neuromuscular disease caused by low levels of the Survival Motor Neuron (SMN) protein. Motor neuron degeneration is the central hallmark of the disease. However, the SMN protein is ubiquitously expressed and depletion of the protein in peripheral tissues results in intrinsic disease manifestations, including muscle defects, independent of neurodegeneration. The approved SMN-restoring therapies have led to remarkable clinical improvements in SMA patients. Yet, the presence of a significant number of non-responders stresses the need for complementary therapeutic strategies targeting processes which do not rely solely on restoring SMN. Dysregulated cell death pathways are candidates for SMN-independent pathomechanisms in SMA. Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 have been widely recognized as critical therapeutic targets of necroptosis, an important form of programmed cell death. In addition, Caspase-1 plays a fundamental role in inflammation and cell death. In this study, we evaluate the role of necroptosis, particularly RIPK3 and Caspase-1, in the Smn 2B/- mouse model of SMA. We have generated a triple mutant (TKO), the Smn 2B/-; Ripk3 -/-; Casp1 -/- mouse. TKO mice displayed a robust increase in survival and improved motor function compared to Smn 2B/- mice. While there was no protection against motor neuron loss or neuromuscular junction pathology, larger muscle fibers were observed in TKO mice compared to Smn 2B/- mice. Our study shows that necroptosis modulates survival, motor behavior and muscle fiber size independent of SMN levels and independent of neurodegeneration. Thus, small-molecule inhibitors of necroptosis as a combinatorial approach together with SMN-restoring drugs could be a future strategy for the treatment of SMA.

SMN Depleted Mice Offer a Robust and Rapid Onset Model of Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is considered a health epidemic with potential devastating effects on the patients and the healthcare systems. Current preclinical models of NAFLD are invariably imperfect and generally take a long time to develop. A mouse model of survival motor neuron (SMN) depletion (Smn2B/- mice) was recently shown to develop significant hepatic steatosis in less than 2 weeks from birth. The rapid onset of fatty liver in Smn2B/- mice provides an opportunity to identify molecular markers of NAFLD. Here, we investigated whether Smn2B/- mice display typical features of NAFLD/nonalcoholic steatohepatitis (NASH). METHODS: Biochemical, histologic, electron microscopy, proteomic, and high-resolution respirometry were used. RESULTS: The Smn2B/- mice develop microvesicular steatohepatitis within 2 weeks, a feature prevented by AAV9-SMN gene therapy. Although fibrosis is not overtly apparent in histologic sections of the liver, there is molecular evidence of fibrogenesis and presence of stellate cell activation. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in hepatic dysfunction in protein output, complement, coagulation, iron homeostasis, and insulin-like growth factor-1 metabolism. The NAFLD phenotype is likely due to non-esterified fatty acid overload from peripheral lipolysis subsequent to hyperglucagonemia compounded by reduced muscle use and insulin resistance. Despite the low hepatic mitochondrial content, isolated mitochondria show enhanced ß-oxidation, likely as a compensatory response, resulting in the production of reactive oxygen species. In contrast to typical NAFLD/NASH, the Smn2B/- mice lose weight because of their associated neurological condition (spinal muscular atrophy) and develop hypoglycemia. CONCLUSIONS: The Smn2B/- mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early-onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations.

Bin1 SRC homology 3 domain acts as a scaffold for myofiber sarcomere assembly.

In skeletal muscle development, the genes and regulatory factors that govern the specification of myocytes are well described. Despite this knowledge, the mechanisms that regulate the coordinated assembly of myofiber proteins into the functional contractile unit or sarcomere remain undefined. Here we explored the hypothesis that modular domain proteins such as Bin1 coordinate protein interactions to promote sarcomere formation. We demonstrate that Bin1 facilitates sarcomere organization through protein-protein interactions as mediated by the Src homology 3 (SH3) domain. We observed a profound disorder in myofiber size and structural organization in a murine model expressing the Bin1 SH3 region. In addition, satellite cell-derived myogenesis was limited despite the accumulation of skeletal muscle-specific proteins. Our experiments revealed that the Bin1 SH3 domain formed transient protein complexes with both actin and myosin filaments and the pro-myogenic kinase Cdk5. Bin1 also associated with a Cdk5 phosphorylation domain of titin. Collectively, these observations suggest that Bin1 displays protein scaffold-like properties and binds with sarcomeric factors important in directing sarcomere protein assembly and myofiber maturation.

Transgenic expression of the activating natural killer receptor Ly49H confers resistance to cytomegalovirus in genetically susceptible mice.

Natural resistance to infection with mouse cytomegalovirus (MCMV) is controlled by a dominant locus, Cmv1. Cmv1 is linked to the Ly49 family of natural killer receptors on distal chromosome 6. While some studies localized Cmv1 as distal to the Ly49 gene cluster, genetic and functional analysis identified Ly49h as a pivotal factor in resistance to MCMV. The role of these two independent genomic domains in MCMV resistance was evaluated by functional complementation using transgenesis of bacterial artificial chromosomes (BAC) in genetically susceptible mice. Phenotypic and genetic characterization of the transgenic animals traced the resistance gene to a single region spanning the Ly49h gene. The appearance of the Ly49H protein in NK cells of transgenic mice coincided with the emergence of MCMV resistance, and there was a threshold Ly49H protein level associated with full recovery. Finally, transgenic expression of Ly49H in the context of either of the two independent susceptibility alleles, Cmv1(sBALB) or Cmv1(sFVB), conferred resistance to MCMV infection. These results demonstrate that Ly49h is necessary and sufficient to confer MCMV resistance, and formally demonstrate allelism between Cmv1 and Ly49h. This panel of transgenic animals provides a unique resource to study possible pleiotropic effect of Cmv1.

Production of mouse chimeras by injection of embryonic stem cells into the perivitelline space of one-cell stage embryos.

Generation of mouse chimeras is useful for the elucidation of gene function. In the present report, we describe a new technique for the production of chimeras by injection of R1 embryonic stem (ES) cells into the perivitelline space of one-cell stage mouse embryos. One-cell embryos are injected with 2-6 ES cells into the perivitelline space under the zona pellucida without laser-assistance. Our embryo culture experiments reveal that ES cells injected at the one-cell stage embryo start to be incorporated into the blastomeres beginning at the 8-cell stage and form a chimeric blastocyst after 4 days. We have used this approach to successfully produce a high rate of mouse chimeras in two different mouse genetic backgrounds permitting the establishment of germ line transmitters. This method allows for the earlier introduction of ES cells into mouse embryos, and should free up the possibility of using frozen one-cell embryos for this purpose.

Mice with podocyte-specific overexpression of wild type alpha-actinin-4 are healthy controls for K256E-alpha-actinin-4 mutant transgenic mice.

Mutations in the gene ACTN4 encoding the actin bundling protein-alpha-actinin-4 underlie an inherited form of kidney lesions known as focal segmental glomerulosclerosis (FSGS). Previously, we developed a model for this condition by generating mice with podocyte-specific overexpression of a disease-causing mutant alpha-actinin-4 (K256E-ACTN4 (pod+)). However, whether alpha-actinin-4 overexpression artifacts and not the gain of affinity effects of the mutation accounted for the robust FSGS phenotype in these mice was unclear. To address this question, we developed a control line of mice with podocyte-specific overexpression of wildtype alpha-actinin-4 (wt-ACTN4 (pod+)). An 8.3 kb fragment of the mouse nephrin promoter (NPHS1) was used to drive expression of a hemagglutinin (HA)-tagged wildtype alpha-actinin-4 coding sequence in mice. Five founder lines expressing the HA-tagged alpha-actinin-4 protein in a podocyte-specific manner were obtained, as determined by co-immunofluorescence with HA and synaptopodin antibodies. Quantitative PCR revealed that renal transgene mRNA levels of wt-ACTN4 (pod+) mice are similar to K256E-ACTN4 (pod+) mice. In contrast to K256E-ACTN4 (pod+) mice which exhibit albuminuria, podocyte foot process effacement and glomerular scarring, wt-ACTN4 (pod+) mice are healthy and indistinguishable from non-transgenic littermates. These findings suggest that the K256E mutation itself and not overexpression of alpha-actinin-4 protein per se accounts for the FSGS phenotype in our transgenic model.

Characterization of gastrointestinal pathologies in the dystonia musculorum mouse model for hereditary sensory and autonomic neuropathy type VI.

BACKGROUND: Dystonia musculorum (Dstdt ) is a murine disease caused by recessive mutations in the dystonin (Dst) gene. Loss of dorsal root ganglion (DRG) sensory neurons, ataxia, and dystonic postures before death by postnatal day 18 (P18) is a hallmark feature. Recently we observed gas accumulation and discoloration in the small intestine and cecum in Dstdt mice by P15. The human disease resulting from dystonin loss-of-function, known as hereditary sensory and autonomic neuropathy type VI (HSAN-VI), has also been associated with gastrointestinal (GI) symptoms including chronic diarrhea and abdominal pain. As neuronal dystonin isoforms are expressed in the GI tract, we hypothesized that dystonin loss-of-function in Dstdt-27J enteric nervous system (ENS) neurons resulted in neurodegeneration associated with the GI abnormalities. METHODS: We characterized the nature of the GI abnormalities observed in Dstdt mice through histological analysis of the gut, assessing the ENS for signs of neurodegeneration, evaluation of GI motility and absorption, and by profiling the microbiome. KEY RESULTS: Though gut histology, ENS viability, and GI absorption were normal, slowed GI motility, thinning of the colon mucous layer, and reduced microbial richness/evenness were apparent in Dstdt-27J mice by P15. Parasympathetic GI input showed signs of neurodegeneration, while sympathetic did not. CONCLUSIONS & INFERENCES: Dstdt-27J GI defects are not linked to ENS neurodegeneration, but are likely a result of an imbalance in autonomic control over the gut. Further characterization of HSAN-VI patient GI symptoms is necessary to determine potential treatments targeting symptom relief.

Metformin promotes CNS remyelination and improves social interaction following focal demyelination through CBP Ser436 phosphorylation.

Individuals with demyelinating diseases often experience difficulties during social interactions that are not well studied in preclinical models. Here, we describe a novel juvenile focal corpus callosum demyelination murine model exhibiting a social interaction deficit. Using this preclinical murine demyelination model, we discover that application of metformin, an FDA-approved drug, in this model promotes oligodendrocyte regeneration and remyelination and improves the social interaction. This beneficial effect of metformin acts through stimulating Ser436 phosphorylation in CBP, a histone acetyltransferase. In addition, we found that metformin acts through two distinct molecular pathways to enhance oligodendrocyte precursor (OPC) proliferation and differentiation, respectively. Metformin enhances OPC proliferation through early-stage autophagy inhibition, while metformin promotes OPC differentiation into mature oligodendrocytes through activating CBP Ser436 phosphorylation. In summary, we identify that metformin is a promising remyelinating agent to improve juvenile demyelination-associated social interaction deficits by promoting oligodendrocyte regeneration and remyelination.

XIAP Protects Retinal Ganglion Cells in the Mutant ND4 Mouse Model of Leber Hereditary Optic Neuropathy.

Purpose: Leber hereditary optic neuropathy (LHON) is a genetic form of vision loss that occurs primarily owing to mutations in the nicotinamide adenine dinucleotide dehydrogenase (ND) subunits that make up complex I of the electron transport chain. LHON mutations result in the apoptotic death of retinal ganglion cells. We tested the hypothesis that gene therapy with the X-linked inhibitor of apoptosis (XIAP) would prevent retinal ganglion cell apoptosis and reduce disease progression in a vector-induced mouse model of LHON that carries the ND4 mutation. Methods: Adeno-associated virus (AAV) encoding full length hemagglutinin-tagged XIAP (AAV2.HA-XIAP) or green fluorescent protein (AAV2.GFP) was injected into the vitreous of DBA/1J mice. Two weeks later, the LHON phenotype was induced by AAV delivery of mutant ND4 (AAV2.mND4FLAG) to the vitreous. Retinal function was assessed by pattern electroretinography. Optic nerves were harvested at 4 months, and the effects of XIAP therapy on nerve fiber layer and optic nerve integrity were evaluated using immunohistochemistry, transmission electron microscopy and magnetic resonance imaging. Results: During LHON disease progression, retinal ganglion cell axons are lost. Apoptotic cell bodies are seen in the nuclei of astrocytes or oligodendrocytes in the optic nerve, and there is thinning of the optic nerve and the nerve fiber layer of the retina. At 4 months after disease onset, XIAP gene therapy protects the nerve fiber layer and optic nerve architecture by preserving axon health. XIAP also decreases nuclear fragmentation in resident astrocytes or oligodendrocytes and decreases glial cell infiltration. Conclusions: XIAP therapy improves optic nerve health and delays disease progression in LHON.