The Evolution of Medical Countermeasures for Ebola Virus Disease: Lessons Learned and Next Steps.

The Ebola virus disease outbreak that occurred in Western Africa from 2013-2016, and subsequent smaller but increasingly frequent outbreaks of Ebola virus disease in recent years, spurred an unprecedented effort to develop and deploy effective vaccines, therapeutics, and diagnostics. This effort led to the U.S. regulatory approval of a diagnostic test, two vaccines, and two therapeutics for Ebola virus disease indications. Moreover, the establishment of fieldable diagnostic tests improved the speed with which patients can be diagnosed and public health resources mobilized. The United States government has played and continues to play a key role in funding and coordinating these medical countermeasure efforts. Here, we describe the coordinated U.S. government response to develop medical countermeasures for Ebola virus disease and we identify lessons learned that may improve future efforts to develop and deploy effective countermeasures against other filoviruses, such as Sudan virus and Marburg virus.

Loss of TDP-43 function and rimmed vacuoles persist after T cell depletion in a xenograft model of sporadic inclusion body myositis.

Sporadic inclusion body myositis (IBM) is the most common acquired muscle disease in adults over age 50, yet it remains unclear whether the disease is primarily driven by T cell­mediated autoimmunity. IBM muscle biopsies display nuclear clearance and cytoplasmic aggregation of TDP-43 in muscle cells, a pathologic finding observed initially in neurodegenerative diseases, where nuclear loss of TDP-43 in neurons causes aberrant RNA splicing. Here, we show that loss of TDP-43­mediated splicing repression, as determined by inclusion of cryptic exons, occurs in skeletal muscle of subjects with IBM. Of 119 muscle biopsies tested, RT-PCR­mediated detection of cryptic exon inclusion was able to diagnose IBM with 84% sensitivity and 99% specificity. To determine the role of T cells in pathogenesis, we generated a xenograft model by transplanting human IBM muscle into the hindlimb of immunodeficient mice. Xenografts from subjects with IBM displayed robust regeneration of human myofibers and recapitulated both inflammatory and degenerative features of the disease. Myofibers in IBM xenografts showed invasion by human, oligoclonal CD8+ T cells and exhibited MHC-I up-regulation, rimmed vacuoles, mitochondrial pathology, p62-positive inclusions, and nuclear clearance and cytoplasmic aggregation of TDP-43, associated with cryptic exon inclusion. Reduction of human T cells within IBM xenografts by treating mice intraperitoneally with anti-CD3 (OKT3) suppressed MHC-I up-regulation. However, rimmed vacuoles and loss of TDP-43 function persisted. These data suggest that T cell depletion does not alter muscle degenerative pathology in IBM.

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2rγnull (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

The expression of select genes necessary for membrane-associated estrogen receptor signaling differ by sex in adult rat hippocampus.

17ß-estradiol can rapidly modulate neuron function via membrane estrogen receptors (ERs) in a sex-specific manner. For example, female rat hippocampal neurons express palmitoylated versions of ERα and ERß that associate with the plasma membrane. These membrane-associated ERs are organized by caveolin proteins into functional signaling microdomains with metabotropic glutamate receptors (mGluRs). ER/mGluR signaling mediates several sex-specific estradiol actions on hippocampal neuron function. An important unanswered question regards the mechanism by which sex-specific membrane-associated ER signaling is generated, especially since it has been previously demonstrated that mGluR action is not sex-specific. One possibility is that the genes necessary for the ER membrane complex are differentially expressed between males and females, including genes that encode ERα and ß, caveolin 1 and 3, and/or the palmitoylacyltransferases DHHC-7 and -21. Thus we used qPCR to test the hypothesis that these genes show sex differences in expression in neonatal and adult rat hippocampus. As an additional control we tested the expression of the 20 other DHHC palmitoylacyltransferases with no known connections to ER. In neonatal hippocampus, no sex differences were detected in gene expression. In adult hippocampus, the genes that encode caveolin 1 and DHHC-7 showed decreased expression in females compared to males. Thus, select genes differ by sex at specific developmental stages, arguing for a more nuanced model than simple widespread perinatal emergence of sex differences in all genes enabling sex-specific estradiol action. These findings enable the generation of new hypotheses regarding the mechanisms by which sex differences in membrane-associated ER signaling are programmed.

TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle.

A dominant histopathological feature in neuromuscular diseases, including amyotrophic lateral sclerosis and inclusion body myopathy, is cytoplasmic aggregation of the RNA-binding protein TDP-43. Although rare mutations in TARDBP-the gene that encodes TDP-43-that lead to protein misfolding often cause protein aggregation, most patients do not have any mutations in TARDBP. Therefore, aggregates of wild-type TDP-43 arise in most patients by an unknown mechanism. Here we show that TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, which we call myo-granules, during regeneration of skeletal muscle in mice and humans. Myo-granules bind to mRNAs that encode sarcomeric proteins and are cleared as myofibres mature. Although myo-granules occur during normal skeletal-muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro and are increased in a mouse model of inclusion body myopathy. Therefore, increased assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates that commonly occur in neuromuscular disease.

New Developments in the Genetics of Inclusion Body Myositis.

PURPOSE OF REVIEW: Our goal is to review the recent literature pertaining to the genetics of sporadic inclusion body myositis (IBM). RECENT FINDINGS: In a study of 252 IBM patients, the class II MHC allele HLA-DRB1*03:01 showed the most significant association with IBM, and that risk could be largely attributed to amino acids within the peptide-binding pocket. Candidate gene sequencing identified rare missense variants in proteins regulating protein homeostasis including VCP and SQSTM1. An unbiased approach employing exome sequencing of genes encoding rimmed vacuole proteins identified FYCO1 variants in IBM. Ongoing GWAS approaches may shed new light on genetic risk factors for IBM. Many variants have been reported at an increased frequency in IBM in small studies; however, only HLA association has shown genome-wide significance. Future studies are needed to validate variants in larger cohorts and to understand the molecular roles these risk factors play in IBM.

Palmitoylation of estrogen receptors is essential for neuronal membrane signaling.

In addition to activating nuclear estrogen receptor signaling, 17ß-estradiol can also regulate neuronal function via surface membrane receptors. In various brain regions, these actions are mediated by the direct association of estrogen receptors (ERs) activating metabotropic glutamate receptors (mGluRs). These ER/mGluR signaling partners are organized into discrete functional microdomains via caveolin proteins. A central question that remains concerns the underlying mechanism by which these subpopulations of ERs are targeted to the surface membrane. One candidate mechanism is S-palmitoylation, a posttranscriptional modification that affects the subcellular distribution and function of the modified protein, including promoting localization to membranes. Here we test for the role of palmitoylation and the necessity of specific palmitoylacyltransferase proteins in neuronal membrane ER action. In hippocampal neurons, pharmacological inhibition of palmitoylation eliminated 17ß-estradiol-mediated phosphorylation of cAMP response element-binding protein, a process dependent on surface membrane ERs. In addition, mutation of the palmitoylation site on estrogen receptor (ER) α blocks ERα-mediated cAMP response element-binding protein phosphorylation. Similar results were obtained after mutation of the palmitoylation site on ERß. Importantly, mutation of either ERα or ERß did not affect the ability of the reciprocal ER to signal at the membrane. In contrast, membrane ERα and ERß signaling were both dependent on the expression of the palmitoylacyltransferase proteins DHHC-7 and DHHC-21. Neither mGluR activity nor caveolin or ER expression was affected by knockdown of DHHC-7 and DHHC-21. These data collectively suggest discrete mechanisms that regulate specific isoform or global membrane ER signaling in neurons separate from mGluR activity or nuclear ER function.