Enhanced BDNF and ROS in Mucosa of Lower Motor Neuron Lesioned Dog Bladder Following Somatic Motor Nerve Transfer

AUTHORS

Nagat Frara, Kais Jawawdeh, Dania Giaddui, Istvan P. Tamas, Ryan P. Gares, Elizabeth R. McGonagle, Brendan A. Hilliard, Mikhail A. Kolpakov, Lewis Bright-Rowe, Alan S. Braverman, Justin M. Brown, Michael R. Ruggieri, Sr., Mary F. Barbe

ABSTRACT

Neurotrophic factors and reactive oxygen species (ROS) modulate neuronal plasticity. In a model of a lower motor neuron lesioned bladder, somatic nerve transfer was used as a reinnervation strategy. Levels of neurotrophins, ROS, and TNF-α in bladder mucosa and muscle layers collected from three groups of adult female dogs: (1) Decentralized, via bilateral transection of coccygeal and sacral spinal roots, lumbar 7 dorsal roots, and hypogastric nerves, then 6–21 mo recovery; (2) reinnervated (ObNT-Reinn), after similar decentralization for 12 mo, then bilateral obturator-to-vesical nerve transfer and 8–12 mo recovery; and (3) Controls. In mucosa, BDNF and ROS levels were highest in ObNT-Reinn bladders, GDNF and TNF-α levels were restored to Control levels in ObNT-Reinn bladders (lowest in Decentralized). NT-3 and ARTN were lower in ObNT-Reinn and Decentralized bladders versus Controls. In muscle, ROS was lower in ObNT-Reinn muscle versus Controls. BDNF mucosa levels correlated with bladder axonal density and detrusor layer thickness; and GDNF mucosal correlated with bladder contraction after vesical or transferred obturator nerve electrical stimulation, as did BDNF and GDNF muscle levels. The increased BDNF and GDNF in bladders that underwent somatic nerve transfer with subsequent recovery suggest that BDNF and GDNF may help promote the reestablishment of bladder innervation.

Effects of Short-Term Treatment of Rabbit Extraocular Muscle With Ciliary Neurotrophic Factor

AUTHORS

Jolene C. Rudell; Linda K. McLoon

ABSTRACT

Purpose: Little is known about the effect of ciliary neurotrophic factor (CNTF) on extraocular muscles, but microarray studies suggested CNTF might play a role in the development and/or maintenance of strabismus. The effect of short-term treatment of adult rabbit extraocular muscle with injected CNTF was examined for its ability to alter muscle characteristics.

Methods: Eight adult New Zealand white rabbits received an injection into one superior rectus muscle of 2 µg/100 µL CNTF on 3 consecutive days. One week after the first injection, the rabbits were euthanized, and the treated and contralateral superior rectus muscles were assessed for force generation capacity and contraction characteristics using an in vitro stimulation protocol and compared to naïve control superior rectus muscles. All muscles were analyzed to determine mean cross-sectional areas and expression of slow twitch myosin heavy chain isoform.

Results: Short-term treatment of rabbit superior rectus muscles with CNTF resulted in a significant decrease in muscle force generation, but only at the higher stimulation frequencies. Significantly decreased myofiber cross-sectional areas of the treated muscles correlated with the decreased generated force. In addition, there were significant changes to contractile properties of the treated muscles, as well as a decrease in the number of myofibers expressing slow twitch myosin heavy chain.

Conclusions: We show that short-term treatment of a single rabbit superior rectus muscle results in decreased myofiber size, decreased force, and altered contractile characteristics. Further studies are needed to determine if it can play a role in improving alignment in animal models of strabismus.

FTO Stabilizes MIS12 to Inhibit Vascular Smooth Muscle Cell Senescence in Atherosclerotic Plaque

AUTHORS

Sun J, Wang M, Jia F, Song J, Ren J, Hu B

ABSTRACT

Purpose: Atherosclerosis is the main cause of atherosclerotic cardiovascular disease (CVD). Here, we aimed to uncover the role and mechanisms of fat mass and obesity-associated genes (FTO) in the regulation of vascular smooth muscle cell (VSMC) senescence in atherosclerotic plaques.

Methods: ApoE−/− mice fed a high-fat diet (HFD) were used to establish an atherosclerotic animal model. Immunohistochemistry, and the staining of hematoxylin-eosin, Oil Red O, Sirius red, and Masson were performed to confirm the role of FTO in atherosclerosis in vivo. Subsequently, FTO expression in primary VSMCs is either upregulated or downregulated. Oxidized low-density lipoprotein (ox-LDL) was used to treat VSMCs, followed by EdU staining, flow cytometry, senescence-associated β-galactosidase (SA-β-gal) staining, immunofluorescence, telomere detection, RT-qPCR, and Western blotting to determine the molecular mechanisms by which FTO inhibits VSMC senescence.

Results: Decreased FTO expression was observed in progressive atherosclerotic plaques of ApoE−/− mice fed with HFD. FTO upregulation inhibits atherosclerotic lesions in mice. FTO inhibits VSMC aging in atherosclerotic plaques by helping VSMC withstand ox-LDL-induced cell cycle arrest and senescence. This process is achieved by stabilizing the MIS12 protein in VSMC through a proteasome-mediated pathway.

Conclusion: FTO inhibits VSMC senescence and subsequently slows the progression of atherosclerotic plaques by stabilizing the MIS12 protein.

Gene therapy with bidridistrogene xeboparvovec for limb-girdle muscular dystrophy type 2E/R4: phase 1/2 trial results

AUTHORS

Jerry R. Mendell, Eric R. Pozsgai, Sarah Lewis, Danielle A. Griffin, Linda P. Lowes, Lindsay N. Alfano, Kelly J. Lehman, Kathleen Church, Natalie F. Reash, Megan A. Iammarino, Brenna Sabo, Rachael Potter, Sarah Neuhaus, Xiaoxi Li, Herb Stevenson & Louise R. Rodino-Klapac

ABSTRACT

Limb-girdle muscular dystrophy 2E/R4 is caused by mutations in the β-sarcoglycan (SGCB) gene, leading to SGCB deficiency and consequent muscle loss. We developed a gene therapy approach based on functional replacement of the deficient SCB protein. Here we report interim results from a first-in-human, open-label, nonrandomized, phase 1/2 trial evaluating the safety and efficacy of bidridistrogene xeboparvovec, an adeno-associated virus-based gene therapy containing a codon-optimized, full-length human SGCB transgene. Patients aged 4–15 years with confirmed SGCB mutations at both alleles received one intravenous infusion of either 1.85 × 1013 vector genome copies kg−1 (Cohort 1, n = 3) or 7.41 × 1013 vector gene copies kg−1 (Cohort 2, n = 3). Primary endpoint was safety, and secondary endpoint was change in SGCB expression in skeletal muscle from baseline to Day 60. We report interim Year 2 results (trial ongoing). The most frequent treatment-related adverse events were vomiting (four of six patients) and gamma-glutamyl transferase increase (three of six patients). Serious adverse events resolved with standard therapies. Robust SGCB expression was observed: Day 60 mean (s.d.) percentage of normal expression 36.2% (2.7%) in Cohort 1 and 62.1% (8.7%) in Cohort 2. Post hoc exploratory analysis showed preliminary motor improvements using the North Star Assessment for Limb-girdle Type Muscular Dystrophies maintained through Year 2. The 2-year safety and efficacy of bidridistrogene xeboparvovec support clinical development advancement. Further studies are necessary to confirm the long-term safety and efficacy of this gene therapy. ClinicalTrials.gov registration: NCT03652259.

Klotho regulates the myogenic response of muscle to mechanical loading and exercise

AUTHORS

Eisuke Ochi, Alice Barrington, Michelle Wehling-Henricks, Marcus Avila, Makoto Kuro-o, James G. Tidball

ABSTRACT

Muscle growth is influenced by changes in the mechanical environment that affect the expression of genes that regulate myogenesis. We tested whether the hormone Klotho could influence the response of muscle to mechanical loading. Applying mechanical loads to myoblasts in vitro increased RNA encoding transcription factors that are expressed in activated myoblasts (Myod) and in myogenic cells that have initiated terminal differentiation (Myog). However, application of Klotho to myoblasts prevented the loading-induced activation of Myog without affecting loading-induced activation of Myod. This indicates that elevated Klotho inhibits mechanically-induced differentiation of myogenic cells. Elevated Klotho also reduced the transcription of genes encoding proteins involved in the canonical Wnt pathway or their target genes (Wnt9a, Wnt10a, Ccnd1). Because the canonical Wnt pathway promotes differentiation of myogenic cells, these findings indicate that Klotho inhibits the differentiation of myogenic cells experiencing mechanical loading. We then tested whether these effects of Klotho occurred in muscles of mice experiencing high-intensity interval training (HIIT) by comparing wild-type mice and klotho transgenic mice. The expression of a klotho transgene combined with HIIT synergized to tremendously elevate numbers of Pax7+ satellite cells and activated MyoD+ cells. However, transgene expression prevented the increase in myogenin+ cells caused by HIIT in wild-type mice. Furthermore, transgene expression diminished the HIIT-induced activation of the canonical Wnt pathway in Pax7+ satellite cells. Collectively, these findings show that Klotho inhibits loading- or exercise-induced activation of muscle differentiation and indicate a new mechanism through which the responses of muscle to the mechanical environment are regulated.

Nystagmus Associated With the Absence of MYOD Expression Across the Lifespan in Extraocular and Limb Muscles

AUTHORS

Laura L. Johnson; Sadie Hebert; Rachel B. Kueppers; Linda K. McLoon

ABSTRACT

Purpose: The extraocular muscles (EOMs) undergo significant levels of continuous myonuclear turnover and myofiber remodeling throughout life, in contrast to limb skeletal muscles. Activation of the myogenic pathway in muscle precursor cells is controlled by myogenic transcription factors, such as MYOD. Limb muscles from MyoD−/− mice develop normally but have a regeneration defect, and these mice develop nystagmus. We examined MyoD−/− mice to determine if they have an aging phenotype.

Methods: Eye movements of aging MyoD−/− mice and littermate controls (wild type) were examined using optokinetic nystagmus (OKN). We assessed limb muscle function, changes to myofiber number, mean cross-sectional area, and abundance of the PAX7 and PITX2 populations of myogenic precursor cells.

Results: Aging did not significantly affect limb muscle function despite decreased mean cross-sectional areas at 18+ months. Aging wild type mice had normal OKN responses; all aging MyoD−/− mice had nystagmus. With OKN stimulus present, the MyoD−/− mice at all ages had shorter slow phase durations compared to wild type age matched controls. In the dark, the MyoD−/− mice had a shorter slow phase duration with age. This correlated with significantly decreased fiber numbers and cross-sectional areas. The EOM in MyoD−/− mice had increased numbers of PAX7-positive satellite cells and significantly decreased PITX2-positive myonuclei.

Conclusions: The absence of MYOD expression in aging mice causes a decrease in on-going myofiber remodeling, EOM fiber size, and number, and is associated with the development of spontaneous nystagmus. These results suggest that muscle-specific mutations can result in nystagmus, with increasing aging-related changes in the MyoD−/− EOM.