Transplantation of dorsal root ganglia overexpressing the NaChBac sodium channel improves locomotion after complete SCI.

Spinal cord injury (SCI) is a debilitating condition currently lacking treatment. Severe SCI causes the loss of most supraspinal inputs and neuronal activity caudal to the injury, which, coupled with the limited endogenous capacity for spontaneous regeneration, can lead to complete functional loss even in anatomically incomplete lesions. We hypothesized that transplantation of mature dorsal root ganglia (DRGs) genetically modified to express the NaChBac sodium channel could serve as a therapeutic option for functionally complete SCI. We found that NaChBac expression increased the intrinsic excitability of DRG neurons and promoted cell survival and neurotrophic factor secretion in vitro. Transplantation of NaChBac-expressing dissociated DRGs improved voluntary locomotion 7 weeks after injury compared to control groups. Animals transplanted with NaChBac-expressing DRGs also possessed higher tubulin-positive neuronal fiber and myelin preservation, although serotonergic descending fibers remained unaffected. We observed early preservation of the corticospinal tract 14 days after injury and transplantation, which was lost 7 weeks after injury. Nevertheless, transplantation of NaChBac-expressing DRGs increased the neuronal excitatory input by an increased number of VGLUT2 contacts immediately caudal to the injury. Our work suggests that the transplantation of NaChBac-expressing dissociated DRGs can rescue significant motor function, retaining an excitatory neuronal relay activity immediately caudal to injury.

Proteasomal activity-based probes mark protein homeostasis in muscles.

BACKGROUND: Protein homeostasis, primarily regulated by the ubiquitin-proteasome system is crucial for proper function of cells. In tissues of post-mitotic cells, the impaired ubiquitin-proteasome system is found in a wide range of neuromuscular disorders. Activity-based probes (ABPs) measure proteasomal proteolytic subunits and can be used to report protein homeostasis. Despite the crucial role of the proteasome in neuromuscular pathologies, ABPs were not employed in muscle cells and tissues, and measurement of proteasomal activity was carried out in vitro using low-throughput procedures. METHODS: We screened six ABPs for specific application in muscle cell culture using high throughput call-based imaging procedures. We then determined an in situ proteasomal activity in myofibers of muscle cryosections. RESULTS: We demonstrate that LWA300, a pan-reactive proteasomal probe, is most suitable to report proteasomal activity in muscle cells using cell-based bio-imaging. We found that proteasomal activity is two-fold and three-fold enhanced in fused muscle cell culture compared with non-fused cells. Moreover, we found that proteasomal activity can discriminate between muscles. Across muscles, a relative higher proteasomal activity was found in hybrid myofibers whereas fast-twitch myofibers displayed lower activity. CONCLUSIONS: Our study demonstrates that proteasomal activity differ between muscles and between myofiber types. We suggest that ABPs can be used to report disease progression and treatment efficacy.

PABPN1-Dependent mRNA Processing Induces Muscle Wasting.

Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3'-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting.

Systematic Analyses of Substrate Preferences of 20S Proteasomes Using Peptidic Epoxyketone Inhibitors.

Cleavage analyses of 20S proteasomes with natural or synthetic substrates allowed to infer the substrate specificities of the active sites and paved the way for the rational design of high-affinity proteasome inhibitors. However, details of cleavage preferences remained enigmatic due to the lack of appropriate structural data. In a unique approach, we here systematically examined substrate specificities of yeast and human proteasomes using irreversibly acting α',ß'epoxyketone (ep) inhibitors. Biochemical and structural analyses provide unique insights into the substrate preferences of the distinct active sites and highlight differences between proteasome types that may be considered in future inhibitor design efforts. (1) For steric reasons, epoxyketones with Val or Ile at the P1 position are weak inhibitors of all active sites. (2) Identification of the ß2c selective compound Ac-LAE-ep represents a promising starting point for the development of compounds that discriminate between ß2c and ß2i. (3) The compound Ac-LAA-ep was found to favor subunit ß5c over ß5i by three orders of magnitude. (4) Yeast ß1 and human ß1c subunits preferentially bind Asp and Leu in their S1 pockets, while Glu and large hydrophobic residues are not accepted. (5) Exceptional structural features in the ß1/2 substrate binding channel give rise to the ß1 selectivity of compounds featuring Pro at the P3 site. Altogether, 23 different epoxyketone inhibitors, five proteasome mutants, and 43 crystal structures served to delineate a detailed picture of the substrate and ligand specificities of proteasomes and will further guide drug development efforts toward subunit-specific proteasome inhibitors for applications as diverse as cancer and autoimmune disorders.

Toward understanding induction of oxidative stress and apoptosis by proteasome inhibitors.

SIGNIFICANCE: Proteasome inhibitors (PIs) are used in the clinic for the treatment of hematopoietic malignancies. PI inhibitors induce endoplasmatic reticulum (ER) stress and oxidative stress, disruption of signaling pathways, mitochondrial dysfunction, and, eventually, cell death by apoptosis. PIs designated as clinical candidates include natural product derivatives and compounds developed by rational design and feature a wide diversity of structural elements. The vast amount of literature on this topic underscores PIs significance in driving basic research alongside therapeutic benefit. RECENT ADVANCES: Research in recent years has brought an in-depth insight into the molecular mechanisms of PI-induced apoptosis. However, there are some paradoxes and controversies in the literature. In this review, the advances and uncertainties, in particular on the time course events that make cells commit to apoptosis, are discussed. In addition, some mechanisms of evolved PI resistance are presented, and speculations on the difference in sensitivity between cell or tumor types are brought forward. The review concludes by giving an outlook of recent methods that may be employed to describe the system biology of how PIs impact cell survival decisions. CRITICAL ISSUES: The biology of ER stress, reactive oxygen species (ROS) production, and apoptosis as induced by PIs is not well understood. Absorbed by the strong focus on PIs, one might overlook the importance of proteasome activity activators or modulators and the study of enzymatic pathways that lie up- or downstream from the proteasome function. FUTURE DIRECTIONS: An increased understanding of the systems biology at mRNA and protein levels and the kinetics behind the interaction between PIs and cells is imperative. The design and synthesis of subunit specific inhibitors for each of the seven known proteasome activities and for the enzymes associated to proteasomes will aid in unraveling biology of the ubiquitin-proteasome system in relation to ER stress, ROS production, and apoptosis and will generate leads for therapeutic intervention.