Survival motor neuron protein deficiency impairs myotube formation by altering myogenic gene expression and focal adhesion dynamics.

While spinal muscular atrophy (SMA) is characterized by motor neuron degeneration, it is unclear whether and how much survival motor neuron (SMN) protein deficiency in muscle contributes to the pathophysiology of the disease. There is increasing evidence from patients and SMA model organisms that SMN deficiency causes intrinsic muscle defects. Here we investigated the role of SMN in muscle development using muscle cell lines and primary myoblasts. Formation of multinucleate myotubes by SMN-deficient muscle cells is inhibited at a stage preceding plasma membrane fusion. We found increased expression and reduced induction of key muscle development factors, such as MyoD and myogenin, with differentiation of SMN-deficient cells. In addition, SMN-deficient muscle cells had impaired cell migration and altered organization of focal adhesions and the actin cytoskeleton. Partially restoring SMN inhibited the premature expression of muscle differentiation markers, corrected the cytoskeletal abnormalities and improved myoblast fusion. These findings are consistent with a role for SMN in myotube formation through effects on muscle differentiation and cell motility.

Survival motor neuron protein in motor neurons determines synaptic integrity in spinal muscular atrophy.

The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein and results in severe muscle weakness. In SMA mice, synaptic dysfunction of both neuromuscular junctions (NMJs) and central sensorimotor synapses precedes motor neuron cell death. To address whether this synaptic dysfunction is due to SMN deficiency in motor neurons, muscle, or both, we generated three lines of conditional SMA mice with tissue-specific increases in SMN expression. All three lines of mice showed increased survival, weights, and improved motor behavior. While increased SMN expression in motor neurons prevented synaptic dysfunction at the NMJ and restored motor neuron somal synapses, increased SMN expression in muscle did not affect synaptic function although it did improve myofiber size. Together these data indicate that both peripheral and central synaptic integrity are dependent on motor neurons in SMA, but SMN may have variable roles in the maintenance of these different synapses. At the NMJ, it functions at the presynaptic terminal in a cell-autonomous fashion, but may be necessary for retrograde trophic signaling to presynaptic inputs onto motor neurons. Importantly, SMN also appears to function in muscle growth and/or maintenance independent of motor neurons. Our data suggest that SMN plays distinct roles in muscle, NMJs, and motor neuron somal synapses and that restored function of SMN at all three sites will be necessary for full recovery of muscle power.

Increased IGF-1 in muscle modulates the phenotype of severe SMA mice.

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1(NEG) and mIGF-1(POS) SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients.

Single-incision laparoscopic sigmoid resection: a technical video of a standardized approach.

BACKGROUND: Initially described in 2008, single-incision laparoscopic colectomy has evolved into a safe and feasible surgical approach. Noted advantages include elimination of trocar-site incisions and improved cosmesis. Additional benefits including reduced abdominal wall trauma, diminished pain, and shorter length of hospitalization have been proposed. Following utilization in over 150 colectomies, we present a standardized approach and describe our technique for single-incision laparoscopic sigmoid resection through a single-port access device. TECHNIQUE: A 2.5-cm umbilical incision is used for insertion of the single-incision access device. A 30° 5-mm camera with a right-angle light cord adaptor and 2 bowel graspers are inserted through the access device. Exploration and lysis of adhesions are performed before placing the patient in a steep Trendelenburg position with 20° left-sided elevation. Dissection commences in a medial-to-lateral fashion, developing the presacral avascular plane while ensuring nerve preservation. The retroperitoneal plane is established from the sacral promontory to the lateral peritoneal reflection. After identification of the left ureter and isolation of the vascular pedicle, the inferior mesenteric artery is isolated and ligated. The lateral attachments of the left colon and rectosigmoid are then divided, followed by additional pelvic dissection along the presacral avascular plane. The mesentery of the distal resection margin is divided before transection of the corresponding bowel using a stapling device. The bowel is then extracted and resected at the site of the single-incision access device. An intracorporeal primary end-to-end anastomosis is fashioned. CONCLUSION: We present a dynamic article with video illustrating a standardized medial-to-lateral approach for single-incision laparoscopic sigmoid resection. The technique effectively avoids the use of multiple trocar sites, maintains basic oncologic principles of resection, and affords the benefits of minimally invasive surgery.

Regulation of Hsp90 client proteins by a Cullin5-RING E3 ubiquitin ligase.

We report a link between Cullin5 (Cul5) E3 ubiquitin ligase and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the folding of its client proteins into their functional conformation. Many Hsp90 clients have been reported to be aberrantly expressed in a number of cancers. We demonstrate Cul5 interaction with members of the Hsp90 chaperone complex as well as the Hsp90 client, ErbB2. We observed recruitment of Cul5 to the site of ErbB2 at the plasma membrane and subsequent induction of polyubiquitination and proteasomal degradation. We also demonstrate Cul5 involvement in regulation of another Hsp90 client, Hif-1alpha. We observed Cul5 degradation of ErbB2 to occur independently of ElonginB-ElonginC function. The involvement of Cul5 in Hsp90 client regulation has implications in the effectiveness of Hsp90 targeted chemotherapy, which is currently undergoing clinical trials. The link between Cul5 and Hsp90 client regulation may represent an avenue for cancer drug development.

IL-6 induces regionally selective spinal cord injury in patients with the neuroinflammatory disorder transverse myelitis.

Transverse myelitis (TM) is an immune-mediated spinal cord disorder associated with inflammation, demyelination, and axonal damage. We investigated the soluble immune derangements present in TM patients and found that IL-6 levels were selectively and dramatically elevated in the cerebrospinal fluid and directly correlated with markers of tissue injury and sustained clinical disability. IL-6 was necessary and sufficient to mediate cellular injury in spinal cord organotypic tissue culture sections through activation of the JAK/STAT pathway, resulting in increased activity of iNOS and poly(ADP-ribose) polymerase (PARP). Rats intrathecally infused with IL-6 developed progressive weakness and spinal cord inflammation, demyelination, and axonal damage, which were blocked by PARP inhibition. Addition of IL-6 to brain organotypic cultures or into the cerebral ventricles of adult rats did not activate the JAK/STAT pathway, which is potentially due to increased expression of soluble IL-6 receptor in the brain relative to the spinal cord that may antagonize IL-6 signaling in this context. The spatially distinct responses to IL-6 may underlie regional vulnerability of different parts of the CNS to inflammatory injury. The elucidation of this pathway identifies specific therapeutic targets in the management of CNS autoimmune conditions.

Improvement of neuromuscular synaptic phenotypes without enhanced survival and motor function in severe spinal muscular atrophy mice selectively rescued in motor neurons.

In the inherited childhood neuromuscular disease spinal muscular atrophy (SMA), lower motor neuron death and severe muscle weakness result from the reduction of the ubiquitously expressed protein survival of motor neuron (SMN). Although SMA mice recapitulate many features of the human disease, it has remained unclear if their short lifespan and motor weakness are primarily due to cell-autonomous defects in motor neurons. Using Hb9(Cre) as a driver, we selectively raised SMN expression in motor neurons in conditional SMAΔ7 mice. Unlike a previous study that used choline acetyltransferase (ChAT(Cre+) ) as a driver on the same mice, and another report that used Hb9(Cre) as a driver on a different line of conditional SMA mice, we found no improvement in survival, weight, motor behavior and presynaptic neurofilament accumulation. However, like in ChAT(Cre+) mice, we detected rescue of endplate size and mitigation of neuromuscular junction (NMJ) denervation status. The rescue of endplate size occurred in the absence of an increase in myofiber size, suggesting endplate size is determined by the motor neuron in these animals. Real time-PCR showed that the expression of spinal cord SMN transcript was sharply reduced in Hb9(Cre+) SMA mice relative to ChAT(Cre+) SMA mice. This suggests that our lack of overall phenotypic improvement is most likely due to an unexpectedly poor recombination efficiency driven by Hb9(Cre) . Nonetheless, the low levels of SMN were sufficient to rescue two NMJ structural parameters indicating that these motor neuron cell autonomous phenotypes are very sensitive to changes in motoneuronal SMN levels. Our results directly suggest that even those therapeutic interventions with very modest effects in raising SMN in motor neurons may provide mitigation of neuromuscular phenotypes in SMA patients.

Robotic-assisted laparoscopic primary repair of acute iatrogenic colonic perforation: case report.

BACKGROUND: Colonic perforation during colonoscopy is a rare complication and is usually considered a surgical emergency. Traditionally, such perforations have required laparotomy with repair or resection. Minimally invasive approaches have recently been successfully implemented. We describe our initial experience with a robotic-assisted laparoscopic technique for primary colorrhaphy following colonoscopic perforation. METHODS: An 84 year-old female presented with an acute sigmoid perforation identified during colonoscopy. Laparoscopic exploration revealed a full-thickness tear into the sigmoid mesentery, which was primarily repaired using robotic-assisted technique. RESULTS: The procedure was successfully completed in 135 min with an estimated blood loss of 25 ml. There were no intraoperative complications or need for open conversion. The patient was discharged after 4 days without further hospitalization or secondary surgical intervention. CONCLUSION: In the presented case, robotic primary colorrhaphy was demonstrated to be a safe and feasible alternative for the management of acute colonoscopic perforation and may warrant consideration in the emergency setting.

Mutations in TRPV4 cause Charcot-Marie-Tooth disease type 2C.

Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. We sequenced all genes in this region and identified two heterozygous missense mutations in the TRPV4 gene, C805T and G806A, resulting in the amino acid substitutions R269C and R269H. TRPV4 is a well-known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously associated with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of the peripheral nerves. The CMT2C-associated mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this phenotypic variability may be due to differential effects on regulatory protein-protein interactions.

Correction of humoral derangements from mutant superoxide dismutase 1 spinal cord.

OBJECTIVE: We sought to define molecular and cellular participants that mediate motor neuron injury in amyotrophic lateral sclerosis using a coculture system. METHODS: We cocultured embryonic stem cell-derived motor neurons with organotypic slice cultures from wild-type or SOD1G93A (MT) mice. We examined axon lengths and cell survival of embryonic stem cell-derived motor neurons. We defined and quantified the humoral factors that differed between wild-type and MT organotypic cultures, and then corrected these differences in cell culture. RESULTS: MT spinal cord organotypic slices were selectively toxic to motor neurons as defined by axonal lengths and cell survival. MT spinal cord organotypic slices secreted higher levels of nitric oxide, interleukin (IL)-1beta, IL-6, and IL-12p70 and lower levels of vascular endothelial growth factor. The toxicity of MT spinal cord organotypic cultures was reduced and axonal lengths were restored to near normal by coculturing in the presence of reactive oxygen species scavenger, vascular endothelial growth factor, and neutralizing antibodies to IL-1beta, IL-6, and IL-12. INTERPRETATION: MT spinal cord organotypic cultures overexpress certain factors and underexpress others, creating a nonpermissive environment for cocultured motor neurons. Correction of these abnormalities as a group, but not individually, restores axonal length to near normal. Such a "cocktail" approach to the treatment of amyotrophic lateral sclerosis should be investigated further.

Recovery from paralysis in adult rats using embryonic stem cells.

OBJECTIVE: We explored the potential of embryonic stem cell-derived motor neurons to functionally replace those cells destroyed in paralyzed adult rats. METHODS: We administered a phosphodiesterase type 4 inhibitor and dibutyryl cyclic adenosine monophosphate to overcome myelin-mediated repulsion and provided glial cell-derived neurotrophic factor within the sciatic nerve to attract transplanted embryonic stem cell-derived axons toward skeletal muscle targets. RESULTS: We found that these strategies significantly increased the success of transplanted axons extending out of the spinal cord into ventral roots. Furthermore, transplant-derived axons reached muscle, formed neuromuscular junctions, were physiologically active, and mediated partial recovery from paralysis. INTERPRETATION: We conclude that restoration of functional motor units by embryonic stem cells is possible and represents a potential therapeutic strategy for patients with paralysis. To our knowledge, this is the first report of the anatomical and functional replacement of a motor neuron circuit within the adult mammalian host.

Correlation of transcriptional repression by p21(SNFT) with changes in DNA.NF-AT complex interactions.

p21(SNFT) (21-kDa small nuclear factor isolated from T cells) is a novel human protein of the basic leucine zipper family. The overexpression of p21(SNFT) leads to the significant and specific repression of transcription from the interleukin-2 promoter as well as from several essential activator protein 1 (AP-1)-driven composite promoter elements. One example is the distal nuclear factor of activated T cells (NF-AT)/AP-1 element where the AP-1 (Fos/Jun) basic leucine zipper heterodimer interacts with members of the NF-AT family. p21(SNFT) has been shown to replace Fos in dimerization with Jun on a consensus AP-1 binding site (12-O-tetradecanolyphorbol-13-acetate response element (TRE)) and to interact with Jun and NF-AT at the distal NF-AT/AP-1 enhancer element. A detailed biochemical analysis presented here compares interactions involving p21(SNFT) with those involving Fos. The results demonstrate that a p21(SNFT)/Jun dimer binds a TRE similarly to AP-1 and like AP-1 binds cooperatively with NF-AT at the NF-AT/AP-1 composite element. However, Fos interacts significantly more efficiently than p21(SNFT) with Jun and NF-AT, and the replacement of Fos by p21(SNFT) in the trimolecular complex drastically alters protein-DNA contacts. The data suggest that p21(SNFT) may repress transcriptional activity by inducing a unique conformation in the transcription factor complex.