Cytoskeletal Stability Research Articles

Abstract 15489: Altered Biomechanical Properties of PKP2-deficient Hl-1 Cardiac Cells

Arrhythmogenic cardiomyopathy (AC) is a genetic disease caused by mutations in genes encoding the intercalated disk (ID) proteins. ID is a multiprotein complex providing cell-cell junction and connecting the nucleus to the extracellular matrix, and playing a pivotal role for maintaining the structural integrity of the heart tissue. Mutations in PKP2 encoding plakophilin 2 (PKP2) are the most common causes of AC. The objective of this study was to investigate the biomechanical properties of the PKP2-knock down HL-1 mouse cardiac myocytes (PKP2-KD). PKP2 protein levels were reduced by 66 to 75% upon shRNA-targeting of the Pkp2 mRNA. Nuclear stiffness (Young’s modulus) and the force required to deform the nucleus were assessed by atomic force microscopy (AFM). Nuclear stiffness was decreased significantly (5 times) in PKP2-KD HL-1 myocytes as compared to wild-type (WT) myocytes, indicating an increased elasticity and/or susceptibility of the nucleus to be deformed (2 times). Interestingly, cell adhesion was also reduced (10 times) in the PKP2-KD myocytes, indicating a pivotal role of ID proteins in maintaining cell adhesion and mechanical integrity in cardiomyocytes. In the stress-relaxation test, while the decay time value was comparable between WT and PKP2-KD cells, relaxation force and cell deformation values were decreased 3 and 8 times, respectively, in PKP2-KD HL-1 myocytes, confirming the finding in the Young’s modulus. Moreover, the relaxation test revealed a reduced viscosity, highlighting a compromised cytoskeletal stability induced by the PKP2 deficiency. RNA-Sequencing and Ingenuity Canonical Pathway analysis identified integrin, tight junction, and the canonical Wnt signaling, among the most disturbed pathways in the PKP2-KD HL-1 myocytes. The findings indicate that the deficiency of a component of the ID protein markedly affects the whole-cellular biomechanics, which is expected to affect tissue stability, leading to activation of the mechano-transduction pathways.

Mediators and mechanisms of heat shock protein 70 based cytoprotection in obstructive nephropathy.

Urinary heat shock protein 70 (Hsp70) is rapidly increased in patients with clinical acute kidney injury, indicating that it constitutes a component of the endogenous stress response to renal injury. Moreover, experimental models have demonstrated that Hsp70 activation is associated with the cytoprotective actions of several drugs following obstruction, including nitric oxide (NO) donors, geranylgeranylacetone, vitamin D, and rosuvastatin. Discrete and synergistic effects of the biological activities of Hsp70 may explain its cytoprotective role in obstructive nephropathy. Basic studies point to a combination of effects including inhibition of apoptosis and inflammation, repair of damaged proteins, prevention of unfolded protein aggregation, targeting of damaged protein for degradation, and cytoskeletal stabilization as primary effectors of Hsp70 action. This review summarizes our understanding of how the biological actions of Hsp70 may affect renal cytoprotection in the context of obstructive injury. The potential of Hsp70 to be of central importance to the mechanism of action of various drugs that modify the genesis of experimental obstructive nephropathy is considered.

The Cytoskeleton Regulates Cell Attachment Strength

Quantitative information about adhesion strength is a fundamental part of our understanding of cell-extracellular matrix (ECM) interactions. Adhesion assays should measure integrin-ECM bond strength, but reports now suggest that cell components remain behind after exposure to acute force for radial shear assays in the presence of divalent cations that increase integrin-ECM affinity. Here, we show that focal adhesion proteins FAK, paxillin, and vinculin but not the cytoskeletal protein actin remain behind after shear-induced detachment of HT1080 fibrosarcoma cells. Cytoskeletal stabilization increased attachment strength by eightfold, whereas cross-linking integrins to the substrate only caused a 1.5-fold increase. Reducing temperature—only during shear application—also increased attachment strength eightfold, with detachment again occurring between focal adhesion proteins and actin. Detachment at the focal adhesion-cytoskeleton interface was also observed in mouse and human fibroblasts and was ligand-independent, highlighting the ubiquity of this mode of detachment in the presence of divalent cations. These data show that the cytoskeleton and its dynamic coupling to focal adhesions are critically important for cell adhesion in niche with divalent cations.

Functional and Genetic Analysis of Neuronal Isoforms of BPAG1.

The neuronal isoforms of bullous pemphigoid antigen 1 (BPAG1, and also known as dystonin) are a group of large cytoskeletal linker proteins predominantly expressed in sensory neurons. The major neuronal isoforms consist of the spectraplakins (BPAG1/dystonin-a1, -a2, -a3), which have an N-terminus actin-binding domain and a C-terminus microtubule-binding domain. These proteins have crucial roles in cytoskeletal organization and stability, organelle integrity, and intracellular transport. BPAG1 loss-of-function in mice results in a lethal movement disorder known as dystonia musculorum (dt), which is likely caused by rapid sensory neuron degeneration. A human disease termed hereditary and sensory autonomic neuropathy type VI was also identified to be associated with mutations in the BPAG1 gene (DST). This chapter provides an overview of the type of experiments used for analysis of the different isoforms of BPAG1.

Protein Kinase G and VASP in the Control of Vascular Smooth Muscle Cell Migration

Uncontrolled migration of vascular smooth muscle cells (VSMCs) is a mechanistic foundation of cardiovascular disease, the number one killer of Americans and individuals worldwide. Unfortunately, current strategies aimed at preventing pathologic VSMC migration are largely inefficient. This study was designed to characterize a potential novel target capable of controlling VSMC migration, and our hypothesis is that protein kinase G (PKG)-stimulated vasodilator-activated serum phosphoprotein (VASP) serves to inhibit VSMC migration through enhanced actin polymerization and increased cytoskeletal stability. In human primary and rat commercial VSMCs, using pharmacologic and mechanical stimulation as well as a newly developed confocal and laser capture microdissection-assisted migration assay, results show that PKG phosphorylates VASP at Ser239, a reported PKG-sensitive site, and that this correlates with reduced globular to filamentous actin (G:F), and inhibited cell migration. In complement, using a rat carotid artery injury model, PKG activation and subsequent VASP activation significantly diminished neointima formation and vascular wall remodeling. These findings suggest that vascular protection can be accomplished through PKG and downstream phosphorylation of the actin binding protein VASP, which may serve to increase cytoskeletal stability and offset the capacity of VSMCs to migrate as an underpinning of abnormal cell migration during cardiovascular disease.

Cytosolic calreticulin inhibits microwave radiation-induced microvascular endothelial cell injury through the integrin-focal adhesion kinase pathway.

To determine the effects of cytosolic CRT on MR-induced MMEC injury, and the underlying mechanism. MMECs were randomized into eight groups: control, AdCRT (infected with pAdCMV/V5-DEST-CRT adenovirus), stCRT (transfected with rCRT-siRNAs), Mock (transfected with scrambled siRNAs), MR (exposed to MR for six minutes), AdCRT+MR, stCRT+MR, and Mock+MR. The magnitude of cell injury were assessed by Annexin V-PI staining, LDH activity in culture medium, MMEC migration ability, ultrastructure and cytoskeletal stability. Subcellular colocalization of CRT and ConA or integrin were evaluated by immunocytochemistry. The mRNA and protein expression levels of target genes were examined by qRT-PCR and western blotting, respectively. MR-induced cytotoxicity was dose-dependent. Overexpression of cytosolic CRT suppressed MR injury, shown as decreased cell apoptosis, reduced LDH activity, enhanced cell migration capability, and maintenance of ultrastructure and cytoskeleton integrity. Conversely, CRT deficiency aggravated MR-induced injury. Exposure of AdCRT MMECs to MR promoted membrane translocation of CRT and the interaction of CRT-integrin-α. Correlation analysis revealed that integrin-α expression or FAK phosphorylation was positively associated with cytosolic CRT expression. Cytosolic CRT inhibits MR-induced MMEC injury through activation of the integrin-FAK pathway.

Abstract 378: Macrophage ß3 Integrin is an Important Player in Inflammation and Cellular Plasticity

Communication between cells and the surrounding environment is a crucial mechanism for survival. Integrins are membrane-bound molecules that are involved in signaling between the cells and the extracellular matrix, thereby influencing cytoskeletal stability and intracellular signaling. β3 integrin and its binding partner αv form the αvβ3 heterodimer that is expressed in various cells. We and others have described the consequences of its absence in inflammation, atherosclerosis and cancer in vivo. However, the distinct role of this integrin as a signaling molecule and the consequences of its absence for macrophage structure remain mostly elusive. Our aim is to further characterize the phenotype of β3-deficient (β3-/-) bone marrow-derived macrophages (BMDM) under stimulatory conditions (LPS and LDLs) compared to control cells in vitro. qPCR, WB, ELISA, migration, proliferation assays were used to investigate β3-/- BMDM and controls (wt BMDM and Raw 264.7). LPS was described to be not only pro- but also anti-inflammatory in a time-dependent manner. We show that LPS stimulation leads to high expression of pro-inflammatory cytokines (IL-1β and TNFα) shortly after treatment, while expression of anti-inflammatory cytokine (IL-10) arises at a later stage (12h post stimulation). Interestingly, β3-/- BMDM express more IL-1β than controls. IL-10 expression appears much earlier in β3-/- BMDM (6h post stimulation) but is reduced after 12h, indicating a faster and higher cellular response in the absence of the β3 integrin. OxLDL, the leading cause to foam cell formation, stimulates the expression of IL-1β in controls and β3-/- BMDM with the latter expressing significantly less of this cytokine indicating that lack of β3 causes differential cellular responses after LPS and oxLDL stimulation. Other LDL forms tested (nLDL, acLDL, cLDL) did not have any effect on IL-1β expression. In addition, we identified a higher proliferation rate in the β3-/- BMDM when cultured with M-CSF and a migration deficit in response to LPS, M-CSF and VEGF. Taken together, our results show that macrophage β3 deficiency causes differential cellular plasticity depending on the stimulus, with functional consequences that could be essential in inflammation and atherosclerosis.

Investigating the Relationship between Cellular Mechanics and Beta Amyloid in Alzheimer's Disease

The beta amyloid (Aβ) peptide was first implicated in the formation of amyloid plaques, a characteristic of Alzheimer's disease (AD), nearly 30 years ago. Misfolding of the Aβ peptide into nonnative conformations promotes the formation of aggregates such as oligomers, annular aggregates and fibrils that can impair cellular function. Although these aggregates are known to be toxic, the exact mechanism of their cytotoxicity remains unclear. As aging is associated with AD, we explored how cytoskeletal degradation, associated with the aging process, modulates a cell's ability to cope with exposure to exogenous Aβ. In this study, hypothalamic neurons, of the GT1-7 cell line, were treated using a variety of cytoskeletal altering drugs. The mechanics of the altered neurons were examined by atomic force microscopy-based techniques (force-distance curve and force volume analysis) and the altered neurons were then exposed to the Aβ peptide to determine how the cytoskeletal network affects peptide binding and toxicity. Binding studies were performed using fluorescence activated cell sorting and toxicity was examined using a variety of biochemical assays. Finally, the topography and mechanics of the the aged neurons exposed to Aβ were examined to determine the impact of Aβ binding. This research gives a mechanistic understanding of AD pathology in relation to the cytoskeleton and examines the potential for cytoskeletal stabilization as a therapeutic intervention for combating AD.

Roles of BMP6/7 in Actin Dynamics in Amyloid β-Induced Neurotoxicity

Actin dynamics plays an important role in many physiological functions such as long-term potentiation, neurotransmission, regulatory proteins translocation, ATP cycle, etc. When amyloid β (Aβ)-induced neurotoxicity occurs, the imbalance of actin dynamics leads to dystrophy of dendrites, which is characteristic pathology in Alzheimer’s disease (AD). Transient and persistent Aβ neurotoxicity provokes distinct manifestations of actin dynamics and causes opposing effects in AD. It has been shown that bone morphogenetic protein 6/7 (BMP6/7) protects neuronal morphology against Aβ-induced neurotoxicity, and can directly bind to LIM kinase1 (LIMK1), being one part of the upstream regulatory pathway of actin dynamics. This review aims to discuss a potential mechanism of BMPs underlying maintainance of cytoskeletal stabilization in neurite.

Loss of the spectraplakin short stop activates the DLK injury response pathway in Drosophila.

The MAPKKK dual leucine zipper-containing kinase (DLK, Wallenda in Drosophila) is an evolutionarily conserved component of the axonal injury response pathway. After nerve injury, DLK promotes degeneration of distal axons and regeneration of proximal axons. This dual role in coordinating degeneration and regeneration suggests that DLK may be a sensor of axon injury, and so understanding how DLK is activated is important. Two mechanisms are known to activate DLK. First, increasing the levels of DLK via overexpression or loss of the PHR ubiquitin ligases that target DLK activate DLK signaling. Second, in Caenorhabditis elegans, a calcium-dependent mechanism, can activate DLK. Here we describe a new mechanism that activates DLK in Drosophila: loss of the spectraplakin short stop (shot). In a genetic screen for mutants with defective neuromuscular junction development, we identify a hypomorphic allele of shot that displays synaptic terminal overgrowth and a precocious regenerative response to nerve injury. We demonstrate that both phenotypes are the result of overactivation of the DLK signaling pathway. We further show that, unlike mutations in the PHR ligase Highwire, loss of function of shot activates DLK without a concomitant increase in the levels of DLK. As a spectraplakin, Shot binds to both actin and microtubules and promotes cytoskeletal stability. The DLK pathway is also activated by downregulation of the TCP1 chaperonin complex, whose normal function is to promote cytoskeletal stability. These findings support the model that DLK is activated by cytoskeletal instability, which is a shared feature of both spectraplakin mutants and injured axons.

Abstract C131: HSP27 is necessary in cells showing oncogene hyper-activation.

Abstract The small heat shock protein of 27 KDa (HSP27) encoded by the HSPB1 gene, is a molecular chaperone with anti-aggregation property, it participates in sequestering damaged proteins, and is involved in the proteasomal degradation of certain proteins under stress conditions. Besides its role in stressed condition, HSP27 plays crucial roles within the cell under unstressed conditions and it provides cytoskeletal structural stability and exerts important anti-apoptotic function by binding apoptotic proteins. Clinically, Hsp27 is highly expressed in many cancers including breast, ovarian and prostate carcinomas and is associated with aggressive tumor behaviour, metastasis, poor prognosis and resistance to chemotherapeutics. Using LV driven expression of HSP27 specific shRNA, HSP27 suppression in three gastric cancer cell lines (GTL-16, MKN-45 and EBC-1) showing addiction to MET because of MET gene amplification was able to induce cell death. Moreover, HSP27 silencing synergized in inducing apoptosis with the MET inhibitor JNJ-38877605, which alone was only able to induce cell cycle blockade. Similarly, (H3255) lung carcinoma and (DiFi) colorectal carcinoma cell lines which carry EGFR amplification and are susceptible to EGFR inhibition were exquisitely susceptible to HSP27 silencing, that induced cell death. HSP27 silenced in twenty human colorectal (CRC) cell lines with either wt KRAS or harbouring KRAS mutations (SW620, LS513, LS1034) or BRAF mutations (OXCO-1, COLO741, COLO-201, COLO-205), Among them the LS513 and LS1034 cells and the COLO-201 and COLO-205, addicted to KRAS and BRAF, respectively, came out to be also addicted to HSP27, as they undergo apoptosis by HSP27 silencing. Taken together, these data suggest that cells addicted to oncogene over-activation require HSP27 for survival and that HSP27 interferes with the effectiveness of targeted agents. Due to its involvement in cancer development, the inhibition of HSP27 has been proposed as a potential cancer treatment strategy. Antisense and peptide aptamer strategies have shown that targeting HSP27 increases cancer cell death in vitro and in vivo in preclinical models. More important, an antisense drug inhibiting HSP27 is available for human therapy and is ongoing Phase II clinical trials. Therefore, HSP27 knockdown in combination with targeted therapies can be envisaged as a viable therapeutic approach for clinical application. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C131. Citation Format: Johndavid Konda, Martina Olivero, Daniele Musiani, Simona Lamba, Alberto Bardelli, Federica Di Nicolantonio, Maria Flavia Di Renzo. HSP27 is necessary in cells showing oncogene hyper-activation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C131.

Sparing of the Dystrophin-Deficient Cranial Sartorius Muscle Is Associated with Classical and Novel Hypertrophy Pathways in GRMD Dogs

Both Duchenne and golden retriever muscular dystrophy (GRMD) are caused by dystrophin deficiency. The Duchenne muscular dystrophy sartorius muscle and orthologous GRMD cranial sartorius (CS) are relatively spared/hypertrophied. We completed hierarchical clustering studies to define molecular mechanisms contributing to this differential involvement and their role in the GRMD phenotype. GRMD dogs with larger CS muscles had more severe deficits, suggesting that selective hypertrophy could be detrimental. Serial biopsies from the hypertrophied CS and other atrophied muscles were studied in a subset of these dogs. Myostatin showed an age-dependent decrease and an inverse correlation with the degree of GRMD CS hypertrophy. Regulators of myostatin at the protein (AKT1) and miRNA (miR-539 and miR-208b targeting myostatin mRNA) levels were altered in GRMD CS, consistent with down-regulation of myostatin signaling, CS hypertrophy, and functional rescue of this muscle. mRNA and proteomic profiling was used to identify additional candidate genes associated with CS hypertrophy. The top-ranked network included α-dystroglycan and like-acetylglucosaminyltransferase. Proteomics demonstrated increases in myotrophin and spectrin that could promote hypertrophy and cytoskeletal stability, respectively. Our results suggest that multiple pathways, including decreased myostatin and up-regulated miRNAs, α-dystroglycan/like-acetylglucosaminyltransferase, spectrin, and myotrophin, contribute to hypertrophy and functional sparing of the CS. These data also underscore the muscle-specific responses to dystrophin deficiency and the potential deleterious effects of differential muscle involvement.

L-Threonine induces heat shock protein expression and decreases apoptosis in heat-stressed intestinal epithelial cells

ObjectivesOsmotically acting amino acids can be cytoprotective following injury. As threonine (THR) induces osmotic cell swelling, our aim was to investigate the potential for THR to induce cellular protection in intestinal epithelial cells and evaluate possible mechanisms of protection. MethodsCells treated with a range of THR doses were evaluated following heat stress (HS) injury. Alpha-aminoisobutyric acid (AIB), a non-metabolizable amino acid analog, was used as an osmotic control. MTS assays were used to assess cell survival. Heat shock protein (HSP) expression and cleaved caspase-3 (CC3) were evaluated via Western blot. Cell morphology and cell size were analyzed via microscopy. ResultFollowing HS, THR treatment increased cell viability in a dose dependent manner vs. non-THR treated cells (CT). The non-metabolized amino acid analogue, AIB, also increased cell survival in heat-stressed cells versus HS controls. HSP70 and HSP25 expression increased with THR and AIB treatment versus HS controls. THR also increased HSP25 in non-stressed cells. Microscopic evaluation revealed both THR and AIB preserved the structural integrity of the actin cytoskeleton in heat-stressed cells versus HS controls. THR, but not AIB, enhanced nuclear translocation of HSP25 during HS. This nuclear translocation was associated with a 60% decrease in apoptosis in heat-stressed cells with THR. No antiapoptotic effect was observed with AIB. ConclusionsThis is the first demonstration that THR increases HSP70 and HSP 25 and protects cells from HS. THR's mechanism of protection may involve cytoskeletal stabilization, HSP up-regulation and nuclear translocation, and decreased apoptosis. THR's protection appears to involve both cell-swelling–dependent and –independent processes.

Abstract B3: HSP27 is required for invasion and metastasis triggered by hepatocyte growth factor

Abstract The Hepatocyte Growth Factor (HGF) also known as Scatter Factor, activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27KDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the motility in vitro and the remodeling of actin filaments induced by HGF. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired both the ability of ovarian cancer cells to form experimental haematogenous metastases and to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic Paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the pro-invasive and pro-metastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy. Grant sponsors: This work has been supported by grants to M.F.D.: 2012 IG grant and 2010 Special Program Molecular Clinical Oncology 5xMille of the Italian Association of Cancer Research (AIRC), Project n° 9970, and Grant of the CARIPLO Foundation. Citation Format: Simona Pavan, Daniele Musiani, Erica Torchiaro, Giorgia Migliardi, Jessica Erriquez, Martina Olivero, Maria Flavia Di Renzo. HSP27 is required for invasion and metastasis triggered by hepatocyte growth factor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B3.

HSP27 is required for invasion and metastasis triggered by hepatocyte growth factor

The hepatocyte growth factor (HGF) also known as scatter factor activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27 kDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the remodeling of actin filaments induced by HGF and that motility in vitro depended on the p38MAPK-MK2 axis. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired the ability of circulating ovarian cancer cells to home to the lungs and to form experimental hematogenous metastases and the capability of cancer cells to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the proinvasive and prometastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy.

Microtubule Alterations Occur Early in Experimental Parkinsonism and The Microtubule Stabilizer Epothilone D Is Neuroprotective

The role of microtubule (MT) dysfunction in Parkinson's disease is emerging. It is still unknown whether it is a cause or a consequence of neurodegeneration. Our objective was to assess whether alterations of MT stability precede or follow axonal transport impairment and neurite degeneration in experimental parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in C57Bl mice. MPTP induced a time- and dose-dependent increase in fibres with altered mitochondria distribution, and early changes in cytoskeletal proteins and MT stability. Indeed, we observed significant increases in neuron-specific βIII tubulin and enrichment of deTyr tubulin in dopaminergic neurons. Finally, we showed that repeated daily administrations of the MT stabilizer Epothilone D rescued MT defects and attenuated nigrostriatal degeneration induced by MPTP. These data suggest that alteration of ΜΤs is an early event specifically associated with dopaminergic neuron degeneration. Pharmacological stabilization of MTs may be a viable strategy for the management of parkinsonism.

Vascular-targeted therapies for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and an X-linked recessive, progressive muscle wasting disease caused by the absence of a functional dystrophin protein. Dystrophin has a structural role as a cytoskeletal stabilization protein and protects cells against contraction-induced damage. Dystrophin also serves a signaling role through mechanotransduction of forces and localization of neuronal nitric oxide synthase (nNOS), which produces nitric oxide (NO) to facilitate vasorelaxation. In DMD, the signaling defects produce inadequate tissue perfusion caused by functional ischemia due to a diminished ability to respond to shear stress induced endothelium-dependent dilation. Additionally, the structural defects seen in DMD render myocytes with an increased susceptibility to mechanical stress. The combination of both defects is necessary to generate myocyte damage, which induces successive rounds of myofiber degeneration and regeneration, loss of calcium homeostasis, chronic inflammatory response, fibrosis, and myonecrosis. In individuals with DMD, these processes inevitably cause loss of ambulation shortly after the first decade and an abbreviated life with death in the third or fourth decade due to cardio-respiratory anomalies. There is no known cure for DMD, and although the culpable gene has been identified for more than twenty years, research on treatments has produced few clinically relevant results. Several recent studies on novel DMD therapeutics are vascular targeted and focused on attenuating the inherent functional ischemia. One approach improves vasorelaxation capacity through pharmaceutical inhibition of either phosphodiesterase 5 (PDE5) or angiotensin-converting enzyme (ACE). Another approach increases the density of the underlying vascular network by inducing angiogenesis, and this has been accomplished through either direct delivery of vascular endothelial growth factor (VEGF) or by downregulating the VEGF decoy-receptor type 1 (VEGFR-1 or Flt-1). The pro-angiogenic approaches also seem to be pro-myogenic and could resolve the age-related decline in satellite cell (SC) quantity seen in mdx models through expansion of the SC juxtavascular niche. Here we review these four vascular targeted treatment strategies for DMD and discuss mechanisms, proof of concept, and the potential for clinical relevance associated with each therapy.

C‐Jun N‐terminal Kinase (JNK) maintains tissue integrity during cell rearrangement in the gut

Tissue elongation is a fundamental morphogenetic process that generates the proper anatomical topology of the body plan and vital organs. In vertebrates, c‐Jun N‐terminal Kinase (JNK) is a downstream effector of Wnt/PCP signaling that is required for tissue elongation, but the precise cellular role of JNK in this context has remained elusive. Here, we show that JNK activity is indispensable for the rearrangement of endoderm cells that underlies the elongation of the Xenopus gut tube. Whereas other Wnt/PCP mediators, such as Rho, are necessary to induce cell intercalation and remodel adhesive contacts, we found that JNK is required to maintain cell‐cell adhesion and establish parallel microtubule arrays; without JNK activity, the reorganizing endoderm dissociates. Depleting microtubules phenocopies JNK inhibition, consistent with a model in which JNK regulates microtubule architecture to preserve adhesive contacts between rearranging cells. Thus, in contrast to Rho, which generates actomyosin‐based tension and cell movement, JNK signaling is required to establish cytoskeletal stability and maintain tissue cohesion; both factors are required to achieve proper cell rearrangement and gut extension. Our results shed new light on the means by which intra‐ and intercellular forces are balanced to promote topological change, while preserving structural integrity, in numerous morphogenetic contexts.

Cytoskeletal regulation dominates proteomic changes associated with hibernation in 13‐lined ground squirrels

13‐lined ground squirrels are obligate hibernators that transition from summer homeothermy to winter heterothermy – wherein they exploit episodic torpor bouts. To explore the elements of winter neuroprotection, we applied 2D gel electrophoresis coupled with LC‐MS/MS to brain extracts of squirrels in two summer, four winter and fall transition states. Only 88 protein spots differed significantly among groups. The abundance pattern of the brain proteome was reciprocal between cold‐torpid and euthermic animals. The brain proteome of fall warm‐, but not cold‐torpid squirrels strongly resembled the homeotherms, indicating that the changes observed in torpid hibernators are defined by body temperature, not torpor per se. Metabolic enzymes were largely unchanged despite varied metabolic activity across annual and torpor‐arousal cycles. Proteins elevated in summer and aroused winter squirrels represented pathway enrichments in neural growth/differentiation and synaptic transmission. Differential cytoskeletal regulation across torpor‐arousal cycles is revealed by changes in microtubule dis/assembly regulating protein abundance (STMN1, DPYSL2), suggesting a mechanism for cytoskeletal stabilization during torpor and rapid reorganization on return to euthermy. Funded by NIH R01HL089049.

Thyroid Hormones and Peripheral Nerve Regeneration

Peripheral nerve regeneration is a unique process in which cellular rather than tissue response is involved. Depending on the extent and proximity of the lesion and the age and type of the neuronal soma, the cell body may either initiate a reparative response or may die. Microsurgical intervention may alter the prognosis after a peripheral nerve injury but to a certain extent. By altering the biochemical microenvironment of the neuron, we can increase the proportion of neurons that survive the injury and initiate the reparative response. Thyroid hormone critically regulates tissue growth and differentiation and plays a crucial role during organ development. Furthermore, recent research has provided new insight into thyroid hormone cellular action. Thyroid hormone regulates stress response intracellular signaling and targets molecules important for cytoskeletal stability and cell integrity. Changes in thyroid hormone signaling occur in nerve and other tissues, with important physiological consequences. The interest in thyroid hormone in the context of nerve regeneration has recently been revived.