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The dynamic remodeling of the cytoskeletal network of vimentin intermediate filaments supports various cellular functions, including cell morphology, elasticity, migration, organelle localization, and resistance against mechanical or pathological stress. Currently available chemicals targeting vimentin predominantly induce network reorganization and shrinkage around the nucleus. Effective tools for long-term manipulation of vimentin network dispersion in living cells are still lacking, limiting in-depth studies on vimentin function and potential therapeutic applications. Here, we verified that a commercially available small molecule, trametinib, is capable of inducing spatial spreading of the cellular vimentin network without affecting its transcriptional or Translational regulation. Further evidence confirmed its low cytotoxicity and similar effects on different cell types. Importantly, Trametinib has no impact on the other two cytoskeletal systems, actin filaments and the microtubule network. Moreover, Trametinib regulates vimentin network dispersion rapidly and efficiently, with effects persisting for up to 48 h after drug withdrawal. We also ruled out the possibility that Trametinib directly affects the phosphorylation level of vimentin. In summary, we identified an unprecedented regulator Trametinib, which is capable of spreading the vimentin network toward the cell periphery, and thus complemented the existing repertoire of vimentin remodeling drugs in the field of cytoskeletal research., (© The Author(s) (2024). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

Cancer is one of the deadliest illness globally. Searching for new solutions in cancer treatments is essential because commonly used mixed, targeted and personalized therapies are sometimes not sufficient or are too expensive for common patients. Sugar fatty acid esters (SFAEs) are already well-known as promising candidates for an alternative medical tool. The manuscript brings the reader closer to methods of obtaining various SFAEs using combined biological, chemical and enzymatic methods. It presents how modification of SFAE's hydrophobic chains can influence their cytotoxicity against human skin melanoma and prostate cancer cell lines. The compound's cytotoxicity was determined by an MTT assay, which followed an assessment of SFAEs' potential metastatic properties in concentrations below IC 50 values. Despite relatively high IC 50 values (63.3-1737.6 μM) of the newly synthesized SFAE, they can compete with other sugar esters already described in the literature. The chosen bioactives caused low polymerization of microtubules and the depolymerization of actin filaments in nontoxic levels, which suggest an apoptotic rather than metastatic process. Altogether, cancer cells showed no propensity for metastasis after treating them with SFAE. They confirmed that lactose-based compounds seem the most promising surfactants among tested sugar esters. This manuscript creates a benchmark for creation of novel anticancer agents based on 3-hydroxylated fatty acids of bacterial origin.

(1) Background: Triple negative breast cancer (TNBC) is a highly aggressive tumor, associated with high rates of early distant recurrence and short survival times, and treatment may require surgery, and thus anesthesia. The effects of anesthetic drugs on cancer progression are under scrutiny, but published data are controversial, and the involved mechanisms unclear. Anesthetic agents have been shown to modulate several molecular cascades, including PI3K/AKT/mTOR. AKT isoforms are frequently amplified in various malignant tumors and associated with malignant cell survival, proliferation and invasion. Their activation is often observed in human cancers and is associated with decreased survival rate. Certain anesthetics are known to affect hypoxia cell signaling mechanisms by upregulating hypoxia-inducible factors (HIFs). (2) Methods: MCF-10A and MDA-MB 231 cells were cultivated and CellTiter-Blue ® Cell Viability assay, 2D and 3D matrigel assay, immunofluorescence assays and gene expressions assay were performed after exposure to different sevoflurane concentrations. (3) Results: Sevoflurane exposure of TNBC cells results in morphological and behavioral changes. Sevoflurane differently influences the AKT isoforms expression in a time-dependent manner, with an important early AKT3 upregulation. The most significant effects occur at 72 h after 2 mM sevoflurane treatment and consist in increased viability, proliferation and aggressiveness and increased vimentin and HIF expression. (4) Conclusions: Sevoflurane exposure during surgery may contribute to cancer recurrence via AKT3 induced epithelial-mesenchymal transition (EMT) and by all three AKT isoforms enhanced cancer cell survival and proliferation.

Background: Administration of d-galactose (d-gal) has been used to create animal models of neurodegenerative diseases, and huperzine A has been used to treat the neurodegenerative diseases such as Alzheimer disease., Methods: An animal model of hearing dysfunction was established by administration of d-gal in the rats, and the effect of huperzine A on d-gal-induced abnormal hearing function and cochlear damage was investigated. Senescence of the cochlear tissues was examined by β-galactase staining, and messenger RNA expression of inflammatory cytokines was quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR)., Results: It was found that d-gal significantly increased auditory brainstem response (ABR) threshold and cellular senescence and decreased neurofilament in the cochlear tissues. Huperzine A could significantly attenuate d-gal-induced increase of ABR threshold and cellular senescence as well as reduction of neurofilament. Moreover, huperzine A could inhibit d-gal-induced activation of nuclear factor kappa-B (NF-κB) in Schwann cells and significantly blocked d-gal-stimulated gene expression of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α., Conclusion: These findings suggested that d-gal causes hearing dysfunction by inflammatory injury of cochlear neurons and that huperzine A could prevent hearing loss by protecting d-gal-induced physical damage of cochlear tissues.

Excessive neuroinflammatory responses play important roles in the development of postoperative cognitive dysfunction (POCD). Neurofilaments (NFs) were essential to the structure of axon and nerve conduction; and the abnormal degradation of NFs were always accompanied with degenerative diseases, which were also characterized by excessive neuroinflammatory responses in brain. However, it is still unclear whether the NFs were involved in the POCD. In this study, the LC-MS/MS method was used to explore the neuroinflammatory response and NFs of POCD in aged rats. Moreover, trichostatin A (TSA), an inflammation-related drug, was selected to test whether it could improve the surgery-induced cognitive dysfunction, inflammatory responses and NFs. Evident cognitive dysfunction, excessive microglia activation, neuroinflammatory responses and upregulated NFs in hippocampus were observed in the POCD group. TSA pretreatment could significantly mitigate these changes. The KEGG analysis revealed that nine pathways were enriched in the TSA + surgery group (versus the surgery group). Among them, two signaling pathways were closely related with the changes of NFs proteins. In conclusion, surgery could impair the cognitive function and aggravate neuroinflammation and NFs. The TSA could significantly improve these changes which might be related to the activation of the "focal adhesion" and "ECM-receptor interaction" pathways.

This study highlighted the effect of Carbendazim on the testicular micro and intermediate filaments adult male African catfish (Clarias gariepinus). Previous studies related to carbendazim toxicity in fish have been limited to mortality patterns and degree of sensitivity across species. Literature on actual pathology in fish  is scanty. The fish were exposed to a pre-determined sub-lethal concentration (1.4 mg/L) of Carbendazim for seven and fourteen days, 10 fish were  sedated by cold shock, and sacrificed on days seven and 14. Another untreated group (control) were sacrificed at the same periods. The testes were harvested and weighed.  Testicular actin microfilament, cytokeratin, desmin and vimentin intermediate filaments were determined using standard immunohistochemistry protocols. Variations in the intensity and pattern of immuno-expression of the testicular actin, cytokeratin, desmin and vimentin were significant in a phase dependent manner with day 14 being more pronounced. Immunohistochemical features of degenerated and necrotic germinal and Sertoli cells in the treated group, with loss of wire-mesh network which supported the mature germinal cells in the testicular lumen were also observed.   A sub-lethal dose of carbendazim exposure for either seven or 14 days, induced deliterous  changes in the testicular micro- and intermediate filaments, of the African catfish. This portends a reduction in the male reproductive potentials of the exposed species and resultant negative impact on production.

The mechanical state of cells is a critical part of their healthy functioning and it is controlled primarily by cytoskeletal networks (actin, microtubules and intermediate filaments). Drug-based strategies targeting the assembly of a given cytoskeletal network are often used to pinpoint their role in cellular function. Unlike actin and microtubules, there has been limited interest in the role of intermediate filaments, and fewer drugs have thus been identified and characterised as modulators of its assembly. Here, we evaluate whether Withaferin-A (WFA), an established disruptor of vimentin filaments, can also be used to modulate keratin filament assembly. Our results show that in keratinocytes, which are keratin-rich but vimentin-absent, Withaferin-A disrupts keratin filaments. Importantly, the dosages required are similar to those previously reported to disrupt vimentin in other cell types. Furthermore, Withaferin-A-induced keratin disassembly is accompanied by changes in cell stiffness and migration. Therefore, we propose that WFA can be repurposed as a useful drug to disrupt the keratin cytoskeleton in epithelial cells.

Heat shock protein 27 (HSP27) is commonly involved in cellular stress. Increased levels of HSP27 as well as autoantibodies against this protein were previously detected in glaucoma patients. Moreover, systemic immunization with HSP27 induced glaucoma-like damage in rodents. Now, for the first time, the direct effects of an intravitreal HSP27 application were investigated. For this reason, HSP27 or phosphate buffered saline (PBS, controls) was applied intravitreally in rats ( n = 12/group). The intraocular pressure (IOP) as well as the electroretinogram recordings were comparable in HSP27 and control eyes 21 days after the injection. However, significantly fewer retinal ganglion cells (RGCs) and amacrine cells were observed in the HSP27 group via immunohistochemistry and western blot analysis. The number of bipolar cells, on the other hand, was similar in both groups. Interestingly, a stronger neurofilament degeneration was observed in HSP27 optic nerves, while no differences were noted regarding the myelination state. In summary, intravitreal HSP27 injection led to an IOP-independent glaucoma-like damage. A degeneration of RGCs as well as their axons and amacrine cells was noted. This suggests that high levels of extracellular HSP27 could have a direct damaging effect on RGCs.

Targeted inhibition of mutated kinases using selective MAP kinase inhibitors in malignant melanoma often results in temporary improvement of clinical symptoms followed by rapid development of resistance. To gain insights in molecular processes that govern resistance, we performed SILAC-based quantitative proteomics profiling of vemurafenib-resistant and -sensitive melanoma cells. Among downregulated proteins in vemurafenib-resistant cell lines we detected multiple proteins involved in cytoskeletal organization and signaling, including the intermediate filament nestin, which was one of the most downregulated proteins. Previous studies showed that nestin is expressed in various types of solid tumors and its abundance correlates with malignant phenotype of transformed cells. However, the role of nestin in cancer cells regarding acquired resistance is still poorly understood. We performed CRISPR/Cas9 knockout of the nestin gene ( NES ) in vemurafenib-sensitive cells and showed that loss of nestin leads to increased cellular proliferation and colony formation upon treatment with BRAF V600E and MEK inhibitors. Moreover, nestin depletion led to increased invasiveness and metalloproteinase activity like the phenotype of melanoma cells with acquired resistance to the BRAF inhibitor. Finally, phosphoproteome analysis revealed that nestin depletion influenced signaling through integrin and PI3K/AKT/mTOR pathways and led to increased focal adhesion kinase abundance and phosphorylation. Taken together, our results reveal that nestin is associated with acquired vemurafenib resistance in melanoma cells., (© 2019 Schmitt et al.)

Myelination leads to the formation of myelin sheaths surrounding neuronal axons and is crucial for function, plasticity and repair of the central nervous system (CNS). It relies on the interaction of the axons and the oligodendrocytes: the glial cells producing CNS myelin. Here, we have investigated the role of a crucial component of the Sonic hedgehog (Shh) signalling pathway, the co-receptor Boc, in developmental and repairing myelination. During development, Boc mutant mice display a transient decrease in oligodendroglial cell density together with delayed myelination. Despite recovery of oligodendroglial cells at later stages, adult mutants still exhibit a lower production of myelin basic protein correlated with a significant decrease in the calibre of callosal axons and a reduced amount of the neurofilament NF-M. During myelin repair, the altered OPC differentiation observed in the mutant is reminiscent of the phenotype observed after blockade of Shh signalling. In addition, Boc mutant microglia/macrophages unexpectedly exhibit the apparent inability to transition from a highly to a faintly ramified morphology in vivo Altogether, these results identify Boc as an important component of myelin formation and repair., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Idiopathic pulmonary fibrosis (IPF) is a progressive disease, with a median survival of 3-5 years following diagnosis. Lung remodeling by invasive fibroblasts is a hallmark of IPF. In this study, we demonstrate that inhibition of vimentin intermediate filaments (VimIFs) decreases the invasiveness of IPF fibroblasts and confers protection against fibrosis in a murine model of experimental lung injury. Increased expression and organization of VimIFs contribute to the invasive property of IPF fibroblasts in connection with deficient cellular autophagy. Blocking VimIF assembly by pharmacologic and genetic means also increases autophagic clearance of collagen type I. Furthermore, inhibition of expression of collagen type I by siRNA decreased invasiveness of fibroblasts. In a bleomycin injury model, enhancing autophagy in fibroblasts by an inhibitor of VimIF assembly, withaferin A (WFA), protected from fibrotic lung injury. Additionally, in 3D lung organoids, or pulmospheres, from patients with IPF, WFA reduced the invasiveness of lung fibroblasts in the majority of subjects tested. These studies provide insights into the functional role of vimentin, which regulates autophagy and restricts the invasiveness of lung fibroblasts.

Notochordal cells (NCs), characterized by their vacuolated morphology and coexpression of cytokeratin and vimentin intermediate filaments (IFs), form the immature nucleus pulposus (NP) of the intervertebral disc. As humans age, NCs give way to mature NP cells, which do not possess a vacuolated morphology and typically only express vimentin IFs. In light of their concomitant loss, we investigated the relationship between cytosolic vacuoles and cytokeratin IFs, specifically those containing cytokeratin-8 proteins, using a human chordoma cell line as a model for NCs. We demonstrate that the chemical disruption of IFs with acrylamide, F-actin with cytochalasin-D, and microtubules with nocodazole all result in a significant (p < 0.001) decrease in vacuolation. However, vacuole loss was the greatest in acrylamide-treated cells. Examination of the individual roles of vimentin and cytokeratin-8 IFs in the existence of vacuoles was accomplished using small interfering RNA-mediated RNA interference to knock down either vimentin or cytokeratin-8 expression. Reduction of cytokeratin-8 expression was associated with a less-vacuolated cell morphology. These data demonstrate that cytokeratin-8 IFs are involved in stabilizing vacuoles and that their diminished expression could play a role in the loss of vacuolation in NCs during aging. A better understanding of the NCs may assist in preservation of this cell type for NP maintenance and regeneration., (© 2018 Wiley Periodicals, Inc.)

The enrichment of intermediate filaments in the apical cytoplasm of intestinal cells is evolutionarily conserved, forming a sheath that is anchored to apical junctions and positioned below the microvillar brush border, which suggests a protective intracellular barrier function. To test this, we used Caenorhabditis elegans , the intestinal cells of which are endowed with a particularly dense intermediate filament-rich layer that is referred to as the endotube. We found alterations in endotube structure and intermediate filament expression upon infection with nematicidal B. thuringiensis or treatment with its major pore-forming toxin crystal protein Cry5B. Endotube impairment due to defined genetic mutations of intermediate filaments and their regulators results in increased Cry5B sensitivity as evidenced by elevated larval arrest, prolonged time of larval development and reduced survival. Phenotype severity reflects the extent of endotube alterations and correlates with reduced rescue upon toxin removal. The results provide in vivo evidence for a major protective role of a properly configured intermediate filament network as an intracellular barrier in intestinal cells. This notion is further supported by increased sensitivity of endotube mutants to oxidative and osmotic stress., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: Amyotrophic lateral sclerosis (ALS) represents a devastating, progressive, heterogeneous, and the most common motor neuron (MN) disease. To date, no cure has been available for the condition. Studies with transgenic mice have yielded significant results that help us understand the underlying mechanisms of ALS. Nonetheless, none of more than 30 large clinical trials over the past 20 years proved successful, which led some researchers to challenge the validity of the preclinical models., Methods: Human-induced pluripotent cells (iPSCs) were established by introducing Sendai virus into fibroblast cells. We established TDP-43 HES by inserting CAG-TDP43 (G298S) cassette or the CAG-EGFP cassette into PPP1R12C-locus of human embryonic stem cells (ESC, H9) by TALEN-mediated homologous recombination. iPSCs or HESC were differentiated to motor neurons and non-motor neuron as control. Relevant biomarkers were detected in different differentiated stages. TDP-43 aggregates, neurofilament, and mitochondria analyses were performed., Results: In this study, using iPSCs-derived human MN from an ALS patient with a TDP43 G298S mutation and two sporadic ALS patients, we showed that both sporadic and familial ALS were characterized by TDP-43 aggregates in the surviving MN. Significantly higher neurofilament (NF) inclusion was also found in ALS MN compared with wild-type (WT) GM15 controls (P < 0.05). The neurite mitochondria density was significantly lower in ALS MN than that in the control MNs. Transgenesis of TDP-43 G298S into AAVS locus in human embryonic stem cells reproduced phenotype of patient-derived G289S MN. By challenging MNs with a proteasome inhibitor, we found that MNs were more vulnerable to MG132, with some accompanying phenotype changes, such as TDP43 translocation, NF inclusion, mitochondria distribution impairment, and activation of caspase3., Conclusions: Our results suggested that changes in TDP43 protein, NF inclusion, and distribution impairment of mitochondria are common early pathology both in familial and sporadic ALS. These findings will help us gain insight into the pathogenesis of the condition and screen relevant drugs for the disease.

Cytoskeletal proteins are increasingly recognized as having important roles as a target of the action of different neurotoxins. In the last years, several works of our group have shown that quinolinic acid (QUIN) was able to disrupt the homeostasis of the cytoskeleton of neural cells and this was associated with cell dysfunction and neurodegeneration. QUIN is an excitotoxic metabolite of tryptophan metabolism and its accumulation is associated with several neurodegenerative diseases. In the present review, we provide a comprehensive view of the actions of QUIN upstream of glutamate receptors, eliciting kinase/phosphatase signaling cascades that disrupt the homeostasis of the phosphorylation system associated with intermediate filament proteins of astrocytes and neurons. We emphasize the critical role of calcium in these actions and the evidence that misregulated cytoskeleton takes part of the cell response to the injury resulting in neurodegeneration in different brain regions, disrupted cell signaling in acute tissue slices, and disorganized cytoskeleton with altered cell morphology in primary cultures. We also discuss the interplay among misregulated cytoskeleton, oxidative stress, and cell-cell contact through gap junctions mediating the quinolinic acid injury in rat brain. The increasing amount of cross talks identified between cytoskeletal proteins and cellular signaling cascades reinforces the exciting possibility that cytoskeleton could be a new target in the neurotoxicity of QUIN and further studies will be necessary to develop strategies to protect the cytoskeleton and counteracts the cytotoxicity of this metabolite.

Human natural killer-1 (HNK-1) cell antigen is a glycan epitope involved in several neural events, such as neuritogenesis, myelination, synaptic plasticity and regeneration of the nervous system after injury. We have recently identified the small organic compound ursolic acid (UA) as a HNK-1 mimetic with the aim to test its therapeutic potential in the central nervous system. UA, a plant-derived pentacyclic triterpenoid, is well known for its multiple biological functions, including neuroprotective, antioxidant and anti-inflammatory activities. In the present study, we evaluated its functions in a mouse model of spinal cord injury (SCI) and explored the molecular mechanisms underlying its positive effects. Oral administration of UA to mice 1 h after SCI and thereafter once daily for 6 weeks enhanced the regaining of motor functions and axonal regrowth, and decreased astrogliosis. UA administration decreased levels of proinflammatory markers, including interleukin-6 and tumor necrosis factor-α, in the injured spinal cord at the acute phase of inflammation and activated the mitogen-activated protein kinase and phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathways in the injured spinal cord. Taken together, these results suggest that UA may be a candidate for treatment of nervous system injuries., (Copyright © 2017. Published by Elsevier Inc.)

Epileptic seizure has been reported to enhance adult neurogenesis and induce aberrant synaptic reorganization in the human dentate gyrus in the hippocampal formation. However, adult neurogenesis in the extrahippocampal regions has not been well studied. To investigate seizure-enhanced neurogenesis in the extrahippocampal regions, we performed histological and immunohistochemical as well as western blot analyses on the cerebrum of Sprague-Dawley rats (n = 51, male, 7 weeks old, body weight 250-300 g) treated with intraperitoneal injection of kainic acid (KA, 10 mg/kg) to induce status epilepticus (SE) (n = 36) or normal saline solution (n = 15) followed by 5'-bromo-2-deoxyuridine (BrdU) injection to label newborn cells. Even though severe neuronal damage was found in the piriform cortex of rats having SE, immunohistochemistry for double cortin (DCX) revealed an increase in the number of immature neurons in the piriform cortex. Double immunofluorescence staining demonstrated that DCX-positive cells in the piriform cortex were positive for both BrdU and neuronal nuclear antigen. Immunohistochemistry and western blotting revealed increased expressions of synaptophysin and postsynaptic density protein 95 in the piriform cortex of rat having SE. These results suggested the enhanced neurogenesis and possible synaptic reorganization in the piriform cortex of the KA-treated rat., (© 2017 Japanese Society of Neuropathology.)

Neurobasal®/B27 is a gold standard culture media used to study primary neurons in vitro. An alternative media (BrainPhys®/SM1) was recently developed which robustly enhances neuronal activity vs. Neurobasal® or DMEM. To the best of our knowledge BrainPhys® has not been explored in the setting of neuronal injury. Here we characterized the utility of BrainPhys® in a model of in vitro mechanical-stretch injury., Methods/results: Primary rat cortical neurons were maintained in classic Neurobasal®, or sequentially maintained in Neurocult® followed by BrainPhys® (hereafter simply referred to as "BrainPhys® maintained neurons"). The levels of axonal markers and proteins involved in neurotransmission were compared on day in vitro 10 (DIV10). BrainPhys® maintained neurons had higher levels of GluN2B, GluR1, Neurofilament light/heavy chain (NF-L & NF-H), and protein phosphatase 2 A (PP2A) vs. neurons in Neurobasal®. Mechanical stretch-injury (50ms/54% biaxial stretch) to BrainPhys® maintained neurons modestly (albeit significantly) increased 24h lactate dehydrogenase (LDH) levels but markedly decreased axonal NF-L levels post-injury vs. uninjured controls or neurons given a milder 38% stretch-injury. Furthermore, two 54% stretch-injuries (in tandem) exacerbated 24h LDH release, increased α-spectrin breakdown products (SBDPs), and decreased Tau levels. Also, BrainPhys® maintained cultures had decreased markers of cell damage 24h after a single 54% stretch-injury vs. neurons in Neurobasal®. Finally, we tested the hypothesis that lentivirus mediated overexpression of the pro-death protein RBM5 exacerbates neuronal and/or axonal injury in primary CNS cultures. RBM5 overexpression vs. empty-vector controls increased 24h LDH release, and SBDP levels, after a single 54% stretch-injury but did not affect NF-L levels or Tau., Conclusion: BrainPhys® is a promising new reagent which facilities the investigation of molecular targets involved in axonal and/or neuronal injury in vitro., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

In response to central nervous system (CNS) injury, astrocytes upregulate intermediate filament (nanofilament) proteins GFAP and vimentin. Whereas the intermediate filament upregulation in astrocytes is important for neuroprotection in the acute phase of injury, in some contexts it might inhibit some of the regenerative processes later on. Thus, timely modulation of the astrocyte intermediate filaments was proposed as a strategy to promote brain repair. We used clomipramine, epoxomicin and withaferin A, drugs reported to decrease the expression of GFAP, and assessed their effect on neurosupportive properties and resilience of astrocytes to oxygen and glucose deprivation (OGD). Clomipramine decreased protein levels of GFAP, as well as vimentin and nestin, and did not affect astrocyte resilience to oxidative stress. Withaferin A sensitized astrocytes to OGD. Both clomipramine and epoxomicin promoted the attachment and survival of neurons co-cultured with astrocytes under standard culture conditions. Moreover, epoxomicin increased neurosupportive properties of astrocytes after OGD. Our data point to clomipramine and epoxomicin as potential candidates for astrocyte modulation to improve outcome after CNS injury., (Copyright © 2017 Elsevier Inc. All rights reserved.)

High temperature requirement A1 (HtrA1) belongs to an ancient protein family that is linked to various human disorders. The precise role of exon 1-encoded N-terminal domains and how these influence the biological functions of human HtrA1 remain elusive. In this study, we traced the evolutionary origins of these N-terminal domains to a single gene fusion event in the most recent common ancestor of vertebrates. We hypothesized that human HtrA1 is implicated in unfolded protein response. In highly secretory cells of the retinal pigmented epithelia, endoplasmic reticulum (ER) stress upregulated HtrA1. HtrA1 co-localized with vimentin intermediate filaments in highly arborized fashion. Upon ER stress, HtrA1 tracked along intermediate filaments, which collapsed and bundled in an aggresome at the microtubule organizing center. Gene silencing of HtrA1 altered the schedule and amplitude of adaptive signaling and concomitantly resulted in apoptosis. Restoration of wild-type HtrA1, but not its protease inactive mutant, was necessary and sufficient to protect from apoptosis. A variant of HtrA1 that harbored exon 1 substitutions displayed reduced efficacy in rescuing cells from proteotoxicity. Our results illuminate the integration of HtrA1 in the toolkit of mammalian cells against protein misfolding and the implications of defects in HtrA1 in proteostasis.

Geminiviral single-stranded circular DNA genomes replicate in nuclei so that the progeny DNA has to cross both the nuclear envelope and the plasmodesmata for systemic spread within plant tissues. For intra- and intercellular transport, two proteins are required: a nuclear shuttle protein (NSP) and a movement protein (MP). New characteristics of ectopically produced Abutilon mosaic virus (AbMV) MP (MP AbMV ), either authentically expressed or fused to a yellow fluorescent protein or epitope tags, respectively, were determined by localization studies in mammalian cell lines in comparison to plant cells. Wild-type MP AbMV and the distinct MP AbMV : reporter protein fusions appeared as curled threads throughout mammalian cells. Co-staining with cytoskeleton markers for actin, intermediate filaments, or microtubules identified these threads as re-organized microtubules. These were, however, not stabilized by the viral MP, as demonstrated by nocodazole treatment. The MP of a related bipartite New World begomovirus, Cleome leaf crumple virus (ClLCrV), resulted in the same intensified microtubule bundling, whereas that of a nanovirus did not. The C-terminal section of MP AbMV , i.e., the protein's oligomerization domain, was dispensable for the effect. However, MP expression in plant cells did not affect the microtubules network. Since plant epidermal cells are quiescent whilst mammalian cells are proliferating, the replication-associated protein Rep AbMV protein was then co-expressed with MP AbMV to induce cell progression into S-phase, thereby inducing distinct microtubule bundling without MP recruitment to the newly formed threads. Co-immunoprecipitation of MP AbMV in the presence of Rep AbMV , followed by mass spectrometry identified potential novel MP AbMV -host interaction partners: the peptidyl-prolyl cis-trans isomerase NIMA-interacting 4 (Pin4) and stomatal cytokinesis defective 2 (SCD2) proteins. Possible roles of these putative interaction partners in the begomoviral life cycle and cytoskeletal association modes are discussed., Competing Interests: The authors declare no conflict of interest.

Acori Tatarinowii Rhizoma (ATR), the rhizome of Acorus tatarinowii Schott, is a common traditional Chinese medicine being used clinically for mental disorder. However, other Acorus species herbs are all having the same Chinese name 'Chang Pu', making the confusion in herbal market. Acori Graminei Rhizoma (AGR) and Acori Calami Rhizoma (ACR) are common adulterants of ATR. Here, we aim to provide a comparative analysis between ATR, AGR, and ACR in potentiating neuronal differentiation. Volatile oil, derived from Acorus species, was applied onto cultured PC12 cells, and various parameters were determined: (i) transcriptional activation of neurofilament promoters was determined by the promoter-driven luciferase activity assay; (ii) the neurite outgrowth of PC12 cells was captured and measured; and (iii) the neurofilament expression and its underlying mechanism were analyzed by western blotting. The co-treatment of ATR, AGR, or ACR volatile oil with low concentration of nerve growth factor (NGF) could potentiate the NGF-induced neuronal differentiation in cultured PC12 cells. In addition, application of protein kinase A inhibitor H89 in cultures blocked the induction of neurofilament. Among these three Acorus species, ATR volatile oil showed the highest NGF-induced induction in neurite outgrowth and neurofilament expression, as compared with that of AGR and ACR. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Curing spinal cord injury (SCI) in mammals is a daunting task because of the lack of permissive mechanisms and strong inhibitory responses at and around the lesion. The neural cell adhesion molecule L1CAM (L1) has been shown to favor axonal regrowth and enhance neuronal survival and synaptic plasticity but delivery of full-length L1 or its extracellular domain could encounter difficulties in translation to therapy in humans. We have, therefore, identified several small organic compounds that bind to L1 and stimulate neuronal survival, neuronal migration and neurite outgrowth in an L1-dependent manner. Here, we assessed the functions of two L1 mimetics, trimebutine and honokiol, in regeneration following SCI in young adult mice. Using the Basso Mouse Scale (BMS) score, we found that ground locomotion in trimebutine-treated mice recovered better than honokiol-treated or vehicle-receiving mice. Enhanced hindlimb locomotor functions in the trimebutine group were observed at 6 weeks after SCI. Immunohistology of the spinal cords rostral and caudal to the lesion site showed reduced areas and intensities of glial fibrillary acidic protein immunoreactivity in both trimebutine and honokiol groups, whereas increased regrowth of axons was observed only in the trimebutine-treated group. Both L1- and L1 mimetic-mediated intracellular signaling cascades in the spinal cord lesion sites were activated by trimebutine and honokiol, with trimebutine being more effective than honokiol. These observations suggest that trimebutine and, to a lesser extent under the present experimental conditions, honokiol have a potential for therapy in regeneration of mammalian spinal cord injuries., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The purpose of this study was to explore how liraglutide affects AD-like pathology and cognitive function in APP/PS1/Tau triple transgenic (3 × Tg) Alzheimer disease (AD) model mice. Male 3 × Tg mice and C57BL/6 J mice were treated for 8 weeks with liraglutide (300 μg/kg/day, subcutaneous injection) or saline. Levels of phosphorylated tau, neurofilaments (NFs), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) in brain tissues were assessed with western blots. Fluoro-Jade-B labeling were applied to detect pathological changes. The Morris water maze (MWM) was used to assess the spatial learning and memory. Liraglutide decreased levels of hyperphosphorylated tau and NFs in 3 × Tg liraglutide-treated (Tg + LIR) mice, increased ERK phosphorylation, and decreased JNK phosphorylation. Liraglutide also decreased the number of degenerative neurons in the hippocampus and cortex of Tg + LIR mice, and shortened their escape latencies and increased their hidden platform crossings in the MWM task. Liraglutide did not significantly affect the animals' body weight (BW) or fasting blood glucose. Liraglutide can reduce hyperphosphorylation of tau and NFs and reduce neuronal degeneration, apparently through alterations in JNK and ERK signaling, which may be related to its positive effects on AD-like learning and memory impairment.

Aims: To optimise the ELISA method for the avidity of IgG antibodies against neurofilament heavy chain (NfH) and to determine the levels and avidity of anti-NfH antibodies in patients with Alzheimer's disease (AD) and a healthy control group., Methods: Various dilutions of sera and concentrations of urea and sodium chloride as chaotropic reagents were tested in the process of the ELISA optimisation. The levels and avidity of anti-NfH antibodies were determined in 30 patients with Alzheimer's disease and 30 age-matched cognitively normal elderly adults., Results: Sera dilution 1:200 and urea as a chaotrope in a concentration 6 mol/L were chosen to be the most suitable for the avidity assay of anti-NfH antibodies by ELISA. The results showed no differences in either level or avidity of IgG anti-NfH antibodies between AD patients and cognitively normal persons. The levels of anti-NfH IgG antibodies inversely correlated with their avidities., Conclusions: We optimised the ELISA method for the determination of anti-NfH antibody avidity determination which is suitable for research of anti-NfH antibody avidity in patients with neurological diseases associated with neurocytoskeletal defects. The determination of serum anti-NfH antibody avidity in AD patients seems to have limited diagnostic significance.

Clostridium perfringens epsilon toxin (ETX), the most potent toxin produced by this bacteria, plays a key role in the pathogenesis of enterotoxaemia in ruminants, causing brain edema and encephalomalacia. Studies of animals suffering from ETX intoxication describe severe neurological disorders that are thought to be the result of vasogenic brain edemas and indirect neuronal toxicity, killing oligodendrocytes but not astrocytes, microglia, or neurons in vitro. In this study, by means of intravenous and intracerebroventricular delivery of sub-lethal concentrations of ETX, the histological and ultrastructural changes of the brain were studied in rats and mice. Histological analysis showed degenerative changes in neurons from the cortex, hippocampus, striatum and hypothalamus. Ultrastructurally, necrotic neurons and apoptotic cells were observed in these same areas, among axons with accumulation of neurofilaments and demyelination as well as synaptic stripping. Lesions observed in the brain after sub-lethal exposure to ETX, result in permanent behavioral changes in animals surviving ETX exposure, as observed individually in several animals and assessed in the Inclined Plane Test and the Wire Hang Test. Pharmacological studies showed that dexamethasone and reserpine but not ketamine or riluzole were able to reduce the brain lesions and the lethality of ETX. Cytotoxicity was not observed upon neuronal primary cultures in vitro. Therefore, we hypothesize that ETX can affect the brain of animals independently of death, producing changes on neurons or glia as the result of complex interactions, independently of ETX-BBB interactions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Intermediate filaments (IF) can be altered under disorders such as neurodegenerative diseases. Kainic acid (KA) induce behavioral changes and histopathological alterations of the spinal cord of injected rats. Our goal was to evaluate the IF expression in neurons during this injury model. Animals were injected with KA at the C5 segment of the cervical spinal cord and euthanized at 1, 3 and 7 post injection (pi) days. Neuronal cell counting showed a significant loss of neurons at the injection site when compared with those of sham and non-operated animals. Immunohistochemistry for vimentin and neurofilament showed positive labeling of perikarya in sham and KA-injected animals since day 1 pi that lasted for the remaining experimental days. Colocalization analysis between enolase and vimentin or neurofilament confirmed a high index of colocalization in both experimental groups at day 1 pi. This index decreased in sham animals by day 3 pi whereas that of KA-injected animals remained high throughout the experiment. These results may suggest that perikarya initiate an unconventional IF expression, which may respond to the neuronal damage induced by the mechanical injury and the excitotoxic effect of KA. It seems that vimentin and neurofilament expression may be a necessary change to promote recovery of the damaged tissue., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The obligatory role of carnitine palmitoyltransferase-I (CPT-I) in mediating mitochondrial lipid transport is well established, a process attenuated by malonyl-CoA (M-CoA). However, the necessity of reducing M-CoA concentrations to promote lipid oxidation has recently been challenged, suggesting external regulation on CPT-I. Since previous work in hepatocytes suggests the involvement of the intermediate filament fraction of the cytoskeleton in regulating CPT-I, we investigated in skeletal muscle if CPT-I sensitivity for M-CoA inhibition could be regulated by the intermediate filaments, and whether AMP-activated protein kinase (AMPK) could be involved in this process. Chemical disruption (3,3'-iminodipropionitrile, IDPN) of the intermediate filaments did not alter mitochondrial respiration or sensitivity for numerous substrates (palmitoyl-CoA, ADP, palmitoyl carnitine and pyruvate). In contrast, IDPN reduced CPT-I sensitivity for M-CoA inhibition in permeabilized muscle fibers, identifying M-CoA kinetics as a specific target for intermediate filament regulation. Importantly, exercise mimicked the effect of IDPN on M-CoA sensitivity, suggesting that intermediate filament disruption in vivo is physiologically important for CPT-I regulation. To ascertain a potential mechanism, since AMPK is activated during exercise, AMPK β1β2-KO mice were utilized in an attempt to ablate the observed exercise response. Unexpectedly, these mice displayed drastic attenuation in resting M-CoA sensitivity, such that exercise and IDPN could not further alter M-CoA sensitivity. These data suggest that AMPK is not required for the regulation of the intermediate filament interaction with CPT-I. Altogether, these data highlight that M-CoA sensitivity is important for regulating mitochondrial lipid transport. Moreover, M-CoA sensitivity appears to be regulated by intermediate filament interaction with CPT-I, a process that is important when metabolic homeostasis is challenged., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Paclitaxel (PT)-induced neurotoxicity is a significant problem associated with successful treatment of cancers. Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor and plays an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Whether IGF-1 has protective effects on neurite growth, cell viability, neuronal apoptosis and neuronal phenotypes in DRG neurons with PT-induced neurotoxicity is still unclear. In this study, primary cultured rat DRG neurons were used to assess the effects of IGF-1 on DRG neurons with PT-induced neurotoxicity. The results showed that PT exposure caused neurite retraction in a dose-dependent manner. PT exposure caused a decrease of cell viability and an increase in the ratio of apoptotic cells which could be reversed by IGF-1. The percentage of calcitonin gene-related peptide immunoreactive (CGRP-IR) neurons and neurofilament (NF)-200-IR neurons, mRNA, and protein levels of CGRP and NF-200 decreased significantly after treatment with PT. IGF-1 administration had protective effects on CGRP-IR neurons, but not on NF-200-IR neurons. Either extracellular signal-regulated protein kinase (ERK1/2) inhibitor PD98059 or phosphatidylinositol 3-kinase (PI3 K) inhibitor LY294002 blocked the effect of IGF-1. The results imply that IGF-1 may attenuate apoptosis to improve neuronal cell viability and promote neurite growth of DRG neurons with PT-induced neurotoxicity. Moreover, these results support an important neuroprotective role of exogenous IGF-1 on distinct subpopulations of DRG neurons which is responsible for skin sensation. The effects of IGF-1 might be through ERK1/2 or PI3 K/Akt signaling pathways. These findings provide experimental evidence for IGF-1 administration to alleviate neurotoxicity of distinct subpopulations of DRG neurons induced by PT.

Background: The survival of motor neurons is dependent upon neurotrophic factors both during childhood and adolescence and during adult life. In disease conditions, such as in patients with amyotrophic lateral sclerosis (ALS), the mRNA levels of trophic factors like brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor are downregulated. This was replicated in our in vivo experimental system following the injection of cerebral spinal fluid (CSF) of sporadic ALS (ALS-CSF) patients., Objective: To evaluate the protective role of BDNF in a model of sporadic ALS patients., Methods: The expressions of endogenous BDNF, its receptor TrkB, the enzyme choline acetyl transferase (ChAT), and phosphorylated neurofilaments were studied in NSC-34 cells. The calcium-buffering and proapoptotic effects were assessed by calbindin-D28K and caspase-3 expression, respectively., Results: ALS-CSF considerably depleted the endogenous BDNF protein, while its effect on IGF-1 and FGF-2 was inconsequential; this indirectly indicates a key role for BDNF in supporting motor neuronal survival. The exogenous supplementation of BDNF reversed autocrine expression; however, it may not be completely receptor mediated, as the TrkB levels were not restored. BDNF completely revived ChAT expression. It may inhibit apoptosis by restoring Ca2+ homeostasis, since caspase-3 and calbindin-D28K expression was back to normal. The organellar ultrastructural changes were only partially reversed., Conclusion: Our study provides evidence that BDNF supplementation ameliorates most but not all degenerative changes. The incomplete revival at the ultrastructural level signifies the requirement of factors other than BDNF for near-total protection of motor neurons, and, to an extent, it explains why only a partial success is achieved in clinical trials with BDNF in ALS patients., (© 2016 S. Karger AG, Basel.)

After spinal cord injury (SCI), there is an acute phase of alternatively activated (M2) macrophage infiltration, followed by a long-lasting phase of classically activated (M1) macrophage accumulation in the wound, which is believed to derail healing and compromize organ functions. Thus, agents which are able to modulate macrophage phenotypes may provide significant benefits to SCI patients. In the present study, we demonstrate that the niacin receptor HCA 2 is specifically expressed on the cell surface of M1 but not M2 macrophages. Treatment of M1 macrophages with niacin (300μM) resulted in down-regulation of the p65 NF-κB phosphorylation, associated with a marked decrease in the levels of M1 markers, including CD86, IL-12, and IL-6, and a significant increase in the expressions of M2 markers, such as CD206, IL-10, and IL-13, suggesting that niacin causes a shift of M1 to M2. Moreover, treatment of the M1-oligodendrocyte precursor cell (OPC) co-cultures with niacin markedly promoted the expression of myelin binding protein (MBP). After SCI in C57/BL6 mice for a week, a marked accumulation of M1 macrophages, which expressed HCA 2 receptor, was evident in the wound. Treatment of the SCI mice with niacin (100mg/kg) resulted in a dramatic decrease in the number of M1 macrophages and a significant increase in the number of M2 macrophages in the wound. This was associated with a robust inflammation resolution, attenuation of demyelination and neurofilament loss, and significant improvement of locomotor function. Thus, HCA 2 receptor may serve as a therapeutic target to promote post-SCI recovery., (Copyright © 2016. Published by Elsevier B.V.)

It has been previously described the presence of GnRH receptor in spinal cord neurons of rat embryos and adult rats. However, the functional role of these receptors has not been studied. In this work, the effect of GnRH on neurite outgrowth and cytoskeletal protein expression in cultured spinal cord neurons of rat embryos was analyzed. Specifically, neurofilaments of 68 and 200 kDa by immunoblot assays and spinophilin mRNA expression by RT-PCR. Results show that GnRH stimulates neurite outgrowth in addition to an increase in neurofilaments and spinophilin expression. These findings suggest that GnRH may play a role as neuromodulator in neuronal plasticity and that could be considered as a potential factor for neuronal regeneration in spinal cord injuries.

Reactive gliosis is an early pathological feature common to most neurodegenerative diseases, yet its regulation and impact remain poorly understood. Normally astrocytes maintain a critical homeostatic balance. After stress or injury they undergo rapid parainflammatory activation, characterized by hypertrophy, and increased polymerization of type III intermediate filaments (IFs), particularly glial fibrillary acidic protein and vimentin. However, the consequences of IF dynamics in the adult CNS remains unclear, and no pharmacologic tools have been available to target this mechanism in vivo. The mammalian retina is an accessible model to study the regulation of astrocyte stress responses, and their influence on retinal neuronal homeostasis. In particular, our work and others have implicated p38 mitogen-activated protein kinase (MAPK) signaling as a key regulator of glutamate recycling, antioxidant activity and cytokine secretion by astrocytes and related Müller glia, with potent influences on neighboring neurons. Here we report experiments with the small molecule inhibitor, withaferin A (WFA), to specifically block type III IF dynamics in vivo. WFA was administered in a model of metabolic retinal injury induced by kainic acid, and in combination with a recent model of debridement-induced astrocyte reactivity. We show that WFA specifically targets IFs and reduces astrocyte and Müller glial reactivity in vivo. Inhibition of glial IF polymerization blocked p38 MAPK-dependent secretion of TNF-α, resulting in markedly reduced neuronal apoptosis. To our knowledge this is the first study to demonstrate that pharmacologic inhibition of IF dynamics in reactive glia protects neurons in vivo.

Preovulatory granulosa cells express the low-molecular-mass MAP2D variant of microtubule-associated protein 2 (MAP2). Activation of the luteinizing hormone choriogonadotropin receptor by human choriogonadotropin (hCG) promotes dephosphorylation of MAP2D on Thr256 and Thr259. We sought to evaluate the association of MAP2D with the cytoskeleton, and the effect of hCG on this association. MAP2D partially colocalized, as assessed by confocal immunofluorescence microscopy, with the vimentin intermediate filament and microtubule cytoskeletons in naive cells. In vitro binding studies showed that MAP2D bound directly to vimentin and β-tubulin. Phosphorylation of recombinant MAP2D on Thr256 and Thr259, which mimics the phosphorylation status of MAP2D in naive cells, reduces binding of MAP2D to vimentin and tubulin by two- and three-fold, respectively. PKA-dependent phosphorylation of vimentin (Ser32 and Ser38) promoted binding of vimentin to MAP2D and increased contraction of granulosa cells with reorganization of vimentin filaments and MAP2D from the periphery into a thickened layer surrounding the nucleus and into prominent cellular extensions. Chemical disruption of vimentin filament organization increased progesterone production. Taken together, these results suggest that hCG-stimulated dephosphorylation of MAP2D at Thr256 and Thr259, phosphorylation of vimentin at Ser38 and Ser72, and the resulting enhanced binding of MAP2D to vimentin might contribute to the progesterone synthetic response required for ovulation., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Chronic acrylamide (ACR) exposure induces peripheral-central axonopathy in occupational workers and laboratory animals, but the underlying mechanisms remain unclear. In this study, we first investigated the effects of ACR on slow axonal transport of neurofilaments in cultured rat dorsal root ganglia (DRG) neurons through live-cell imaging approach. Then for the underlying mechanisms exploration, the protein level of neurofilament subunits, motor proteins kinesin and dynein, and dynamitin subunit of dynactin in DRG neurons were assessed by western blotting and the concentrations of ATP was detected using ATP Assay Kit. The results showed that ACR treatment results in a dose-dependent decrease of slow axonal transport of neurofilaments. Furthermore, ACR intoxication significantly increases the protein levels of the three neurofilament subunits (NF-L, NF-M, NF-H), kinesin, dynein, and dynamitin subunit of dynactin in DRG neurons. In addition, ATP level decreased significantly in ACR-treated DRG neurons. Our findings indicate that ACR exposure retards slow axonal transport of NF-M, and suggest that the increase of neurofilament cargoes, motor proteins, dynamitin of dynactin, and the inadequate ATP supply contribute to the ACR-induced retardation of slow axonal transport.

Methyl viologen is a highly effective contact herbicide, a neurotoxic compound and an inducer of reactive oxygen species, which generate oxidative stress in cells. Reactive oxygen species has been known to function as an important messenger in cell differentiation. In this study, we investigated the effect of methyl viologen on neural cell differentiation using pluripotent mouse embryonal carcinoma P19 cells, which reportedly differentiate into neural cells upon exposure to retinoic acid. Methyl viologen, an inducer of intracellular reactive oxygen species, induced the differentiation of P19 cells into neural cells in the presence of neurofilament. Reduced glutathione, an eliminator of reactive oxygen species, also induced neural differentiation in P19 cells. These results suggest that P19 cells differentiate into neural cells, conceivably independent of intracellular reactive oxygen species.

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E2) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E2 (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K(+) depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E2 treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells' population growth by inducing maturation and differentiation.

The ubiquitous α-catulin acts as a scaffold for distinct signalosomes including RhoA/ROCK; however, its function is not well understood. While α-catulin has homology to the cytoskeletal linkers α-catenin and vinculin, it appears to be functionally divergent. Here we further investigated α-catulin function in pulmonary vascular endothelial cells (VEC) on the premise that α-catulin has a unique cytoskeletal role. Examination of endogenous α-catulin intracellular localization by immunofluorescence revealed a highly organized cytosolic filamentous network suggestive of a cytoskeletal system in a variety of cultured VEC. Double-immunofluorescence analyses of VEC showed endogenous α-catulin co-localization with vimentin intermediate filaments. Similar to vimentin, α-catulin was found to distribute into detergent-soluble and -insoluble fractions. Treatment of VEC with withaferinA, an agent that targets vimentin filaments, disrupted the α-catulin network distribution and altered α-catulin solubility. Vimentin participates in cell migration, and withaferinA was found to inhibit VEC migration in vitro; similarly, α-catulin knock-down reduced VEC migration. Based on previous reports showing that ROCK modulates vimentin, we found that ROCK depletion attenuated VEC migration; furthermore, α-catulin depletion was shown to reduce ROCK-induced signaling. These findings indicate that α-catulin has a unique function in co-localization with vimentin filaments that contributes to VEC migration via a pathway that may involve ROCK signaling. J. Cell. Physiol. 231: 934-943, 2016. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

Spinal cord injury (SCI) often results in some form of paralysis. Recently, SCI therapy has been focused on preventing secondary injury to reduce both neuroinflammation and lesion size so that functional outcome after an SCI may be improved. Previous studies have shown that adenosine receptors (AR) are a major regulator of inflammation after an SCI. The current study was performed to examine the effect of caffeine, a pan-AR blocker, on spontaneous functional recovery after an SCI. Animals were assigned into 3 groups randomly, including sham, PBS and caffeine groups. The rat SCI was generated by an NYU impactor with a 10 g rod dropped from a 25 mm height at thoracic 9 spinal cord level. Caffeine and PBS were injected daily during the experiment period. Hind limb motor function was evaluated by the Basso, Beattie, Bresnahan (BBB) locomotor rating scale at 1 week and 4 weeks after the SCI. Spinal cord segments were collected after final behavior evaluation for morphological analysis. The tissue sparing was evaluated by luxol fast blue staining. Immunofluorescence stain was employed to assess astrocyte activation and neurofilament positioning, while microglia activation was examined by immunohistochemistry stain.The results showed that spontaneous functional recovery was blocked after the animals were subjected caffeine daily. Moreover, caffeine administration increased the demyelination area, promoted astrocyte and microglia activation and decreased the quantity of neurofilaments. These findings suggest that the neurotoxicity effect of caffeine may be associated with the inhibition of neural repair and the promotion of neuroinflammation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Butylbenzyl phthalate (BBP), a suspected endocrine disruptor, adversely affects male reproductive function. In this study, morphological alterations of prepubertal rat testes caused by single administration of BBP, were examined by light microscopy. Three-week-old male rats were given a single dose of 500 mg/kg BBP by oral gavage and sacrificed at 3, 12, and 24 h after administration. Histopathological examination revealed progressive detachment and sloughing of spermatogenic cells into the lumen, and a significant increase in the number of TUNEL-positive (apoptotic) spermatogenic cells in the treated groups, compared to the control. Semithin sections confirmed the apoptotic cells by their prominent basophilia, condensed chromatin, and shrunken cytoplasm, hallmarks of apoptotic cell death. Immunohistochemistry identified disruption of Sertoli cell vimentin and actin filaments in the treated groups. To elucidate the recovery effects of BBP, rats were treated in the same way and were sacrificed at D1-12h after administration. The apoptotic index returned to normal at D9. While, the testes revealed lower weight gain until D12. These results show for the first time that BBP induces collapse of vimentin filaments in Sertoli cells which may lead to disruption of Sertoli-spermatogenic cell physical interaction and induces spermatogenic cell apoptosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Vimentin expression correlates well with migratory and invasive potential of the carcinoma cells. The molecular mechanism by which vimentin regulates cell motility is not yet clear. Here, we addressed this issue by depleting vimentin in oral squamous cell carcinoma derived cell line. Vimentin knockdown cells showed enhanced adhesion and spreading to laminin-5. However, we found that they were less invasive as compared to the vector control cells. In addition, signaling associated with adhesion behavior of the cell was increased in vimentin knockdown clones. These findings suggest that the normal function of β4 integrin as mechanical adhesive device is enhanced upon vimentin downregulation. As a proof of principle, the compromised invasive potential of vimentin depleted cells could be rescued upon blocking with β4 integrin adhesion-blocking (ASC-8) antibody or downregulation of β4 integrin in vimentin knockdown background. Interestingly, plectin which associates with α6β4 integrin in the hemidesmosomes, was also found to be upregulated in vimentin knockdown clones. Furthermore, experiments on lysosome and proteasome inhibition revealed that perhaps vimentin regulates the turnover of β4 integrin and plectin. Moreover, an inverse association was observed between vimentin expression and β4 integrin in oral squamous cell carcinoma (OSCC). Collectively, our results show a novel role of vimentin in modulating cell motility by destabilizing β4 integrin-mediated adhesive interactions. Further, vimentin-β4 integrin together may prove to be useful markers for prognostication of human oral cancer., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Chrysin (CHR) is a natural flavonoid and is present in high concentration in honey, propolis and many plant extracts. The aim of the present study was to evaluate the effects of CHR to reduce cardiomyocyte apoptosis and loss of intermediate filaments in a mouse model of mitoxantrone cardiotoxicity. Morphology of the cardiomyocytes was determined by optic and transmission electron microscopy and biochemistry methods. The expression of Bcl-2, Bax and Caspase-3 were assessed by immunofluorecence. Tunel assay was used to assess apoptosis in cardiomyocytes. In addition, the distribution of desmin protein was evaluated using immunohistochemistry. Our results show that MTX treatment significantly increased serum levels of creatine kinase isoenzyme (CK-MB), indicator of cardiac injury and withdrawn under CHR protection. Expression levels of Bcl-2 decreased, while those of Bax and caspase-3 increased following MTX treatment. 50 mg/kg of daily CHR intake reduced Bax and caspase-3 immunopositivity and restored Bcl-2 levels to a value comparable to the control. TUNEL (+) cardiomyocyte nuclei of MTX group showed typical signs of apoptosis which almost completely disappeared in response to 50 mg/kg CHR treatment. In parallel, an irregular distribution and a weak expression of desmin is associated with MTX induced cardiotoxic effects which was also restored by CHR treatment. In conclusion chrysin inhibits MTX-triggered cardiomyocyte apoptosis via multiple pathways, including decrease of the Bax/Bcl-2 ratio and caspase-3 expression along with preservation of the desmin disarray.

Ethanol exposure to offspring during pregnancy and lactation leads to developmental disorders, including central nervous system dysfunction. In the present work, we have studied the effect of chronic ethanol exposure during pregnancy and lactation on the phosphorylating system associated with the astrocytic and neuronal intermediate filament (IF) proteins: glial fibrillary acidic protein (GFAP), and neurofilament (NF) subunits of low, medium, and high molecular weight (NFL, NFM, and NFH, respectively) in 9- and 21-day-old pups. Female rats were fed with 20% ethanol in their drinking water during pregnancy and lactation. The homeostasis of the IF phosphorylation was not altered in the cerebral cortex, cerebellum, or hippocampus of 9-day-old pups. However, GFAP, NFL, and NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. PKA had been activated in the hippocampus, and Ser55 in the N-terminal region of NFL was hyperphosphorylated. In addition, JNK/MAPK was activated and KSP repeats in the C-terminal region of NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. Decreased NFH immunocontent but an unaltered total NFH/phosphoNFH ratio suggested altered stoichiometry of NFs in the hippocampus of ethanol-exposed 21-day-old pups. In contrast to the high susceptibility of hippocampal cytoskeleton in developing rats, the homeostasis of the cytoskeleton of ethanol-fed adult females was not altered. Disruption of the cytoskeletal homeostasis in neural cells supports the view that regions of the brain are differentially vulnerable to alcohol insult during pregnancy and lactation, suggesting that modulation of JNK/MAPK and PKA signaling cascades target the hippocampal cytoskeleton in a window of vulnerability in 21-day-old pups. Our findings are relevant, since disruption of the cytoskeleton in immature hippocampus could contribute to later hippocampal damage associated with ethanol toxicity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca²⁺-free CZB medium containing 10 mM SrCl₂) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion, and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF., (© 2014 by the Society for the Study of Reproduction, Inc.)

Most experiments of peripheral nerve repair after injury have been conducted in the rodent model but the translation of findings from rodent studies to clinical practice is needed partly because the nerve regeneration must occur over much longer distances in humans than in rodents. The reconstruction of long distance nerve injuries still represents a great challenge to surgeons who is engaged in peripheral nerve surgery. Here we used the functional nerve conduit (collagen scaffolds incorporated with neurocytokines CNTF and bFGF) to bridge a 35 mm long facial nerve gap in minipig models. At 6 months after surgery, electrophysiology assessment and histological examination were conducted to evaluate the regeneration of peripheral facial nerves. Based on functional and histological observations, the results indicated that the functional collagen scaffolds promoted nerve reconstruction. The number and arrangement of regenerated nerve fibers, myelination, and nerve function reconstruction was better in the CNTF + bFGF conduit group than the single factor CNTF or bFGF conduit group. The functional composite conduit, which exhibited favorable mechanical properties, may promote facial nerve regeneration in minipigs effectively., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases., (© FASEB.)

We report on the heterogeneity and diversity of lipid droplets (LDs) in early stages of adipogenesis by elucidating the cell and molecular biology of amphiphilic and cytoskeletal proteins regulating and stabilizing the generation of LDs in human adipose cells. A plethora of distinct and differently sized LDs was detected by a brief application of adipocyte differentiation medium and additional short treatment with oleic acid. Using these cells and highly specific antibodies for LD-binding proteins of the perilipin (PLIN) family, we could distinguish between endogenously derived LDs (endogenous LDs) positive for perilipin from exogenously induced LDs (exogenous LDs) positive for adipophilin, TIP47 and S3-12. Having optimized these stimulation conditions, we used early adipogenic differentiation stages to investigate small-sized LDs and concentrated on LD-protein associations with the intermediate-sized filament (IF) vimentin. This IF protein was described earlier to surround lipid globules, showing spherical, cage-like structures. Consequently - by biochemical methods, by immunofluorescence microscopy and by electron- and immunoelectron microscopy - various stages of emerging lipid globules were revealed with perilipin as linking protein between LDs and vimentin. For this LD-PLIN-Vimentin connection, a model is now proposed, suggesting an interaction of proteins via opposed charged amino acid domains respectively. In addition, multiple sheaths of smooth endoplasmic reticulum cisternae surrounding concentrically nascent LDs are shown. Based on our comprehensive localization studies we present and discuss a novel pathway for the LD formation.

Evidence from our group supports that diphenyl ditelluride (PhTe)2 neurotoxicity depends on modulation of signaling pathways initiated at the plasma membrane. The (PhTe)2-evoked signal is transduced downstream of voltage-dependent Ca(2+) channels (VDCC), N-methyl-D-aspartate receptors (NMDA), or metabotropic glutamate receptors activation via different kinase pathways (protein kinase A, phospholipase C/protein kinase C, mitogen-activated protein kinases (MAPKs), and Akt signaling pathway). Among the most relevant cues of misregulated signaling mechanisms evoked by (PhTe)2 is the cytoskeleton of neural cells. The in vivo and in vitro exposure to (PhTe)2 induce hyperphosphorylation/hypophosphorylation of neuronal and glial intermediate filament (IF) proteins (neurofilaments and glial fibrillary acidic protein, resp.) in different brain structures of young rats. Phosphorylation of IFs at specific sites modulates their association/disassociation and interferes with important physiological roles, such as axonal transport. Disrupted cytoskeleton is a crucial marker of neurodegeneration and is associated with reactive astrogliosis and apoptotic cell death. This review focuses the current knowledge and important results on the mechanisms of (PhTe)2 neurotoxicity with special emphasis on the cytoskeletal proteins and their differential regulation by kinases/phosphatases and Ca(2+)-mediated mechanisms in developmental rat brain. We propose that the disrupted cytoskeletal homeostasis could support brain damage provoked by this neurotoxicant.

Lipid droplets (LDs) are spherical accumulations of apolar lipids and other hydrophobic substances and are generally surrounded by a thin cortical layer of specific amphiphilic proteins (APs). These APs segregate the LDs from the mostly polar components of the cytoplasm. We have studied LDs in epithelium-derived cell cultures and in particular characterized proteins from the perilipin (PLIN) gene family - in mammals consisting of the proteins Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5). Using a large number of newly generated and highly specific mono- and polyclonal antibodies specific for individual APs, and using improved LD isolation methods, we have enriched and characterized APs in greater detail and purity. The majority of lipid-AP complexes could be obtained in the top layer fractions of density gradient centrifugation separations of cultured cells, but APs could also be detected in other fractions within such separations. The differently sized LD complexes were analyzed using various biochemical methods and mass spectrometry as well as immunofluorescence and electron- in particular immunoelectron-microscopy. Moreover, by immunoprecipitation, protein-protein binding assays and by immunoelectron microscopy we identified a direct linkage between LD-binding proteins and the intermediate-sized filaments (IF) cytokeratins 8 and 18 (also designated as keratins K8 and K18). Specifically, in gradient fractions of higher density supposedly containing small LDs, we received as co-precipitations cytidylyl-, palmitoyl- and cholesterol transferases and other specific enzymes involved in lipid metabolism. So far, common proteomic studies have used LDs from top layer fractions only and did not report on these transferases and other enzymes. In addition to findings of short alternating hydrophobic/hydrophilic segments within the PLIN protein family, we propose and discuss a model for the interaction of LD-coating APs with IF proteins.

Toxic carbonyls such as acrolein participate in many degenerative diseases. Although the nucleophilic vasodilatory drug hydralazine readily traps such species under "test-tube" conditions, whether these reactions adequately explain its efficacy in animal models of carbonyl-mediated disease is uncertain. We have previously shown that hydralazine attacks carbonyl-adducted proteins in an "adduct-trapping" reaction that appears to take precedence over direct "carbonyl-sequestering" reactions, but how this reaction conferred cytoprotection was unclear. This study explored the possibility that by increasing the bulkiness of acrolein-adducted proteins, adduct-trapping might alter the redistribution of chaperones to damaged cytoskeletal proteins that are known targets for acrolein. Using A549 lung adenocarcinoma cells, the levels of chaperones heat shock protein (Hsp) 40, Hsp70, Hsp90, and Hsp110 were measured in intermediate filament extracts prepared after a 3-h exposure to acrolein. Exposure to acrolein alone modestly increased the levels of all four chaperones. Coexposure to hydralazine (10-100 μM) strongly suppressed cell ATP loss while producing strong adduct-trapping in intermediate filaments. Most strikingly, hydralazine selectively boosted the levels of cytoskeletal-associated Hsp90, including a high-mass species that was sensitive to the Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin. Biochemical fractionation of acrolein- and hydralazine-treated cells revealed that hydralazine likely promoted Hsp90 migration from cytosol into other subcellular compartments. A role for Hsp90 mobilization in cytoprotection was confirmed by the finding that brief heat shock treatment suppressed acute acrolein toxicity in A549 cells. Taken together, these findings suggest that by increasing the steric bulk of carbonyl-adducted proteins, adduct-trapping drugs trigger the intracellular mobilization of the key molecular chaperone Hsp90.