Search

We recently demonstrated that acute oral ketone monoester intake induces a stimulation of postprandial myofibrillar protein synthesis rates comparable to that elicited following the ingestion of 10 g whey protein or their coingestion. The present investigation aimed to determine the acute effects of ingesting a ketone monoester, whey protein, or their coingestion on mechanistic target of rapamycin (mTOR)-related protein-protein colocalization and intracellular trafficking in human skeletal muscle. In a randomized, double-blind, parallel group design, 36 healthy recreationally active young males (age: 24.2 ± 4.1 yr) ingested either: 1 ) 0.36 g·kg -1 bodyweight of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 2 ) 10 g whey protein (PRO), or 3 ) the combination of both (KET + PRO). Muscle biopsies were obtained in the overnight postabsorptive state (basal conditions), and at 120 and 300 min in the postprandial period for immunofluorescence assessment of protein translocation and colocalization of mTOR-related signaling molecules. All treatments resulted in a significant (Interaction: P < 0.0001) decrease in tuberous sclerosis complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) colocalization at 120 min versus basal; however, the decrease was sustained at 300 min versus basal ( P < 0.0001) only in KET + PRO. PRO and KET + PRO increased (Interaction: P < 0.0001) mTOR-Rheb colocalization at 120 min versus basal; however, KET + PRO resulted in a sustained increase in mTOR-Rheb colocalization at 300 min that was greater than KET and PRO. Treatment intake increased mTOR-wheat germ agglutinin (WGA) colocalization at 120 and 300 min (Time: P = 0.0031), suggesting translocation toward the fiber periphery. These findings demonstrate that ketone monoester intake can influence the spatial mechanisms involved in the regulation of mTORC1 in human skeletal muscle. NEW & NOTEWORTHY We explored the effects of a ketone monoester (KET), whey protein (PRO), or their coingestion (KET + PRO) on mTOR-related protein-protein colocalization and intracellular trafficking in human muscle. All treatments decreased TSC2-Rheb colocalization at 120 minutes; however, KET + PRO sustained the decrease at 300 min. Only PRO and KET + PRO increased mTOR-Rheb colocalization; however, the increase at 300 min was greater in KET + PRO. Treatment intake increased mTOR-WGA colocalization, suggesting translocation to the fiber periphery. Ketone bodies influence the spatial regulation of mTOR.

Autosomal Recessive Spastic Ataxia of the Charlevoix Saguenay (ARSACS) is caused by mutation in the SACS gene resulting in loss of function of the protein sacsin. A key feature is the formation of abnormal bundles of neurofilaments (NF) in neurons and vimentin intermediate filaments (IF) in cultured fibroblasts, suggesting a role of sacsin in IF homeostasis. Sacsin contains a J domain (SacsJ) homologous to Hsp40, that can interact with Hsp70 chaperones. The SacsJ domain resolved NF bundles in cultured Sacs -/- neurons. Having studied the mechanism using NF assembled in vitro from purified NF proteins, we report that the SacsJ domain interacts with NF proteins to disassemble NFL filaments, and to inhibit their initial assembly. A cell-penetrating peptide derived from this domain, SacsJ-myc-TAT was efficient in disassembling NF bundles in cultured Sacs -/- motor neurons, restoring the NF network; however, there was some loss of vimentin IF and NF in cultured Sacs +/+ fibroblasts and motor neurons, respectively. These results suggest that sacsin through its SacsJ domain is a key regulator of NF and vimentin IF networks in cells.

Mutations in the HADHB gene lead to Mitochondrial Trifunctional Protein (MTP) deficiency. MTP deficiency is a rare autosomal recessive disorder affecting long-chain fatty acid oxidation. Patients affected by MTP deficiency are unable to metabolize long-chain fatty-acids and suffer a variety of symptoms exacerbated during fasting. The three phenotypes associated with complete MTP deficiency are an early-onset cardiomyopathy and early death, an intermediate form with recurrent hypoketotic hypoglycemia and a sensorimotor neuropathy with episodic rhabdomyolysis with small amount of residual enzyme activities. This review aims to discuss the pathophysiological mechanisms and clinical manifestations of each phenotype, which appears different and linked to HADHB expression levels. Notably, the pathophysiology of the sensorimotor neuropathy is relatively unknown and we provide a hypothesis on the qualitative aspect of the role of acylcarnitine buildup in Schwann cells in MTP deficiency patients. We propose that acylcarnitine may exit the Schwann cell and alter membrane properties of nearby axons leading to axonal degeneration based on recent findings in different metabolic disorders., Competing Interests: Declaration of Competing Interest The authors have no relevant conflicts of interest to disclose., (Copyright © 2021. Published by Elsevier Inc.)

In mammals, adipose tissue is an active secretory tissue that responds to mild hypothermia and as such is a genuine model to study molecular and cellular adaptive responses to cold-stress. A recent study identified a mammal-specific protein of the endoplasmic reticulum that is strongly induced in the inguinal subcutaneous white adipocyte upon exposure to cold, calsyntenin 3β (CLSTN3β). CLSTN3β regulates sympathetic innervation of thermogenic adipocytes and contributes to adaptive non-shivering thermogenesis. The calcium- and zinc-binding S100B is a downstream effector in the CLSTN3β pathways. We review, here, the literature on the transcriptional regulation of the S100b gene in adipocyte cells. We also rationalize the interactions of the S100B protein with its recognized or hypothesized intracellular (p53, ATAD3A, CYP2E1, AHNAK) and extracellular (Receptor for Advanced Glycation End products (RAGE), RPTPσ) target proteins in the context of adipocyte differentiation and adaptive thermogenesis. We highlight a chaperon-associated function for the intracellular S100B and point to functional synergies between the different intracellular S100B target proteins. A model of non-classical S100B secretion involving AHNAK/S100A10/annexin2-dependent exocytosis by the mean of exosomes is also proposed. Implications for related areas of research are noted and suggestions for future research are offered.

Neurofilaments (NFs), a major cytoskeletal component of motor neurons, play a key role in the differentiation, establishment and maintenance of their morphology and mechanical strength. The de novo assembly of these neuronal intermediate filaments requires the presence of the neurofilament light subunit (NEFL), whose expression is reduced in motor neurons in amyotrophic lateral sclerosis (ALS). This study used zebrafish as a model to characterize the NEFL homologue neflb , which encodes two different isoforms via a splicing of the primary transcript ( neflbE4 and neflbE3 ). In vivo imaging showed that neflb is crucial for proper neuronal development, and that disrupting the balance between its two isoforms specifically affects the NF assembly and motor axon growth, with resultant motor deficits. This equilibrium is also disrupted upon the partial depletion of TDP-43 (TAR DNA-binding protein 43), an RNA-binding protein encoded by the gene TARDBP that is mislocalized into cytoplasmic inclusions in ALS. The study supports the interaction of the NEFL expression and splicing with TDP-43 in a common pathway, both biologically and pathogenetically.

Upregulation of heat shock proteins (HSPs) is an approach to treatment of neurodegenerative disorders with impaired proteostasis. Many neurons, including motor neurons affected in amyotrophic lateral sclerosis (ALS), are relatively resistant to stress-induced upregulation of HSPs. This study demonstrated that histone deacetylase (HDAC) inhibitors enable the heat shock response in cultured spinal motor neurons, in a stress-dependent manner, and can improve the efficacy of HSP-inducing drugs in murine spinal cord cultures subjected to thermal or proteotoxic stress. The effect of particular HDAC inhibitors differed with the stress paradigm. The HDAC6 (class IIb) inhibitor, tubastatin A, acted as a co-inducer of Hsp70 (HSPA1A) expression with heat shock, but not with proteotoxic stress induced by expression of mutant SOD1 linked to familial ALS. Certain HDAC class I inhibitors (the pan inhibitor, SAHA, or the HDAC1/3 inhibitor, RGFP109) were HSP co-inducers comparable to the hydroxyamine arimoclomol in response to proteotoxic stress, but not thermal stress. Regardless, stress-induced Hsp70 expression could be enhanced by combining an HDAC inhibitor with either arimoclomol or with an HSP90 inhibitor that constitutively induced HSPs. HDAC inhibition failed to induce Hsp70 in motor neurons expressing ALS-linked mutant FUS, in which the heat shock response was suppressed; yet SAHA, RGFP109, and arimoclomol did reduce loss of nuclear FUS, a disease hallmark, and HDAC inhibition rescued the DNA repair response in iPSC-derived motor neurons carrying the FUS P525L mutation, pointing to multiple mechanisms of neuroprotection by both HDAC inhibiting drugs and arimoclomol.

Loss of sacsin, a large 520 kDa multidomain protein, causes autosomal recessive spastic ataxia of the Charlevoix-Saguenay, one of the most common childhood-onset recessive ataxias. A prominent feature is abnormal bundling of neurofilaments in many neuronal populations. This study shows the direct involvement of sacsin domains in regulating intermediate filament assembly and dynamics and identifies important domains for alleviating neurofilament bundles in neurons lacking sacsin. Peptides encoding sacsin internal repeat (SIRPT) 1, J-domains, and ubiquitin-like domain modified neurofilament assembly in vivo. The domains with chaperone homology, the SIRPT and the J-domain, had opposite effects, promoting and preventing filament assembly, respectively. In cultured Sacs -/- motor neurons, both the SIRPT1 and J-domain resolved preexisting neurofilament bundles. Increasing expression of heat shock proteins also resolved neurofilament bundles, indicating that this endogenous chaperone system can compensate to some extent for sacsin deficiency.-Gentil, B. J., Lai, G.-T., Menade, M., Larivière, R., Minotti, S., Gehring, K., Chapple, J.-P., Brais, B., Durham, H. D. Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease with paralysis resulting from dysfunction and loss of motor neurons. A common neuropathological finding is attrition of motor neuron dendrites, which make central connections vital to motor control. The chromatin remodelling complex, neuronal Brahma-related gene 1 (Brg1)-associated factor complex (nBAF), is critical for neuronal differentiation, dendritic extension and synaptic function. We have identified loss of the crucial nBAF subunits Brg1, Brg1-associated factor 53b and calcium responsive transactivator in cultured motor neurons expressing FUS or TAR-DNA Binding Protein 43 (TDP-43) mutants linked to familial ALS. When plasmids encoding wild-type or mutant human FUS or TDP-43 were expressed in motor neurons of dissociated spinal cord cultures prepared from E13 mice, mutant proteins in particular accumulated in the cytoplasm. Immunolabelling of nBAF subunits was reduced in proportion to loss of nuclear FUS or TDP-43 and depletion of Brg1 was associated with nuclear retention of Brg1 mRNA. Dendritic attrition (loss of intermediate and terminal dendritic branches) occurred in motor neurons expressing mutant, but not wild-type, FUS or TDP-43. This attrition was delayed by ectopic over-expression of Brg1 and was reproduced by inhibiting Brg1 activity either through genetic manipulation or treatment with the chemical inhibitor, (E)-1-(2-Hydroxyphenyl)-3-((1R, 4R)-5-(pyridin-2-yl)-2, 5-diazabicyclo[2.2.1]heptan-2-yl)prop-2-en-1-one, demonstrating the importance of Brg1 to maintenance of dendritic architecture. Loss of nBAF subunits was also documented in spinal motor neurons in autopsy tissue from familial amyotrophic sclerosis (chromosome 9 open reading frame 72 with G4C2 nucleotide expansion) and from sporadic cases with no identified mutation, pointing to dysfunction of nBAF chromatin remodelling in multiple forms of ALS., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mutations in FIG4, coding for a phosphoinositol(3,5) bisphosphate 5' phosphatase and involved in vesicular trafficking and fusion, have been shown causing a recessive form of Charcot-Marie-Tooth (CMT). We have identified a novel intronic mutation in the FIG4 in a wheel-chair bound patient presenting with a severe form of CMT4J and provide a longitudinal study. Investigations indicated a demyelinating sensorimotor polyneuropathy with diffuse active denervation and severe axonal loss. Genetic testing revealed that the patient is heterozygous for 2 FIG4 mutations, p.I41T and a T > G transversion at IVS17-10, the latter predicted to cause a splicing defect. FIG4 was severely diminished in patient's fibroblasts indicating loss-of-function. Consistent with FIG4's function in phosphoinositol homeostasis and vesicular trafficking, fibroblasts contained multiple large vacuoles and vesicular organelles were abnormally dispersed. FIG4 deficiency has implications for turnover of membrane proteins. The transient receptor cation channel, TRPV4, accumulated at the plasma membrane of patient's fibroblasts due to slow turnover. Knocking down Fig4 in murine cultured motor neurons resulted in vacuolation and cell death. Inhibiting TRPV4 activity significantly preserved viability, although not correcting vesicular trafficking. In conclusion, we demonstrate a new FIG4 intronic mutation and, importantly, a functional interaction between FIG4 and TRPV4., (© 2017 American Association of Neuropathologists, Inc. All rights reserved.)

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the gene SACS, encoding the 520 kDa protein sacsin. Although sacsin's physiological role is largely unknown, its sequence domains suggest a molecular chaperone or protein quality control function. Consequences of its loss include neurofilament network abnormalities, specifically accumulation and bundling of perikaryal and dendritic neurofilaments. To investigate if loss of sacsin affects intermediate filaments more generally, the distribution of vimentin was analysed in ARSACS patient fibroblasts and in cells where sacsin expression was reduced. Abnormal perinuclear accumulation of vimentin filaments, which sometimes had a cage-like appearance, occurred in sacsin-deficient cells. Mitochondria and other organelles were displaced to the periphery of vimentin accumulations. Reorganization of the vimentin network occurs in vitro under stress conditions, including when misfolded proteins accumulate. In ARSACS patient fibroblasts HSP70, ubiquitin and the autophagy-lysosome pathway proteins Lamp2 and p62 relocalized to the area of the vimentin accumulation. There was no overall increase in ubiquitinated proteins, suggesting the ubiquitin-proteasome system was not impaired. There was evidence for alterations in the autophagy-lysosome pathway. Specifically, in ARSACS HDFs cellular levels of Lamp2 were elevated while levels of p62, which is degraded in autophagy, were decreased. Moreover, autophagic flux was increased in ARSACS HDFs under starvation conditions. These data show that loss of sacsin effects the organization of intermediate filaments in multiple cell types, which impacts the cellular distribution of other organelles and influences autophagic activity., (© The Author 2017. Published by Oxford University Press.)

Neurons are extremely polarised cells in which the cytoskeleton, composed of microtubules, microfilaments and neurofilaments, plays a crucial role in maintaining structure and function. Neurofilaments, the 10-nm intermediate filaments of neurons, provide structure and mechanoresistance but also provide a scaffolding for the organization of the nucleus and organelles such as mitochondria and ER. Disruption of neurofilament organization and expression or metabolism of neurofilament proteins is characteristic of certain neurological syndromes including Amyotrophic Lateral Sclerosis, Charcot-Marie-Tooth sensorimotor neuropathies and Giant Axonal Neuropathy. Microfluorometric live imaging techniques have been instrumental in revealing the dynamics of neurofilament assembly and transport and their functions in organizing intracellular organelle networks. The insolubility of neurofilament proteins has limited identifying interactors by conventional biochemical techniques but yeast two-hybrid experiments have revealed new roles for oligomeric, nonfilamentous structures including vesicular trafficking. Although having long half-lives, new evidence points to degradation of subunits by the ubiquitin-proteasome system as a mechanism of normal turnover. Although certain E3-ligases ubiquitinating neurofilament proteins have been identified, the overall process of neurofilament degradation is not well understood. We review these mechanisms of neurofilament homeostasis and abnormalities in motor neuron and peripheral nerve disorders. Much remains to discover about the disruption of processes that leads to their pathological aggregation and accumulation and the relevance to pathogenesis. Understanding these mechanisms is crucial for identifying novel therapeutic strategies.

The diaphragm is a unique skeletal muscle designed to be rhythmically active throughout life, such that its sustained inactivation by the medical intervention of mechanical ventilation (MV) represents an unanticipated physiological state in evolutionary terms. Within a short period after initiating MV, the diaphragm develops muscle atrophy, damage, and diminished strength, and many of these features appear to arise from mitochondrial dysfunction. Notably, in response to metabolic perturbations, mitochondria fuse, divide, and interact with neighboring organelles to remodel their shape and functional properties-a process collectively known as mitochondrial dynamics. Using a quantitative electron microscopy approach, here we show that diaphragm contractile inactivity induced by 6 h of MV in mice leads to fragmentation of intermyofibrillar (IMF) but not subsarcolemmal (SS) mitochondria. Furthermore, physical interactions between adjacent organellar membranes were less abundant in IMF mitochondria during MV. The profusion proteins Mfn2 and OPA1 were unchanged, whereas abundance and activation status of the profission protein Drp1 were increased in the diaphragm following MV. Overall, our results suggest that mitochondrial morphological abnormalities characterized by excessive fission-fragmentation represent early events during MV, which could potentially contribute to the rapid onset of mitochondrial dysfunction, maladaptive signaling, and associated contractile dysfunction of the diaphragm., (Copyright © 2015 the American Physiological Society.)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS [MIM 270550]) is an early-onset neurodegenerative disorder caused by mutations in the SACS gene. Over 170 SACS mutations have been reported worldwide and are thought to cause loss of function of sacsin, a poorly characterized and massive 520 kDa protein. To establish an animal model and to examine the pathophysiological basis of ARSACS, we generated Sacs knockout (Sacs(-/-)) mice. Null animals displayed an abnormal gait with progressive motor, cerebellar and peripheral nerve dysfunctions highly reminiscent of ARSACS. These clinical features were accompanied by an early onset, progressive loss of cerebellar Purkinje cells followed by spinal motor neuron loss and peripheral neuropathy. Importantly, loss of sacsin function resulted in abnormal accumulation of non-phosphorylated neurofilament (NF) bundles in the somatodendritic regions of vulnerable neuronal populations, a phenotype also observed in an ARSACS brain. Moreover, motor neurons cultured from Sacs(-/-) embryos exhibited a similar NF rearrangement with significant reduction in mitochondrial motility and elongated mitochondria. The data points to alterations in the NF cytoskeleton and defects in mitochondrial dynamics as the underlying pathophysiological basis of ARSACS., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Peripherin is a type III intermediate filament protein, the expression of which is associated with the acquisition and maintenance of a terminally differentiated neuronal phenotype. Peripherin up-regulation occurs during acute neuronal injury and in degenerating motor neurons of amyotrophic lateral sclerosis. The functional role(s) of peripherin during normal, injurious, and disease conditions remains unknown, but may be related to differential expression of spliced isoforms. To better understand peripherin function, we performed a yeast two-hybrid screen on a mouse brain cDNA library using an assembly incompetent peripherin isoform, Per-61, as bait. We identified new peripherin interactors with roles in vesicular trafficking, signal transduction, DNA/RNA processing, protein folding, and mitochondrial metabolism. We focused on the interaction of Per-61 and the constitutive isoform, Per-58, with SNAP25 interacting protein 30 (SIP30), a neuronal protein involved in SNAP receptor-dependent exocytosis. We found that peripherin and SIP30 interacted through coiled-coil domains and colocalized in cytoplasmic aggregates in SW13vim(-) cells. Interestingly, Per-61 and Per-58 differentially altered the subcellular distribution of SIP30 and SNAP25 in primary motor neurons. Our findings suggest a novel role of peripherin in vesicle trafficking., (© 2014 International Society for Neurochemistry.)

Excitotoxicity and disruption of Ca(2+) homeostasis have been implicated in amyotrophic lateral sclerosis (ALS) and limiting Ca(2+) entry is protective in models of ALS caused by mutation of SOD1. Lomerizine, an antagonist of L- and T-type voltage-gated calcium channels and transient receptor potential channel 5 transient receptor potential channels, is well tolerated clinically, making it a potential therapeutic candidate. Lomerizine reduced glutamate excitotoxicity in cultured motor neurons by reducing the accumulation of cytoplasmic Ca(2+) and protected motor neurons against multiple measures of mutant SOD1 toxicity: Ca(2+) overload, impaired mitochondrial trafficking, mitochondrial fragmentation, formation of mutant SOD1 inclusions, and loss of viability. To assess the utility of lomerizine in other forms of ALS, calcium homeostasis was evaluated in culture models of disease because of mutations in the RNA-binding proteins transactive response DNA-binding protein 43 (TDP-43) and Fused in Sarcoma (FUS). Calcium did not play the same role in the toxicity of these mutant proteins as with mutant SOD1 and lomerizine failed to prevent cytoplasmic accumulation of mutant TDP-43, a hallmark of its pathology. These experiments point to differences in the pathogenic pathways between types of ALS and show the utility of primary culture models in comparing those mechanisms and effectiveness of therapeutic strategies., (© 2014 International Society for Neurochemistry.)

Heat shock proteins (HSPs) are attractive therapeutic targets for neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), characterized by aberrant formation of protein aggregates. Although motor neurons have a high threshold for activation of HSP genes, HSP90 inhibitors are effective inducers. This study evaluated NXD30001, a novel, small molecule HSP90 inhibitor based on the radicicol backbone, for its ability to induce neuronal HSPs and for efficacy in an experimental model of ALS based on mutations in superoxide-dismutase 1 (SOD1). In motor neurons of dissociated murine spinal cord cultures, NXD30001-induced expression of HSP70/HSPA1 (iHSP70) and its co-chaperone HSP40/DNAJ through activation of HSF1 and exhibited a protective profile against SOD1(G93A) similar to geldanamycin, but with less toxicity. Treatment prevented protein aggregation, mitochondrial fragmentation, and motor neuron death, important features of mutant SOD1 toxicity, but did not effectively prevent aberrant intracellular Ca(2+) accumulation. NXD30001 distributed to brain and spinal cord of wild-type and SOD1(G93A) transgenic mice following intraperitoneal injection; however, unlike in culture, in vivo levels of SOD1 were not reduced. NXD30001-induced expression of iHSP70 in skeletal and cardiac muscle and, to a lesser extent, in kidney, but not in liver, spinal cord, or brain, with either single or repeated administration. NXD30001 is a very useful experimental tool in culture, but these data point to the complex nature of HSP gene regulation in vivo and the necessity for early evaluation of the efficacy of novel HSP inducers in target tissues in vivo.

Clusterin, a protein chaperone found at high levels in physiological fluids, is expressed in nervous tissue and upregulated in several neurological diseases. To assess relevance to amyotrophic lateral sclerosis (ALS) and other motor neuron disorders, clusterin expression was evaluated using long-term dissociated cultures of murine spinal cord and SOD1(G93A) transgenic mice, a model of familial ALS. Motor neurons and astrocytes constitutively expressed nuclear and cytoplasmic forms of clusterin, and secreted clusterin accumulated in culture media. Although clusterin can be stress inducible, heat shock failed to increase levels in these neural cell compartments despite robust upregulation of stress-inducible Hsp70 (HspA1) in non-neuronal cells. In common with HSPs, clusterin was upregulated by treatment with the Hsp90 inhibitor, geldanamycin, and thus could contribute to the neuroprotection previously identified for such compounds in disease models. Clusterin expression was not altered in cultured motor neurons expressing SOD1(G93A) by gene transfer or in presymptomatic SOD1(G93A) transgenic mice; however, clusterin immunolabeling was weakly increased in lumbar spinal cord of overtly symptomatic mice. More striking, mutant SOD1 inclusions, a pathological hallmark, were strongly labeled by anti-clusterin. Since secreted, as well as intracellular, mutant SOD1 contributes to toxicity, the extracellular chaperoning property of clusterin could be important for folding and clearance of SOD1 and other misfolded proteins in the extracellular space. Evaluation of chaperone-based therapies should include evaluation of clusterin as well as HSPs, using experimental models that replicate the control mechanisms operant in the cells and tissue of interest.

A unique property of mitochondria in mammalian cells is their ability to physically interact and undergo dynamic events of fusion/fission that remodel their morphology and possibly their function. In cultured cells, metabolic perturbations similar to those incurred during exercise influence mitochondrial fusion and fission processes, but it is unknown whether exercise acutely alters mitochondrial morphology and/or membrane interactions in vivo. To study this question, we subjected mice to a 3-h voluntarily exercise intervention following their normal physical activity patterns, and quantified mitochondrial morphology and membrane interactions in the soleus using a quantitative electron microscopy approach. A single exercise bout effectively decreased blood glucose (P < 0.05) and intramyocellular lipid content (P < 0.01), indicating increased muscle metabolic demand. The number of mitochondria spanning Z-lines and proportion of electron-dense contact sites (EDCS) between adjacent mitochondrial membranes were increased immediately after exercise among both subsarcolemmal (+116%, P < 0.05) and intermyofibrillar mitochondria (+191%, P < 0.001), indicating increased physical interactions. Mitochondrial morphology, and abundance of the mitochondrial pro-fusion proteins Mfn2 and OPA1 were unchanged. Collectively, these results support the notion that mitochondrial membrane dynamics are actively remodelled in skeletal muscle, which may be regulated by contractile activity and the metabolic state. Future studies are required to understand the implications of mitochondrial dynamics in skeletal muscle physiology during exercise and inactivity.

Aberrant aggregation of neurofilament proteins is a common feature of neurodegenerative diseases. For example, neurofilament light protein (NEFL) mutants causing Charcot-Marie-Tooth disease induce misassembly of neurofilaments. This study demonstrated that mutations in different functional domains of NEFL have different effects on filament assembly and susceptibility to interventions to restore function. The mouse NEFL mutants, NEFL(Q333P) and NEFL(P8R), exhibited different assembly properties in SW13-cells, cells lacking endogenous intermediate filaments, indicating different consequences of these mutations on the biochemical properties of NEFL. The p.Q333P mutation caused reversible misfolding of the protein. NEFL(Q333P) could be refolded and form coil-coiled dimers, in vitro using chaotropic agent, and in cultured cells by induction of HSPA1 and HSPB1. Celastrol, an inducer of chaperone proteins, induced HSPA1 expression in motor neurons and prevented the formation of neurofilament inclusions and mitochondrial shortening induced by expression of NEFL(Q333P), but not in sensory neurons. Conversely, celastrol had a protective effect against the toxicity of NEFL(P8R), a mutant which is sensitive to HSBP1 but not HSPA1 chaperoning, only in large-sized sensory neurons, not in motor neurons. Importantly, sensory and motor neurons do not respond identically to celastrol and different chaperones are upregulated by the same treatment. Thus, effective therapy of CMT not only depends on the identity of the mutated gene, but the consequences of the specific mutation on the properties of the protein and the neuronal population targeted., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In response to cellular and environmental stresses, mitochondria undergo morphology transitions regulated by dynamic processes of membrane fusion and fission. These events of mitochondrial dynamics are central regulators of cellular activity, but the mechanisms linking mitochondrial shape to cell function remain unclear. One possibility evaluated in this review is that mitochondrial morphological transitions (from elongated to fragmented, and vice-versa) directly modify canonical aspects of the organelle's function, including susceptibility to mitochondrial permeability transition, respiratory properties of the electron transport chain, and reactive oxygen species production. Because outputs derived from mitochondrial metabolism are linked to defined cellular signaling pathways, fusion/fission morphology transitions could regulate mitochondrial function and retrograde signaling. This is hypothesized to provide a dynamic interface between the cell, its genome, and the fluctuating metabolic environment.

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. It comprises a group of diseases caused by mutations in genes involved in Schwann cells homeostasis and neuronal function that affect the peripheral nerves. So far mutations in more than 33 genes have been identified causing either the demyelinating form (CMT1) or the axonal form (CMT2). Genes involving a large variety of unrelated functions may lead to the same phenotype when mutated. Our review will focus on the common link between genes causing axonal phenotypes like MFN2, KIF1B, DYNC1H1, Rab7, TRPV4, ARSs, NEFL, HSPB1, MPZ, and HSPB8. While KIF1B and DYNC1H1, two genes coding for molecular motors, are directly linked to axonal transport, the involvement of the other CMT2-causing genes in this function is less obvious. However, the last years have seen a growing list of evidence demonstrating that intracellular trafficking and mitochondrial dynamics might be dysfunctional in CMT2, and these mechanisms might present a common link between dissimilar CMT2-causing genes. The involvement of impaired transport in the pathogenesis of other rare neurological diseases or recessive CMT2 is also discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Intermediate filaments serve important structural roles, but other cellular functions are increasingly recognized. This study demonstrated normal function of the low-molecular-weight neurofilament protein (NFL) in mitochondrial dynamics and disruption in Charcot-Marie-Tooth disease (CMT) due to mutations in the Nefl gene. In motor neurons of spinal cord cultured from Nefl-knockout mice, mitochondrial length and the rate of fusion were decreased concomitant with increased motility. These parameters were normalized after expression of NFL(wt) on the Nefl(-/-) background, but not by overexpression of the profusion protein, mitofusin 2 (MFN2). The effects of CMT-causing NFL mutants bore similarities to and differences from Nefl knockout. In the early phase of toxicity before disruption of the neurofilament network, NFL(Q333P) and NFL(P8R) integrated into neurofilaments and had effects on mitochondria similar to those with Nefl knockout. The reduction of fusion rate by NFL(Q333P) was partly due to interference with the function of the profusion protein MFN2, which is mutated in CMT2A, functionally linking these forms of CMT. In the later phase of toxicity, mitochondria essentially stopped moving in neurons expressing NFL mutants, probably a consequence of cytoskeletal disruption. Overall, the data point to important functions of neurofilaments in mitochondrial dynamics as well as primary involvement in CMT2E/1F.

Mutations in NEFL encoding the light neurofilament subunit (NFL) cause Charcot-Marie-Tooth disease type 2E (CMT2E), which affects both motor and sensory neurons. We expressed the disease-causing mutants NFL and NFL in motor neurons of dissociated spinal cord-dorsal root ganglia and demonstrated that they are incorporated into the preexisting neurofilament network but eventually disrupt neurofilaments without causing significant motor neuron death. Importantly, rounding of mitochondria and reduction in axonal diameter occurred before disruption of the neurofilament network, indicating that mitochondrial dysfunction contributes to the pathogenesis of CMT2E, as well as to CMT caused by mitofusin mutations. Heat shock proteins (HSPs) are involved in the formation of the neurofilament network and in protecting cells from misfolded mutant proteins. Cotransfection of HSPB1 with mutated NEFL maintained the neurofilament network, axonal diameter, and mitochondrial length in motor neurons expressing NFL, but not NFL. Conversely, HSPA1 cotransfection was effective in motor neurons expressing NFL, but not NFL. Thus, there are NFL mutant-specific differences in the ability of individual HSPs to prevent neurofilament abnormalities, reduction in axonal caliber, and disruption of mitochondrial morphology in motor neurons. These results suggest that HSP inducers have therapeutic potential for CMT2E but that their efficacy would depend on the profile of HSPs induced and the type of NEFL mutation.

Mutations in the ALS2 gene has recently been linked to cases of juvenile amyotrophic lateral sclerosis, juvenile primary lateral sclerosis and ascending hereditary spastic paralysis. All reported mutations predict the production of truncated forms of Alsin suggesting a loss of function mechanism for these motor neuron disorders. Here we used the tetracycline-regulated expression system to overexpress the full-length and truncated forms of Alsin in different cell lines. Alsin overexpression caused severe phenotypic changes in monkey COS-7 cells including the enlargement and accumulation of early endosomes, impairment of mitochondria trafficking and fragmentation of the Golgi apparatus. Our results further demonstrate the requirement of the Alsin VPS9 domain for occurrence of the vacuolation process and the role of Alsin as a guanine nucleotide exchange factor for Rab5. Transfected human SW13 cells exhibited an unexpected centrosomal localization for Alsin that was linked to the presence of the c-terminal part of the protein. Immunofluorescence staining revealed a colocalization of Alsin with the centrosomal markers gamma-tubulin and A kinase anchoring protein (AKAP-450). Similar results were obtained with human LA-N-2 and SK-N-SH neuronal cells. Moreover endogenous Alsin was detected in a centrosome preparation purified from human cortical brain. Considering the crucial role of centrosome in the production of microtubules required for intracellular transport, these findings are of potential relevance for unravelling the disease mechanisms linked to Alsin mutations.

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. Because disruption of the BBB may contribute to many brain disorders, they are of considerable interests in the identification of the molecular mechanisms of BBB development and integrity. We here report that the giant protein AHNAK is expressed at the plasma membrane of endothelial cells (ECs) forming specific blood-tissue barriers, but is absent from the endothelium of capillaries characterized by extensive molecular exchanges between blood and extracellular fluid. In the brain, AHNAK is widely distributed in ECs with BBB properties, where it co-localizes with the tight junction protein ZO-1. AHNAK is absent from the permeable brain ECs of the choroid plexus and is down-regulated in permeable angiogenic ECs of brain tumors. In the choroid plexus, AHNAK accumulates at the tight junctions of the choroid epithelial cells that form the blood-cerebrospinal fluid (CSF) barrier. In EC cultures, the regulation of AHNAK expression and its localization corresponds to general criteria of a protein involved in barrier organization. AHNAK is up-regulated by angiopoietin-1 (Ang-1), a morphogenic factor that regulates brain EC permeability. In bovine cerebral ECs co-cultured with glial cells, AHNAK relocates from the cytosol to the plasma membrane when endothelial cells acquire BBB properties. Our results identify AHNAK as a protein marker of endothelial cells with barrier properties., (Copyright 2004 Wiley-Liss, Inc.)

The S100B protein belongs to the S100 family of EF-hand calcium binding proteins implicated in cell growth and differentiation. Here, we show that in the developing and the adult mouse brain, S100B is expressed in oligodendroglial progenitor cells (OPC) committed to differentiate into the oligodendrocyte (OL) lineage. Nuclear S100B accumulation in OPC correlates with the transition from the fast dividing multipotent stage to the morphological differentiated, slow proliferating, pro-OL differentiation stage. In the adult, S100B expression is down-regulated in mature OLs that have established contacts with their axonal targets, suggesting a nuclear S100B function during oligodendroglial cells maturation. In vitro, the morphological transformation and maturation of pro-OL cells are delayed in the absence of S100B. Moreover, mice lacking S100B show an apparent delay in OPC maturation in response to demyelinating insult. We propose that nuclear S100B participates in the regulation of oligodendroglial cell maturation.

Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell-cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca(2+)-dependent cell-cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

The S100 family consists of 19 members, which function as transducers of calcium signals in a tissue-specific manner. Upon calcium binding, the conformation of many S100 proteins changes dramatically. Several hydrophobic residues are exposed, allowing the S100 proteins to interact with their target proteins, and thereby to transduce calcium signals into specific biological responses. To further elucidate the exact contribution of the S100 calciproteins in the calcium signalling pathways, several groups have applied the yeast two-hybrid technology to identify putative target proteins for the various S100 calciproteins. Two-hybrid large screens using S100 proteins as baits have confirmed the biochemical and structural feature of S100, which enable them to form homodimers and the ability of some members to form specific heterodimers in vivo. Yeast two-hybrid investigations have allowed the identification of conserved hydrophobic residues and domains that are crucial for the stabilization of S100 homo- and heterodimers. Furthermore, this method clearly underlines that the homo- and heterodimerization mechanisms differ among the members of the S100 family. However, several lines of evidence strongly suggest that two-hybrid methodology is limited to the analysis of interactions that are calcium-independent, since no target proteins other than S100 family members themselves have been detected with this methodology., (Copyright 2003 Wiley-Liss, Inc.)

Here we report a detailed analysis of the expression and localization of the giant protein AHNAK in adult mouse tissues. We show that AHNAK is widely expressed in muscle cells, including cardiomyocytes, smooth muscle cells, skeletal muscle, myoepithelium, and myofibroblasts. AHNAK is also specifically expressed in epithelial cells of most lining epithelium, but is absent in epithelium with more specialized secretory or absorptive functions. In all adult tissues, the main localization of AHNAK is at the plasma membrane. A role for AHNAK in the specific organization and the structural support of the plasma membrane common to muscle and lining epithelium is discussed.

The Zn(2+)- and Ca(2+)-binding S100B protein is implicated in multiple intracellular and extracellular regulatory events. In glial cells, a relationship exists between cytoplasmic S100B accumulation and cell morphological changes. We have identified the IQGAP1 protein as the major cytoplasmic S100B target protein in different rat and human glial cell lines in the presence of Zn(2+) and Ca(2+). Zn(2+) binding to S100B is sufficient to promote interaction with IQGAP1. IQ motifs on IQGAP1 represent the minimal interaction sites for S100B. We also provide evidence that, in human astrocytoma cell lines, S100B co-localizes with IQGAP1 at the polarized leading edge and areas of membrane ruffling and that both proteins relocate in a Ca(2+)-dependent manner within newly formed vesicle-like structures. Our data identify IQGAP1 as a potential target protein of S100B during processes of dynamic rearrangement of cell membrane morphology. They also reveal an additional cellular function for IQGAP1 associated with Zn(2+)/Ca(2+)-dependent relocation of S100B.

Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val(135) is reverted by ectopic expression of the calcium- and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca(2+)-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca(2+)-dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca(2+)- and Zn(2+)-dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn(2+) equivalents/mol S100B enhances Ca(2+)-dependent S100B-AHNAK interaction and that the effect of Zn(2+) relies on Zn(2+)-dependent regulation of S100B affinity for Ca(2+). Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca(2+) homeostasis.