Acute effects of a ketone monoester, whey protein, or their co-ingestion on mTOR trafficking and protein-protein co-localization in human skeletal muscle.

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 co-ingestion. The present investigation aimed to determine the acute effects of ingesting a ketone monoester, whey protein, or their co-ingestion on mTOR-related protein-protein co-localization 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 y) 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-minutes in the postprandial period for immunofluorescence assessment of protein translocation and co-localization 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) co-localization at 120-minutes vs. basal; however, the decrease was sustained at 300-minutes vs. basal (P<0.0001) only in KET+PRO. PRO and KET+PRO increased (Interaction: P<0.0001) mTOR-Rheb co-localization at 120-minutes vs. basal; however, KET+PRO resulted in a sustained increase in mTOR-Rheb co-localization at 300-minutes that was greater than KET and PRO. Treatment intake increased mTOR-wheat germ agglutinin (WGA) co-localization at 120- and 300-minutes (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.

The J Domain of Sacsin Disrupts Intermediate Filament Assembly.

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.

MTP deficiency caused by HADHB mutations: Pathophysiology and clinical manifestations.

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.

The S100B Protein and Partners in Adipocyte Response to Cold Stress and Adaptive Thermogenesis: Facts, Hypotheses, and Perspectives.

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 3ß (CLSTN3ß). CLSTN3ß 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 CLSTN3ß 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.

Functional Characterization of Neurofilament Light Splicing and Misbalance in Zebrafish.

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.

Depending on the stress, histone deacetylase inhibitors act as heat shock protein co-inducers in motor neurons and potentiate arimoclomol, exerting neuroprotection through multiple mechanisms in ALS models.

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 FUSP525Lmutation, pointing to multiple mechanisms of neuroprotection by both HDAC inhibiting drugs and arimoclomol.

Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics.

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.

Dysregulation of chromatin remodelling complexes in amyotrophic lateral sclerosis.

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.

A New Mutation in FIG4 Causes a Severe Form of CMT4J Involving TRPV4 in the Pathogenic Cascade.

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.

Altered organization of the intermediate filament cytoskeleton and relocalization of proteostasis modulators in cells lacking the ataxia protein sacsin.

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.

Mechanical ventilation triggers abnormal mitochondrial dynamics and morphology in the diaphragm.

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.

Neurofilament dynamics and involvement in neurological disorders.

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.

Sacs knockout mice present pathophysiological defects underlying autosomal recessive spastic ataxia of Charlevoix-Saguenay.

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.

A two-hybrid screen identifies an unconventional role for the intermediate filament peripherin in regulating the subcellular distribution of the SNAP25-interacting protein, SIP30.

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.

The voltage-gated calcium channel blocker lomerizine is neuroprotective in motor neurons expressing mutant SOD1, but not TDP-43.

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.

A novel small molecule HSP90 inhibitor, NXD30001, differentially induces heat shock proteins in nervous tissue in culture and in vivo.

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.

Acute exercise remodels mitochondrial membrane interactions in mouse skeletal muscle.

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.

Expression of the protein chaperone, clusterin, in spinal cord cells constitutively and following cellular stress, and upregulation by treatment with Hsp90 inhibitor.

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.

Heterogeneity in the properties of NEFL mutants causing Charcot-Marie-Tooth disease results in differential effects on neurofilament assembly and susceptibility to intervention by the chaperone-inducer, celastrol.

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.

Mitochondrial morphology transitions and functions: implications for retrograde signaling?

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.