Insulin-like growth factor 1 signaling in motor neuron and polyglutamine diseases: From molecular pathogenesis to therapeutic perspectives

The pleiotropic peptide insulin-like growth factor 1 (IGF-I) regulates human body homeostasis and cell growth. IGF-I activates two major signaling pathways, namely phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB/Akt) and Ras/extracellular signal-regulated kinase (ERK), which contribute to brain development, metabolism and function as well as to neuronal maintenance and survival. In this review, we discuss the general and tissue-specific effects of the IGF-I pathways. In addition, we present a comprehensive overview examining the role of IGF-I in neurodegenerative diseases, such as spinal and muscular atrophy, amyotrophic lateral sclerosis, and polyglutamine diseases. In each disease, we analyze the disturbances of the IGF-I pathway, the modification of the disease protein by IGF-I signaling, and the therapeutic strategies based on the use of IGF-I developed to date. Lastly, we highlight present and future considerations in the use of IGF-I for the treatment of these disorders

Disease mechanism, biomarker and therapeutics for spinal and bulbar muscular atrophy (SBMA)

Spinal and bulbar muscular atrophy (SBMA) is a hereditary neuromuscular disorder caused by CAG trinucleotide expansion in the gene encoding the androgen receptor (AR). In the central nervous system, lower motor neurons are selectively affected, whereas pathology of patients and animal models also indicates involvement of skeletal muscle including loss of fast-twitch type 2 fibres and increased slow-twitch type 1 fibres, together with a glycolytic-to-oxidative metabolic switch. Evaluation of muscle and fat using MRI, in addition to biochemical indices such as serum creatinine level, are promising biomarkers to track the disease progression. The serum level of creatinine starts to decrease before the onset of muscle weakness, followed by the emergence of hand tremor, a prodromal sign of the disease. Androgen-dependent nuclear accumulation of the polyglutamine-expanded AR is an essential step in the pathogenesis, providing therapeutic opportunities via hormonal manipulation and gene silencing with antisense oligonucleotides. Animal studies also suggest that hyperactivation of Src, alteration of autophagy and a mitochondrial deficit underlie the neuromuscular degeneration in SBMA and provide alternative therapeutic targets

Beyond motor neurons: expanding the clinical spectrum in Kennedy’s disease

Kennedy's disease, or spinal and bulbar muscular atrophy (SBMA), is an X-linked neuromuscular condition clinically characterised by weakness, atrophy and fasciculations of the limb and bulbar muscles, as a result of lower motor neuron degeneration. The disease is caused by an abnormally expanded triplet repeat expansions in the ubiquitously expressed androgen receptor gene, through mechanisms which are not entirely elucidated. Over the years studies from both humans and animal models have highlighted the involvement of cell populations other than motor neurons in SBMA, widening the disease phenotype. The most compelling aspect of these findings is their potential for therapeutic impact: muscle, for example, which is primarily affected in the disease, has been recently shown to represent a valid alternative target for therapy to motor neurons. In this review, we discuss the emerging study of the extra-motor neuron involvement in SBMA, which, besides increasingly pointing towards a multidisciplinary approach for affected patients, deepens our understanding of the pathogenic mechanisms and holds potential for providing new therapeutic targets for this disease

Trehalose prodegradative role on AR aggregates in a muscle model of Spinal and Bulbar Muscular Atrophy.

Spinal and Bulbar Muscular Atrophy (SBMA) is a motor neuronal disease whose onset and progression have been recently linked also to a muscular defect. SBMA is caused by a polyglutammine tract in the exon 1 of the androgen receptor protein (ARpolyQ). When AR is activated by testosterone a fraction of the protein misfolds and become toxic to cells. Moreover if ARpolyQ is not correctly removed from cellular environment it also forms aggregates that could damage many cellular process. In this work we have studied the protein quality control system (composed of a chaperone network and two main degradative pathways: proteasome and autophagy) in a cellular muscular model of SBMA. We use C2C12 stably transfected with ARwt or ARpolyQ bearing an elongation of 100 glutammine. Initially we performed a filter trap assay (FTA) on both cell line treated with testosterone. We observed that testosterone triggers the aggregation of ARpolyQ but not of ARwt. We also observed that testosterone treatment caused mortality in C2C12 with ARpolyQ. By real time PCR we found that there was not activation of the PQC system in presence of ARpolyQ but that the expression of AchR was significantly lower than in control cell. These data suggest that ARpolyQ led to muscular atrophy. We investigate degradative systems that degrade AR and found that autophagy is highly involved in AR degradation. We facilitate autophagy towards the overexpression of HspB8. We observed that HspB8 counteracted testosterone dependent aggregation of ARpolyQ. We know that trehalose in motorneuronal model of SBMA induce the expression of HspB8. We then enhanced autophagy with trehalose and found that ARpolyQ aggregation was almost completely reverted. We co-treated cells with trehalose and bafilomycin and found that this condition abolished trehalose effect. We demonstrated that trehalose effect depend upon an efficient autophagic flux. By rtPCR we observed that trehalose enhance the expression of a wide range of genes related to autophagy. Interestingly we also found VCP overexpression in presence of trehalose. The valosin containing protein VCP is a multi-functional protein involved also in the ERAD pathway. So we inhibit VCP with DBEQ a specific inhibitor of the ATPase activity of VCP and found that testosterone dependent aggregation was significantly increased. Interestingly we found that trehalose treatment counteracted this DBEQ associated aggregation. In conclusion we characterized C2C12 as a reliable muscle model of SBMA. we also found that autophagy is highly involved in ARpolyQ degradation and consequently we demonstrated that autophagy activation rescues ARpolyQ aggregation in muscle cells. We finally observed that also the ERAD pathway plays an important role in ARpolyQ degradation. AFM-TELETHON; FONDAZIONE TELETHON; FONDAZIONE CARIPLO; FONDAZIONE ARISLA; Ministero della Sanità; Joint Programme Neurodegenerative Disease (JPND)

Ablation of the UPR-mediator CHOP restores motor function and reduces demyelination in Charcot-Marie-Tooth 1 B mice

Deletion of serine 63 from PO glycoprotein (POS63del) causes Charcot-Marie-Tooth 1 B neuropathy in humans, and POS63del produces a similar demyelinating neuropathy in transgenic mice. POS63del is retained in the endoplasmic reticulum and fails to be incorporated into myelin. Here we report that POS63clel is mis-folded and Schwann cells mount a consequential canonical unfolded protein response (UPR), including expression of the transcription factor CHOP, previously associated with apoptosis in ER-stressed cells. UPR activation and CHOP expression respond dynamically to POS63del levels and are reversible but are associated with only limited apoptosis of Schwann cells. Nonetheless, Chop ablation in S63del mice completely rescues their motor deficit and reduces active demyelination 2-fold. This indicates that signaling through the CHOP arm of the UPR provokes demyelination in inherited neuropathy. S63del mice also provide an opportunity to explore how cells can dysfunction yet survive in prolonged ER stress-important for neurodegeneration related to misfolded proteins

Increased mitophagy in the skeletal muscle of spinal and bulbar muscular atrophy patients

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by polyglutamine expansion in the androgen receptor (AR) and characterized by the loss of lower motor neurons. Here we investigated pathological processes occurring in muscle biopsy specimens derived from SBMA patients and, as controls, age-matched healthy subjects and patients suffering from amyotrophic lateral sclerosis (ALS) and neurogenic atrophy. We detected atrophic fibers in the muscle of SBMA, ALS and neurogenic atrophy patients. In addition, SBMA muscle was characterized by the presence of a large number of hypertrophic fibers, with oxidative fibers having a larger size compared with glycolytic fibers. Polyglutamine-expanded AR expression was decreased in whole muscle, yet enriched in the nucleus, and localized to mitochondria. Ultrastructural analysis revealed myofibrillar disorganization and streaming in zones lacking mitochondria and degenerating mitochondria. Using molecular (mtDNA copy number), biochemical (citrate synthase and respiratory chain enzymes) and morphological (dark blue area in nicotinamide adenine dinucleotide-stained muscle cross-sections) analyses, we found a depletion of the mitochondria associated with enhanced mitophagy. Mass spectrometry analysis revealed an increase of phosphatidylethanolamines and phosphatidylserines in mitochondria isolated from SBMA muscles, as well as a 50% depletion of cardiolipin associated with decreased expression of the cardiolipin synthase gene. These observations suggest a causative link between nuclear polyglutamine-expanded AR accumulation, depletion of mitochondrial mass, increased mitophagy and altered mitochondrial membrane composition in SBMA muscle patients. Given the central role of mitochondria in cell bioenergetics, therapeutic approaches toward improving the mitochondrial network are worth considering to support SBMA patients

Autophagic and proteasomal mediated removal of mutant androgen receptor in muscle models of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease (MND) caused by a mutant androgen receptor (AR) containing an elongated polyglutamine (polyQ) tract. ARpolyQ toxicity is triggered by androgenic AR ligands, which induce aberrant conformations (misfolding) of the ARpolyQ protein that aggregates. Misfolded proteins perturb the protein quality control (PQC) system leading to cell dysfunction and death. Spinal cord motoneurons, dorsal root ganglia neurons and skeletal muscle cells are affected by ARpolyQ toxicity. Here, we found that, in stabilized skeletal myoblasts (s-myoblasts), ARpolyQ formed testosterone-inducible aggregates resistant to NP-40 solubilization; these aggregates did not affect s-myoblasts survival or viability. Both wild type AR and ARpolyQ were processed via proteasome, but ARpolyQ triggered (and it was also cleared via) autophagy. ARpolyQ reduced two pro-autophagic proteins expression (BAG3 and VCP), leading to decreased autophagic response in ARpolyQ s-myoblasts. Overexpression of two components of the chaperone assisted selective autophagy (CASA) complex (BAG3 and HSPB8), enhanced ARpolyQ clearance, while the treatment with the mTOR independent autophagy activator trehalose induced complete ARpolyQ degradation. Thus, trehalose has beneficial effects in SBMA skeletal muscle models even when autophagy is impaired, possibly by stimulating CASA to assist the removal of ARpolyQ misfolded species/aggregates

Motor neuron degeneration in spinal and bulbar muscular atrophy: molecular approaches to counteract mutant androgen receptor neurotoxicity

The neuromuscular disease Spinal bulbar muscular atrophy (SBMA) associates with loss of bulbar or spinal motoneurons and skeletal muscle atrophy. SBMA is caused by a mutation of the androgen receptor (AR) gene resulting in a protein with an elongated polyglutamine (polyQ) tract. ARpolyQ acquires nuclear toxicity after binding testosterone, which induces AR nuclear translocation and ARpolyQ misfolding. Misfolded ARpolyQ is prone to aggregate, a process counteracted by the protein quality control (PQC) system. This system comprises chaperones and the degradative pathways (proteasome and autophagy). Several data suggest that misfolded ARpolyQ is mainly process via autophagy, and causes autophagy flux blockage. Restoration of a functional autophagy is beneficial to cells expressing misfolded ARpolyQ. A peculiar form of autophagy is the "chaperone-assisted selective autoghagy" (CASA), which relies on dynein-mediated retrograde transport of the CASA (HSPB8-BAG3-HSC70-CHIP) complex. This complex binds misfolded ARpolyQ enhancing its clearance. In immortalized motoneurons (MNs) and MNs derived from SBMA iPSCs we found that inhibition of dynein-mediated retrograde transport reduces ARpolyQ accumulation enhancing its clearance. This process is mediated by the HSC70 co-chaperone BAG1 which activate a compensatory mechanism alternative to HSPB8/BAG3. In the knock-in ARQ113 SBMA mouse model (KIARQ113), we found that in affected muscle both BAG1 and BAG3 are upregulated, with an increased BAG3:BAG1 ratio which preferentially routes misfolded ARpolyQ to autophagy. On these basis and on our previous in vitro studies, we tested bicalutamide (an antiandrogen which prevent AR nuclear translocation) and trehalose (an autophagy activator) in KIARQ113 mouse. We found found that mice survival was not significantly modified by the treatments, but an apparent positive trend was present. In Rotarod test KIARQ113 mice the impaired motor coordination was completely recovered by trehalose treatment. Bicalutamide administered at early stages worsened this phenotype (probably because of its anti-anabolic effects on muscle development), but recovered the motor coordination phenotype when administered at later stages (when muscle reached the adulthood stage). Grip strength test in KI mice showed decreased forelimb muscle force, which was further decreased by early, but not late bicalutamide treatment. Hystopathological analyses of mouse gastrocnemius reveal no variation associated to treatments in Feret min and max diameter of the KI mouse muscle fibers. Molecular analyses of gastrocnemius muscle of treated mice showed an increased PGC1alpha expression, paralleled by an increased mitochondrial DNA content and enhanced mitochondrial complex levels (particularly of complex V - ATP synthase subunits and complex III). Thus, the combined trehalose/bicalutamide treatment ameliorates muscle energy production and counteracts ARpolyQ mediated toxicity in vivo