ROLE OF THE PROTEIN QUALITY CONTOL SYSTEM IN MOTOR NEURON DISEASES: THE CASE OF MUSCLE CELLS

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA), are characterized by the progressive loss of motor neurons, and patients die for respiratory failure after the paralysis of voluntary muscle. Muscle system is, thus, highly affected but it is still unclear whether they play a role in disease onset or if they are a secondary target of toxicity. In my PhD period, I analyzed the behaviour in muscle cells of misfolded protein causing neurodegenerative diseases. I found that misfolded protein aggregates also in muscle cells. As a second step I studied the activation of the protein quality control system and its role at basal level in presence of misfolded proteins. I found that autophagy and proteasome are differently involved in the degradation of misfolded proteins in both muscle and motor neurons. Nevertheless, increasing the degradation of misfolded proteins by overexpressing key molecular chaperone or treating cells with autophagy inhibitors rescue the formation of aggregates in both models of disease

The role of the PQC system in managing misfolded protein in motoneuronal and muscular models of ALS and SBMA

Motorneuronal diseases are fatal neurodegenerative diseases which include spinobulbar muscular atrophy (SBMA). Recent studies have demonstrated that both motorneuron and muscle cells are directly affected in SBMA patients. SBMA is caused by a poliglutammine stretch in the N-terminal region of the androgen receptor (AR) protein. In presence of testosterone ARpolyQ lose the proper conformation, misfolds and becomes toxic to cells. The protein quality control (PQC) system is in charge of protein homeostasis. The chaperone system maintains proteins in the proper conformation; if it fails misfolded proteins are directed to the degradative systems: the ubiquitin proteasome system (UPS) and the autophagy. We have already demonstrated that autophagy stimulation reduces protein aggregation in motorneuronal models of SBMA. Here we studied the involvement of the PQC system in the muscular response to misfolded toxic proteins and in the removal of aggregates, hallmark of the disease. We tried to potentiate the autophagic response with trehalose in muscle C2C12 cells stably expressing AR with a stretch of 100 glutamine (C2C12_ARQ100). By filter retardation assay (FRA) we observed that ARpolyQ aggregation was reverted by trehalose. By RTq-PCR analysis we found that trehalose increased the expression of the small heat shock protein B8 a molecular chaperone involved in the autophagic machinery. Overexpression of HspB8 in C2C12_ARQ100, interestingly, caused a significative reduction of ARpolyQ aggregation in FRA. We extend our findings to a SBMA-related motorneuronal disease: the amyotrophic lateral sclerosis (ALS). As model of sporadic ALS we studied the TAR-DNA-Binding Protein of 43 kDa (TDP43) and its truncated form TDP43-25 that aggregates in the cytoplasm. Trehalose treatment of muscle C2C12 cells expressing both TDP43 and TDP43-25 caused no aggregate reduction. In conclusion we demonstrate that the enhancement of autophagy is a possible therapeutical strategy for treating SBMA; in the case of sporadic ALS other degradative pathways should to be investigated as autophagic facilitation can not prevent aggregate formation. GRANTS: Regione Lombardia; AFM-TELETHON, FRANCE; FONDAZIONE TELETHON, ITALY; FONDAZIONE CARIPLO, ITALY; FONDAZIONE ARISLA, ITALY; Ministero della Sanit\ue0, ITALY

Dual role of autophagy on docetaxel-sensitivity in prostate cancer cells

Prostate cancer (PC) is one of the leading causes of death in males. Available treatments often lead to the appearance of chemoresistant foci and metastases, with mechanisms still partially unknown. Within tumour mass, autophagy may promote cell survival by enhancing cancer cells tolerability to different cell stresses, like hypoxia, starvation or those triggered by chemotherapic agents. Because of its connection with the apoptotic pathways, autophagy has been differentially implicated, either as prodeath or prosurvival factor, in the appearance of more aggressive tumours. Here, in three PC cells (LNCaP, PC3, and DU145), we tested how different autophagy inducers modulate docetaxel-induced apoptosis. We selected the mTOR-independent disaccharide trehalose and the mTOR-dependent macrolide lactone rapamycin autophagy inducers. In castration-resistant PC (CRPC) PC3 cells, trehalose specifically prevented intrinsic apoptosis in docetaxel-treated cells. Trehalose reduced the release of cytochrome c triggered by docetaxel and the formation of aberrant mitochondria, possibly by enhancing the turnover of damaged mitochondria via autophagy (mitophagy). In fact, trehalose increased LC3 and p62 expression, LC3-II and p62 (p62 bodies) accumulation and the induction of LC3 puncta. In docetaxel-treated cells, trehalose, but not rapamycin, determined a perinuclear mitochondrial aggregation (mito-aggresomes), and mitochondria specifically colocalized with LC3 and p62-positive autophagosomes. In PC3 cells, rapamycin retained its ability to activate autophagy without evidences of mitophagy even in presence of docetaxel. Interestingly, these results were replicated in LNCaP cells, whereas trehalose and rapamycin did not modify the response to docetaxel in the ATG5-deficient (autophagy resistant) DU145 cells. Therefore, autophagy is involved to alter the response to chemotherapy in combination therapies and the response may be influenced by the different autophagic pathways utilized and by the type of cancer cells

Enhanced Clearance of Neurotoxic Misfolded Proteins by the Natural Compound Berberine and Its Derivatives

Background: Accumulation of misfolded proteins is a common hallmark of several neurodegenerative disorders (NDs) which results from a failure or an impairment of the proteinquality control (PQC) system. The PQC system is composed by chaperones and the degradative systems (proteasome and autophagy). Mutant proteins that misfold are potentially neurotoxic, thus strategies aimed at preventing their aggregation or at enhancing their clearance are emerging as interesting therapeutic targets for NDs. Methods: We tested the natural alkaloid berberine (BBR) and some derivatives for their capability to enhance misfolded protein clearance in cell models of NDs, evaluating which degradative pathway mediates their action. Results: We found that both BBR and its semisynthetic derivatives promote degradation of mutant androgen receptor (ARpolyQ) causative of spinal and bulbar muscular atrophy, acting mainly via proteasome and preventing ARpolyQ aggregation. Overlapping effects were observed on other misfolded proteins causative of amyotrophic lateral sclerosis, frontotemporal-lobar degeneration or Huntington disease, but with selective and specific action against each different mutant protein. Conclusions: BBR and its analogues induce the clearance of misfolded proteins responsible for NDs, representing potential therapeutic tools to counteract these fatal disorders

The contribution of protein quality control in the pathogenesis of SBMA

Spinal and bulbar muscular atrophy (SBMA) is a motoneuronal diseases caused by an elogated polyglutamine (polyQ) tract in the androgen receptor (AR). The intracellular accumulation of ARpolyQ, induced by the ligand testosterone, altered the protein quality control system (PQC) and impaired the protective mechanisms deputed to refolding and clearance of misfolded proteins. Emerging evidence reveal that ARpolyQ toxicity is not related only to motoneuron degeneration but also to skeletal muscle damage. Using SBMA knock-in mice (113Q SBMA mice), we analysed the role of PQC in skeletal muscle. All mice were analysed both at a pre-symptomatic stage (8 weeks) and at symptomatic stage (24 weeks). At symptomatic stages, the skeletal muscle of SBMA mice showed an increased expression of muscular markers (MYOG, TGF-beta1, AchR) suggesting that there is atrophy accompanied by denervation. In this condition, we have analysed the transcriptional regulation of several proteins involved in the PQC system. We found no variations of the autophagic master key regulator TFEB expression, while all autophagic markers analysed were specifically induced in skeletal muscle of symptomatic tg SBMA male mice (p62, LC3 Beclin-1 ATG10). Moreover, we have analysed the expression of HSPB8, a pro-autophagic chaperone, and of the co-chaperones BAG3 and BAG1, involved in the autophagic and proteasomal removal of the misfolded proteins, respectively. We found that HSPB8, BAG1 and BAG3 were transcriptionally up-regulated in symptomatic tg SBMA male mice. Moreover, the ratio BAG3:BAG1, index of which PQC degradative pathways is preferred to clear misfolded proteins, was increased in favor of the BAG3. Collectively, these data might suggest that in the skeletal muscle of SBMA mice autophagy is highly activated and the data might elucidate how muscle responds to ARpolyQ toxicity. GRANTS: Fondazione AriSLA; Fondazione Cariplo; AFM Telethon France; Regione Lombardia; UNIMI; Telethon Italy

TDP25 aggregation in motor neuron and muscle cells is rescued by chaperone overexpression

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving both upper and lower motor neurons (MNs). As target of MNs, muscle cells role in ALS has always been investigated. ALS can appear as familial or sporadic form, and in the vast majority of patients motor neurons have been observed proteinaceous inclusions containing TDP43. TDP43 inside cytoplasmic aggregates has been found cleaved into C-terminal fragments of 35 and 25 kDa highly aggregation prone. In this work we firstly investigate the aggregation propensity of TDP43 and its ALS-associated fragments TDP35 and TDP25 in both motor neuron like (NSC34) and muscle like cells (C2C12). We found that TDP43 forms physiological oligomers retained in filter retardation assay (FRA) due to the interaction of the N-terminal domain in the nucleus. To correctly visualized TDP25 aggregates we used the NP40 extraction, and we observed the greatest fraction of TDP25 isolated in the NP40 insoluble fraction. Then we studied the degradation of TDPs species, noting that they were mainly degraded via proteasome, while autophagy contribution is less important. Targeting the degradation of aggregation-prone species is a promising strategy to counteract ALS and we increase protein degradation by mean of particular chaperones such as Bag1, Bag3 and HspB8. These proteins, in complex with Hsp70, direct cargos alternatively to proteasome (Bag1) or to autophagy (HspB8 and Bag3, in complex). Overexpressing Bag1 we found that both in NSC34 and C2C12 TDP25 aggregation was rescued, due to an increased degradation via the proteasome. Similar results were obtained in both models targeting autophagy by overexpressing HspB8 and Bag3. Concluding, we showed that muscle cells are a site of misfolded protein aggregation as well as MNs. Importantly, we demonstrated that indirectly targeting degradative pathways by overexpressing chaperones could be beneficial against the formation of TDP25 aggregates both in MNs and muscle cells

Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration

Macroautophagy/autophagy, a defense mechanism against aberrant stresses, in neurons counteracts aggregate-prone misfolded protein toxicity. Autophagy induction might be beneficial in neurodegenerative diseases (NDs). The natural compound trehalose promotes autophagy via TFEB (transcription factor EB), ameliorating disease phenotype in multiple ND models, but its mechanism is still obscure. We demonstrated that trehalose regulates autophagy by inducing rapid and transient lysosomal enlargement and membrane permeabilization (LMP). This effect correlated with the calcium-dependent phosphatase PPP3/calcineurin activation, TFEB dephosphorylation and nuclear translocation. Trehalose upregulated genes for the TFEB target and regulator Ppargc1a, lysosomal hydrolases and membrane proteins (Ctsb, Gla, Lamp2a, Mcoln1, Tpp1) and several autophagy-related components (Becn1, Atg10, Atg12, Sqstm1/p62, Map1lc3b, Hspb8 and Bag3) mostly in a PPP3- and TFEB-dependent manner. TFEB silencing counteracted the trehalose prodegradative activity on misfolded protein causative of motoneuron diseases. Similar effects were exerted by trehalase-resistant trehalose analogs, melibiose and lactulose. Thus, limited lysosomal damage might induce autophagy, perhaps as a compensatory mechanism, a process that is beneficial to counteract neurodegeneration

BAG1 prevents misfolded proteins accumulation when autophagy flux is blocked in neurodegenerative disorders

Different disease associated proteins, as SOD1 and TDP-43 in familial and sporadic amyotrophic lateral sclerosis and frontotem- poral dementia, or androgen receptor (AR) in spinal and bulbar muscular atrophy, tend to misfold and accumulate into aggregates in neurons. Protein quality control system prevents their aggregation and toxicity by enhancing their degradation via proteasome and/or autophagy. An efficient dynein mediated transport of misfolded proteins to the site of degradation is required as key point to control their aggregation and degradation. HSPB8 is a protective protein that reduces disease associated proteins aggregation by autophagy facilitation. Here we evaluated the HSPB8 effects on the recently discovered RAN translated poly-di-peptides (DPRs) from C9ORF72 gene. Using filter trap and western blot we observed that HSPB8 over-expression facilitates DPRs clearance even when proteasome is blocked. when we blocked the dynein retrograde transport by EHNA we found an alteration of SQSTM1/p62 and LC3 expression and localization. However, dynein inhibition reduced SQSTM1/p62 and LC3 levels induced by trehalose and drastically reduced the number of autophagosome per cell. Moreover, EHNA reduced the PBS insoluble fraction of mutated misfolded proteins and DPRs also when autophagy is blocked. This effect was counteracted by proteasome inhibition. Notably, EHNA selectively increased BAG1 mRNA (responsible for misfolded protein degradation via protea- some) in NSC34 and motoneuron derived from iPS cells, while exogenous BAG1 overexpression reduced misfolded species aggre- gation and BAG1 down-regulation blocked the EHNA effect. Moreover, EHNA increased mRNA and protein levels of chaperone mediated autophagy receptor Lamp2A, suggesting that CMA can restore the degradation of misfolded proteins with KFERQ-like motif that are internalized into lysosome by Lamp2A. Collectively, these data suggest that when autophagy flux is blocked, misfolded proteins can be re-routed by BAG1 to alternative degradative pathways

Clearance of misfolded proteins in motoneuron disease: the case of Spinal and Bulbar Muscular Atrophy

In order to control the cellular homeostasis and survival, the newly synthesized proteins are directed to Protein Quality Control system (PQC) to ensure the correct folding. PQC requires the coordinated action of molecular chaperones and proteolytic systems, the ubiquitin\u2013proteasome system (UPS) and the autophago-lysosomal pathway (ALP). Although the PQC system efficiently remove all the intracellular proteins, some misfolded proteins might escape from PQC defence. The accumulation of misfolded and aggregated proteins is a common hallmark of several motoneuron diseases (MNDs), including spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS). SBMA is caused by a polyglutamine-expanded tract (polyQ) in the androgen receptor protein (AR). The binding of the ligand testosterone to the ARpolyQ induces protein misfolding and aggregation. The expanded polyQ tract confers to mutant AR a toxic gain-of-function that alters a cascade of several downstream pathways, including the PQC. Using SBMA cellular and knock-in mice models, we observed that insufficient or impaired PQC activity contribute to ARpolyQ accumulation. In particular, ALP plays a crucial role in the clearance of ARpolyQ. We studied the activity of HSPB8-BAG3 complex, which acting with Hsc70 and CHIP, can direct ARpolyQ to ALP-mediated clearance. Analysing the expression of these genes in muscle of symptomatic SBMA mice, we found that HSPB8-PQC machinery is highly increased suggesting that ALP might be the preferential degradative pathway rather than UPS. In this line, we analysed compounds able to activate or potentiate the HSPB8 machinery and ALP, in SBMA cell model. We have already tested the disaccharide trehalose, an m- TOR independent ALP inducer. Even the mechanism of action of trehalose is still unclear, we demonstrate that it is able removed the ARpolyQ misfolding also via induction of HSPB8 expression. However, the enzyme trehalase digest trehalose into glucose, reducing its bioavailability. Therefore, we used two trehalase-indigestible dysaccharides, lactulose and melibiose. We found that lactulose and melibiose counteract ARpolyQ aggregation with effects comparable to trehalose. Recently, we also tested another interesting compound Berberine, a traditional herbal medicine. Berberine reduced the ARpolyQ aggregates facilitating the clearance of the mutant proteins activating the PQC system. These data suggest that the autophagy/PQC inducers might have therapeutic potential for SBMA and other misfolding-related MNDs. GRANTS: Ministero Sanit\ue0 (GR-2011-02347198); AFM (16406); Fondazione ARISLA (ALS_GRANULOPATHY); Fondazione Cariplo (2014-0686); JPND (Project: Cure ALS); Regione Lombardia; Fondazione Regionale per la Ricerca Biomedica (TRANS_ALS) Telethon Italy (GGP14039)