HSPB8 frameshift mutant aggregates weaken chaperone-assisted selective autophagy in neuromyopathies.

Chaperone-assisted selective autophagy (CASA) is a highly selective pathway for the disposal of misfolding and aggregating proteins. In muscle, CASA assures muscle integrity by favoring the turnover of structural components damaged by mechanical strain. In neurons, CASA promotes the removal of aggregating substrates. A crucial player of CASA is HSPB8 (heat shock protein family B (small) member 8), which acts in a complex with HSPA, their cochaperone BAG3, and the E3 ubiquitin ligase STUB1. Recently, four novel HSPB8 frameshift (fs) gene mutations have been linked to neuromyopathies, and encode carboxy-terminally mutated HSPB8, sharing a common C-terminal extension. Here, we analyzed the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. We demonstrated that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Notably, all HSPB8 frameshift mutants retain their ability to interact with CASA members but sequester them into the HSPB8-positive aggregates together with two autophagy receptors SQSTM1/p62 and TAX1BP1. This copartitioning process negatively affects the CASA capability to remove its clients and causes a general failure in proteostasis response. Further analyses revealed that the aggregation of the HSPB8_fs mutants occurs independently of the other CASA members or from the autophagy receptors interaction, but it is an intrinsic feature of the mutated amino acid sequence. HSPB8_fs mutants aggregation alters the differentiation capacity of muscle cells and impairs sarcomere organization. Collectively, these results shed light on a potential pathogenic mechanism shared by the HSPB8_fs mutants described in neuromuscular diseases.Abbreviations : ACD: α-crystallin domain; ACTN: actinin alpha; BAG3: BAG cochaperone 3; C: carboxy; CASA: chaperone-assisted selective autophagy; CE: carboxy-terminal extension; CLEM: correlative light and electron microscopy; CMT2L: Charcot-Marie-Tooth type 2L; CTR: carboxy-terminal region; dHMNII: distal hereditary motor neuropathy type II; EV: empty vector; FRA: filter retardation assay; fs: frameshift; HSPA/HSP70: heat shock protein family A (Hsp70); HSPB1/Hsp27: heat shock protein family B (small) member 1; HSPB8/Hsp22: heat shock protein family B (small) member 8; HTT: huntingtin; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MD: molecular dynamics; MTOC: microtubule organizing center; MYH: myosin heavy chain; MYOG: myogenin; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; NSC34: Neuroblastoma X Spinal Cord 34; OPTN: optineurin; polyQ: polyglutamine; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1: Tax1 binding protein 1; TUBA: tubulin alpha; WT: wild-type.

Bicalutamide and Trehalose Ameliorate Spinal and Bulbar Muscular Atrophy Pathology in Mice.

Spinal and bulbar muscular atrophy (SBMA) is characterized by motor neuron (MN) degeneration that leads to slowly progressive muscle weakness. It is considered a neuromuscular disease since muscle has a primary role in disease onset and progression. SBMA is caused by a CAG triplet repeat expansion in the androgen receptor (AR) gene. The translated poly-glutamine (polyQ) tract confers a toxic gain of function to the mutant AR altering its folding, causing its aggregation into intracellular inclusions, and impairing the autophagic flux. In an in vitro SBMA neuronal model, we previously showed that the antiandrogen bicalutamide and trehalose, a natural disaccharide stimulating autophagy, block ARpolyQ activation, reduce its nuclear translocation and toxicity and facilitate the autophagic degradation of cytoplasmic AR aggregates. Here, in a knock-in SBMA mouse model (KI AR113Q), we show that bicalutamide and trehalose ameliorated SBMA pathology. Bicalutamide reversed the formation of the AR insoluble forms in KI AR113Q muscle, preventing autophagic flux blockage. We demonstrated that apoptosis is activated in KI AR113Q muscle, and that both compounds prevented its activation. We detected a decrease of mtDNA and an increase of OXPHOS enzymes, already at early symptomatic stages; these alterations were reverted by trehalose. Overall, bicalutamide and/or trehalose led to a partial recovery of muscle morphology and function, and improved SBMA mouse motor behavior, inducing an extension of their survival. Thus, bicalutamide and trehalose, by counteracting ARpolyQ toxicity in skeletal muscle, are valuable candidates for future clinical trials in SBMA patients.

Identification of HSPB8 modulators counteracting misfolded protein accumulation in neurodegenerative diseases.

AIMS: The small Heat Shock Protein B8 (HSPB8) is the core component of the Chaperone-Assisted Selective Autophagy (CASA) complex. This complex selectively targets, transports, and tags misfolded proteins for their recognition by autophagy receptors and insertion into the autophagosome for clearance. CASA is essential to maintain intracellular proteostasis, especially in heart, muscle, and brain often exposed to various types of cell stresses. In neurons, HSPB8 protects against neurotoxicity caused by misfolded proteins in several models of neurodegenerative diseases; by facilitating autophagy, HSPB8 assists misfolded proteins degradation also counteracting proteasome overwhelming and inhibition. MATERIALS AND METHODS: To enhance HSPB8 protective activity, we screened a library of approximately 120,000 small molecules to identify compounds capable of increasing HSPB8 gene transcription, translation, or protein stability. KEY FINDINGS: We found 83 active compounds active in preliminary dose-response assays and further classified them in 19 chemical classes by medicinal chemists' visual inspection. Of these 19 prototypes, 14 induced HSPB8 mRNA and protein levels in SH-SY5Y cells. Out of these 14 compounds, 3 successfully reduced the aggregation propensity of a disease-associated mutant misfolded superoxide dismutase 1 (SOD1) protein in a flow cytometry-based aggregation assay (Flow cytometric analysis of Inclusions and Trafficking (FloIT)) and induced the expression (mRNA and protein) of some autophagy receptors. Notably, the 3 hits were inactive in HSPB8-depleted cells, confirming that their protective activity is mediated by and requires HSPB8. SIGNIFICANCE: These compounds may be highly relevant for a therapeutic approach in several human disorders, including neurodegenerative diseases, in which enhancement of CASA exerts beneficial activities.

HSPB8 counteracts tumor activity of BRAF- and NRAS-mutant melanoma cells by modulation of RAS-prenylation and autophagy.

Cutaneous melanoma is one of the most aggressive and lethal forms of skin cancer. Some specific driver mutations have been described in multiple oncogenes including BRAF and NRAS that are mutated in 60-70% and 15-20% of melanoma, respectively. The aim of this study was to evaluate the role of Small Heat Shock Protein B8 (HSPB8) on cell growth and migration of both BLM (BRAFwt/NRASQ61R) and A375 (BRAFV600E/NRASwt) human melanoma cell lines. HSPB8 is a member of the HSPB family of chaperones involved in protein quality control (PQC) system and contributes to chaperone assisted selective autophagy (CASA) as well as in the regulation of mitotic spindle. In cancer, HSPB8 has anti- or pro-tumoral action depending on tumor type. In melanoma cell lines characterized by low HSPB8 levels, we demonstrated that the restoration of HSPB8 expression causes cell growth arrest, reversion of EMT (Epithelial-Mesenchymal Transition)-like phenotype switching and antimigratory effect, independently from the cell mutational status. We demonstrated that HSPB8 regulates the levels of the active prenylated form of NRAS in NRAS-mutant and NRAS-wild-type melanoma cell lines. Consequently, the inhibition of NRAS impairs the activation of Akt/mTOR pathway inducing autophagy activation. Autophagy can play a dual role in regulating cell death and survival. We have therefore demonstrated that HSPB8-induced autophagy is a crucial event that counteracts cell growth in melanoma. Collectively, our results suggest that HSPB8 has an antitumoral action in melanoma cells characterized by BRAF and NRAS mutations.

The Role of Small Heat Shock Proteins in Protein Misfolding Associated Motoneuron Diseases.

Motoneuron diseases (MNDs) are neurodegenerative conditions associated with death of upper and/or lower motoneurons (MNs). Proteostasis alteration is a pathogenic mechanism involved in many MNDs and is due to the excessive presence of misfolded and aggregated proteins. Protein misfolding may be the product of gene mutations, or due to defects in the translation process, or to stress agents; all these conditions may alter the native conformation of proteins making them prone to aggregate. Alternatively, mutations in members of the protein quality control (PQC) system may determine a loss of function of the proteostasis network. This causes an impairment in the capability to handle and remove aberrant or damaged proteins. The PQC system consists of the degradative pathways, which are the autophagy and the proteasome, and a network of chaperones and co-chaperones. Among these components, Heat Shock Protein 70 represents the main factor in substrate triage to folding, refolding, or degradation, and it is assisted in this task by a subclass of the chaperone network, the small heat shock protein (sHSPs/HSPBs) family. HSPBs take part in proteostasis by bridging misfolded and aggregated proteins to the HSP70 machinery and to the degradative pathways, facilitating refolding or clearance of the potentially toxic proteins. Because of its activity against proteostasis alteration, the chaperone system plays a relevant role in the protection against proteotoxicity in MNDs. Here, we discuss the role of HSPBs in MNDs and which HSPBs may represent a valid target for therapeutic purposes.

The role of autophagy-lysosomal pathway in motor neuron diseases.

Motor neuron diseases (MNDs) include a broad group of diseases in which neurodegeneration mainly affects upper and/or lower motor neurons (MNs). Although the involvement of specific MNs, symptoms, age of onset, and progression differ in MNDs, the main pathogenic mechanism common to most MNDs is represented by proteostasis alteration and proteotoxicity. This pathomechanism may be directly related to mutations in genes encoding proteins involved in the protein quality control system, particularly the autophagy-lysosomal pathway (ALP). Alternatively, proteostasis alteration can be caused by aberrant proteins that tend to misfold and to aggregate, two related processes that, over time, cannot be properly handled by the ALP. Here, we summarize the main ALP features, focusing on different routes utilized to deliver substrates to the lysosome and how the various ALP pathways intersect with the intracellular trafficking of membranes and vesicles. Next, we provide an overview of the mutated genes that have been found associated with MNDs, how these gene products are involved in different steps of ALP and related processes. Finally, we discuss how autophagy can be considered a valid therapeutic target for MNDs treatment focusing on traditional autophagy modulators and on emerging approaches to overcome their limitations.

Pathogenic variants of Valosin-containing protein induce lysosomal damage and transcriptional activation of autophagy regulators in neuronal cells.

AIM: Mutations in the valosin-containing protein (VCP) gene cause various lethal proteinopathies that mainly include inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). Different pathological mechanisms have been proposed. Here, we define the impact of VCP mutants on lysosomes and how cellular homeostasis is restored by inducing autophagy in the presence of lysosomal damage. METHODS: By electron microscopy, we studied lysosomal morphology in VCP animal and motoneuronal models. With the use of western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence and filter trap assay, we evaluated the effect of selected VCP mutants in neuronal cells on lysosome size and activity, lysosomal membrane permeabilization and their impact on autophagy. RESULTS: We found that VCP mutants induce the formation of aberrant multilamellar organelles in VCP animal and cell models similar to those found in patients with VCP mutations or with lysosomal storage disorders. In neuronal cells, we found altered lysosomal activity characterised by membrane permeabilization with galectin-3 redistribution and activation of PPP3CB. This selectively activated the autophagy/lysosomal transcriptional regulator TFE3, but not TFEB, and enhanced both SQSTM1/p62 and lipidated MAP1LC3B levels inducing autophagy. Moreover, we found that wild type VCP, but not the mutants, counteracted lysosomal damage induced either by trehalose or by a mutant form of SOD1 (G93A), also blocking the formation of its insoluble intracellular aggregates. Thus, chronic activation of autophagy might fuel the formation of multilamellar bodies. CONCLUSION: Together, our findings provide insights into the pathogenesis of VCP-related diseases, by proposing a novel mechanism of multilamellar body formation induced by VCP mutants that involves lysosomal damage and induction of lysophagy.

Lysosomes Dysfunction Causes Mitophagy Impairment in PBMCs of Sporadic ALS Patients.

Mitochondria alterations are present in tissues derived from patients and animal models, but no data are available for peripheral blood mononuclear cells (PBMCs) of ALS patients. This work aims to investigate mitophagy in PBMCs of sporadic (sALS) patients and how this pathway can be tuned by using small molecules. We found the presence of morphologically atypical mitochondria by TEM and morphological abnormalities by MitoTracker™. We found a decreased number of healthy mitochondria in sALS PBMCs and an impairment of mitophagy with western blot and immunofluorescence. After rapamycin treatment, we found a higher increase in the LC3 marker in sALS PBMCs, while after NH4Cl treatment, we found a lower increase in the LC3 marker. Finally, mTOR-independent autophagy induction with trehalose resulted in a significant decrease in the lysosomes level sALS PBMCs. Our data suggest that the presence of morphologically altered mitochondria and an inefficient turnover of damaged mitochondria in PBMCs of sALS patients rely on the impairment of the mitophagy pathway. We also found that the induction of the mTOR-independent autophagy pathway leads to a decrease in lysosomes level, suggesting a more sensitivity of sALS PBMCs to trehalose. Such evidence suggests that trehalose could represent an effective treatment for ALS patients.

Valosin Containing Protein (VCP): A Multistep Regulator of Autophagy.

Valosin containing protein (VCP) has emerged as a central protein in the regulation of the protein quality control (PQC) system. VCP mutations are causative of multisystem proteinopathies, which include neurodegenerative diseases (NDs), and share various signs of altered proteostasis, mainly associated with autophagy malfunctioning. Autophagy is a complex multistep degradative system essential for the maintenance of cell viability, especially in post-mitotic cells as neurons and differentiated skeletal muscle cells. Interestingly, many studies concerning NDs have focused on autophagy impairment as a pathological mechanism or autophagy activity boosting to rescue the pathological phenotype. The role of VCP in autophagy has been widely debated, but recent findings have defined new mechanisms associated with VCP activity in the regulation of autophagy, showing that VCP is involved in different steps of this pathway. Here we will discuss the multiple activity of VCP in the autophagic pathway underlying its leading role either in physiological or pathological conditions. A better understanding of VCP complexes and mechanisms in regulating autophagy could define the altered mechanisms by which VCP directly or indirectly causes or modulates different human diseases and revealing possible new therapeutic approaches for NDs.

Neurodegenerative Disease-Associated TDP-43 Fragments Are Extracellularly Secreted with CASA Complex Proteins.

Extracellular vesicles (EVs) play a central role in neurodegenerative diseases (NDs) since they may either spread the pathology or contribute to the intracellular protein quality control (PQC) system for the cellular clearance of NDs-associated proteins. Here, we investigated the crosstalk between large (LVs) and small (SVs) EVs and PQC in the disposal of TDP-43 and its FTLD and ALS-associated C-terminal fragments (TDP-35 and TDP-25). By taking advantage of neuronal cells (NSC-34 cells), we demonstrated that both EVs types, but particularly LVs, contained TDP-43, TDP-35 and TDP-25. When the PQC system was inhibited, as it occurs in NDs, we found that TDP-35 and TDP-25 secretion via EVs increased. In line with this observation, we specifically detected TDP-35 in EVs derived from plasma of FTLD patients. Moreover, we demonstrated that both neuronal and plasma-derived EVs transported components of the chaperone-assisted selective autophagy (CASA) complex (HSP70, BAG3 and HSPB8). Neuronal EVs also contained the autophagy-related MAP1LC3B-II protein. Notably, we found that, under PQC inhibition, HSPB8, BAG3 and MAP1LC3B-II secretion paralleled that of TDP-43 species. Taken together, our data highlight the role of EVs, particularly of LVs, in the disposal of disease-associated TDP-43 species, and suggest a possible new role for the CASA complex in NDs.

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation.

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

Retinoic Acid Downregulates HSPB8 Gene Expression in Human Breast Cancer Cells MCF-7.

Breast cancer (BC) is a serious and widespread disease for which different treatments have been developed. In addition to the classic therapies, the treatment with retinoic acid (RA) is still being clinically investigated. RA reduces cancer cells proliferation and migration, but its molecular mechanism of action is not clear. In tumor development, autophagy promotes cancer cell survival and prevents apoptosis. Small heat shock protein B8 (HSPB8) acts together with its co-chaperone BCL-2 associated athanogene 3 (BAG3) stimulating BC proliferation and migration. We analyzed whether direct correlations exist between RA and HSPB8 or BAG3 and how this may play a role in BC. We measured HSPB8 and BAG3 gene expression in MCF-7 BC cells and we analyzed the potential correlation between the antiproliferative and antimigratory effect of RA with the expression level of HSPB8. We found that in MCF-7 cells RA reduces both HSPB8 and BAG3 gene expression and it alters the mitotic spindle organization. Notably, the effects of RA on HSPB8 levels are exerted at both transcriptional and translational levels. RA effects are possibly mediated by miR-574-5p that targets the HSPB8 transcript. Our results suggest that therapeutic doses of RA can efficiently counteract the adverse effects of HSPB8 in BC progression.

The Role of HSPB8, a Component of the Chaperone-Assisted Selective Autophagy Machinery, in Cancer.

The cellular response to cancer-induced stress is one of the major aspects regulating cancer development and progression. The Heat Shock Protein B8 (HSPB8) is a small chaperone involved in chaperone-assisted selective autophagy (CASA). CASA promotes the selective degradation of proteins to counteract cell stress such as tumor-induced stress. HSPB8 is also involved in (i) the cell division machinery regulating chromosome segregation and cell cycle arrest in the G0/G1 phase and (ii) inflammation regulating dendritic cell maturation and cytokine production. HSPB8 expression and role are tumor-specific, showing a dual and opposite role. Interestingly, HSPB8 may be involved in the acquisition of chemoresistance to drugs. Despite the fact the mechanisms of HSPB8-mediated CASA activation in tumors need further studies, HSPB8 could represent an important factor in cancer induction and progression and it may be a potential target for anticancer treatment in specific types of cancer. In this review, we will discuss the molecular mechanism underlying HSPB8 roles in normal and cancer conditions. The basic mechanisms involved in anti- and pro-tumoral activities of HSPB8 are deeply discussed together with the pathways that modulate HSPB8 expression, in order to outline molecules with a beneficial effect for cancer cell growth, migration, and death.

A Crucial Role for the Protein Quality Control System in Motor Neuron Diseases.

Motor neuron diseases (MNDs) are fatal diseases characterized by loss of motor neurons in the brain cortex, in the bulbar region, and/or in the anterior horns of the spinal cord. While generally sporadic, inherited forms linked to mutant genes encoding altered RNA/protein products have also been described. Several different mechanisms have been found altered or dysfunctional in MNDs, like the protein quality control (PQC) system. In this review, we will discuss how the PQC system is affected in two MNDs-spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS)-and how this affects the clearance of aberrantly folded proteins, which accumulate in motor neurons, inducing dysfunctions and their death. In addition, we will discuss how the PQC system can be targeted to restore proper cell function, enhancing the survival of affected cells in MNDs.

Multiple Roles of Transforming Growth Factor Beta in Amyotrophic Lateral Sclerosis.

Transforming growth factor beta (TGFB) is a pleiotropic cytokine known to be dysregulated in many neurodegenerative disorders and particularly in amyotrophic lateral sclerosis (ALS). This motor neuronal disease is non-cell autonomous, as it affects not only motor neurons but also the surrounding glial cells, and the target skeletal muscle fibers. Here, we analyze the multiple roles of TGFB in these cell types, and how TGFB signaling is altered in ALS tissues. Data reported support a crucial involvement of TGFB in the etiology and progression of ALS, leading us to hypothesize that an imbalance of TGFB signaling, diminished at the pre-symptomatic stage and then increased with time, could be linked to ALS progression. A reduced stimulation of the TGFB pathway at the beginning of disease blocks its neuroprotective effects and promotes glutamate excitotoxicity. At later disease stages, the persistent activation of the TGFB pathway promotes an excessive microglial activation and strengthens muscular dysfunction. The therapeutic potential of TGFB is discussed, in order to foster new approaches to treat ALS.

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 protein quality 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 Role of Sex and Sex Hormones in Neurodegenerative Diseases.

Neurodegenerative diseases (NDs) are a wide class of disorders of the central nervous system (CNS) with unknown etiology. Several factors were hypothesized to be involved in the pathogenesis of these diseases, including genetic and environmental factors. Many of these diseases show a sex prevalence and sex steroids were shown to have a role in the progression of specific forms of neurodegeneration. Estrogens were reported to be neuroprotective through their action on cognate nuclear and membrane receptors, while adverse effects of male hormones have been described on neuronal cells, although some data also suggest neuroprotective activities. The response of the CNS to sex steroids is a complex and integrated process that depends on (i) the type and amount of the cognate steroid receptor and (ii) the target cell type-either neurons, glia, or microglia. Moreover, the levels of sex steroids in the CNS fluctuate due to gonadal activities and to local metabolism and synthesis. Importantly, biochemical processes involved in the pathogenesis of NDs are increasingly being recognized as different between the two sexes and as influenced by sex steroids. The aim of this review is to present current state-of-the-art understanding on the potential role of sex steroids and their receptors on the onset and progression of major neurodegenerative disorders, namely, Alzheimer's disease, Parkinson's diseases, amyotrophic lateral sclerosis, and the peculiar motoneuron disease spinal and bulbar muscular atrophy, in which hormonal therapy is potentially useful as disease modifier.

HSC70 expression is reduced in lymphomonocytes of sporadic ALS patients and contributes to TDP-43 accumulation.

Aim: The demonstration that chaperone-mediated autophagy (CMA) contributes to the degradation of TDP-43, the main constituent of cytoplasmic inclusions typically found in motor neurons of patients with sporadic amyotrophic lateral sclerosis (sALS), has pointed out a possible involvement of CMA in aggregate formation. To explore this possibility, in this study, we verified the presence of a possible systemic CMA alteration in sALS patients and its effect on TDP-43 expression. Materials and methods: Gene and protein expression of the cytosolic chaperone HSC70 and the lysosome receptor LAMP2A, the two pivotal mediators of CMA, was assessed in peripheral blood mononuclear cells (PBMCs) derived from 30 sALS patients and 30 healthy controls. The expression of TDP-43 and co-chaperones BAG1 and BAG3 was also analyzed. Results: We found reduced HSC70 expression in patient cells, with no change in LAMP2A, and increased insoluble TDP-43 protein levels, with an aberrant intracellular localization. We also observed an unbalanced expression of co-chaperones BAG1 and BAG3. HSC70 down-regulation was confirmed in immortalized lymphoblastoid cell lines derived from sporadic and TARDBP mutant ALS patients. Lastly, we demonstrated that HSC70 silencing directly increases TDP-43 protein levels in human neuroblastoma cells. Discussion: Our results do not support the existence of a systemic CMA alteration in sALS patients but indicate a direct involvement of HSC70 alterations in ALS pathogenesis.

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.

Differential effects of red yeast rice, Berberis aristata and Morus alba extracts on PCSK9 and LDL uptake.

BACKGROUND AND AIMS: The novel nutraceutical combination containing red yeast rice (monacolin K 3.3 mg), Berberis aristata cortex extract (Berberine 531.25 mg) and Morus alba leaves extract (1-deoxynojirimycin 4 mg) is effective in the management of elevated plasma low-density lipoprotein cholesterol (LDL-C) levels. The aim of the present study was to investigate the effects of the three components on proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of LDL receptor (LDLR) expression, in hepatocyte cell lines and to compare their effects on LDL cellular uptake. METHODS AND RESULTS: HepG2 and Huh7 cells were incubated with B. aristata cortex extract (BCE), red yeast rice (RYR) and M. alba leaves extract (MLE) alone or in combination for 24 h. RYR (50 µg/mL) increased PCSK9 protein expression (Western blot analysis and ELISA), PCSK9 mRNA (qPCR) and its promoter activity (luciferase reporter assay). BCE (40 µg/mL) reduced instead PCSK9 expression, mRNA levels and promoter activity. MLE determined a concentration-dependent reduction of PCSK9 at the mRNA and protein levels, with a maximal reduction at 1 mg/mL, without significant changes of PCSK9 promoter activity. MLE also downregulated the expression of 3-hydroxy-3-methyl-3-glutaryl coenzyme A reductase and fatty acid synthase mRNA levels. The combination of RYR, BCE and MLE reduced the PCSK9 mRNA and protein levels, as well as the promoter activity. Finally, the single components and their combination induced LDL receptor and LDL uptake by the hepatocytes. CONCLUSION: The positive effect of MLE on PCSK9 supports the rationale of using the nutraceutical combination of RYR, BCE and MLE to control hyperlipidemic conditions.