Neratinib is a TFEB and TFE3 activator that potentiates autophagy and unbalances energy metabolism in ERBB2+ breast cancer cells.

Neratinib (NE) is an irreversible pan-ERBB tyrosine kinase inhibitor used to treat breast cancers (BCa) with amplification of the ERBB2/HER2/Neu gene or overexpression of the ERBB2 receptor. However, the mechanisms behind this process are not fully understood. Here we investigated the effects of NE on critical cell survival processes in ERBB2+ cancer cells. By kinome array analysis, we showed that NE time-dependently inhibited the phosphorylation of two distinct sets of kinases. The first set, including ERBB2 downstream signaling kinases such as ERK1/2, ATK, and AKT substrates, showed inhibition after 2 h of NE treatment. The second set, which comprised kinases involved in DNA damage response, displayed inhibition after 72 h. Flow cytometry analyses showed that NE induced G0/G1 cell cycle arrest and early apoptosis. By immunoblot, light and electron microscopy, we revealed that NE also transiently induced autophagy, mediated by increased expression levels and nuclear localization of TFEB and TFE3. Altered TFEB/TFE3 expression was accompanied by dysregulation of mitochondrial energy metabolism and dynamics, leading to a decrease in ATP production, glycolytic activity, and a transient downregulation of fission proteins. Increased TFEB and TFE3 expression was also observed in ERBB2-/ERBB1 + BCa cells, supporting that NE may act through other ERBB family members and/or other kinases. Overall, this study highlights NE as a potent activator of TFEB and TFE3, leading to the suppression of cancer cell survival through autophagy induction, cell cycle arrest, apoptosis, mitochondrial dysfunction and inhibition of DNA damage response.

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.

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.

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.

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.

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.

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.

Transforming growth factor beta 1 signaling is altered in the spinal cord and muscle of amyotrophic lateral sclerosis mice and patients.

Gender differences characterize amyotrophic lateral sclerosis (ALS). Because ALS patients have increased circulating levels of transforming growth factor beta 1 (TGFB1), here we analyzed gender and disease progression-related modification of TGFB1 and its related signaling molecules in the spinal cord and skeletal muscle of ALS mice and in muscle biopsies from sporadic ALS patients. At presymptomatic stage, Tgfb1 mRNA expression is reduced in the mouse spinal cord but is increased selectively in the male skeletal muscle. At symptomatic stage, it is induced both in the mouse spinal cord and muscle, as well as in the muscle of ALS patients. Tgfbr2 levels are induced only in the mouse spinal cord. Smad2 and Smad4 mRNAs are decreased in the mouse spinal cord and muscle, but SMAD2 protein levels are augmented selectively in the male mouse muscle. Smad3 mRNA and SMAD3 protein are increased in the mouse muscle. The expression of genes controlled by TGFB1 in the muscle (Pax7, Collagen1a1, and Fibronectin) are reduced both in male and female ALS mice at symptomatic stage. Thus, TGFB1 modulation may serve as a novel therapeutic target for ALS.

The Regulation of the Small Heat Shock Protein B8 in Misfolding Protein Diseases Causing Motoneuronal and Muscle Cell Death.

Misfolding protein diseases are a wide class of disorders in which the aberrantly folded protein aggregates accumulate in affected cells. In the brain and in the skeletal muscle, misfolded protein accumulation induces a variety of cell dysfunctions that frequently lead to cell death. In motoneuron diseases (MNDs), misfolded proteins accumulate primarily in motoneurons, glial cells and/or skeletal muscle cells, altering motor function. The deleterious effects of misfolded proteins can be counteracted by the activity of the protein quality control (PQC) system, composed of chaperone proteins and degradative systems. Here, we focus on a PQC system component: heat shock protein family B (small) member 8 (HSPB8), a chaperone induced by harmful stressful events, including proteotoxicity. In motoneuron and muscle cells, misfolded proteins activate HSPB8 transcription and enhance HSPB8 levels, which contributes to prevent aggregate formation and their harmful effects. HSPB8 acts not only as a chaperone, but also facilitates the autophagy process, to enable the efficient clearance of the misfolded proteins. HSPB8 acts as a dimer bound to the HSP70 co-chaperone BAG3, a scaffold protein that is also capable of binding to HSP70 (associated with the E3-ligase CHIP) and dynein. When this complex is formed, it is transported by dynein to the microtubule organization center (MTOC), where aggresomes are formed. Here, misfolded proteins are engulfed into nascent autophagosomes to be degraded via the chaperone-assisted selective autophagy (CASA). When CASA is insufficient or impaired, HSP70 and CHIP associate with an alternative co-chaperone, BAG1, which routes misfolded proteins to the proteasome for degradation. The finely tuned equilibrium between proteasome and CASA activity is thought to be crucial for maintaining the functional cell homeostasis during proteotoxic stresses, which in turn is essential for cell survival. This fine equilibrium seems to be altered in MNDs, like Amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA), contributing to the onset and the progression of disease. Here, we will review how misfolded proteins may affect the PQC system and how the proper activity of this system can be restored by boosting or regulating HSPB8 activity, with the aim to ameliorate disease progression in these two fatal MNDs.

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 pro-degradative 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. Abbreviations: ALS: amyotrophic lateral sclerosis; AR: androgen receptor; ATG: autophagy related; AV: autophagic vacuole; BAG3: BCL2-associated athanogene 3; BECN1: beclin 1, autophagy related; CASA: chaperone-assisted selective autophagy; CTSB: cathepsin b; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; EGFP: enhanced green fluorescent protein; fALS, familial amyotrophic lateral sclerosis; FRA: filter retardation assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLA: galactosidase, alpha; HD: Huntington disease; hIPSCs: human induced pluripotent stem cells; HSPA8: heat shock protein A8; HSPB8: heat shock protein B8; IF: immunofluorescence analysis; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LGALS3: lectin, galactose binding, soluble 3; LLOMe: L-leucyl-L-leucine methyl ester; LMP: lysosomal membrane permeabilization; Lys: lysosomes; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MCOLN1: mucolipin 1; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; NDs: neurodegenerative diseases; NSC34: neuroblastoma x spinal cord 34; PBS: phosphate-buffered saline; PD: Parkinson disease; polyQ: polyglutamine; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PPP3CB: protein phosphatase 3, catalytic subunit, beta isoform; RT-qPCR: real-time quantitative polymerase chain reaction; SBMA: spinal and bulbar muscular atrophy; SCAs: spinocerebellar ataxias; siRNA: small interfering RNA; SLC2A8: solute carrier family 2, (facilitated glucose transporter), member 8; smNPCs: small molecules neural progenitors cells; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STUB1: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TFEB: transcription factor EB; TPP1: tripeptidyl peptidase I; TREH: trehalase (brush-border membrane glycoprotein); WB: western blotting; ZKSCAN3: zinc finger with KRAB and SCAN domains 3.

Concurrent AFG3L2 and SPG7 mutations associated with syndromic parkinsonism and optic atrophy with aberrant OPA1 processing and mitochondrial network fragmentation.

Mitochondrial dynamics and quality control are crucial for neuronal survival and their perturbation is a major cause of neurodegeneration. m-AAA complex is an ATP-dependent metalloprotease located in the inner mitochondrial membrane and involved in protein quality control. Mutations in the m-AAA subunits AFG3L2 and paraplegin are associated with autosomal dominant spinocerebellar ataxia (SCA28) and autosomal recessive hereditary spastic paraplegia (SPG7), respectively. We report a novel m-AAA-associated phenotype characterized by early-onset optic atrophy with spastic ataxia and L-dopa-responsive parkinsonism. The proband carried a de novo AFG3L2 heterozygous mutation (p.R468C) along with a heterozygous maternally inherited intragenic deletion of SPG7. Functional analysis in yeast demonstrated the pathogenic role of AFG3L2 p.R468C mutation shedding light on its pathogenic mechanism. Analysis of patient's fibroblasts showed an abnormal processing pattern of OPA1, a dynamin-related protein essential for mitochondrial fusion and responsible for most cases of hereditary optic atrophy. Consistently, assessment of mitochondrial morphology revealed a severe fragmentation of the mitochondrial network, not observed in SCA28 and SPG7 patients' cells. This case suggests that coincidental mutations in both components of the mitochondrial m-AAA protease may result in a complex phenotype and reveals a crucial role for OPA1 processing in the pathogenesis of neurodegenerative disease caused by m-AAA defects.

Tdp-25 Routing to Autophagy and Proteasome Ameliorates its Aggregation in Amyotrophic Lateral Sclerosis Target Cells.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motoneurons, while non-neuronal cells may contribute to disease onset and progression. Most ALS cases are characterized by the mislocalization and aggregation of the TAR DNA binding protein 43 (TDP-43) in affected cells. TDP-43 aggregates contain C-terminal TDP-43 fragments of 35 kDa (TDP-35) and 25 kDa (TDP-25) and have been mainly studied in motoneurons, while little is currently known about their rate of accumulation and clearance in myoblasts. Here, we performed a comparative study in immortalized motoneuronal like (NSC34; i-motoneurons) cells and stabilized myoblasts (C2C12; s-myoblasts) to evaluate if these two cell types differentially accumulate and clear TDP forms. The most aggregating specie in i-motoneurons is the TDP-25 fragment, mainly constituted by the "prion-like" domain of TDP-43. To a lower extent, TDP-25 also aggregates in s-myoblasts. In both cell types, all TDP species are cleared by proteasome, but TDP-25 impairs autophagy. Interestingly, the routing of TDP-25 fragment to proteasome, by overexpressing BAG1, or to autophagy, by overexpressing HSPB8 or BAG3 decreased its accumulation in both cell types. These results demonstrate that promoting the chaperone-assisted clearance of ALS-linked proteins is beneficial not only in motoneurons but also in myoblasts.