RESUMO
The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, but its role in disease pathogenesis is unknown. Here, we show alterations in glucose metabolic pathways and ATP levels in the brains of asymptomatic C9-BAC mice. We find that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also show that one of the arginine-rich DPRs (PR) could directly contribute to glucose metabolism and metabolic stress by inhibiting glucose uptake in neurons. Our findings provide a potential mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and suggest a feedforward loop model with potential opportunities for therapeutic intervention.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Demência Frontotemporal , Glucose , Fenótipo , Proteína ran de Ligação ao GTP , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Modelos Animais de Doenças , Expansão das Repetições de DNA/genética , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Glucose/metabolismo , Camundongos Transgênicos , Neurônios/metabolismo , Biossíntese de Proteínas , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder whose exact causative mechanisms are still under intense investigation. Several lines of evidence suggest that the anatomical and temporal propagation of pathological protein species along the neural axis could be among the main driving mechanisms for the fast and irreversible progression of ALS pathology. Many ALS-associated proteins form intracellular aggregates as a result of their intrinsic prion-like properties and/or following impairment of the protein quality control systems. During the disease course, these mutated proteins and aberrant peptides are released in the extracellular milieu as soluble or aggregated forms through a variety of mechanisms. Internalization by recipient cells may seed further aggregation and amplify existing proteostatic imbalances, thus triggering a vicious cycle that propagates pathology in vulnerable cells, such as motor neurons and other susceptible neuronal subtypes. Here, we provide an in-depth review of ALS pathology with a particular focus on the disease mechanisms of seeding and transmission of the most common ALS-associated proteins, including SOD1, FUS, TDP-43, and C9orf72-linked dipeptide repeats. For each of these proteins, we report historical, biochemical, and pathological evidence of their behaviors in ALS. We further discuss the possibility to harness pathological proteins as biomarkers and reflect on the implications of these findings for future research.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Humanos , Dobramento de Proteína , Proteína FUS de Ligação a RNA/metabolismo , Superóxido Dismutase-1/metabolismoRESUMO
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.
Assuntos
Berberina/farmacologia , Produtos Biológicos/farmacologia , Síndromes Neurotóxicas/tratamento farmacológico , Deficiências na Proteostase/tratamento farmacológico , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular , Demência Frontotemporal/tratamento farmacológico , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Humanos , Chaperonas Moleculares/genética , Proteínas Mutantes/genética , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/patologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/genética , Deficiências na Proteostase/genética , Deficiências na Proteostase/patologiaRESUMO
Aggregation of TAR-DNA-binding protein 43 (TDP-43) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragments of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. We report that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in Drosophila melanogaster Overexpression of HSP67Bc, the functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, we report a novel Drosophila model expressing TDP-35, and show that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively, our data demonstrate that HSPB8 upregulation mitigates TDP-43 fragment mediated toxicity, in mammalian neuronal cells and flies.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas de Choque Térmico/genética , Fragmentos de Peptídeos/genética , Proteínas Serina-Treonina Quinases/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/genética , Olho/crescimento & desenvolvimento , Olho/fisiopatologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/biossíntese , Humanos , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Fragmentos de Peptídeos/metabolismo , Agregação Patológica de Proteínas/genética , Proteínas Serina-Treonina Quinases/biossíntese , Pupa/genética , Pupa/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Atrofia Bulboespinal Ligada ao X , Atrofia Muscular Espinal , Camundongos , Animais , Atrofia Bulboespinal Ligada ao X/tratamento farmacológico , Atrofia Bulboespinal Ligada ao X/genética , Trealose/farmacologia , Trealose/uso terapêutico , Receptores Androgênicos/genética , Anilidas/farmacologia , Camundongos TransgênicosRESUMO
Translation of the hexanucleotide G4C2 expansion associated with C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) produces five different dipeptide repeat protein (DPR) species that can confer toxicity. There is yet much to learn about the contribution of a single DPR to disease pathogenesis. We show here that a short repeat length is sufficient for the DPR poly-GR to confer neurotoxicity in vitro, a phenomenon previously unobserved. This toxicity is also reported in vivo in our novel knock-in mouse model characterized by widespread central nervous system (CNS) expression of the short-length poly-GR. We observe sex-specific chronic ALS/FTD-like phenotypes in these mice, including mild motor neuron loss, but no TDP-43 mis-localization, as well as motor and cognitive impairments. We suggest that this model can serve as the foundation for phenotypic exacerbation through second-hit forms of stress.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/metabolismo , Animais , Arginina/genética , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Dipeptídeos/metabolismo , Feminino , Demência Frontotemporal/patologia , Glicina/genética , Masculino , Camundongos , FenótipoRESUMO
A G4C2 hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of ALS and FTLD (C9-ALS/FTLD) with cytoplasmic TDP-43 inclusions observed in regions of neurodegeneration. The accumulation of repetitive RNAs and dipeptide repeat protein (DPR) are two proposed mechanisms of toxicity in C9-ALS/FTLD and linked to impaired nucleocytoplasmic transport. Nucleocytoplasmic transport is regulated by the phenylalanine-glycine nucleoporins (FG nups) that comprise the nuclear pore complex (NPC) permeability barrier. However, the relationship between FG nups and TDP-43 pathology remains elusive. Our studies show that nuclear depletion and cytoplasmic mislocalization of one FG nup, NUP62, is linked to TDP-43 mislocalization in C9-ALS/FTLD iPSC neurons. Poly-glycine arginine (GR) DPR accumulation initiates the formation of cytoplasmic RNA granules that recruit NUP62 and TDP-43. Cytoplasmic NUP62 and TDP-43 interactions promotes their insolubility and NUP62:TDP-43 inclusions are frequently found in C9orf72 ALS/FTLD as well as sporadic ALS/FTLD postmortem CNS tissue. Our findings indicate NUP62 cytoplasmic mislocalization contributes to TDP-43 proteinopathy in ALS/FTLD.
Assuntos
Esclerose Lateral Amiotrófica , Degeneração Lobar Frontotemporal , Esclerose Lateral Amiotrófica/metabolismo , Proteína C9orf72/genética , Expansão das Repetições de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dipeptídeos/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Glicina/genética , HumanosRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais/fisiologia , Medula Espinal/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Medula Espinal/patologia , Fator de Crescimento Transformador beta1/genéticaRESUMO
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.
RESUMO
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.
Assuntos
Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Calcineurina/metabolismo , Lisossomos/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Trealose/farmacologia , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/enzimologia , Autofagossomos/metabolismo , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Atrofia Bulboespinal Ligada ao X/tratamento farmacológico , Atrofia Bulboespinal Ligada ao X/metabolismo , Calcineurina/genética , Cálcio/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Regulação para Baixo/genética , Humanos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios Motores/enzimologia , Neurônios Motores/ultraestrutura , Neuroproteção/efeitos dos fármacos , Neuroproteção/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Trealose/análogos & derivados , Tripeptidil-Peptidase 1 , Resposta a Proteínas não Dobradas/genéticaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Docetaxel/farmacologia , Mitofagia/fisiologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Trealose/farmacologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Linhagem Celular Tumoral , Citocromos c/metabolismo , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/biossíntese , Mitocôndrias/patologia , Mitofagia/efeitos dos fármacos , Células PC-3 , Proteínas de Ligação a RNA/biossíntese , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Autofagia , Proteínas de Ligação a DNA/metabolismo , Fragmentos de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Esclerose Lateral Amiotrófica/etiologia , Autofagia/genética , Proteínas de Ligação a DNA/genética , Humanos , Neurônios Motores/metabolismo , Células Musculares/metabolismo , Fragmentos de Peptídeos/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases in which similar pathogenic mechanisms are involved. Both diseases associate to the high propensity of specific misfolded proteins, like TDP-43 or FUS, to mislocalize and aggregate. This is partly due to their intrinsic biophysical properties and partly as a consequence of failure of the neuronal protein quality control (PQC) system. Several familial ALS/FTD cases are linked to an expansion of a repeated G4C2 hexanucleotide sequence present in the C9ORF72 gene. The G4C2, which localizes in an untranslated region of the C9ORF72 transcript, drives an unconventional repeat-associated ATG-independent translation. This leads to the synthesis of five different dipeptide repeat proteins (DPRs), which are not "classical" misfolded proteins, but generate aberrant aggregation-prone unfolded conformations poorly removed by the PQC system. The DPRs accumulate into p62/SQSTM1 and ubiquitin positive inclusions. Here, we analyzed the biochemical behavior of the five DPRs in immortalized motoneurons. Our data suggest that while the DPRs are mainly processed via autophagy, this system is unable to fully clear their aggregated forms, and thus they tend to accumulate in basal conditions. Overexpression of the small heat shock protein B8 (HSPB8), which facilitates the autophagy-mediated disposal of a large variety of classical misfolded aggregation-prone proteins, significantly decreased the accumulation of most DPR insoluble species. Thus, the induction of HSPB8 might represent a valid approach to decrease DPR-mediated toxicity and maintain motoneuron viability.
Assuntos
Proteína C9orf72/metabolismo , Dipeptídeos/metabolismo , Proteínas de Choque Térmico HSP20/metabolismo , Neurônios Motores/patologia , Doenças Neurodegenerativas/metabolismo , Agregados Proteicos , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Transformada , Inativação Gênica , Camundongos , Neurônios Motores/metabolismo , Doenças Neurodegenerativas/patologia , Inibidores de Proteassoma/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Perturbations in stress granule (SG) dynamics may be at the core of amyotrophic lateral sclerosis (ALS). Since SGs are membraneless compartments, modeling their dynamics in human motor neurons has been challenging, thus hindering the identification of effective therapeutics. Here, we report the generation of isogenic induced pluripotent stem cells carrying wild-type and P525L FUS-eGFP. We demonstrate that FUS-eGFP is recruited into SGs and that P525L profoundly alters their dynamics. With a screening campaign, we demonstrate that PI3K/AKT/mTOR pathway inhibition increases autophagy and ameliorates SG phenotypes linked to P525L FUS by reducing FUS-eGFP recruitment into SGs. Using a Drosophila model of FUS-ALS, we corroborate that induction of autophagy significantly increases survival. Finally, by screening clinically approved drugs for their ability to ameliorate FUS SG phenotypes, we identify a number of brain-penetrant anti-depressants and anti-psychotics that also induce autophagy. These drugs could be repurposed as potential ALS treatments.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Drosophila/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína FUS de Ligação a RNA/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Antidepressivos/farmacologia , Antipiréticos/farmacologia , Autofagia/genética , Sistemas CRISPR-Cas , Drosophila , Avaliação Pré-Clínica de Medicamentos , Proteínas de Fluorescência Verde/genética , Humanos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genéticaRESUMO
Breast cancer (BC) is one of the major causes of cancer death in women and is closely related to hormonal dysregulation. Estrogen receptor (ER)-positive BCs are generally treated with anti hormone therapy using antiestrogens or aromatase inhibitors. However, BC cells may become resistant to endocrine therapy, a process facilitated by autophagy, which may either promote or suppress tumor expansion. The autophagy facilitator HSPB8 has been found overexpressed in some BC. Here we found that HSPB8 is highly expressed and differentially modulated by natural or synthetic selective ER modulators (SERMs), in the triple-positive hormone-sensitive BC (MCF-7) cells, but not in triple-negative MDA-MB-231 BC cells. Specific SERMs induced MCF-7 cells proliferation in a HSPB8 dependent manner whereas, did not modify MDA-MB-231 cell growth. ER expression was unaffected in HSPB8-depleted MCF-7 cells. HSPB8 over-expression did not alter the distribution of MCF-7 cells in the various phases of the cell cycle. Conversely and intriguingly, HSPB8 downregulation resulted in an increased number of cells resting in the G0/G1 phase, thus possibly reducing the ability of the cells to pass through the restriction point. In addition, HSPB8 downregulation reduced the migratory ability of MCF-7 cells. None of these modifications were observed, when another small HSP (HSPB1), also expressed in MCF-7 cells, was downregulated. In conclusion, our data suggest that HSPB8 is involved in the mechanisms that regulate cell cycle and cell migration in MCF-7 cells.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Proteínas de Choque Térmico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico/genética , Células Hep G2 , Humanos , Células MCF-7 , Chaperonas Moleculares , Invasividade Neoplásica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
Motoneuron diseases, like spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS), are associated with proteins that because of gene mutation or peculiar structures, acquire aberrant (misfolded) conformations toxic to cells. To prevent misfolded protein toxicity, cells activate a protein quality control (PQC) system composed of chaperones and degradative pathways (proteasome and autophagy). Inefficient activation of the PQC system results in misfolded protein accumulation that ultimately leads to neuronal cell death, while efficient macroautophagy/autophagy-mediated degradation of aggregating proteins is beneficial. The latter relies on an active retrograde transport, mediated by dynein and specific chaperones, such as the HSPB8-BAG3-HSPA8 complex. Here, using cellular models expressing aggregate-prone proteins involved in SBMA and ALS, we demonstrate that inhibition of dynein-mediated retrograde transport, which impairs the targeting to autophagy of misfolded species, does not increase their aggregation. Rather, dynein inhibition correlates with a reduced accumulation and an increased clearance of mutant ARpolyQ, SOD1, truncated TARDBP/TDP-43 and expanded polyGP C9ORF72 products. The enhanced misfolded protein clearance is mediated by the proteasome, rather than by autophagy and correlates with the upregulation of the HSPA8 cochaperone BAG1. In line, overexpression of BAG1 increases the proteasome-mediated clearance of these misfolded proteins. Our data suggest that when the misfolded proteins cannot be efficiently transported toward the perinuclear region of the cells, where they are either degraded by autophagy or stored into the aggresome, the cells activate a compensatory mechanism that relies on the induction of BAG1 to target the HSPA8-bound cargo to the proteasome in a dynein-independent manner.
Assuntos
Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Dobramento de Proteína , Animais , Autofagia , Transporte Biológico , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Dineínas/metabolismo , Inativação Gênica , Proteínas de Choque Térmico HSP20/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Células PC12 , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Interferente Pequeno/metabolismo , Ratos , Superóxido Dismutase/metabolismo , Fatores de Transcrição , Ubiquitina/metabolismo , Regulação para CimaRESUMO
Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is an X-linked disease associated with the expansion of the CAG triplet repeat present in exon 1 of the androgen receptor (AR) gene. This results in the production of a mutant AR containing an elongated polyglutamine tract (polyQ) in its N-terminus. Interestingly, the ARpolyQ becomes toxic only after its activation by the natural androgenic ligands, possibly because of aberrant androgen-induced conformational changes of the ARpolyQ, which generate misfolded species. These misfolded ARpolyQ species must be cleared from motoneurons and muscle cells, and this process is mediated by the protein quality control (PQC) system. Experimental evidence suggested that failure of the PQC pathways occurs in disease, leading to ARpolyQ accumulation and toxicity in the target cells. In this review, we summarized the overall impact of mutant and misfolded ARpolyQ on the PQC system and described how molecular chaperones and the degradative pathways (ubiquitin-proteasome system (UPS), the autophagy-lysosome pathway (ALP), and the unfolded protein response (UPR), which activates the endoplasmic reticulum-associated degradation (ERAD)) are differentially affected in SBMA. We also extensively and critically reviewed several molecular and pharmacological approaches proposed to restore a global intracellular activity of the PQC system. Collectively, these data suggest that the fine and delicate equilibrium existing among the different players of the PQC system could be restored in a therapeutic perspective by the synergic/additive activities of compounds designed to tackle sequential or alternative steps of the intracellular defense mechanisms triggered against proteotoxic misfolded species.
Assuntos
Atrofia Bulboespinal Ligada ao X/metabolismo , Receptores Androgênicos/metabolismo , Resposta a Proteínas não Dobradas , Animais , Atrofia Bulboespinal Ligada ao X/genética , Humanos , Peptídeos/química , Receptores Androgênicos/química , Receptores Androgênicos/genética , Expansão das Repetições de TrinucleotídeosRESUMO
Spinal and bulbar muscular atrophy (SBMA) is characterized by loss of motoneurons and sensory neurons, accompanied by atrophy of muscle cells. SBMA is due to an androgen receptor containing a polyglutamine tract (ARpolyQ) that misfolds and aggregates, thereby perturbing the protein quality control (PQC) system. Using SBMA AR113Q mice we analyzed proteotoxic stress-induced alterations of HSPB8-mediated PQC machinery promoting clearance of misfolded proteins by autophagy. In muscle of symptomatic AR113Q male mice, we found expression upregulation of Pax-7, myogenin, E2-ubiquitin ligase UBE2Q1 and acetylcholine receptor (AchR), but not of MyoD, and of two E3-ligases (MuRF-1 and Cullin3). TGFß1 and PGC-1α were also robustly upregulated. We also found a dramatic perturbation of the autophagic response, with upregulation of most autophagic markers (Beclin-1, ATG10, p62/SQSTM1, LC3) and of the HSPB8-mediated PQC response. Both HSPB8 and its co-chaperone BAG3 were robustly upregulated together with other specific HSPB8 interactors (HSPB2 and HSPB3). Notably, the BAG3:BAG1 ratio increased in muscle suggesting preferential misfolded proteins routing to autophagy rather than to proteasome. Thus, mutant ARpolyQ induces a potent autophagic response in muscle cells. Alteration in HSPB8-based PQC machinery may represent muscle-specific biomarkers useful to assess SBMA progression in mice and patients in response to pharmacological treatments.