1,721,180 research outputs found
Alteration of the protein quality control system in motor neuron and muscle expressing mutant proteins causing ALS and SBMA
No full textMotor neuronal system and muscle tissue are two districts differentially affected at onset and during the progression of diseases like amyotrophic lateral sclerosis (ALS) or spinal and bulbar muscular atrophy (SBMA).
The bases of these diseases are linked to mutated proteins: fALS is commonly caused by mutations in the SOD1 or the TDP43 genes and SBMA is caused by a of CAG repeat in the AR gene. A fraction of these proteins can not reach a mature conformation and misfolds. The Protein Quality Control (PQC) system is responsible for the correct protein homeostasis: the chaperones maintain proteins in their correct conformations. If they fail, mutated proteins are directed to the proteasome or the macroautophagy. When misfolded proteins are not correctly removed, they aggregate in nucleus and cytoplasm. To understand cellular behavior in presence of misfolded toxic proteins we investigate the different activation of the PQC system in the two mayor tissues involved.
Initially we investigated the differences in PQC activation between NSC34 motor neuronal and C2C12 muscular cell models. Using RTq PCR, western blot and immunocytochemical analysis for p62 and LC3 expression, localization, and turnover we demonstrated that C2C12 cells have a more active autophagic system than NSC34 cells.
Then, we compared the two models in presence of misfolded protein inhibiting degradative systems. With Filter Retardation Assay, we observed that these proteins tend to aggregate when PQC system is impaired. Then, we potentiated the PQC response to reduce the insoluble species. By overexpressing the small heat shock protein B8 in both systems we demonstrated that AR polyQ and SODG93A insoluble species were reduced.
Also autophagy activation by trehalose caused a reduction in protein aggregation in both cell models.
In conclusion misfolded protein aggregates can be reduced by modulating macroautophagy and this could represent a new therapeutical strategy for fatal disease like SBMA and ALS
Involvement of the ubiquitin-proteasome pathway and autophagy in the clearence of mutant androgen receptor in the spinal and bulbar muscular atrophy
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Motorneuron degeneration in Spinal and Bulbar Muscular Atrophy: from the molecular mechanisms to the potential therapeutical approaches
No full textSpinobulbar muscular atrophy (SBMA) is an X-linked motorneuronal disease, caused by a polyglutamine (polyQ) elongation in the androgen receptor (AR) protein, which induces motor neuron loss in the spinal cord and in the bulbar region. In SBMA, the mutant ARpolyQ become neurotoxic after its interaction with the physiological ligand testosterone (T). The binding with T, is known to induce AR activation, dimerization and translocation into the nucleus. In the ARpolyQ, this mechanism is possibly associated with the acquisition of aberrant conformations (misfolding) and T induces ARpolyQ aggregation. The aggregates contain components of the ubiquitin/proteasome pathway (UPS) and Heat shock proteins (Hsps), suggesting a possibile involvement of UPS in SBMA.
Using SBMA motor neuronal cell models (NSC34) expressing wt and mutant AR, we found that the non-activated and soluble ARpolyQ impaired UPS activity. T-activated ARpolyQ aggregated resulting in an improvement of UPS activity. Thus ARpolyQ aggregates might serve to segregate misfolded species into physically defined intracellular compartments (the aggregates) waiting for an alternative degradative pathway, such as autophagy. This was confirmed by analysing the intracellular distribution of the autophagic marker LC3. In fact, in presence of the T-activated mutant ARpolyQ, LC3 showed an intense punctate staining, suggesting that autophagy is involved in the clearance of this mutant protein.
We then used two different approaches to counteract ARpolyQ toxicity by increasing its clearance via the two intracellular proteolytic systems.
In the first approach, we overexpressed the small HspB8, and found that this chaperone decreased the intracellular levels of the mutant ARpolyQ and completely counteracted ARpolyQ aggregation. We demonstrated that HspB8 acts without affecting the UPP, but facilitates the ARpolyQ clearance via the autophagic pathway. In fact, HspB8 stimulated the LC3-II formation, and increased the number of autophagosomes. LC3 silencing correlated with the loss of HspB8 prodegradative effects on ARpolyQ. HspB8 action appeared to be mediated by a heterocomplex with other chaperones, as Bag3, Hsc70 and CHIP.
In the scond approach, we analysed the effects of the Hsp90 inhibitor 17-Allylamino-17-demethoxygeldanamycin (17-AAG) on ARpolyQ degradation. 17-AAG has already been found capable to reduce ARpolyQ toxicity in SBMA transgenic mice. In our SBMA model, 17-AAG reduced ARpolyQ aggregation by stimulation ARpolyQ clearance without altering the UPS activity. 17-AAG induced LC3 expression, increased LC3 turnover and LC3 punctate distribution both in absence and in presence of T. Moreover, 17-AAG action is blocked both by pharmacological autophagic inhibition (3-MA) and by LC3-silencing, suggesting a relevant role for 17-AAG in the induction of autophagy.
Together these data suggest that compounds capable to potentiate ARpolyQ clearance via autophagy may have useful application in SBMA
PHARMACOLOGICAL AND MOLECULAR APPROACHES TO REMOVE MUTANT MISFOLDED ANDROGEN RECEPTOR IN SPINAL AND BULBAR MUSCULAR ATROPHY
No full textEXPRESSION MODULATION OF A SMALL HEAT SHOCK PROTEIN INVOLVED IN NEURODEGENERATIVE DISEASES
No full text17-(ALLYLAMINO)-17-DEMETHOXYGELDANAMYCIN (17 AAG) A PHARMACOLOGICAL APPROACH TO INDUCE DEGRADATION OF MISFOLDED PROTEINS
No full textThe protein quality control in spinal and bulbar muscular atrophy
No full textThe protein misfolding and aggregation, typical of several neurodegenerative disease, are the results of an impairment of Protein Quality Control system (PQC). The PQC is composed by molecular chaperones, the Ubiquitin Proteasome System (UPS) and the autophagic pathway. Understanding the link between neurodegenerative diseases and the PQC might help to identify potential target for these diseases. To this purpose, we have studied the contribution of PQC in a motoneuron disease, the spinal and bulbar muscular atrophy (SBMA). SBMA is caused by a polyglutamine-expanded tract (polyQ) in the androgen receptor protein (AR). The binding of the ligand testosterone to the ARpolyQ induces protein misfolding and aggregation. The expanded polyQ tract confers a toxic gain-of-function to mutant AR that alters a cascade of several downstream pathways, including the PQC.
Using SBMA motoneuronal cell line, we have already demonstrated that both the UPS and the autophagic pathway are altered or impaired.
Thus, we studied the ability of different compounds of promoting ARpolyQ clearance through the potentiation of the PQC activity. We found that a combination of an autophagic inducer, trehalose, with an anti-androgen Bicalutamide, have synergic activity in the autophagic clearance of ARpolyQ., Also Berberine, a natural compound, was able to induce the ARpolyQ clearance possibly through the UPS.
Moreover, using a SBMA knock-in mice model, we observed that in the spinal cord and in the skeletal muscle, the primary sites of pathogenesis, the UPS and the autophagic pathway seem to respond with a different degradative power to the ARpolyQ toxicity.
These data suggest that the up-regulation of PQC may be beneficial for the treatment of SBMA, and these compounds might represent therapeutic candidates in SBMA and in other misfolding-related diseases.
GRANTS: Fondazione AriSLA; Fondazione Cariplo; AFM Telethon France; Regione Lombardia; UNIMI; Telethon Italy
THE INVOLVEMENT OF SMALL HEAT SHOCK PROTEIN B8 IN THE DEGRADATION OF MUTANT PROTEINS IN MOTOR NEURONAL DISEASE
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