sirolimus and Protein-Aggregation--Pathological

Many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), are characterised by the intracellular appearance of protein aggregates or insoluble materials. Accelerated removal of related toxic proteins might be beneficial for these diseases. Here we describe an inducible role of Beclin1, an essential regulator for autophagy, in degradation of the familial ALS-linked Cu/Zn superoxide dismutase 1 (SOD1) mutant. We confirmed that the SOD1 mutant exhibited an increased RIPA (radioimmune precipitation assay buffer, containing NP40 and sodium deoxycholate)-insolubility compared with SOD1 wild-type (WT). Also, the insoluble fraction formed by SOD1 mutant was greatly reduced by coexpressing Beclin1 in both neuronal and non-neuronal cell lines. Pharmacological inhibition of autophagy diminished the effect of Beclin1 and resulted in an accumulation of insoluble SOD1. Our results support the role of Beclin1 in the involvement of autophagic degradation of SOD1 mutant. We propose Beclin1 enhances autophagy and presents a possible therapeutic strategy for familial ALS.

Topics: Ammonium Chloride; Amyotrophic Lateral Sclerosis; Animals; Autophagy; Beclin-1; Cells, Cultured; Humans; Mice; Mutation; Protein Aggregation, Pathological; Radioimmunoassay; Sirolimus; Solubility; Superoxide Dismutase; Superoxide Dismutase-1; Transfection; Up-Regulation

Cell-to-cell propagation of aggregated alpha synuclein (aSyn) has been suggested to play an important role in the progression of alpha synucleinopathies. A critical step for the propagation process is the accumulation of extracellular aSyn within recipient cells. Here, we investigated the trafficking of distinct exogenous aSyn forms and addressed the mechanisms influencing their accumulation in recipient cells. The aggregated aSyn species (oligomers and fibrils) exhibited more pronounced accumulation within recipient cells than aSyn monomers. In particular, internalized extracellular aSyn in the aggregated forms was able to seed the aggregation of endogenous aSyn. Following uptake, aSyn was detected along endosome-to-lysosome and autophagosome-to-lysosome routes. Intriguingly, aggregated aSyn resulted in lysosomal activity impairment, accompanied by the accumulation of dilated lysosomes. Moreover, analysis of autophagy-related protein markers suggested decreased autophagosome clearance. In contrast, the endocytic pathway, proteasome activity, and mitochondrial homeostasis were not substantially affected in recipient cells. Our data suggests that extracellularly added aggregated aSyn primarily impairs lysosomal activity, consequently leading to aSyn accumulation within recipient cells. Importantly, the autophagy inducer trehalose prevented lysosomal alterations and attenuated aSyn accumulation within aSyn-exposed cells. Our study underscores the importance of lysosomes for the propagation of aSyn pathology, thereby proposing these organelles as interventional targets.

Topics: alpha-Synuclein; Animals; Autophagy; Cell Line, Tumor; Escherichia coli; Glioma; Humans; Lysosomes; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Rats; Rats, Wistar; Recombinant Proteins; Sirolimus; Trehalose

Autophagy is an intracellular catabolic mechanism essential for recycling intracellular unfolding protein and eliminating toxic protein aggregates. Several studies have shown that deficient autophagy is implicated in the development of Alzheimer's disease (AD) progression. To date, rapidly emerging evidence suggests that neurosteroid progesterone (PG) may play an important role in ameliorating AD. However, the role of PG and its neuroprotective mechanism in regulating autophagy still require further investigation. Here, we investigated the protective effects of PG against Aβ-induced inflammatory responses in astrocytes and its underlying mechanism in mediating autophagy. Remarkably, Aβ induced astrocyte dysfunction in autophagic activation and up-regulated inflammatory secretion. However, the autophagy inducer rapamycin (RAPA) significantly suppressed Aβ-induced inflammation in astrocytes. In astrocytes, treatment with Aβ caused autophagy deficiency, whereas PG significantly increased autophagy activation. Finally, PG suppressed Aβ-induced neuroinflammatory production via enhancing autophagy together with regulating mTOR signaling. Taken together, these results show that autophagy is a vital mechanism against Aβ-induced neuroinflammatory responses in astrocytes and demonstrate the potential neuroprotective mechanism of PG in suppressing neuroinflammatory responses by enhancing autophagy. Therefore, uncovering the neuroprotective mechanism of PG may provide new insight into novel therapies for the amelioration of AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Astrocytes; Autophagy; Cells, Cultured; Humans; Neurogenic Inflammation; Peptide Fragments; Progesterone; Protein Aggregation, Pathological; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases