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.

MicroRNAs are a class of sophisticated regulators of gene expression, acting as post-transcriptional inhibitors that recognize their target mRNAs through base pairing with short regions along the 3UTRs. Several microRNAs are tissue specific, suggesting a specialized role in tissue differentiation or maintenance, and quite a few are critically involved in tumorigenesis. We studied miR-128, a brain-enriched microRNA, in retinoic acid-differentiated neuroblastoma cells, and we found that this microRNA is up-regulated in treated cells, where it down-modulates the expression of two proteins involved in the migratory potential of neural cells: Reelin and DCX. Consistently, miR-128 ectopic overexpression suppressed Reelin and DCX, whereas the LNA antisensemediated miR-128 knockdown caused the two proteins to increase. Ectopic miR-128 overexpression reduced neuroblastoma cell motility and invasiveness, and impaired cell growth. Finally, the analysis of a small series of primary human neuroblastomas showed an association between high levels of miR-128 expression and favorable features, such as favorable Shimada category or very young age at diagnosis. Thus, we provide evidence for a role for miR-128 in the molecular events modulating neuroblastoma progression and aggressiveness.-Evangelisti, C., Florian, M. C., Massimi, I., Dominici, C., Giannini, G., Galardi, S., Buè, M. C., Massalini, S., McDowell, H. P., Messi, E., Gulino, A., Farace, M. G., Ciafrè, S. A. MiR-128 up-regulation inhibits Reelin and DCX expression and reduces neuroblastoma cell motility and invasiveness. FASEB J. 23, 4276 -4287 (2009). www.fasebj.org

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.

. We identified two compounds, colchicine and doxorubicin, that robustly up-regulated HSPB8 expression. Both colchicine and doxorubicin increased the expression of the master regulator of autophagy TFEB, the autophagy linker p62/SQSTM1 and the autophagosome component LC3. In line, both drugs counteracted the accumulation of TDP-43 and TDP-25 misfolded species responsible for motoneuronal death in sALS. Thus, analogs of colchicine and doxorubicin able to induce HSPB8 and with better safety and tolerability may result beneficial in NDs models.Different human diseases have been associated with an insufficient or an excessive intracellular response to proteotoxic stress. Proteins that lose the capability to acquire and/or to maintain the proper folding, after genetic mutations or damages in their structures (e.g.: specific amino acids modifications due to free radical exposure, etc.), become aberrantly folded or misfolded and can trigger a proteotoxic stress 1,2 . Misfolded proteins may oligomerize into aggregates that accumulate in cells. Misfolded proteins are potentially toxic by affecting several fundamental intracellular functions, either/both in their monomeric or/and in their aggregated forms, particularly in post-mitotic cells like neurons 3,4 . For these reasons, misfolded proteins are physiologically cleared from the cells by the protein quality control (PQC) system. The PQC system is composed of two main arms: the molecular chaperone, mainly represented by the heat shock proteins (HSPs) 5-7 and the degradative pathways, including the proteasome, the autophagic response and the unfolded protein response (UPR) 8,9 . A specific degradative pathway for a given misfolded protein is selected by defined class of chaperones, with the assistance of co-chaperones 5 .

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.