SUMMARY Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer’s disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles. The molecular mechanisms leading to tau accumulation remain unclear and more needs to be done to elucidate them. Age is a major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Here, we use multiple animal models and complementary genetic and pharmacological approaches to show that the mammalian target of rapamycin (mTOR) regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Mechanistically, we provide compelling evidence that the association between mTOR and tau is linked to GSK3β and autophagy function. In summary, we show that increasing mTOR signaling facilitates tau pathology while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence showing that reducing mTOR signaling increases lifespan and health span, the data presented here have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide pre-clinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies.

Cancer and neurodegeneration are different classes of diseases that share the involvement of mitochondria in their pathogenesis. Whereas the high glycolytic rate (the so-called Warburg metabolism) and the suppression of apoptosis are key elements for the establishment and maintenance of cancer cells, mitochondrial dysfunction and increased cell death mark neurodegeneration. As a main actor in the regulation of cell metabolism and apoptosis, VDAC may represent the common point between these two broad families of pathologies. Located in the outer mitochondrial membrane, VDAC forms channels that control the flux of ions and metabolites across the mitochondrion thus mediating the organelle's cross-talk with the rest of the cell. Furthermore, the interaction with both pro-apoptotic and anti-apoptotic factors makes VDAC a gatekeeper for mitochondria-mediated cell death and survival signaling pathways. Unfortunately, the lack of an evident druggability of this protein, since it has no defined binding or active sites, makes the quest for VDAC interacting molecules a difficult tale. Pharmacologically active molecules of different classes have been proposed to hit cancer and neurodegeneration. In this work, we provide an exhaustive and detailed survey of all the molecules, peptides, and microRNAs that exploit VDAC in the treatment of the two examined classes of pathologies. The mechanism of action and the potential or effectiveness of each compound are discussed.

Reducing the mammalian target of rapamycin (mTOR) activity increases lifespan and health span in a variety of organisms. Alterations in protein homeostasis and mTOR activity and signaling have been reported in several neurodegenerative disorders, including Alzheimer disease (AD); however, the causes of such deregulations remain elusive. Here, we show that mTOR activity and signaling are increased in cell lines stably transfected with mutant amyloid precursor protein (APP) and in brains of 3xTg-AD mice, an animal model of AD. In addition, we show that in the 3xTg-AD mice, mTOR activity can be reduced to wild type levels by genetically preventing A␤ accumulation. Similarly, intrahippocampal injections of an anti-A␤ antibody reduced A␤ levels and normalized mTOR activity, indicating that high A␤ levels are necessary for mTOR hyperactivity in 3xTg-AD mice. We also show that the intrahippocampal injection of naturally secreted A␤ is sufficient to increase mTOR signaling in the brains of wild type mice. The mechanism behind the A␤-induced mTOR hyperactivity is mediated by the proline-rich Akt substrate 40 (PRAS40) as we show that the activation of PRAS40 plays a key role in the A␤-induced mTOR hyperactivity. Taken together, our data show that A␤ accumulation, which has been suggested to be the culprit of AD pathogenesis, causes mTOR hyperactivity by regulating PRAS40 phosphorylation. These data further indicate that the mTOR pathway is one of the pathways by which A␤ exerts its toxicity and further support the idea that reducing mTOR signaling in AD may be a valid therapeutic approach.Amyloid plaques and neurofibrillary tangles are hallmark neuropathological lesions of Alzheimer disease (AD), 3 the most common form of neurodegenerative disorder (1). Neurofibrillary tangles are intracellular inclusions formed of hyperphosphorylated Tau (2-4). Plaques are extracellular inclusions mainly formed of a small peptide called amyloid-␤ (A␤) (5, 6). Clinically, AD is characterized by profound memory loss and cognitive dysfunction (7). Growing evidence is converging on soluble A␤ as a mediator of early cognitive decline in AD (8, 9). Although the molecular mechanisms underlying A␤-induced cognitive decline remain elusive, soluble A␤ oligomers have been shown to alter signal transduction pathways that are key for learning and memory, suggesting that alterations in such pathways may underlie the onset of cognitive decline in AD (10).The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two multiprotein complexes known as mTOR complex (mTORC) 1 and 2 (11). mTORC1 controls protein homeostasis; its activity is inhibited by rapamycin, and it contains mTOR, raptor, proline-rich Akt substrate 40 kDa (PRAS40), and mLT8. mTORC2, which is insensitive to rapamycin, controls cellular shape by modulating actin function and contains mTOR, rictor, mLST8, and hSIN (11, 12). In mTORC1, raptor binds to mTOR substrates and is necessary for mTOR activity (13). PRAS40 is another mTOR regulatory protein, which inhibits mTOR...

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This

Background/Aims: Voltage-dependent anion channels (VDAC), also known as eukaryotic porins, are located in the outer mitochondrial membrane and allow the flux of ions and small metabolites. While the pore-forming ability of recombinant VDAC1 and VDAC2 has been extensively studied during the last decades, a clear-cut ion conducting channel activity has not been assigned to the VDAC3 isoform. Methods: Electrophysiological characterization of the recombinant protein purified and refolded was obtained after incorporation into planar lipid bilayers. Results: Here we report for the first time that recombinant hVDAC3, upon expression in E.coli and purification-refolding, shows a channel activity with a very small conductance (90 pS in 1 M KCl) with respect to the conductance of hVDAC1 (>3500 pS in 1 M KCl). Purified hVDAC3 allowed the passage of both chloride and gluconate anions and did not distinguish between potassium, sodium and calcium used as cations. In contrast to VDAC1, the channel was active also at transmembrane voltages higher than +/-40 mV and displayed a relatively high open probability even at +/-80 mV. hVDAC3 was only slightly voltage-dependent, displaying a tendency to adopt lower-conductance states at positive voltages applied to the cis chamber. In accordance with the small conductance of the pore, expression of hVDAC3 in a porin-less yeast strain allowed only partial recovery of the growth under non-permissive conditions. Conclusion: The observed electrophysiological properties of hVDAC3 are surprisingly different from the other isoforms and are discussed in relation to the proposed physiological role of the protein in mammalian cells.

VDACs three isoforms (VDAC1, VDAC2, VDAC3) are integral proteins of the outer mitochondrial membrane whose primary function is to permit the communication and exchange of molecules related to the mitochondrial functions. We have recently reported about the peculiar over-oxidation of VDAC3 cysteines. In this work we have extended our analysis, performed by tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS, to the other two isoforms VDAC1 and VDAC2 from rat liver mitochondria, and we have been able to find also in these proteins over-oxidation of cysteines. Further PTM of cysteines as succination has been found, while the presence of selenocysteine was not detected. Unfortunately, a short sequence stretch containing one genetically encoded cysteine was not covered both in VDAC2 and in VDAC3, raising the suspect that more, unknown modifications of these proteins exist. Interestingly, cysteine over-oxidation appears to be an exclusive feature of VDACs, since it is not present in other transmembrane mitochondrial proteins eluted by hydroxyapatite. The assignment of a functional role to these modifications of VDACs will be a further step towards the full understanding of the roles of these proteins in the cell.

Superoxide Dismutase 1 mutants associate with 20–25% of familial Amyotrophic Lateral Sclerosis (ALS) cases, producing toxic aggregates on mitochondria, notably in spinal cord. The Voltage Dependent Anion Channel isoform 1 (VDAC1) in the outer mitochondrial membrane is a docking site for SOD1 G93A mutant in ALS mice and the physiological receptor of Hexokinase I (HK1), which is poorly expressed in mouse spinal cord. Our results demonstrate that HK1 competes with SOD1 G93A for binding VDAC1, suggesting that in ALS spinal cord the available HK1-binding sites could be used by SOD1 mutants for docking mitochondria, producing thus organelle dysfunction. We tested this model by studying the action of a HK1-N-terminal based peptide (NHK1). This NHK1 peptide specifically interacts with VDAC1, inhibits the SOD1 G93A binding to mitochondria and restores the viability of ALS model NSC34 cells. Altogether, our results suggest that NHK1 peptide could be developed as a therapeutic tool in ALS, predicting an effective role also in other proteinopathies.

This review offers a description of the most promising therapeutic strategies acting on VDAC1, for the treatment of neurodegenerative diseases.