Urolithin A reduces amyloid-beta load and improves cognitive deficits uncorrelated with plaque burden in a mouse model of Alzheimer's disease.

In the present study, we investigated the effects of urolithin A (UA), a metabolite generated from ellagic acid via its metabolism by gut bacteria, as an autophagy activator with potential neuroprotective activity. WT and 3xTg-AD mice were administered long-term intermittent dietary supplementation with UA. UA was found to prevent deficits in spatial memory, cued fear response, and exploratory behavior in this model. It also decreased the Aß plaque burden in areas of the hippocampus where these protein deposits are prominent in the model. Interestingly, correlation analyses demonstrate that Aß plaque burden positively correlates with enhanced spatial memory in 3xTg-AD mice on a control diet but not in those supplemented with UA. In contrast, Aß42 abundance in cortical and hippocampal homogenates negatively correlate with spatial memory in UA-fed mice. Our data suggest that plaque formation may be a protective mechanism against neurodegeneration and cognitive decline and that targeting the generation of proteotoxic Aß species might be a more successful approach in halting disease progression. UA was also found to extend lifespan in normal aging mice. Mechanistically, we demonstrate that UA is able to induce autophagy and to increase Aß clearance in neuronal cell lines. In summary, our studies reveal UA, likely via its actions as a autophagy inducer, is capable of removing Aß from neurons and its dietary administration prevents the onset of cognitive deficits associated with pathological Aß deposition in the 3xTg-AD mouse model as well as extending lifespan in normal aging mice.

mTORC2: The other mTOR in autophagy regulation.

The mechanistic target of rapamycin (mTOR) has gathered significant attention as a ubiquitously expressed multimeric kinase with key implications for cell growth, proliferation, and survival. This kinase forms the central core of two distinct complexes, mTORC1 and mTORC2, which share the ability of integrating environmental, nutritional, and hormonal cues but which regulate separate molecular pathways that result in different cellular responses. Particularly, mTORC1 has been described as a major negative regulator of endosomal biogenesis and autophagy, a catabolic process that degrades intracellular components and organelles within the lysosomes and is thought to play a key role in human health and disease. In contrast, the role of mTORC2 in the regulation of autophagy has been considerably less studied despite mounting evidence this complex may regulate autophagy in a different and perhaps complementary manner to that of mTORC1. Genetic ablation of unique subunits is currently being utilized to study the differential effects of the two mTOR complexes. RICTOR is the best-described subunit specific to mTORC2 and as such has become a useful tool for investigating the specific actions of this complex. The development of complex-specific inhibitors for mTORC2 is also an area of intense interest. Studies to date have demonstrated that mTORC1/2 complexes each signal to a variety of exclusive downstream molecules with distinct biological roles. Pinpointing the particular effects of these downstream effectors is crucial toward the development of novel therapies aimed at accurately modulating autophagy in the context of human aging and disease.

MITF and TFEB cross-regulation in melanoma cells.

The MITF, TFEB, TFE3 and TFEC (MiT-TFE) proteins belong to the basic helix-loop-helix family of leucine zipper transcription factors. MITF is crucial for melanocyte development and differentiation, and has been termed a lineage-specific oncogene in melanoma. The three related proteins MITF, TFEB and TFE3 have been shown to be involved in the biogenesis and function of lysosomes and autophagosomes, regulating cellular clearance pathways. Here we investigated the cross-regulatory relationship of MITF and TFEB in melanoma cells. Like MITF, the TFEB and TFE3 genes are expressed in melanoma cells as well as in melanoma tumors, albeit at lower levels. We show that the MITF and TFEB proteins, but not TFE3, directly affect each other's mRNA and protein expression. In addition, the subcellular localization of MITF and TFEB is subject to regulation by the mTOR signaling pathway, which impacts their cross-regulatory relationship at the transcriptional level. Our work shows that the relationship between MITF and TFEB is multifaceted and that the cross-regulatory interactions of these factors need to be taken into account when considering pathways regulated by these proteins.

Mechanism of conditional partner selectivity in MITF/TFE family transcription factors with a conserved coiled coil stammer motif.

Interrupted dimeric coiled coil segments are found in a broad range of proteins and generally confer selective functional properties such as binding to specific ligands. However, there is only one documented case of a basic-helix-loop-helix leucine zipper transcription factor-microphthalmia-associated transcription factor (MITF)-in which an insertion of a three-residue stammer serves as a determinant of conditional partner selectivity. To unravel the molecular principles of this selectivity, we have analyzed the high-resolution structures of stammer-containing MITF and an engineered stammer-less MITF variant, which comprises an uninterrupted symmetric coiled coil. Despite this fundamental difference, both MITF structures reveal identical flanking in-phase coiled coil arrangements, gained by helical over-winding and local asymmetry in wild-type MITF across the stammer region. These conserved structural properties allow the maintenance of a proper functional readout in terms of nuclear localization and binding to specific DNA-response motifs regardless of the presence of the stammer. By contrast, MITF heterodimer formation with other bHLH-Zip transcription factors is only permissive when both factors contain either the same type of inserted stammer or no insert. Our data illustrate a unique principle of conditional partner selectivity within the wide arsenal of transcription factors with specific partner-dependent functional readouts.

TP53 codon 72 polymorphism affects accumulation of mtDNA damage in human cells.

Human TP53 gene is characterised by a polymorphism at codon 72 leading to an Arginine-to-Proline (R/P) substitution. The two resulting p53 isoforms have a different subcellular localisation after stress (more nuclear or more mitochondrial for the P or R isoform, respectively). p53P72 variant is more efficient than p53R72 in inducing the expression of genes involved in nuclear DNA repair. Since p53 is involved also in mitochondrial DNA (mtDNA) maintenance, we wondered whether these p53 isoforms are associated with different accumulation of mtDNA damage. We observed that cells bearing p53R72 accumulate lower amount of mtDNA damage upon rotenone stress with respect to cells bearing p53P72, and that p53R72 co-localises with polymerase gamma more than p53P72. We also analysed the in vivo accumulation of heteroplasmy in a 300 bp fragment of mtDNA D-loop of 425 aged subjects. We observed that subjects with heteroplasmy higher than 5% are significantly less than expected in the p53R72/R72 group. On the whole, these data suggest that the polymorphism of TP53 at codon 72 affects the accumulation of mtDNA mutations, likely through the different ability of the two p53 isoforms to bind to polymerase gamma, and may contribute to in vivo accumulation of mtDNA mutations.