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Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by protein accumulation in the brain as a main neuropathological hallmark. Among them, Aß42 peptides tend to aggregate and create oligomers and plaques. Macroautophagy, a form of autophagy characterized by a double-membrane vesicle, plays a crucial role in maintaining neuronal homeostasis by degrading protein aggregates and dysfunctional organelles as a quality control process. Recently, DEF8, a relatively uncharacterized protein, has been proposed as a participant in vesicular traffic and autophagy pathways. We have reported increased DEF8 levels in lymphocytes from mild cognitive impairment (MCI) and early-stage AD patients and a neuronal profile in a murine transgenic AD model. Here, we analyzed DEF8 localization and levels in the postmortem frontal cortex of AD patients, finding increased levels compared to healthy controls. To evaluate the potential function of DEF8 in the nervous system, we performed an in silico assessment of its expression and network profiles, followed by an in vivo evaluation of a neuronal Def8 deficient model using a Drosophila melanogaster model of AD based on Aß42 expression. Our findings show that DEF8 is an essential protein for maintaining cellular homeostasis in the nervous system, and it is upregulated under stress conditions generated by Aß42 aggregation. This study suggests DEF8 as a novel actor in the physiopathology of AD, and its exploration may lead to new treatment avenues.

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Epidemiological studies show that having a history of cancer protects from the development of Alzheimer's Disease (AD), and vice versa, AD protects from cancer. The mechanism of this mutual protection is unknown. We have reported that the peripheral blood mononuclear cells (PBMC) of amnestic cognitive impairment (aMCI) and Alzheimer's Disease (AD) patients have increased susceptibility to oxidative cell death compared to control subjects, and from the opposite standpoint a cancer history is associated with increased resistance to oxidative stress cell death in PBMCs, even in those subjects who have cancer history and aMCI (Ca + aMCI). Cellular senescence is a regulator of susceptibility to cell death and has been related to the pathophysiology of AD and cancer. Recently, we showed that cellular senescence markers can be tracked in PBMCs of aMCI patients, so we here investigated whether these senescence markers are dependent on having a history of cancer. Senescence-associated ßeta-galactosidase (SA-ß-Gal) activity, G0-G1 phase cell-cycle arrest, p16 and p53 were analyzed by flow cytometry; phosphorylated H2A histone family member X (γH2AX) by immunofluorescence; IL-6 and IL-8 mRNA by qPCR; and plasmatic levels by ELISA. Senescence markers that were elevated in PBMCs of aMCI patients, such as SA-ß-Gal, Go-G1 arrested cells, IL-6 and IL-8 mRNA expression, and IL-8 plasmatic levels, were decreased in PBMCs of Ca + aMCI patients to levels similar to those of controls or of cancer survivors without cognitive impairment, suggesting that cancer in the past leaves a fingerprint that can be peripherally traceable in PBMC samples. These results support the hypothesis that the senescence process might be involved in the inverse association between cancer and AD.

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Introduction: Neuronal Ca2+ signals generated through the activation of Ca2+-induced Ca2+ release in response to activity-generated Ca2+ influx play a significant role in hippocampal synaptic plasticity, spatial learning, and memory. We and others have previously reported that diverse stimulation protocols, or different memory-inducing procedures, enhance the expression of endoplasmic reticulum-resident Ca2+ release channels in rat primary hippocampal neuronal cells or hippocampal tissue. Methods and Results: Here, we report that induction of long-term potentiation (LTP) by Theta burst stimulation protocols of the CA3-CA1 hippocampal synapse increased the mRNA and protein levels of type-2 Ryanodine Receptor (RyR2) Ca2+ release channels in rat hippocampal slices. Suppression of RyR channel activity (1 h preincubation with 20 µM ryanodine) abolished both LTP induction and the enhanced expression of these channels; it also promoted an increase in the surface expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR1 and GluR2 and caused a moderate but significant reduction of dendritic spine density. In addition, training rats in the Morris water maze induced memory consolidation, which lasted for several days after the end of the training period, accompanied by an increase in the mRNA levels and the protein content of the RyR2 channel isoform. Discussion: We confirm in this work that LTP induction by TBS protocols requires functional RyR channels. We propose that the increments in the protein content of RyR2 Ca2+ release channels, induced by LTP or spatial memory training, play a significant role in hippocampal synaptic plasticity and spatial memory consolidation.

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Recent studies suggest that cellular senescence plays a role in Alzheimer's Disease (AD) pathogenesis. We hypothesize that cellular senescence markers might be tracked in the peripheral tissues of AD patients. Senescence hallmarks, including altered metabolism, cell-cycle arrest, DNA damage response (DDR) and senescence secretory associated phenotype (SASP), were measured in peripheral blood mononuclear cells (PBMCs) of healthy controls (HC), amnestic mild cognitive impairment (aMCI) and AD patients. Senescence-associated ßeta-galactosidase (SA-ß-Gal) activity, G0-G1 phase cell-cycle arrest, p16 and p53 were analyzed by flow cytometry, while IL-6 and IL-8 mRNA were analyzed by qPCR, and phosphorylated H2A histone family member X (γH2AX) was analyzed by immunofluorescence. Senescent cells in the brain tissue were determined with lipofuscin staining. An increase in the number of senescent cells was observed in the frontal cortex and hippocampus of advanced AD patients. PBMCs of aMCI patients, but not in AD, showed increased SA-ß-Gal compared with HCs. aMCI PBMCs also had increased IL-6 and IL8 mRNA expression and number of cells arrested at G0-G1, which were absent in AD. Instead, AD PBMCs had significantly increased p16 and p53 expression and decreased γH2Ax activity compared with HC. This study reports that several markers of cellular senescence can be measured in PBMCs of aMCI and AD patients.

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Alzheimer's disease (AD) is the most prevalent age-associated neurodegenerative disease. A decrease in autophagy during aging contributes to brain disorders by accumulating potentially toxic substrates in neurons. Rubicon is a well-established inhibitor of autophagy in all cells. However, Rubicon participates in different pathways depending on cell type, and little information is currently available on neuronal Rubicon's role in the AD context. Here, we investigated the cell-specific expression of Rubicon in postmortem brain samples from AD patients and 5xFAD mice and its impact on amyloid ß burden in vivo and neuroblastoma cells. Further, we assessed Rubicon levels in human-induced pluripotent stem cells (hiPSCs), derived from early-to-moderate AD and in postmortem samples from severe AD patients. We found increased Rubicon levels in AD-hiPSCs and postmortem samples and a notable Rubicon localization in neurons. In AD transgenic mice lacking Rubicon, we observed intensified amyloid ß burden in the hippocampus and decreased Pacer and p62 levels. In APP-expressing neuroblastoma cells, increased APP/amyloid ß secretion in the medium was found when Rubicon was absent, which was not observed in cells depleted of Atg5, essential for autophagy, or Rab27a, required for exosome secretion. Our results propose an uncharacterized role of Rubicon on APP/amyloid ß homeostasis, in which neuronal Rubicon is a repressor of APP/amyloid ß secretion, defining a new way to target AD and other similar diseases therapeutically.

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Gene Regulatory Networks (GRNs) allow the study of regulation of gene expression of whole genomes. Among the most relevant advantages of using networks to depict this key process, there is the visual representation of large amounts of information and the application of graph theory to generate new knowledge. Nonetheless, despite the many uses of GRNs, it is still difficult and expensive to assign Transcription Factors (TFs) to the regulation of specific genes. ChIP-Seq allows the determination of TF Binding Sites (TFBSs) over whole genomes, but it is still an expensive technique that can only be applied one TF at a time and requires replicates to reduce its noise. Once TFBSs are determined, the assignment of each TF and its binding sites to the regulation of specific genes is not trivial, and it is often performed by carrying out site-specific experiments that are unfeasible to perform in all possible binding sites. Here, we addressed these relevant issues with a two-step methodology using Drosophila melanogaster as a case study. First, our protocol starts by gathering all transcription factor binding sites (TFBSs) determined with ChIP-Seq experiments available at ENCODE and FlyBase. Then each TFBS is used to assign TFs to the regulation of likely target genes based on the TFBS proximity to the transcription start site of all genes. In the final step, to try to select the most likely regulatory TF from those previously assigned to each gene, we employ GENIE3, a random forest-based method, and more than 9,000 RNA-seq experiments from D. melanogaster. Following, we employed known TF protein-protein interactions to estimate the feasibility of regulatory events in our filtered networks. Finally, we show how known interactions between co-regulatory TFs of each gene increase after the second step of our approach, and thus, the consistency of the TF-gene assignment. Also, we employed our methodology to create a network centered on the Drosophila melanogaster gene Hr96 to demonstrate the role of this transcription factor on mitochondrial gene regulation.

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BACKGROUND: Disturbances in the autophagy/endolysosomal systems are proposed as early signatures of Alzheimer's disease (AD). However, few studies are available concerning autophagy gene expression in AD patients. OBJECTIVE: To explore the differential expression of classical genes involved in the autophagy pathway, among them a less characterized one, DEF8 (Differentially expressed in FDCP 8), initially considered a Rubicon family member, in peripheralblood mononuclear cells (PBMCs) from individuals with mild cognitive impairment (MCI) and probable AD (pAD) and correlate the results with the expression of DEF8 in the brain of 5xFAD mice. METHOD: By real-time PCR and flow cytometry, we evaluated autophagy genes levels in PBMCs from MCI and pAD patients. We evaluated DEF8 levels and its localization in brain samples of the 5xFAD mice by real-time PCR, western blot, and immunofluorescence. RESULTS: Transcriptional levels of DEF8 were significantly reduced in PBMCs of MCI and pAD patients compared with healthy donors, correlating with the MoCA and MoCA-MIS cognitive tests scores. DEF8 protein levels were increased in lymphocytes from MCI but not pAD, compared to controls. In the case of brain samples from 5xFAD mice, we observed a reduced mRNA expression and augmented protein levels in 5xFAD compared to age-matched wild-type mice. DEF8 presented a neuronal localization. CONCLUSION: DEF8, a protein proposed to act at the final step of the autophagy/endolysosomal pathway, is differentially expressed in PBMCs of MCI and pAD and neurons of 5xFAD mice. These results suggest a potential role for DEF8 in the pathophysiology of AD.

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BACKGROUND: Amyloid-ß peptide (Aß) deposition in Alzheimer's disease (AD) is due to an imbalance in its production/clearance rate. Aß is transported across the blood-brain barrier by LRP1 and P-gp as efflux transporters and RAGE as influx transporter. Vitamin D deficit and polymorphisms of the vitamin D receptor (VDR) gene are associated with high prevalence of mild cognitive impairment (MCI) and AD. Further, vitamin D promotes the expression of LRP1 and P-gp in AD-animal model brains. OBJECTIVE: To associate VDR polymorphisms Apa I (rs7975232), Taq I (rs731236), and Fok I (rs2228570) with the risk of developing MCI in a Chilean population, and to evaluate the relationship of these polymorphisms to the expression of VDR and Aß-transporters in peripheral blood mononuclear cells (PBMCs). METHODS: VDR polymorphisms Apa I, Taq I, and Fok I were determined in 128 healthy controls (HC) and 66 MCI patients. mRNA levels of VDR and Aß-transporters were evaluated in subgroups by qPCR. RESULTS: Alleles A of Apa I and C of Taq I were associated with a lower risk of MCI. HC with the Apa I AA genotype had higher mRNA levels of P-gp and LRP1, while the expression of VDR and RAGE were higher in MCI patients and HC. For Fok I, the TC genotype was associated with lower expression levels of Aß-transporters in both groups. CONCLUSION: We propose that the response to vitamin D treatment will depend on VDR polymorphisms, being more efficient in carriers of protective alleles of Apa I polymorphism.

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Nicotinamide (vitamin B3) is a key component in the cellular production of Nicotinamide Adenine Dinucleotide (NAD) and has long been associated with neuronal development, survival and death. Numerous data suggest that nicotinamide may offer therapeutic benefits in neurodegenerative disorders, including Alzheimer's Disease (AD). Beyond its effect in NAD+ stores, nicotinamide is an inhibitor of Poly [ADP-ribose] polymerase 1 (PARP-1), an enzyme with multiple cellular functions, including regulation of cell death, energy/metabolism and inflammatory response. PARP-1 functions as a DNA repair enzyme but under intense DNA damage depletes the cell of NAD+ and ATP and leads to a non-apoptotic type of cell death called Parthanatos, which has been associated with the pathogenesis of neurodegenerative diseases. Moreover, NAD+ availability might potentially improve mitochondrial function, which is severely impaired in AD. PARP-1 inhibition may also exert a protective effect against neurodegeneration by its action to diminish neuroinflammation and microglial activation which are also implicated in the pathogenesis of AD. Here we discuss the evidence supporting the use of nicotinamide as adjunctive therapy for the treatment of early stages of AD based on the inhibitory effect of nicotinamide on PARP-1 activity. The data support evaluating nicotinamide as an adjunctive treatment for AD at early stages of the disease not only to increase NAD+ stores but as a PARP-1 inhibitor, raising the hypothesis that other PARP-1 inhibitors - drugs that are already approved for breast cancer treatment - might be explored for the treatment of AD.

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Tumor necrosis factor alpha (TNF) triggers regulated necrosis of mycobacterium-infected macrophages through of mitochondrial reactive oxygen species (mitoROS) production in a RIPK1/3-dependent manner. To explain that, Roca and colleagues describe an inter-orgallenar circuit which involves the lysosomal ceramide production, mitoROS, BAX activation and RyR Ca2+ efflux from the endoplasmic reticulum into the mitochondrion.

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