A Haloarchaeal Transcriptional Regulator That Represses the Expression of CRISPR-Associated Genes.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) systems provide acquired heritable protection to bacteria and archaea against selfish DNA elements, such as viruses. These systems must be tightly regulated because they can capture DNA fragments from foreign selfish elements, and also occasionally from self-chromosomes, resulting in autoimmunity. Most known species from the halophilic archaeal genus Haloferax contain type I-B CRISPR-Cas systems, and the strongest hotspot for self-spacer acquisition by H. mediterranei was a locus that contained a putative transposable element, as well as the gene HFX_2341, which was a very frequent target for self-targeting spacers. To test whether this gene is CRISPR-associated, we investigated it using bioinformatics, deletion, over-expression, and comparative transcriptomics. We show that HFX_2341 is a global transcriptional regulator that can repress diverse genes, since its deletion results in significantly higher expression of multiple genes, especially those involved in nutrient transport. When over-expressed, HFX_2341 strongly repressed the transcript production of all cas genes tested, both those involved in spacer acquisition (cas1, 2 and 4) and those required for destroying selfish genetic elements (cas3 and 5-8). Considering that HFX_2341 is highly conserved in haloarchaea, with homologs that are present in species that do not encode the CRISPR-Cas system, we conclude that it is a global regulator that is also involved in cas gene regulation, either directly or indirectly.

Chronic and acute exposure to rotenone reveals distinct Parkinson's disease-related phenotypes in human iPSC-derived peripheral neurons.

Peripheral autonomic nervous system (P-ANS) dysfunction is a critical non-motor phenotype of Parkinson's disease (PD). The majority of PD cases are sporadic and lack identified PD-associated genes involved. Epidemiological and animal model studies suggest an association with pesticides and other environmental toxins. However, the cellular mechanisms underlying toxin induced P-ANS dysfunctions remain unclear. Here, we mapped the global transcriptome changes in human induced pluripotent stem cell (iPSC) derived P-ANS sympathetic neurons during inhibition of the mitochondrial respiratory chain by the PD-related pesticide, rotenone. We revealed distinct transcriptome profiles between acute and chronic exposure to rotenone. In the acute stage, there was a down regulation of specific cation channel genes, known to mediate electrophysiological activity, while in the chronic stage, the human P-ANS neurons exhibited dysregulation of anti-apoptotic and Golgi apparatus-related pathways. Moreover, we identified the sodium voltage-gated channel subunit SCN3A/Nav1.3 as a potential biomarker in human P-ANS neurons associated with PD. Our analysis of the rotenone-altered coding and non-coding transcriptome of human P-ANS neurons may thus provide insight into the pathological signaling events in the sympathetic neurons during PD progression.

Calcium regulation of the Arabidopsis Na+/K+ transporter HKT1;1 improves seed germination under salt stress.

Calcium is known to improve seed-germination rates under salt stress. We investigated the involvement of calcium ions (Ca2+) in regulating HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1; 1), which encodes a Na+/K+ transporter, and its post-translational regulator TYPE 2C PROTEIN PHOSPHATASE 49 (PP2C49), in germinating Arabidopsis (Arabidopsis thaliana) seedlings. Germination rates of hkt1 mutant seeds under salt stress remained unchanged by CaCl2 treatment in wild-type Arabidopsis, whereas pp2c49 mutant seeds displayed improved salt-stress tolerance in the absence of CaCl2 supplementation. Analysis of HKT1;1 and PP2C49 promoter activity revealed that CaCl2 treatment results in radicle-focused expression of HKT1;1 and reduction of the native radicle-exclusive expression of PP2C49. Ion-content analysis indicated that CaCl2 treatment improves K+ retention in germinating wild-type seedlings under salt stress, but not in hkt1 seedlings. Transgenic seedlings designed to exclusively express HKT1;1 in the radicle during germination displayed higher germination rates under salt stress than the wild type in the absence of CaCl2 treatment. Transcriptome analysis of germinating seedlings treated with CaCl2, NaCl, or both revealed 118 upregulated and 94 downregulated genes as responsive to the combined treatment. Bioinformatics analysis of the upstream sequences of CaCl2-NaCl-treatment-responsive upregulated genes revealed the abscisic acid response element CACGTGTC, a potential CaM-binding transcription activator-binding motif, as most prominent. Our findings suggest a key role for Ca2+ in mediating salt-stress responses during germination by regulating genes that function to maintain Na+ and K+ homeostasis, which is vital for seed germination under salt stress.

A heterodimer of α and ß hemoglobin chains functions as an innate anticancer agent.

Metastatic (as well as tumor) microenvironments contain both cancer-promoting and cancer-restraining factors. The balance between these opposing forces determines the fate of cancer cells that disseminate to secondary organ sites. In search for microenvironmental drivers or inhibitors of metastasis, we identified, in a previous study, the beta subunit of hemoglobin (HBB) as a lung-derived antimetastatic factor. In the present study, exploring mechanisms regulating melanoma brain metastasis, we discovered that brain-derived factors restrain proliferation and induce apoptosis and necrosis of brain-metastasizing melanoma cells. Employing various purification procedures, we identified a heterodimer composed of hemoglobin alpha and beta chains that perform these antimetastatic functions. Neither the alpha nor the beta subunit alone was inhibitory. An alpha/beta chain dimer chemically purified from human hemoglobin inhibited the cell viability of primary melanomas, melanoma brain metastasis (MBM), and breast cancer cell lines. The dimer-induced DNA damage, cell cycle arrest at the SubG1 phase, apoptosis, and significant necrosis in four MBM cell lines. Proteomic analysis of dimer-treated MBM cells revealed that the dimer downregulates the expression of BRD4, GAB2, and IRS2 proteins, playing crucial roles in cancer cell sustainability and progression. Thus, we hypothesize that the hemoglobin dimer functions as a resistance factor against brain-metastasizing cancer cells.

Heterogeneity in the Metastatic Microenvironment: JunB-Expressing Microglia Cells as Potential Drivers of Melanoma Brain Metastasis Progression.

Reciprocal signaling between melanoma brain metastatic (MBM) cells and microglia reprograms the phenotype of both interaction partners, including upregulation of the transcription factor JunB in microglia. Here, we aimed to elucidate the impact of microglial JunB upregulation on MBM progression. For molecular profiling, we employed RNA-seq and reverse-phase protein array (RPPA). To test microglial JunB functions, we generated microglia variants stably overexpressing JunB (JunBhi) or with downregulated levels of JunB (JunBlo). Melanoma-derived factors, namely leukemia inhibitory factor (LIF), controlled JunB upregulation through Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling. The expression levels of JunB in melanoma-associated microglia were heterogeneous. Flow cytometry analysis revealed the existence of basal-level JunB-expressing microglia alongside microglia highly expressing JunB. Proteomic profiling revealed a differential protein expression in JunBhi and JunBlo cells, namely the expression of microglia activation markers Iba-1 and CD150, and the immunosuppressive molecules SOCS3 and PD-L1. Functionally, JunBhi microglia displayed decreased migratory capacity and phagocytic activity. JunBlo microglia reduced melanoma proliferation and migration, while JunBhi microglia preserved the ability of melanoma cells to proliferate in three-dimensional co-cultures, that was abrogated by targeting leukemia inhibitory factor receptor (LIFR) in control microglia-melanoma spheroids. Altogether, these data highlight a melanoma-mediated heterogenous effect on microglial JunB expression, dictating the nature of their functional involvement in MBM progression. Targeting microglia highly expressing JunB may potentially be utilized for MBM theranostics.

Oculopharyngeal muscular dystrophy mutations link the RNA-binding protein HNRNPQ to autophagosome biogenesis.

Autophagy is an intracellular degradative process with an important role in cellular homeostasis. Here, we show that the RNA binding protein (RBP), heterogeneous nuclear ribonucleoprotein Q (HNRNPQ)/SYNCRIP is required to stimulate early events in autophagosome biogenesis, in particular the induction of VPS34 kinase by ULK1-mediated beclin 1 phosphorylation. The RBPs HNRNPQ and poly(A) binding protein nuclear 1 (PABPN1) form a regulatory network that controls the turnover of distinct autophagy-related (ATG) proteins. We also show that oculopharyngeal muscular dystrophy (OPMD) mutations engender a switch from autophagosome stimulation to autophagosome inhibition by impairing PABPN1 and HNRNPQ control of the level of ULK1. The overexpression of HNRNPQ in OPMD patient-derived cells rescues the defective autophagy in these cells. Our data reveal a regulatory mechanism of autophagy induction that is compromised by PABPN1 disease mutations, and may thus further contribute to their deleterious effects.

Kynurenic acid, a key L-tryptophan-derived metabolite, protects the heart from an ischemic damage.

BACKGROUND: Renal injury induces major changes in plasma and cardiac metabolites. Using a small- animal in vivo model, we sought to identify a key metabolite whose levels are significantly modified following an acute kidney injury (AKI) and to analyze whether this agent could offer cardiac protection once an ischemic event has occurred. METHODS AND RESULTS: Metabolomics profiling of cardiac lysates and plasma samples derived from rats that underwent AKI 1 or 7 days earlier by 5/6 nephrectomy versus sham-operated controls was performed. We detected 26 differential metabolites in both heart and plasma samples at the two selected time points, relative to sham. Out of which, kynurenic acid (kynurenate, KYNA) seemed most relevant. Interestingly, KYNA given at 10 mM concentration significantly rescued the viability of H9C2 cardiac myoblast cells grown under anoxic conditions and largely increased their mitochondrial content and activity as determined by flow cytometry and cell staining with MitoTracker dyes. Moreover, KYNA diluted in the drinking water of animals induced with an acute myocardial infarction, highly enhanced their cardiac recovery according to echocardiography and histopathology. CONCLUSION: KYNA may represent a key metabolite absorbed by the heart following AKI as part of a compensatory mechanism aiming at preserving the cardiac function. KYNA preserves the in vitro myocyte viability following exposure to anoxia in a mechanism that is mediated, at least in part, by protection of the cardiac mitochondria. A short-term administration of KYNA may be highly beneficial in the treatment of the acute phase of kidney disease in order to attenuate progression to reno-cardiac syndrom and to reduce the ischemic myocardial damage following an ischemic event.

Serine peptidases and increased amounts of soluble proteins contribute to heat priming of the plant pathogenic fungus Botrytis cinerea.

Botrytis cinerea causes gray mold disease in leading crop plants. The disease develops only at cool temperatures, but the fungus remains viable in warm climates and can survive periods of extreme heat. We discovered a strong heat priming effect in which the exposure of B. cinerea to moderately high temperatures greatly improves its ability to cope with subsequent, potentially lethal temperature conditions. We showed that priming promotes protein solubility during heat stress and discovered a group of priming-induced serine-type peptidases. Several lines of evidence, including transcriptomics, proteomics, pharmacology, and mutagenesis data, link these peptidases to the B. cinerea priming response, highlighting their important roles in regulating priming-mediated heat adaptation. By imposing a series of sub-lethal temperature pulses that subverted the priming effect, we managed to eliminate the fungus and prevent disease development, demonstrating the potential for developing temperature-based plant protection methods by targeting the fungal heat priming response. IMPORTANCE Priming is a general and important stress adaptation mechanism. Our work highlights the importance of priming in fungal heat adaptation, reveals novel regulators and aspects of heat adaptation mechanisms, and demonstrates the potential of affecting microorganisms, including pathogens through manipulations of the heat adaptation response.

The Vicious Cycle of Melanoma-Microglia Crosstalk: Inter-Melanoma Variations in the Brain-Metastasis-Promoting IL-6/JAK/STAT3 Signaling Pathway.

Previous studies from our lab demonstrated that the crosstalk between brain-metastasizing melanoma cells and microglia, the macrophage-like cells of the central nervous system, fuels progression to metastasis. In the present study, an in-depth investigation of melanoma-microglia interactions elucidated a pro-metastatic molecular mechanism that drives a vicious melanoma-brain-metastasis cycle. We employed RNA-Sequencing, HTG miRNA whole transcriptome assay, and reverse phase protein arrays (RPPA) to analyze the impact of melanoma-microglia interactions on sustainability and progression of four different human brain-metastasizing melanoma cell lines. Microglia cells exposed to melanoma-derived IL-6 exhibited upregulated levels of STAT3 phosphorylation and SOCS3 expression, which, in turn, promoted melanoma cell viability and metastatic potential. IL-6/STAT3 pathway inhibitors diminished the pro-metastatic functions of microglia and reduced melanoma progression. SOCS3 overexpression in microglia cells evoked microglial support in melanoma brain metastasis by increasing melanoma cell migration and proliferation. Different melanomas exhibited heterogeneity in their microglia-activating capacity as well as in their response to microglia-derived signals. In spite of this reality and based on the results of the present study, we concluded that the activation of the IL-6/STAT3/SOCS3 pathway in microglia is a major mechanism by which reciprocal melanoma-microglia signaling engineers the interacting microglia to reinforce the progression of melanoma brain metastasis. This mechanism may operate differently in different melanomas.

Antiviral activity of glucosylceramide synthase inhibitors in alphavirus infection of the central nervous system.

Virus-induced CNS diseases impose a considerable human health burden worldwide. For many viral CNS infections, neither antiviral drugs nor vaccines are available. In this study, we examined whether the synthesis of glycosphingolipids, major membrane lipid constituents, could be used to establish an antiviral therapeutic target. We found that neuroinvasive Sindbis virus altered the sphingolipid levels early after infection in vitro and increased the levels of gangliosides GA1 and GM1 in the sera of infected mice. The alteration in the sphingolipid levels appears to play a role in neuroinvasive Sindbis virus replication, as treating infected cells with UDP-glucose ceramide glucosyltransferase (UGCG) inhibitors reduced the replication rate. Moreover, the UGCG inhibitor GZ-161 increased the survival rates of Sindbis-infected mice, most likely by reducing the detrimental immune response activated by sphingolipids in the brains of Sindbis virus-infected mice. These findings suggest a role for glycosphingolipids in the host immune response against neuroinvasive Sindbis virus and suggest that UGCG inhibitors should be further examined as antiviral therapeutics for viral infections of the CNS.

A Tumor Microenvironment-Driven Network Regulated by STAT3 and p65 Negatively Controls the Enrichment of Cancer Stem Cells in Human HR+/HER2- Breast Cancer.

Hormone receptor-positive and HER2-negative (HR+/HER2-; luminal A) tumors are prevalent in breast cancer. Our past studies demonstrated that "TME Stimulation" (estrogen + TNFα + EGF, representing three arms of the tumor microenvironment, TME) has enriched metastasis-forming cancer stem cells (CSCs) in HR+/HER2- human breast cancer cells. Here, following information obtained by RNAseq analyses of TME-stimulated CSCs and Non-CSCs, we found that TME Stimulation has induced the activation of S727-STAT3, Y705-STAT3, STAT1 and p65. Upon TME Stimulation, stattic (STAT3 inhibitor) usage demonstrated that Y705-STAT3 activation negatively controlled CSC enrichment and epithelial-to-mesenchymal transition (EMT) traits, while inducing CXCL8 (IL-8) and PD-L1 expression. However, STAT3 knock-down (siSTAT3) had no effect on these functions; in terms of CSC enrichment, p65 had down-regulatory roles that compensated for the loss of an entire STAT3 protein. Y705-STAT3 and p65 acted additively in reducing CSC enrichment, and Y705A-STAT3 variant + sip65 has enriched chemo-resistant CSCs. Clinical data analyses revealed an inverse correlation between Y705-STAT3 + p65 phosphorylation and CSC signature in luminal A patients, and connection to improved disease course. Overall, we find regulatory roles for Y705-STAT3 and p65 in TME-stimulated HR+/HER2- tumors, with the ability to limit CSC enrichment. These findings raise concerns about using inhibitors of STAT3 and p65 as therapeutic strategies in the clinic.

Tomato receptor-like cytoplasmic kinase Fir1 is involved in flagellin signaling and preinvasion immunity.

Detection of bacterial flagellin by the tomato (Solanum lycopersicum) receptors Flagellin sensing 2 (Fls2) and Fls3 triggers activation of pattern-triggered immunity (PTI). We identified the tomato Fls2/Fls3-interacting receptor-like cytoplasmic kinase 1 (Fir1) protein that is involved in PTI triggered by flagellin perception. Fir1 localized to the plasma membrane and interacted with Fls2 and Fls3 in yeast (Saccharomyces cerevisiae) and in planta. CRISPR/Cas9-generated tomato fir1 mutants were impaired in several immune responses induced by the flagellin-derived peptides flg22 and flgII-28, including resistance to Pseudomonas syringae pv. tomato (Pst) DC3000, production of reactive oxygen species, and enhanced PATHOGENESIS-RELATED 1b (PR1b) gene expression, but not MAP kinase phosphorylation. Remarkably, fir1 mutants developed larger Pst DC3000 populations than wild-type plants, whereas no differences were observed in wild-type and fir1 mutant plants infected with the flagellin deficient Pst DC3000ΔfliC. fir1 mutants failed to close stomata when infected with Pst DC3000 and Pseudomonas fluorescens and were more susceptible to Pst DC3000 than wild-type plants when inoculated by dipping, but not by vacuum-infiltration, indicating involvement of Fir1 in preinvasion immunity. RNA-seq analysis detected fewer differentially expressed genes in fir1 mutants and altered expression of jasmonic acid (JA)-related genes. In support of JA response deregulation in fir1 mutants, these plants were similarly susceptible to Pst DC3000 and to the coronatine-deficient Pst DC3118 strain, and more resistant to the necrotrophic fungus Botrytis cinerea following PTI activation. These results indicate that tomato Fir1 is required for a subset of flagellin-triggered PTI responses and support a model in which Fir1 negatively regulates JA signaling during PTI activation.

Mesenchymal Stem Cell-Derived Extracellular Vesicles as Proposed Therapy in a Rat Model of Cerebral Small Vessel Disease.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been employed in the past decade as therapeutic agents in various diseases, including central nervous system (CNS) disorders. We currently aimed to use MSC-EVs as potential treatment for cerebral small vessel disease (CSVD), a complex disorder with a variety of manifestations. MSC-EVs were intranasally administrated to salt-sensitive hypertension prone SBH/y rats that were DOCA-salt loaded (SBH/y-DS), which we have previously shown is a model of CSVD. MSC-EVs accumulated within brain lesion sites of SBH/y-DS. An in vitro model of an inflammatory environment in the brain demonstrated anti-inflammatory properties of MSC-EVs. Following in vivo MSC-EV treatment, gene set enrichment analysis (GSEA) of SBH/y-DS cortices revealed downregulation of immune system response-related gene sets. In addition, MSC-EVs downregulated gene sets related to apoptosis, wound healing and coagulation, and upregulated gene sets associated with synaptic signaling and cognition. While no specific gene was markedly altered upon treatment, the synergistic effect of all gene alternations was sufficient to increase animal survival and improve the neurological state of affected SBH/y-DS rats. Our data suggest MSC-EVs act as microenvironment modulators, through various molecular pathways. We conclude that MSC-EVs may serve as beneficial therapeutic measure for multifactorial disorders, such as CSVD.

Immune cell C/EBPß deficiency is associated with hepatic mononuclear defects and spontaneous hepatitis but not steatohepatitis induced liver fibrosis.

BACKGROUND: CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor known to be involved in macrophage differentiation and function, steatohepatitis and liver fibrosis. METHODS: Immune restricted C/EBPß deficient and control mice were investigated in steady-state and in the CDA-HFD steatohepatitis model. Mice were assessed for weight change, liver biochemical profile, histology and hepatic phagocytes composition. RESULTS: Flow cytometry analysis of hepatic nonparenchymal cells revealed reduced numbers of hepatic monocytes and Kupffer cells and an increase in hepatic MHC class II positive myeloid cells in immune cells restricted C/EBPß deficient mice. Immune-restricted C/EBPß deficiency resulted in decreased weight gain and appearance of mild spontaneous liver inflammation. Nevertheless, In the CDA-HFD steatohepatitis model, immune restricted C/EBPß deficient and proficient mice exhibit similar grade of hepatic steatosis, liver enzymes levels and fibrosis stage. CONCLUSIONS: Immune-restricted C/EBPß deficiency leads to significant alteration in hepatic mononuclear phagocytes composition associated with spontaneous mild hepatitis. Steatohepatitis associated fibrosis is not dependent on C/EBPß expression by immune cells.

Deconvolution of the epigenetic age discloses distinct inter-personal variability in epigenetic aging patterns.

BACKGROUND: The epigenetic age can now be extrapolated from one of several epigenetic clocks, which are based on age-related changes in DNA methylation levels at specific multiple CpG sites. Accelerated aging, calculated from the discrepancy between the chronological age and the epigenetic age, has shown to predict morbidity and mortality rate. We assumed that deconvolution of epigenetic age to its components could shed light on the diversity of epigenetic, and by inference, on inter-individual variability in the causes of biological aging. RESULTS: Using the Horvath original epigenetic clock, we identified several CpG sites linked to distinct genes that quantitatively explain much of the inter-personal variability in epigenetic aging, with CpG sites related to secretagogin and malin being the most variable. We show that equal epigenetic age in different subjects can result from variable contribution size of the same CpG sites to the total epigenetic age. In a healthy cohort, the most variable CpG sites are responsible for accelerated and decelerated epigenetic aging, relative to chronological age. CONCLUSIONS: Of the 353 CpG sites that form the basis for the Horvath epigenetic age, we have found the CpG sites that are responsible for accelerated and decelerated epigenetic aging in healthy subjects. However, the relative contribution of each site to aging varies between individuals, leading to variable personal aging patterns. Our findings pave the way to form personalized aging cards allowing the identification of specific genes related to CpG sites, as aging markers, and perhaps treatment of these targets in order to hinder undesirable age drifting.

Genomic alterations drive metastases formation in pancreatic ductal adenocarcinoma cancer: deciphering the role of CDKN2A and CDKN2B in mediating liver tropism.

Metastases are often the direct cause of death from pancreatic ductal adenocarcinoma (PDAC). The role of genomic alterations (GA) in mediating tropism and metastasis formation by PDAC cells is currently unknown. We aimed to identify GAs predisposing colonization of PDAC cells to the liver and decipher mechanisms enabling this process. In order to reveal specific genes, we studied the frequency of GA in 8,880 local and 7,983 metastatic PDAC samples. We observed differential pattern of GA in the local tumor and specific metastatic sites, with liver metastases characterized by deletion of CDKN2A/B (encoding p16/p15, respectively). The role of CDKN2A/B in promoting liver metastasis was evidenced by enhanced tumorigenic phenotype of p15/p16-deleted PDAC cells when exposed to hepatocytes conditioned media. The liver is characterized by high-ammonia low-glutamine environment and transcriptomic assays indicated unique adaptation of PDAC cells to these conditions, including regulation of genes leading to reduced glutaminolysis, like overexpression of GLUL and reduction in GLS2. Furthermore, metabolic assays indicated an increase in glutamate derived from [U-13C]-glucose in p15/p16-deleted cells. Importantly, these cells thrived under high ammonia condition. These data suggest a unique role for genomic alterations in mediating tropism of PDAC. Among these alterations, p15/16 deletion was identified as a promoter of liver metastases. Further studies indicated a unique role for p15/16 in regulating glutaminolysis. These findings reveal vulnerabilities in PDAC cells, which may pave the way for the development of novel therapeutic strategies aiming at the prevention of liver metastases formation.

Novel ADNP Syndrome Mice Reveal Dramatic Sex-Specific Peripheral Gene Expression With Brain Synaptic and Tau Pathologies.

BACKGROUND: ADNP is essential for embryonic development. As such, de novo ADNP mutations lead to an intractable autism/intellectual disability syndrome requiring investigation. METHODS: Mimicking humans, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 editing produced mice carrying heterozygous Adnp p.Tyr718∗ (Tyr), a paralog of the most common ADNP syndrome mutation. Phenotypic rescue was validated by treatment with the microtubule/autophagy-protective ADNP fragment NAPVSIPQ (NAP). RESULTS: RNA sequencing of spleens, representing a peripheral biomarker source, revealed Tyr-specific sex differences (e.g., cell cycle), accentuated in females (with significant effects on antigen processing and cellular senescence) and corrected by NAP. Differentially expressed, NAP-correctable transcripts, including the autophagy and microbiome resilience-linked FOXO3, were also deregulated in human patient-derived ADNP-mutated lymphoblastoid cells. There were also Tyr sex-specific microbiota signatures. Phenotypically, Tyr mice, similar to patients with ADNP syndrome, exhibited delayed development coupled with sex-dependent gait defects. Speech acquisition delays paralleled sex-specific mouse syntax abnormalities. Anatomically, dendritic spine densities/morphologies were decreased with NAP amelioration. These findings were replicated in the Adnp+/- mouse, including Foxo3 deregulation, required for dendritic spine formation. Grooming duration and nociception threshold (autistic traits) were significantly affected only in males. Early-onset tauopathy was accentuated in males (hippocampus and visual cortex), mimicking humans, and was paralleled by impaired visual evoked potentials and correction by acute NAP treatment. CONCLUSIONS: Tyr mice model ADNP syndrome pathology. The newly discovered ADNP/NAP target FOXO3 controls the autophagy initiator LC3 (microtubule-associated protein 1 light chain 3), with known ADNP binding to LC3 augmented by NAP, protecting against tauopathy. NAP amelioration attests to specificity, with potential for drug development targeting accessible biomarkers.

MicroRNA 132-3p Is Upregulated in Laron Syndrome Patients and Controls Longevity Gene Expression.

The growth hormone (GH)-insulin-like growth factor-1 (IGF1) endocrine axis is a central player in normal growth and metabolism as well as in a number of pathologies, including cancer. The GH-IGF1 hormonal system, in addition, has emerged as a major determinant of lifespan and healthspan. Laron syndrome (LS), the best characterized entity under the spectrum of the congenital IGF1 deficiencies, results from mutation of the GH receptor (GHR) gene, leading to dwarfism, obesity and other defects. Consistent with the key role of IGF1 in cellular proliferation, epidemiological studies have shown that LS patients are protected from cancer development. While reduced expression of components of the GH-IGF1 axis is associated with enhanced longevity in animal models, it is still unknown whether LS is associated with an increased lifespan. MicroRNAs (miRs) are endogenous short non-coding RNAs that regulate the expression of complementary mRNAs. While a number of miRs involved in the regulation of IGF components have been identified, no previous studies have investigated the differential expression of miRs in congenital IGF1 deficiencies. The present study was aimed at identifying miRs that are differentially expressed in LS and that might account for the phenotypic features of LS patients, including longevity. Our genomic analyses provide evidence that miR-132-3p was highly expressed in LS. In addition, we identified SIRT1, a member of the sirtuin family of histone deacetylases, as a target for negative regulation by miR-132-3p. The data was consistent with the notion that low concentrations of IGF1 in LS lead to elevated miR-132-3p levels, with ensuing reduction in SIRT1 gene expression. The impact of the IGF1-miR-132-3p-SIRT1 loop on aging merits further investigation.

Autophagy is induced and modulated by cholesterol depletion through transcription of autophagy-related genes and attenuation of flux.

Perturbations to cellular homeostasis, including reduction of the cholesterol level, induce autophagy, a self-digestion process of cellular constituents through an autophagosomal-lysosomal pathway. In accord with its function as a membrane organizer and metabolic sentinel, the cellular response to cholesterol depletion comprises multiple phenomena, including the activation of transcriptional responses, accumulation of reactive oxygen species (ROS), and activation of stress-related signaling pathways. However, the molecular mechanisms by which cholesterol depletion regulates autophagy and the putative involvement of transcriptional responses, ROS and/or stress-related signaling in autophagy regulation in this biological context are not fully understood. Here, we find that cholesterol depletion regulates autophagy at three different levels. First, employing RNA-seq, we show that cholesterol depletion increases the expression of autophagy-related genes independent of ROS or JNK activity. Second, analysis of LC3 lipidation and intracellular localization, and of p62 levels and degradation kinetics, reveals that cholesterol depletion mediates autophagy induction while interfering with autophagic flux. Of note, only the latter depends on ROS accumulation and JNK activity. In view of the common use of cholesterol-reducing drugs as therapeutic agents, our findings have important implications for multiple cellular settings in which autophagy plays a prominent role.