Alteration of gene expression and protein solubility of the PI 5-phosphatase SHIP2 are correlated with Alzheimer's disease pathology progression.

A recent large genome-wide association study has identified EGFR (encoding the epidermal growth factor EGFR) as a new genetic risk factor for late-onset AD. SHIP2, encoded by INPPL1, is taking part in the signalling and interactome of several growth factor receptors, such as the EGFR. While INPPL1 has been identified as one of the most significant genes whose RNA expression correlates with cognitive decline, the potential alteration of SHIP2 expression and localization during the progression of AD remains largely unknown. Here we report that gene expression of both EGFR and INPPL1 was upregulated in AD brains. SHIP2 immunoreactivity was predominantly detected in plaque-associated astrocytes and dystrophic neurites and its increase was correlated with amyloid load in the brain of human AD and of 5xFAD transgenic mouse model of AD. While mRNA of INPPL1 was increased in AD, SHIP2 protein undergoes a significant solubility change being depleted from the soluble fraction of AD brain homogenates and co-enriched with EGFR in the insoluble fraction. Using FRET-based flow cytometry biosensor assay for tau-tau interaction, overexpression of SHIP2 significantly increased the FRET signal while siRNA-mediated downexpression of SHIP2 significantly decreased FRET signal. Genetic association analyses suggest that some variants in INPPL1 locus are associated with the level of CSF pTau. Our data support the hypothesis that SHIP2 is an intermediate key player of EGFR and AD pathology linking amyloid and tau pathologies in human AD.

miR-483-5p orchestrates the initiation of protein synthesis by facilitating the decrease in phosphorylated Ser209eIF4E and 4E-BP1 levels.

Eukaryotic initiation factor 4E (eIF4E) is a pivotal protein involved in the regulatory mechanism for global protein synthesis in both physiological and pathological conditions. MicroRNAs (miRNAs) play a significant role in regulating gene expression by targeting mRNA. However, the ability of miRNAs to regulate eIF4E and its phosphorylation remains relatively unknown. In this study, we predicted and experimentally verified targets for miR-483-5p, including eukaryotic translation initiation factor eIF4E and its binding proteins, 4E-BPs, that regulate protein synthesis. Using the Web of Science database, we identified 28 experimentally verified miR-483-5p targets, and by the TargetScan database, we found 1818 predicted mRNA targets, including EIF4E, EIF4EBP1, and EIF4EBP2. We verified that miR-483-5p significantly reduced ERK1 and MKNK1 mRNA levels in HEK293 cells. Furthermore, we discovered that miR-483-5p suppressed EIF4EBP1 and EIF4EBP2, but not EIF4E. Finally, we found that miR-483-5p reduced the level of phosphorylated eIF4E (pSer209eIF4E) but not total eIF4E. In conclusion, our study suggests that miR-483-5p's multi-targeting effect on the ERK1/ MKNK1 axis modulates the phosphorylation state of eIF4E. Unlike siRNA, miRNA can have multiple targets in the pathway, and thereby exploring the role of miR-483-5p in various cancer models may uncover therapeutic options.

Mesenchymal stem cell engineering by ARCA analog-capped mRNA.

We previously have shown that mRNA-based engineering may enhance mesenchymal stem cell (MSC) trafficking. However, optimal conditions for in vitro mRNA engineering of MSCs are unknown. Here, we investigated several independent variables: (1) transfection factor (Lipofectamine 2000 vs. TransIT), (2) mRNA purification method (spin column vs. high-performance liquid chromatography [HPLC] column), and (3) mRNA capping (ARCA vs. ß-S-ARCA D1 and ß-S-ARCA D2). Dependent variables included protein production based on mRNA template (measured by the bioluminescence of reporter gene luciferase over hours), MSC metabolic activity corresponding with their wellbeing measured by CCK-8 over days, and endogenous expression of genes by RT-qPCR related to innate intracellular immune response and decapping at two time points: days 2 and 5. We have found that Lipofectamine 2000 outperforms TransIT, and used it throughout the study. Then, we showed that mRNA must be purified by HPLC to be relatively neutral to MSCs in terms of metabolic activity and endogenous protein production. Ultimately, we demonstrated that ß-S-ARCA D1 enables higher protein production but at the cost of lower MSC metabolic activity, with no impact on RT-qPCR results. Thus Lipofectamine 2000-based in vitro transfection of HPLC-purified and ARCA- or ß-S-ARCA D1-capped mRNA is optimal for MSC engineering.

PICALM and Alzheimer's Disease: An Update and Perspectives.

Genome-wide association studies (GWAS) have identified the PICALM (Phosphatidylinositol binding clathrin-assembly protein) gene as the most significant genetic susceptibility locus after APOE and BIN1. PICALM is a clathrin-adaptor protein that plays a critical role in clathrin-mediated endocytosis and autophagy. Since the effects of genetic variants of PICALM as AD-susceptibility loci have been confirmed by independent genetic studies in several distinct cohorts, there has been a number of in vitro and in vivo studies attempting to elucidate the underlying mechanism by which PICALM modulates AD risk. While differential modulation of APP processing and Aß transcytosis by PICALM has been reported, significant effects of PICALM modulation of tau pathology progression have also been evidenced in Alzheimer's disease models. In this review, we summarize the current knowledge about PICALM, its physiological functions, genetic variants, post-translational modifications and relevance to AD pathogenesis.

RNA sensor response in HeLa cells for transfected mRNAs prepared in vitro by SP6 and HiT7 RNA polymerases: A comparative study.

In vitro transcribed (IVT) synthetic mRNAs are in high demand due to their attractive bench to clinic translational processes. Mainly, the procedure to make IVT mRNA using bacteriophage RNA polymerases (RNAP) is relatively uncomplicated and scalable to produce large quantities in a short time period. However, IVT mRNA preparations are accompanied by contaminants such as double-stranded RNA (dsRNA) as by-products that elicit undesired cellular immune responses upon transfections. Therefore, removing dsRNA contaminants is critical in IVT mRNA preparations for therapeutic applications. One such method to minimize dsRNA contaminants is to use genetically modified thermostable bacteriophage polymerase, HiT7 RNAP that performs IVT reaction at a higher temperature than typically used. However, the cellular RNA sensor response for IVT mRNA preparations by HiT7 RNAP is not characterized. Here, we compared the cellular RNA sensor response for mRNAs prepared by HiT7 RNAP (at 50°C) and SP6 RNAP (at 37°C) in HeLa cells. We show that IVT mRNA preparations by HiT7 RNAP reduced the dsRNA levels and dsRNA specific RNA sensor response (retinoic acid-inducible gene I, RIG-I and melanoma differentiation-associated 5, MDA5) compared to the IVT mRNA preparations by SP6 RNAP. Similarly, the incorporation of pseudouridine nucleotides instead of uridine nucleotides reduced dsRNA sensor response and increased the mRNA translation. Overall, the least dsRNA mediated RNA sensor response is observed when mRNA is synthesized by HiT7 RNAP and incorporated with pseudouridine nucleotides.

Candidate Alzheimer's Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression.

MicroRNAs have been demonstrated as key regulators of gene expression in the etiology of a range of diseases including Alzheimer's disease (AD). Recently, we identified miR-483-5p as the most upregulated miRNA amongst a panel of miRNAs in blood plasma specific to prodromal, early-stage Alzheimer's disease patients. Here, we investigated the functional role of miR-483-5p in AD pathology. Using TargetScan and miRTarBase, we identified the microtubule-associated protein MAPT, often referred to as TAU, and the extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), known to phosphorylate TAU, as predicted direct targets of miR-483-5p. Employing several functional assays, we found that miR-483-5p regulates ERK1 and ERK2 at both mRNA and protein levels, resulting in lower levels of phosphorylated forms of both kinases. Moreover, miR-483-5p-mediated repression of ERK1/2 resulted in reduced phosphorylation of TAU protein at epitopes associated with TAU neurofibrillary pathology in AD. These results indicate that upregulation of miR-483-5p can decrease phosphorylation of TAU via ERK pathway, representing a compensatory neuroprotective mechanism in AD pathology. This miR-483-5p/ERK1/TAU axis thus represents a novel target for intervention in AD.

TDP-43: A Key Therapeutic Target beyond Amyotrophic Lateral Sclerosis.

Accumulation of TDP-43 in the cytoplasm of diseased neurons is the pathological hallmark of frontotemporal dementia-TDP (FTLD-TDP) and amyotrophic lateral sclerosis (ALS), two diseases that lack efficacious medicine to prevent or to stop disease progression. The discovery of mutations in the TARDBP gene (encoding the nuclear protein known as TDP-43) in both FTLD and ALS patients provided evidence for a link between TDP-43 alterations and neurodegeneration. Our understanding of TDP-43 function has advanced profoundly in the past several years; however, its complete role and the molecular mechanisms that lead to disease are not fully understood. Here we summarize the recent studies of this protein, its relation to neurodegenerative diseases, and the therapeutic strategies for restoring its homeostasis with small molecules. Finally, we briefly discuss the available cellular and animal models that help to shed light on TDP-43 pathology and could serve as tools for the discovery of pharmacological agents for the treatment of TDP-43-related diseases.

microRNA diagnostic panel for Alzheimer's disease and epigenetic trade-off between neurodegeneration and cancer.

microRNAs (miRNAs) have been extensively studied as potential biomarkers for Alzheimer's disease (AD). Their profiles have been analyzed in blood, cerebrospinal fluid (CSF) and brain tissue. However, due to the high variability between the reported data, stemming from the lack of methodological standardization and the heterogeneity of AD, the most promising miRNA biomarker candidates have not been selected. Our literature review shows that out of 137 miRNAs found to be altered in AD blood, 36 have been replicated in at least one independent study, and out of 166 miRNAs reported as differential in AD CSF, 13 have been repeatedly found. Only 3 miRNAs have been consistently reported as altered in three analyzed specimens: blood, CSF and the brain (hsa-miR-146a, hsa-miR-125b, hsa-miR-135a). Nonetheless, all 36 repeatedly differential miRNAs in AD blood are promising as components of the diagnostic panel. Given their predicted functions, such miRNA panel may report multiple pathways contributing to AD pathology, enabling the design of personalized therapies. In addition, the analysis revealed that the miRNAs dysregulated in AD overlap highly with miRNAs implicated in cancer. However, the directions of the miRNA changes are usually opposite in cancer and AD, indicative of an epigenetic trade-off between the two diseases.

Recapitulation of Pathological TDP-43 Features in Immortalized Lymphocytes from Sporadic ALS Patients.

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disorder of still unknown etiology that results in loss of motoneurons, paralysis, and death, usually between 2 and 4 years from onset. There are no currently available ALS biomarkers to support early diagnosis and to facilitate the assessment of the efficacy of new treatments. Since ALS is considered a multisystemic disease, here we have investigated the usefulness of immortalized lymphocytes from sporadic ALS patients to study TDP-43 homeostasis as well as to provide a convenient platform to evaluate TDP-43 phosphorylation as a novel therapeutic approach for ALS. We report here that lymphoblasts from ALS patients recapitulate the hallmarks of TDP-43 processing in affected motoneurons, such as increased phosphorylation, truncation, and mislocalization of TDP-43. Moreover, modulation of TDP-43 by an in-house designed protein casein kinase-1δ (CK-1δ) inhibitor, IGS3.27, reduced phosphorylation of TDP-43, and normalized the nucleo-cytosol translocation of TDP-43 in ALS lymphoblasts. Therefore, we conclude that lymphoblasts, easily accessible cells, from ALS patients could be a useful model to study pathological features of ALS disease and a suitable platform to test the effects of potential disease-modifying drugs even in a personalized manner.

Profile of 6 microRNA in blood plasma distinguish early stage Alzheimer's disease patients from non-demented subjects.

Alzheimer's disease (AD) is the most common age-related dementia. Among its major challenges is identifying molecular signatures characteristic for the early AD stage in patients with Mild Cognitive Impairment (MCI-AD), which could serve for deciphering the AD pathomechanism and also as non-invasive, easy-to-access biomarkers. Using qRT-PCR we compared the microRNA (miRNA) profiles in blood plasma of 15 MCI-AD patients, whose diagnoses were confirmed by cerebrospinal fluid (CSF) biomarkers, with 20 AD patients and 15 non-demented, age-matched individuals (CTR).To minimize methodological variability, we adhered to standardization of blood and CSF assays recommended by the international Joint Programming for Neurodegenerative Diseases (JPND) BIOMARKAPD consortium, and we employed commercially available Exiqon qRT-PCR-assays. In the first screening, we assessed 179 miRNAs of plasma. We confirmed 23 miRNAs reported earlier as AD biomarker candidates in blood and found 26 novel differential miRNAs between AD and control subjects. For representative 15 differential miRNAs, the TargetScan, MirTarBase and KEGG database analysis indicated putative protein targets among such AD hallmarks as MAPT (Tau), proteins involved in amyloidogenic proteolysis, and in apoptosis. These 15 miRNAs were verified in separate, subsequent subject groups. Finally, 6 miRNAs (3 not yet reported in AD context and 3 reported in AD blood) were selected as the most promising biomarker candidates differentiating early AD from controls with the highest fold changes (from 1.32 to 14.72), consistent significance, specificities from 0.78 to 1 and sensitivities from 0.75 to 1. (patent pending, PCT/IB2016/052440).