Mitigation of TDP-43 toxic phenotype by an RGNEF fragment in amyotrophic lateral sclerosis models.

Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As TDP-43 aggregation and dysregulation are causative of neuronal death, there is a special interest in targeting this protein as a therapeutic approach. Previously, we found that TDP-43 extensively co-aggregated with the dual function protein GEF (guanine exchange factor) and RNA-binding protein rho guanine nucleotide exchange factor (RGNEF) in ALS patients. Here, we show that an N-terminal fragment of RGNEF (NF242) interacts directly with the RNA recognition motifs of TDP-43 competing with RNA and that the IPT/TIG domain of NF242 is essential for this interaction. Genetic expression of NF242 in a fruit fly ALS model overexpressing TDP-43 suppressed the neuropathological phenotype increasing lifespan, abolishing motor defects and preventing neurodegeneration. Intracerebroventricular injections of AAV9/NF242 in a severe TDP-43 murine model (rNLS8) improved lifespan and motor phenotype, and decreased neuroinflammation markers. Our results demonstrate an innovative way to target TDP-43 proteinopathies using a protein fragment with a strong affinity for TDP-43 aggregates and a mechanism that includes competition with RNA sequestration, suggesting a promising therapeutic strategy for TDP-43 proteinopathies such as ALS and FTD.

Association of plasma biomarkers with cognition, cognitive decline, and daily function across and within neurodegenerative diseases: Results from the Ontario Neurodegenerative Disease Research Initiative.

INTRODUCTION: We investigated whether novel plasma biomarkers are associated with cognition, cognitive decline, and functional independence in activities of daily living across and within neurodegenerative diseases. METHODS: Glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), phosphorylated tau (p-tau)181 and amyloid beta (Aß)42/40 were measured using ultra-sensitive Simoa immunoassays in 44 healthy controls and 480 participants diagnosed with Alzheimer's disease/mild cognitive impairment (AD/MCI), Parkinson's disease (PD), frontotemporal dementia (FTD) spectrum disorders, or cerebrovascular disease (CVD). RESULTS: GFAP, NfL, and/or p-tau181 were elevated among all diseases compared to controls, and were broadly associated with worse baseline cognitive performance, greater cognitive decline, and/or lower functional independence. While GFAP, NfL, and p-tau181 were highly predictive across diseases, p-tau181 was more specific to the AD/MCI cohort. Sparse associations were found in the FTD and CVD cohorts and for Aß42/40 . DISCUSSION: GFAP, NfL, and p-tau181 are valuable predictors of cognition and function across common neurodegenerative diseases, and may be useful in specialized clinics and clinical trials.

Dynamic Localization of Paraspeckle Components under Osmotic Stress.

Paraspeckles are nuclear condensates formed by NEAT1_2 lncRNA and different RNA-binding proteins. In general, these membraneless organelles function in the regulation of gene expression and translation and in miRNA processing, and in doing this, they regulate cellular homeostasis and mediate pro-survival in the cell. Despite evidence showing the importance of paraspeckles in the stress response, the dynamics of paraspeckles and their components under conditions of osmotic stress remain unknown. We exposed HEK293T cells to sorbitol and examined NEAT1_2 expression using real-time PCR. Localization and quantification of the main paraspeckle components, NEAT1_2, PSPC1, NONO, and SFPQ, in different cellular compartments was performed using smFISH and immunofluorescence. Our findings showed a significant decrease in total NEAT1_2 expression in cells after osmotic stress. Sorbitol shifted the subcellular localization of NEAT1_2, PSPC1, NONO, and SFPQ from the nucleus to the cytoplasm and decreased the number and size of NEAT1_2 foci in the nucleus. PSPC1 formed immunoreactive cytoplasmic fibrils under conditions of osmotic stress, which slowly disassembled under recovery. Our study deepens the paraspeckle dynamics in response to stress, suggesting a novel role for NEAT1_2 in the cytoplasm in osmotic stress and physiological conditions.

New metal-organic framework coated sodium alginate for the delivery of curcumin as a sustainable drug delivery and cancer therapy system.

The utilization of biocompatible drug delivery systems with extended drug release capabilities is highly advantageous in cancer therapy, as they can mitigate adverse effects. To establish such a biocompatible system with prolonged drug release behavior, researchers developed an innovative drug carrier. In this study, a sustainable approach was employed to synthesize a new zinc-based metal-organic framework (Zn-MOF) through the reaction between synthesized Schiff base ligands and zinc ions. Comprehensive analyses, including FT-IR, XRD, SEM, BET surface area, and TGA techniques, were employed to thoroughly characterize the frameworks. Following comprehensive characterization, curcumin (CUR) was loaded onto the Zn-MOF, resulting in CUR entrapment efficiency and loading capacity of 79.23 % and 26.11 %, respectively. In vitro evaluations of CUR release from CUR@MOF exhibited controlled release patterns, releasing 78.9 % and 50.0 % of CUR at pH 5.0 and pH 7.4, respectively. To mitigate initial burst release, a coating of the biopolymer sodium alginate (SA) was applied to CUR@Zn-MOF. In vitro CUR release tests indicated that SA/CUR@Zn-MOF outperformed pristine CUR@Zn-MOF. The release of CUR conformed to the Korsmeyer-Peppas model, displaying non-Fickian diffusion. Furthermore, an in vitro cytotoxicity study clearly demonstrated the potent anti-tumor activity of the synthesized CUR@Zn-MOF attributed to its controlled release of CUR. This led to the induction of apoptotic effects and cell death across HeLa, HEK293, and SH-SY5Y cell lines. These findings strongly suggest that the developed pH-sensitive carriers hold remarkable potential as targeted vehicles for drug delivery in cancer therapy.

A technique for repeated blood and cerebrospinal fluid sampling from individual rats over time without the need for repeated anesthesia.

Ethical animal use follows the 3R's: Replacement, Reduction and Refinement. Here, we present the use of simultaneous jugular vein and cisterna magna catheterization via a port system in rats for repeated fluid sampling for 14 consecutive days without loss of catheter patency. This technique allows repeated intra-animal sampling without anesthesia and, if used with pooling samples from a cohort of animals, replaces the need for terminal collections for sufficient sample volumes.

A methodological primer of extracellular vesicles isolation and characterization via different techniques.

We present four different protocols of varying complexity for the isolation of cell culture-derived extracellular vesicles (EVs)/exosome-enriched fractions with the objective of providing researchers with easily conducted methods that can be adapted for many different uses in various laboratory settings and locations. These protocols are primarily based on polymer precipitation, filtration and/or ultracentrifugation, as well as size-exclusion chromatography (SEC) and include: (i) polyethylene glycol and sodium chloride supplementation of the conditioned medium followed by low-speed centrifugation; (ii) ultracentrifugation of conditioned medium; (iii) filtration of conditioned media through a 100-kDa exclusion filter; and (iv) isolation using a standard commercial kit. These techniques can be followed by further purification by ultracentrifugation, sucrose density gradient centrifugation, or SEC if needed and the equipment is available. HEK293 and SH-SY5Y cell cultures were used to generate conditioned medium containing exosomes. This medium was then depleted of cells and debris, filtered through a 0.2-µM filter, and supplemented with protease and RNAse inhibitors prior to exosomal isolation. The purified EVs can be used immediately or stably stored at 4°C (up to a week for imaging or using intact EVS downstream) or at -80°C for extended periods and then used for biochemical study. Our aim is not to compare these methodologies but to present them with descriptors so that researchers can choose the "best method" for their work under their individual conditions.