MicroRNA Implications across Neurodevelopment and Neuropathology

MicroRNAs (miRNAs) have rapidly emerged as biologically important mediators of posttranscriptional and epigenetic regulation in both plants and animals. miRNAs function through a variety of mechanisms including mRNA degradation and translational repression; additionally, miRNAs may guide gene expression by serving as transcription factors. miRNAs are highly expressed in human brain. Tissue and cell type-specific enrichments of certain miRNAs within the nervous system argue for a biological significance during neurodevelopmental stages. On the other hand, a large number of studies have reported links between alterations of miRNA homeostasis and pathologic conditions such as cancer, heart diseases, and neurodegeneration. Thus, profiles of distinct or aberrant miRNA signatures have most recently surged as one of the most fascinating interests in current biology. Here, the most recent insights into the involvement of miRNAs in the biology of the nervous system and the occurrence of neuropathological disorders are reviewed and discussed

Absence of Metabolic Cross-correction in Tay-Sachs Cells: IMPLICATIONS FOR GENE THERAPY

We have investigated the ability of a receptor-mediated gene transfer strategy (cross-correction) to restore ganglioside metabolism in fibroblasts from Tay-Sachs (TS) patients in vitro. TS disease is a GM2 gangliosidosis attributed to the deficiency of the lysosomal enzyme beta-hexosaminidase A (HexA) (beta-N-acetylhexosaminidase, EC ). The hypothesis is that transduced cells overexpressing and secreting large amounts of the enzyme would lead to a measurable activity in defective cells via a secretion-recapture mechanism. We transduced NIH3T3 murine fibroblasts with the LalphaHexTN retroviral vector carrying the cDNA encoding for the human Hex alpha-subunit. The Hex activity in the medium from transduced cells was approximately 10-fold higher (up to 75 milliunits) than observed in non-transduced cells. TS cells were cultured for 72 h in the presence of the cell medium derived from the transduced NIH3T3 cells, and they were analyzed for the presence and catalytic activity of the enzyme. Although TS cells were able to efficiently uptake a large amount of the soluble enzyme, the enzyme failed to reach the lysosomes in a sufficient quantity to hydrolyze the GM2 ganglioside to GM3 ganglioside. Thus, our results showed that delivery of the therapeutic HexA was not sufficient to correct the phenotype of TS cells

Novel bicistronic lentiviral vectors correct β-Hexosaminidase deficiency in neural and hematopoietic stem cells and progeny: implications for in vivo and ex vivo gene therapy of GM2 gangliosidosis.

Abstract The favorable outcome of in vivo and ex vivo gene therapy approaches in several Lysosomal Storage Diseases suggests that these treatment strategies might equally benefit GM2 gangliosidosis. Tay-Sachs and Sandhoff disease (the main forms of GM2 gangliosidosis) result from mutations in either the HEXA or HEXB genes encoding, respectively, the α- or β-subunits of the lysosomal β-Hexosaminidase enzyme. In physiological conditions, α- and β-subunits combine to generate β-Hexosaminidase A (HexA, αβ) and β-Hexosaminidase B (HexB, ββ). A major impairment to establishing in vivo or ex vivo gene therapy for GM2 gangliosidosis is the need to synthesize the α- and β-subunits at high levels and with the correct stoichiometric ratio, and to safely deliver the therapeutic products to all affected tissues/organs. Here, we report the generation and in vitro validation of novel bicistronic lentiviral vectors (LVs) encoding for both the murine and human codon optimized Hexa and Hexb genes. We show that these LVs drive the safe and coordinate expression of the α- and β-subunits, leading to supranormal levels of β-Hexosaminidase activity with prevalent formation of a functional HexA in SD murine neurons and glia, murine bone marrow-derived hematopoietic stem/progenitor cells (HSPCs), and human SD fibroblasts. The restoration/overexpression of β-Hexosaminidase leads to the reduction of intracellular GM2 ganglioside storage in transduced and in cross-corrected SD murine neural progeny, indicating that the transgenic enzyme is secreted and functional. Importantly, bicistronic LVs safely and efficiently transduce human neurons/glia and CD34+ HSPCs, which are target and effector cells, respectively, in prospective in vivo and ex vivo GT approaches. We anticipate that these bicistronic LVs may overcome the current requirement of two vectors co-delivering the α- or β-subunits genes. Careful assessment of the safety and therapeutic potential of these bicistronic LVs in the SD murine model will pave the way to the clinical development of LV-based gene therapy for GM2 gangliosidosis

Piezo1 – serine/threonine-protein phosphatase 2A – Cofilin1 biochemical mechanotransduction axis controls F-actin dynamics and cell migration

This study sheds light on a ground-breaking biochemical mechanotransduction pathway and reveals how Piezo1 channels orchestrate cell migration. We observed an increased cell migration rate in HEK293T (HEK) cells treated with Yoda1, a Piezo1 agonist, or in HEK cells overexpressing Piezo1 (HEK+P). Conversely, a significant reduction in cell motility was observed in HEK cells treated with GsMTx4 (a channel inhibitor) or upon silencing Piezo1 (HEK-P). Our findings establish a direct correlation between alterations in cell motility, Piezo1 expression, abnormal F-actin microfilament dynamics, and the regulation of Cofilin1, a protein involved in severing F-actin microfilaments. Here, the conversion of inactive pCofilin1 to active Cofilin1, mediated by the serine/threonine-protein phosphatase 2A catalytic subunit C (PP2AC), resulted in increased severing of F-actin microfilaments and enhanced cell migration in HEK+P cells compared to HEK controls. However, this effect was negligible in HEK-P and HEK cells transfected with hsa-miR-133b, which post-transcriptionally inhibited PP2AC mRNA expression. In summary, our study suggests that Piezo1 regulates cell migration through a biochemical mechanotransduction pathway involving PP2AC-mediated Cofilin1 dephosphorylation, leading to changes in F-actin microfilament dynamics

Critical issues for the proper diagnosis of Metachromatic Leukodystrophy

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Development of a New Tool for 3D Modeling for Regenerative Medicine

The effectiveness of therapeutic treatment based on regenerative medicine for degenerative diseases (i.e., neurodegenerative or cardiac diseases) requires tools allowing the visualization and analysis of the three-dimensional (3D) distribution of target drugs within the tissue. Here, we present a new computational procedure able to overcome the limitations of visual analysis emerging by the examination of a molecular signal within images of serial tissue/organ sections by using the conventional techniques. Together with the 3D anatomical reconstitution of the tissue/organ, our framework allows the detection of signals of different origins (e.g., marked generic molecules, colorimetric, or fluorimetric substrates for enzymes; microRNA; recombinant protein). Remarkably, the application does not require the employment of specific tracking reagents for the imaging analysis. We report two different representative applications: the first shows the reconstruction of a 3D model of mouse brain with the analysis of the distribution of the β-Galactosidase, the second shows the reconstruction of a 3D mouse heart with the measurement of the cardiac volume

A comparison of lysosomal enzymes expression levels in peripheral blood of mild- and severe-Alzheimer's disease and MCI patients: implications for regenerative medicine approaches

The association of lysosomal dysfunction and neurodegeneration has been documented in several neurodegenerative diseases, including Alzheimer's Disease (AD). Herein, we investigate the association of lysosomal enzymes with AD at different stages of progression of the disease (mild and severe) or with mild cognitive impairment (MCI). We conducted a screening of two classes of lysosomal enzymes: glycohydrolases (\u3b2-Hexosaminidase, \u3b2-Galctosidase, \u3b2-Galactosylcerebrosidase, \u3b2-Glucuronidase) and proteases (Cathepsins S, D, B, L) in peripheral blood samples (blood plasma and PBMCs) from mild AD, severe AD, MCI and healthy control subjects. We confirmed the lysosomal dysfunction in severe AD patients and added new findings enhancing the association of abnormal levels of specific lysosomal enzymes with the mild AD or severe AD, and highlighting the difference of AD from MCI. Herein, we showed for the first time the specific alteration of \u3b2-Galctosidase (Gal), \u3b2-Galactosylcerebrosidase (GALC) in MCI patients. It is notable that in above peripheral biological samples the lysosomes are more sensitive to AD cellular metabolic alteration when compared to levels of A\u3b2-peptide or Tau proteins, similar in both AD groups analyzed. Collectively, our findings support the role of lysosomal enzymes as potential peripheral molecules that vary with the progression of AD, and make them useful for monitoring regenerative medicine approaches for AD

Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy

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Biochemical Pathways of Cellular Mechanosensing/Mechanotransduction and Their Role in Neurodegenerative Diseases Pathogenesis

In this review, we shed light on recent advances regarding the characterization of biochemical pathways of cellular mechanosensing and mechanotransduction with particular attention to their role in neurodegenerative disease pathogenesis. While the mechanistic components of these pathways are mostly uncovered today, the crosstalk between mechanical forces and soluble intracellular signaling is still not fully elucidated. Here, we recapitulate the general concepts of mechanobiology and the mechanisms that govern the mechanosensing and mechanotransduction processes, and we examine the crosstalk between mechanical stimuli and intracellular biochemical response, highlighting their effect on cellular organelles’ homeostasis and dysfunction. In particular, we discuss the current knowledge about the translation of mechanosignaling into biochemical signaling, focusing on those diseases that encompass metabolic accumulation of mutant proteins and have as primary characteristics the formation of pathological intracellular aggregates, such as Alzheimer’s Disease, Huntington’s Disease, Amyotrophic Lateral Sclerosis and Parkinson’s Disease. Overall, recent findings elucidate how mechanosensing and mechanotransduction pathways may be crucial to understand the pathogenic mechanisms underlying neurodegenerative diseases and emphasize the importance of these pathways for identifying potential therapeutic targets

Above the Epitranscriptome: RNA Modifications and Stem Cell Identity

Sequence databases and transcriptome-wide mapping have revealed different reversible and dynamic chemical modifications of the nitrogen bases of RNA molecules. Modifications occur in coding RNAs and noncoding-RNAs post-transcriptionally and they can influence the RNA structure, metabolism, and function. The result is the expansion of the variety of the transcriptome. In fact, depending on the type of modification, RNA molecules enter into a specific program exerting the role of the player or/and the target in biological and pathological processes. Many research groups are exploring the role of RNA modifications (alias epitranscriptome) in cell proliferation, survival, and in more specialized activities. More recently, the role of RNA modifications has been also explored in stem cell biology. Our understanding in this context is still in its infancy. Available evidence addresses the role of RNA modifications in self-renewal, commitment, and differentiation processes of stem cells. In this review, we will focus on five epitranscriptomic marks: N6-methyladenosine, N1-methyladenosine, 5-methylcytosine, Pseudouridine (Ψ) and Adenosine-to-Inosine editing. We will provide insights into the function and the distribution of these chemical modifications in coding RNAs and noncoding-RNAs. Mainly, we will emphasize the role of epitranscriptomic mechanisms in the biology of naïve, primed, embryonic, adult, and cancer stem cells