Wild-Type and Mutant FUS Expression Reduce Proliferation and Neuronal Differentiation Properties of Neural Stem Progenitor Cells.

Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.

Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons.

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a "cholinergic locus", and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

Phosphodiesterase Inhibitors: Could They Be Beneficial for the Treatment of COVID-19?

In March 2020, the World Health Organization declared the severe acute respiratory syndrome corona virus 2 (SARS-CoV2) infection to be a pandemic disease. SARS-CoV2 was first identified in China and, despite the restrictive measures adopted, the epidemic has spread globally, becoming a pandemic in a very short time. Though there is growing knowledge of the SARS-CoV2 infection and its clinical manifestations, an effective cure to limit its acute symptoms and its severe complications has not yet been found. Given the worldwide health and economic emergency issues accompanying this pandemic, there is an absolute urgency to identify effective treatments and reduce the post infection outcomes. In this context, phosphodiesterases (PDEs), evolutionarily conserved cyclic nucleotide (cAMP/cGMP) hydrolyzing enzymes, could emerge as new potential targets. Given their extended distribution and modulating role in nearly all organs and cellular environments, a large number of drugs (PDE inhibitors) have been developed to control the specific functions of each PDE family. These PDE inhibitors have already been used in the treatment of pathologies that show clinical signs and symptoms completely or partially overlapping with post-COVID-19 conditions (e.g., thrombosis, inflammation, fibrosis), while new PDE-selective or pan-selective inhibitors are currently under study. This review discusses the state of the art of the different pathologies currently treated with phosphodiesterase inhibitors, highlighting the numerous similarities with the disorders linked to SARS-CoV2 infection, to support the hypothesis that PDE inhibitors, alone or in combination with other drugs, could be beneficial for the treatment of COVID-19.

Transcriptional regulation of adult neural stem/progenitor cells: tales from the subventricular zone.

In rodents, well characterized neurogenic niches of the adult brain, such as the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampus, support the maintenance of neural/stem progenitor cells (NSPCs) and the production of new neurons throughout the lifespan. The adult neurogenic process is dependent on the intrinsic gene expression signatures of NSPCs that make them competent for self-renewal and neuronal differentiation. At the same time, it is receptive to regulation by various extracellular signals that allow the modulation of neuronal production and integration into brain circuitries by various physiological stimuli. A drawback of this plasticity is the sensitivity of adult neurogenesis to alterations of the niche environment that can occur due to aging, injury or disease. At the core of the molecular mechanisms regulating neurogenesis, several transcription factors have been identified that maintain NSPC identity and mediate NSPC response to extrinsic cues. Here, we focus on REST, Egr1 and Dbx2 and their roles in adult neurogenesis, especially in the subventricular zone. We review recent work from our and other laboratories implicating these transcription factors in the control of NSPC proliferation and differentiation and in the response of NSPCs to extrinsic influences from the niche. We also discuss how their altered regulation may affect the neurogenic process in the aged and in the diseased brain. Finally, we highlight key open questions that need to be addressed to foster our understanding of the transcriptional mechanisms controlling adult neurogenesis.

Extracellular Vesicle-Induced Differentiation of Neural Stem Progenitor Cells.

Neural stem progenitor cells (NSPCs) from E13.5 mouse embryos can be maintained in culture under proliferating conditions. Upon growth-factor removal, they may differentiate toward either neuronal or glial phenotypes or both. Exosomes are small extracellular vesicles that are part of the cell secretome; they may contain and deliver both proteins and genetic material and thus play a role in cell-cell communication, guide axonal growth, modulate synaptic activity and regulate peripheral nerve regeneration. In this work, we were interested in determining whether NSPCs and their progeny can produce and secrete extracellular vesicles (EVs) and if their content can affect cell differentiation. Our results indicate that cultured NSPCs produce and secrete EVs both under proliferating conditions and after differentiation. Treatment of proliferating NSPCs with EVs derived from differentiated NSPCs triggers cell differentiation in a dose-dependent manner, as demonstrated by glial- and neuronal-marker expression.

Metabolic role of cGMP in S. cerevisiae: the murine phosphodiesterase-5 activity affects yeast cell proliferation by altering the cAMP/cGMP equilibrium.

In higher eukaryotes, cAMP and cGMP are signal molecules of major transduction pathways while phosphodiesterases (PDE) are a superfamily of cAMP/cGMP hydrolysing enzymes, modulatory components of these routes. Saccharomyces cerevisiae harbours two genes for PDE: Pde2 is a high affinity cAMP-hydrolysing enzyme, while Pde1 can hydrolyse both cAMP and cGMP. To gain insight into the metabolic role of cGMP in the physiology of yeast, the murine Pde5a1 gene encoding a specific cGMP-hydrolysing enzyme, was expressed in S. cerevisiae pdeΔ strains. pde1Δ and pde2Δ PDE5A1-transformed strain displayed opposite growth-curve profiles; while PDE5A1 recovered the growth delay of pde1Δ, PDE5A1 reversed the growth profile of pde2Δ to that of the untransformed pde1Δ. Growth test analysis and the use of Adh2 and Adh1 as respiro-fermentative glycolytic flux markers confirmed that PDE5A1 altered the metabolism by acting on Pde1-Pde2/cyclic nucleotides content and also on the TORC1 nutrient-sensing cascade. cGMP is required during the log-phase of cell proliferation to adjust/modulate cAMP levels inside well-defined ranges. A model is presented proposing the role of cGMP in the cAMP/PKA pathway. The expression of the PDE5A1 cassette in other mutant strains might constitute the starting tool to define cGMP metabolic role in yeast nutrient signaling.

Egr-1 Maintains NSC Proliferation and Its Overexpression Counteracts Cell Cycle Exit Triggered by the Withdrawal of Epidermal Growth Factor.

In adult mammals, neural stem cells (NSCs) reside in specialized niches at the level of selected CNS regions, such as the subventricular zone (SVZ). The signaling pathways that reg-ulate NSC proliferation and differentiation remain poorly understood. Early growth response protein 1 (Egr-1) is an important transcription factor, widely studied in the adult mammalian brain, mediating the activation of target genes by a variety of extracellular stimuli. In our study, we aimed at testing how Egr-1 regulates adult NSCs derived from mouse SVZ and, in particular, the interplay between Egr-1 and the proliferative factor EGF. We demonstrate that Egr-1 expression in NSCs is induced by growth factor stimulation, and its level decreases after EGF deprivation or by using AG1478, an inhibitor of the EGF/EGFR signaling pathway. We also show that Egr-1 overexpression rescues the cell proliferation decrease observed either after EGF removal or upon treatment with AG1478, suggesting that Egr-1 works downstream of the EGF pathway. To better understand this mechanism, we investigated targets downstream of both the EGF pathway and Egr-1, and found that they regulate genes involved in NSC proliferation, such as cell cycle regulators, cyclins, and cyclin-dependent kinase inhibitors.

5-Hydroxytryptamine 1A and 2B serotonin receptors in neurite outgrowth: involvement of early growth response protein 1.

Neurotransmitters play important roles in neurogenesis; in particular, acetylcholine and serotonin may regulate neurite elongation. Acetylcholine may also activate transcription factors such as early growth response protein 1 (EGR-1), which plays a role in neurite extension. N18TG2 neuroblastoma cells (which do not produce neurotransmitters and constitutively express muscarinic acetylcholine receptors) were transfected with constructs containing the cDNA for choline acetyltransferase, 5-hydroxytryptamine 1A (5-HT1A) and 5-HT2B serotonin receptors to study acetylcholine and serotonin interplay in neurite outgrowth. 5-HT1A receptor stimulation causes a decrease in EGR-1 levels and inhibition of neurite outgrowth; 5-HT2B stimulation, however, has no effect. Muscarinic cholinergic stimulation, on the other end, increases EGR-1 levels and fiber outgrowth. Inhibition of EGR-1 binding reduces fiber outgrowth activity. When both cholinergic and 5-HT1A receptors are stimulated, fiber outgrowth is restored; therefore, acetylcholine counterbalances the inhibitory effect of serotonin on neurite outgrowth. These results suggest that EGR-1 plays a role in the interplay of acetylcholine and serotonin in the regulation of neurite extension during development.

Acetylcholine-induced neuronal differentiation: muscarinic receptor activation regulates EGR-1 and REST expression in neuroblastoma cells.

Neurotransmitters are considered part of the signaling system active in nervous system development and we have previously reported that acetylcholine (ACh) is capable of enhancing neuronal differentiation in cultures of sensory neurons and N18TG2 neuroblastoma cells. To study the mechanism of ACh action, in this study, we demonstrate the ability of choline acetyltransferase-transfected N18TG2 clones (e.g. 2/4 clone) to release ACh. Analysis of muscarinic receptors showed the presence of M1-M4 subtypes and the activation of both IP(3) and cAMP signal transduction pathways. Muscarinic receptor activation increases early growth response factor-1 (EGR-1) levels and treatments with agonists, antagonists, and signal transduction enzyme inhibitors suggest a role for M3 subtype in EGR-1 induction. The role of EGR-1 in the enhancement of differentiation was investigated transfecting in N18TG2 cells a construct for EGR-1. EGR-1 clones show increased neurite extension and a decrease in Repressor Element-1 silencing transcription factor (REST) expression: both these features have also been observed for the 2/4 clone. Transfection of this latter with EGR zinc-finger domain, a dominant negative inhibitor of EGR-1 action, increases REST expression, and decreases fiber outgrowth. The data reported suggest that progression of the clone 2/4 in the developmental program is dependent on ACh release and the ensuing activation of muscarinic receptors, which in turn modulate the level of EGR-1 and REST transcription factors.

Acetylcholine induces neurite outgrowth and modulates matrix metalloproteinase 2 and 9.

The matrix metalloproteinases (MMPs), responsible for the degradation of extracellular matrix (ECM) proteins, may regulate brain cellular functions. Choline acetyltransferase (ChAT) transfected murine neuroblastoma cell line N18TG2, that synthesize acetylcholine and show enhancement of several neurospecific markers (i.e., sinapsin I, voltage gated Na(+) channels, high affinity choline uptake) and fiber outgrowth, were studied for the MMP regulation during neuronal differentiation. Zymography of N18TG2 culture medium revealed no gelatinolytic activity, whereas after carbachol treatment of cells both MMP-9 and activated MMP-2 forms were detected. ChAT-transfected clone culture medium contains three MMP forms at 230, 92, and 66kDa. Carbachol treatment increased MMP-2 and MMP-9 gene expression in N18TG2 cells and higher levels for both genes were also observed in ChAT transfected cells. The data are consistent with the hypothesis that acetylcholine brings about the activation of an autocrine loop modulating MMP expression.