Overexpression of the X-Linked Inhibitor of Apoptosis Protein (XIAP) in Neurons Improves Cell Survival and the Functional Outcome after Traumatic Spinal Cord Injury

Mechanical trauma to the spinal cord causes extensive neuronal death, contributing to the loss of sensory-motor and autonomic functions below the injury location. Apoptosis affects neurons after spinal cord injury (SCI) and is associated with increased caspase activity. Cleavage of X-linked inhibitor of apoptosis protein (XIAP) after SCI may contribute to this rise in caspase activity. Accordingly, we have shown that the elevation of XIAP resulted in increased neuronal survival after SCI and improved functional recovery. Therefore, we hypothesise that neuronal overexpression of XIAP can be neuroprotective after SCI with improved functional recovery. In line with this, studies of a transgenic mice with overexpression of XIAP in neurons revealed that higher levels of XIAP after spinal cord trauma favours neuronal survival, tissue preservation, and motor recovery after the spinal cord trauma. Using human SH-SY5Y cells overexpressing XIAP, we further showed that XIAP reduced caspase activity and apoptotic cell death after pro-apoptotic stimuli. In conclusion, this study shows that the levels of XIAP expression are an important factor for the outcome of spinal cord trauma and identifies XIAP as an important therapeutic target for alleviating the deleterious effects of SCI

Evaluation of Poly(N-Ethyl Pyrrolidine Methacrylamide) (EPA) and Derivatives as Polymeric Vehicles for miRNA Delivery to Neural Cells

MicroRNAs (miRNAs) are endogenous, short RNA oligonucleotides that regulate the expression of hundreds of proteins to control cells' function in physiological and pathological conditions. miRNA therapeutics are highly specific, reducing the toxicity associated with off-target effects, and require low doses to achieve therapeutic effects. Despite their potential, applying miRNA-based therapies is limited by difficulties in delivery due to their poor stability, fast clearance, poor efficiency, and off-target effects. To overcome these challenges, polymeric vehicles have attracted a lot of attention due to their ease of production with low costs, large payload, safety profiles, and minimal induction of the immune response. Poly(N-ethyl pyrrolidine methacrylamide) (EPA) copolymers have shown optimal DNA transfection efficiencies in fibroblasts. The present study aims to evaluate the potential of EPA polymers as miRNA carriers for neural cell lines and primary neuron cultures when they are copolymerized with different compounds. To achieve this aim, we synthesized and characterized different copolymers and evaluated their miRNA condensation ability, size, charge, cytotoxicity, cell binding and internalization ability, and endosomal escape capacity. Finally, we evaluated their miRNA transfection capability and efficacy in Neuro-2a cells and rat primary hippocampal neurons. The results indicate that EPA and its copolymers, incorporating β-cyclodextrins with or without polyethylene glycol acrylate derivatives, can be promising vehicles for miRNA administration to neural cells when all experiments on Neuro-2a cells and primary hippocampal neurons are considered together.This research was supported by the Council of Education, Culture and Sports of the Regional Government of Castilla La Mancha (Spain) and Co-financed by the European Union (FEDER) “A way to make Europe” (project references SBPLY/17/000376 and SBPLY/21/180501/000097) and by the Ministerio de Ciencia, Innovación y Universidades (RTI2018-096328-B-I00). Altea Soto was funded by the Council of Education, Culture and Sports of the Regional Government of Castilla La Mancha (Spain) M. Asunción Barreda-Manso is funded by the Council of Health of the Regional Government of Castilla La Mancha (Spain), through: “Convocatoria de Ayudas Regionales a la Investigación en Biomedicina y Ciencias de la Salud” (II-2020_05). Irene Novillo Algaba is funded by the Next Generation Funds of the European Union through the “Programa Investigo”.Peer reviewe

MicroRNA Dysregulation in the Spinal Cord following Traumatic Injury

Spinal cord injury (SCI) triggers a multitude of pathophysiological events that are tightly regulated by the expression levels of specific genes. Recent studies suggest that changes in gene expression following neural injury can result from the dysregulation of microRNAs, short non-coding RNA molecules that repress the translation of target mRNA. To understand the mechanisms underlying gene alterations following SCI, we analyzed the microRNA expression patterns at different time points following rat spinal cord injury

Climate seasonality limits leaf carbon assimilation and wood productivity in tropical forests

The seasonal climate drivers of the carbon cycle in tropical forests remain poorly known, although these forests account for more carbon assimilation and storage than any other terrestrial ecosystem. Based on a unique combination of seasonal pan-tropical data sets from 89 experimental sites (68 include aboveground wood productivity measurements and 35 litter productivity measurements), their associated canopy photosynthetic capacity (enhanced vegetation index, EVI) and climate, we ask how carbon assimilation and aboveground allocation are related to climate seasonality in tropical forests and how they interact in the seasonal carbon cycle. We found that canopy photosynthetic capacity seasonality responds positively to precipitation when rainfall is  < 2000 mm yr⁻¹ (water-limited forests) and to radiation otherwise (light-limited forests). On the other hand, independent of climate limitations, wood productivity and litterfall are driven by seasonal variation in precipitation and evapotranspiration, respectively. Consequently, light-limited forests present an asynchronism between canopy photosynthetic capacity and wood productivity. First-order control by precipitation likely indicates a decrease in tropical forest productivity in a drier climate in water-limited forest, and in current light-limited forest with future rainfall  < 2000 mm yr⁻¹

Factors promoting neurite outgrowth during deer antler regeneration

Every year male deers completely regenerate their antlers. During this process, antlers are reinnervated by sensory fibers, growing at the highest rate recorded for any adult mammal. Despite its clinical potential, only a few studies have dealt with this fascinating phenomenon. Among the possible factors underlying fast growth of the antler's innervation, the effects of the antler's endocrine and paracrine factors were evaluated, using an in vitro assay for sensory neurite growth. We found that soluble molecules secreted by the velvet, the modified skin that covers the antler, strongly promote neurite outgrowth. Using specific blocking antibodies, we demonstrated that nerve growth factor is partially responsible for these effects, although other unidentified molecules are also involved. On the contrary, neither endocrine serum factors nor antler substrates promoted neurite outgrowth, although antler substrata from deep velvet layers cause neurite outgrowth orientation. Taken together, our results point to the existence in the deep velvet of an environment that promotes oriented axon growth, in agreement with the distribution of the antler innervation. © 2010 Wiley-Liss, Inc.Peer Reviewe

Gene expression of axon growth promoting factors in the deer antler

The annual regeneration cycle of deer (Cervidae, Artiodactyla) antlers represents a unique model of epimorphic regeneration and rapid growth in adult mammals. Regenerating antlers are innervated by trigeminal sensory axons growing through the velvet, the modified form of skin that envelopes the antler, at elongation velocities that reach one centimetre per day in the common deer (Cervus elaphus). Several axon growth promoters like NT-3, NGF or IGF-1 have been described in the antler. To increase the knowledge on the axon growth environment, we have combined different gene-expression techniques to identify and characterize the expression of promoting molecules not previously described in the antler velvet. Cross-species microarray analyses of deer samples on human arrays allowed us to build up a list of 90 extracellular or membrane molecules involved in axon growth that were potentially being expressed in the antler. Fifteen of these genes were analysed using PCR and sequencing techniques to confirm their expression in the velvet and to compare it with the expression in other antler and skin samples. Expression of 8 axon growth promoters was confirmed in the velvet, 5 of them not previously described in the antler. In conclusion, our work shows that antler velvet provides growing axons with a variety of promoters of axon growth, sharing many of them with deer's normal and pedicle skin. © 2010 Pita-Thomas et al.Peer Reviewe

In Silico and In Vitro Analyses Validate Human MicroRNAs Targeting the SARS-CoV-2 3′-UTR

COVID-19 pandemic is caused by betacoronavirus SARS-CoV-2. The genome of this virus is composed of a single strand of RNA with 5′ and 3′-UTR flanking a region of protein-coding ORFs closely resembling cells’ mRNAs. MicroRNAs are endogenous post-transcriptional regulators that target mRNA to modulate protein expression and mediate cellular functions, including antiviral defense. In the present study, we carried out a bioinformatics screening to search for endogenous human microRNAs targeting the 3′-UTR of SARS-CoV-2. Results from the computational techniques allowed us to identify 10 potential candidates. The capacity of 3 of them, together with hsa-miR-138-5p, to target the SARS-CoV-2 3′-UTR was validated in vitro by gene reporter assays. Available information indicates that two of these microRNAs, namely, hsa-miR-3941 and hsa-miR-138-5p, combine effective targeting of SARS-CoV-2 genome with complementary antiviral or protective effects in the host cells that make them potential candidates for therapeutic treatment of most, if not all, COVID-19 variants known to date. All information obtained while conducting the present analysis is available at Open Science Framework repository

MiR-138-5p Upregulation during Neuronal Maturation Parallels with an Increase in Neuronal Survival

Neuronal maturation is a process that plays a key role in the development and regeneration of the central nervous system. Although embryonic brain development and neurodegeneration have received considerable attention, the events that govern postnatal neuronal maturation are less understood. Among the mechanisms influencing such neuronal maturation processes, apoptosis plays a key role. Several regulators have been described to modulate apoptosis, including post-transcriptional regulation by microRNAs. This study aimed to analyze endogenous expression changes of miR-138-5p, as well as its main validated pro-apoptotic target caspase3, during the maturation of neuronal cultures and their response under apoptotic challenge. Our results point out that the observed opposite expression of miR-138-5p and its target caspase3 might modulate apoptosis favoring neuronal survival at distinct maturation stages. The unchanged expression of miR-138-5p in mature neurons contrasts with the significant downregulation in immature neurons upon apoptotic stimulation. Similarly, immunoblot and individual cellular assays confirmed that during maturation, not only the expression but processing of CASP-3 and caspase activity is reduced after apoptotic stimulation which results in a reduction of neuronal death. Further studies would be needed to determine a more detailed role of miR-138-5p in apoptosis during neuronal maturation and the synergistic action of several microRNAs acting cooperatively on caspase3 or other apoptotic targets

miR-182-5p Regulates Nogo-A Expression and Promotes Neurite Outgrowth of Hippocampal Neurons In Vitro

Nogo-A protein is a key myelin-associated inhibitor of axonal growth, regeneration, and plasticity in the central nervous system (CNS). Regulation of the Nogo-A/NgR1 pathway facilitates functional recovery and neural repair after spinal cord trauma and ischemic stroke. MicroRNAs are described as effective tools for the regulation of important processes in the CNS, such as neuronal differentiation, neuritogenesis, and plasticity. Our results show that miR-182-5p mimic specifically downregulates the expression of the luciferase reporter gene fused to the mouse Nogo-A 3&prime;UTR, and Nogo-A protein expression in Neuro-2a and C6 cells. Finally, we observed that when rat primary hippocampal neurons are co-cultured with C6 cells transfected with miR-182-5p mimic, there is a promotion of the outgrowth of neuronal neurites in length. From all these data, we suggest that miR-182-5p may be a potential therapeutic tool for the promotion of axonal regeneration in different diseases of the CNS

Overexpression of the X-Linked Inhibitor of Apoptosis Protein (XIAP) in Neurons Improves Cell Survival and the Functional Outcome after Traumatic Spinal Cord Injury

Mechanical trauma to the spinal cord causes extensive neuronal death, contributing to the loss of sensory-motor and autonomic functions below the injury location. Apoptosis affects neurons after spinal cord injury (SCI) and is associated with increased caspase activity. Cleavage of X-linked inhibitor of apoptosis protein (XIAP) after SCI may contribute to this rise in caspase activity. Accordingly, we have shown that the elevation of XIAP resulted in increased neuronal survival after SCI and improved functional recovery. Therefore, we hypothesise that neuronal overexpression of XIAP can be neuroprotective after SCI with improved functional recovery. In line with this, studies of a transgenic mice with overexpression of XIAP in neurons revealed that higher levels of XIAP after spinal cord trauma favours neuronal survival, tissue preservation, and motor recovery after the spinal cord trauma. Using human SH-SY5Y cells overexpressing XIAP, we further showed that XIAP reduced caspase activity and apoptotic cell death after pro-apoptotic stimuli. In conclusion, this study shows that the levels of XIAP expression are an important factor for the outcome of spinal cord trauma and identifies XIAP as an important therapeutic target for alleviating the deleterious effects of SCI