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Neural stem cells (NSCs) exhibit a remarkable capacity for self-renewal and have the potential to differentiate into various neural lineage cells, which makes them pivotal in the management of neurological disorders. Harnessing the inherent potential of endogenous NSCs for enhancing nerve repair and regeneration represents an optimal approach to addressing diseases of the nervous system. In this study, we explored the potential of a novel benzophenone derivative named Digirseophene A (DGA), which was isolated from the endophytic fungus Corydalis tomentella. Previous experiments have extensively identified and characterized DGA, revealing its unique properties. Our findings demonstrate the remarkable capability of DGA to stimulate neural stem cell proliferation, both in vitro and in vivo. Furthermore, we established a model of radiation-induced cerebellar injury to assess the effects of DGA on the distribution of different cell subpopulations within the damaged cerebellum, thereby suggesting its beneficial role in cerebellar repair. In addition, our observations on a primary NSCs model revealed that DGA significantly increased cellular oxygen consumption, indicating increased energy and metabolic demands. By utilizing various pathway inhibitors in combination with DGA, we successfully demonstrated its ability to counteract the suppressive impacts of AMPK and GSK3ß inhibitors on NSC proliferation. Collectively, our research results strongly suggest that DGA, as an innovative compound, exerts its role in activating NSCs and promoting injury repair through the regulation of the AMPK/AKT/GSK3ß pathway.

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Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here, we define the specific metabolic pathway by which O-GlcNAc transferase (ogt-1) loss of function mediates increased regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans, we find that glycolysis, serine synthesis pathway (SSP), one-carbon metabolism (OCM), and the downstream transsulfuration metabolic pathway (TSP) are all essential in this process. The regenerative effects of ogt-1 mutation are abrogated by genetic and/or pharmacological disruption of OCM and the SSP linking OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.

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Aging causes numerous age-related diseases, leading the human species to death. Nevertheless, rejuvenating strategies based on cell epigenetic modifications are a possible approach to counteract disease progression while getting old. Cell reprogramming of adult somatic cells toward pluripotency ought to be a promising tool for age-related diseases. However, researchers do not have control over this process as cells lose their fate, and cause potential cancerous cells or unexpected cell phenotypes. Direct and partial reprogramming were introduced in recent years with distinctive applications. Although direct reprogramming makes cells lose their identity, it has various applications in regeneration medicine. Temporary and regulated in vivo overexpression of Yamanaka factors has been shown in several experimental contexts to be achievable and is used to rejuvenate mice models. This regeneration can be accomplished by altering the epigenetic adult cell signature to the signature of a younger cell. The greatest advantage of partial reprogramming is that this method does not allow cells to lose their identity when they are resetting their epigenetic clock. It is a regimen of short-term Oct3/4, Sox2, Klf4, and c-Myc expression in vivo that prevents full reprogramming to the pluripotent state and avoids both tumorigenesis and the presence of unwanted undifferentiated cells. We know that many neurological age-related diseases, such as Alzheimer's disease, stroke, dementia, and Parkinson's disease, are the main cause of death in the last decades of life. Therefore, scientists have a special tendency regarding neuroregeneration methods to increase human life expectancy.

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In this work, the glycine-based acryloyl monomer is polymerized to obtain a neurogenic polymeric hydrogel for regenerative applications. The synthesized poly(N-acryloylglycine-acrylamide) [poly(NAG-b-A)] nanohydrogel exhibits high swelling (∼1500%) and is mechanically very stable, biocompatible, and proliferative in nature. The poly(NAG-b-A) nanohydrogel provides a stable 3D extracellular mimetic environment and promotes healthy neurite growth for primary cortical neurons by facilitating cellular adhesion, proliferation, actin filament stabilization, and neuronal differentiation. Furthermore, the protective role of the poly(NAG-b-A) hydrogel for the neurons in oxidative stress conditions is revealed and it is found that it is a clinically relevant material for neuronal regenerative applications, such as for promoting nerve regeneration via GSK3ß inhibition. This hydrogel additionally plays an important role in modulating the biological microenvironment, either as an agonist and antagonist or as an antioxidant. Furthermore, it favors the physiological responses and eases the neurite growth efficiency. Additionally, we found out that the conversion of glycine-based acryloyl monomers into their corresponding polymer modulates the mechanical performance, mimics the cellular microenvironment, and accelerates the self-healing capability due to the responsive behavior towards reactive oxygen species (ROS). Thus, the p(NAG-b-A) hydrogel could be a potential candidate to induce neuronal regeneration since it provides a physical cue and significantly boosts neurite outgrowth and also maintains the microtubule integrity in neuronal cells.

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Background: Retinal diseases characterized with irreversible loss of retinal nerve cells, such as optic atrophy and retinal degeneration, are the main causes of blindness. Current treatments for these diseases are very limited. An emerging treatment strategy is to induce the reprogramming of Müller glial cells to generate new retinal nerve cells, which could potentially restore vision. Main text: Müller glial cells are the predominant glial cells in retinae and play multiple roles to maintain retinal homeostasis. In lower vertebrates, such as in zebrafish, Müller glial cells can undergo cell reprogramming to regenerate new retinal neurons in response to various damage factors, while in mammals, this ability is limited. Interestingly, with proper treatments, Müller glial cells can display the potential for regeneration of retinal neurons in mammalian retinae. Recent studies have revealed that dozens of genetic and epigenetic regulators play a vital role in inducing the reprogramming of Müller glial cells in vivo. This review summarizes these critical regulators for Müller glial cell reprogramming and highlights their differences between zebrafish and mammals. Conclusions: A number of factors have been identified as the important regulators in Müller glial cell reprogramming. The early response of Müller glial cells upon acute retinal injury, such as the regulation in the exit from quiescent state, the initiation of reactive gliosis, and the re-entry of cell cycle of Müller glial cells, displays significant difference between mouse and zebrafish, which may be mediated by the diverse regulation of Notch and TGFß (transforming growth factor-ß) isoforms and different chromatin accessibility.

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Electrical stimulation is a powerful strategy to improve the differentiation of neural stem cells into neurons. Such an approach can be implemented, in association with biomaterials and nanotechnology, for the development of new therapies for neurological diseases, including direct cell transplantation and the development of platforms for drug screening and disease progression evaluation. Poly(aniline):camphorsulfonic acid (PANI:CSA) is one of the most well-studied electroconductive polymers, capable of directing an externally applied electrical field to neural cells in culture. There are several examples in the literature on the development of PANI:CSA-based scaffolds and platforms for electrical stimulation, but no review has examined the fundamentals and physico-chemical determinants of PANI:CSA for the design of platforms for electrical stimulation. This review evaluates the current literature regarding the application of electrical stimulation to neural cells, specifically reviewing: (1) the fundamentals of bioelectricity and electrical stimulation; (2) the use of PANI:CSA-based systems for electrical stimulation of cell cultures; and (3) the development of scaffolds and setups to support the electrical stimulation of cells. Throughout this work, we critically evaluate the revised literature and provide a steppingstone for the clinical application of the electrical stimulation of cells using electroconductive PANI:CSA platforms/scaffolds.

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A conditioning lesion of the peripheral sensory axon triggers robust central axon regeneration in mammals. We trigger conditioned regeneration in the Caenorhabditis elegans ASJ neuron by laser surgery or genetic disruption of sensory pathways. Conditioning upregulates thioredoxin-1 (trx-1) expression, as indicated by trx-1 promoter-driven expression of green fluorescent protein and fluorescence in situ hybridization (FISH), suggesting trx-1 levels and associated fluorescence indicate regenerative capacity. The redox activity of trx-1 functionally enhances conditioned regeneration, but both redox-dependent and -independent activity inhibit non-conditioned regeneration. Six strains isolated in a forward genetic screen for reduced fluorescence, which suggests diminished regenerative potential, also show reduced axon outgrowth. We demonstrate an association between trx-1 expression and the conditioned state that we leverage to rapidly assess regenerative capacity.

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Olfactory disorders, which are closely related to cognitive deterioration, can be caused by several factors, including infections, such as COVID-19; aging; and environmental chemicals. Injured olfactory receptor neurons (ORNs) regenerate after birth, but it is unclear which receptors and sensors are involved in ORN regeneration. Recently, there has been great focus on the involvement of transient receptor potential vanilloid (TRPV) channels, which are nociceptors expressed on sensory nerves during the healing of damaged tissues. The localization of TRPV in the olfactory nervous system has been reported in the past, but its function there are unclear. Here, we investigated how TRPV1 and TRPV4 channels are involved in ORN regeneration. TRPV1 knockout (KO), TRPV4 KO, and wild-type (WT) mice were used to model methimazole-induced olfactory dysfunction. The regeneration of ORNs was evaluated using olfactory behavior, histologic examination, and measurement of growth factors. Both TRPV1 and TRPV4 were found to be expressed in the olfactory epithelium (OE). TRPV1, in particular, existed near ORN axons. TRPV4 was marginally expressed in the basal layer of the OE. The proliferation of ORN progenitor cells was reduced in TRPV1 KO mice, which delayed ORN regeneration and the improvement of olfactory behavior. Postinjury OE thickness improved faster in TRPV4 KO mice than WT mice but without acceleration of ORN maturation. The nerve growth factor and transforming growth factor ß levels in TRPV1 KO mice were similar to those in WT mice, and the transforming growth factor ß level was higher than TRPV4 KO mice. TRPV1 was involved in stimulating the proliferation of progenitor cells. TRPV4 modulated their proliferation and maturation. ORN regeneration was regulated by the interaction between TRPV1 and TRPV4. However, in this study, TRPV4 involvement was limited compared with TRPV1. To our knowledge, this is the first study to demonstrate the involvement of TRPV1 and TRPV4 in OE regeneration.

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Hearing loss affects over 460 million people worldwide and is a major socioeconomic burden. Both genetic and environmental factors (i.e., noise overexposure, ototoxic drug treatment and ageing), promote the irreversible degeneration of cochlear hair cells and associated auditory neurons, leading to sensorineural hearing loss. In contrast to birds, fish and amphibians, the mammalian inner ear is virtually unable to regenerate due to the limited stemness of auditory progenitors, and no causal treatment is able to prevent or reverse hearing loss. As of today, a main limitation for the development of otoprotective or otoregenerative therapies is the lack of efficient preclinical models compatible with high-throughput screening of drug candidates. Currently, the research field mainly relies on primary organotypic inner ear cultures, resulting in high variability, low throughput, high associated costs and ethical concerns. We previously identified and characterized the phoenix auditory neuroprogenitors (ANPGs) as highly proliferative progenitor cells isolated from the A/J mouse cochlea. In the present study, we aim at identifying the signaling pathways responsible for the intrinsic high stemness of phoenix ANPGs. A transcriptomic comparison of traditionally low-stemness ANPGs, isolated from C57Bl/6 and A/J mice at early passages, and high-stemness phoenix ANPGs was performed, allowing the identification of several differentially expressed pathways. Based on differentially regulated pathways, we developed a reprogramming protocol to induce high stemness in presenescent ANPGs (i.e., from C57Bl6 mouse). The pharmacological combination of the WNT agonist (CHIR99021) and TGFß/Smad inhibitors (LDN193189 and SB431542) resulted in a dramatic increase in presenescent neurosphere growth, and the possibility to expand ANPGs is virtually limitless. As with the phoenix ANPGs, stemness-induced ANPGs could be frozen and thawed, enabling distribution to other laboratories. Importantly, even after 20 passages, stemness-induced ANPGs retained their ability to differentiate into electrophysiologically mature type I auditory neurons. Both stemness-induced and phoenix ANPGs resolve a main bottleneck in the field, allowing efficient, high-throughput, low-cost and 3R-compatible in vitro screening of otoprotective and otoregenerative drug candidates. This study may also add new perspectives to the field of inner ear regeneration.

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Femtosecond lasers are capable of precise ablation that produces surgical dissections in vivo. The transverse and axial resolutions of the laser damage inside the bulk are important parameters of ablation. The transverse resolution is routinely quantified; but the axial resolution is more difficult to measure and is less commonly performed. Using a 1040-nm, 400-fs pulsed laser, and a 1.4-NA objective, we performed ablation inside agarose and glass, producing clear, and persistent damage spots. Near the ablation threshold of both media, we found that the axial resolution is similar to the transverse resolution. We also ablated neuron cell bodies and fibers in Caenorhabditis elegans and demonstrate submicrometer resolution in both the transverse and axial directions, consistent with our results in agarose and glass. Using simple yet rigorous methods, we define the resolution of laser ablation in transparent media along all directions.

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Auditory neurons connect the sensory hair cells from the inner ear to the brainstem. These bipolar neurons are relevant targets for pharmacological intervention aiming at protecting or improving the hearing function in various forms of sensorineural hearing loss. In the research laboratory, neurotrophic compounds are commonly used to improve survival and to promote regeneration of auditory neurons. One important roadblock delaying eventual clinical applications of these strategies in humans is the lack of powerful in vitro models allowing high throughput screening of otoprotective and regenerative compounds. The recently discovered auditory neuroprogenitors (ANPGs) derived from the A/J mouse with an unprecedented capacity to self-renew and to provide mature auditory neurons offer the possibility to overcome this bottleneck. In the present study, we further characterized the new phoenix ANPGs model and compared it to the current gold-standard spiral ganglion organotypic explant (SGE) model to assay neurite outgrowth, neurite length and glutamate-induced Ca2+ response in response to neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF) treatment. Whereas both, SGEs and phoenix ANPGs exhibited a robust and sensitive response to neurotrophins, the phoenix ANPGs offer a considerable range of advantages including high throughput suitability, lower experimental variability, single cell resolution and an important reduction of animal numbers. The phoenix ANPGs in vitro model therefore provides a robust high-throughput platform to screen for otoprotective and regenerative neurotrophic compounds in line with 3R principles and is of interest for the field of auditory neuroscience.

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Background: Generalized anxiety disorder (GAD) is one of the most common types of anxiety disorders with unclear pathogenesis. Our team's previous research found that extensive neuronal apoptosis and neuronal regeneration disorders occur in the hippocampus of GAD rats. Danzhi Xiaoyao (DZXYS) Powder can improve the anxiety behavior of rats, but its molecular mechanism is not well understood. Objective: This paper discusses whether the pathogenesis of GAD is related to the abnormal expression of Notch signal pathway, and whether the anti-anxiety effect of DZXYS promotes nerve regeneration in the hippocampus by regulating the Notch signaling pathway. Methods: The animal model of GAD was developed by the chronic restraint stress and uncertain empty bottle stimulation method. After the model was successfully established, the rats in the model preparation group were divided into the buspirone, DZXYS, DZXYS + DAPT, and model groups, and were administered the corresponding drug intervention. The changes in body weight and food intake of rats were continuously monitored throughout the process. The changes in anxiety behavior of rats were measured by open field experiment and elevated plus-maze test, and morphological changes and regeneration of neurons in the rat hippocampus were observed by HE staining and double immunofluorescence staining. Changes in the expression of key targets of the Notch signaling pathway in the hippocampus were monitored by real-time fluorescence quantitative PCR and western blotting. Results: In this study, we verified that the GAD model was stable and reliable, and found that the key targets of the Notch signaling pathway (Notch1, Hes1, Hes5, etc.) in the hippocampus of GAD rats were significantly upregulated, leading to the increased proliferation of neural stem cells in the hippocampus and increased differentiation into astrocytes, resulting in neuronal regeneration. DZXYS intervention in GAD rats can improve appetite, promote weight growth, and significantly reverse the anxiety behavior of GAD rats, which can inhibit the upregulation of key targets of the Notch signaling pathway, promote the differentiation of neural stem cells in the hippocampus into neurons, and inhibit their differentiation into astrocytes, thus alleviating anxiety behavior. Conclusion: The occurrence of GAD is closely related to the upregulation of the Notch signaling pathway, which hinders the regeneration of normal neurons in the hippocampus, while DZXYS can downregulate the Notch signaling pathway and promote neuronal regeneration in the hippocampus, thereby relieving anxiety behavior.

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miRNA(miR)-124 is an important regulator of neurogenesis, but its upregulation in SOD1G93A motor neurons (mSOD1 MNs) was shown to associate with neurodegeneration and microglia activation. We used pre-miR-124 in wild-type (WT) MNs and anti-miR-124 in mSOD1 MNs to characterize the miR-124 pathological role. miR-124 overexpression in WT MNs produced a miRNA profile like that of mSOD1 MNs (high miR-125b; low miR-146a and miR-21), and similarly led to early apoptosis. Alterations in mSOD1 MNs were abrogated with anti-miR-124 and changes in their miRNAs mostly recapitulated by their secretome. Normalization of miR-124 levels in mSOD1 MNs prevented the dysregulation of neurite network, mitochondria dynamics, axonal transport, and synaptic signaling. Same alterations were observed in WT MNs after pre-miR-124 transfection. Secretome from mSOD1 MNs triggered spinal microglia activation, which was unno-ticed with that from anti-miR-124-modulated cells. Secretome from such modulated MNs, when added to SC organotypic cultures from mSOD1 mice in the early symptomatic stage, also coun-teracted the pathology associated to GFAP decrease, PSD-95 and CX3CL1-CX3CR1 signaling im-pairment, neuro-immune homeostatic imbalance, and enhanced miR-124 expression levels. Data suggest that miR-124 is implicated in MN degeneration and paracrine-mediated pathogenicity. We propose miR-124 as a new therapeutic target and a promising ALS biomarker in patient sub-populations.

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Aging is known to slow the neurogenic capacity of the hippocampus, one of only two mammalian adult neurogenic niches. The reduction of adult-born neurons with age may initiate cognitive decline progression which is exacerbated in chronic neurodegenerative disorders, e.g., Alzheimer's disease (AD). With physiologic neurogenesis diminished, but still viable in aging, non-invasive therapeutic modulation of this neuron regeneration process remains possible. The discovery of truly novel neuron regenerative therapies could be identified through phenotypic screening of small molecules that promote adult-born neurons from human neural progenitor cells (hNPCs). By identifying neuron-generating therapeutics and potentially novel mechanism of actions, therapeutic benefit could be confirmed through in vivo proof-of-concept studies. The key aging and longevity mTOR/p70S6 kinase axis, a commonly targeted pathway, is substrate for potential selective kinase modulators to promote new hippocampal neurons from NPCs. The highly regulated downstream substrate of mTOR, p70S6 kinase, directly controls pleiotropic cellular activities, including translation and cell growth. Stimulating this kinase, selectively in an adult neurogenic niche, should promote NPC proliferation, and cell growth and survival in the hippocampus. Studies of kinase profiling and immunocytochemistry of human progenitor neurogenesis suggest that the novel small molecule NNI-362 stimulates p70S6 kinase phosphorylation, which, in turn, promotes proliferation and differentiation of NPCs to neurons. NNI-362 promoted the associative reversal of age- and disease-related cognitive deficits in aged mice and Down syndrome-modeled mice. This oral, allosteric modulator may ultimately be beneficial for age-related neurodegenerative disorders involving hippocampal-dependent cognitive impairment, specifically AD, by promoting endogenous hippocampal regeneration.

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Biomaterial scaffold designs are needed for self-organizing features related to tissue formation while also simplifying the fabrication processes involved. Toward this goal, silk protein-based self-folding scaffolds to support 3D cell culture, while providing directional guidance and promotion of cell growth and differentiation, are reported. A simple and robust one-step self-folding approach is developed using bilayers consisting of a hydrogel and silk film in aqueous solution. The 3D silk rolls, with patterns transferred from the initially prepared 2D films, guide the directional outgrowth of neurites and also promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The osteogenic outcomes are further supported by enhanced biomechanical performance. By utilizing this self-folding method, cocultures of neurons and hMSCs are achieved by patterning cells on silk films and then converting these materials into a 3D format with rolling, mimicking aspects of the structure of osteons and providing physiologically relevant structures to promote bone regeneration. These results demonstrate the utility of self-folded silk rolls as efficient scaffold systems for tissue regeneration, while exploiting relatively simple 2D designs programmed to form more complex 3D structures.

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BACKGROUND: Inflammatory damage following stroke aggravates brain damage, resulting in long-term neurological sequelae. The purpose of this study was to identify ways to reduce inflammatory reactions and to accelerate neuron regeneration after cerebral apoplexy. METHODS: We formulated a biomimetic vesicle, the leukosome, constituted by liposome, artificial long intergenic noncoding RNA (lincRNA)-EPS, and membrane proteins derived from macrophages and their physical-chemical characteristics were evaluated. Migration distance and cytotoxic levels were measured to determine the effect of lncEPS-leukosomes on lipopolysaccharide-activated microglia. An in vivo transient middle cerebral artery occlusion/reperfusion (tMCAO) model was established in mice, which were treated with lncEPS-leukosomes. Vesicle seepage, infiltration of inflammatory cells, cytotoxic levels in the cerebrospinal fluid, and neural stem cell (NSC) density were measured. RESULTS: Biomimetic vesicles with a homogeneous size increased lincRNA-EPS levels in activated microglia by 77.9%. In vitro studies showed that lincRNA-EPS inhibited the migration and cytotoxic levels of activated microglia by 63.2% and 43.6%, respectively, which promoted NSC proliferation and anti-apoptotic ability. In vivo data showed that leukosomes targeted to inflamed sites and lncEPS-leukosomes decreased the infiltration of inflammatory cells and cytotoxic levels by 81.3% and 48.7%, respectively. In addition, lncEPS-leukosomes improved neuron density in the ischemic core and boundary zone after tMCAO. CONCLUSIONS: The biomimetic vesicles formulated in this study targeted inflammatory cells and accelerated neuron regeneration by promoting inflammation resolution. This study may provide a promising treatment approach for accelerated neuron regeneration after cerebral apoplexy.

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Generation of new neurons by utilizing the regenerative potential of adult neural stem cells (NSCs) and neuroblasts is an emerging therapeutic strategy to treat various neurodegenerative diseases, including neuronal loss after stroke. Committed to neuronal lineages, neuroblasts are differentiated from NSCs and have a lower proliferation rate. In stroke the proliferation of the neuroblasts in the neurogenic areas is increased, but the limiting factor for regeneration is the poor survival of migrating neuroblasts. Survival of neuroblasts can be promoted by small molecules; however, new drug delivery methods are needed to specifically target these cells. Herein, to achieve specific targeting, we have engineered biofunctionalized porous silicon nanoparticles (PSi NPs) conjugated with a specific antibody against polysialylated neural cell adhesion molecule (PSA-NCAM). The PSi NPs loaded with a small molecule drug, SC-79, were able to increase the activity of the Akt signaling pathway in doublecortin positive neuroblasts both in cultured cells and in vivo in the rat brain. This study opens up new possibilities to target drug effects to migrating neuroblasts and facilitate differentiation, maturation and survival of developing neurons. The conjugated PSi NPs are a novel tool for future studies to develop new therapeutic strategies aiming at regenerating functional neurocircuitry after stoke.

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Neural tissue engineering has emerged as a promising technology to cure neural damages. Although various synthetic polymers with good biocompatibility and biodegradability have been adopted as candidate materials for scaffolds, most of them require the incorporation of biomolecules or conductive materials to promote the growth of long axons. Herein we demonstrate for the first time a unique peptide-based polyelectrolyte that is ionically conductive and contains a neurotransmitter, glutamic acid. The designed polymer, sodium salt of poly(γ-benzyl-l-glutamate)-r-poly(l-glutamic acid) (PBGA20-Na), was synthesized and fabricated into a 3D fibrous scaffold with aligned fibers. Neuron-like rat pheochromocytoma (PC12) cells were cultured on the scaffolds to evaluate cell proliferation and differentiation with or without electrical stimulation. The results show that with both electrical and biochemical cues presented in the polyelectrolyte, PBGA20-Na promotes longer neurite outgrowth compared with the neutral poly(γ-benzyl-l-glutamate) (PBG) and the poly(γ-benzyl-l-glutamate)-r-poly(l-glutamic acid) (PBGA20). Furthermore, the neurite length of the cells cultured on PBGA20-Na is more than twice as long compared with the conventional biopolymer, polycaprolactone. In conclusion, PBGA20-Na is a promising biomaterial for neural tissue engineering and drug-screening platforms.

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The irrefutable change in the expression of brain-enriched microRNAs (miRNAs) following ischemic stroke has promoted the development of radical miRNA-based therapeutics encompassing neuroprotection and neuronal restoration. Our previous report on the systems-level prediction of miR-9 in post-stroke-induced neurogenesis served as a premise to experimentally uncover the functional role of miR-9 in post-ischemic neuronal survival and regeneration. The oxygen-glucose deprivation (OGD) in SH-SY5Y cells significantly reduced miR-9 expression, while miR-9 mimic transfection enhanced post-ischemic neuronal cell viability. The next major objective involved the execution of a drug repositioning strategy to augment miR-9 expression via structure-based screening of Food and Drug Administration (FDA)-approved drugs that bind to Histone Deacetylase 4 (HDAC4), a known miR-9 target. Glucosamine emerged as the top hit and its binding potential to HDAC4 was verified by Molecular Dynamics (MD) Simulation, Drug Affinity Responsive Target Stability (DARTS) assay, and MALDI-TOF MS. It was intriguing that the glucosamine treatment 1-h post-OGD was associated with the increased miR-9 level as well as enhanced neuronal viability. miR-9 mimic or post-OGD glucosamine treatment significantly increased the cellular proliferation (BrdU assay), while the neurite outgrowth assay displayed elongated neurites. The enhanced BCL2 and VEGF parallel with the reduced NFκB1, TNF-α, IL-1ß, and iNOS mRNA levels in miR-9 mimic or glucosamine-treated cells further substantiated their post-ischemic neuroprotective and regenerative efficacy. Hence, this study unleashes a potential therapeutic approach that integrates neuronal survival and regeneration via small-molecule-based regulation of miR-9 favoring long-term recovery against ischemic stroke.

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Biodegradable nanoparticles (PEG-PCL) decorated with tetra peptides (IKRG) on the surface were evaluated for their potential to deliver drugs into neurons for neural regeneration. The chosen 4-amino-acid peptide sequence was reported previously to mimic the function of BDNF (brain-derived neurotrophic factor) and target TrkB receptors that are present in abundance in neurons. Enhanced uptake for peptide-modified nanoparticles was observed in TrkB-positive PC12 cells but not in TrkB-negative HeLa cells. The modified nanoparticles were internalized selectively into neurons of dorsal root ganglion (DRG) and at a significantly higher rate. VO-OHpic, an inhibitor of PTEN (Phosphatase and tension homolog deleted on chromosome 10), was encapsulated into the modified nanoparticles and released over 14 days. Prolonged and enhanced neural regenerative effect, as confirmed by increased pAKT expression and increased neurite density, was observed when DRGs were treated with drug-containing nanoparticles. This was attributed to the increased and targeted cellular uptake and sustainable release by the peptide-modified nanoparticles. Furthermore, nanoparticle encapsulation was found to reduce the cytotoxicity of the free drug to the neurons. Our findings support that the BDNF-derived peptide modified PEG-PCL nanoparticles are promising carriers for localized and controlled drug delivery to peripheral neurons.

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