The prevalence of central nervous system (CNS) dysfunction as a result of disease or trauma remains a clinically unsolved problem which is raising increased awareness in our aging society. Human Dental Pulp Stem Cells (hDPSCs) are excellent candidates to be used in tissue engineering and regenerative therapies of the CNS due to their neural differentiation ability and lack of tumorigenicity. Accordingly, they have been successfully used in animal models of spinal cord injury, stroke and peripheral neuropathies. The ideal therapy in brain injury should combine strategies aiming to protect the damaged lesion and, at the same time, accelerate brain tissue regeneration, thus promoting fast recovery while minimizing side or long-term effects. The use of bioresorbable nanopatterned poly(lactide-co-ɛ-caprolactone) (PLCL) polymeric scaffolds as hDPCSs carriers can represent an advantage for tissue regeneration. In this chapter, we describe the surgical procedures to implant functionalized bioresorbable scaffolds loaded with hDPSCs to improve the brain lesion microenvironment in an intracranial stab wound injury model severing the rostral migratory stream (RMS) that connects the brain subventricular zone (SVZ) and the olfactory bulb in nude mice. Additionally, we also describe the technical steps after animal sacrifice for histological tissue observation and characterization.

Several evidence demonstrated that glucagon-like peptide 1 receptor agonists (GLP1-RAs) reduce the risk of dementia in type 2 diabetes patients by improving memory, learning, and overcoming cognitive impairment. In this study, we elucidated the molecular processes underlying the protective effect of Tirzepatide (TIR), a dual glucose-dependent insulinotropic polypeptide receptor agonist (GIP-RA)/ GLP-1RA, against learning and memory disorders.
We investigated the effects of TIR on markers of neuronal growth (CREB and BDNF), apoptosis (BAX/Bcl2 ratio) differentiation (pAkt, MAP2, GAP43, and AGBL4), and insulin resistance (GLUT1, GLUT4, GLUT3 and SORBS1) in a neuroblastoma cell line (SHSY5Y) exposed to normal and high glucose concentration. The potential role on DNA methylation of genes involved in neuroprotection and epigenetic modulators of neuronal growth (miRNA 34a), apoptosis (miRNA 212), and differentiation (miRNA 29c) was also investigated. The cell proliferation was detected by measuring Ki-67 through flow cytometry. The data were analysed by SPSS IBM Version 23 or GraphPad Prism 7.0 software and expressed as the means ± SEM. Differences between the mean values were considered significant at a p-value of < 0.05. GraphPad Prism software was used for drawing figures.
For the first time, it was highlighted: (a) the role of TIR in the activation of the pAkt/CREB/BDNF pathway and the downstream signaling cascade; (b) TIR efficacy in neuroprotection; (c) TIR counteracting of hyperglycemia and insulin resistance-related effects at the neuronal level.
We demonstrated that TIR can ameliorate high glucose-induced neurodegeneration and overcome neuronal insulin resistance. Thus, this study provides new insight into the potential role of TIR in improving diabetes-related neuropathy.

Since the 1980s, it has been known that the administration of ganglioside GM1 to cultured cells induced or enhanced neuronal differentiation. GM1 mechanism of action relies on its direct interaction and subsequent activation of the membrane tyrosine kinase receptor, TrkA, which naturally serves as NGF receptor. This process is mediated by the sole oligosaccharide portion of GM1, the pentasaccharide β-Gal-(1-3)-β-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-β-Gal-(1-4)-β-Glc. Here we detailed the minimum structural requirements of the oligosaccharide portion of GM1 for mediating the TrkA dependent neuritogenic processing. By in vitro and in silico biochemical approaches, we demonstrated that the minimal portion of GM1 required for the TrkA activation is the inner core of the ganglioside\'s oligosaccharide β-Gal-(1-3)-β-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-β-Gal. The addition of a sialic acid residue at position 3 of the outer galactose of the GM1 oligosaccharide, which forms the oligosaccharide of GD1a, prevented the interaction with TrkA and the resulting neuritogenesis. On the contrary, the addition of a fucose residue at position 2 of the outer galactose, forming the Fucosyl-GM1 oligosaccharide, did not prevent the TrkA-mediated neuritogenesis.

UNASSIGNED: Human induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the neurodifferentiation process.
UNASSIGNED: In this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter.
UNASSIGNED: The retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types.
UNASSIGNED: Our results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the neurodifferentiation process in our mixed human neural cultures.

Alzheimer\'s disease (AD) is a progressive and complex neurodegenerative disease. Acetylcholinesterase inhibitors (AChEIs) are a major class of drugs used in AD therapy. ROCK2, another promising target for AD, has been associated with the induction of neurogenesis via PTEN/AKT. This study aimed to characterize the therapeutic potential of a novel donepezil-tacrine hybrid compound (TA8Amino) to inhibit AChE and ROCK2 protein, leading to the induction of neurogenesis in SH-SY5Y cells. Experiments were carried out with undifferentiated and neuron-differentiated SH-SY5Y cells submitted to treatments with AChEIs (TA8Amino, donepezil, and tacrine) for 24 h or 7 days. TA8Amino was capable of inhibiting AChE at non-cytotoxic concentrations after 24 h. Following neuronal differentiation for 7 days, TA8Amino and donepezil increased the percentage of neurodifferentiated cells and the length of neurites, as confirmed by β-III-tubulin and MAP2 protein expression. TA8Amino was found to participate in the activation of PTEN/AKT signaling. In silico analysis showed that TA8Amino can stably bind to the active site of ROCK2, and in vitro experiments in SH-SY5Y cells demonstrate that TA8Amino significantly reduced the expression of ROCK2 protein, contrasting with donepezil and tacrine. Therefore, these results provide important information on the mechanism underlying the action of TA8Amino with regard to multi-target activities.

Recently, the scientific community has started to focus on the neurogenic potential of cannabinoids. The phytocompound cannabidiol (CBD) shows different mechanism of signaling on cannabinoid receptor 1 (CB1), depending on its concentration. In this study, we investigated if CBD may induce in vitro neuronal differentiation after treatment at 5 µM and 10 µM. For this purpose, we decided to use the spinal cord × neuroblastoma hybrid cell line (NSC-34) because of its proliferative and undifferentiated state. The messenger RNAs (mRNAs) expression profiles were tested using high-throughput sequencing technology and Western blot assay was used to determine the number of main proteins in different pathways. Interestingly, the treatment shows different genes associated with neurodifferentiation statistically significant, such as Rbfox3, Tubb3, Pax6 and Eno2. The CB1 signaling pathway is responsible for neuronal differentiation at 10 µM, as suggested by the presence of p-ERK and p-AKT, but not at 5 µM. A new correlation between CBD, neurodifferentiation and retinoic acid receptor-related orphan receptors (RORs) has been observed.

High-contrast visual stimulation promotes retinal regeneration and visual function, but the underlying mechanism is not fully understood. Here, we hypothesized that Müller cells (MCs), which express neurotrophins such as brain-derived neurotrophic factor (BDNF), could be key players in this retinal plasticity process. This hypothesis was tested by conducting in vivo and in vitro high-contrast stimulation of adult mice and MCs. Following stimulation, we examined the expression of BDNF and its inducible factor, VGF, in the retina and MCs. We also investigated the alterations in the expression of VGF, nuclear factor kappa B (NF-κB) and pro-inflammatory mediators in MCs, as well as their capacity to proliferate and develop a neurogenic or reactive gliosis phenotype after high-contrast stimulation and treatment with BDNF. Our results showed that high-contrast stimulation upregulated BDNF levels in MCs in vivo and in vitro. The additional BDNF treatment significantly augmented VGF production in MCs and their neuroprotective features, as evidenced by increased MC proliferation, neurodifferentiation, and decreased expression of the pro-inflammatory factors and the reactive gliosis marker GFAP. These results demonstrate that high-contrast stimulation activates the neurotrophic and neuroprotective properties of MCs, suggesting their possible direct involvement in retinal neuronal survival and improved functional outcomes in response to visual stimulation.

Human Dental Pulp Stem Cells (hDPSCs) are one of the most promising stem cell sources for tissue engineering and regeneration, due to their extraordinary multi-lineage differentiation ability, ease of extraction from biological waste in dental clinics, safe non-tumorigenic phenotype, immune-tolerance upon in vivo transplantation, and great possibilities of application in autologous tissue reconstruction. The in vitro manipulation of hDPSCs paves the way for drug screening and tailor-made regeneration of damaged tissues, in the context of personalized medicine. The neural crest phenotype of these stem cells gives them the capacity to differentiate to a large variety of cell types, including neural-lineage cells. In this chapter, we describe various culture methods to generate different cellular phenotypes from hDPSCs, which can not only grow as mesenchymal-like plastic adherent cells, but also as non-adherent neurogenic dentospheres in serum-free medium. Floating dentospheres can be used to generate large populations of mature neuronal and glial marker expressing cells, which may be cultured over a substrate of nanopatterned scaffold based on biodegradable poly(lactide-co-caprolactone) (PLCL) to induce a controlled alignment of neurites and cell migration, to generate in vivo biocompatible constructs for nerve tissue bioengineering.

Vascular cognitive impairment and dementia (VCID) is a neurodegenerative disease that is recognized as the second leading cause of dementia after Alzheimer\'s disease (AD). The underlying pathological mechanism of VCID include crebromicrovascular dysfunction, blood-brain barrier (BBB) disruption, neuroinflammation, capillary rarefaction, and microhemorrhages, etc. Despite the high incidence of VCID, no effective therapies are currently available for preventing or delaying its progression. Recently, pathophysiological microRNAs (miRNAs) in VCID have shown promise as novel diagnostic biomarkers and therapeutic targets. Studies have revealed that miRNAs can regulate the function of the BBB, affect apoptosis and oxidative stress (OS) in the central nervous system, and modulate neuroinflammation and neurodifferentiation. Thus, this review summarizes recent findings on VCID and miRNAs, focusing on their correlation and contribution to the development of VCID pathology.

During adult neurogenesis, neuronal stem cells differentiate into mature neurons that are functionally integrated into the existing network. One hallmark during the late phase of this neurodifferentiation process is the formation of dendritic spines. These morphological specialized structures form the basis of most excitatory synapses in the brain, and are essential for neuronal communication. Additionally, dendritic spines are affected in neurological disorders, such as Alzheimer\'s disease or schizophrenia. However, the mechanisms underlying spinogenesis, as well as spine pathologies, are poorly understood. Plasticity-related Gene 5 (PRG5), a neuronal transmembrane protein, has previously been linked to spinogenesis in vitro. Here, we analyze endogenous expression of the PRG5 protein in different mouse brain areas, as well as on a subcellular level. We found that native PRG5 is expressed dendritically, and in high abundance in areas characterized by their regenerative capacity, such as the hippocampus and the olfactory bulb. During adult neurogenesis, PRG5 is specifically expressed in a late phase after neuronal cell-fate determination associated with dendritic spine formation. On a subcellular level, we found PRG5 not to be localized at the postsynaptic density, but at the base of the synapse. In addition, we showed that PRG5-induced formation of membrane protrusions is independent from neuronal activity, supporting a possible role in the morphology and stabilization of spines.