Currently, the construction of novel biomimetic reduced graphene oxide (RGO)-based nanocomposites to induce neurite sprouting and repair the injured neurons represents a promising strategy in promoting neuronal development or treatment of cerebral anoxia or ischemia. Here, we present an effective method for constructing palladium-reduced graphene oxide (Pd-RGO) nanocomposites by covalently bonding Pd onto RGO surfaces to enhance neurite sprouting of cultured neurons. As described, the Pd-RGO nanocomposites exhibit the required physicochemical features for better biocompatibility without impacting cell viability. Primary neurons cultured on Pd-RGO nanocomposites had significantly increased number and length of neuronal processes, including both axons and dendrites, compared with the control. Western blotting showed that Pd-RGO nanocomposites improved the expression levels of growth associate protein-43 (GAP-43), as well as β-III tubulin, Tau-1, microtubule-associated protein-2 (MAP2), four proteins that are involved in regulating neurite sprouting and outgrowth. Importantly, Pd-RGO significantly promoted neurite length and complexity under oxygen-glucose deprivation/re-oxygenation (OGD/R) conditions, an in vitro cellular model of ischemic brain damage, that closely relates to neuronal GAP-43 expression. Furthermore, using the middle cerebral artery occlusion (MCAO) model in rats, we found Pd-RGO effectively reduced the infarct area, decreased neuronal apoptosis in the brain, and improved the rats\' behavioral outcomes after MCAO. Together, these results indicate the great potential of Pd-RGO nanocomposites as a novel excellent biomimetic material for neural interfacing that shed light on its applications in brain injuries.

The molecular mechanisms underlying neurite formation include multiple crosstalk between pathways such as membrane trafficking, intracellular signaling, and actin cytoskeletal rearrangement. To study the proteins involved in such complex pathways, we present a detailed workflow of the sample preparation for mass spectrometry-based proteomics and data analysis. We have also included steps to perform label-free quantification of proteins that will help researchers quantify changes in the expression levels of key regulators of neuronal morphogenesis on a global scale.

Here, we establish that plasticity exists within the postnatal enteric nervous system by demonstrating the reinnervation potential of post-mitotic enteric neurons (ENs). Employing BAF53b-Cre mice for selective neuronal tracing, the reinnervation capabilities of mature postnatal ENs are shown across multiple model systems. Isolated ENs regenerate neurites in vitro, with neurite complexity and direction influenced by contact with enteric glial cells (EGCs). Nerve fibers from transplanted ENs exclusively interface and travel along EGCs within the muscularis propria. Resident EGCs persist after Cre-dependent ablation of ENs and govern the architecture of the myenteric plexus for reinnervating ENs, as shown by nerve fiber projection tracing. Transplantation and optogenetic experiments in vivo highlight the rapid reinnervation potential of post-mitotic neurons, leading to restored gut muscle contractile activity within 2 weeks. These studies illustrate the structural and functional reinnervation capacity of post-mitotic ENs and the critical role of EGCs in guiding and patterning their trajectories.

Spinal and bulbar muscular atrophy (SBMA) is a slowly progressing neuromuscular disease caused by a polyglutamine (polyQ)-encoding CAG trinucleotide repeat expansion in the androgen receptor (AR) gene, leading to AR aggregation, lower motor neuron death, and muscle atrophy. AR is a ligand-activated transcription factor that regulates neuronal architecture and promotes axon regeneration; however, whether AR transcriptional functions contribute to disease pathogenesis is not fully understood. Using a differentiated PC12 cell model of SBMA, we identified dysfunction of polyQ-expanded AR in its regulation of neurite growth and maintenance. Specifically, we found that in the presence of androgens, polyQ-expanded AR inhibited neurite outgrowth, induced neurite retraction, and inhibited neurite regrowth. This dysfunction was independent of polyQ-expanded AR transcriptional activity at androgen response elements (ARE). We further showed that the formation of polyQ-expanded AR intranuclear inclusions promoted neurite retraction, which coincided with reduced expression of the neuronal differentiation marker β-III-Tubulin. Finally, we revealed that cell death is not the primary outcome for cells undergoing neurite retraction; rather, these cells become senescent. Our findings reveal that mechanisms independent of AR canonical transcriptional activity underly neurite defects in a cell model of SBMA and identify senescence as a pathway implicated in this pathology. These findings suggest that in the absence of a role for AR canonical transcriptional activity in the SBMA pathologies described here, the development of SBMA therapeutics that preserve this activity may be desirable. This approach may be broadly applicable to other polyglutamine diseases such as Huntington\'s disease and spinocerebellar ataxias.

The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.

The accumulation of amyloid-β (Aβ) in the brain is the first pathological mechanism to initiate Alzheimer\'s disease (AD) pathogenesis. However, the precise role of Aβ in the disease progression remains unclear. Through decades of research, prolonged inflammation has emerged as an important core pathology in AD. Previously, a study has demonstrated the neurotoxic effect of Aβ-induced neuroinflammation in neuron-glia co-culture at 72 h. Here, we hypothesise that initial stage Aβ may trigger microglial inflammation, synergistically contributing to the progression of neurite lesions relevant to AD progression. In the present study, we aimed to determine whether olanzapine, an antipsychotic drug possessing anti-inflammatory properties, can ameliorate Aβ-induced progressive neurite lesions. Our study reports that Aβ induces neurite lesions with or without inflammatory microglial cells in vitro. More intriguingly, the present study revealed that Aβ exacerbates neurite lesions in synergy with microglia. Moreover, the time course study revealed that Aβ promotes microglia-mediated neurite lesions by eliciting the secretion of pro-inflammatory cytokines. Furthermore, our study shows that olanzapine at lower doses prevents Aβ-induced microglia-mediated progressive neurite lesions. The increase in pro-inflammatory cytokines induced by Aβ is attenuated by olanzapine administration, associated with a reduction in microglial inflammation. Finally, this study reports that microglial senescence induced by Aβ was rescued by olanzapine. Thus, our study provides the first evidence that 1 µM to 5 µM of olanzapine can effectively prevent Aβ-induced microglia-mediated progressive neurite lesions by modulating microglial inflammation. These observations reinforce the potential of targeting microglial remodelling to slow disease progression in AD.

Spinal cord injury induces significant cellular stress responses. The Heat Shock Protein 90 (HSP90) plays a pivotal role as a molecular chaperone and is crucial for protein folding, stabilization, and cellular signaling pathways. Despite its important function in stress adaptation, the specific expression patterns and functional roles of HSP90 after nerve injury remain unclear. This study aimed to elucidate the expression dynamics and functional implications of HSP90 following central nervous system (CNS) injury. Using western blotting and immunohistochemical analyses, we observed upregulation of HSP90 expression in spinal cord tissues and within injured neurons in a spinal cord contusion injury model. Additionally, HSP90 was found to enhance neurite outgrowth in primary cortical neurons cultured in vitro. Furthermore, in a glutamate-induced neuronal injury model, the expression of HSP90 was up-regulated, and overexpression of HSP90 promoted neurite re-growth in damaged neurons. Overall, our findings highlight the critical involvement of HSP90 in the neural response to injury and offer valuable insights into potential therapeutic strategies for CNS repair.

Previous reports indicated that zinc deficiency could increase the risk of infectious diseases and developmental retardation in children. In experimental study, it has been reported that zinc deficiency during the embryonic period inhibited fetal growth, and disturbed neural differentiation and higher brain function later in adulthood. Although it has been suggested that zinc deficiency during development can have significant effects on neuronal differentiation and maturation, the molecular mechanisms of the effects of low zinc on neuronal differentiation during development have not been elucidated in detail. This study was performed to determine the effects of low zinc status on neurite outgrowth and collapsin response mediator protein 2 (CRMP2) signal pathway. Low zinc suppressed neurite outgrowth, and caused increase levels of phosphorylated CRMP2 (pCRMP2) relative to CRMP2, and decrease levels of phosphorylated glycogen synthase kinase 3β (pGSK3β) relative to GSK3β in human neuroblastoma cell line (SH-SY5Y) cells on days 1, 2, and 3 of neuronal differentiation induction. Neurite outgrowth inhibited by low zinc was restored by treatment with the GSK3β inhibitor CHIR99021. These results suggested that low zinc causes neurite outgrowth inhibition via phosphorylation of CRMP2 by GSK3β. In conclusion, this study is the first to demonstrate that CRMP signaling is involved in the suppression of neurite outgrowth by low zinc.

We examined neurite orientation dispersion and density imaging in patients with migraine. We found that patients with medication overuse headache exhibited lower orientation dispersion than those without. Moreover, orientation dispersion in the body of the corpus callosum was statistically negatively correlated with migraine attack frequencies. These findings indicate that neurite dispersion is damaged in patients with chronic migraine. Our study results indicate the orientation preference of neurite damage in migraine.

Brain functional impairment after stroke is common; however, the molecular mechanisms of post-stroke recovery remain unclear. It is well-recognized that age is the most important independent predictor of poor outcomes after stroke as older patients show poorer functional outcomes following stroke. Mounting evidence suggests that axonal regeneration and angiogenesis, the major forms of brain plasticity responsible for post-stroke recovery, diminished with advanced age. Previous studies suggest that Ras-related C3 botulinum toxin substrate (Rac) 1 enhances stroke recovery as activation of Rac1 improved behavior recovery in a young mice stroke model. Here, we investigated the role of Rac1 signaling in long-term functional recovery and brain plasticity in an aged (male, 18 to 22 months old C57BL/6J) brain after ischemic stroke. We found that as mice aged, Rac1 expression declined in the brain. Delayed overexpression of Rac1, using lentivirus encoding Rac1 injected day 1 after ischemic stroke, promoted cognitive (assessed using novel object recognition test) and sensorimotor (assessed using adhesive removal tests) recovery on days 14-28. This was accompanied by the increase of neurite and proliferative endothelial cells in the peri-infarct zone assessed by immunostaining. In a reverse approach, pharmacological inhibition of Rac1 by intraperitoneal injection of Rac1 inhibitor NSC23766 for 14 successive days after ischemic stroke worsened the outcome with the reduction of neurite and proliferative endothelial cells. Furthermore, Rac1 inhibition reduced the activation of p21-activated kinase 1, the protein level of brain-derived neurotrophic factor, and increased the protein level of glial fibrillary acidic protein in the ischemic brain on day 28 after stroke. Our work provided insight into the mechanisms behind the diminished plasticity after cerebral ischemia in aged brains and identified Rac1 as a potential therapeutic target for improving functional recovery in the older adults after stroke.