Stimulate Neurite Outgrowth Research Articles

AAV1.tMCK.NT-3 gene therapy improves phenotype in Sh3tc2-/- mouse model of Charcot-Marie-Tooth Type 4C.

Charcot-Marie-Tooth Type 4C (CMT4C) is associated with mutations in the SH3 domain and tetratricopeptide repeats 2 (SH3TC2) gene, primarily expressed in Schwann cells (SCs). Neurotrophin-3 (NT-3) is an important autocrine factor for SC survival and differentiation, and it stimulates neurite outgrowth and myelination. In this study, scAAV1.tMCK.NT-3 was delivered intramuscularly to 4-week-old Sh3tc2-/- mice, a model for CMT4C, and treatment efficacy was assessed at 6-month post-gene delivery. Efficient transgene production was verified with the detection of NT-3 in serum from the treated cohort. NT-3 gene therapy improved functional and electrophysiological outcomes including rotarod, grip strength and nerve conduction velocity. Qualitative and quantitative histopathological studies showed that hypomyelination of peripheral nerves and denervated status of neuromuscular junctions at lumbrical muscles were also improved in the NT-3-treated mice. Morphometric analysis in mid-sciatic and tibial nerves showed treatment-induced distally prominent regenerative activity in the nerve and an increase in the estimated SC density. This indicates that SC proliferation and differentiation, including the promyelination stage, are normal in the Sh3tc2-/- mice, consistent with the previous findings that Sh3tc2 is not involved in the early stages of myelination. Moreover, in size distribution histograms, the number of myelinated axons within the 3- to 6-µm diameter range increased, suggesting that treatment resulted in continuous radial growth of regenerating axons over time. In conclusion, this study demonstrates the efficacy of AAV1.NT-3 gene therapy in the Sh3tc2-/- mouse model of CMT4C, the most common recessively inherited demyelinating CMT subtype.

The Bioactive Gamma-Oryzanol from Oryza sativa L. Promotes Neuronal Differentiation in Different In Vitro and In Vivo Models.

Gamma-oryzanol (ORY), found in rice (Oryza sativa L.), is a mixture of ferulic acid esters with triterpene alcohols, well-known for its antioxidant and anti-inflammatory properties. Our past research demonstrated its positive impact on cognitive function in adult mice, influencing synaptic plasticity and neuroprotection. In this study, we explored whether ORY can exert neuro-differentiating effects by using different experimental models. For this purpose, chemical characterization identified four components that are most abundant in ORY. In human neuroblastoma cells, we showed ORY's ability to stimulate neurite outgrowth, upregulating the expression of GAP43, BDNF, and TrkB genes. In addition, ORY was found to guide adult mouse hippocampal neural progenitor cells (NPCs) toward a neuronal commitment. Microinjection of ORY in zebrafish Tg (-3.1 neurog1:GFP) amplified neurog1-GFP signal, islet1, and bdnf mRNA levels. Zebrafish nrf2a and nrf2b morphants (MOs) were utilized to assess ORY effects in the presence or absence of Nrf2. Notably, ORY's ability to activate bdnf was nullified in nrf2a-MO and nrf2b-MO. Furthermore, computational analysis suggested ORY's single components have different affinities for the Keap1-Kelch domain. In conclusion, although more in-depth studies are needed, our findings position ORY as a potential source of bioactive molecules with neuro-differentiating potential involving the Nrf2 pathway.

The bidirectional role of GABAA and GABAB receptors during the differentiation process of neural precursor cells of the subventricular zone.

The intricate process of neuronal differentiation integrates multiple signals to induce transcriptional, morphological, and electrophysiological changes that reshape the properties of neural precursor cells during their maturation and migration process. An increasing number of neurotransmitters and biomolecules have been identified as molecular signals that trigger and guide this process. In this sense, taurine, a sulfur-containing, non-essential amino acid widely expressed in the mammal brain, modulates the neuronal differentiation process. In this study, we describe the effect of taurine acting via the ionotropic GABAA receptor and the metabotropic GABAB receptor on the neuronal differentiation and electrophysiological properties of precursor cells derived from the subventricular zone of the mouse brain. Taurine stimulates the number of neurites and favors the dendritic complexity of the neural precursor cells, accompanied by changes in the somatic input resistance and the strength of inward and outward membranal currents. At the pharmacological level, the blockade of GABAA receptors inhibits these effects, whereas the stimulation of GABAB receptors has no positive effects on the taurine-mediated differentiation process. Strikingly, the blockade of the GABAB receptor with CGP533737 stimulates neurite outgrowth, dendritic complexity, and membranal current kinetics of neural precursor cells. The effects of taurine on the differentiation process involve Ca2+ mobilization and the activation of intracellular signaling cascades since chelation of intracellular calcium with BAPTA-AM, and inhibition of the CaMKII, ERK1/2, and Src kinase inhibits the neurite outgrowth of neural precursor cells of the subventricular zone.

Abstract 5390: Exploring signaling pathways of tumor-nerve interactions in prostate and breast cancer

Abstract A significant portion of prostate cancer (PCa) and breast cancer (BCa) patients develop bone metastatic disease. In some cases, cancer spreads through the nervous system, a process known as perineural invasion (PNI). Neurite outgrowth is believed to be a precursor to PNI. Snail is an important gene which regulates the epithelial-mesenchymal transition (EMT) process in which tumor cells at the invasive front undergo this transition to promote invasion, migration, and subsequent metastasis. We recently published that Snail promotes neurite outgrowth in PCa cells. We hypothesize that Snail expression will stimulate neurite outgrowth in both PCa and BCa cells through extracellular vesicles from cancer cells interacting with neurons. To test this hypothesis, we first collected conditioned media from PCa and BCa cells expressing Snail and isolated exosomes. Exosomal markers were detected using western blot to confirm the isolated exosomes. Additionally, we used Transmission Electron Microscopy (TEM) to observe the secretion of exosomes. Proteomics was performed to analyze proteins expressed within exosomes of LNCaP Snail overexpressing or C4-2 Snail knockdown PCa cells. Our results showed that Snail-expressing cells secrete exosomes containing Talin1, specifically, the proteolyzed C-terminal rod domain, while full-length Talin1 is found in whole cell lysates from Snail-expressing cells. Talin1 has previously been associated with neurite outgrowth. Neurite outgrowth assays were then performed using conditioned medium collected from C4-2 PCa cells with Snail knockdown and MCF-7 BCa cells with Snail overexpression. Increased neurite outgrowth is observed in PC-12 cells and NPC (Induced Pluripotent Stem Cells differentiated neuronal progenitor cells) when cultured with conditioned medium collected from PCa and BCa cells expressing high levels of Snail. AKT activation was observed in the neuronal cells in response to conditioned media from Snail-expressing cells. Furthermore, we found that mH4, a proprietary small molecule inhibitor of Talin1, reduces Snail-mediated neurite outgrowth and AKT activation (p-AKT Thr308 and Ser473). Overall, we have uncovered a pathway whereby Snail transcription factor promotes secretion of exosomes containing Talin1 which promotes AKT signaling in neuronal cells and neurite outgrowth. Talin1 small molecule inhibitor shows promise for therapeutic targeting of tumor-nerve interactions. Acknowledgements: The authors acknowledge the use of core facilities supported by the National Institute on Minority Health and Health Disparities through grant number 5U54MD013376 and 1U54GM128729-01-DaCCoTA Scholars Program (PI: Rezvani) National Institute of General Medical Sciences (NIGMS). Citation Format: Bor-Jang Hwang, Gabrielle J. Edwards, Cole Davis Knoblich, Khosrow Rezvani, Valerie Odero-Marah. Exploring signaling pathways of tumor-nerve interactions in prostate and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5390.

Herpes simplex virus type 1 modifies the protein composition of extracellular vesicles to promote neurite outgrowth and neuroinfection.

The highly prevalent herpes simplex virus type 1 (HSV-1) causes a range of diseases, including cold sores, blinding keratitis, and life-threatening encephalitis. HSV-1 initially replicates in epithelial cells, enters the peripheral nervous system via neurites, and establishes lifelong infection in the neuronal cell bodies. Neurites are highly dynamic structures that grow or retract in response to attractive or repulsive cues, respectively. Here, we show that infection with HSV-1, but not with a mutant virus lacking glycoprotein G (gG), reduced the repulsive effect of epithelial cells on neurite outgrowth and facilitated HSV-1 invasion of neurons. HSV-1 gG was required and sufficient to induce neurite outgrowth by modifying the protein composition of extracellular vesicles, increasing the amount of neurotrophic and neuroprotective proteins, including galectin-1. Antibodies directed against galectin-1 neutralized the capacity of extracellular vesicles released from HSV-1-infected cells to promote neurite outgrowth. Our study provides new insights into the neurotropism of HSV-1 and identifies a viral protein that modifies the protein composition of extracellular vesicles to stimulate neurite outgrowth and invasion of the nervous system.IMPORTANCEHerpes simplex virus type 1 (HSV-1) must infect neurites (or nerve endings) to establish a chronic infection in neurons. Neurites are highly dynamic structures that retract or grow in the presence of repulsive or attractive proteins. Some of these proteins are released by epithelial cells in extracellular vesicles and act upon interaction with their receptor present on neurites. We show here that HSV-1 infection of epithelial cells modulated their effect on neurites, increasing neurite growth. Mechanistically, HSV-1 glycoprotein G (gG) modifies the protein composition of extracellular vesicles released by epithelial cells, increasing the amount of attractive proteins that enhance neurite outgrowth and facilitate neuronal infection. These results could inform of therapeutic strategies to block HSV-1 induction of neurite outgrowth and, thereby, neuronal infection.

HT22 cell differentiation reduces insulin receptor levels

Purpose: The brain is an insülin-sensitive organ and has widespread insulin receptor (IR) expression. IR signaling in the brain is essential for neuronal development, feeding behavior, body weight, and cognitive processes such as attention, learning, and memory. HT22 cells, which are derived from parent HT4 cells that are immortalized from primary mouse hippocampal neuronal cells are used in research related to insulin signaling. However, the role of these cells in insulin signaling is not known. In this study, we aimed to examine IR levels in cells differentiated using neurobasal medium. Material and methods: For the study, briefly, the cells were seeded in 6-well plates at 2x105 cells/well for 24 h. After the cells reached 80% confluence, the normal growth medium was replaced with a differentiation medium and the cells were incubated for 72 hours at 37 0C in 5% CO2. Western blot procedure was used to determine the expression of the IR. Result: Our results show that differentiation of HT22 cells stimulates neurite outgrowth. Furthermore, IR protein levels were significantly downregulated in differentiated HT22 cells. Conclusion: This finding may require careful consideration of the use of neurobasal medium in conditions where IR signaling is important.

Small Organic Compounds Mimicking the Effector Domain of Myristoylated Alanine-Rich C-Kinase Substrate Stimulate Female-Specific Neurite Outgrowth.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a critical member of a signaling cascade that influences disease-relevant neural functions such as neural growth and plasticity. The effector domain (ED) of MARCKS interacts with the extracellular glycan polysialic acid (PSA) through the cell membrane to stimulate neurite outgrowth in cell culture. We have shown that a synthetic ED peptide improves functional recovery after spinal cord injury in female but not male mice. However, peptides themselves are unstable in therapeutic applications, so we investigated more pharmacologically relevant small organic compounds that mimic the ED peptide to maximize therapeutic potential. Using competition ELISAs, we screened small organic compound libraries to identify molecules that structurally and functionally mimic the ED peptide of MARCKS. Since we had shown sex-specific effects of MARCKS on spinal cord injury recovery, we assayed neuronal viability as well as neurite outgrowth from cultured cerebellar granule cells of female and male mice separately. We found that epigallocatechin, amiodarone, sertraline, tegaserod, and nonyloxytryptamine bind to a monoclonal antibody against the ED peptide, and compounds stimulate neurite outgrowth in cultured cerebellar granule cells of female mice only. Therefore, a search for compounds that act in males appears warranted.

Neonatal malnutrition impacts fibroblast growth factor 21-induced neuron neurite outgrowth and growth hormone-releasing hormone secretion in neonatal mouse brain

Neonatal malnutrition is one of the most common causes of neurological disorders. However, the mechanism of action of the factors associated with neonatal nutrition in the brain remains unclear. In this study, we focused on fibroblast growth factor (FGF) 21 to elucidate the effects of malnutrition on the neonatal brain. FGF21 is an endocrine factor produced by the liver during lactation which is the main source of nutrition during the neonatal period. In this study, malnourishment during nursing mice induced decreased levels of Fgf21 mRNA in the liver and decreased levels of FGF21 in the serum. RNA-seq analysis of neonatal mouse brain tissue revealed that FGF21 controlled the expression of Kalrn-201 in the neonatal mouse brain. Kalrn-201 is a transcript of Kalirin, a Ras homologous guanine nucleotide exchange factor at the synapse. In mouse neurons, FGF21 induced the expression of Kalirin-7 (a Kalirin isoform) by down-regulating Kalrn-201. FGF21-induced Kalirin-7 stimulated neurite outgrowth in Neuro-2a cells. FGF21 also induced Growth hormone-releasing hormone (GHRH) expression in Neuro-2a cells. Kalirin-7 and GHRH expression induced by FGF21 was altered by inhibiting the activity of SH2-containing tyrosine phosphatase (SHP2) which is located downstream of the FGF receptor (FGFR). Additionally, malnourished nursing induced intron retention of the SHP2 gene (Ptpn11), resulting in the alteration of Kalirin-7 and GHRH expression by FGF21 signaling. Ptpn11 intron retention is suggested to be involved in regulating SHP2 activity. Taken together, these results suggest that FGF21 plays a critical role in the induction of neuronal neurite outgrowth and GHRH secretion in the neonatal brain, and this mechanism is regulated by SHP2. Thus, Ptpn11 intron retention induced by malnourished nursing may be involved in SHP2 activity.

Multiple Metabolites Derived from Mushrooms and Their Beneficial Effect on Alzheimer's Diseases.

Mushrooms with edible and medicinal potential have received widespread attention because of their diverse biological functions, nutritional value, and delicious taste, which are closely related to their rich active components. To date, many bioactive substances have been identified and purified from mushrooms, including proteins, carbohydrates, phenols, and vitamins. More importantly, molecules derived from mushrooms show great potential to alleviate the pathological manifestations of Alzheimer's disease (AD), which seriously affects the health of elderly people. Compared with current therapeutic strategies aimed at symptomatic improvement, it is particularly important to identify natural products from resource-rich mushrooms that can modify the progression of AD. This review summarizes recent investigations of multiple constituents (carbohydrates, peptides, phenols, etc.) isolated from mushrooms to combat AD. In addition, the underlying molecular mechanisms of mushroom metabolites against AD are discussed. The various mechanisms involved in the antiAD activities of mushroom metabolites include antioxidant and anti-neuroinflammatory effects, apoptosis inhibition, and stimulation of neurite outgrowth, etc. This information will facilitate the application of mushroom-derived products in the treatment of AD. However, isolation of new metabolites from multiple types of mushrooms and further in vivo exploration of the molecular mechanisms underlying their antiAD effect are still required.

Single-cell RNA-sequencing analysis reveals enhanced non-canonical neurotrophic factor signaling in the subacute phase of traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of long-term disability in young adults and induces complex neuropathological processes. Cellular autonomous and intercellular changes during the subacute phase contribute substantially to the neuropathology of TBI. However, the underlying mechanisms remain elusive. In this study, we explored the dysregulated cellular signaling during the subacute phase of TBI. Single-cell RNA-sequencing data (GSE160763) of TBI were analyzed to explore the cell-cell communication in the subacute phase of TBI. Upregulated neurotrophic factor signaling was validated in a mouse model of TBI. Primary cell cultures and cell lines were used as in vitro models to examine the potential mechanisms affecting signaling. Single-cell RNA-sequencing analysis revealed that microglia and astrocytes were the most affected cells during the subacute phase of TBI. Cell-cell communication analysis demonstrated that signaling mediated by the non-canonical neurotrophic factors midkine (MDK), pleiotrophin (PTN), and prosaposin (PSAP) in the microglia/astrocytes was upregulated in the subacute phase of TBI. Time-course profiling showed that MDK, PTN, and PSAP expression was primarily upregulated in the subacute phase of TBI, and astrocytes were the major source of MDK and PTN after TBI. In vitro studies revealed that the expression of MDK, PTN, and PSAP in astrocytes was enhanced by activated microglia. Moreover, MDK and PTN promoted the proliferation of neural progenitors derived from human-induced pluripotent stem cells (iPSCs) and neurite growth in iPSC-derived neurons, whereas PSAP exclusively stimulated neurite growth. The non-canonical neurotrophic factors MDK, PTN, and PSAP were upregulated in the subacute phase of TBI and played a crucial role in neuroregeneration.

Vertigoheel promotes rodent cognitive performance in multiple memory tests.

Cognitive impairment associated with old age or various brain disorders may be very disabling for affected individuals, placing their carers and public health services under considerable stress. The standard-of-care drugs produce only transient improvement of cognitive impairment in older people, so the search for novel, safe and effective therapeutics that would help to reverse or delay cognitive impairment is warranted. Repurposing pharmacological therapies with well-established safety record for additional indications is a promising recent trend in drug development. Vertigoheel (VH-04), a multicomponent drug made of Ambra grisea, Anamirta cocculus L., Conium maculatum, and Petroleum rectificatum, has been successfully used for several decades in the treatment of vertigo. Here, we investigated effects of VH-04 on cognitive performance in standard behavioral tests assessing different types of memory and explored cellular and molecular underpinnings of VH-04's biological activity. In the majority of behavioral experiments, namely in the spontaneous and rewarded alternation tests, passive avoidance test, contextual/cued fear conditioning, and social transmission of food preference, we examined the ability of single and repeated intraperitoneal administrations of VH-04 to improve cognitive parameters of mice and rats disrupted by the application of the muscarinic antagonist scopolamine. In addition, we also assessed how VH-04 affected novel object recognition and influenced performance of aged animals in Morris water maze. Furthermore, we also studied the effects of VH-04 on primary hippocampal neurons in vitro and mRNA expression of synaptophysin in the hippocampus. Administration of VH-04 positively influenced visual recognition memory in the novel object recognition test and alleviated the impairments in spatial working memory and olfactory memory caused by the muscarinic antagonist scopolamine in the spontaneous alternation and social transmission of food preference tests. In addition, VH-04 improved retention of the spatial orientation memory of old rats in the Morris water maze. In contrast, VH-04 did not have significant effects on scopolamine-induced impairments in tests of fear-aggravated memory or rewarded alternation. Experiments in vitro showed that VH-04 stimulated neurite growth and possibly reversed the age-dependent decrease in hippocampal synaptophysin mRNA expression, which implies that VH-04 may preserve synaptic integrity in the aging brain. Our findings allow a cautious conclusion that in addition to its ability to alleviate manifestations of vertigo, VH-04 may be also used as a cognitive enhancer.

"Turning up the heat": Role of neurotrophic batokines in the postnatal maturation and remodelling of brown adipose tissue in deer mice.

Activation of brown adipose tissue (BAT) thermogenesis impacts energy balance and must be tightly regulated. Several neurotrophic factors, expressed in BAT of adult laboratory rodents, have been implicated in remodelling the sympathetic neural network to enhance thermogenesis (e.g., NGF, NRG4 and S100b). Here, we compare for the first time the relative roles of three neurotropic 'batokines' in establishing/remodelling innervation during postnatal development and adult cold stress. We used lab-reared P. maniculatus, which rely heavily on BAT-based thermogenesis for survival in the wild, beginning between postnatal days (P) 8 and 10. BAT sympathetic innervation was enhanced from P6-10, and exogenous NGF, NRG4, and S100b stimulated neurite outgrowth from P6 sympathetic neurons. Endogenous BAT protein stores and/or gene expression of NRG4, S100b, and calsyntenin-3ß (which regulates S100b secretion), remained high and constant during development. However, endogenous NGF was low and ngf mRNA was undetectable. Conditioned media (CM) from cultured P10 BAT slices stimulated neurite outgrowth from sympathetic neurons in vitro that was inhibited by antibodies against all three growth factors. P10 CM had significant amounts of secreted NRG4 and S100b protein, but not NGF. By contrast, BAT slices from cold-acclimated adults released significant amounts of all three factors relative to thermoneutral controls. These data suggest that while neurotrophic batokines regulate sympathetic innervation in vivo, their relative contributions differ depending on life stage. They also provide novel insight into the regulation of BAT remodelling and BAT's secretory role, which are both critical to our understanding of mammalian energy homeostasis.

Injectable Hydrogel Guides Neurons Growth with Specific Directionality.

Visual disabilities affect more than 250 million people, with 43 million suffering from irreversible blindness. The eyes are an extension of the central nervous system which cannot regenerate. Neural tissue engineering is a potential method to cure the disease. Injectability is a desirable property for tissue engineering scaffolds which can eliminate some surgical procedures and reduce possible complications and health risks. We report the development of the anisotropic structured hydrogel scaffold created by a co-injection of cellulose nanofiber (CNF) solution and co-polypeptide solution. The positively charged poly (L-lysine)-r-poly(L-glutamic acid) with 20 mol% of glutamic acid (PLLGA) is crosslinked with negatively charged CNF while promoting cellular activity from the acid nerve stimulate. We found that CNF easily aligns under shear forces from injection and is able to form hydrogel with an ordered structure. Hydrogel is mechanically strong and able to support, guide, and stimulate neurite growth. The anisotropy of our hydrogel was quantitatively determined in situ by 2D optical microscopy and 3D X-ray tomography. The effects of PLLGA:CNF blend ratios on cell viability, neurite growth, and neuronal signaling are systematically investigated in this study. We determined the optimal blend composition for stimulating directional neurite growth yielded a 16% increase in length compared with control, reaching anisotropy of 30.30% at 10°/57.58% at 30°. Using measurements of calcium signaling in vitro, we found a 2.45-fold increase vs. control. Based on our results, we conclude this novel material and unique injection method has a high potential for application in neural tissue engineering.

Ontogeny and Trophic Factor Sensitivity of Gastrointestinal Projecting Vagal Sensory Cell Types.

Vagal sensory neurons (VSNs) located in the nodose ganglion provide information, such as stomach stretch or the presence of ingested nutrients, to the caudal medulla via specialized cell types expressing unique marker genes. Here, we leverage VSN marker genes identified in adult mice to determine when specialized vagal subtypes arise developmentally and the trophic factors that shape their growth. Experiments to screen for trophic factor sensitivity revealed that brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) robustly stimulate neurite outgrowth from VSNs in vitro Perinatally, BDNF was expressed by neurons of the nodose ganglion itself, while GDNF was expressed by intestinal smooth muscle cells. Thus, BDNF may support VSNs locally, whereas GDNF may act as a target-derived trophic factor supporting the growth of processes at distal innervation sites in the gut. Consistent with this, expression of the GDNF receptor was enriched in VSN cell types that project to the gastrointestinal tract. Last, the mapping of genetic markers in the nodose ganglion demonstrates that defined vagal cell types begin to emerge as early as embryonic day 13, even as VSNs continue to grow to reach gastrointestinal targets. Despite the early onset of expression for some marker genes, the expression patterns of many cell type markers appear immature in prenatal life and mature considerably by the end of the first postnatal week. Together, the data support location-specific roles for BDNF and GDNF in stimulating VSN growth, and a prolonged perinatal timeline for VSN maturation in male and female mice.

Bile Acids Induce Neurite Outgrowth in Nsc-34 Cells via TGR5 and a Distinct Transcriptional Profile.

Increasing evidence supports a neuroprotective role for bile acids in major neurodegenerative disorders. We studied major human bile acids as signaling molecules for their two cellular receptors, farnesoid X receptor (FXR or NR1H4) and G protein-coupled bile acid receptor 1 (GPBAR1 or TGR5), as potential neurotrophic agents. Using quantitative image analysis, we found that 20 μM deoxycholic acid (DCA) could induce neurite outgrowth in NSC-34 cells that was comparable to the neurotrophic effects of the culture control 1 μM retinoic acid (RA), with lesser effects observed for chenodexoycholic acid (CDCA) at 20 μM, and similar though less robust neurite outgrowth in SH-SY5Y cells. Using chemical agonists and antagonists of FXR, LXR, and TGR5, we found that TGR5 agonism was comparable to DCA stimulation and stronger than RA, and that neither FXR nor liver X receptor (LXR) inhibition could block bile acid-induced neurite growth. RNA sequencing identified a core set of genes whose expression was regulated by DCA, CDCA, and RA. Our data suggest that bile acid signaling through TGR5 may be a targetable pathway to stimulate neurite outgrowth.

Supramolecular presentation of bioinstructive peptides on soft multilayered nanobiomaterials stimulates neurite outgrowth.

Peptide amphiphiles (PAs) have emerged as effective molecular building blocks for creating self-assembling nanobiomaterials for multiple biomedical applications. Herein, we report a straightforward approach to assemble soft bioinstructive platforms to recreate the native neural extracellular matrix (ECM) aiming for neuronal regeneration based on the electrostatic-driven supramolecular presentation of laminin-derived IKVAV-containing self-assembling PA (IKVAV-PA) on biocompatible multilayered nanoassemblies. Spectroscopic and microscopic techniques show that the co-assembly of positively charged low-molecular-weight IKVAV-PA with oppositely charged high-molecular-weight hyaluronic acid (HA) triggers the formation of ordered β-sheet structures denoting a one-dimensional nanofibrous network. The successful functionalization of poly(L-lysine)/HA layer-by-layer nanofilms with an outer positively charged layer of self-assembling IKVAV-PA is demonstrated by the quartz crystal microbalance with dissipation monitoring and the nanofibrous morphological properties revealed by atomic force microscopy. The bioactive ECM-mimetic supramolecular nanofilms promote the enhancement of primary neuronal cells' adhesion, viability, and morphology when compared to the PA without the IKVAV sequence and PA-free biopolymeric multilayered nanofilms, and stimulate neurite outgrowth. The nanofilms hold great promise as bioinstructive platforms for enabling the assembly of customized and robust multicomponent supramolecular biomaterials for neural tissue regeneration.

Functionalized hydrogel-microsphere composites stimulating neurite outgrowth for vascularized bone regeneration.

Neurovascularized bone regeneration remains an enormous challenge in the clinic. Biomaterials mimicking the developmental microenvironment might be promising tools to enhance tissue regeneration. In this study, functionalized hydrogel-microsphere composites are developed to enhance bone regeneration via a recapitulating neurovascularized microenvironment. RGD peptide and the porous structure generated by the degradation of gelatin microspheres (GMs) are beneficial for the proliferation and migration of human mesenchymal stem cells (hMSCs); mesoporous silica nanoparticles (MSNs) promote osteogenic differentiation of hMSCs through the delivery of BFP-1 peptide; the QK peptide from the GMs is sustained-released to recruit endogenous endothelial cells (ECs), and IK19 peptide grafted on the hydrogel guides the neurite outgrowth. The in vivo results show that the hydrogel-microsphere composites not only promote new bone formation, but also facilitate nerve infiltration and angiogenesis. Furthermore, the neurovascularized niche created by this composite stimulated neurite growth through MAPK, PI3K, IL17 and TNF signaling pathways, enabling vascularized bone regeneration. The findings suggest a novel bioengineering approach to guide the construction of neurovascularized bone repair materials, which is beneficial for achieving functional bone regeneration and repair.

The novel FKBP51‐Hsp90 interaction inhibitor attenuates high‐fat diet induced cognitive impairment

AbstractBackgroundObesity increases the risk of several chronic diseases, such as type 2 diabetes mellitus, insulin resistance, and Alzheimer disease. As one of the co‐chaperones of heat shock protein 90 (Hsp90), FK506‐binding protein 51 (FKBP51) is an important regulator of metabolic and cognitive dysfunction. FKBP51 knockout mice are resistant to diet‐induced obesity, exhibit elevated glucose and insulin tolerance and have less endogenous tau. FKBP51 overexpression preserves tau and impairs spatial reversal learning and memory. Our group has identified a new family of FKBP51 inhibitors that can disrupt FKBP51‐Hsp90 interactions. Previous study has shown that our inhibitors can dose‐dependently reduce lipid accumulation in adipocytes differentiated from primary human mesenchymal stem cells and stimulate neurite outgrowth in differentiated neuroblastoma SH‐SY5Y cells. This study aims to investigate the potential roles of our inhibitors on metabolic and cognitive dysfunction in vivo.MethodsPharmacokinetic studies have been performed at first. Plasma and brain samples were collected and analyzed by liquid chromatography‐mass spectrometry. Male C57BL/6J mice were fed either the control or high‐fat diet (HFD) for 4 weeks and then treated with vehicle or inhibitor E8 (20 mg/kg, s.c.) and their respective diets for another 4 weeks. Behaviors, body composition measuremnets by EchoMRI, fasting glucose/insulin detections and glucose/insulin tolerance tests were performed during the last week. Hippocampus was collected for western blot, RNA sequencing and quantitative real‐time PCR analysis.ResultsAfter subcutaneous injection, E8 can be quickly absorbed into the blood and penetrate the blood‐brain barrier. In HFD model, E8 can attenuate the impairment of locomotor activity and short‐term memory. But it could neither reduce body weight gain nor improve insulin sensitivity and glucose/insulin tolerance. In the hippocampus, E8 reduces the elevated p‐tau (Ser214)/tau levels induced by HFD but does not show any effects on insulin signaling. It suggests that E8 reverses the behavioral impairments independently of metabolic regulation in vivo. The underlying mechanisms were further investigated by RNA sequencing and quantitative real‐time PCR analysis.ConclusionWe showed that inhibiting FKBP51‐Hsp90 interactions with small molecules provides novel therapeutic targets on cognitive dysfunction in obesity.

Transepithelial Effect of Probiotics in a Novel Model of Gut Lumen to Nerve Signaling.

Recent studies have shown that the gut microbiome changes brain function, behavior, and psychiatric and neurological disorders. The Gut-Brain Axis (GBA) provides a neuronal pathway to explain this. But exactly how do commensal bacteria signal through the epithelial layer of the large intestine to activate GBA nerve afferents? An in vitro model is described. We differentiated two human cell lines: Caco2Bbe1 into mature epithelium on 0.4-micron filters and then SH-SY5Y into mature neurons in 24-well plates. These were co-cultured by placing the epithelium-laden filters 1 mm above the neurons. Twenty-four hours later they were tri-cultured by apical addition of 107Lactobacillus rhamnosus or Lactobacillus fermentum which settled on the epithelium. Alone, the Caco2bbe1 cells stimulated neurite outgrowth in underlying SH-SY5Y. Beyond this, the lactobacilli were well tolerated and stimulated further neurite outgrowth by 24 h post-treatment, though not passing through the filters. The results provide face validity for a first-of-kind model of transepithelial intestinal lumen-to nerve signaling. The model displays the tight junctional barrier characteristics found in the large intestine while at the same time translating stimulatory signals from the bacteria through epithelial cells to attracted neurons. The model is easy to set-up with components widely available.

Molecular and Functional Characterization of BDNF-Overexpressing Human Retinal Pigment Epithelial Cells Established by Sleeping Beauty Transposon-Mediated Gene Transfer.

More and more patients suffer from multifactorial neurodegenerative diseases, such as age-related macular degeneration (AMD). However, their pathological mechanisms are still poorly understood, which complicates the development of effective therapies. To improve treatment of multifactorial diseases, cell-based gene therapy can be used to increase the expression of therapeutic factors. To date, there is no approved therapy for dry AMD, including late-stage geographic atrophy. We present a treatment option for dry AMD that transfers the brain-derived neurotrophic factor (BDNF) gene into retinal pigment epithelial (RPE) cells by electroporation using the plasmid-based Sleeping Beauty (SB) transposon system. ARPE-19 cells and primary human RPE cells were co-transfected with two plasmids encoding the SB100X transposase and the transposon carrying a BDNF transcription cassette. We demonstrated efficient expression and secretion of BDNF in both RPE cell types, which were further increased in ARPE-19 cell cultures exposed to hydrogen peroxide. BDNF-transfected cells exhibited lower apoptosis rates and stimulated neurite outgrowth in human SH-SY5Y cells. This study is an important step in the development of a cell-based BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat dry AMD or other degenerative retinal diseases.