P75 Neurotrophin Receptor Research Articles

Sexual Dimorphism in Balance and Coordination in p75NTRexonIII Knock-Out Mice.

The p75 neurotrophin receptor (p75NTR) is implicated in various biological functions during development and adulthood. Several animal models have been developed to identify the roles of p75NTR in vivo and in vitro. P75NTRExonIII knock-out mice are widely used to study the neurotrophin receptor and its signaling pathways. Similar to other models of p75NTR knock-out (p75NTRExon IV KO) or conditional knock-out (p75NTRfl/fl) mice, p75NTRExonIII knock-out mice present severe abnormalities in walking, gait, balance and strength. The present study identifies a sexual dimorphism in the p75NTRExonIII knock-out strain regarding balance and coordination. Using Kondziela’s inverted grid test, we observed that p75NTRExonIII knock-out males performed poorly at the task, whereas p75NTRExonIII knock-out females did not exhibit any defects. We also observed that female p75NTRExonIII knock-out mice performed significantly better than male p75NTRExonIII knock-out mice at the beam balance test. There were no differences in strength, skin innervation, or the number of ulcers on the toes between p75NTRExonIII knock-out males and females. The literature regarding the role of p75NTR in behavior is controversial; our results suggest that studies investigating the role of p75NTR in vivo using p75NTR knock-out mice should systematically report data from males and females.

Exploring the Correlation Between the Regulation of Macrophages by Regulatory T Cells and Peripheral Neuropathic Pain.

ObjectiveIntractable pain after peripheral nerve injury has become a major concern in the field of pain. Current evidence shows that routine medications or surgical treatment is associated with inconsistent results and different curative effects. Stable and effective treatment methods in clinical practice are also lacking. To date, there is no consensus on the pathophysiological mechanisms of pain. The present study investigates the potential regulatory role of regulatory T cells in the differentiation of macrophages on dorsal root ganglion (DRG) and explores the mechanism of nociceptive signals in the signal transfer station. The findings are expected to guide the prevention of various types of peripheral neuropathic pain.MethodsThirty-six male Sprague Dawley (SD) rats and 18 male Nude rats, of equal weight (250–300g), were used in this study. The rats were divided into 3 groups: SD rat sciatic nerve transection group (SNT group, n = 18), SD rat nerve transection experimental group (SNT/RAPA group, n = 18) and Nude rat nerve transection experimental group (SNT/NUDE group, n = 18). The behavior related to neuropathic pain of animals were comprehensively evaluated in all groups. Furthermore, we analyzed the degree of neuroma development, histology, gene, and protein expression, and compared their correlation with the ultrastructural changes of M1/M2 type differentiation of macrophages in DRG.ResultsSciatic nerve transection (SNT), induced the aggregation of several types of macrophages in lumbar DRG of SD rats leading to a higher ratio of M1/M2. Following the inhibition of the M1 type polarization of macrophages, axon outgrowth increased significantly. A significantly lower average autotomy score was reported in the SNT/NUDE group (*p < 0.05) and the SNT/RAPA group (@p < 0.05) as compared to that of the SNT group. The SNT/NUDE group showed no noticeable neuroma formation 30 days after the nerve transection. However, bulbous neuromas were observed in the nerve stumps of both the SNT control and SNT/RAPA groups. Immunofluorescence staining revealed a significant decrease in the proportion of M1/M2 macrophages in lumbar DRG of the SNT/NUDE group (**p < 0.001) and the SNT/RAPA group (@p < 0.05) compared to the SNT group. The expression of pain-related proteins was also decreased (@p < 0.05, *p < 0.05,**p < 0.001). Also, the expression of alpha-smooth muscle actin (α-SMA), neurofilament 200 (NF-200), and nerve growth factor low-affinity receptor p75 were significantly down-regulated in the nerve tissue (@p < 0.05, @@p < 0.001, **p < 0.001).ConclusionM1/M2 type differentiation of macrophages on DRG plays a significant role in the formation of traumatic painful neuroma after neurotomy. In combination with our previous study, the results of this study suggest that regulatory T cells reduce the ratio of M1/M2 macrophages and alleviate the pain of neuroma by regulating the polarization direction of macrophages on neuroma. These findings provide key insights into developing new strategies to manage painful neuroma.

Oligodendroglia Generated From Adult Rat Adipose Tissue by Direct Cell Conversion.

Obtaining oligodendroglial cells from dispensable tissues would be of great interest for autologous or immunocompatible cell replacement therapy in demyelinating diseases, as well as for studying myelin-related pathologies or testing therapeutic approaches in culture. We evaluated the feasibility of generating oligodendrocyte precursor cells (OPCs) from adult rat adipose tissue by expressing genes encoding transcription factors involved in oligodendroglial development. Adipose-derived mesenchymal cells were lentivirally transduced with tetracycline-inducible Sox10, Olig2, Zfp536, and/or Nkx6.1 transgenes. Immunostaining with the OPC-specific O4 monoclonal antibody was used to mark oligodendroglial induction. O4- and myelin-associated glycoprotein (MAG)-positive cells emerged after 3 weeks when using the Sox10 + Olig2 + Zfp536 combination, followed in the ensuing weeks by GFAP-, O1 antigen-, p75NTR (low-affinity NGF receptor)-, and myelin proteins-positive cells. The O4+ cell population progressively expanded, eventually constituting more than 70% of cells in culture by 5 months. Sox10 transgene expression was essential for generating O4+ cells but was insufficient for inducing a full oligodendroglial phenotype. Converted cells required continuous transgene expression to maintain their glial phenotype. Some vestigial characteristics of mesenchymal cells were maintained after conversion. Growth factor withdrawal and triiodothyronine (T3) supplementation generated mature oligodendroglial phenotypes, while FBS supplementation produced GFAP+- and p75NTR+-rich cultures. Converted cells also showed functional characteristics of neural-derived OPCs, such as the expression of AMPA, NMDA, kainate, and dopaminergic receptors, as well as similar metabolic responses to differentiation-inducing drugs. When co-cultured with rat dorsal root ganglion neurons, the converted cells differentiated and ensheathed multiple axons. We propose that functional oligodendroglia can be efficiently generated from adult rat mesenchymal cells by direct phenotypic conversion.

Structural study of regulated intramembrane proteolysis of the p75 neurotrophin receptor by γ-secretase

Programmed cell death (PCD) maintains the tissue size in a multicellular organism. Uncontrolled PCD in neurons has long been implicated in neurodegenerative diseases, e.g. Alzheimer's disease. p75 neurotrophin receptor (p75NTR) acts as a switch of a neuron's fate in maintaining neuronal growth as well as triggering apoptosis. p75NTR, the founding member of tumor necrosis factor (TNF) receptor superfamily, is a type I transmembrane protein. The membrane complex formation of the pro-NGF, p75NTR and sortilin initiates the apoptotic signaling.

Protective Effects of a synthetic glycosaminoglycan mimetic (OTR4132) in a rat immunotoxic lesion model of septohippocampal cholinergic degeneration

Using a partial hippocampal cholinergic denervation model, we assessed the effects of the RGTA® named OTR4132, a synthetic heparan-mimetic biopolymer with neuroprotective/neurotrophic properties. Long-Evans male rats were injected with the cholinergic immunotoxin 192 IgG-saporin into the medial septum/diagonal band of Broca (0.37 µg); vehicle injections served as controls. Immediately after surgery, OTR4132 was injected into the lateral ventricles (0.25 µg/5 µl/rat) or intramuscularly (1.5 mg/kg). To determine whether OTR4132 reached the lesion site, some rats received intracerebroventricular (ICV) or intramuscular (I.M.) injections of fluorescent OTR4132. Rats were sacrificed at 4, 10, 20, or 60 days post-lesion (DPL). Fluorescein-labeled OTR4132 injected ICV or I.M. was found in the lesion from 4 to 20 DPL. Rats with partial hippocampal cholinergic denervation showed decreases in hippocampal acetylcholinesterase reaction products and in choline acetyltransferase-positive neurons in the medial septum. These lesions were the largest at 10 DPL and then remained stable until 60 DPL. Both hippocampal acetylcholinesterase reaction products and choline acetyltransferase-positive neurons in the medial septum effects were significantly attenuated in OTR4132-treated rats. These effects were not related to competition between OTR4132 and 192 IgG-saporin for the neurotrophin receptor P75 (p75NTR), as OTR4132 treatment did not alter the internalization of Cy3-labelled 192 IgG. OTR4132 was more efficient at reducing the acetylcholinesterase reaction products and choline acetyltransferase-positive neurons than a comparable heparin dose used as a comparator. Using the slice superfusion technique, we found that the lesion-induced decrease in muscarinic autoreceptor sensitivity was abolished by intramuscular OTR4132. After partial cholinergic damage, OTR4132 was able to concentrate at the brain lesion site possibly due to the disruption of the blood-brain barrier and to exert structural and functional effects that hold promises for neuroprotection/neurotrophism.

Abstract TMP120: Brain Plasticity Modulator P75 Neurotrophin Receptor And Its Mechanistically Linked Signaling Molecules Predict Clinical Outcome Across Different Acute Brain Injuries

Introduction: Brain plasticity and synaptic regeneration are important processes in recovering after acute brain injuries such as aneurysmal subarachnoid hemorrhage (aSAH), ischemic stroke (IS) and traumatic brain injury (TBI). p75 neurotrophic receptor (p75NTR) is a consequential receptor modulating brain plasticity and apoptosis. Hypotheses: Measurable changes in serum levels of p75NTR mechanistically linked proteins are associated to recovery of acute brain injuries. Differentially expressed (DE) miRNAs after acute brain injuries target to p75NTR pathway and have association to outcome. Methods: Concentrations of p75NTR, NGF, sortilin, IL1β, TNFα and cyclophilin were measured from serum using ELISA. Prospective cohort (n=75) consisted of IS (n=30), aSAH (n=31) and TBI (n=14) patients. Serum samples were collected 24-48h after insults. Late samples (120-192h) were also taken from 12 aSAH patients. Outcome was measured 90 days after injury (mRS favorable 0-4, unfavorable 5-6). We generated characteristic curves and area under the curve (AUC) for weighted linear combination of the biomarkers with a linear discriminant analysis. MiRNAs were extracted from the cohort (early and late samples, n=53). Results: In the whole cohort (n=75) combination of cyclophilin, IL1β, sortilin, p75NTR, NGF and TNFα predicted unfavorable mRS (AUC 0.69, p=0.01). In IS group combination of cyclophilin, sortilin, p75NTR, NGF and TNFα predicted unfavorable mRS (AUC 0.84, p=0.02). In aSAH group combination of IL1β, p75NTR and NGF predicted unfavorable mRS (AUC 0.77, p=0.07). In 12 aSAH patient sub-group (late samples included) combination of cyclophilin, IL1β, sortilin, p75NTR and TNFα predicted unfavorable mRS (AUC 1.0, p=0.07). In TBI group combination of cyclophilin, IL1β, p75NTR, NGF and TNFα predicted unfavorable mRS (AUC 0.79, p=0.08). In the aSAH group 2 DE miRNAs were detected, 4 in the IS group and 7 in the TBI group (p&lt;0.05, FDR corrected). MiRNAs targeted p75NTR , NGF , IL1 β , cyclophilin ( PPIA ), and sortilin ( SORT1 ). Conclusions: p75NTR and mechanistically linked proteins predict outcome across different types of brain injuries suggesting a common mechanism irrespective of the type of brain insult. Identified DE miRNAs targeted studied molecules.

Astrocytic p75NTR expression provoked by ischemic stroke exacerbates the blood-brain barrier disruption.

The disruption of the blood-brain barrier (BBB) plays a critical role in the pathology of ischemic stroke. p75 neurotrophin receptor (p75NTR ) contributes to the disruption of the blood-retinal barrier in retinal ischemia. However, whether p75NTR influences the BBB permeability after acute cerebral ischemia remains unknown. The present study investigated the role and underlying mechanism of p75NTR on BBB integrity in an ischemic stroke mouse model, middle cerebral artery occlusion (MCAO). After 24 h of MCAO, astrocytes and endothelial cells in the infarct-affected brain area up-regulated p75NTR . Genetic p75NTR knockdown (p75NTR+/- ) or pharmacological inhibition of p75NTR using LM11A-31, a selective inhibitor of p75NTR , both attenuated brain damage and BBB leakage in MCAO mice. Astrocyte-specific conditional knockdown of p75NTR mediated with an adeno-associated virus significantly ameliorated BBB disruption and brain tissue damage, as well as the neurological functions after stroke. Further molecular biological examinations indicated that astrocytic p75NTR activated NF-κB and HIF-1α signals, which upregulated the expression of MMP-9 and vascular endothelial growth factor (VEGF), subsequently leading to tight junction degradation after ischemia. As a result, increased leukocyte infiltration and microglia activation exacerbated brain injury after stroke. Overall, our results provide novel insight into the role of astrocytic p75NTR in BBB disruption after acute cerebral ischemia. The p75NTR may therefore be a potential therapeutic target for the treatment of ischemic stroke.

Urinary neopterin: A novel biomarker of disease progression in amyotrophic lateral sclerosis.

The aim was to evaluate urinary neopterin, a marker of pro-inflammatory state, as a potential biomarker of disease prognosis and progression in amyotrophic lateral sclerosis (ALS); and to compare its utility to urinary neurotrophin receptor p75 extracellular domain (p75ECD ). This was an observational study including 21 healthy controls and 46 people with ALS, 29 of whom were sampled longitudinally. Neopterin and p75ECD were measured using enzyme-linked immunoassays. Baseline and longitudinal changes in clinical measures, neopterin and urinary p75ECD were examined, and prognostic utility was explored by survival analysis. At baseline, urinary neopterin was higher in ALS compared to controls (181.7±78.9μmol/mol creatinine vs. 120.4±60.8μmol/mol creatinine, p=0.002, Welch's t test) and correlated with the Revised ALS Functional Rating Scale (r=-0.36, p=0.01). Combining previously published urinary p75ECD results from 22 ALS patients with a further 24 ALS patients, baseline urinary p75ECD was also higher compared to healthy controls (6.0±2.7 vs. 3.2±1.0ng/mg creatinine, p<0.0001) and correlated with the Revised ALS Functional Rating Scale (r=-0.36, p=0.01). Urinary neopterin and p75ECD correlated with each other at baseline (r=0.38, p=0.009). In longitudinal analysis, urinary neopterin increased on average (±SE) by 6.8±1.1µmol/mol creatinine per month (p<0.0001) and p75ECD by 0.19±0.02ng/mg creatinine per month (p<0.0001) from diagnosis in 29 ALS patients. Urinary neopterin holds promise as marker of disease progression in ALS and is worthy of future evaluation for its potential to predict response to anti-inflammatory therapies.

Transplanted neural lineage cells derived from dental pulp stem cells promote peripheral nerve regeneration.

Cell therapy for peripheral nerve injury is a promising strategy as regenerative medicine that restores neurological function. However, challenges remain in producing suitable and sufficient amounts of autologous cells for promoting nerve regeneration. This study aimed to identify the characteristics of neural lineage cells (NLCs) differentiated from dental pulp stem cells (DPSCs) and reveal their effect on functional recovery and nerve regeneration after cell transplantation into an immunodeficient rat using a nerve guide conduit. Here we report a protocol of neural induction in monolayer culture and characterize NLCs in vitro. Furthermore, NLCs were transplanted into an immunodeficient rat model with a 10-mm sciatic nerve defect, and cell survival and differentiation were investigated in vivo. Outcomes of nerve regeneration were also assessed using the remyelinated axon numbers, myelin sheath thickness, electrophysiological activities, and gastrocnemius muscle mass. NLCs comprised neuronal, astrocyte, oligodendrocyte, and neural crest lineage cells. NLCs enhanced the activities of endothelial cells, Schwann cells, and neurons in a paracrine-dependent manner in vitro. At 2weeks post-transplantation, numerous transplanted NLCs differentiated into platelet-derived growth factor receptor alpha (PDGFRα) + oligodendrocyte progenitor cells (OPCs) and a few PDGFRα + /p75 neurotrophin receptor + Schwann cell-like cells derived from OPCs were observed. At 12weeks post-transplantation, human Schwann cell-like cells survived, and axon growth, remyelination, electrophysiological activities, and muscle atrophy were improved. This study demonstrates the broad application of our protocol of neural induction of DPSCs and portrays the efficacy of transplantation of NLCs derived from human DPSCs as a promising strategy for peripheral nerve regeneration.

Brain-Derived Neurotrophic Factor Suppressed Proinflammatory Cytokines Secretion and Enhanced MicroRNA(miR)-3168 Expression in Macrophages

We investigated the role of brain-derived neurotrophic factor (BDNF) and its signaling pathway in the proinflammatory cytokines production of macrophages. The effects of different concentrations of BDNF on proinflammatory cytokines expression and secretion in U937 cell-differentiated macrophages, and human monocyte-derived macrophages were analyzed using enzyme-linked immunosorbent assay and real-time polymerase chain reaction. The CRISPR-Cas9 system was used to knockout p75 neurotrophin receptor (p75NTR), one of the BDNF receptors. Next-generation sequencing (NGS) was conducted to search for BDNF-regulated microRNA. A very low concentration of BDNF (1 ng/mL) could suppress the secretion of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 in lipopolysaccharide (LPS)-stimulated macrophages but did not change their mRNA expression. BDNF suppressed IL-1β and IL-6 secretion in human monocyte-derived macrophages. In U937 cells, BDNF suppressed the phosphorylation of JNK and c-Jun. The p75NTR knockout strongly suppressed IL-1β, IL-6, and TNF-α secretion in macrophages and LPS-stimulated macrophages. BDNF regulated the expression of miR-3168 with Ras-related protein Rab-11A as its target. In conclusion, BDNF suppressed proinflammatory cytokines secretion in macrophages and inhibited the phosphorylation of JNK. Knockout of p75NTR suppressed proinflammatory cytokines expression and secretion. BDNF upregulated the expression of miR-3168. The inhibition of p75NTR could be a potential strategy to control inflammation.

Neurotrophin3 promotes hepatocellular carcinoma apoptosis through the JNK and P38 MAPK pathways.

Although liver cancer is a malignant tumor with the highest mortality across the world, its pathogenesis and therapeutic targets remain unclear. Apoptosis, a natural cell death mechanism, is an important target of anticancer therapy. The discovery of effective apoptotic regulators can lead to the identification of novel therapeutic targets for treating cancer. Neurotrophin 3 (NTF3) is a member of the nerve growth factor (NGF) family that is involved in the progression of various cancers, including medulloblastoma, primitive neuroectodermal brain tumors, and breast cancer. NTF3 is under-expressed in human hepatocellular carcinoma (HCC), albeit its specific effects and the action mechanism have not been elucidated. Here, we confirmed that NTF3 expression was significantly low in HCC with reference to the GSEA database. By collecting patient data from our center and performing qRT-PCR analysis, we found that NTF3 expression was significantly downregulated in 74 patients with HCC. Low NTF3 expression was associated with a shorter overall survival (OS), recurrence-free survival (RFS), progression-free survival (PFS), and disease-specific survival (DSS). Both in vivo and in vitro experiments revealed that NTF3 considerably inhibited the progression of HCC cells. We found that the ligand NTF3 is regulated by c-Jun and binds to the p75 neurotrophin receptor (p75NTR) and then activates the JNK and P38 MAPK pathways to induce apoptosis. Entinostat (the target of HDAC1/HDAC3) can activate the NTF3/p75NTR pathway. These results indicate that NTF3 is a tumor suppressor, and that its low expression can help in predict poor clinical outcomes in HCC. Therefore, NTF3 can be used as a potential treatment molecule for HCC.

Ultrasound delivery of a TrkA agonist confers neuroprotection to Alzheimer-associated pathologies.

Early degeneration of basal forebrain cholinergic neurons contributes substantially to cognitive decline in Alzheimer’s disease. Evidence from preclinical models of neuronal injury and aging support a pivotal role for nerve growth factor (NGF) in neuroprotection, resilience, and cognitive function. However, whether NGF can provide therapeutic benefit in the presence of Alzheimer’s disease-related pathologies still unresolved. Perturbations in the NGF signalling system in Alzheimer’s disease may render neurons unable to benefit from NGF administration. Additionally, challenges related to brain delivery remain for clinical translation of NGF-based therapies in Alzheimer’s disease. To be safe and efficient, NGF-related agents should stimulate the NGF receptor, tropomyosin receptor kinase A (TrkA), avoid activation through the p75 neurotrophin receptor (p75NTR), and be delivered non-invasively to targeted brain areas using real-time monitoring. We addressed these limitations using MRI-guided focused ultrasound (MRIgFUS) to increase blood–brain barrier permeability locally and transiently, allowing an intravenously administered TrkA agonist that does not activate p75NTR, termed D3, to enter targeted brain areas. Here, we report the therapeutic potential of selective TrkA activation in a transgenic mouse model that recapitulates numerous Alzheimer’s disease-associated pathologies. Repeated MRIgFUS-mediated delivery of D3 (D3/FUS) improved cognitive function in the TgCRND8 model of Alzheimer’s disease. Mechanistically, D3/FUS treatment effectively attenuated cholinergic degeneration and promoted functional recovery. D3/FUS treatment also resulted in widespread reduction of brain amyloid pathology and dystrophic neurites surrounding amyloid plaques. Furthermore, D3/FUS markedly enhanced hippocampal neurogenesis in TgCRND8 mice, implicating TrkA agonism as a novel therapeutic target to promote neurogenesis in the context of Alzheimer’s disease-related pathology. Thus, this study provides evidence that selective TrkA agonism confers neuroprotection to effectively counteract Alzheimer’s disease-related vulnerability. Recent clinical trials demonstrate that non-invasive blood–brain barrier modulation using MRIgFUS is safe, feasible and reversible in Alzheimer’s disease patients. TrkA receptor agonists coupled with MRIgFUS delivery constitute a promising disease-modifying strategy to foster brain health and counteract cognitive decline in Alzheimer’s disease.

Morphological and ultrastructural characterization of neurospheres spontaneously generated in the culture from sheep ovarian cortical cells.

Neurospheres (NS) derived from adult stem cells of non-neural tissues represent a promising source of neural stem cells (NSCs) and neural progenitor cells (NPCs) for autologous cell therapy. Knowing the fine structure of NS cells is essential for characterizing them during differentiation or oncogenic transformation. NS are generated by culturing ovarian cortical cells (OCCs) under specific conditions. To establish whether these OCCs exhibited a similar morphophenotype as those from the central nervous system (CNS) reported in the literature, sheep OCCs were cultured for 21 days to generate NS. Expression levels of pluripotency (Nanog, octamer-binding transcription factor 4 [Oct4], and SRY-box transcription factor 2 [Sox2]) and NSCs/NPCs (nestin, paired box 6 [Pax6], and p75 neurotrophin receptor [P75NTR]) transcripts were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the NSC/NPC antigens were immunolocalized, and structural and ultrastructural analyses were performed in OCC-NS on Days 10, 15, and 21 in culture. Spheroids expressed transcripts and antigens of pluripotency as well as NSCs/NPCs. Cells were arranged into an inner core, with frequent apoptotic and degenerative events, and a peripheral epithelial-like cover with abundant cytoplasmic organelles, apical microvilli, and filament bundles of cytoskeleton elements. Adherens junctions and apical tight and lateral loose interdigitations were found in peripheral cells that eventually lost apical-basal polarization, which might indicate their disengaging/aggregating from/to the NS. We can conclude that OCC-NS shares the most structural and ultrastructural characteristics with CNS-NS.

Post-Stroke Administration of the p75 Neurotrophin Receptor Modulator, LM11A-31, Attenuates Chronic Changes in Brain Metabolism, Increases Neurotransmitter Levels, and Improves Recovery.

The aim of this study was to test whether poststroke oral administration of a small molecule p75 neurotrophin receptor (p75NTR) modulator (LM11A-31) can augment neuronal survival and improve recovery in a mouse model of stroke. Mice were administered LM11A-31 for up to 12 weeks, beginning 1 week after stroke. Metabolomic analysis revealed that after 2 weeks of daily treatment, mice that received LM11A-31 were distinct from vehicle-treated mice by principal component analysis and had higher levels of serotonin, acetylcholine, and dopamine in their ipsilateral hemisphere. LM11A-31 treatment also improved redox homeostasis by restoring reduced glutathione. It also offset a stroke-induced reduction in glycolysis by increasing acetyl-CoA. There was no effect on cytokine levels in the infarct. At 13 weeks after stroke, adaptive immune cell infiltration in the infarct was unchanged in LM11A-31-treated mice, indicating that LM11A-31 does not alter the chronic inflammatory response to stroke at the site of the infarct. However, LM11A-31-treated mice had less brain atrophy, neurodegeneration, tau pathology, and microglial activation in other regions of the ipsilateral hemisphere. These findings correlated with improved recovery of motor function on a ladder test, improved sensorimotor and cognitive abilities on a nest construction test, and less impulsivity in an open field test. These data support small molecule modulation of the p75NTR for preserving neuronal health and function during stroke recovery. SIGNIFICANCE STATEMENT: The findings from this study introduce the p75 neurotrophin receptor as a novel small molecule target for promotion of stroke recovery. Given that LM11A-31 is in clinical trials as a potential therapy for Alzheimer's disease, it could be considered as a candidate for assessment in stroke or vascular dementia studies.

Live-Cell Imaging of Single Neurotrophin Receptor Molecules on Human Neurons in Alzheimer's Disease.

Neurotrophin receptors such as the tropomyosin receptor kinase A receptor (TrkA) and the low-affinity binding p75 neurotrophin receptor p75NTR play a critical role in neuronal survival and their functions are altered in Alzheimer’s disease (AD). Changes in the dynamics of receptors on the plasma membrane are essential to receptor function. However, whether receptor dynamics are affected in different pathophysiological conditions is unexplored. Using live-cell single-molecule imaging, we examined the surface trafficking of TrkA and p75NTR molecules on live neurons that were derived from human-induced pluripotent stem cells (hiPSCs) of presenilin 1 (PSEN1) mutant familial AD (fAD) patients and non-demented control subjects. Our results show that the surface movement of TrkA and p75NTR and the activation of TrkA- and p75NTR-related phosphoinositide-3-kinase (PI3K)/serine/threonine-protein kinase (AKT) signaling pathways are altered in neurons that are derived from patients suffering from fAD compared to controls. These results provide evidence for altered surface movement of receptors in AD and highlight the importance of investigating receptor dynamics in disease conditions. Uncovering these mechanisms might enable novel therapies for AD.

Structural study of regulated intramembrane proteolysis of p75NTR by γ-secretase.

Programmed cell death (PCD) maintains the tissue size in a multicellular organism. Uncontrolled PCD in neurons has long been implicated in neurodegenerative diseases, e.g. Alzheimer's disease. p75 neurotrophin receptor (p75NTR) plays an important role in regulating neuronal growth and also involves in apoptotic signaling, which leads to morphological changes in cells in PCD. When binding to nerve growth factor (NGF), p75NTR maintains cell survival and proliferation; on the contrary, p75NTR induces apoptosis when the NGF precursor (pro-NGF) binds onto p75NTR. The complex formation of the pro-NGF, p75NTR, and sortilin initiates the apoptotic signaling. The subsequent proteolytic processing on p75NTR by γ-secretase relays the signaling to the neuronal apoptosis. p75NTR, the founding member of tumor necrosis factor (TNF) receptor superfamily, is a type I transmembrane protein, involved in the downstream cell death signaling after the release of intracellular domain (ICD) through regulated intramembrane proteolysis (RIP) executed by γ-secretase. Detailed molecular mechanism of how the γ-secretase cleaves p75NTR and releases its death domain is still unknown. To answer this question, our ultimate goal is to study the molecular mechanism of the p75NTR cleavage by γ-secretase using cryogenic electron microscopy (cryo-EM). We generated four p75NTR mutants with various truncated mutants in Sf9 insect cells to determine the cleavage site for the 𝛾-secretase. We characterized the bindings using size-exclusion chromatography, SDS-PAGE, and negative-stain EM. p75NTR mutants generated and characterized their bindings with γ -secretase. We will stabilize the complex formation and probe the interaction between these proteins using single-particle cryo-EM. The result will gain a fundamental understanding of how p75NTR cleavage triggers the neuronal apoptosis signaling.

Small molecule modulation of TrkB and TrkC neurotrophin receptors prevents cholinergic neuron atrophy in an Alzheimer's disease mouse model at an advanced pathological stage

Degeneration of basal forebrain cholinergic neurons (BFCNs) in the nucleus basalis of Meynert (NBM) and vertical diagonal band (VDB) along with their connections is a key pathological event leading to memory impairment in Alzheimer's disease (AD). Aberrant neurotrophin signaling via Trks and the p75 neurotrophin receptor (p75NTR) contributes importantly to BFCN dystrophy. While NGF/TrkA signaling has received the most attention in this regard, TrkB and TrkC signaling also provide trophic support to BFCNs and these receptors may be well located to preserve BFCN connectivity. We previously identified a small molecule TrkB/TrkC ligand, LM22B-10, that promotes cell survival and neurite outgrowth in vitro and activates TrkB/TrkC signaling in the hippocampus of aged mice when given intranasally, but shows poor oral bioavailability. An LM22B-10 derivative, PTX-BD10–2, with improved oral bioavailability has been developed and this study examined its effects on BFCN atrophy in the hAPPLond/Swe (APPL/S) AD mouse model. Oral delivery of PTX-BD10–2 was started after appreciable amyloid and cholinergic pathology was present to parallel the clinical context, as most AD patients start treatment at advanced pathological stages. PTX-BD10–2 restored cholinergic neurite integrity in the NBM and VDB, and reduced NBM neuronal atrophy in symptomatic APPL/S mice. Dystrophy of cholinergic neurites in BF target regions, including the cortex, hippocampus, and amygdala, was also reduced with treatment. Finally, PTX-BD10–2 reduced NBM tau pathology and improved the survival of cholinergic neurons derived from human induced pluripotent stem cells (iPSCs) after amyloid-β exposure. These data provide evidence that targeting TrkB and TrkC signaling with PTX-BD10–2 may be an effective disease-modifying strategy for combating cholinergic dysfunction in AD. The potential for clinical translation is further supported by the compound's reduction of AD-related degenerative processes that have progressed beyond early stages and its neuroprotective effects in human iPSC-derived cholinergic neurons.

Computational model of brain endothelial cell signaling pathways predicts therapeutic targets for cerebral pathologies

Brain endothelial cells serve many critical homeostatic functions. In addition to sensing and regulating blood flow, they maintain blood-brain barrier function, including precise control of nutrient exchange and efflux of xenobiotics. Many signaling pathways in brain endothelial cells have been implicated in both health and disease; however, our understanding of how these signaling pathways functionally integrate is limited. A model capable of integrating these signaling pathways could both advance our understanding of brain endothelial cell signaling networks and potentially identify promising molecular targets for endothelial cell-based drug or gene therapies. To this end, we developed a large-scale computational model, wherein brain endothelial cell signaling pathways were reconstructed from the literature and converted into a network of logic-based differential equations. The model integrates 63 nodes (including proteins, mRNA, small molecules, and cell phenotypes) and 82 reactions connecting these nodes. Specifically, our model combines signaling pathways relating to VEGF-A, BDNF, NGF, and Wnt signaling, in addition to incorporating pathways relating to focused ultrasound as a therapeutic delivery tool. To validate the model, independently established relationships between selected inputs and outputs were simulated, with the model yielding correct predictions 73% of the time. We identified influential and sensitive nodes under different physiological or pathological contexts, including altered brain endothelial cell conditions during glioma, Alzheimer's disease, and ischemic stroke. Nodes with the greatest influence over combinations of desired model outputs were identified as potential druggable targets for these disease conditions. For example, the model predicts therapeutic benefits from inhibiting AKT, Hif-1α, or cathepsin D in the context of glioma – each of which are currently being studied in clinical or pre-clinical trials. Notably, the model also permits testing multiple combinations of node alterations for their effects on the network and the desired outputs (such as inhibiting AKT and overexpressing the P75 neurotrophin receptor simultaneously in the context of glioma), allowing for the prediction of optimal combination therapies. In all, our approach integrates results from over 100 past studies into a coherent and powerful model, capable of both revealing network interactions unapparent from studying any one pathway in isolation and predicting therapeutic targets for treating devastating brain pathologies.

ProBDNF expression induces apoptosis and inhibits synaptic regeneration by regulating the RhoA-JNK pathway in an in vitro post-stroke depression model

Brain-derived neurotrophic factor (BDNF) plays an important role in the pathophysiology of post-stroke depression (PSD). However, the precise function and potential mechanism of proBDNF, the precursor form of BDNF, are unknown. In our study, a PSD-like model was established by treating neuronal cells with oxygen-glucose deprivation and corticosterone. We found that the protein proBDNF levels were significantly higher in the cortex and hippocampus in the PSD group than in the control group, suggesting that proBDNF plays a role in the pathophysiology of PSD. Furthermore, we re-established the PSD-like cell model using recombinant p75 neurotrophin receptor (p75NTR) or silencing c-Jun N-terminal kinase (JNK), and found that the PSD-induced upregulation of proBDNF was inhibited by recombinant p75NTR and JNK silencing (siJNK), and increased cellular apoptosis. Moreover, the application of recombinant p75NTR and siJNK in the PSD-like cell model significantly reversed the expression of apoptosis-related and depression-related proteins and decreased cellular apoptosis. Our findings suggest that proBDNF is involved in neural plasticity in PSD in vitro. The RhoA-JNK signaling pathway is activated after proBDNF binds to the p75NTR receptor, followed by the expression of apoptosis-related proteins (PSD95, synaptophysin, and P-cofilin), which contribute to PSD progression. The mechanism might involve the promotion of cellular apoptosis and the inhibition of nerve synapses regeneration by proBDNF.

A fast and efficient method for isolating Schwann cells from sciatic nerves of neonatal mice

BackgroundSchwann cells (SCs) isolation is one of the basic techniques for study of peripheral nervous system and peripheral neuropathy. A combined and effective method of isolating SCs from sciatic nerves of newborn mice with high yield and purity is still lacking. New methodsSciatic nerves from neonatal mice aged 3–5 days serve as the source of SCs. Removal of adjacent connective tissue and epineurium, treatment with arabinoside hydrochloride and differential cell detachment technique were applied to eliminate fibroblast contamination and increase the purity of SCs. Combined use of collagenase/dispase and trypsin was chosen to increase the yield of SCs. Culture dishes precoated with poly-l-lysine and laminin, culture medium supplemented with heregulin β-1 and forskolin, and reasonable cell seeding density were implemented to increase the growth and proliferation of cultured SCs. Immunostaining of S100β and p75 neurotrophin receptor was used to identify the purity of SCs. ResultsOur method is able to obtain high-yield SCs with a purity of 90% within five days and a purity more than 99% within seven days from sciatic nerves of neonatal mice. Comparison with existing methodsPrevious SCs isolation mostly focused on rats or adult mice and have a few limitations due to fibroblasts contamination, low yield and time-consuming. Our method permits SCs isolation from neonatal mice with a high yield and purity of primary SCs within 7 days. ConclusionWe described a fast, efficient and step-by-step method of isolating SCs from sciatic nerves of neonatal mice with high yield and purity.