Brain-derived Neurotrophic Factor Precursor Research Articles

Effect of electroacupuncture on proBDNF/mBDNF and synaptic plasticity in rats with learning and memory impairment after cerebral ischemia-reperfusion.

To observe the effect of electroacupuncture (EA) at "Baihui" (GV20) and "Shenting" (GV24) on the rats' behavior and the transforming precursor of brain-derived neurotrophic factor (proBDNF) into mature brain-derived neurotrophic factor (mBDNF) in the hippocampus of rats with learning and memory impairment induced by cerebral ischemia-reperfusion (IR), so as to explore its mechanisms underlying improvement of learning and memory ability. SD rats were randomly divided into blank, sham operation, model, and EA groups, with 6 rats in each group. The model of IR was established by occlusion of the middle cerebral artery. EA (1 Hz/20 Hz) was applied to GV24 and GV20 for 30 min, once daily for 14 days. The neurological function was evaluated according to the Zea Longa's score criteria 24 h after modeling and after intervention. Morris water maze test was used to detect the learning and memory function of the rats. TTC staining was used to evaluate the cerebral infarction volume on the affected side. The protein expression levels of proBDNF, mBDNF, tissue plasminogen activator (tPA), tyrosine kinase receptor B (TrkB) and p75 neurotrophin receptor (p75NTR) in hippocampal tissue were detected by Western blot. Compared with the sham operation group, the neurological function score, the percentage of cerebral infarction volume and the expression levels of proBDNF and p75NTR protein in hippocampus were increased (P<0.01), while the times of crossing the original platform and the total distance in the target quadrant, the expression levels of mBDNF, TrkB and tPA protein and the ratio of mBDNF/proBDNF were decreased (P<0.01, P<0.05) in the model group. Compared with the model group, the neurological function score, the percentage of cerebral infarction volume, and the expression levels of proBDNF and p75NTR protein in hippocampus were decreased (P<0.01, P<0.05), while the times of crossing the original platform, the total distance in the target quadrant, and the expression levels of mBDNF, TrkB and tPA protein and the ratio of mBDNF/proBDNF were increased (P<0.05, P<0.01) in the EA group. EA can alleviate learning and memory impairment in IR rats, which may be related to its function in up-regulating the expression of tPA protein and promoting the transformation of proBDNF to mBDNF, thus improving the synaptic plasticity.

The expression of ProBDNF and its high affinity receptor P75NTR in the neurons of emotion-related brain regions of post-stroke depression rats

ObjectiveTo investigate the expression of the precursor of brain-derived neurotrophic factor (proBDNF) and its high-affinity receptor p75NTR in neurons of emotion-related brain areas (prefrontal cortex, hippocampus, and amygdala) in rats with post-stroke depression (PSD), and to explore the expression levels of proBDNF and p75NTR in neurons of emotion-related brain areas by injecting tissue plasminogen activator (t-PA) into the lateral ventricle of PSD rats, this significantly improved the stress-induced depression-like behavior,thus further validating the above results. MethodsRats were randomly divided into four groups: a normal control group (n = 8), a depression group (n = 8), a stroke group (n = 8), and a PSD group (n = 8). The rat model of stroke was established by thread embolism, and the PSD animal model was induced by chronic unpredictable mild stress (CUMS) and solitary feeding. Behavioral tests were conducted, including weight measurement, open field tests, and sucrose preference tests. Immunofluorescence double labeling was used to detect the expression of proBDNF and p75NTR in neurons of emotion-related brain regions in the PSD rat model. Four weeks after CUMS treatment, the PSD group was selected. Rats were infused with t-PA (3 μg dissolved in 6 μL saline, Boehringer Ingelheim), proBDNF (3 μg dissolved in 6 μL saline, Abcam), or equal-volume NS once per day for 7 consecutive days using the syringe pump connecting to injection needles. After 7 days of continuous administration, animal behavior was assessed through scoring, and the expression of proBDNF and p75NTR in the emotion-related brain regions of the PSD rat model was detected using immunofluorescence double labeling. ResultsCompared with the normal control group and the stroke group, the body weight, sucrose water consumption, and vertical movement distance in the PSD group were significantly lower (P < 0.05). In contrast, when compared with the proBDNF injection group and saline injection group, the weight, sucrose water consumption, field horizontal movement, and vertical movement distance of the t-PA injection group significantly increased after PSD lateral ventricle intubation.Double immunofluorescence revealed a higher neuronal expression of proBDNF as well as p75NTR in the prefrontal cortex and hippocampus of PSD rats compared to control animals (P < 0.05). In the amygdala, the expression levels of proBDNF and P75NTR were significantly reduced in the PSD group compared to the control group (P < 0.05). The results of the expression levels of proBDNF and P75NTR in the emotion-related brain regions of PSD rats injected with t-PA showed that proBDNF and P75NTR was significantly reduced in the prefrontal cortex, hippocampus, and amygdala of PSD rats compared to those of the NS and proBDNF groups (P < 0.05). ConclusionsThe increased expression of the brain-derived neurotrophic factor precursor proBDNF and its receptor p75NTR in neurons of emotion-related brain regions may play an important role in the pathogenesis of PSD.t-PA reduced the expression of proBDNF and its receptor p75NTR in neurons emotion-related brain regions and significantly improved the stress-induced depression-like behavior. Therefore, it is reasonable to assume that exogenous injection of t-PA may alleviate the depressive symptoms of PSD patients.Reducing the expression of proBDNF by injecting t-PA may provide a novel therapeutic approach for the treatment of stress-related mood disorders.

ProBDNF contributed to patrolling monocyte infiltration and renal damage in systemic lupus erythematosus

Monocyte aberrations have been increasingly recognized as contributors to renal damage in systemic lupus erythematosus (SLE), however, recognition of the underlying mechanisms and modulating strategies is at an early stage. Our studies have demonstrated that brain-derived neurotrophic factor precursor (proBDNF) drives the progress of SLE by perturbing antibody-secreting B cells, and proBDNF facilitates pro-inflammatory responses in monocytes. By utilizing peripheral blood from patients with SLE, GEO database and spontaneous MRL/lpr lupus mice, we demonstrated in the present study that CX3CR1+ patrolling monocytes (PMo) numbers were decreased in SLE. ProBDNF was specifically expressed in CX3CR1+ PMo and was closely correlated with disease activity and the degree of renal injury in SLE patients. In MRL/lpr mice, elevated proBDNF was found in circulating PMo and the kidney, and blockade of proBDNF restored the balance of circulating and kidney-infiltrating PMo. This blockade also led to the reversal of pro-inflammatory responses in monocytes and a noticeable improvement in renal damage in lupus mice. Overall, the results indicate that the upregulation of proBDNF in PMo plays a crucial role in their infiltration into the kidney, thereby contributing to nephritis in SLE. Targeting of proBDNF offers a potential therapeutic role in modulating monocyte-driven renal damage in SLE.

Genetic distinctiveness of an endangered falcon: Implications for conservation in Europe.

In the Falconidae, the genus Falco comprises species of large birds of prey with wide distribution worldwide. However, the European lanner falcon Falco biarmicus feldeggii is rapidly heading for global extinction following a dramatic decline caused by anthropogenic interference. Conservation projects are currently underway with the main purpose of increasing its population size in the Mediterranean basin through captive breeding and release of birds into the wild. To support the projects, and strengthen the legitimacy of conservation efforts consistently with the Evolutionary Significant Unit concept, we explored the possibility of characterising the gene pool of the European lanner and reliably distinguishing it from other falcon taxa inhabiting the Mediterranean area, which show morphological and genetic similarities. To address the issue, we examined genetic variability at the nuclear level through the analysis of 12 neutral Short Tandem Repeat loci, and, for the first time in these taxa, two single-copy functional genes, coding for the brain-derived neurotrophic factor precursor and the oocyte maturation factor, respectively. The second exon of the major histocompatibility complex class II B gene was also investigated. Additionally, to frame our data with previously published data, we assess variation at the mitochondrial level by sequencing portions of the cytochrome b, 12S rRNA gene, and the control region. Our results showed that the European lanner is highly distinct from other falcon taxa, as revealed by nuclear, but not by mitochondrial DNA. We discuss our findings focusing on their implications for the preservation of this highly endangered European bird, and highlighted the critical role of genetic information in planning and monitoring concrete interventions.

Brain-derived neurotrophic factor knock-out mice develop non-alcoholic steatohepatitis.

While brain-derived neurotrophic factor (BDNF), which is a growth factor associated with cognitive improvement and the alleviation of depression symptoms, is known to regulate food intake and body weight, the role of BDNF in peripheral disease is not fully understood. Here, we show that reduced BDNF expression is associated with weight gain and the chronic liver disease non-alcoholic steatohepatitis (NASH). At 10 months of age, BDNF-heterozygous (BDNF+/- ) mice developed symptoms of NASH: centrilobular/perivenular steatosis, lobular inflammation with infiltration of neutrophils, ballooning hepatocytes, and fibrosis of the liver. Obesity and higher serum levels of glucose and insulin - major pathologic features in human NASH - were dramatic. Dying adipocytes were surrounded by macrophages in visceral fat, suggesting that chronic inflammation occurs in peripheral organs. RNA sequencing (RNA-seq) studies of the liver revealed that the most significantly enriched Gene Ontology term involved fatty acid metabolic processes and the modulation of neutrophil aggregation, pathologies that well characterise NASH. Gene expression analysis by RNA-seq also support the notion that BDNF+/- mice are under oxidative stress, as indicated by alterations in the expression of the cytochrome P450 family and a reduction in glutathione S-transferase p, an antioxidant enzyme. Histopathologic phenotypes of NASH were also observed in a knock-in mouse (BDNF+/pro ), in which the precursor BDNF is inefficiently converted into the mature form of BDNF. Lastly, as BDNF reduction causes overeating and subsequent obesity, a food restriction study was conducted in BDNF+/pro mice. Pair-fed BDNF+/pro mice developed hepatocellular damage and showed infiltration of inflammatory cells, including neutrophils in the liver, despite having body weights and blood parameters that were comparable to those of controls. This is the first report demonstrating that reduced BDNF expression plays a role in the pathogenic mechanism of NASH, which is a hepatic manifestation of metabolic syndrome. © 2023 The Pathological Society of Great Britain and Ireland.

Early treatment with an M1 and sigma-1 receptor agonist prevents cognitive decline in a transgenic rat model displaying Alzheimer-like amyloid pathology

The application of the selective allosteric M1 muscarinic and sigma-1 receptor agonist, AF710B (aka ANAVEX3-71), has shown to attenuate Alzheimer’s disease-like hallmarks in McGill-R-Thy1-APP transgenic rats when administered at advanced pathological stages. It remains unknown whether preventive treatment strategies applying this compound may be equally effective. We tested whether daily oral administration of AF710B (10 µg/kg) in 7-month-old, preplaque, McGill-R-Thy1-APP rats for 7 months, followed by a 4-week washout period, could prevent Alzheimer’s disease-like pathological hallmarks. Long-term AF710B treatment prevented the cognitive impairment of McGill-R-Thy1-APP rats. The effect was accompanied by a reduction in the number of amyloid plaques in the hippocampus and the levels of Aβ42 and Aβ40 peptides in the cerebral cortex. AF710B treatment also reduced microglia and astrocyte recruitment toward CA1 hippocampal Aβ-burdened neurons compared to vehicle-treated McGill-R-Thy1-APP rats, also altering the inflammatory cytokines profile. Lastly, AF710B treatment rescued the conversion of brain-derived neurotrophic factor precursor to its mature and biologically active form. Overall, these results suggest preventive and disease-modifying properties of the compound.

Neonatal oxytocin treatment alters levels of precursor and mature BDNF forms and modifies the expression of neuronal markers in the male rat hippocampus

Neuropeptide oxytocin appears to be involved in the formation of hippocampal circuitry, underlying social memory and behaviour. Recent studies point to the role of oxytocin in regulating the levels of nerve growth factors that could influence neurogenesis and neuritogenesis during the early stages of brain development. Therefore, the aim of the present study was to evaluate the early developmental effect of oxytocin administration (P2 and P3 days, two doses, 5 μg/pup, s.c.) on the expression of 1) brain-derived neurotrophic factor (BDNF) isoforms and 2) GABAergic and glutamatergic markers in the male rat hippocampus. Furthermore, we evaluated the branching of dendrites of primary hippocampal GABAergic and glutamatergic neurons in response to incubation with oxytocin (1 μM). We found that after oxytocin administration, levels of proBDNF increased on P5 and mBDNF on P7 in the CA1 hippocampal region. We also observed a reduction in the expression of glutamatergic marker (VGluT2) on P7 compared to P5 in control and oxytocin treated rats. During the early developmental stages (P5, P7, P9) the expression of GABAergic markers (Gad65 and Gad67) decreased regardless of oxytocin treatment. Incubation in a presence of oxytocin reduced branching of glutamatergic hippocampal neurons and the opposite stimulatory effect of oxytocin was observed in GABAergic neurons. These findings suggest that oxytocin affects neurotrophin isoforms in the male rat hippocampus in the early stages of development, which could explain changes in glutamatergic neurons and their morphology.

Elevated Plasma Levels of Mature Brain-Derived Neurotrophic Factor in Major Depressive Disorder Patients with Higher Suicidal Ideation.

Several pieces of evidence show that signaling via brain-derived neurotrophic factor (BDNF) and its receptor, tropomycin receptor kinase B (TrkB), as well as inflammation, play a crucial part in the pathophysiology of depression. The purpose of our study was to evaluate plasma levels of BDNF-TrkB signaling, which are inflammatory factors in major depressive disorder (MDD) patients, and assess their associations with clinical performance. This study recruited a total sample of 83 MDD patients and 93 healthy controls (CON). All the participants were tested with the Hamilton Depression Scale (HAMD), the Beck Scale for Suicide Ideation, and the NEO Five-Factor Inventory. The plasma level of selected BDNF-TrkB signaling components (mature BDNF (mBDNF), precursor BDNF (proBDNF), tyrosine kinase B (TrkB), and tissue plasminogen activator (tPA)) and selected inflammatory factors (interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)) were measured using an enzyme-linked immunosorbent assay (ELISA). Further, we performed correlation analysis to indicate the relationship between the plasma levels of the factors and clinical characteristics. Results: (i) A higher level of mBDNF and lower openness were observed in MDD patients with higher suicidal ideation than patients with lower suicidal ideation. (ii) In MDD patients, mBDNF was positively correlated with the sum score of the Beck Scale for Suicide Ideation (BSS). (iii) The levels of mBDNF, tPA, IL-1 β and IL-6 were significantly higher in all MDD subjects compared to the healthy controls, while the levels of TrkB and proBDNF were lower in MDD subjects. Conclusion: Our study provides novel insights regarding the potential role of mBDNF in the neurobiology of the association between depression and suicidal ideation and, in particular, the relationship between BDNF-TrkB signaling, inflammatory factors, and clinical characteristics in MDD.

Omega-3 Polyunsaturated Fatty Acid Eicosapentaenoic Acid or Docosahexaenoic Acid Improved Ageing-Associated Cognitive Decline by Regulating Glial Polarization.

Neuroinflammation induced by microglial and astrocyte polarizations may contribute to neurodegeneration and cognitive impairment. Omega (n)-3 polyunsaturated fatty acids (PUFAs) have anti-inflammatory and neuroprotective effects, but conflicting results were reported after different n-3 PUFA treatments. This study examined both the change in glial polarizations in ageing rats and the differential effects of two omega-3 PUFAs. The results showed that both PUFAs improved spatial memory in ageing rats, with docosahexaenoic acid (DHA) being more effective than eicosapentaenoic acid (EPA). The imbalance between microglial M1/M2 polarizations, such as up-regulating ionized calcium binding adaptor molecule 1 (IBA1) and down-regulating CD206 and arginase-1 (ARG-1) was reversed in the hippocampus by both n-3 PUFAs, while the DHA effect on CD206 was stronger. Astrocyte A1 polarization presented increasing S100B and C3 but decreasing A2 parameter S100A10 in the ageing brain, which were restored by both PUFAs, while DHA was more effective on S100A10 than EPA. Consistent with microglial M1 activation, the concentration of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were significantly increased, which were attenuated by DHA, while EPA only suppressed IL-6. In correlation with astrocyte changes, brain-derived neurotrophic factor precursor was increased in ageing rats, which was more powerfully down-regulated by DHA than EPA. In summary, enhanced microglial M1 and astrocytic A1 polarizations may contribute to increased brain pro-inflammatory cytokines, while DHA was more powerful than EPA to alleviate ageing-associated neuroimmunological changes, thereby better-improving memory impairment.

An Imbalance in the Pro/mature BDNF Ratio Occurs in Multiple Brain Regions During Normal Ageing in Wild-Type Mice.

The early transition to Alzheimer's disease is characterized by a period of accelerated brain atrophy that exceeds normal ageing. Identifying the molecular basis of this atrophy could facilitate the discovery of novel drug targets. The precursor of brain-derived neurotrophic factor, a well characterized neurotrophin, is increased in the hippocampus of aged rodents, while its mature isoform is relatively stable. This imbalance could increase the risk of Alzheimer's disease by precipitating its pathological hallmarks. However, less is known about how relative levels of these isoforms change in middle-aged mice. In addition, the underlying mechanisms that might cause an imbalance are unknown. The main aim of this study was to determine how precursor brain-derived neurotrophic factor changes relative to its mature isoform with normal brain ageing in wild type mice. A secondary aim was to determine if signaling through the neurotrophin receptor, p75 influences this ratio. An increasing ratio was identified in several brain regions, except the hippocampus, suggesting a neurotrophic imbalance occurs as early as middle age. Some changes in receptors that mediate the isoforms effects were also identified, but these did not correspond with trends in the isoforms. Relative amounts of precursor brain-derived neurotrophic factor were mostly unchanged in mutant p75 mice. The lack of changes suggested that signaling through the receptor had no influence on the ratio.

α7 Nicotinic acetylcholine receptor potentiation downregulates chemotherapy-induced inflammatory overactivation by overlapping intracellular mechanisms.

We studied, using a combination of animal andcellular models, the glial mechanisms underlying the anti-neuropathic and anti-inflammatory properties of PAM-2 [(E)-3-furan-2-yl-N-p-tolyl-acrylamide], a positive allosteric modulator of α7 nicotinic acetylcholine receptors (nAChRs). In mice, PAM-2 decreased the inflammatory process induced by the combination of oxaliplatin (OXA), a chemotherapeutic agent, and interleukin-1β (IL-1β), a pro-inflammatory molecule. In the brain and spinal cord of treated animals, PAM-2 reduced pro-inflammatory cytokines/chemokines by mechanisms involving mRNA downregulation of factors in the toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB pathway, and increased the precursor of brain-derived neurotrophic factor (proBDNF). To determine the molecular mechanisms underlying the anti-inflammatory activity of PAM-2, both human C20 microglia and normal human astrocytes (NHA) were used. The results showed that PAM-2-induced potentiation of glial α7 nAChRs decreases OXA/IL-1β-induced overexpression of inflammatory molecules by different mechanisms, including mRNA downregulation of factors in the NF-κB pathway (in microglia and astrocyte) and ERK (only in microglia). The OXA/IL-1β-mediated reduction in proBDNF was prevented by PAM-2 in microglia, but not in astrocytes. Our findings also indicate that OXA/IL-1β-induced organic cation transporter 1 (OCT1) expression is decreased by PAM-2, suggesting that decreased OXA influx may be involved in the protective effects of PAM-2. The α7-selective antagonist methyllycaconitine blocked the most important effects mediated by PAM-2 at both animal and cellular levels, supporting a mechanism involving α7 nAChRs. In conclusion, glial α7 nAChR stimulation/potentiation downregulates neuroinflammatory targets, and thereby remains a promising therapeutic option for cancer chemotherapy-induced neuroinflammation and neuropathic pain.

ProBDNF and its receptors in immune-mediated inflammatory diseases: novel insights into the regulation of metabolism and mitochondria.

Immune-mediated inflammatory diseases (IMIDs) consist of a common and clinically diverse group of diseases. Despite remarkable progress in the past two decades, no remission is observed in a large number of patients, and no effective treatments have been developed to prevent organ and tissue damage. Brain-derived neurotrophic factor precursor (proBDNF) and receptors, such as p75 neurotrophin receptor (p75NTR) and sortilin, have been proposed to mediate intracellular metabolism and mitochondrial function to regulate the progression of several IMIDs. Here, the regulatory role of proBDNF and its receptors in seven typical IMIDs, including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, allergic asthma, type I diabetes, vasculitis, and inflammatory bowel diseases, was investigated.

Association between inflammatory cytokines and symptoms of major depressive disorder in adults.

This study investigated the association between inflammatory cytokines and major depressive disorder. Plasma biomarkers were measured by enzyme-linked immunosorbent assay (ELISA). Statistical analysis of baseline biomarkers in the major depression disorder (MDD) group and healthy controls (HC) group, and differences in biomarkers before and after treatment. Spearman analysis was performed to correlate baseline and after treatment MDD biomarkers with the 17-item Hamilton Depression Rating Scale (HAMD-17) total scores. Receiver operator characteristic (ROC) curves were analyzed for the effect of biomarkers on MDD and HC classification and diagnosis. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were significantly higher in the MDD group than in the HC group, while high mobility group protein 1 (HMGB1) levels were significantly lower in the MDD group. The AUCs for HMGB1, TNF-α, and IL-6 were 0.375, 0.733, and 0.783, respectively, according to the ROC curves. MDD patients with brain-derived neurotrophic factor precursor (proBDNF) levels were positively correlated with total HAMD-17 scores. The levels of proBDNF levels were positively correlated with the total HAMD-17 score in male MDD patients, and brain-derived neurotrophic factor (BDNF) and interleukin 18 (IL-18) levels were negatively correlated with the total HAMD-17 score in female MDD patients. Inflammatory cytokines are associated with the severity of MDD, and TNF-α and IL-6 have the potential as objective biomarkers to aid in the diagnosis of MDD.

ProBDNF signaling is involved in periodontitis-induced depression-like behavior in mouse hippocampus

ObjectiveIncreasing evidence supports the association between periodontitis and depression. However, the specific mechanisms remain to be further elucidated. The present study aimed to mechanistically investigate the regional roles of proBDNF (the precursor of brain-derived neurotrophic factor) in periodontitis induced depression-like behavior in mice. MethodsExperimental periodontitis model was established by periodontal injection of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) in 8-week-old male Bdnf-HA/HA mice for 3 weeks. The depression-like behaviors, spontaneous exploratory activity and the level of anxiety were assessed by behavior tests. The activation of microglia and astrocytes, as well as the expression of Interleukin (IL)-1β and Tumor necrosis factor (TNF)-α in the hippocampus, prefrontal cortex, and cortex were further assessed by immunofluorescence and western blots. The levels of IL-1β in blood serum and expression of occludin as well as claudin5 in the hippocampus, prefrontal cortex, and cortex were further determined by enzyme-linked immunosorbent assay and western blot. Finally, the expression of proBDNF, its receptors, and mature BDNF (mBDNF), as well as neuronal activity were measured by western blots and immunofluorescence. ResultsPg-LPS successfully induced periodontitis in mice and caused obvious depression-like behavior. Furthermore, we observed an increased activation of astrocytes and microglia, as well as a significant increase in expression of IL-1β and TNF-α in the hippocampus of mice treated with Pg-LPS, with elevated level of IL-1β in serum and decreased expression of occludin and claudin5 in the hippocampus. Importantly, we found that the levels of proBDNF and its receptors, SorCS2 and p75NTR, were increased significantly; however, the level of mBDNF was decreased, therefor leading to greater ratio of proBDNF/mBDNF. In addition, we also detected decreased neuronal activity in the hippocampus of mice treated with Pg-LPS. ConclusionsOur results indicate that Pg-LPS-induced periodontitis could cause depression-like behaviors in mice, and the proBDNF signaling is involved in the process.

Synaptic proteins in neuron-derived extracellular vesicles as biomarkers for Alzheimer's disease: novel methodology and clinical proof of concept.

Blood biomarkers can improve drug development for Alzheimer's disease (AD) and its treatment. Neuron-derived extracellular vesicles (NDEVs) in plasma offer a minimally invasive platform for developing novel biomarkers that may be used to monitor the diverse pathogenic processes involved in AD. However, NDEVs comprise only a minor fraction of circulating extracellular vesicles (EVs). Most published studies have leveraged the L1 cell adhesion molecule (L1CAM) for NDEV immunocapture. We aimed to develop and optimize an alternative, highly specific immunoaffinity method to enrich blood NDEVs for biomarker development. After screening multiple neuronal antigens, we achieved NDEV capture with high affinity and specificity using antibodies against Growth-Associated Protein (GAP) 43 and Neuroligin 3 (NLGN3). The EV identity of the captured material was confirmed by electron microscopy, western blotting, and proteomics. The specificity for neuronal origin was demonstrated by showing enrichment for neuronal markers (proteins, mRNA) and recovery of spiked neuronal EVs. We performed NDEV isolation retrospectively from plasma samples from two cohorts of early AD patients (N = 19 and N = 40) and controls (N = 20 and N = 19) and measured p181-Tau, amyloid-beta (Aβ) 42, brain-derived neurotrophic factor (BDNF), precursor brain-derived neurotrophic factor (proBDNF), glutamate receptor 2 (GluR2), postsynaptic density protein (PSD) 95, GAP43, and syntaxin-1. p181-Tau, Aβ42, and NRGN were elevated in AD samples, whereas proBDNF, GluR2, PSD95, GAP43, and Syntaxin-1 were reduced. Differences for p181-Tau, proBDNF, and GluR2 survived multiple-comparison correction and were correlated with cognitive scores. A model incorporating biomarkers correctly classified 94.7% of AD participants and 61.5% of control participants. The observed differences in NDEVs-associated biomarkers are consistent with previous findings. NDEV isolation by GAP43 and NLGN3 immunocapture offers a robust novel platform for biomarker development in AD, suitable for large-scale validation.

Up-Regulation of ProBDNF/p75NTR Signaling in Spinal Cord Drives Inflammatory Pain in Male Rats.

The spinal cord expresses brain-derived neurotrophic factor precursor (proBDNF) and its receptor pan neurotrophin receptor 75 (p75NTR). However, the role of spinal proBDNF signaling in the pathogenesis of inflammatory pain remains unknown. Rats were locally injected with complete Freund's adjuvant (CFA) to induce inflammatory pain. The proBDNF signal expression was detected by double-labeled immunofluorescence. ProBDNF protein, p75NTR extracellular domain (p75NTR-ECD), or monoclonal anti-proBDNF (McAb-proB) were administrated by intrathecal injection to investigate their effects on pain behavior. Paw withdrawal thermal latency (PWL) and paw withdrawal mechanical threshold (PWT) were performed to evaluate pain behavior. Immunoblotting, immunohistochemistry, and immunofluorescence were used to assess inflammation-induced biochemical changes. CFA induced a rapid increase in proBDNF in the ipsilateral spinal cord, and immunofluorescence revealed that CFA-enhanced proBDNF was expressed in NeuN positive neurons and GFAP positive astrocytes. The administration of furin cleavage-resistant proBDNF via intrathecal injection (I.t.) significantly decreased the PWT and PWL, whereas McAb-proB by I.t. alleviated CFA-induced pain-like hypersensitivity in rats. Meanwhile, CFA administration triggered the activation of p75NTR and its downstream signaling extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor (NF)-kappaB p65 in the spinal cord. I.t. administration of p75NTR-ECD suppressed CFA-induced pain and neuroinflammation, including the expression of p-ERK1/2, p-p65, and the gene expression of tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6). Our study reveals that the activatedproBDNF/p75NTRsignaling in the spinal cord contributes to the development of CFA-induced inflammatory pain. McAb-proB and p75NTR-ECD appear to be promising therapeutic agents for inflammatory pain.

Systemic blockade of proBDNF inhibited the expansion and altered the transcriptomic expression in CD3+B220+ cells in MRL/lpr lupus mice

ObjectivesThe overexpansion of CD3+B220+ cells is the hallmark and main pathological mechanism of clinical manifestations of spontaneously developed MRL/lpr mice, which are primarily used as a mouse model of SLE....

Downregulation of Fat Mass and Obesity-Related Protein in the Anterior Cingulate Cortex Participates in Anxiety- and Depression-Like Behaviors Induced by Neuropathic Pain.

N6-methyladenosine (m6A) is the most abundant methylation modification on mRNA in mammals. Fat mass and obesity-related protein (FTO) is the main RNA m6A demethylase. FTO is involved in the occurrence and maintenance of neuropathic pain (NP). NP often induces mental disorders. We found that NP downregulated the expression of FTO in the anterior cingulate cortex (ACC), inhibited the expression of matrix metalloproteinase-9 (MMP-9) in the ACC, maladjusted the brain-derived neurotrophic factor precursor (proBDNF) and mature brain-derived neurotrophic factor (mBDNF) levels in the ACC, and induced anxiety- and depression-like behaviors in mice. Blocking the downregulation of FTO in the ACC induced by peripheral nerve injury could reverse the anxiety- and depression-like behaviors of mice. Contrarily, downregulation of simulated FTO induced anxiety- and depression-like behaviors in mice. After peripheral nerve injury, the binding of FTO to MMP-9 mRNA decreased and the enrichment of m6A on MMP-9 mRNA increased. In conclusion, downregulation of FTO in ACC by regulating MMP-9 mRNA methylation level contributes to the occurrence of anxiety- and depression-like behaviors in NP mice.

ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses.

The effects of brain-derived neurotrophic factor (BDNF) processing by-products (proBDNF and BDNF prodomain) on the activity of mouse neuromuscular junctions (NMJs) were studied in synapses formed during the reinnervation of extensor digitorum longus muscle (m. EDL) and mature synapses of the diaphragm. The parameters of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were analyzed in presence of each of the BDNF maturation products (both – 1 nM). In newly formed NMJs, proBDNF caused an increase in the resting membrane potential of muscle fibers and a decrease in the frequency of MEPPs, which was prevented by tertiapin-Q, a G-protein-coupled inwardly rectifying potassium channels (GIRK) blocker but not by p75 receptor signaling inhibitor TAT-Pep5. proBDNF had no effect on the parameters of EPPs. BDNF prodomain in newly formed synapses had effects different from those of proBDNF: it increased the amplitude of MEPPs, which was prevented by vesamicol, an inhibitor of vesicular acetylcholine (ACh) transporter; and reduced the quantal content of EPPs. In mature NMJs, proBDNF did not influence MEPPs parameters, but BDNF prodomain suppressed both spontaneous and evoked ACh release: decreased the frequency and amplitude of MEPPs, and the amplitude and quantal content of EPPs. This effect of the BDNF prodomain was prevented by blocking GIRK channels, by TAT-Pep5 or by Rho-associated protein kinase (ROCK) inhibitor Y-27632. At the same time, the BDNF prodomain did not show any inhibitory effects in diaphragm motor synapses of pannexin 1 knockout mice, which have impaired purinergic regulation of neuromuscular transmission. The data obtained suggest that there is a previously unknown mechanism for the acute suppression of spontaneous and evoked ACh release in mature motor synapses, which involves the activation of p75 receptors, ROCK and GIRK channels by BDNF prodomain and requires interaction with metabotropic purinoreceptors. In general, our results show that both the precursor of BDNF and the product of its maturation have predominantly inhibitory effects on spontaneous and evoked ACh release in newly formed or functionally mature neuromuscular junctions, which are mainly opposite to the effects of BDNF. The inhibitory influences of both proteins related to brain neurotrophin are mediated via GIRK channels of mouse NMJs.

ProBDNF/p75NTR promotes rheumatoid arthritis and inflammatory response by activating proinflammatory cytokines.

P75 pan-neurotrophin receptor (p75NTR) is an important receptor for the role of neurotrophins in survival and death of neurons during development and after nerve injury. Our previous research found that the precursor of brain-derived neurotrophic factor (proBDNF) regulates pain as an inflammatory mediator. The current understanding of the role of proBDNF/p75NTR signaling pathway in inflammatory arthritis pain and rheumatoid arthritis (RA) is unclear. We recruited 20 RA patients, 20healthy donors (HDs), and 10 osteoarthritis (OA) patients. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) of proBDNF and p75NTR in synovial membrane were performed and evaluated. We next examined the mRNA and protein expression of proBDNF/p75NTR signaling pathway in peripheral blood mononuclear cells (PBMCs) and synovial tissue. ELISA and flow cytometry were assessed between the blood of RA patients and HD. To induce RA, collagen-induced arthritis (CIA) were induced in mice. We found over-synovitis of RA synovial membrane compared to OA controls in histologic sections. P75NTR and sortilin mRNA, and proBDNF protein level were significantly increased in PBMCs of RA patients compared with the HD. Consistently, ELISA showed that p75NTR, sortilin, tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) levels in the serum of RA patients were increased compared with HD and p75NTR, sortilin were positively correlated with Disease Activity Score in 28 joints (DAS28). In addition, using flow cytometry we showed that the increased levels of proBDNF and p75NTR characterized in CD4+ and CD8+ T cells of RA patients were subsequently reversed with methotrexate (MTX) treatment. Furthermore, we found pathological changes, inflammatory pain, upregulation of the mRNA and protein expression of proBDNF/p75NTR signaling pathway, and upregulation of inflammatory cytokines in spinal cord using a well-established CIA mouse model. We showed intravenous treatment of recombinant p75ECD-Fc that biologically blocked all inflammatory responses and relieved inflammatory pain of animals with CIA. Our findings showed the involvement of proBDNF/p75NTR pathway in the RA inflammatory response and how blocking it with p75ECD-Fc may be a promising therapeutic treatment for RA.