Upregulation of proBDNF/p75NTR signaling in immune cells and its correlation with inflammatory markers in patients with major depression.

ProBDNF is the precursor protein of brain-derived neurotrophic factor (BDNF) expressed in the central nervous system and peripheral tissues. Previous studies showed that the blood levels of both proBDNF and p75 neurotrophic receptors (p75NTR) in major depressive disorder (MDD) were increased, but which blood cell types express proBDNF and its receptors is not known. Furthermore, the relationship between proBDNF/p75NTR and inflammatory cytokines in peripheral blood of MDD is unclear. Peripheral blood mononuclear cells (PBMCs) and serum were obtained from depressive patients (n = 32) and normal donors (n = 20). We examined the expression of proBDNF and inflammatory markers and their correlative relationship in patients with major depression. Using flow cytometry analysis, we examined which blood cells express proBDNF and its receptors. Finally, the role of proBDNF/p75NTR signal in inflammatory immune activity of PBMCs was verified in vitro experiments. Inflammatory cytokines in PBMC from MDD patients were increased and correlated with the major depression scores. The levels of IL-1ß and IL-10 were also positively correlated with the major depression scores, while the levels of TNF-α and IL-6 were negatively correlated with the major depression scores. Intriguingly, the levels of sortilin were positively correlated with IL-1ß. Q-PCR and Western blots showed proBDNF, p75NTR, and sortilin levels were significantly increased in PBMCs from MDD patients compared with that from the normal donors. Flow cytometry studies showed that proBDNF and p75NTR were present mainly in CD4+ and CD8+ T cells. The number of proBDNF and p75NTR positive CD4+ and CD8+ T cells from MDD patients was increased and subsequently reversed after therapeutic management. Exogenous proBDNF protein or p75ECD-Fc treatment of cultured PBMC affected the release of inflammatory cytokines in vitro. ProBDNF promoted the expression of inflammatory cytokines, while p75ECD-Fc inhibited the expression of inflammatory cytokines. Given there was an inflammatory response of lymphocytes to proBDNF, it is suggested that proBDNF/p75NTR signaling may upstream inflammatory cytokines in MDD. Our data suggest that proBDNF/p75NTR signaling may not only serve as biomarkers but also may be a potential therapeutic target for MDD.

SorCS2 is involved in promoting periodontitis-induced depression-like behaviour in mice.

BACKGROUND: Emerging evidence supports the association between periodontitis and depression, although the mechanisms are unclear. This study investigated the role of SorCS2 in the pathogenesis of periodontitis-induced depression. MATERIALS AND METHODS: An experimental periodontitis model was established using SorCS2 knockout mice and their wild-type littermates, and depression-like behaviour was evaluated. The expression of proBDNF signalling, neuronal activity, and glutamate-associated signalling pathways were further measured by western blotting and immunofluorescence. In addition, neuroinflammatory status, astrocytic and microglial markers, and the expression of corticosterone-related factors were measured by immunofluorescence, western blotting, and enzyme-linked immunosorbent assays. RESULTS: SorCS2 deficiency alleviated periodontitis-induced depression-like behaviour in mice. Further results suggested that SorCS2 deficiency downregulated the expression of pro-BDNF and glutamate signalling and restored neuronal activities in mice with periodontitis. Neuroinflammation in the mouse hippocampus was triggered by experimental periodontitis but was not affected by SorCS2 deficiency. The levels of corticosterone and the expression of glucocorticoid receptors were also not altered. CONCLUSION: Our study, for the first time, reveals the critical role of SorCS2 in the pathogenesis of periodontitis-induced depression. The underlying mechanism involves proBDNF and glutamate signalling in the hippocampus, providing a novel therapeutic target for periodontitis-associated depression.

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, 20 healthy 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-1ß (IL-1ß), 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.

ProBDNF Dependence of LTD and Fear Extinction Learning in the Amygdala of Adult Mice.

Neurotrophins are secreted proteins that control survival, differentiation, and synaptic plasticity. While mature neurotrophins regulate these functions via tyrosine kinase signaling (Trk), uncleaved pro-neurotrophins bind preferentially to the p75 neurotrophin receptor (p75NTR) and often exert opposite effects to those of mature neurotrophins. In the amygdala, brain-derived neurotrophic factor (BDNF) enables long-term potentiation as well as fear and fear extinction learning. In the present study, we focused on the impact of mature BDNF and proBDNF signaling on long-term depression (LTD) in the lateral amygdala (LA). Hence, we conducted extracellular field potential recordings in an in vitro slice preparation and recorded LTD in cortical and thalamic afferents to the LA. LTD was unchanged by acute block of BDNF/TrkB signaling. In contrast, LTD was inhibited by blocking p75NTR signaling, by disinhibition of the proteolytic cleavage of proBDNF into mature BDNF, and by preincubation with a function-blocking anti-proBDNF antibody. Since LTD-like processes in the amygdala are supposed to be related to fear extinction learning, we locally inhibited p75NTR signaling in the amygdala during or after fear extinction training, resulting in impaired fear extinction memory. Overall, these results suggest that in the amygdala proBDNF/p75NTR signaling plays a pivotal role in LTD and fear extinction learning.

Untangling the Conformational Plasticity of V66M Human proBDNF Polymorphism as a Modifier of Psychiatric Disorder Susceptibility.

The human genetic variant BDNF (V66M) represents the first example of neurotrophin family member that has been linked to psychiatric disorders. In order to elucidate structural differences that account for the effects in cognitive function, this hproBDNF polymorph was expressed, refolded, purified, and compared directly to the WT variant for the first time for differences in their 3D structures by DSF, limited proteolysis, FT-IR, and SAXS measurements in solution. Our complementary studies revealed a deep impact of V66M polymorphism on hproBDNF conformations in solution. Although the mean conformation in solution appears to be more compact in the V66M variant, overall, we demonstrated a large increase in flexibility in solution upon V66M mutation. Thus, considering that plasticity in IDR is crucial for protein function, the observed alterations may be related to the functional alterations in hproBDNF binding to its receptors p75NTR, sortilin, HAP1, and SorCS2. These effects can provoke altered intracellular neuronal trafficking and/or affect proBDNF physiological functions, leading to many brain-associated diseases and conditions such as cognitive impairment and anxiety. The structural alterations highlighted in the present study may pave the way to the development of drug discovery strategies to provide greater therapeutic responses and of novel pharmacologic strategy in human populations with this common polymorphism, ultimately guiding personalized medicine for neuropsychiatric disorders.

ProBDNF induces sustained elevation of intracellular Ca2+ possibly mediated by TRPM7 channels in rodent microglial cells.

Microglia are intrinsic immune cells that release factors including pro- and anti-inflammatory cytokines, nitric oxide (NO) and neurotrophins following activation in the brain. Elevation of intracellular Ca2+ concentration ([Ca2+ ]i) is important for microglial functions, such as the release of cytokines or NO from activated microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia. Interestingly, proBDNF, the precursor form of mature BDNF, and mature BDNF elicit opposing neuronal responses in the brain. Mature BDNF induces sustained intracellular Ca2+ elevation through the upregulation of the surface expression of TRPC3 channels in rodent microglial cells. In addition, TRPC3 channels are important for the BDNF-induced suppression of NO production in activated microglia. In this study, we observed that proBDNF and mature BDNF have opposite effects on the relative expression of surface p75 neurotrophin receptor (p75NTR ) in rodent microglial cells. ProBDNF induces a sustained elevation of [Ca2+ ]i through binding to the p75NTR , which is possibly mediated by Rac 1 activation and TRPM7 channels in rodent microglial cells. Flow cytometry showed that proBDNF increased the relative surface expression of TRPM7. Although proBDNF did not affect either mRNA expression of pro- and anti-inflammatory cytokines or the phagocytic activity, proBDNF potentiates the generation of NO induced by IFN-γ and TRPM7 channels could be involved in the proBDNF-induced potentiation of IFN-γ-mediated production of NO. We show direct evidence that rodent microglial cells are able to respond to proBDNF, which might be important for the regulation of inflammatory responses in the brain.

The regulatory role of ProBDNF in monocyte function: Implications in Stanford type-A aortic dissection disease.

Brain-derived neurotrophic factor precursor (proBDNF) has been reported to strengthen the dysfunction of monocytes/macrophages in animal studies. However, it is still unknown the roles of proBDNF in the dysfunction of monocytes in the inflammatory diseases in humans. In the present study, we showed that proBDNF and pan neurotrophic receptor p75 were significantly upregulated in monocytes from healthy donors (HD) after lipopolysaccharide treatment. Exogenous proBDNF treatment upregulated CD40 and proinflammatory cytokines expression in monocytes including interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α. In Stanford type-A acute aortic dissection (AAD) patients, proBDNF was upregulated in CD14+ CD163+ CX3CR1+ M2- but not CD14+ CD68+ CCR2+ M1-like monocytes. In addition, sera from AAD patients activated gene expression of proinflammatory cytokines in cultured PBMCs from HD, which was attenuated by proBDNF monoclonal antibody (Ab-proB) treatment. These findings suggested that upregulation of proBDNF in M2-like monocytes may contribute to the proinflammatory response in the AAD.

The impact of antipsoriatic treatment on serum pro-BDNF, BDNF levels, depression, anxiety scores, and quality of life.

Depression is a comorbidity of psoriasis. Suppression of neurotrophins has been proposed to cause depression. Peripheral brain-derived neurotrophic factor (BDNF) and its precursor, pro-BDNF have been shown to be altered in depression. To compare serum pro-BDNF and BDNF levels, depression, anxiety, and quality of life (QoL) in psoriasis patients, diseased, and healthy controls, to assess impact of 12-week antipsoriatic treatment on abovementioned markers. At baseline, all groups completed Beck Depression Inventory (BDI), Spielberger State-Trait Anxiety Inventory-II (STAI-II) and DLQI; serum BDNF, proBDNF levels were measured. These were repeated after 3-months of treatment in psoriasis patients. Depression and anxiety were significantly higher, QoL was poorer in psoriasis. ProBDNF and proBDNF/BDNF ratios were not different among groups at baseline but significantly decreased after treatment in psoriasis. Depression and QoL improved significantly, BDNF and anxiety scores did not change. Altered pro-BDNF and proBDNF/BDNF ratios may have a role in depression pathogenesis in psoriasis. Antipsoriatic treatment causes improvement in depression, QoL, and reduction of proBDNF and proBDNF/BDNF ratios. Effective disease control may reverse dysregulated neurotrophin pathways and its consequences like depression.

No association of both serum pro-brain-derived neurotrophic factor (proBDNF) and BDNF concentrations with depressive state in community-dwelling elderly people.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is involved in emotional and cognitive function. Low-BDNF levels occur in patients with depression, while proBDNF, a precursor of BDNF with the opposite physiological function, increases in major depression. However, it is unclear whether BDNF and proBDNF are associated with depression in the elderly. The present study aimed to investigate whether serum proBDNF and BDNF are associated with depressive state in community-dwelling elderly people. METHODS: This was a cross-sectional study conducted in Kurogawa-cho Imari, Saga Prefecture, Japan, in people aged ≥65 years. Depressive state was assessed using the Geriatric Depression Scale-Short Form (Japanese version) (GDS). Of the 274 patients who undertook the GDS, those with a medical history affecting cognitive function were excluded, as were those with Mini-Mental State Examination score ≥ 24 or a Clinical Dementia Rating < 0.5. Further, we used delayed recall of 'logical memory A' from the Wechsler Memory Scale-Revised (LMII-DR) for memory assessment. RESULTS: The final sample consisted of 155 individuals (mean age 75.4 ± 6.8 years; 55 men, mean age 74.8 ± 5.9 years; 100 women, mean age 76.3 ± 7.1 years). In the GDS, 139 participants showed a normal score (0-4) and 16 showed depressive tendencies or depression (score: ≥ 5). After examining confounders of the GDS, logistic regression using categorical covariates showed a negative significant difference between depressive state and serum BDNF in the low-BDNF group only, with a positive correlation in the trend test. None of the analyses showed any association between GDS and proBDNF levels. CONCLUSION: ProBDNF and BDNF levels seemed not to be associated with depressive state in community-dwelling elderly people.

Up-regulation of the p75 neurotrophin receptor is an essential mechanism for HIV-gp120 mediated synaptic loss in the striatum.

Reduced synaptodendritic complexity appears to be a key feature in human immunodeficiency virus (HIV)-associated neurological disorder (HAND). Viral proteins, and in particular the envelope protein gp120, play a role in the pathology of synapses. Gp120 has been shown to increase both in vitro and in vivo the proneurotrophin brain-derived neurotrophic factor, which promotes synaptic simplification through the activation of the p75 neurotrophin receptor (p75NTR). To provide evidence that p75NTR plays a role in gp120-mediated loss of synapses in vivo, we intercrossed gp120tg mice with p75NTR null mice and used molecular, histological and behavioral analyses to establish a link between p75NTR and gp120-mediated synaptic simplification. Synaptosomes obtained from the striatum of gp120tg mice exhibited a significant increase in p75NTR levels concomitantly to a decrease in synaptic markers such as TrkB and PSD95. Analysis of striatal dendritic spines by Golgi staining revealed that gp120tg mice display a reduced proportion of mushroom-type spines in addition to fewer spines overall, when compared to wild type or gp120tg lacking one or two p75NTR alleles. Moreover, removal of one p75NTR allele in gp120 transgenic mice abolished the gp120-driven impairment on a task of striatal-dependent motor learning. These data indicate that p75NTR could be a key player in HIV-mediated synaptic simplification in the striatum.

Investigation of Mature BDNF and proBDNF Signaling in a Rat Photothrombotic Ischemic Model.

Treatment with mature brain-derived neurotrophic factor (mBDNF) promotes functional recovery after ischemia in animal trials but the possible role of its precursor protein proBDNF and its receptors or the factors responsible for the conversion of proBDNF to mBDNF in ischemic stroke are not known. The main aim of this study was to characterize the time-dependent expression of genes and/or proteins related to BDNF processing and signaling after ischemia as well as the sensorimotor behavioral dysfunction in a photothrombotic ischemic model in rats. Characterization of different genes and proteins related to BDNF processing and signaling was performed using qPCR, immunoblotting and enzyme-linked immunosorbent assays. We showed in this study that some sensory and motor functional deficiencies appeared in the ischemic group at day 1 and persisted until day 14. Most changes in gene expression of BDNF and its processing enzymes occurred within the first 24 h in the ipsilateral cortex, but not in the contralateral cortex. At the protein level, proBDNF expression was increased at 6 h, mBDNF expression was increased between 15 h and 1 day while p75 receptor protein expression was increased between 6 h and 3 days in the ipsilateral cortex, but not in the contralateral cortex. Therefore, cerebral ischemia in rats led to the up-regulation of genes and/or proteins of BDNF, proBDNF and their processing enzymes and receptors in a time-dependent manner. We propose that the balance between BDNF and proBDNF and their associated proteins may play an important role in the pathogenesis and recovery from ischemia.

Growth factors and hormones pro-peptides: the unexpected adventures of the BDNF prodomain.

Most growth factors and hormones are synthesized as pre-pro-proteins which are processed to the biologically active mature protein. The pre- and prodomains are cleaved from the precursor protein in the secretory pathway or, in some cases, extracellularly. The canonical functions of these prodomains are to assist in folding and stabilization of the mature domain, to direct intra and extracellular localization, to facilitate storage, and to regulate bioavailability of their mature counterpart. Recently, exciting evidence has revealed that prodomains of certain growth factors, after cleaved from the precursor pro-protein, can act as independent active signaling molecules. In this review, we discuss the various classical functions of prodomains, and the biological consequences of these pro-peptides acting as ligands. We will focus our attention on the brain-derived neurotrophic factor prodomain (pBDNF), which has been recently described as a novel secreted ligand influencing neuronal morphology and physiology.

Region-specific expression of precursor and mature brain-derived neurotrophic factors after chronic alcohol exposure.

BACKGROUND: Alcohol abuse is a serious health problem worldwide that causes a variety of physical and mental disorders. Research has shown that the brain-derived neurotrophic factor (BDNF) plays an important role in alcohol addiction. The BDNF precursor (proBDNF) exhibits different actions than BDNF through separate receptors and pathways in the central nervous system. However, the effects of proBDNF and BDNF in alcohol addiction are not fully known. OBJECTIVES: The objective was to identify the expression patterns and effects of proBDNF and BDNF after chronic alcohol exposure. METHODS: A total of 40 male adult mice were studied. A mouse psychomotor sensitization (PS) model was established to explore the effects of BDNF and proBDNF treatment following chronic alcohol exposure. Reverse transcription PCR (RT-PCR) was performed to measure mRNA levels for BDNF, TrkB, P75NTR, and sortilin in the prefrontal cortex, hippocampus, and dorsal striatum of Kunming mice after chronic alcohol exposure. RESULTS: In Kunming mice, chronic alcohol exposure up-regulated BDNF and TrkB mRNA levels in the prefrontal cortex, but decreased sortilin and P75 mRNA levels in the dorsal striatum. No changes in mRNA levels were found in other measured brain regions in the alcohol and control groups. CONCLUSION: Chronic alcohol exposure induced the region-specific expression of BDNF and proBDNF and their respective receptors in the brain. These results suggest that BDNF and proBDNF signaling pathways may play major roles in alcohol preference and addiction.

[The Effects of Chronic Alcoholization on the Expression of Brain-Derived Neurotrophic Factor and Its Receptors in the Brains of Mice Genetically Predisposed to Depressive-Like Behavior].

Brain-derived neurotropic factor (BDNF) plays an important role in mechanisms of depression. Precursor protein of this factor (proBDNF) can initiate apoptosis in the brain, while the mature form of BDNF is involved in neurogenesis. It is known that chronic alcoholization leads to the activation of apoptotic processes, neurodegeneration, brain injury, and cognitive dysfunction. In this work, we have studied the influence of long-term ethanol exposure on the proBDNF and BDNF protein levels, as well as on the expression of genes that encode these proteins in the brain structures of ASC mice with genetic predisposition to depressive-like behavior and in mice from parental nondepressive CBA strain. It was shown that chronic alcoholization results in a reduction of the BDNF level in the hippocampus and an increase in the amount of TrkB and p75 receptors in the frontal cortex of nondepressive CBA mice. At the same time, the long-term alcoholization of depressive ASC mice results in an increase of the proBDNF level in the frontal cortex and a reduction in the p75 protein level in the hippocampus. It has also been shown that, in depressive ASC mice, proBDNF and BDNF levels are significantly lower in the hippocampus and the frontal cortex compared with nondepressive CBA strain. However, no significant differences in the expression of genes encoding the studied proteins were observed. Thus, changes in the expression patterns of proBDNF, BDNF, and their receptors under the influence of alcoholization in the depressive ASC strain and nondepressive CBA strain mice are different.

proBDNF and p75NTR Control Excitability and Persistent Firing of Cortical Pyramidal Neurons.

Persistent firing of entorhinal cortex (EC) pyramidal neurons is a key component of working and spatial memory. We report here that a pro-brain-derived neurotrophic factor (proBDNF)-dependent p75NTR signaling pathway plays a major role in excitability and persistent activity of pyramidal neurons in layer V of the EC. Using electrophysiological recordings, we show that proBDNF suppresses persistent firing in entorhinal slices from wild-type mice but not from p75NTR-null mice. Conversely, function-blocking proBDNF antibodies enhance excitability of pyramidal neurons and facilitate their persistent firing, and acute exposure to function-blocking p75NTR antibodies results in enhanced firing activity of pyramidal neurons. Genetic deletion of p75NTR specifically in neurons or during adulthood also induces enhanced excitability and persistent activity, indicating that the proBDNF-p75NTR signaling cascade functions within adult neurons to inhibit pyramidal activity. Phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive transient receptor potential canonical channels play a critical role in mediating persistent firing in the EC and we hypothesized that proBDNF-dependent p75NTR activation regulates PIP2 levels. Accordingly, proBDNF decreases cholinergic calcium responses in cortical neurons and affects carbachol-induced depletion of PIP2. Further, we show that the modulation of persistent firing by proBDNF relies on a p75NTR-Rac1-PI4K pathway. The hypothesis that proBDNF and p75NTR maintain network homeostasis in the adult CNS was tested in vivo and we report that p75NTR-null mice show improvements in working memory but also display an increased propensity for severe seizures. We propose that the proBDNF-p75NTR axis controls pyramidal neuron excitability and persistent activity to balance EC performance with the risk of runaway activity. SIGNIFICANCE STATEMENT: Persistent firing of entorhinal cortex (EC) pyramidal neurons is required for working memory. We report here that pro-brain-derived neurotrophic factor (proBDNF) activates p75NTR to induce a Rac1-dependent and phosphatidylinositol 4,5-bisphosphate-dependent signaling cascade that suppresses persistent activity. Conversely, using loss-of-function approaches, we find that endogenous proBDNF or p75NTR activation strongly decreases pyramidal neuron excitability and persistent firing, suggesting that a physiological role of this proBDNF-p75NTR cascade may be to regulate working memory in vivo. Consistent with this, mice rendered null for p75NTR during adulthood show improvements in working memory but also display an increased propensity for severe seizures. We propose that by attenuating EC network performance, the proBDNF-p75NTR signaling cascade reduces the probability of epileptogenesis.

Nerve growth factor (NGF) and pro-NGF increase low-density lipoprotein (LDL) receptors in neuronal cells partly by different mechanisms: role of LDL in neurite outgrowth.

Low-density lipoprotein receptors (LDLRs) mediate the uptake of lipoprotein particles into cells, as studied mainly in peripheral tissues. Here, we show that nerve growth factor (NGF) increases LDLR levels in PC6.3 cells and in cultured septal neurons from embryonic rat brain. Study of the mechanisms showed that NGF enhanced transcription of the LDLR gene, acting mainly via Tropomyosin receptor kinase A receptors. Simvastatin, a cholesterol-lowering drug, also increased the LDLR expression in PC6.3 cells. In addition, pro-NGF and pro-brain-derived neurotrophic factor, acting via the p75 neurotrophin receptor (p75NTR) also increased LDLRs. We further observed that Myosin Regulatory Light Chain-Interacting Protein/Inducible Degrader of the LDLR (Mylip/Idol) was down-regulated by pro-NGF, whereas the other LDLR regulator, proprotein convertase subtilisin kexin 9 (PCSK9) was not significantly changed. On the functional side, NGF and pro-NGF increased lipoprotein uptake by neuronal cells as shown using diacetyl-labeled LDL. The addition of serum-derived lipoprotein particles in conjunction with NGF or simvastatin enhanced neurite outgrowth. Collectively, these results show that NGF and simvastatin are able to stimulate lipoprotein uptake by neurons with a positive effect on neurite outgrowth. Increases in LDLRs and lipoprotein particles in neurons could play a functional role during brain development, in neuroregeneration and after brain injuries. Nerve growth factor (NGF) and pro-NGF induce the expression of low-density lipoprotein receptors (LDLRs) in neuronal cells leading to increased LDLR levels. Pro-NGF also down-regulated myosin regulatory light chain-interacting protein/inducible degrader of the LDLR (Mylip/Idol) that is involved in the degradation of LDLRs. NGF acts mainly via Tropomyosin receptor kinase A (TrkA) receptors, whereas pro-NGF stimulates p75 neurotrophin receptor (p75NTR). Elevated LDLRs upon NGF and pro-NGF treatments enhanced lipoprotein uptake by neurons. Addition of LDL particles further led to the stimulation of neurite outgrowth in PC6.3 cells after NGF or simvastatin treatments, suggesting a stimulatory role of lipoproteins on neuronal differentiation. In contrast, pro-NGF had no effect on neurite outgrowth either in the absence or presence of LDL particles. The precise mechanisms by which increased lipoproteins uptake can affect neurite outgrowth warrant further studies.

Activation of a synapse weakening pathway by human Val66 but not Met66 pro-brain-derived neurotrophic factor (proBDNF).

This study describes a fundamental functional difference between the two main polymorphisms of the pro-form of brain-derived neurotrophic factor (proBDNF), providing an explanation as to why these forms have such different age-related neurological outcomes. Healthy young carriers of the Met66 form (present in ∼30% Caucasians) have reduced hippocampal volume and impaired hippocampal-dependent memory function, yet the same polymorphic population shows enhanced cognitive recovery after traumatic brain injury, delayed cognitive dysfunction during aging, and lower risk of late-onset Alzheimer's disease (AD) compared to those with the more common Val66 polymorphism. To examine the differences between the protein polymorphisms in structure, kinetics of binding to proBDNF receptors and in vitro function, we generated purified cleavage-resistant human variants. Intriguingly, we found no statistical differences in those characteristics. As anticipated, exogenous application of proBDNF Val66 to rat hippocampal slices dysregulated synaptic plasticity, inhibiting long-term potentiation (LTP) and facilitating long-term depression (LTD). We subsequently observed that this occurred via the glycogen synthase kinase 3ß (GSK3ß) activation pathway. However, surprisingly, we found that Met66 had no such effects on either LTP or LTD. These novel findings suggest that, unlike Val66, the Met66 variant does not facilitate synapse weakening signaling, perhaps accounting for its protective effects with aging.

Regional differences in the expression of brain-derived neurotrophic factor (BDNF) pro-peptide, proBDNF and preproBDNF in the brain confer stress resilience.

Using learned helplessness (LH) model of depression, we measured protein expression of brain-derived neurotrophic factor (BDNF) pro-peptide, BDNF precursors (proBDNF and preproBDNF) in the brain regions of LH (susceptible) and non-LH rats (resilience). Expression of preproBDNF, proBDNF and BDNF pro-peptide in the medial prefrontal cortex of LH rats, but not non-LH rats, was significantly higher than control rats, although expression of these proteins in the nucleus accumbens of LH rats was significantly lower than control rats. This study suggests that regional differences in conversion of BDNF precursors into BDNF and BDNF pro-peptide by proteolytic cleavage may contribute to stress resilience.

Protective effects of polysialic acid on proteolytic cleavage of FGF2 and proBDNF/BDNF.

Polysialic acid (polySia) is a linear polymer of sialic acid that modifies neural cell adhesion molecule (NCAM) in the vertebrate brain. PolySia is a large and exclusive molecule that functions as a negative regulator of cell-cell interactions. Recently, we demonstrated that polySia can specifically bind fibroblast growth factor 2 (FGF2) and BDNF; however, the protective effects of polySia on the proteolytic cleavage of these proteins remain unknown, although heparin/heparan sulfate has been shown to impair the cleavage of FGF2 by trypsin. Here, we analyzed the protective effects of polySia on the proteolytic cleavage of FGF2 and proBDNF/BDNF. We found that polySia protected intact FGF2 from tryptic activity via the specific binding of extended polySia chains on NCAM to FGF2. Oligo/polySia also functioned to impair the processing of proBDNF by plasmin via binding of oligo/polySia chains on NCAM. In addition, the polySia structure synthesized by mutated polysialyltransferase, ST8SIA2/STX(SNP7), which was previously identified from a schizophrenia patient, was impaired for these functions compared with polySia produced by normal ST8SIA2. Taken together, these data suggest that the protective effects of polySia toward FGF2 and proBDNF may be involved in the regulation of the concentrations of these neurologically active molecules.

Development of mature BDNF-specific sandwich ELISA.

Mature brain-derived neurotrophic factor (mBDNF) plays a vital role in the nervous system, whereas proBDNF elicits neurodegeneration and neuronal apoptosis. Although current enzyme-linked immunosorbent assay (ELISA) has been widely used to measure BDNF levels, it cannot differentiate mBDNF from proBDNF. As the function of proBDNF differs from mBDNF, it is necessary to establish an ELISA assay specific for the detection of mBDNF. Therefore, we aimed to establish a new mBDNF-specific sandwich ELISA. In this study, we have screened and found a combination of antibodies for a sandwich ELISA. A monoclonal antibody and sheep anti-BDNF were chosen as capture and detection antibody for sandwich ELISA respectively. The new ELISA showed no cross-reactivity to human recombinant NT-3, NT-4, nerve growth factor and negligible cross-reactivity (0.99-4.99%) for proBDNF compared to commercial ELISA kits (33.18-91.09%). The application of the new mBDNF ELISA was shown through the measurement of mBDNF levels in different brain regions of rats and in the brain of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1)(-/-) and WT mice and compared to western blot. Overall, this new ELISA will be useful for the measurement of mBDNF levels with high specificity. As the function of proBDNF differs from mBDNF (mature BDNF), it is necessary to establish an ELISA specific for the detection of mBDNF. Here, we present a novel sandwich ELISA which detects mBDNF with high specificity. This new ELISA will be useful for the measurement of mBDNF levels with high specificity in various human and animal tissues. proBDNF, precursor of BDNF; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; NGF, nerve growth factor.