Human BDNF Research Articles

Correlation between human BDNF rs6265 gene polymorphism and type-2 diabetes and diabetic peripheral neuropathy

Background & objective: Diabetes mellitus is quite common and globally prevalent condition affecting patients’ quality of life. Patients who have type 2 diabetes mellitus (T2DM), are more likely to get diabetic peripheral neuropathy (DPN), which may affect feet, legs, hand, and arm. A BDNF gene rs6265 polymorphism in humans results in the substitution of valine with methionine at codon 66 (Val66Met). We aimed to find the possible link between human BDNF rs6265 gene polymorphism and T2DM with and without peripheral neuropathy and compare it with healthy subjects in Kirkuk City, Iraq. Methodology: One hundred subjects were chosen to participate in this research, aged from 35 to 75 y and divided into three groups: 35 DPN, 35 T2DM patients, and 30 healthy controls were selected randomly for BDNF gene rs6265 SNP screening by using conventional PCR with specific sets of primers. Products of PCR for patients and control groups were run on the gel electrophoresis to detect the SNP rs6265 fragment. A nerve-conducting study was used to examine DPN. Results: The observed results demonstrated the presence of two types of alleles identified as genotypic variants (GG and GA) among all participants in this investigation. By applying the Hardy-Weinberg Equilibrium principle (HWE) for both patient and control groups, the results proved that there was a statistically significant variance (P < 0.05) in the genotypic frequencies between each of the groups that had been studied. The wild homozygous GG genotype in type 2 diabetic without neuropathy, with DPN, and healthy control group were 51.4%, 40% and 80% respectively. The heterozygous GA genotype were 48.6%, 60% and 20%, respectively in the three groups. Conclusion: The present study showed that the BDNF (Val66Met) rs6265 polymorphism is associated with type 2 diabetes mellitus and diabetic peripheral neuropathy in heterozygous allele G/A (Met/Met) and homozygous allele GG (Val/Val) genotype. Also, the current study found the absence of the mutant genotype AA, possibly due to the evidence that the distribution of the BDNF polymorphisms varies widely among different ethnic groups. Abbreviations: BDNF - Brain-derived neurotrophic factor; DPN - Diabetic peripheral neuropathy Keywords: Type2 diabetes mellitus; Diabetic peripheral neuropathy; Brain-Derived Neurotrophic Factor; BDNF rs6265 SNP; BDNF Val66Met polymorphism. Citation: Hamid R, Al-Wasiti E, Jabbar AM. Correlation between human BDNF rs6265 gene polymorphism and type-2 diabetes and diabetic peripheral neuropathy. Anaesth. pain intensive care 2024;28(4):752−756. DOI: 10.35975/apic.v28i4.2517 Received: March 08, 2024; Reviewed: April 20, 2024; Accepted: April 28, 2024

Evaluation of dihydrotestosterone and dihydroprogesterone levels and gene expression of genes involved in neurosteroidogenesis in the SH-SY5Y Alzheimer disease cell model.

Alzheimer's disease (AD) is the most common form of dementia worldwide. This study investigated the effects of lipopolysaccharide on neurosteroidogenesis and its relationship to growth and differentiation using SH-SY5Y cells. In this study, we used the MTT assay to assess the impact of LPS on SH-SY5Y cell viability. We also evaluated apoptotic effects using FITC Annexin V staining to detect phosphatidylserine in the cell membrane. To identify gene expression related to human neurogenesis, we utilized the RT2 Profiler TM PCR array human neurogenesis PAHS-404Z. Our study found that LPS had an IC50 level of 0.25 μg/mL on the SH-SY5Y cell line after 48 h. We observed Aβ deposition in SH-SY5Y cells treated with LPS, and a decrease in DHT and DHP levels in the cells. Our analysis showed that the total rate of apoptosis varied with LPS dilution: 4.6% at 0.1 μg/mL, 10.5% at 10 μg/mL, and 44.1% at 50 μg/mL. We also observed an increase in the expression of several genes involved in human neurogenesis, including ASCL1, BCL2, BDNF, CDK5R1, CDK5RAP2, CREB1, DRD2, HES1, HEYL, NOTCH1, STAT3, and TGFB1, after treatment with LPS at 10 μg/mL and 50 μg/mL. LPS at 50 μg/mL increased the expression of FLNA and NEUROG2, as well as the other genes mentioned. Our study showed that LPS treatment altered the expression of human neurogenesis genes and decreased DHT and DHP levels in SH-SY5Y cells. These findings suggest that targeting LPS, DHT, and DHP could be potential therapeutic strategies to treat AD or improve its symptoms.

Anxiety Disorders and Prediction of Their Development in Patients with Hypothyroidism and Autoimmune Thyroiditis

Objective: Since the association between thyroid dysfunction and neuropsychiatric disorders has been known for many years, it is important to analyze the associations of the BDNF gene polymorphism (rs6265), the VDR gene polymorphism (rs2228570), and the NMDA gene polymorphism (rs4880213) with the anxiety in patients with autoimmune thyroiditis and hypothyroidism in the Western Ukrainian population and predict the development of anxiety disorders in these patients.
 Methods: The study involved a total of 153 patients with various forms of thyroid pathology. BDNF levels in the sera of the patients and healthy individuals were quantified using an enzyme-linked immunosorbent assay with a highly sensitive Human BDNF ELISA Kit (Elabscience®, United States, Catalog No: E-EL-H0010) on E.I.A. Reader Sirio S (Seac, Italy). Genotyping of the VDR (rs2228570), BDNF (rs6265), and NMDA (rs4880213) gene polymorphism using TaqMan probes and TaqMan Genotyping Master Mix (4371355) on CFX96™Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., USA). Polymerase chain reaction (PCR) for TaqMan genotyping was carried out according to the kit instructions (Applied Biosystems, USA).
 Results: According to the data obtained when comparing the presence of anxiety on the Hamilton scale on SNP statistically significant differences were revealed depending on BDNF gene polymorphism (rs6265) (p < 0.001). When comparing the presence of anxiety on the Hamilton scale on SNP depending on VDR gene polymorphism (rs2228570), NMDA gene polymorphism (rs4880213) no statistically significant differences were revealed (p = 0.782, p = 0.677 respectively) We identified an inverse strong correlation between the presence of anxiety on Hamilton scale and BDNF, 25-OH Vitamin D levels, and fT4 in the blood (p <0.001) and a direct moderate correlation between the presence of anxiety on Hamilton scale and TSH, GRIN2B, and anti-TPO (p <0.001).
 Conclusion: Indicators such as BDNF, GRIN2B, fT4, anti-TG, and 25-OH levels of vitamin D are prognostically significant risk criteria for anxiety.

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.

Prediction of the Development of Depression in Patients with Autoimmune Thyroiditis and Hypothyroidism

BACKGROUND: Hypothyroidism is frequently accompanied by depression symptoms, whereas depression is considered the most common mental disorder. AIM: It is crucial to analyze the associations of the brain neurotrophic factor (BDNF) gene polymorphism (rs6265), the VDR gene polymorphism (rs2228570), and the NMDA gene polymorphism (rs4880213) with the depression in patients with autoimmune thyroiditis and hypothyroidism in the Western Ukrainian population and predict the development of depressive disorders in these patients. METHODS: The study involved a total of 153 patients with various forms of thyroid pathology. BDNF levels in the sera of the patients and healthy individuals were quantified using enzyme-linked immunosorbent assay with highly sensitive Human BDNF ELISA Kit (Elabscience®, United States, Catalog No: E-EL-H0010) on E.I.A. Reader Sirio S (Seac, Italy). Genotyping of the VDR (rs2228570), BDNF (rs6265), and NMDA (rs4880213) gene polymorphism using TaqMan probes and TaqMan Genotyping Master Mix (4371355) on CFX96™Real-Time Polymerase chain reaction (PCR) Detection System (Bio-Rad Laboratories, Inc., USA). PCR for TaqMan genotyping was carried out according to the kit instructions (Applied Biosystems, USA). We used the Student’s t-test, ANOVA, Pearson’s Chi-square test, ROC-analysis, odds ratio test, relative odds ratio test. The odds ratio and 95% confidence interval were computed by binary logistic regression. RESULTS: When comparing the presence of depression on the Hamilton scale, statistically significant differences were found depending on BDNF gene polymorphism (rs6265) (p < 0.001), and non-statistically noticeable differences were detected depending on the VDR gene polymorphism (rs2228570) and NMDA gene polymorphism (rs4880213). Our study revealed a marked inverse connection between depression and BDNF levels (p < 0.001) and a reverse moderate correlation between depression and fT4 (p < 0.001) and 25-OH Vitamin D levels (p < 0.001). In addition, we identified a direct moderate correlation between pronounced levels of depression and thyroid-stimulating hormone (TSH) in the blood (p < 0.001). CONCLUSIONS: Indicators such as BDNF, TSH, fT4, anti-TPO, and 25-OH Vitamin D levels were found to be prognostically significant criteria for the risk of developing depression.

Temporal dissection of Rai1 function reveals brain-derived neurotrophic factor as a potential therapeutic target for Smith-Magenis syndrome.

Haploinsufficiency of retinoic acid-induced 1 (RAI1) is responsible for Smith-Magenis syndrome (SMS), a childhood neurodevelopmental disorder associated with hyperphagia, obesity and autistic features. We previously showed that constitutive inactivation of one or both copies of Rai1 in the germline or developing brain induces SMS-like neurobehavioral deficits and obesity in mice. By contrast, the postnatal function of Rai1 is unclear. Here, we globally deleted one or both copies of Rai1 during two postnatal developmental windows by generating an inducible Rai1 knockout mouse model. We found that delayed Rai1 deletion at 3 or 8weeks of age had no effect on neurobehavioral functions but resulted in adult-onset obesity and decreased expression of brain-derived neurotrophic factor (Bdnf) in the hypothalamus. Remarkably, genetic overexpression of human Bdnf in Rai1 heterozygous mice reversed SMS-like obesity, hyperphagia, metabolic syndrome-like features and hyposociability. Increasing Bdnf signaling in the paraventricular nucleus of the hypothalamus or the ventromedial nucleus of the hypothalamus was sufficient to mediate the anti-obesity effect. Our work identifies the function of Rai1 in different temporal windows after birth and provides in vivo evidence that increasing Bdnf signaling is therapeutically effective in a preclinical mouse model of SMS.

Roflumilast Ameliorates Isoflurane-Induced Inflammation in Astrocytes via the CREB/BDNF Signaling Pathway.

Background and purpose: Astrocyte-mediated neuroinflammation plays an important role in anesthetic isoflurane-induced cognitive impairment. Roflumilast, a selective inhibitor of phosphodiesterase-4 (PDE-4) used for the treatment of chronic obstructive pulmonary disease (COPD), has displayed a wide range of anti-inflammatory capacity in different types of cells and tissues. In the current study, we aimed to investigate whether roflumilast possesses a protective effect against isoflurane-induced insults in mouse primary astrocytes. Methods: Primary astrocytes were isolated from the cerebral cortices of immature rats. The production of NO was determined using DAF-FM DA staining assay. QRT-PCR and western blot were used to evaluate the expression levels of iNOS, COX-2, and BDNF in the astrocytes treated with different therapies. The gene expressions and concentrations of IL-6 and MCP-1 released by the astrocytes were detected using qRT-PCR and ELISA, respectively. The expression levels of phosphorylated CREB and PGE2 were determined using western blot and ELISA, respectively. H89 was introduced to evaluate the function of CREB. Recombinant human BDNF and ANA-12 were used to verify the role of BDNF. Results: The upregulated iNOS, excessive production of NO, IL-6, and MCP-1, and activated COX-2/PGE2 signaling pathways in the astrocytes induced by isoflurane were significantly reversed by the introduction of roflumilast, in a dose-dependent manner. Subsequently, we found that BDNF could be upregulated by roflumilast, which was verified to be related to the activation of CREB and blocked by H89 (a CREB inhibitor). In addition, the COX-2/PGE2 signaling pathway activated by isoflurane can be inactivated by recombinant human BDNF. Finally, the regulatory effect of roflumilast against the isoflurane-activated COX-2/PGE2 signaling pathway was significantly blocked by ANA-12, which is a BDNF inhibitor. Conclusion: Roflumilast might ameliorate isoflurane-induced inflammation in astrocytes via the CREB/BDNF signaling pathway.

Loss of STEP61 couples disinhibition to N-methyl-d-aspartate receptor potentiation in rodent and human spinal pain processing.

Dysregulated excitability within the spinal dorsal horn is a critical mediator of chronic pain. In the rodent nerve injury model of neuropathic pain, BDNF-mediated loss of inhibition (disinhibition) gates the potentiation of excitatory GluN2B N-methyl-d-aspartate receptor (NMDAR) responses at lamina I dorsal horn synapses. However, the centrality of this mechanism across pain states and species, as well as the molecular linker involved, remain unknown. Here, we show that KCC2-dependent disinhibition is coupled to increased GluN2B-mediated synaptic NMDAR responses in a rodent model of inflammatory pain, with an associated downregulation of the tyrosine phosphatase STEP61. The decreased activity of STEP61 is both necessary and sufficient to prime subsequent phosphorylation and potentiation of GluN2B NMDAR by BDNF at lamina I synapses. Blocking disinhibition reversed the downregulation of STEP61 as well as inflammation-mediated behavioural hypersensitivity. For the first time, we characterize GluN2B-mediated NMDAR responses at human lamina I synapses and show that a human ex vivo BDNF model of pathological pain processing downregulates KCC2 and STEP61 and upregulates phosphorylated GluN2B at dorsal horn synapses. Our results demonstrate that STEP61 is the molecular brake that is lost following KCC2-dependent disinhibition and that the decrease in STEP61 activity drives the potentiation of excitatory GluN2B NMDAR responses in rodent and human models of pathological pain. The ex vivo human BDNF model may thus form a translational bridge between rodents and humans for identification and validation of novel molecular pain targets.

An increase in plasma brain derived neurotrophic factor levels is related to n-3 polyunsaturated fatty acid efficacy in first episode schizophrenia: secondary outcome analysis of the OFFER randomized clinical trial

RationaleN−3 polyunsaturated fatty acids (n−3 PUFA) influence multiple biochemical mechanisms postulated in the pathogenesis of schizophrenia that may influence BDNF synthesis.ObjectivesA randomized placebo-controlled study was designed to compare the efficacy of a 26-week intervention composed of either 2.2 g/day of n−3 PUFA or olive oil placebo, with regard to symptom severity in first-episode schizophrenia patients. The secondary outcome measure of the study was to describe the association between n−3 PUFA clinical effect and changes in peripheral BDNF levels.MethodsSeventy-one patients aged 16–35 were enrolled in the study and randomly assigned to the following study arms: 36 to the EPA + DHA group and 35 to the placebo group. Plasma BDNF levels were assessed three times, at baseline and at weeks 8 and 26 of the intervention. BDNF levels were determined in plasma samples using Quantikine Human BDNF ELISA kit. Plasma BDNF level changes were further correlated with changes in the severity of symptoms in different clinical domains.ResultsA significantly greater increase in plasma BDNF levels was observed in the intervention compared to the placebo group (Cohen’s d = 1.54). Changes of BDNF levels inversely correlated with change in depressive symptoms assessed using the Calgary Depression Rating Scale in Schizophrenia (Pearson’s r = − 0.195; p = 0.018).ConclusionsThe efficacy of a six-month intervention with n−3 PUFA observed in first-episode schizophrenia may be related to an increase in BDNF levels, which may be triggered by the activation of intracellular signaling pathways including transcription factors such as cAMP-reactive element binding protein.

Comprehensive In silico Analysis of Single Nucleotide Polymorphisms (SNPs) in Human BDNF Gene

Since a number of single nucleotide polymorphisms (SNPs) are associated with the BDNF gene mutations involved in different diseases. Hence, it is important to find out the probable functional SNPs before planning a larger population study. So, to look out for the functional nsSNPs (non-synonymous single nucleotide polymorphisms) in BDNF gene, existing data present in the dbSNP database and different bioinformatics tools like SIFT, PolyPhen, nsSNPAnalyzer, F-SNP, I-mutant, Pymol were used for the analysis. The results showed that out of the total 2929 SNPs, 58 were nsSNPs (non-synonymous single nucleotide polymorphisms), 44 occurred in the mRNA 3′ UTR, 108 occurred in 5′ UTR region, 257 occurred in intronic regions and the rest were other types of SNPs. SIFT and PolyPhen programs predicted 3 out of 58 nsSNPs (rs8192466, rs1048218, and rs77787410) as not tolerable, deleterious and damaging. F-SNP revealed that the rs1048221, rs1048220, rs1048218, rs6265 and rs8192466 SNP in the non-synonymous coding region may have protein-coding and splicing regulator functions. PDBSum and UniProtKB predicted the number of protein structures, sharing 100% similarity with the BDNF amino acid sequence. Moreover, I-Mutant and nsSNPAnalyzer showed a decrease in stability for these nsSNPs upon mutation. Protein structural analysis with these amino acid variants was performed by using I-Mutant and PyMOL. This study suggested that three nsSNPs, rs8192466, rs1048218, and rs77787410 were identified as deleterious, thus destabilizing the protein stability, amino acid interactions, and hydrogen bond networks of the protein directly or indirectly. Hence, these SNPs can explain the functional deviation of protein to some extent. Keywords: BDNF, F-SNP, I-Mutant, nsSNPAnalyzer, PolyPhen, SIFT, SNP

Long noncoding RNA BDNF-AS is downregulated in cervical cancer and has anti-cancer functions by negatively associating with BDNF

PurposeWe investigated expression and mechanism long noncoding RNA BDNF-AS in human cervical cancer (CC). MethodsBDNF-AS expressions were examined by qPCR in CC cell lines and human CC tumors. CC cell lines, SiHa and DoTc2-4510 were transduced with lentivirus to ectopically overexpress BDNF-AS. Possible anti-cancer effects of BDNF overexpression were examined on CC in vitro proliferation and migration, and in vivo transplantation. Human BDNF gene expression was also examined in CC cell lines and tumors. In CC cells with overexpressed BDNF-AS, BDNF was upregulated to examine its direct effect in NDNF-AS-modulated CC proliferation and migration. ResultsBDNF was downregulated in both CC cells and human CC tumors. In CC cells, BDNF-AS overexpression is anti-cancer by inhibiting proliferation and migration in vitro, and transplantation in vivo. BDNF was inversely expressed as BDNF-AS in CC. Upregulation of BDNF in BDNF-AS-overexpressed CC cells reversed the anti-cancer effects of BDNF-AS. ConclusionBDNF-AS is downregulated in CC. Overexpressing BDNF-AS may inhibit CC, possibly through inverse regulation on BDNF.

The BDNF val-66-met Polymorphism Affects Neuronal Morphology and Synaptic Transmission in Cultured Hippocampal Neurons from Rett Syndrome Mice.

Brain-derived neurotrophic factor (Bdnf) has been implicated in several neurological disorders including Rett syndrome (RTT), an X-linked neurodevelopmental disorder caused by loss-of-function mutations in the transcriptional modulator methyl-CpG-binding protein 2 (MECP2). The human BDNF gene has a single nucleotide polymorphism (SNP)—a methionine (met) substitution for valine (val) at codon 66—that affects BDNF’s trafficking and activity-dependent release and results in cognitive dysfunction. Humans that are carriers of the met-BDNF allele have subclinical memory deficits and reduced hippocampal volume and activation. It is still unclear whether this BDNF SNP affects the clinical outcome of RTT individuals. To evaluate whether this BDNF SNP contributes to RTT pathophysiology, we examined the consequences of expression of either val-BDNF or met-BDNF on dendrite and dendritic spine morphology, and synaptic function in cultured hippocampal neurons from wildtype (WT) and Mecp2 knockout (KO) mice. Our findings revealed that met-BDNF does not increase dendritic growth and branching, dendritic spine density and individual spine volume, and the number of excitatory synapses in WT neurons, as val-BDNF does. Furthermore, met-BDNF reduces dendritic complexity, dendritic spine volume and quantal excitatory synaptic transmission in Mecp2 KO neurons. These results suggest that the val-BDNF variant contributes to RTT pathophysiology, and that BDNF-based therapies should take into consideration the BDNF genotype of the RTT individuals.

Candidate SNP markers of social dominance, which may affect the affinity of the TATA-binding protein for human gene promoters

The following heuristic hypothesis has been proposed: if an excess of a protein in several animal organs was experimentally identified as a physiological marker of increased aggressiveness, and if a single nucleotide polymorphism (SNP) can cause the overexpression of a human gene homologous to the animal gene encoding this protein, then this polymorphism can be a candidate SNP marker of social dominance. In turn, a deficient expression would correspond to subordinate behavior. Within this hypothesis, we analyzed 21 human genes, ADORA2A, BDNF, CC2D1A, CC2D1B, ESR2, FEV, FOS, GH1, GLTSCR2, GRIN1, HTR1B, HTR1A, HTR2A, HTR2C, LGI4, LEP, MAOA, SLC17A7, SLC6A3, SNCA, and TH, which determine the functions of the proteins known as the physiological markers of aggressive behavior in animals. These genes encode for hormones and their receptors, biosynthetic enzymes, receptors of neurotransmitters, and transcription and neurotrophic factors. These proteins have been postulated to play important roles in determining the hierarchical relationships in social animals. Using our previously developed Web-service SNP_TATA_Comparator (http://beehive.bionet.nsc.ru/cgi-bin/mgs/tatascan/start.pl), we analyzed 381 SNPs within the [–70;–20] region preceding the start of the protein-coding transcripts, obtained from the database dbSNP, v.147. This is the region for all the known TATA-binding protein (TBP) binding sites. As a result, we found 45 and 47 candidate SNP markers of dominance and subordination, respectively (e.g., rs373600960 and rs747572588). Within the proposed heuristic hypotheses and dbSNP database v.147, we found statistically significant (α 0.35). The proposed hypothesis, the candidate SNP markers obtained on its basis, and the observed regularities of the effects of their natural selection on the human genome are discussed in comparison with the published data with respect to whether these markers can have an effect on the expression of social dominance in human society. We conclude that our candidate SNPs, identified with a computational model, require further experimental verification.

A method for reproducible measurements of serum BDNF: comparison of the performance of six commercial assays.

Brain-Derived Neurotrophic Factor (BDNF) has attracted increasing interest as potential biomarker to support the diagnosis or monitor the efficacy of therapies in brain disorders. Circulating BDNF can be measured in serum, plasma or whole blood. However, the use of BDNF as biomarker is limited by the poor reproducibility of results, likely due to the variety of methods used for sample collection and BDNF analysis. To overcome these limitations, using sera from 40 healthy adults, we compared the performance of five ELISA kits (Aviscera-Bioscience, Biosensis, Millipore-ChemiKineTM, Promega-Emax®, R&D-System-Quantikine®) and one multiplexing assay (Millipore-Milliplex®). All kits showed 100% sample recovery and comparable range. However, they exhibited very different inter-assay variations from 5% to 20%. Inter-assay variations were higher than those declared by the manufacturers with only one exception which also had the best overall performance. Dot-blot analysis revealed that two kits selectively recognize mature BDNF, while the others reacted with both pro-BDNF and mature BDNF. In conclusion, we identified two assays to obtain reliable measurements of human serum BDNF, suitable for future clinical applications.

Alteration of the Centromedial Amygdala Glutamatergic Synapses by the BDNF Val66Met Polymorphism.

Fear expression is mediated by an activation of the centromedial amygdala (CEm), the major output nucleus of the amygdaloid complex. Consistently, fear extinction is associated with an increased synaptic inhibition as well as a suppression of the excitability of the CEm neurons. However, little is known about the role of CEm glutamatergic synapses in fear regulation and anxiety-like behaviors. The BDNF Val66Met, a single-nucleotide polymorphism in the human BDNF gene, impairs fear extinction and leads to anxiety-like symptoms. To determine whether the BDNF Val66Met polymorphism affects the CEm excitatory synapses, we examined basal glutamatergic synaptic transmission and plasticity in the CEm neurons of BDNF Val66Met knock-in (BDNF(Met/Met)) mice. The BDNF Val66Met single-nucleotide polymorphism exerted an opposite effect on non-NMDA and NMDA receptor transmission with a potentiation of the former and a suppression of the latter. In addition, the decay time of NMDA currents was decreased in BDNF(Met/Met) mice, suggesting a modification of NMDA receptor subunit composition. Unlike the wild-type mice that exhibited a potentiation of non-NMDA receptor transmission following fear conditioning and a depotentiation upon fear extinction, BDNF(Met/Met) mice failed to show this experience-dependent synaptic plasticity in the CEm neurons. Our results suggest that the elevated non-NMDA receptor transmission, the suppression of NMDA receptor transmission, and an impairment of synaptic plasticity in the CEm neurons might contribute to the fear extinction deficit and increased anxiety-like symptoms in BDNF Val66Met carriers.

Rescue of retinal function by BDNF in a mouse model of glaucoma.

Vision loss in glaucoma is caused by progressive dysfunction of retinal ganglion cells (RGCs) and optic nerve atrophy. Here, we investigated the effectiveness of BDNF treatment to preserve vision in a glaucoma experimental model. As an established experimental model, we used the DBA/2J mouse, which develops chronic intraocular pressure (IOP) elevation that mimics primary open-angle glaucoma (POAG). IOP was measured at different ages in DBA/2J mice. Visual function was monitored using the steady-state Pattern Electroretinogram (P-ERG) and visual cortical evoked potentials (VEP). RGC alterations were assessed using Brn3 immunolabeling, and confocal microscope analysis. Human recombinant BDNF was dissolved in physiological solution (0.9% NaCl); the effects of repeated intravitreal injections and topical eye BDNF applications were independently evaluated in DBA/2J mice with ocular hypertension. BDNF level was measured in retinal homogenate by ELISA and western blot. We found a progressive decline of P-ERG and VEP responses in DBA/2J mice between 4 and 7 months of age, in relationship with the development of ocular hypertension and the reduction of Brn3 immunopositive RGCs. Conversely, repeated intravitreal injections (BDNF concentration = 2 µg/µl, volume = 1 µl, for each injection; 1 injection every four days, three injections over two weeks) and topical eye application of BDNF eye-drops (12 µg/µl, 5 µl eye-drop every 48 h for two weeks) were able to rescue visual responses in 7 month DBA/2J mice. In particular, BDNF topical eye treatment recovered P-ERG and VEP impairment increasing the number of Brn3 immunopositive RGCs. We showed that BDNF effects were independent of IOP reduction. Thus, topical eye treatment with BDNF represents a promisingly safe and feasible strategy to preserve visual function and diminish RGC vulnerability to ocular hypertension.

Alteration in BDNF and its receptors, full-length and truncated TrkB and p75NTR following penetrating traumatic brain injury

The evidence that BDNF is involved in neuroprotection, neuronal repair and recovery after traumatic brain injury (TBI) is substantial. We have previously shown that the polymorphism of the human BDNF gene predicts cognitive recovery and outcome following penetrating TBI. The distribution of expression of BDNF and its receptors after penetrating TBI has not been investigated. In this study we examined the expression of these genes in a rat model of penetrating TBI. The injury is produced by a controlled penetration of a 2mm thick needle-shaped object, which is accelerated with a pellet from an air gun. We used in situ hybridization and investigated the mRNA expression of BDNF and its receptors: the full-length and the truncated TrkB and p75NTR, from 1 day to 8 weeks following penetrating TBI. In addition, the protein level of BDNF in frontal cortex and hippocampus was measured by reverse phase protein microarray (RPPM). The mRNA expression of BDNF and its receptors decreased in the hippocampus in the border zone ipsilateral to the injury while there was an increase in mRNA expression at the contralateral side. The increase in BDNF mRNA expression in the hippocampus was sustained for 2 weeks following injury, with the highest expression noted in the CA3 cell layer. Furthermore, the protein analysis by RPPM showed increased levels of BDNF in the frontal cortex and the hippocampus up to 2 weeks after TBI. At 8 weeks following injury there was an intense labeling of the truncated TrkB receptor and the p75NTR in the area surrounding the cavity. Our study is the first report on the expression of BDNF and its receptors following penetrating TBI and suggests that their expression is altered long after the acute phase of injury. Further studies are needed to investigate if the late expressions of these receptors are beneficial or deleterious. In either case it indicates the possibility to influence the recovery after brain injury during the chronic phase and the development of treatments that may improve the outcome of TBI patients.

TGF-Beta-Guided Inflammatory Signaling Originating in Visceral Adipose Tissue Is Intrinsically Involved in the Etiology of Depression in Obese Patients with Non-alcoholic Fatty Liver Disease (NAFLD)

Purpose: Recent studies have provided compelling evidence linking depression and cytokine imbalance. Numerous studies have shown that the visceral adipose tissue (VAT) of obese NAFLD patients is heavily involved in cytokine release, and particularly overall pathway commitment to Th1/Th2/Th17 type signaling. Our aim was to demonstrate a role for visceral adipose signaling, particularly of adipocytic release of TGF-b, one of the master regulators of Th1/Th2/Th17, in the cytokine imbalance associated with depression. Methods: Patients undergoing bariatric surgery for morbid obesity were included. Diagnosis of depression was based on clinical diagnosis or with documented use of anti-depressants. Diagnosis of NAFLD was based on liver biopsy. Visceral adipose tissue was collected at the time of bariatric surgery, and was used for gene expression analysis. 1 μg of RNA was extracted from frozen VAT of patients with liver biopsy-proven NAFLD, Bio-Rad's aurum total RNA fatty and fibrous tissue kit was used. cDNA was synthesized with the SABiosciences' RT2 first strand kit. cDNA samples were used in 10 μL q-RTPCR reactions. Frozen serum from patients were tested with the Biorad Bio-Plex Pro TGF-b1 and 17-plex cytokine assays. Serum levels of serotonin and BDNF were analyzed using the ALPCO human serotonin ELISA kit and the human BDNF quantikine ELISA kit. Group differences were assessed using Mann-Whitney tests and chi-square test. Co-expression patterns were analyzed using Spearman's rank-sum correlation. Results: A total of 241 biopsy-proven NAFLD patients (39.1% NASH, 20.8% with type 2 diabetes, age 43.61+/-11.41 years, BMI 46.39+/-10.91, depression/anxiety 42.6%) were assayed. In the group-wise comparison of VAT-derived mRNAs, TGFb1 (p<0.007) and UCP1 (p<0.004) transcripts were significantly higher in the depressed versus non-depressed cohort, whereas the levels of serum serotonin (p<0.00017), IL-1B (p<0.04), IL-7 (p<0.023), IL-13 (p<0.008), and G-CSF (p<0.034) were significantly lower. Analysis of correlations further confirmed these findings, and showed a positive association between serotonin levels and degree of ballooning in liver biopsies (r=0.14 p<0.03428). Interestingly, there was no correlation between serum levels of TGFb1, serotonin, or BDNF and depression/anxiety. Additionally, there was no correlation between serum levels of TGFb1 and TGFb1 receptor transcript levels in VAT, suggesting VAT as the site of origin for TGFb1 signal, rather than its target. Conclusion: Inflammatory signaling originating in VAT seems to be intrinsically involved in the etiology of depression, particularly in morbidly obese NAFLD patients. The molecular networks leading to these mood disorders need to be further elucidated.

Therapeutic Effect of BDNF-Overexpressing Human Neural Stem Cells (HB1.F3.BDNF) in a Rodent Model of Middle Cerebral Artery Occlusion

Ischemic stroke mainly caused by middle cerebral artery occlusion (MCAo) represents the major type of stroke; however, there are still very limited therapeutic options for the stroke-damaged patients. In this study, we evaluated the neurogenic and therapeutic potentials of human neural stem cells (NSCs) overexpressing brain-derived neurotrophic factor (HB1.F3.BDNF) following transplantation into a rodent model of MCAo. F3.BDNF human NSCs (F3.BDNF) were transplanted into the contralateral side of striatum at 7 days after MCAo, and the transplanted animals were monitored up to 8 weeks using animal MRI and various behavioral tests before they were sacrificed for immunohistochemical analysis. Interestingly, animal MRI results indicate that the majority of contralaterally transplanted neural stem cells were migrated to the peri-infarct area, showing a pathotropism. Transplanted animals exhibited significant behavioral improvements in stepping, rotarod, and modified neurological severity score (mNSS) tests. We also found that the transplanted human cells were colocalized with nestin, DCX, MAP2, DARPP-32, TH, GAD65/67-positive cells, of which results can be correlated with neural regeneration and behavioral recovery in the transplanted animals. More importantly, we were able to detect high levels of human BDNF protein expression, presumably derived from the transplanted F3.BDNF. Taken together, these results provide strong evidence that human neural stem cells (F3.BDNF) are effective in treating stroke animal models.

Abstract 89: Bdnf val66met polymorphism is Associated with Enhanced Motor Recovery Following Stroke

Background and Purpose: Genetics is one of several key factors that influence stroke recovery. A single nucleotide polymorphism (SNP) of the bdnf gene (Val66Met) is common in humans, ranging from 20-30% in Caucasians up to 70% in the Asian population. Previous studies show several disadvantages to carrying this allele in humans. However, the high rate of SNP occurrence raises the intriguing possibility of a selective advantage to carrying the Met allele, such as motor function. The current study investigates the impact of bdnf val66met SNP on long term recovery after stroke. Methods: Mice with a genetic knock-in of the human BDNF variant (val66met) were generated. Adult male BDNF +/+ (WT) and BDNF Met/Met (M/M) mice were subjected to the proximal middle cerebral artery occlusion. Motor and gait functions were assessed by rotarod and Catwalk analysis at acute phase and recovery phase. Anatomical volume analysis was evaluated by cresyl violet and Golgi staining at 6 months after stroke. Results: As previously reported (Qin et al., 2011), there was greater acute impairment in motor behavioral outcomes in M/M mice after stroke, compared to WT mice. However, M/M mice displayed significantly enhanced rotarod performance at 2 weeks and the enhancement was sustained up to 6 months post-stroke. Similarly, Catwalk gait analyses in swing speed and stride length showed that M/M mice exhibited enhanced gaits (i.e. faster and longer stride) than WT mice during long-term recovery phases, especially in the ipsilesional right hind limb. Since ipsilesional limbs are controlled by the contralesional hemisphere, sub-regional volumes in this hemisphere were assessed. M/M mice showed specifically increased striatal volume at 6 months post-ischemia. The increased striatal volume was also associated with higher dendritic complexity of medium spiny neurons in this region, which indicated that the contralesional anatomical plasticity may account for enhanced motor recovery in MM mice. Conclusions: The findings suggest that BDNF SNP may have selective advantage in enhancing motor/gait function through the contralesional hemisphere during stroke recovery. The study has clinical importance by providing a means to predict the course of stroke recovery based on an individual’s SNP.