Ann M. Marini

BDNF variation and mood disorders: a novel functional promoter polymorphism and Val66Met are associated with anxiety but have opposing effects.

The brain-derived neurotrophic factor (BDNF) gene is critical for neuronal function and survival, and is likely to be important in psychiatric disorders. In this study, we used single-nucleotide polymorphism (SNP) discovery, functional analyses, and genetic association studies to better understand the potential role of BDNF sequence variation in behavior. Screening 480 unrelated individuals for SNPs and genotyping was performed in US Caucasian, American Indian, and African American populations. Lifetime DSM-III-R psychiatric diagnoses were assigned and the Tridimensional Personality Questionnaire (TPQ) was administered to measure anxious temperament (harm avoidance (HA)) and novelty seeking (NS). A novel SNP (−281 C>A) in promoter 1 was discovered that had decreased DNA binding in vitro and decreased basal reporter gene activity in transfected rat hippocampal neurons. The frequency of the −281 A allele was 0.03 in a Caucasian sample, but was virtually absent in other populations. Association analyses in a community-based sample showed that individuals with the −281 A allele (13 heterozygotes) had lower TPQ HA (F=4.8, p<0.05). In contrast, the Met 66 allele was associated with increased HA (F=4.1, p=0.02) and was most abundant in individuals with both anxiety disorders and major depression (p<0.05). Among the Val66Val homozygotes, individuals who were –281 CA heterozygotes had significantly lower HA than the –281 CC homozygotes (p<0.01). Our results suggest that in this population, the low activity –281 A allele may be protective against anxiety and psychiatric morbidity, whereas Met 66 may be a risk allele.

Activity-dependent release of brain-derived neurotrophic factor underlies the neuroprotective effect of N-methyl-D-aspartate.

The molecular mechanism(s) ofN-methyl-d-aspartate (NMDA) neuroprotective properties were investigated in primary cultures of cerebellar granule cell neurons. Granule cells express the neurotrophin receptor TrkB but not TrkA or TrkC. In these cells, the TrkB ligand brain-derived neurotrophic factor (BDNF) prevents glutamate toxicity. Therefore, we have tested the hypothesis that NMDA activates synthesis and release of BDNF, which may prevent glutamate toxicity by an autocrine loop. Exposure of granule cells for 2 and 5 min to a subtoxic concentration of NMDA (100 μm) evoked an accumulation of BDNF in the medium without concomitant changes in the intracellular levels of BDNF protein or mRNA. The increase in BDNF in the medium is followed by enhanced TrkB tyrosine phosphorylation, suggesting that NMDA increases the release of BDNF and therefore the activity of TrkB receptors. To examine whether BDNF and TrkB signaling play a role in the NMDA-mediated neuroprotective properties, neurons were exposed to soluble trkB receptor-IgG fusion protein, which is known to inhibit the activity of extracellular BDNF, and to K252a, a tyrosine kinase inhibitor. Both compounds blocked the NMDA-mediated TrkB tyrosine phosphorylation and subsequently its neuroprotective properties. We suggest that NMDA activates the TrkB receptor via a BDNF autocrine loop, resulting in neuronal survival.

Nuclear factor κB is a critical determinant in N‐methyl‐d‐aspartate receptor‐mediated neuroprotection

The role of a nuclear factor κB (NF‐κB) in NMDA receptor‐mediated neuroprotection is not known. A candidate sequence from the 5′ flanking region of exon 3 of the rat brain‐derived neurotrophic factor (BDNF) gene was used to show that exposure of rat cerebellar granule cells to 100 μm NMDA activated a specific DNA binding activity that was blocked by the NMDA receptor antagonist MK‐801. Anti‐p65 antibody or anti‐p50 antibody ‘supershifted’ the DNA binding activity, suggesting that the DNA–protein complex was composed of p65 and p50 subunits. NMDA receptor‐mediated neuroprotection was blocked when cerebellar neurons were transfected with a double‐stranded oligonucleotide containing the BDNF gene NF‐κB sequence. Furthermore, nuclear extracts prepared from neurons treated with NMDA and the double‐stranded NF‐κB oligonucleotide showed reduced DNA binding activity to the target sequence, supporting the idea that NF‐κB may be involved in the transcriptional activation of the BDNF gene. To address this issue, we quantified the level of exon 3‐specific BDNF mRNA. Relative to GAPDH mRNA levels and compared with untreated neurons, NMDA increased exon 3‐specific BDNF mRNA twofold. In contrast, pretreatment of neurons with the NF‐κB target DNA abolished the increase in BDNF mRNA following addition of NMDA. We also determined that BDNF itself induced an NF‐κB DNA binding activity. Taken together, these data support a mechanism where NF‐κB plays a critical role in NMDA‐mediated neuroprotection.

The excitoprotective effect of N‐methyl‐d‐aspartate receptors is mediated by a brain‐derived neurotrophic factor autocrine loop in cultured hippocampal neurons

The neuroprotective effect and molecular mechanisms underlying preconditioning with N‐methyl‐D‐aspartate (NMDA) in cultured hippocampal neurons have not been described. Pre‐incubation with subtoxic concentrations of the endogenous neurotransmitter glutamate protects vulnerable neurons against NMDA receptor‐mediated excitotoxicity. As a result of physiological preconditioning, NMDA significantly antagonizes the neurotoxicity resulting from subsequent exposure to an excitotoxic concentration of glutamate. The protective effect of glutamate or NMDA is time‐ and concentration‐dependent, suggesting that sufficient agonist and time are required to establish an intracellular neuroprotective state. In these cells, the TrkB ligand, brain‐derived neurotrophic factor (BDNF) attenuates glutamate toxicity. Therefore, we tested the hypothesis that NMDA protects neurons via a BDNF‐dependent mechanism. Exposure of hippocampal cultures to a neuroprotective concentration of NMDA (50 μm) evoked the release of BDNF within 2 min without attendant changes in BDNF protein or gene expression. The accumulated increase of BDNF in the medium is followed by an increase in the phosphorylation (activation) of TrkB receptors and a later increase in exon 4‐specific BDNF mRNA. The neuroprotective effect of NMDA was attenuated by pre‐incubation with a BDNF‐blocking antibody and TrkB‐IgG, a fusion protein known to inhibit the activity of extracellular BDNF, suggesting that BDNF plays a major role in NMDA‐mediated survival. These results demonstrate that low level stimulation of NMDA receptors protect neurons against glutamate excitotoxicity via a BDNF autocrine loop in hippocampal neurons and suggest that activation of neurotrophin signaling pathways plays a key role in the neuroprotection of NMDA.

Subchronic alpha-linolenic acid treatment enhances brain plasticity and exerts an antidepressant effect: a versatile potential therapy for stroke.

Omega-3 polyunsaturated fatty acids are known to have therapeutic potential in several neurological and psychiatric disorders. However, the molecular mechanisms of action underlying these effects are not well elucidated. We previously showed that alpha-linolenic acid (ALA) reduced ischemic brain damage after a single treatment. To follow-up this finding, we investigated whether subchronic ALA treatment promoted neuronal plasticity. Three sequential injections with a neuroprotective dose of ALA increased neurogenesis and expression of key proteins involved in synaptic functions, namely, synaptophysin-1, VAMP-2, and SNAP-25, as well as proteins supporting glutamatergic neurotransmission, namely, V-GLUT1 and V-GLUT2. These effects were correlated with an increase in brain-derived neurotrophic factor (BDNF) protein levels, both in vitro using neural stem cells and hippocampal cultures and in vivo, after subchronic ALA treatment. Given that BDNF has antidepressant activity, this led us to test whether subchronic ALA treatment could produce antidepressant-like behavior. ALA-treated mice had significantly reduced measures of depressive-like behavior compared with vehicle-treated animals, suggesting another aspect of ALA treatment that could stimulate functional stroke recovery by potentially combining acute neuroprotection with long-term repair/compensatory plasticity. Indeed, three sequential injections of ALA enhanced protection, either as a pretreatment, wherein it reduced post-ischemic infarct volume 24 h after a 1-hour occlusion of the middle cerebral artery or as post-treatment therapy, wherein it augmented animal survival rates by threefold 10 days after ischemia.

AMPA protects cultured neurons against glutamate excitotoxicity through a phosphatidylinositol 3-kinase-dependent activation in extracellular signal-regulated kinase to upregulate BDNF gene expression

The signal transduction and molecular mechanisms underlying α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)‐mediated neuroprotection are unknown. In the present study, we determined a major AMPA receptor‐mediated neuroprotective pathway. Exposure of cerebellar granule cells to AMPA (500 µm) + aniracetam (1 µm), a known blocker of AMPA receptor desensitization, evoked an accumulation of brain‐derived neurotropic factor (BDNF) in the culture medium and enhanced TrkB‐tyrosine phosphorylation following the release of BDNF. AMPA also activated the src‐family tyrosine kinase, Lyn, and the downstream target of the phosphatidylinositol 3‐kinase (PI3‐K) pathway, Akt. Extracellular signal regulated kinase (ERK), a component of the mitogen‐activated protein kinase (MAPK) pathway, was also activated. K252a, a selective inhibitor of neurotrophin signaling, blocked the AMPA‐mediated neuroprotection. The involvement of BDNF release in protecting neurons by AMPA was confirmed using a BDNF‐blocking antibody. AMPA‐mediated neuroprotection is blocked by PP1, an inhibitor of src family kinases, LY294002, a PI3‐K inhibitor, or U0126, a MAPK kinase (MEK) inhibitor. Neuroprotective concentrations of AMPA increased BDNF mRNA levels that was blocked by the AMPA receptor antagonist, 1,2,3,4‐tetrahydro‐6‐nitro‐2,3‐dioxo‐benzo[f]quinoxaline‐7‐sulfonamide (NBQX). The increase in BDNF gene expression appeared to be the downstream target of the PI3‐K‐dependent activation of the MAPK cascade since MEK or the PI3‐K inhibitor blocked the AMPA receptor‐mediated increase in BDNF mRNA. Thus, AMPA receptors protect neurons through a mechanism involving BDNF release, TrkB receptor activation, and a signaling pathway involving a PI3‐K dependent activation of MAPK that increases BDNF expression.

BHLHB2 Controls Bdnf Promoter 4 Activity and Neuronal Excitability

Brain-derived neurotrophic factor (BDNF), via activation of TrkB receptors, mediates vital physiological functions in the brain, ranging from neuronal survival to synaptic plasticity, and has been implicated in the pathophysiology of neurodegenerative disorders. Although transcriptional regulation of the BDNF gene (Bdnf) has been extensively studied, much remains to be understood. We discovered a sequence within Bdnf promoter 4 that binds the basic helix-loop-helix protein BHLHB2 and is a target for BHLHB2-mediated transcriptional repression. NMDA receptor activation de-repressed promoter 4-mediated transcription and correlated with reduced occupancy of the promoter by BHLHB2 in cultured hippocampal neurons. Bhlhb2 gene −/− mice showed increased hippocampal exon 4-specific Bdnf mRNA levels compared with +/+ littermates under basal and activity-dependent conditions. Bhlhb2 knock-out mice also showed increased status epilepticus susceptibility, suggesting that BHLHB2 alters neuronal excitability. Together, these results support a role for BHLHB2 as a new modulator of Bdnf transcription and neuronal excitability.

N-Methyl-D-aspartate and TrkB Receptor Activation in Cerebellar Granule Cells: An In Vitro Model of Preconditioning to Stimulate Intrinsic Survival Pathways in Neurons

Abstract: Delineating the mechanisms of survival pathways that exist in neurons will provide important insight into how neurons utilize intracellular proteins as neuroprotectants against the causes of acute neurodegeneration. We have employed cultured rat cerebellar granule cells as a model for determining the mechanisms of these intraneuronal survival pathways. Glutamate has long been known to kill neurons by an N‐methyl‐d‐aspartate (NMDA) receptor‐mediated mechanism. Paradoxically, subtoxic concentrations of NMDA protect neurons against glutamate‐mediated excitotoxicity. Because NMDA protects neurons in physiologic concentrations of glucose and oxygen, we refer to this phenomenon as physiologic preconditioning. One of the major mechanisms of NMDA neuroprotection involves the activation of NMDA receptors leading to the rapid release of brain‐derived neurotrophic factor (BDNF). BDNF then binds to and activates its cognate receptor, receptor tyrosine kinase B (TrkB). The efficient utilization of these two receptors confers remarkable resistance against millimolar concentrations of glutamate that kill more than eighty percent of the neurons in the absence of preconditioning the neurons with a subtoxic concentration of NMDA. Exactly how the neurons mediate neuroprotection by activation of both receptors is just beginning to be understood. Both NMDA and TrkB receptors activate nuclear factor kappaB (NF‐κB), a transcription factor known to be involved in protecting neurons against many different kinds of toxic insults. By converging on survival transcription factors, such as NF‐κB, NMDA and TrkB receptors protect neurons. Thus, crosstalk between these very different receptors provides a rapid means of neuronal communication to upregulate survival proteins through release and transcriptional activation of messenger RNA.

Preconditioning and neurotrophins: a model for brain adaptation to seizures, ischemia and other stressful stimuli.

Summary.The amino acid glutamate, the major excitatory neurotransmitter in the central nervous system, activates receptors coupled to calcium influx. Excessive activation of glutamate receptors in conditions such as severe epileptic seizures or stroke can kill neurons in a process called excitotoxicity. However, subtoxic levels of activation of the N-methyl-D-aspartate (NMDA) type of glutamate receptor elicit adaptive responses in neurons that enhance their ability to withstand more severe stress. A variety of stimuli induce adaptive responses to protect neurons. For example, sublethal ischemic episodes or a mild epileptic insult can protect neurons in a process referred to as tolerance. The molecular mechanisms that protect neurons by these different stressful stimuli are largely unknown but they share common features such as the transcription factor, nuclear factor kappa B (NF-κB), which is activated by ischemic and epileptic preconditioning as well as exposure to subtoxic NMDA concentrations. In this article, we describe stress-induced neuroprotective mechanisms highlighting the role of brain-derived neurotrophic factor (BDNF), a protein that plays a crucial role in neuronal survival and maintenance, neurogenesis and learning and memory.

N‐methyl‐D‐aspartate and TrkB receptors protect neurons against glutamate excitotoxicity through an extracellular signal‐regulated kinase pathway

N‐Methyl‐D‐aspartate (NMDA) at a subtoxic concentration (100 μM) promotes neuronal survival against glutamate‐mediated excitotoxicity via a brain‐derived neurotrophic factor (BDNF) autocrine loop in cultured cerebellar granule cells. The signal transduction mechanism(s) underlying NMDA neuroprotection, however, remains elusive. The mitogen‐activated protein kinase (MAPK) and phosphatidylinositol‐3 kinase (PI3‐K) pathways alter gene expression and are involved in synaptic plasticity and neuronal survival. This study tested whether neuroprotective activation of NMDA receptors, together with TrkB receptors, coactivated the MAPK or PI3‐K pathways to protect rat cerebellar neurons. NMDA receptor activation caused a concentration‐ and time‐dependent activation of MAPK lasting 24 hr. This activation was blocked by the NMDA receptor antagonist MK‐801 but was attenuated only partially by the tyrosine kinase inhibitor k252a, suggesting that activation of both NMDA and TrkB receptors are required for maximal neuroprotection. The MAPK kinase (MEK) inhibitor U0126 (10 μM) partially blocked NMDA neuroprotection, whereas LY294002, a selective inhibitor of the PI3‐K pathway, did not affect the neuroprotective activity of NMDA. Glutamate excitotoxicity decreased bcl‐2, bcl‐XL, and bax mRNA levels,. NMDA increases Bcl‐2 and Bcl‐XL protein levels and decreases Bax protein levels. NMDA and TrkB receptor activation thus converge on the extracellular signal‐regulated kinase (ERK) 1/2 signaling pathway to protect neurons against glutamate‐mediated excitotoxicity. By increasing antiapoptotic proteins of the Bcl‐2 family, NMDA receptor activation may also promote neuronal survival by preventing apoptosis.