Cortical Brain Injury Causes Retrograde Degeneration of Afferent Basal Forebrain Cholinergic Neurons via the p75NTR.

Traumatic brain injury (TBI) elicits neuronal loss at the site of injury and progressive neuronal loss in the penumbra. However, the consequences of TBI on afferent neurons projecting to the injured tissue from distal locations is unknown. Basal forebrain cholinergic neurons (BFCNs) extend long projections to multiple brain regions including the cortex, regulate many cognitive functions, and are compromised in numerous neurodegenerative disorders. To determine the consequence of cortical injury on these afferent neurons, we used the fluid percussion injury model of traumatic brain injury and assessed the effects on BFCN survival and axon integrity in male and female mice. Survival or death of BF neurons can be regulated by neurotrophins or proneurotrophins, respectively. The injury elicited an induction of proNGF and proBDNF in the cortex and a loss of BFCNs ipsilateral to the injury compared with sham uninjured mice. The p75NTR knock-out mice did not show loss of BFCN neurons, indicating a retrograde degenerative effect of the cortical injury on the afferent BFCNs mediated through p75NTR. In contrast, locus ceruleus neurons, which also project throughout the cortex, were unaffected by the injury, suggesting specificity in retrograde degeneration after cortical TBI. Proneurotrophins (proNTs) provided directly to basal forebrain axons in microfluidic cultures triggered retrograde axonal degeneration and cell death, which did not occur in the absence of p75NTR. This study shows that after traumatic brain injury, proNTs induced in the injured cortex promote BFCN axonal degeneration and retrograde neuron loss through p75NTR.

Proneurotrophins Induce Apoptotic Neuronal Death After Controlled Cortical Impact Injury in Adult Mice.

The p75 neurotrophin receptor (p75NTR) can regulate multiple cellular functions including proliferation, survival, and apoptotic cell death. The p75NTR is widely expressed in the developing brain and is downregulated as the nervous system matures, with only a few neuronal subpopulations retaining expression into adulthood. However, p75NTR expression is induced following damage to the adult brain, including after traumatic brain injury, which is a leading cause of mortality and disability worldwide. A major consequence of traumatic brain injury is the progressive neuronal loss that continues secondary to the initial trauma, which ultimately contributes to cognitive decline. Understanding mechanisms governing this progressive neuronal death is key to developing targeted therapeutic strategies to provide neuroprotection and salvage cognitive function. In this study, we demonstrate that a cortical impact injury to the sensorimotor cortex elicits p75NTR expression in apoptotic neurons in the injury penumbra, confirming previous studies. To establish whether preventing p75NTR induction or blocking the ligands would reduce the extent of secondary neuronal cell death, we used a noninvasive intranasal strategy to deliver either siRNA to block the induction of p75NTR, or function-blocking antibodies to the ligands pro-nerve growth factor and pro-brain-derived neurotrophic factor. We demonstrate that either preventing the induction of p75NTR or blocking the proneurotrophin ligands provides neuroprotection and preserves sensorimotor function.

The p75 Neurotrophin Receptor Facilitates TrkB Signaling and Function in Rat Hippocampal Neurons.

Neurotrophins activate Trk receptor signaling to support neuronal survival and many aspects of neuronal function. Early studies demonstrated that TrkA formed a complex with the p75 neurotrophin receptor (p75 NTR ), which increased the affinity and selectivity of NGF binding, however, whether interaction of p75 NTR with other Trk receptors performs a similar function to enhance ligand binding has not been demonstrated. We investigated the interaction of TrkB with full length p75 NTR in hippocampal neurons in response to BDNF and found that the association of these receptors occurs after ligand binding and requires phosphorylation of TrkB, indicating that formation of this receptor complex was not necessary for ligand binding. Moreover, the interaction of these receptors required internalization and localization to early endosomes. We found that association of TrkB with p75 NTR was necessary for optimal downstream signaling of the PI3K-Akt pathway, but not the Erk pathway, in hippocampal neurons. The absence of p75 NTR impaired the ability of BDNF to rescue hippocampal neurons in a trophic deprivation model, suggesting that p75 NTR facilitates the ability of TrkB to activate specific pathways to promote neuronal survival.

Regulation of BACE1 expression after injury is linked to the p75 neurotrophin receptor.

BACE1 is a transmembrane aspartic protease that cleaves various substrates and it is required for normal brain function. BACE1 expression is high during early development, but it is reduced in adulthood. Under conditions of stress and injury, BACE1 levels are increased; however, the underlying mechanisms that drive BACE1 elevation are not well understood. One mechanism associated with brain injury is the activation of injurious p75 neurotrophin receptor (p75), which can trigger pathological signals. Here we report that within 72 h after controlled cortical impact (CCI) or laser injury, BACE1 and p75 are increased and tightly co-expressed in cortical neurons of mouse brain. Additionally, BACE1 is not up-regulated in p75 null mice in response to focal cortical injury, while p75 over-expression results in BACE1 augmentation in HEK-293 and SY5Y cell lines. A luciferase assay conducted in SY5Y cell line revealed that BACE1 expression is regulated at the transcriptional level in response to p75 transfection. Interestingly, this effect does not appear to be dependent upon p75 ligands including mature and pro-neurotrophins. In addition, BACE1 activity on amyloid precursor protein (APP) is enhanced in SY5Y-APP cells transfected with a p75 construct. Lastly, we found that the activation of c-jun n-terminal kinase (JNK) by p75 contributes to BACE1 up-regulation. This study explores how two injury-induced molecules are intimately connected and suggests a potential link between p75 signaling and the expression of BACE1 after brain injury.

Loss of TrkB Signaling Due to Status Epilepticus Induces a proBDNF-Dependent Cell Death.

Neurotrophins (NTs) are secretory proteins that bind to target receptors and influence many cellular functions, such as cell survival and cell death in neurons. The mammalian NT brain-derived neurotrophic factor (matBDNF) is the C-terminal mature form released by cleavage from the proBDNF precursor. The binding of matBDNF to the tyrosine kinase receptor B (TrkB) activates different signaling cascades and leads to neuron survival and plasticity, while the interaction of proBDNF with the p75 NT receptor (p75NTR)/sortilin receptor complex has been highly involved in apoptosis. Many studies have demonstrated that prolonged seizures such as status epilepticus (SE) induce changes in the expression of NT, pro-NT, and their receptors. We have previously described that the blockage of both matBDNF and proBDNF signaling reduces neuronal death after SE in vivo (Unsain et al., 2008). We used an in vitro model as well as an in vivo model of SE to determine the specific role of TrkB and proBDNF signaling during neuronal cell death. We found that the matBDNF sequestering molecule TrkB-Fc induced an increase in neuronal death in both models of SE, and it also prevented a decrease in TrkB levels. Moreover, SE triggered the interaction between proBDNF and p75NTR, which was not altered by sequestering matBDNF. The intra-hippocampal administration of TrkB-Fc, combined with an antibody against proBDNF, prevented neuronal degeneration. In addition, we demonstrated that proBDNF binding to p75NTR exacerbates neuronal death when matBDNF signaling is impaired through TrkB. Our results indicated that both the mature and the precursor forms of BDNF may have opposite effects depending on the scenario in which they function and the signaling pathways they activate.

A Novel Neuroprotective Mechanism for Lithium That Prevents Association of the p75NTR-Sortilin Receptor Complex and Attenuates proNGF-Induced Neuronal Death In Vitro and In Vivo.

Neurotrophins play critical roles in the survival, maintenance and death of neurons. In particular, proneurotrophins have been shown to mediate cell death following brain injury induced by status epilepticus (SE) in rats. Previous studies have shown that pilocarpine-induced seizures lead to increased levels of proNGF, which binds to the p75NTR-sortilin receptor complex to elicit apoptosis. A screen to identify compounds that block proNGF binding and uptake into cells expressing p75 and sortilin identified lithium citrate as a potential inhibitor of proNGF and p75NTR-mediated cell death. In this study, we demonstrate that low, submicromolar doses of lithium citrate effectively inhibited proNGF-induced cell death in cultured neurons and protected hippocampal neurons following pilocarpine-induced SE in vivo. We analyzed specific mechanisms by which lithium citrate afforded neuroprotection and determined that lithium citrate prevented the association and internalization of the p75NTR-sortilin receptor complex. Our results demonstrate a novel mechanism by which low-dose treatments of lithium citrate are effective in attenuating p75NTR-mediated cell death in vitro and in vivo.

An alkaloid extract obtained from Phlegmariurus Saururus induces neuroprotection after status epilepticus.

BACKGROUND: The brain is exposed to many excitotoxic insults that can lead to neuronal damage. Among these, Epilepsy is a neurological disease that affects a large percentage of world population and is commonly associated with cognitive disorders and excitotoxic neuronal death. Most experimental strategies are focused on preventing Status Epilepticus (SE), but once it has already occurred, the key question is whether it is possible to save neurons. PURPOSE: The aim of this study was to determine if a purified alkaloid extract (AE) obtained from Phlegmariurus saururus, a genus of Lycophyte plants (sometimes known as firmossesor fir club mosses) could induce neuroprotection following SE. METHODS: In vitro and in vivo techniques were applied for this purpose. Protein levels were measured by western blotting procedures. Neuronal death analysis was performed by calcein-ethidium staining and the presence of the NeuN protein as a marker for presence or absence of cells (in vitro experiments) and by Fluoro Jade B staining for the in vivo experiments. RESULTS: The effect of AE in the hippocampal neurons culture was the first determination, where we found an increase in neuronal survival and in the level of pErk and TrkB activation, 24 h after the addition of AE. In a well-established in vitro model of SE, we found that 24 h after being added to the hippocampal neuron-astrocyte co-culture, the AE induces a significant increase in neuronal survival. In addition to this, in the in vivo Li-pilocarpine model of SE, the AE induced a remarkable neuroprotection in areas such as the entorhinal cortex and hippocampal CA1 area. CONCLUSION: These results make the AE an excellent candidate for potential clinical use in neurological disorders where memory impairment and neuronal death occurs.

Calpain-dependent truncated form of TrkB-FL increases in neurodegenerative processes.

Recent findings indicate that the mechanisms that drive reshaping of the nervous system are aberrantly activated in epilepsy and several neurodegenerative diseases. The recurrent seizures in epilepsy, particularly in the condition called status epilepticus, can cause permanent neurological damage, resulting in cognitive dysfunction and other serious neurological conditions. In this study, we used an in vitro model of status epilepticus to examine the role of calpain in the degeneration of hippocampal neurons. We grew neurons on a culture system that allowed studying the dendritic and axonal domains separately from the cell bodies. We found that a recently characterized calpain substrate, the neurotrophin receptor TrkB, is cleaved in the dendritic and axonal domain of neurons committed to die, and this constitutes an early step in the neuronal degeneration process. While the full-length TrkB (TrkB-FL) levels decreased, the truncated form of TrkB (Tc TrkB-FL) concurrently increased, leading to a TrkB-FL/Tc TrkB-FL imbalance, which is thought to be causally linked to neurodegeneration. We further show that the treatment with N-acetyl-Leu-Leu-norleucinal, a specific calpain activity blocker, fully protects the neuronal processes from degeneration, prevents the TrkB-FL/Tc TrkB-FL imbalance, and provides full neuroprotection. Moreover, the use of the TrkB antagonist ANA 12 at the time when the levels of TrkB-FL were significantly decreased, totally blocked neuronal death, suggesting that Tc TrkB-FL may have a role in neuronal death. These results indicate that the imbalance of these neurotrophins receptors plays a key role in neurite degeneration induced by seizures.

Brain-derived neurotrophic factor facilitates TrkB down-regulation and neuronal injury after status epilepticus in the rat hippocampus.

Brain-derived neurotrophic factor (BDNF) is involved in many aspects of neuronal biology and hippocampal physiology. Status epilepticus (SE) is a condition in which prolonged seizures lead to neuronal degeneration. SE-induced in rodents serves as a model of Temporal Lobe Epilepsy with hippocampal sclerosis, the most frequent epilepsy in humans. We have recently described a strong correlation between TrkB decrease and p75ntr increase with neuronal degeneration (Neuroscience 154:978, 2008). In this report, we report that local, acute intra-hippocampal infusion of function-blocking antibodies against BDNF prevented both early TrkB down-regulation and neuronal degeneration after SE. Conversely, the infusion of recombinant human BDNF protein after SE greatly increased neuronal degeneration. The inhibition of BDNF mRNA translation by the infusion of antisense oligonucleotides induced a rapid decrease of BDNF protein levels, and a delayed increase. If seizures were induced at the time endogenous BDNF was decreased, SE-induced neuronal damage was prevented. On the other hand, if seizures were induced at the time endogenous BDNF was increased, SE-induced neuronal damage was exacerbated. These results indicate that under a pathological condition BDNF exacerbates neuronal injury.