Fluoxetine and ketamine trigger the p75NTR proteolytic pathway and enhance extinction memory and brain plasticity through p75NTR.

BACKGROUND: Diverse antidepressants were recently described to bind to TrkB and drive a positive allosteric modulation of endogenous BDNF. Although neurotrophins such as BDNF can bind to the p75 neurotrophin receptor (p75NTR), their precursors are the high affinity p75NTR ligands. While part of an unrelated receptor family capable of inducing completely opposite physiological changes, TrkB and p75NTR feature a cross-like conformation dimer and carry a cholesterol-recognition and alignment consensus in the transmembrane domain. Since such qualities were found crucial for antidepressants to bind to TrkB and drive behavioral and neuroplasticity effects, we hypothesized that their effects might also depend on p75NTR. METHODS: ELISA-based binding assay and NMR spectroscopy were accomplished to assess whether antidepressants would bind to p75NTR. HEK293T cells and a variety of in vitro assays were used to address whether fluoxetine (FLX) or ketamine (KET) would trigger any α- and γ-secretase-dependent p75NTR proteolysis, and lead to p75NTR nuclear localization. Ocular dominance shift was performed with male and female p75KO mice to study the effects of KET and FLX on brain plasticity, in addition to pharmacological interventions to verifying how p75NTR signaling is important for the effects of KET and FLX in enhancing extinction memory in male WT mice and rats. RESULTS: Antidepressants were found binding to p75NTR, FLX and KET triggered the p75NTR proteolytic pathway and induced p75NTR-dependent behavioral/neuroplasticity changes. CONCLUSION: We thus hypothesize that antidepressants co-opt both BDNF/TrkB and proBDNF/p75NTR systems to induce a more efficient activity-dependent synaptic competition, thereby boosting the brain ability for remodeling.

The loop diuretic bumetanide blocks posttraumatic p75NTR upregulation and rescues injured neurons.

Injured neurons become dependent on trophic factors for survival. However, application of trophic factors to the site of injury is technically extremely challenging. Novel approaches are needed to circumvent this problem. Here, we unravel the mechanism of the emergence of dependency of injured neurons on brain-derived neurotrophic factor (BDNF) for survival. Based on this mechanism, we propose the use of the diuretic bumetanide to prevent the requirement for BDNF and consequent neuronal death in the injured areas. Responses to the neurotransmitter GABA change from hyperpolarizing in intact neurons to depolarizing in injured neurons. We show in vivo in rats and ex vivo in mouse organotypic slice cultures that posttraumatic GABA(A)-mediated depolarization is a cause for the well known phenomenon of pathological upregulation of pan-neurotrophin receptor p75(NTR). The increase in intracellular Ca(2+) triggered by GABA-mediated depolarization activates ROCK (Rho kinase), which in turn leads to the upregulation of p75(NTR). We further show that high levels of p75(NTR) and its interaction with sortilin and proNGF set the dependency on BDNF for survival. Thus, application of bumetanide prevents p75(NTR) upregulation and neuronal death in the injured areas with reduced levels of endogenous BDNF.

Mapping of the interaction site between sortilin and the p75 neurotrophin receptor reveals a regulatory role for the sortilin intracellular domain in p75 neurotrophin receptor shedding and apoptosis.

Neurotrophins comprise a group of neuronal growth factors that are essential for the development and maintenance of the nervous system. However, the immature pro-neurotrophins promote apoptosis by engaging in a complex with sortilin and the p75 neurotrophin receptor (p75(NTR)). To identify the interaction site between sortilin and p75(NTR), we analyzed binding between chimeric receptor constructs and truncated p75(NTR) variants by co-immunoprecipitation experiments, surface plasmon resonance analysis, and FRET. We found that complex formation between sortilin and p75(NTR) relies on contact points in the extracellular domains of the receptors. We also determined that the interaction critically depends on an extracellular juxtamembrane 23-amino acid sequence of p75(NTR). Functional studies further revealed an important regulatory function of the sortilin intracellular domain in p75(NTR)-regulated intramembrane proteolysis and apoptosis. Thus, although the intracellular domain of sortilin does not contribute to p75(NTR) binding, it does regulate the rates of p75(NTR) cleavage, which is required to mediate pro-neurotrophin-stimulated cell death.

Erenumab Decreases Headache-Related Sick Leave Days and Health Care Visits: A Retrospective Real-World Study in Working Patients with Migraine.

INTRODUCTION: The prevalence of migraine is highest among working age individuals, and this disease is associated with an increased number of sick leaves and health care visits, as well as lost productivity. Erenumab, the first monoclonal antibody targeting the calcitonin gene-related peptide (CGRP) pathway, is effective in decreasing the monthly number of migraine days, but evidence of its impact on the number of sick leave days and health care visits in patients with migraine is limited. METHODS: This retrospective registry study focused on occupationally active patients with migraine treated with erenumab at a Finnish private health care provider, Terveystalo. Erenumab responders, defined as patients who had at least two unique prescriptions of erenumab and no prescription of other CGRP inhibitor (CGRPi), were followed for 12 months prior to and after erenumab treatment initiation (index), and the change in the number of headache-related and all-cause sick leave days, health care visits and prescriptions for other medications during this period were assessed from the registry data. The same outcomes were assessed in an age- and sex-matched control group of migraine patients not receiving CGRPi to control for potential changes in patient behavior and health care practices during the COVID-19 pandemic. RESULTS: Altogether, 162 patients who were entitled to employer-sponsored health care received erenumab and met the 12-month follow-up requirements. In the responder group (n = 82; 50.1%) headache-related sick leave days were reduced by 73.9% (p = 0.035) and health care visits by 44.6% (p < 0.001) in the 12 months following treatment initiation compared to the period of 12 months prior to treatment. All-cause sick leave days were reduced by 19.4% and all-cause health care visits by 13.5%, but these changes were not statistically significant. Triptan prescriptions decreased by 30.4% (p = 0.012) and other prophylactic treatments by 31.5% (p = 0.004). No significant changes were observed in the corresponding outcomes in the migraine control group during the same period. CONCLUSIONS: The results of this registry study suggest that in addition to the effect on the monthly number of migraine days documented in clinical trials, erenumab can significantly reduce the number of headache-related sick leave days and health care visits in employed patients with migraine managed in routine clinical practice.

Isoflurane produces antidepressant effects and induces TrkB signaling in rodents.

A brief burst-suppressing isoflurane anesthesia has been shown to rapidly alleviate symptoms of depression in a subset of patients, but the neurobiological basis of these observations remains obscure. We show that a single isoflurane anesthesia produces antidepressant-like behavioural effects in the learned helplessness paradigm and regulates molecular events implicated in the mechanism of action of rapid-acting antidepressant ketamine: activation of brain-derived neurotrophic factor (BDNF) receptor TrkB, facilitation of mammalian target of rapamycin (mTOR) signaling pathway and inhibition of glycogen synthase kinase 3ß (GSK3ß). Moreover, isoflurane affected neuronal plasticity by facilitating long-term potentiation in the hippocampus. We also found that isoflurane increased activity of the parvalbumin interneurons, and facilitated GABAergic transmission in wild type mice but not in transgenic mice with reduced TrkB expression in parvalbumin interneurons. Our findings strengthen the role of TrkB signaling in the antidepressant responses and encourage further evaluation of isoflurane as a rapid-acting antidepressant devoid of the psychotomimetic effects and abuse potential of ketamine.

Utilization of in situ ELISA method for examining Trk receptor phosphorylation in cultured cells.

BACKGROUND: Trk receptor tyrosine kinases regulate multiple important neuronal processes during the development and in the adulthood. Tyrosine phosphorylation of Trk serves as the initial step in the Trk signaling pathway and indicates receptor' autocatalytic activity. However, methods allowing simple and large-scale Trk phosphorylation analyses in cultured cells are lacking. NEW METHOD: We describe an in situ phospho-Trk ELISA (enzyme-linked immunosorbent assay) method where cell culture, receptor stimulation and Trk phosphorylation analysis are all performed on the same multiwell plate. RESULTS: In situ phospho-Trk ELISA readily and specifically detects neurotrophin-induced Trk phosphorylation in cultured cells. A proof-of-concept small molecule screening of a library composed of 2000 approved drugs and other bioactive compounds was carried out using this novel method. COMPARISON WITH EXISTING METHODS: In situ phospho-Trk ELISA utilizes the principles and advantages of conventional sandwich ELISA in an in situ context. CONCLUSIONS: We describe a novel method that can be efficiently used to examine Trk receptor phosphorylation in cultured cells. Principally similar methods can be developed to examine the levels and signaling of any intracellular protein.

The impact of Bdnf gene deficiency to the memory impairment and brain pathology of APPswe/PS1dE9 mouse model of Alzheimer's disease.

Brain-derived neurotrophic factor (BDNF) importantly regulates learning and memory and supports the survival of injured neurons. Reduced BDNF levels have been detected in the brains of Alzheimer's disease (AD) patients but the exact role of BDNF in the pathophysiology of the disorder remains obscure. We have recently shown that reduced signaling of BDNF receptor TrkB aggravates memory impairment in APPswe/PS1dE9 (APdE9) mice, a model of AD. The present study examined the influence of Bdnf gene deficiency (heterozygous knockout) on spatial learning, spontaneous exploratory activity and motor coordination/balance in middle-aged male and female APdE9 mice. We also studied brain BDNF protein levels in APdE9 mice in different ages showing progressive amyloid pathology. Both APdE9 and Bdnf mutations impaired spatial learning in males and showed a similar trend in females. Importantly, the effect was additive, so that double mutant mice performed the worst. However, APdE9 and Bdnf mutations influenced spontaneous locomotion in contrasting ways, such that locomotor hyperactivity observed in APdE9 mice was normalized by Bdnf deficiency. Obesity associated with Bdnf deficiency did not account for the reduced hyperactivity in double mutant mice. Bdnf deficiency did not alter amyloid plaque formation in APdE9 mice. Before plaque formation (3 months), BDNF protein levels where either reduced (female) or unaltered (male) in the APdE9 mouse cortex. Unexpectedly, this was followed by an age-dependent increase in mature BDNF protein. Bdnf mRNA and phospho-TrkB levels remained unaltered in the cortical tissue samples of middle-aged APdE9 mice. Immunohistological studies revealed increased BDNF immunoreactivity around amyloid plaques indicating that the plaques may sequester BDNF protein and prevent it from activating TrkB. If similar BDNF accumulation happens in human AD brains, it would suggest that functional BDNF levels in the AD brains are even lower than reported, which could partially contribute to learning and memory problems of AD patients.

The antidepressant-like effects of glutamatergic drugs ketamine and AMPA receptor potentiator LY 451646 are preserved in bdnf⁺/⁻ heterozygous null mice.

Accumulating evidence suggests that biogenic amine-based antidepressants act, at least in part, via regulation of brain-derived neurotrophic factor (BDNF) signaling. Biogenic amine-based antidepressants increase BDNF synthesis and activate its signaling pathway through TrkB receptors. Moreover, the antidepressant-like effects of these molecules are abolished in BDNF deficient mice. Glutamate-based drugs, including the NMDA antagonist ketamine, and the AMPA receptor potentiator LY 451646, mimic the effects of antidepressants in preclinical tests with high predictive validity. In humans, a single intravenous dose of ketamine produces an antidepressant effect that is rapid, robust and persistent. In this study, we examined the role of BDNF in expression of the antidepressant-like effects of ketamine and an AMPA receptor potentiator (LY 451646) in the forced swim test (FST). Ketamine and LY 451646 produced antidepressant-like effects in the FST in mice at 45 min after a single injection, but no effects were observed one week after a single ketamine injection. As previously reported, the effects of imipramine in the forced swim test were blunted in heterozygous BDNF knockout (bdnf(+/-)) mice. However ketamine and LY 451646 produced similar antidepressant-like responses in wildtype and bdnf(+/-) mice. Neither ketamine nor LY 451646 significantly influenced the levels BDNF or TrkB phosphorylation in the hippocampus when assessed at 45 min or 7 days after the drug administration. These data demonstrate that under the conditions tested, neither ketamine nor the AMPA-potentiator LY 451656 activate BDNF signaling, but produce a characteristic antidepressant-like response in heterozygous bdnf(+/-) mice. These data indicate that unlike biogenic amine-based agents, BDNF signaling does not play a pivotal role in the antidepressant effects of glutamate-based compounds. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Impaired TrkB receptor signaling contributes to memory impairment in APP/PS1 mice.

Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal plasticity, learning, and memory. Levels of BDNF and its main receptor TrkB (TrkB.TK) have been reported to be decreased while the levels of the truncated TrkB (TrkB.T1) are increased in Alzheimer's disease. We show here that incubation with amyloid-ß increased TrkB.T1 receptor levels and decreased TrkB.TK levels in primary neurons. In vivo, APPswe/PS1dE9 transgenic mice (APdE9) showed an age-dependent relative increase in cortical but not hippocampal TrkB.T1 receptor levels compared with TrkB.TK. To investigate the role of TrkB isoforms in Alzheimer's disease, we crossed AP mice with mice overexpressing the truncated TrkB.T1 receptor (T1) or the full-length TrkB.TK isoform. Overexpression of TrkB.T1 in APdE9 mice exacerbated their spatial memory impairment while the overexpression of TrkB.TK alleviated it. These data suggest that amyloid-ß changes the ratio between TrkB isoforms in favor of the dominant-negative TrkB.T1 isoform both in vitro and in vivo and supports the role of BDNF signaling through TrkB in the pathophysiology and cognitive deficits of Alzheimer's disease.

Acetylcholinesterase inhibitors rapidly activate Trk neurotrophin receptors in the mouse hippocampus.

Acetylcholinesterase inhibitors are first-line therapies for Alzheimer's disease. These drugs increase cholinergic tone in the target areas of the cholinergic neurons of the basal forebrain. Basal forebrain cholinergic neurons are dependent upon trophic support by nerve growth factor (NGF) through its neurotrophin receptor, TrkA. In the present study, we investigated whether the acetylcholinesterase inhibitors donepezil and galantamine could influence neurotrophin receptor signaling in the brain. Acute administration of donepezil (3 mg/kg, i.p.) led to the rapid autophosphorylation of TrkA and TrkB neurotrophin receptors in the adult mouse hippocampus. Similarly, galantamine dose-dependently (3, 9 mg/kg, i.p.) increased TrkA and TrkB phosphorylation in the mouse hippocampus. Both treatments also increased the phosphorylation of transcription factor CREB and tended to increase the phosphorylation of AKT kinase but did not alter the activity of MAPK42/44. Chronic treatment with galantamine (3 mg/kg, i.p., 14 days), did not induce changes in hippocampal NGF and BDNF synthesis or protein levels. Our findings show that acetylcholinesterase inhibitors are capable of rapidly activating hippocampal neurotrophin signaling and thus suggest that therapies targeting Trk signaling may already be in clinical use in the treatment of AD.