Antidepressant drugs act by directly binding to TRKB neurotrophin receptors.

It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants in vitro and in vivo. We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery.

Mutation in the TRKB Cholesterol Recognition Site that blocks Antidepressant Binding does not Influence the Basal or BDNF-Stimulated Activation of TRKB.

Brain-derived neurotrophic factor (BDNF) acting upon its receptor Neurotrophic tyrosine kinase receptor 2 (NTRK2, TRKB) plays a central role in the development and maintenance of synaptic function and activity- or drug-induced plasticity. TRKB possesses an inverted cholesterol recognition and alignment consensus sequence (CARC), suggesting this receptor can act as a cholesterol sensor. We have recently shown that antidepressant drugs directly bind to the CARC domain of TRKB dimers, and that this binding as well as biochemical and behavioral responses to antidepressants are lost with a mutation in the TRKB CARC motif (Tyr433Phe). However, it is not clear if this mutation can also compromise the receptor function and lead to behavioral alterations. Here, we observed that Tyr433Phe mutation does not alter BDNF binding to TRKB, or BDNF-induced dimerization of TRKB. In this line, primary cultures from embryos of heterozygous Tyr433Phe mutant mice (hTRKB.Tyr433Phe) are responsive to BDNF-induced activation of TRKB, and samples from adult mice do not show any difference on TRKB activation compared to wild-type littermates (TRKB.wt). The behavioral phenotype of hTRKB.Tyr433Phe mice is indistinguishable from the wild-type mice in cued fear conditioning, contextual discrimination task, or the elevated plus maze, whereas mice heterozygous to BDNF null allele show a phenotype in context discrimination task. Taken together, our results indicate that Tyr433Phe mutation in the TRKB CARC motif does not show signs of loss-of-function of BDNF responses, while antidepressant binding to TRKB and responses to antidepressants are lost in Tyr433Phe mutants, making them an interesting mouse model for antidepressant research.

Mechanisms of secretion and spreading of pathological tau protein.

Accumulation of misfolded and aggregated forms of tau protein in the brain is a neuropathological hallmark of tauopathies, such as Alzheimer's disease and frontotemporal lobar degeneration. Tau aggregates have the ability to transfer from one cell to another and to induce templated misfolding and aggregation of healthy tau molecules in previously healthy cells, thereby propagating tau pathology across different brain areas in a prion-like manner. The molecular mechanisms involved in cell-to-cell transfer of tau aggregates are diverse, not mutually exclusive and only partially understood. Intracellular accumulation of misfolded tau induces several mechanisms that aim to reduce the cellular burden of aggregated proteins and also promote secretion of tau aggregates. However, tau may also be released from cells physiologically unrelated to protein aggregation. Tau secretion involves multiple vesicular and non-vesicle-mediated pathways, including secretion directly through the plasma membrane. Consequently, extracellular tau can be found in various forms, both as a free protein and in vesicles, such as exosomes and ectosomes. Once in the extracellular space, tau aggregates can be internalized by neighboring cells, both neurons and glial cells, via endocytic, pinocytic and phagocytic mechanisms. Importantly, accumulating evidence suggests that prion-like propagation of misfolding protein pathology could provide a general mechanism for disease progression in tauopathies and other related neurodegenerative diseases. Here, we review the recent literature on cellular mechanisms involved in cell-to-cell transfer of tau, with a particular focus in tau secretion.

Pharmacologically diverse antidepressants facilitate TRKB receptor activation by disrupting its interaction with the endocytic adaptor complex AP-2.

Several antidepressant drugs activate tropomyosin-related kinase B (TRKB) receptor, but it remains unclear whether these compounds employ a common mechanism for TRKB activation. Here, using MS, we found that a single intraperitoneal injection of fluoxetine disrupts the interaction of several proteins with TRKB in the hippocampus of mice. These proteins included members of adaptor protein complex-2 (AP-2) involved in vesicular endocytosis. The interaction of TRKB with the cargo-docking µ subunit of the AP-2 complex (AP2M) was confirmed to be disrupted by both acute and repeated fluoxetine treatments. Of note, fluoxetine disrupted the coupling between full-length TRKB and AP2M, but not the interaction between AP2M and the TRKB C-terminal region, indicating that the fluoxetine-binding site in TRKB lies outside the TRKB:AP2M interface. ELISA experiments revealed that in addition to fluoxetine, other chemically diverse antidepressants, such as imipramine, rolipram, phenelzine, ketamine, and its metabolite 2R,6R-hydroxynorketamine, also decreased the interaction between TRKB and AP2M in vitro Silencing the expression of AP2M in a TRKB-expressing mouse fibroblast cell line (MG87.TRKB) increased cell-surface expression of TRKB and facilitated its activation by brain-derived neurotrophic factor (BDNF), observed as levels of phosphorylated TRKB. Moreover, animals haploinsufficient for the Ap2m1 gene displayed increased levels of active TRKB, along with enhanced cell-surface expression of the receptor in cultured hippocampal neurons. Taken together, our results suggest that disruption of the TRKB:AP2M interaction is a common mechanism underlying TRKB activation by several chemically diverse antidepressants.

FRMD4A-cytohesin signaling modulates the cellular release of tau.

One of the defining pathological features of Alzheimer's disease is the intraneuronal accumulation of tau (also known as MAPT) protein. Tau is also secreted from neurons in response to various stimuli and accumulates in the cerebrospinal fluid of Alzheimer's disease patients. Tau pathology might spread from cell to cell through a mechanism involving secretion and uptake. Here, we developed an assay to follow cellular release and uptake of tau dimers. Individual silencing of ten common late-onset Alzheimer's disease risk genes in HEK293T cells expressing the tau reporters suggested that FRMD4A is functionally linked to tau secretion. FRMD4A depletion by using RNA interference (RNAi) reduced and overexpression increased tau secretion. The activity of cytohesins, interactors of FRMD4A and guanine-nucleotide-exchange factors of Arf6, was necessary for FRMD4A-induced tau secretion. Increased Arf6 and cell polarity signaling through Par6 and atypical protein kinase Cζ (aPKCζ) stimulated tau secretion. In mature cortical neurons, FRMD4A RNAi or inhibition of cytohesins strongly upregulated secretion of endogenous tau. These results suggest that FRMD4A, a genetic risk factor for late-onset Alzheimer's disease, regulates tau secretion by activating cytohesin-Arf6 signaling. We conclude that genetic risk factors of Alzheimer's disease might modulate disease progression by altering tau secretion.

Microtechnologies to fuel neurobiological research with nanometer precision.

The interface between engineering and molecular life sciences has been fertile ground for advancing our understanding of complex biological systems. Engineered microstructures offer a diverse toolbox for cellular and molecular biologists to direct the placement of cells and small organisms, and to recreate biological functions in vitro: cells can be positioned and connected in a designed fashion, and connectivity and community effects of cells studied. Because of the highly polar morphology and finely compartmentalized functions of neurons, microfabricated cell culture systems and related on-chip technologies have become an important enabling platform for studying development, function and degeneration of the nervous system at the molecular and cellular level. Here we review some of the compartmentalization techniques developed so far to highlight how high-precision control of neuronal connectivity allows new approaches for studying axonal and synaptic biology.

Psychedelics promote plasticity by directly binding to BDNF receptor TrkB.

Psychedelics produce fast and persistent antidepressant effects and induce neuroplasticity resembling the effects of clinically approved antidepressants. We recently reported that pharmacologically diverse antidepressants, including fluoxetine and ketamine, act by binding to TrkB, the receptor for BDNF. Here we show that lysergic acid diethylamide (LSD) and psilocin directly bind to TrkB with affinities 1,000-fold higher than those for other antidepressants, and that psychedelics and antidepressants bind to distinct but partially overlapping sites within the transmembrane domain of TrkB dimers. The effects of psychedelics on neurotrophic signaling, plasticity and antidepressant-like behavior in mice depend on TrkB binding and promotion of endogenous BDNF signaling but are independent of serotonin 2A receptor (5-HT2A) activation, whereas LSD-induced head twitching is dependent on 5-HT2A and independent of TrkB binding. Our data confirm TrkB as a common primary target for antidepressants and suggest that high-affinity TrkB positive allosteric modulators lacking 5-HT2A activity may retain the antidepressant potential of psychedelics without hallucinogenic effects.

Secretion of Tau via an Unconventional Non-vesicular Mechanism.

Tauopathies are characterized by cerebral accumulation of Tau protein aggregates that appear to spread throughout the brain via a cell-to-cell transmission process that includes secretion and uptake of pathological Tau, followed by templated misfolding of normal Tau in recipient cells. Here, we show that phosphorylated, oligomeric Tau clusters at the plasma membrane in N2A cells and is secreted in vesicle-free form in an unconventional process sensitive to changes in membrane properties, particularly cholesterol and sphingomyelin content. Cell surface heparan sulfate proteoglycans support Tau secretion, possibly by facilitating its release after membrane penetration. Notably, secretion of endogenous Tau from primary cortical neurons is mediated, at least partially, by a similar mechanism. We suggest that Tau is released from cells by an unconventional secretory mechanism that involves its phosphorylation and oligomerization and that membrane interaction may help Tau to acquire properties that allow its escape from cells directly through the plasma membrane.

Internalized Tau sensitizes cells to stress by promoting formation and stability of stress granules.

Stress granules are membrane-less RNA- and RNA-binding protein-containing complexes that are transiently assembled in stressful conditions to promote cell survival. Several stress granule-associated RNA-binding proteins have been associated with neurodegenerative diseases. In addition, a close link was recently identified between the stress granule core-nucleating protein TIA-1 and Tau. Tau is a central pathological protein in Alzheimer's disease and other tauopathies, and misfolded, aggregated Tau is capable of propagating pathology via cell-to-cell transmission. Here we show that following internalization hyperphosphorylated extracellular Tau associates with stress granules in a TIA-1 dependent manner. Cytosolic Tau normally only weakly interacts with TIA-1 but mutations mimicking abnormal phosphorylation promote this interaction. We show that internalized Tau significantly delays normal clearance of stress granules in the recipient cells sensitizing them to secondary stress. These results suggest that secreted Tau species may have properties, likely related to its hyperphosphorylation and oligomerization, which promote pathological association of internalized Tau with stress granules altering their dynamics and reducing cell viability. We suggest that stress granules and TIA-1 play a central role in the cell-to-cell transmission of Tau pathology.

Mitochondria and NMDA receptor-dependent toxicity of berberine sensitizes neurons to glutamate and rotenone injury.

The global incidence of metabolic and age-related diseases, including type 2 diabetes and Alzheimer's disease, is on the rise. In addition to traditional pharmacotherapy, drug candidates from complementary and alternative medicine are actively being pursued for further drug development. Berberine, a nutraceutical traditionally used as an antibiotic, has recently been proposed to act as a multi-target protective agent against type 2 diabetes, dyslipidemias, ischemic brain injury and neurodegenerative diseases, such as Parkinson's and Alzheimer's disease. However, the safety profile of berberine remains controversial, as isolated reports suggest risks with acute toxicity, bradycardia and exacerbation of neurodegeneration. We report that low micromolar berberine causes rapid mitochondria-dependent toxicity in primary neurons characterized by mitochondrial swelling, increased oxidative stress, decreased mitochondrial membrane potential and depletion of ATP content. Berberine does not induce caspase-3 activation and the resulting neurotoxicity remains unaffected by pan-caspase inhibitor treatment. Interestingly, inhibition of NMDA receptors by memantine and MK-801 completely blocked berberine-induced neurotoxicity. Additionally, subtoxic nanomolar concentrations of berberine were sufficient to sensitize neurons to glutamate excitotoxicity and rotenone injury. Our study highlights the need for further safety assessment of berberine, especially due to its tendency to accumulate in the CNS and the risk of potential neurotoxicity as a consequence of increasing bioavailability of berberine.