Biphasic regulation of epigenetic state by matrix stiffness during cell reprogramming.

We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.

Reduction of Intracellular Tension and Cell Adhesion Promotes Open Chromatin Structure and Enhances Cell Reprogramming.

The role of transcription factors and biomolecules in cell type conversion has been widely studied. Yet, it remains unclear whether and how intracellular mechanotransduction through focal adhesions (FAs) and the cytoskeleton regulates the epigenetic state and cell reprogramming. Here, it is shown that cytoskeletal structures and the mechanical properties of cells are modulated during the early phase of induced neuronal (iN) reprogramming, with an increase in actin cytoskeleton assembly induced by Ascl1 transgene. The reduction of actin cytoskeletal tension or cell adhesion at the early phase of reprogramming suppresses the expression of mesenchymal genes, promotes a more open chromatin structure, and significantly enhances the efficiency of iN conversion. Specifically, reduction of intracellular tension or cell adhesion not only modulates global epigenetic marks, but also decreases DNA methylation and heterochromatin marks and increases euchromatin marks at the promoter of neuronal genes, thus enhancing the accessibility for gene activation. Finally, micro- and nano-topographic surfaces that reduce cell adhesions enhance iN reprogramming. These novel findings suggest that the actin cytoskeleton and FAs play an important role in epigenetic regulation for cell fate determination, which may lead to novel engineering approaches for cell reprogramming.

Cytosolic domain regulates the calcium sensitivity and surface expression of BEST1 channels in the HEK293 cells.

BEST family is a class of Ca2+-activated Cl- channels evolutionary well conserved from bacteria to human. The human BEST paralogs (BEST1-BEST4) share significant amino acid sequence homology in the N-terminal region, which forms the transmembrane helicases and contains the direct calcium-binding site, Ca2+-clasp. But the cytosolic C-terminal region is less conserved in the paralogs. Interestingly, this domain-specific sequence conservation is also found in the BEST1 orthologs. However, the functional role of the C-terminal region in the BEST channels is still poorly understood. Thus, we aimed to understand the functional role of the C-terminal region in the human and mouse BEST1 channels by using electrophysiological recordings. We found that the calcium-dependent activation of BEST1 channels can be modulated by the C-terminal region. The C-terminal deletion hBEST1 reduced the Ca2+-dependent current activation and the hBEST1-mBEST1 chimera showed a significantly reduced calcium sensitivity to hBEST1 in the HEK293 cells. And the C-terminal domain could regulate cellular expression and plasma membrane targeting of BEST1 channels. Our results can provide a basis for understanding the C-terminal roles in the structure-function of BEST family proteins. [BMB Reports 2023; 56(3): 172-177].

Endocytic BDNF secretion regulated by Vamp3 in astrocytes.

Brain-derived neurotrophic factor (BDNF) regulates diverse brain functions via TrkB receptor signaling. Due to the expression of TrkB receptors, astrocytes can internalize extracellular BDNF proteins via receptor-mediated endocytosis. Endocytosed BDNF can be re-secreted upon stimulation, but the molecular mechanism underlying this phenomenon remains unrecognized. Our study reveals that vesicle-associated membrane protein 3 (Vamp3) selectively regulates the release of endocytic BDNF from astrocytes. By using quantum dot (QD)-conjugated mature BDNF (QD-BDNF) as a proxy for the extracellular BDNF protein, we monitored the uptake, transport, and secretion of BDNF from cultured cortical astrocytes. Our data showed that endocytic QD-BDNF particles were enriched in Vamp3-containing vesicles in astrocytes and that ATP treatment sufficiently triggered either the antero- or retrograde transport and exocytosis of QD-BDNF-containing vesicles. Downregulation of Vamp3 expression disrupted endocytic BDNF secretion from astrocytes but did not affect uptake or transport. Collectively, these results provide evidence of the selective ability of astrocytic Vamp3 to control endocytic BDNF secretion during BDNF recycling.

Astrocytes phagocytose adult hippocampal synapses for circuit homeostasis.

In the adult hippocampus, synapses are constantly formed and eliminated1,2. However, the exact function of synapse elimination in the adult brain, and how it is regulated, are largely unknown. Here we show that astrocytic phagocytosis3 is important for maintaining proper hippocampal synaptic connectivity and plasticity. By using fluorescent phagocytosis reporters, we find that excitatory and inhibitory synapses are eliminated by glial phagocytosis in the CA1 region of the adult mouse hippocampus. Unexpectedly, we found that astrocytes have a major role in the neuronal activity-dependent elimination of excitatory synapses. Furthermore, mice in which astrocytes lack the phagocytic receptor MEGF10 show a reduction in the elimination of excitatory synapses; as a result, excessive but functionally impaired synapses accumulate. Finally, Megf10-knockout mice show defective long-term synaptic plasticity and impaired formation of hippocampal memories. Together, our data provide strong evidence that astrocytes eliminate unnecessary excitatory synaptic connections in the adult hippocampus through MEGF10, and that this astrocytic function is crucial for maintaining circuit connectivity and thereby supporting cognitive function.

Labeling Dual Presynaptic Inputs using cFork Anterograde Tracing System.

Understanding brain function-related neural circuit connectivity is essential for investigating how cognitive functions are decoded in neural circuits. Trans-synaptic viral vectors are useful for identifying neural synaptic connectivity because of their ability to be transferred from transduced cells to synaptically connected cells. However, concurrent labeling of multisynaptic inputs to postsynaptic neurons is impossible with currently available trans-synaptic viral vectors. Here, we report a neural circuit tracing system that can simultaneously label postsynaptic neurons with two different markers, the expression of which is defined by presynaptic input connectivity. This system, called "cFork (see fork)", includes delivering serotype 1-packaged AAV vectors (AAV1s) containing Cre or flippase recombinase (FlpO) into two different presynaptic brain areas, and AAV5 with a dual gene expression cassette in postsynaptic neurons. Our in vitro and in vivo tests showed that selective expression of two different fluorescence proteins, EGFP and mScarlet, in postsynaptic neurons could be achieved by AAV1-mediated anterograde trans-synaptic transfer of Cre or FlpO constructs. When this tracing system was applied to the somatosensory barrel field cortex (S1BF) or striatum innervated by multiple presynaptic inputs, postsynaptic neurons defined by presynaptic inputs were simultaneously labeled with EGFP or mScarlet. Our new anterograde tracing tool may be useful for elucidating the complex multisynaptic connectivity of postsynaptic neurons regulating diverse brain functions.

Microtubule-associated protein 2 mediates induction of long-term potentiation in hippocampal neurons.

Microtubule-associated protein (MAP) 2 has been perceived as a static cytoskeletal protein enriched in neuronal dendritic shafts. Emerging evidence indicates dynamic functions for various MAPs in activity-dependent synaptic plasticity. However, it is unclear how MAP2 is associated with synaptic plasticity mechanisms. Here, we demonstrate that specific silencing of high-molecular-weight MAP2 in vivo abolished induction of long-term potentiation (LTP) in the Schaffer collateral pathway of CA1 pyramidal neurons and in vitro blocked LTP-induced surface delivery of AMPA receptors and spine enlargement. In mature hippocampal neurons, we observed rapid translocation of a subpopulation of MAP2, present in dendritic shafts, to spines following LTP stimulation. Time-lapse confocal imaging showed that spine translocation of MAP2 was coupled with LTP-induced spine enlargement. Consistently, immunogold electron microscopy revealed that LTP stimulation of the Schaffer collateral pathway promoted MAP2 labeling in spine heads of CA1 neurons. This translocation depended on NMDA receptor activation and Ras-MAPK signaling. Furthermore, LTP stimulation led to an increase in surface-expressed AMPA receptors specifically in the neurons with MAP2 spine translocation. Altogether, this study indicates a novel role for MAP2 in LTP mechanisms and suggests that MAP2 participates in activity-dependent synaptic plasticity in mature hippocampal networks.

Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions.

Postsynaptic density protein 95 (PSD-95) is a pivotal postsynaptic scaffolding protein in excitatory neurons. Although the transport and regulation of PSD-95 in synaptic regions is well understood, dendritic transport of PSD-95 before synaptic localization still remains to be clarified. To evaluate the role of KIF5, conventional kinesin, in the dendritic transport of PSD-95 protein, we expressed a transport defective form of KIF5A (ΔMD) that does not contain the N-terminal motor domain. Expression of ΔMD significantly decreased PSD-95 level in the dendrites. Consistently, KIF5 was associated with PSD-95 in in vitro and in vivo assays. This interaction was mediated by the C-terminal tail regions of KIF5A and the third PDZ domain of PSD-95. Additionally, the ADPDZ3 (the association domain of NMDA receptor and PDZ3 domain) expression significantly reduced the levels of PSD-95, glutamate receptor 1 (GluA1) in dendrites. The association between PSD-95 and KIF5A was dose-dependent on Staufen protein, suggesting that the Staufen plays a role as a regulatory role in the association. Taken together, our data suggest a new mechanism for dendritic transport of the AMPA receptor-PSD-95.

Balanced actions of protein synthesis and degradation in memory formation.

Storage of long-term memory requires not only protein synthesis but also protein degradation. In this article, we overview recent publications related to this issue, stressing that the balanced actions of protein synthesis and degradation are critical for long-term memory formation. We particularly focused on the brain-derived neurotrophic factor signaling that leads to protein synthesis; proteasome- and autophagy-dependent protein degradation that removes molecular constraints; the role of Fragile X mental retardation protein in translational suppression; and epigenetic modifications that control gene expression at the genomic level. Numerous studies suggest that an imbalance between protein synthesis and degradation leads to intellectual impairment and cognitive disorders.

Tweety-homolog (Ttyh) Family Encodes the Pore-forming Subunits of the Swelling-dependent Volume-regulated Anion Channel (VRACswell) in the Brain.

In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers Cl-- and osmolytes-efflux via volume-regulated anion channel (VRAC). Although LRRC8 family has been recently proposed as the pore-forming VRAC which is activated by low cytoplasmic ionic strength but not by swelling, the molecular identity of the pore-forming swelling-dependent VRAC (VRACswell) remains unclear. Here we identify and characterize Tweety-homologs (TTYH1, TTYH2, TTYH3) as the major VRACswell in astrocytes. Gene-silencing of all Ttyh1/2/3 eliminated hypo-osmotic-solution-induced Cl- conductance (ICl,swell) in cultured and hippocampal astrocytes. When heterologously expressed in HEK293T or CHO-K1 cells, each TTYH isoform showed a significant ICl,swell with similar aquaporin-4 dependency, pharmacological properties and glutamate permeability as ICl,swell observed in native astrocytes. Mutagenesis-based structure-activity analysis revealed that positively charged arginine residue at 165 in TTYH1 and 164 in TTYH2 is critical for the formation of the channel-pore. Our results demonstrate that TTYH family confers the bona fide VRACswell in the brain.

Adenomatous polyposis coli-stimulated GEF 1 (Asef1) is a negative regulator of excitatory synaptic function.

Guanine nucleotide exchange factors (GEFs) play important roles in many cellular processes, including regulation of the structural plasticity of dendritic spines. A GEF protein, adenomatous polyposis coli-stimulated GEF 1 (Asef1, ARHGEF4) is highly expressed in the nervous system. However, the function of Asef1 has not been investigated in neurons. Here, we present evidence showing that Asef1 negatively regulates the synaptic localization of postsynaptic density protein 95 (PSD-95) in the excitatory synapse by inhibiting Staufen-mediated synaptic localization of PSD-95. Accordingly, Asef1 expression impairs synaptic transmission in hippocampal cultured neurons. In addition, neuronal activity facilitates the dissociation of Asef1 from Staufen in a phosphoinositide 3 kinase (PI3K)-dependent manner. Taken together, our data reveal Asef1 functions as a negative regulator of synaptic localization of PSD-95 and synaptic transmission.

Anterior cingulate cortex and its input to the basolateral amygdala control innate fear response.

Prefrontal brain areas are implicated in the control of fear behavior. However, how prefrontal circuits control fear response to innate threat is poorly understood. Here, we show that the anterior cingulate cortex (ACC) and its input to the basolateral nucleus of amygdala (BLA) contribute to innate fear response to a predator odor in mice. Optogenetic inactivation of the ACC enhances freezing response to fox urine without affecting conditioned freezing. Conversely, ACC stimulation robustly inhibits both innate and conditioned freezing. Circuit tracing and slice patch recordings demonstrate a monosynaptic glutamatergic connectivity of ACC-BLA but no or very sparse ACC input to the central amygdala. Finally, our optogenetic manipulations of the ACC-BLA projection suggest its inhibitory control of innate freezing response to predator odors. Together, our results reveal the role of the ACC and its projection to BLA in innate fear response to olfactory threat stimulus.

Cortical Axonal Secretion of BDNF in the Striatum Is Disrupted in the Mutant-huntingtin Knock-in Mouse Model of Huntington's Disease.

Deficient BDNF signaling is known to be involved in neurodegenerative diseases such as Huntington's disease (HD). Mutant huntingtin (mhtt)-mediated disruption of either BDNF transcription or transport is thought to be a factor contributing to striatal atrophy in the HD brain. Whether and how activity-dependent BDNF secretion is affected by the mhtt remains unclear. In the present study, I provide evidence for differential effects of the mhtt on cortical BDNF secretion in the striatum during HD progression. By two-photon imaging of fluorescent BDNF sensor (BDNF-pHluorin and -EGFP) in acute striatal slices of HD knock-in model mice, I found deficient cortical BDNF secretion regardless of the HD onset, but antisense oligonucleotide (ASO)-mediated reduction of htts only rescues BDNF secretion in the early HD brain before the disease onset. Although secretion modes of individual BDNF-containing vesicle were not altered in the pre-symptomatic brain, the full-fusion and partial-fusion modes of BDNF-containing vesicles were significantly altered after the onset of HD symptoms. Thus, besides abnormal BDNF transcription and transport, our results suggest that mhtt-mediated alteration in activity-dependent BDNF secretion at corticostriatal synapses also contributes to the development of HD.

Channel-mediated astrocytic glutamate modulates hippocampal synaptic plasticity by activating postsynaptic NMDA receptors.

BACKGROUND: Activation of G protein coupled receptor (GPCR) in astrocytes leads to Ca(2+)-dependent glutamate release via Bestrophin 1 (Best1) channel. Whether receptor-mediated glutamate release from astrocytes can regulate synaptic plasticity remains to be fully understood. RESULTS: We show here that Best1-mediated astrocytic glutamate activates the synaptic N-methyl-D-aspartate receptor (NMDAR) and modulates NMDAR-dependent synaptic plasticity. Our data show that activation of the protease-activated receptor 1 (PAR1) in hippocampal CA1 astrocytes elevates the glutamate concentration at Schaffer collateral-CA1 (SC-CA1) synapses, resulting in activation of GluN2A-containing NMDARs and NMDAR-dependent potentiation of synaptic responses. Furthermore, the threshold for inducing NMDAR-dependent long-term potentiation (LTP) is lowered when astrocytic glutamate release accompanied LTP induction, suggesting that astrocytic glutamate is significant in modulating synaptic plasticity. CONCLUSIONS: Our results provide direct evidence for the physiological importance of channel-mediated astrocytic glutamate in modulating neural circuit functions.

Essential role of presynaptic NMDA receptors in activity-dependent BDNF secretion and corticostriatal LTP.

Activation of N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) in postsynaptic dendrites is required for long-term potentiation (LTP) of many excitatory synapses, but the role of presynaptic axonal NMDARs in synaptic plasticity remains to be clarified. Here we report that axonal NMDARs play an essential role in LTP induction at mouse corticostriatal synapses by triggering activity-induced presynaptic secretion of brain-derived neurotrophic factor (BDNF). Genetic depletion of either BDNF or the NMDAR subunit GluN1 specifically in cortical axons abolished corticostriatal LTP in response to theta burst stimulation (TBS). Furthermore, functional axonal NMDARs were required for TBS-triggered prolonged axonal Ca(2+) elevation and BDNF secretion, supporting the notion that activation of axonal NMDARs induces BDNF secretion via enhancing Ca(2+) signals in the presynaptic nerve terminals. These results demonstrate that presynaptic NMDARs are equally important as postsynaptic NMDARs in LTP induction of corticostriatal synapses due to their role in mediating activity-induced presynaptic BDNF secretion.

Spike-timing-dependent BDNF secretion and synaptic plasticity.

In acute hippocampal slices, we found that the presence of extracellular brain-derived neurotrophic factor (BDNF) is essential for the induction of spike-timing-dependent long-term potentiation (tLTP). To determine whether BDNF could be secreted from postsynaptic dendrites in a spike-timing-dependent manner, we used a reduced system of dissociated hippocampal neurons in culture. Repetitive pairing of iontophoretically applied glutamate pulses at the dendrite with neuronal spikes could induce persistent alterations of glutamate-induced responses at the same dendritic site in a manner that mimics spike-timing-dependent plasticity (STDP)-the glutamate-induced responses were potentiated and depressed when the glutamate pulses were applied 20 ms before and after neuronal spiking, respectively. By monitoring changes in the green fluorescent protein (GFP) fluorescence at the dendrite of hippocampal neurons expressing GFP-tagged BDNF, we found that pairing of iontophoretic glutamate pulses with neuronal spiking resulted in BDNF secretion from the dendrite at the iontophoretic site only when the glutamate pulses were applied within a time window of approximately 40 ms prior to neuronal spiking, consistent with the timing requirement of synaptic potentiation via STDP. Thus, BDNF is required for tLTP and BDNF secretion could be triggered in a spike-timing-dependent manner from the postsynaptic dendrite.

Efficient derivation of cortical glutamatergic neurons from human pluripotent stem cells: a model system to study neurotoxicity in Alzheimer's disease.

Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population, and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular, disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors, aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits, we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aß known as "globulomers", which have shown strong correlation with the level of cognitive deficits in AD. Administration of such Aß oligomers yielded signs of the disease, including cell culture age-dependent binding of Aß and cell death in the glutamatergic populations. Furthermore, consistent with previous findings in postmortem human AD brain, Aß-induced toxicity was selective for glutamatergic rather than GABAeric neurons present in our cultures. This in vitro model of cortical glutamatergic neurons thus offers a system for future mechanistic investigation and therapeutic development for AD pathology using human cell types specifically affected by this disease.

High glutamate permeability and distal localization of Best1 channel in CA1 hippocampal astrocyte.

BACKGROUND: Glutamate is the major neurotransmitter that mediates a principal form of excitatory synaptic transmission in the brain. From the presynaptic terminals of neurons, glutamate is released upon exocytosis of the glutamate-packaged vesicles. In recent years, astrocytes are also known to release glutamate via various routes to modulate synaptic transmission. In particular, we have characterized a glutamate-permeable Ca2+-activated anion channel encoded by Bestrophin 1 gene (Best1) that is responsible for Ca2+-dependent, channel-mediated glutamate release in astrocyte. Best1 channel contains a large pore that is readily permeable to large molecules such as glutamate and GABA. In those studies we obtained permeability ratio of glutamate to Cl- in heterologously expressed mouse Best1 in HEK293T cells and in endogenously expressed mouse Best1 in cultured astrocytes. However, up to now, glutamate permeability of the native Best1 channel in vivo has not been reported. FINDINGS: In whole-cell recordings of CA1 hippocampal astrocytes, we found that opening of Best1 channel upon activation of a Gq-coupled GPCR, protease-activated receptor 1 (PAR1) generated the anion current carried by glutamate via Ca2+ increase. This Ca2+-evoked glutamate-mediated anion current was unaffected by pretreatment of the inhibitors for a gap junction hemi-channel or Ca2+-activated K+ conductance. This astrocytic anion conductance carried by glutamate was mediated by Best1 channel expression in CA1 hippocampal astrocytes, because Best1 knock-down by shRNA expression eliminated astrocytic glutamate conductance by PAR-1 activation. However, we found that these astrocytes showed a deviation in reversal potential of Best1-mediated current from the predicted value. By performing dual patch recording, we concluded that the deviation of reversal potential is due to incomplete space clamping arising from extremely leaky membrane (input resistance ranging 1-3 MΩ), very low length constant of astrocytic processes, and the localization of Best1 channel in distal microdomains near synapses. Based on the relative shift of reversal potentials by ion substitutions, we estimated the permeability ratio of glutamate and Cl- (Pglutamate/PCl) as 0.53. CONCLUSIONS: Our study shows that Best1, located at the microdomains near the synaptic junctions, has a significantly high permeability to glutamate in vivo, serving as the prominent glutamate-releasing channel in astrocytes, mediating the release of various gliotransmitters in the brain, and playing an important role in modulating synaptic transmission.

Channel-mediated astrocytic glutamate release via Bestrophin-1 targets synaptic NMDARs.

BACKGROUND: Astrocytes regulate neuronal excitability and synaptic activity by releasing gliotransmitters such as glutamate. Our recent study demonstrated that astrocytes release glutamate upon GPCR activation via Ca2+ activated anion channel, Bestrophin-1 (Best1). The target of Best1-mediated astrocytic glutamate has been shown to be the neuronal NMDA receptors (NMDAR). However, whether it targets synaptically or extra-synaptically localized NMDAR is not known. FINDINGS: We recorded spontaneous miniature excitatory postsynaptic currents (mEPSCs) from CA1 pyramidal cells to test whether Best1-mediated astrocytic glutamate targets synaptic NMDAR. An agonist of protease activated receptor 1 (PAR1) was used to induce astrocytic Ca(2+) increase and glutamate release. Firstly, we found that activation of PAR1 and subsequent release of glutamate from astrocyte does not alone increase the frequency of mEPSCs. Secondly, we found that mEPSC rise time is variable depending on the different electrotonic distances from the somatic recording site to the synaptic region where each mEPSC occurs. Two subgroups of mEPSC from CA1 pyramidal neuron by rise time were selected and analyzed. One group is fast rising mEPSCs with a rise time of 1 ~ 5 ms, representing synaptic activities arising from proximal dendrites. The other group is slowly rising mEPSCs with a rise time of 5 ~ 10 ms, representing synaptic events arising from glutamate release at synapses located in the distal dendrites. We used cell-type specific Best1 gene silencing system by Cre-loxP cleavage to dissociate the effect of neuronal and astrocytic Best1. Astrocytic Best1-mediated glutamate release by PAR1 activation did not affect decay kinetics, frequency, and amplitude of fast rising mEPSC. In contrast, PAR1 activation resulted in an NMDA receptor component to be present on slowly rising mEPSC, but did not alter frequency or amplitude. CONCLUSIONS: Our results indicate that astrocytic glutamate via Best1 channel targets and activates synaptic NMDARs.