Dendrites Of Hippocampal Neurons Research Articles

Hippocampal Crhr1 conditional gene knockout ameliorated the depression-like behavior and pathological damage in male offspring mice caused by chronic stress during pregnancy

Numerous studies have demonstrated that chronic stress during pregnancy (CSDP) can induce depression and hippocampal damage in offspring. It has also been observed that high levels of corticotropin-releasing hormone (CRH) can damage hippocampal neurons, and intraperitoneal injection of a corticotropin releasing hormone receptor 1 (CRHR1) antagonist decreases depression-like behavior and hippocampal neuronal damage in a mouse depression model. However, whether CSDP causes hippocampal damage and depression in offspring through the interaction of CRH and hippocampal CRHR1 remains unknown and warrants further investigation. Therefore, hippocampal Crhr1 conditional gene knockout mice and C57/BL6J mice were used to study these questions. Depression-related indexs in male offspring mice were examined using the forced swim test (FST), sucrose preference test (SPT), tail suspension test (TST) and open field test (OFT). Serum CRH levels were measured by enzyme-linked immunosorbent assay (ELISA). Golgi-Cox staining was used to examine the morphological changes of hippocampal neuronal dendrites. Neuronal apoptosis in the hippocampal CA3 regions was detected by terminal deoxynucleotidy transferase dUTP nick end labeling (TUNEL) staining. The levels of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR) and protein kinase B (AKT) proteins were measured by Western blot analysis. This study showed that CSDP induces depression-like behavior, hippocampal neuronal dendrite damage and apoptosis in male offspring mice. Conditional gene knockout of hippocampal Crhr1 in mice reduced CSDP-induced depression-like behavior, hippocampal neuronal dendrite damage and apoptosis in male offspring, and counteracted the CSDP-induced decreased expression of p-Akt and mTOR activity in male offspring hippocampus. These findings demonstrated that CSDP might inhibit the Akt/mTOR pathway by increasing the levels of CRH, leading to increased CRH-mediated activation of hippocampal CRHR1, thereby inducing synaptic impairment and apoptosis in hippocampal neurons, which in turn leads to depression-like behavior in offspring.

Acupuncture Alleviates Chronic Ischemic White Matter Injury in SHR Rats via JNK-NMDAR Circuit.

To study the protective mechanism of acupuncture at "Jiangya Recipe" on chronic ischemic white matter injury in spontaneously hypertensive rats (SHR) and the regulation of Jun N-terminal kinase-N-methyl-D-aspartate receptor (JNK-NMDAR) loop. A hypertensive white matter injury model was established in 46 male SHR rats aged 11 weeks by bilateral common carotid artery tapering (SHR-2VGO). In the SHR sham operation group, only bilateral common carotid arteries were isolated and in the SHR-2VGO modeling group, 36 rats were used for microcoil spring clip implantation to narrow the common carotid arteries and then, after 2 weeks of modeling, rats with impaired motor function were removed, and SHR-2VGO rats with successful final models were randomly divided into the model group, JNK blocking group, and acupuncture group. The sham operation group, model group, and JNK blocking group underwent the same grasping fixation, and the acupuncture group received acupuncture at acupoints "Jiangya Fang" once daily. In the JNK blocker group, an injection cannula was implanted into the lateral ventricle and sp600125 was injected into the lateral ventricle at 4.5 ul/day for 4 weeks. One week after the end of the intervention, white matter lesions were detected by MRI DWI and T2 imaging, and the learning and memory ability of rats was tested by Y-Maze and Passive Avoidance. Myelin density was detected by luxol fast blue (LFB) staining, also axon arrangement, myelin integrity, and thickness of neurons were detected by electron microscopy; neuronal morphology and the number of Nissl bodies in the hippocampus were detected by Nissl staining, dendritic spine density changes were detected by Golgi staining, and JNK, NMDAR1, and N-methyl-D-receptor 2B (NMDAR2B) in DG, CA3 region of hippocampus were detected by immunohistochemistry, protein expression of p-JNK/JNK, p-NMDAR1/NMDAR1, NMDAR2B, GSK3β protein expression in the fimbria of hippocampus was detected by Western blot. The Y maze test of SHR-2VGO+Acu and SHR-2VGO+ sp600125 group showed that the spontaneous alternating reaction rate increased significantly. At the same time, the incubation period increased significantly and the number of errors decreased significantly in Passive Avoidance. MRI T2WI showed that the white matter high signal of the corpus callosum, internal capsule and hippocampal fimbria in the SHR-2VGO+ sp600125 and SHR-2VGO+Acu groups was significantly lower than that in the SHR-2VGO model group, and the striatum and anterior commissure were not obvious. DWI showed that the SHR-2VGO model group had scattered high signal and limited diffusion movement in both the internal capsule and striatum, but the difference between groups was not obvious. Compared with SHR-2VGO rats, LFB staining of SHR-2VGO + sp600125 and SHR-2VGO +Acu groups showed significant relaxation of myelin porosity in corpus callosum, striatum, inner capsule, anterior commissure and hippocampal fimbria, and electron microscopy showed improved axonal myelin integrity and thickness in corpus callosum region. Also, the number of blue patchy Nissl bodies increased, and the number and complexity of dendritic spines increased significantly in Golgi staining. Immunohistochemical detection showed that JNK levels in DG and CA3 region were increased and NMDAR1 and NMDAR2B levels were decreased in SHR-2VGO+Acu and SHR-2VGO+ sp600125 groups. Meanwhile, protein expressions of GSK3β, NMDAR1/p-NMDAR1 and NMDAR2B in fimbria of hippocampus were increased, and JNK/P-JNK protein expression decreased. Acupuncture can increase the density and thickness of myelin sheath in white matter areas of corpus callosum, anterior commissure and hippocampal fimbria, increase the number and length of hippocampal neuronal dendrites, and improve hypertensive white matter injury and cognitive decline through JNK-NMDAR pathway.

Klotho Regulated by Estrogen Plays a Key Role in Sex Differences in Stress Resilience in Rats.

Klotho (KL) is a glycosyl hydrolase and aging-suppressor gene. Stress is a risk factor for depression and anxiety, which are highly comorbid with each other. The aim of this study is to determine whether KL is regulated by estrogen and plays an important role in sex differences in stress resilience. Our results showed that KL is regulated by estrogen in rat hippocampal neurons in vivo and in vitro and is essential for the estrogen-mediated increase in the number of presynaptic vesicular glutamate transporter 1 (Vglut1)-positive clusters on the dendrites of hippocampal neurons. The role of KL in sex differences in stress response was examined in rats using 3-week chronic unpredictable mild stress (CUMS). CUMS produced a deficit in spatial learning and memory, anhedonic-like behaviors, and anxiety-like behaviors in male but not female rats, which was accompanied by a reduction in KL protein levels in the hippocampus of male but not female rats. This demonstrated the resilience of female rats to CUMS. Interestingly, the knockdown of KL protein levels in the rat hippocampus of both sexes caused a decrease in stress resilience in both sexes, especially in female rats. These results suggest that the regulation of KL by estrogen plays an important role in estrogen-mediated synapse formation and that KL plays a critical role in the sex differences in cognitive deficit, anhedonic-like behaviors, and anxiety-like behaviors induced by chronic stress in rats, highlighting an important role of KL in sex differences in stress resilience.

E-Syt1 Regulates Neuronal Activity-Dependent Endoplasmic Reticulum-Plasma Membrane Junctions and Surface Expression of AMPA Receptors.

Endoplasmic reticulum (ER)-plasma membrane (PM) contact sites/junctions play important roles in cell physiology including signal transduction, ion and lipid transfer, and membrane dynamics. However, little is known about the dynamic regulation and functional roles of ER-PM junctions in neurons. Using a split green fluorescent protein-based membrane contact probe, we find that the density of ER-PM contact sites changes dynamically in the dendrites of hippocampal neurons undergoing long-term synaptic potentiation (LTP). We show that the Ca2±-sensing membrane tethering protein Extended Synaptotagmin 1 (E-Syt1) mediates the formation of ER-PM contact sites during LTP. We also show that E-Syt1 is required for neuronal activity-dependent surface expression of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors. These findings implicate ER-PM junctions in the regulation of neurotransmitter receptor trafficking and synaptic plasticity.

Primary neurons lacking the SNAREs vti1a and vti1b show altered neuronal development

BackgroundNeurons are highly specialized cells with a complex morphology generated by various membrane trafficking steps. They contain Golgi outposts in dendrites, which are formed from somatic Golgi tubules. In trafficking membrane fusion is mediated by a specific combination of SNARE proteins. A functional SNARE complex contains four different helices, one from each SNARE subfamily (R-, Qa, Qb and Qc). Loss of the two Qb SNAREs vti1a and vti1b from the Golgi apparatus and endosomes leads to death at birth in mice with massive neurodegeneration in peripheral ganglia and defective axon tracts.MethodsHippocampal and cortical neurons were isolated from Vti1a−/−Vti1b−/− double deficient, Vti1a−/−Vti1b+/−, Vti1a+/−Vti1b−/− and Vti1a+/−Vti1b+/− double heterozygous embryos. Neurite outgrowth was determined in cortical neurons and after stimulation with several neurotrophic factors or the Rho-associated protein kinase ROCK inhibitor Y27632, which induces exocytosis of enlargeosomes, in hippocampal neurons. Moreover, postsynaptic densities were isolated from embryonic Vti1a−/−Vti1b−/− and Vti1a+/−Vti1b+/− control forebrains and analyzed by western blotting.ResultsGolgi outposts were present in Vti1a−/−Vti1b+/− and Vti1a+/−Vti1b−/− dendrites of hippocampal neurons but not detected in the absence of vti1a and vti1b. The length of neurites was significantly shorter in double deficient cortical neurons. These defects were not observed in Vti1a−/−Vti1b+/− and Vti1a+/−Vti1b−/− neurons. NGF, BDNF, NT-3, GDNF or Y27632 as stimulator of enlargeosome secretion did not increase the neurite length in double deficient hippocampal neurons. Vti1a−/−Vti1b−/− postsynaptic densities contained similar amounts of scaffold proteins, AMPA receptors and NMDA receptors compared to Vti1a+/−Vti1b+/−, but much more TrkB, which is the receptor for BDNF.ConclusionThe absence of Golgi outposts did not affect the amount of AMPA and NMDA receptors in postsynaptic densities. Even though TrkB was enriched, BDNF was not able to stimulate neurite elongation in Vti1a−/−Vti1b−/− neurons. Vti1a or vti1b function as the missing Qb-SNARE together with VAMP-4 (R-SNARE), syntaxin 16 (Qa-SNARE) and syntaxin 6 (Qc-SNARE) in induced neurite outgrowth. Our data show the importance of vti1a or vti1b for two pathways of neurite elongation.

Blue-Light-Blocking Lenses Ameliorate Structural Alterations in the Rodent Hippocampus.

Evidence suggests that prolonged blue-light exposure can impact vision; however, less is known about its impact on non-visual higher-order functions in the brain, such as learning and memory. Blue-light-blocking lenses (BBLs) claim to reduce these potential impacts. Hence, we assessed structural and functional hippocampal alterations following blue-light exposure and the protective efficacy of BBLs. Male Wistar rats were divided into (n = 6 in each group) normal control (NC), blue-light exposure (LE), and blue-light with BBLs (Crizal Prevencia, CP and DuraVision Blue, DB) groups. After 28 days of light exposure (12:12 light: dark cycle), rats were trained for the Morris water maze memory retention test, and brain tissues were sectioned for hippocampal neuronal analysis using Golgi and Cresyl violet stains. The memory retention test was significantly delayed (p < 0.05) in LE compared with DB groups on day 1 of training. Comparison of Golgi-stained neurons showed significant structural alterations, particularly in the basal dendrites of hippocampal neurons in the LE group, with BBLs significantly mitigating these structural changes (p < 0.05). Comparison of Cresyl-violet-stained neurons revealed significantly (p < 0.001) increased degenerated hippocampal neurons in LE rats, with fewer degenerated neurons in the CP lens group for CA1 neurons (p < 0.05), and for both CP and DB groups (p < 0.05) for CA3 neurons. Thus, in addition to documented effects on visual centers, high-level blue-light exposure also results in degeneration in hippocampal neurons with associated behavioral deficits. These changes can be partially ameliorated with blue-light-blocking lenses.

Cytoskeleton Protein EB3 Contributes to Dendritic Spines Enlargement and Enhances Their Resilience to Toxic Effects of Beta-Amyloid.

EB3 protein is expressed abundantly in the nervous system and transiently enters the dendritic spines at the tip of the growing microtubule, which leads to spine enlargement. Nevertheless, the role of dynamic microtubules, and particularly EB3 protein, in synapse function is still elusive. By manipulating the EB3 expression level, we have shown that this protein is required for a normal dendritogenesis. Nonetheless, EB3 overexpression also reduces hippocampal neurons dendritic branching and total dendritic length. This effect likely occurs due to the speeding neuronal development cycle from dendrite outgrowth to the step when dendritic spines are forming. Implementing direct morphometric characterization of dendritic spines, we showed that EB3 overexpression leads to a dramatic increase in the dendritic spine head area. EB3 knockout oppositely reduces spine head area and increases spine neck length and spine neck/spine length ratio. The same effect is observed in conditions of amyloid-beta toxicity, modeling Alzheimer`s disease. Neck elongation is supposed to be a common detrimental effect on the spine’s shape, which makes them biochemically and electrically less connected to the dendrite. EB3 also potentiates the formation of presynaptic protein Synapsin clusters and CaMKII-alpha preferential localization in spines rather than in dendrites of hippocampal neurons, while its downregulation has an opposite effect and reduces the size of presynaptic protein clusters Synapsin and PSD95. EB3′s role in spine development and maturation determines its neuroprotective effect. EB3 overexpression makes dendritic spines resilient to amyloid-beta toxicity, restores altered PSD95 clustering, and reduces CaMKII-alpha localization in spines observed in this pathological state.

LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function.

Lysosomes function not only as degradatory compartments but also as dynamic intracellular calcium ion stores. The transient receptor potential mucolipin 1 (TRPML1) channel mediates lysosomal Ca2+ release, thereby participating in multiple cellular functions. The pentameric Ragulator complex, which plays a critical role in the activation of mTORC1, is also involved in lysosomal trafficking and is anchored to lysosomes through its LAMTOR1 subunit. Here, we report that the Ragulator restricts lysosomal trafficking in dendrites of hippocampal neurons via LAMTOR1‐mediated tonic inhibition of TRPML1 activity, independently of mTORC1. LAMTOR1 directly interacts with TRPML1 through its N‐terminal domain. Eliminating this inhibition in hippocampal neurons by LAMTOR1 deletion or by disrupting LAMTOR1‐TRPML1 binding increases TRPML1‐mediated Ca2+ release and facilitates dendritic lysosomal trafficking powered by dynein. LAMTOR1 deletion in the hippocampal CA1 region of adult mice results in alterations in synaptic plasticity, and in impaired object‐recognition memory and contextual fear conditioning, due to TRPML1 activation. Mechanistically, changes in synaptic plasticity are associated with increased GluA1 dephosphorylation by calcineurin and lysosomal degradation. Thus, LAMTOR1‐mediated inhibition of TRPML1 is critical for regulating dendritic lysosomal motility, synaptic plasticity, and learning.

Regulation of neuronal excitation–transcription coupling by Kv2.1-induced clustering of somatic L-type Ca2+ channels at ER-PM junctions

In mammalian brain neurons, membrane depolarization leads to voltage-gated Ca2+ channel-mediated Ca2+ influx that triggers diverse cellular responses, including gene expression, in a process termed excitation-transcription coupling. Neuronal L-type Ca2+ channels, which have prominent populations on the soma and distal dendrites of hippocampal neurons, play a privileged role in excitation-transcription coupling. The voltage-gated K+ channel Kv2.1 organizes signaling complexes containing the L-type Ca2+ channel Cav1.2 at somatic endoplasmic reticulum-plasma membrane junctions. This leads to enhanced clustering of Cav1.2 channels, increasing their activity. However, the downstream consequences of the Kv2.1-mediated regulation of Cav1.2 localization and function on excitation-transcription coupling are not known. Here, we have identified a region between residues 478 to 486 of Kv2.1's C terminus that mediates the Kv2.1-dependent clustering of Cav1.2. By disrupting this Ca2+ channel association domain with either mutations or with a cell-penetrating interfering peptide, we blocked the Kv2.1-mediated clustering of Cav1.2 at endoplasmic reticulum-plasma membrane junctions and the subsequent enhancement of its channel activity and somatic Ca2+ signals without affecting the clustering of Kv2.1. These interventions abolished the depolarization-induced and L-type Ca2+ channel-dependent phosphorylation of the transcription factor CREB and the subsequent expression of c-Fos in hippocampal neurons. Our findings support a model whereby the Kv2.1-Ca2+ channel association domain-mediated clustering of Cav1.2 channels imparts a mechanism to control somatic Ca2+ signals that couple neuronal excitation to gene expression.

Morphological alteration in rat hippocampal neuronal dendrites following chronic binge prenatal alcohol exposure

Prenatal alcohol exposure (PAE) may result in Fetal Alcohol Spectrum Disorders (FASD). The hippocampus has been recognized as a vulnerable target to alcohol-induced developmental damage. However, the effect of prenatal exposure to alcohol on dendritic morphological adaptations throughout the hippocampal fields in the developing brain still remains largely unknown in the context of FASD. We hypothesized that chronic binge alcohol exposure during pregnancy alters dendrite arborization throughout the developing rat hippocampus. Pregnant Sprague-Dawley rats were assigned to either a pair-fed control (PF-Cont) or a binge alcohol (Alcohol) treatment group. Alcohol dams were acclimatized via a once-daily orogastric gavage of 4.5 g/kg alcohol from gestational day (GD) 5–10 and progressed to 6 g/kg alcohol from GD 11–21. Pair-fed dams similarly received isocaloric maltose dextrin. After parturition, all dams received an ad libitum diet and nursed their offspring until postnatal day (PND) 10 when the pup brains were collected for morphological analysis. PAE increased dendritic arborization and complexities of CA1, CA2/3, and DG neurons in the PND 10 rat hippocampus. The number of primary dendrites, total dendritic length, and number of dendritic branches were significantly increased following PAE, and Sholl analysis revealed significantly more intersections of the dendritic processes in PND 10 offspring following PAE compared with those in the PF-Cont group. We conclude that chronic binge PAE significantly alters hippocampal dendritic morphology in the developing hippocampus. We conjecture that this morphological alteration in postnatal rat hippocampal dendrites following chronic binge prenatal alcohol exposure may play a critical role in FASD neurobiological phenotypes.

Distance-dependent regulation of NMDAR nanoscale organization along hippocampal neuron dendrites

Hippocampal pyramidal neurons are characterized by a unique arborization subdivided in segregated dendritic domains receiving distinct excitatory synaptic inputs with specific properties and plasticity rules that shape their respective contributions to synaptic integration and action potential firing. Although the basal regulation and plastic range of proximal and distal synapses are known to be different, the composition and nanoscale organization of key synaptic proteins at these inputs remains largely elusive. Here we used superresolution imaging and single nanoparticle tracking in rat hippocampal neurons to unveil the nanoscale topography of native GluN2A- and GluN2B-NMDA receptors (NMDARs)-which play key roles in the use-dependent adaptation of glutamatergic synapses-along the dendritic arbor. We report significant changes in the nanoscale organization of GluN2B-NMDARs between proximal and distal dendritic segments, whereas the topography of GluN2A-NMDARs remains similar along the dendritic tree. Remarkably, the nanoscale organization of GluN2B-NMDARs at proximal segments depends on their interaction with calcium/calmodulin-dependent protein kinase II (CaMKII), which is not the case at distal segments. Collectively, our data reveal that the nanoscale organization of NMDARs changes along dendritic segments in a subtype-specific manner and is shaped by the interplay with CaMKII at proximal dendritic segments, shedding light on our understanding of the functional diversity of hippocampal glutamatergic synapses.

Hippocalcin Distribution between the Cytosol and Plasma Membrane of Living Cells

Hippocalcin (HPCA) is a neuronal calcium sensor (NCS) protein that provides intracellular signaling via its Ca2+-dependent translocation from the cytosol to the plasma membrane. Although such translocation of HPCA and other NCS proteins is well established, no quantitative estimates of HPCA distribution between the cytosol and plasma membrane at the basal level of the intracellular free calcium concentration ([Ca2+]i) have been obtained. Thus, it is still unknown whether HPCA regulates its plasma membrane targets under resting conditions. In this work, we have evaluated this distribution in living cells at the basal level of [Ca2+]i, comparing HPCA spatial distribution with that of exogenously expressed membrane protein, EYFP-Mem. Using fluorescence recovery after photobleaching, we showed that the diffusion coefficient of fluorescently tagged HPCA in the dendrites of hippocampal neurons (~40 μm2/sec) is within the range of values typical of cytosolic proteins. The coefficient was about 10-fold higher than that for EYFP-Mem. Both results indicate that HPCA is mainly localized in the cytosol of the dendrites of hippocampal neurons. Next, direct calculations for HPCA fractions in the cytosol and plasma membrane at the basal level of [Ca2+]i demonstrated that the plasma membrane fraction of HPCA does not exceed 8%. Altogether, our results suggest that although being mainly cytosolic, HPCA may potentially signal its plasma membrane targets under resting conditions. New approaches used for obtaining such estimates can be applied for quantitative evaluation of NCS protein distribution in living cells under different experimental conditions and for the development of precise biophysical models of their signaling.

Myosin V regulates synaptopodin clustering and localization in the dendrites of hippocampal neurons.

ABSTRACTThe spine apparatus (SA) is an endoplasmic reticulum-related organelle that is present in a subset of dendritic spines in cortical and pyramidal neurons, and plays an important role in Ca2+ homeostasis and dendritic spine plasticity. The protein synaptopodin is essential for the formation of the SA and is widely used as a maker for this organelle. However, it is still unclear which factors contribute to its localization at selected synapses, and how it triggers local SA formation. In this study, we characterized development, localization and mobility of synaptopodin clusters in hippocampal primary neurons, as well as the molecular dynamics within these clusters. Interestingly, synaptopodin at the shaft-associated clusters is less dynamic than at spinous clusters. We identify the actin-based motor proteins myosin V (herein referring to both the myosin Va and Vb forms) and VI as novel interaction partners of synaptopodin, and demonstrate that myosin V is important for the formation and/or maintenance of the SA. We found no evidence of active microtubule-based transport of synaptopodin. Instead, new clusters emerge inside spines, which we interpret as the SA being assembled on-site.

Direct investigation of charge transfer in neurons by electrostatic force microscopy

Charge transfer plays fundamental roles in information transmission in cells, especially in neurons. To date, direct observation of charge propagation in neurons at nanometer level has not been achieved yet. Herein, a combined charge injection and Electrostatic Force Microscopy (EFM) detection approach is applied to directly study charge propagation and distribution at nanometer resolution in spines and synapses of hippocampal neurons. Charge density, charge mobility and membrane potential in neural signal transmission process through the spines of axons and dendrites of hippocampal neurons were investigated quantitatively. Postsynaptic densities (PSD) in spines of axons and dendrites were revealed and studied. The methods and results from present work provide insights into physiological activities and processes related with electrical properties in nervous system and other biological samples.

S66. Therapeutic effects of an AMPA receptor antagonist on fragile X syndrome and cerebellar coordination disorders/involuntary movements

Introduction To examine the effectiveness of an AMPA receptor antagonist (perampanel: PER) for treating cerebellar coordination disorders/involuntary movements with the genetic loss of FMRP protein, which regulates AMPA receptor expression in Purkinje cells. Fragile X syndrome causes mental retardation and cerebellar motor disorders through CGG repeat expansion in the 5′-untranslated region of the FMR1 gene (chX 27.3). The FMRP protein encoded by the FMR1 gene is responsible for translational repression of the signaling of a metabotropic glutamate receptor, mGluR. In FMRP-protein knockout animals, abnormal phenotypes are caused by mGluR-dependent LTD, resulting in morphological abnormalities of the hippocampal neuronal dendrites, which were improved by mGluR antagonist administration ( Bear et al., 2004 ). In Japan, PER is covered by insurance as a combination therapy for epilepsy. Methods The present study was approved by the ethics committee of our hospital, with consent obtained from patients and their families. Two patients with fragile X syndrome (16-year-old male with FMR1-full mutation and 68-year-old female with FMRI-premutation) were included. One case of Usher syndrome and two cases of LCCA were included as disease controls. The administration of PER was started at 2 mg/day, and maintained at 4 mg/day in week 2 and later to examine effectiveness for 12 weeks. Motor symptoms and involuntary movements were evaluated using an international standard score, ICARS, and MDS-UPDRS part III-3, respectively 15–18. AMPA-R protein (130 m/kDa) expressed on the peripheral blood lymphocytes before drug administration was examined by Western blotting (MyBioSource, AMPA receptor (GluR4) rabbit polyclonal antibody). Results Motor and gait functions were markedly improved in the cases of fragile X syndrome (ICARS-motor; −9, gait; −3). Involuntary movement scores were improved in all cases (−12). AMPA-R protein on lymphocytes was detected in the cases of fragile X syndrome, but was not in the disease controls. Conclusion PER is effective for treating cerebellar involuntary movements in fragile X syndrome, and may have LTP/disinhibiting effects through AMPA-R blocking in Purkinje cells. Changes in lymphocyte AMPA-R expression level suggested that AMPA-R expression pattern might be changed in fragile X syndrome.

Correction: Optogenetic activation of EphB2 receptor in dendrites induced actin polymerization by activating Arg kinase

Erythropoietin-producing hepatocellular (Eph) receptors regulate a wide array of developmental processes by responding to cell-cell contacts. EphB2 is well-expressed in the brain and known to be important for dendritic spine development, as well as for the maintenance of the synapses, although the mechanisms of these functions have not been fully understood. Here we studied EphB2's functions in hippocampal neurons with an optogenetic approach, which allowed us to specify spatial regions of signal activation and monitor in real-time the consequences of signal activation. We designed and constructed OptoEphB2, a genetically encoded photoactivatable EphB2. Photoactivation of OptoEphB2 in fibroblast cells induced receptor phosphorylation and resulted in cell rounding ------- a well-known cellular response to EphB2 activation. In contrast, local activation of OptoEphb2 in dendrites of hippocampal neurons induces rapid actin polymerization, resulting dynamic dendritic filopodial growth. Inhibition of Rac1 and CDC42 did not abolish OptoEphB2-induced actin polymerization. Instead, we identified Abelson tyrosine-protein kinase 2 (Abl2/Arg) as a necessary effector in OptoEphB2-induced filopodia growth in dendrites. These findings provided new mechanistic insight into EphB2's role in neural development and demonstrated the advantage of OptoEphB as a new tool for studying EphB signaling.

Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of CaV1.2 calcium channels in hippocampal neurons

L-type voltage-gated CaV1.2 calcium channels (CaV1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed CaV1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of CaV1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of CaV1.2 are completely unexplored. Here, we examined the dynamic states of CaV1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in CaV1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered CaV1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of CaV1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of CaV1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of CaV1.2 at signaling complexes.

Activity-Dependent Arc Expression and Homeostatic Synaptic Plasticity Are Altered in Neurons from a Mouse Model of Angelman Syndrome.

Angelman syndrome (AS) is a neurodevelopmental disorder that results from deletions or mutations in chromosome 15, which usually includes the UBE3A gene. Ube3A protein is an E3 ubiquitin ligase that ubiquitinates proteins and targets them for degradation. The immediate-early gene Arc, a master regulator of synaptic plasticity, was identified as a putative substrate of Ube3A, but there have been conflicting reports on whether Arc is a bona fide E3 ligase substrate. Using multiple approaches, we found no evidence for a physical interaction between Arc and Ube3A in vivo. Nonetheless, activity-induced subcellular distribution of Arc is altered in brains from Ube3am−/p+ mice, with abnormal concentration of Arc at synapses. Furthermore, although activation of Arc transcription is normal, the stability of Arc protein is enhanced in dendrites of hippocampal neurons cultured from Ube3am−/p+ mice. Finally, homeostatic synaptic scaling of surface AMPA receptors does not occur in Ube3am−/p+ hippocampal neurons, reminiscent of neurons that lack Arc protein. Although Ube3A does not seem to bind Arc in a canonical E3 ligase-substrate interaction, Arc-dependent synaptic plasticity is still altered in Ube3am−/p+ mice, which may underlie the cognitive deficits observed in AS.

Dynein efficiently navigates the dendritic cytoskeleton to drive the retrograde trafficking of BDNF/TrkB signaling endosomes.

The efficient transport of cargoes within axons and dendrites is critical for neuronal function. Although we have a basic understanding of axonal transport, much less is known about transport in dendrites. We used an optogenetic approach to recruit motor proteins to cargo in real time within axons or dendrites in hippocampal neurons. Kinesin-1, a robust axonal motor, moves cargo less efficiently in dendrites. In contrast, cytoplasmic dynein efficiently navigates both axons and dendrites; in both compartments, dynamic microtubule plus ends enhance dynein-dependent transport. To test the predictions of the optogenetic assay, we examined the contribution of dynein to the motility of an endogenous dendritic cargo and found that dynein inhibition eliminates the retrograde bias of BDNF/TrkB trafficking. However, inhibition of microtubule dynamics has no effect on BDNF/TrkB motility, suggesting that dendritic kinesin motors may cooperate with dynein to drive the transport of signaling endosomes into the soma. Collectively our data highlight compartment-specific differences in kinesin activity that likely reflect specialized tuning for localized cytoskeletal determinants, whereas dynein activity is less compartment specific but is more responsive to changes in microtubule dynamics.

Densin-180 Controls the Trafficking and Signaling of L-Type Voltage-Gated Cav1.2 Ca2+ Channels at Excitatory Synapses.

Voltage-gated Cav1.2 and Cav1.3 (L-type) Ca2+ channels regulate neuronal excitability, synaptic plasticity, and learning and memory. Densin-180 (densin) is an excitatory synaptic protein that promotes Ca2+-dependent facilitation of voltage-gated Cav1.3 Ca2+ channels in transfected cells. Mice lacking densin (densin KO) exhibit defects in synaptic plasticity, spatial memory, and increased anxiety-related behaviors-phenotypes that more closely match those in mice lacking Cav1.2 than Cav1.3. Therefore, we investigated the functional impact of densin on Cav1.2. We report that densin is an essential regulator of Cav1.2 in neurons, but has distinct modulatory effects compared with its regulation of Cav1.3. Densin binds to the N-terminal domain of Cav1.2, but not that of Cav1.3, and increases Cav1.2 currents in transfected cells and in neurons. In transfected cells, densin accelerates the forward trafficking of Cav1.2 channels without affecting their endocytosis. Consistent with a role for densin in increasing the number of postsynaptic Cav1.2 channels, overexpression of densin increases the clustering of Cav1.2 in dendrites of hippocampal neurons in culture. Compared with wild-type mice, the cell surface levels of Cav1.2 in the brain, as well as Cav1.2 current density and signaling to the nucleus, are reduced in neurons from densin KO mice. We conclude that densin is an essential regulator of neuronal Cav1 channels and ensures efficient Cav1.2 Ca2+ signaling at excitatory synapses.SIGNIFICANCE STATEMENT The number and localization of voltage-gated Cav Ca2+ channels are crucial determinants of neuronal excitability and synaptic transmission. We report that the protein densin-180 is highly enriched at excitatory synapses in the brain and enhances the cell surface trafficking and postsynaptic localization of Cav1.2 L-type Ca2+ channels in neurons. This interaction promotes coupling of Cav1.2 channels to activity-dependent gene transcription. Our results reveal a mechanism that may contribute to the roles of Cav1.2 in regulating cognition and mood.