Regulation of the Ca2+ Channel CaV1.2 Supports Spatial Memory and Its Flexibility and LTD.

Widespread release of norepinephrine (NE) throughout the forebrain fosters learning and memory via adrenergic receptor (AR) signaling, but the molecular mechanisms are largely unknown. The ß2 AR and its downstream effectors, the trimeric stimulatory Gs-protein, adenylyl cyclase (AC), and the cAMP-dependent protein kinase A (PKA), form a unique signaling complex with the L-type Ca2+ channel (LTCC) CaV1.2. Phosphorylation of CaV1.2 by PKA on Ser1928 is required for the upregulation of Ca2+ influx on ß2 AR stimulation and long-term potentiation induced by prolonged theta-tetanus (PTT-LTP) but not LTP induced by two 1-s-long 100-Hz tetani. However, the function of Ser1928 phosphorylation in vivo is unknown. Here, we show that S1928A knock-in (KI) mice of both sexes, which lack PTT-LTP, express deficiencies during initial consolidation of spatial memory. Especially striking is the effect of this mutation on cognitive flexibility as tested by reversal learning. Mechanistically, long-term depression (LTD) has been implicated in reversal learning. It is abrogated in male and female S1928A knock-in mice and by ß2 AR antagonists and peptides that displace ß2 AR from CaV1.2. This work identifies CaV1.2 as a critical molecular locus that regulates synaptic plasticity, spatial memory and its reversal, and LTD.SIGNIFICANCE STATEMENT We show that phosphorylation of the Ca2+ channel CaV1.2 on Ser1928 is important for consolidation of spatial memory and especially its reversal, and long-term depression (LTD). Identification of Ser1928 as critical for LTD and reversal learning supports the model that LTD underlies flexibility of reference memory.

Deep phenotyping of cardiac function in heart transplant patients using cardiovascular system models.

KEY POINTS: Right heart catheterization data from clinical records of heart transplant patients are used to identify patient-specific models of the cardiovascular system. These patient-specific cardiovascular models represent a snapshot of cardiovascular function at a given post-transplant recovery time point. This approach is used to describe cardiac function in 10 heart transplant patients, five of which had multiple right heart catheterizations allowing an assessment of cardiac function over time. These patient-specific models are used to predict cardiovascular function in the form of right and left ventricular pressure-volume loops and ventricular power, an important metric in the clinical assessment of cardiac function. Outcomes for the longitudinally tracked patients show that our approach was able to identify the one patient from the group of five that exhibited post-transplant cardiovascular complications. ABSTRACT: Heart transplant patients are followed with periodic right heart catheterizations (RHCs) to identify post-transplant complications and guide treatment. Post-transplant positive outcomes are associated with a steady reduction of right ventricular and pulmonary arterial pressures, toward normal levels of right-side pressure (about 20 mmHg) measured by RHC. This study shows that more information about patient progression is obtained by combining standard RHC measures with mechanistic computational cardiovascular system models. The purpose of this study is twofold: to understand how cardiovascular system models can be used to represent a patient's cardiovascular state, and to use these models to track post-transplant recovery and outcome. To obtain reliable parameter estimates comparable within and across datasets, we use sensitivity analysis, parameter subset selection, and optimization to determine patient-specific mechanistic parameters that can be reliably extracted from the RHC data. Patient-specific models are identified for 10 patients from their first post-transplant RHC, and longitudinal analysis is carried out for five patients. Results of the sensitivity analysis and subset selection show that we can reliably estimate seven non-measurable quantities; namely, ventricular diastolic relaxation, systemic resistance, pulmonary venous elastance, pulmonary resistance, pulmonary arterial elastance, pulmonary valve resistance and systemic arterial elastance. Changes in parameters and predicted cardiovascular function post-transplant are used to evaluate the cardiovascular state during recovery of five patients. Of these five patients, only one showed inconsistent trends during recovery in ventricular pressure-volume relationships and power output. At the four-year post-transplant time point this patient exhibited biventricular failure along with graft dysfunction while the remaining four exhibited no cardiovascular complications.

SemGen: a tool for semantics-based annotation and composition of biosimulation models.

SUMMARY: As the number and complexity of biosimulation models grows, so do demands for tools that can help users understand models and compose more comprehensive and accurate systems from existing models. SemGen is a tool for semantics-based annotation and composition of biosimulation models designed to address this demand. A key SemGen capability is to decompose and then integrate models across existing model exchange formats including SBML and CellML. To support this capability, we use semantic annotations to explicitly capture the underlying biological and physical meanings of the entities and processes that are modeled. SemGen leverages annotations to expose a model's biological and computational architecture and to help automate model composition. AVAILABILITY AND IMPLEMENTATION: SemGen is freely available at https://github.com/SemBioProcess/SemGen. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Autophosphorylation of F-actin binding domain of CaMKIIß is required for fear learning.

CaMKII is a pivotal kinase that plays essential roles in synaptic plasticity. Apart from its signaling function, the structural function of CaMKII is becoming clear. CaMKII - F-actin interaction stabilizes actin cytoskeleton in a dendritic spine. A transient autophosphorylation at the F-actin binding region during LTP releases CaMKII from F-actin and opens a brief time-window of actin reorganization. However, the physiological relevance of this finding in learning and memory was not presented. Using a knock-in (KI) mouse carrying phosphoblock mutations in the actin-binding domain of CaMKIIß, we demonstrate that proper regulation of CaMKII - F-actin interaction is important for fear conditioning memory tasks. The KI mice show poor performance in contextual and cued versions of fear conditioning test. These results suggest the importance of CaMKII - F-actin interactions in learning and memory.

Inflammatory mediators ATP and S100A12 activate the NLRP3 inflammasome to induce MUC5AC production in airway epithelial cells.

Danger-associated molecular patterns (DAMPs) play a proinflammatory role in the pathogenesis of airway obstructive diseases such as severe asthma and chronic obstructive pulmonary disease. The NLRP3 inflammasome is a cytosolic multiprotein platform that activates the caspase-1 pathway in response to inflammatory stimuli such as DAMPs. ATP and S100 proteins are newly identified DAMPs that accumulate in inflamed airways. We previously demonstrated that S100A8, S100A9, and S100A12 induce production and secretion of MUC5AC, a major mucin in the conducting airway mucosa. The purpose of this study was to determine the involvement of NLRP3 inflammasome in, and the contribution of ATP to, S100 protein-induced MUC5AC production by NCI-H292 mucoepidermoid carcinoma cells. Stimulation with either S100A12 or ATP led to MUC5AC production at comparable levels. Simultaneous treatment with both stimuli resulted in additive increases in NLRP3, active caspase-1, IL-1ß, NLRP3/caspase-1 colocalization, and MUC5AC. NLRP3 siRNA or inhibitors of NF-κB, NLRP3 inflammasome oligomerization, or caspase-1 nearly completely inhibited ATP- and S100A12-mediated MUC5AC production. Furthermore, S100A12-as well as ATP-mediated MUC5AC production was almost equally blunted by both nonspecific and specific antagonists of the purinergic receptor P2X7, a principal receptor mediating NLRP3 inflammasome activation by ATP. Thus, these two danger signals contribute to MUC5AC production in airway epithelial cells through overlapping signaling pathways for NLRP3 inflammasome activation.

Isoflurane's Effect on Intraoperative Systolic Left Ventricular Performance in Cardiac Valve Surgery Patients.

BACKGROUND: Isoflurane, a common anesthetic for cardiac surgery, reduced myocardial contractility in many experimental studies, few studies have determined isoflurane's direct impact on the left ventricular (LV) contractile function during cardiac surgery. We determined whether isoflurane dose-dependently reduces the peak systolic velocity of the lateral mitral annulus in tissue Doppler imaging (S') in patients undergoing cardiac surgery. METHODS: During isoflurane-supplemented remifentanil-based anesthesia for patients undergoing cardiac surgery with preoperative LV ejection fraction greater than 50% (n = 20), we analyzed the changes of S' at each isoflurane dose increment (1.0, 1.5, and 2.0 minimum alveolar concentration [MAC]: T1, T2, and T3, respectively) with a fixed remifentanil dosage (1.0 µg/min/kg) by using transesophageal echocardiography. RESULTS: Mean S' values (95% confidence interval [CI]) at T1, T2, and T3 were 10.5 (8.8-12.2), 9.5 (8.3-10.8), and 8.4 (7.3-9.5) cm/s, respectively (P < 0.001 in multivariate analysis of variance test). Their mean differences at T1 vs. T2, T2 vs. T3, and T1 vs. T3 were -1.0 (-1.6, -0.3), -1.1 (-1.7, -0.6), and -2.1 (-3.1, -1.1) cm/s, respectively. Phenylephrine infusion rates were significantly increased (0.26, 0.22, and 0.47 µg/kg/min at T1, T2, and T3, respectively, P < 0.001). CONCLUSION: Isoflurane increments (1.0-2.0 MAC) dose-dependently reduced LV systolic long-axis performance during cardiac surgeries with a preserved preoperative systolic function.

Ursolic Acid Induces Apoptosis in Colorectal Cancer Cells Partially via Upregulation of MicroRNA-4500 and Inhibition of JAK2/STAT3 Phosphorylation.

Though ursolic acid (UA) isolated from Oldenlandia diffusa was known to exhibit anti-cancer, anti-inflammatory, and anti-obesity effects, the underlying antitumor mechanism of ursolic acid was not fully understood to date. Thus, in the present study, the apoptotic mechanism of ursolic acid was elucidated in HCT116 and HT29 colorectal cancer cells in association with STAT3 and microRNA-4500 (miR-4500) by MTT assay, Terminal deoxynucleotidyl transferase-dT-mediated dUTP nick end labelling (TUNEL) assay, cell cycle analysis, immunofluorescence, and Western blotting. Ursolic acid significantly exerted cytotoxicity, increased TUNEL positive cells and sub-G1 apoptotic portion, induced cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) and caspase 3 in HCT116 and HT29 cells. Of note, ursolic acid attenuated the expression of anti-apoptotic proteins such as Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) and also blocked nuclear translocation of STAT3 in colorectal cancer cells. Notably, ursolic acid increased the expression level of miR-4500 in HCT116 cells by qRT-PCR analysis and conversely miR-4500 inhibitor reversed cytotoxic, anti-proliferative, and apoptotic effects by increasing TUNEL positive cells, PARP cleavage and inhibiting p-STAT3 in ursolic acid treated colorectal cancer cells. Overall, our findings provide evidence that usolic acid induces apoptosis in colorectal cancer cells partially via upregulation of miR-4500 and inhibition of STAT3 phosphorylation as a potent anti-cancer agent for colorectal cancer therapy.

Interplay of enzymatic and structural functions of CaMKII in long-term potentiation.

Since the discovery of long-term potentiation (LTP) about a half-century ago, Ca2+ /CaM-dependent protein kinase II (CaMKII) has been one of the most extensively studied components of the molecular machinery that regulate plasticity. This unique dodecameric kinase complex plays pivotal roles in LTP by phosphorylating substrates through elaborate regulatory mechanisms, and is known to be both necessary and sufficient for LTP. In addition to acting as a kinase, CaMKII has been postulated to have structural roles because of its extraordinary abundance and diverse interacting partners. It now is becoming clear that these two functions of CaMKII cooperate closely for the induction of both functional and structural synaptic plasticity of dendritic spines. Because of its extraordinary abundance within neuronal cells, calmodulin kinase CaMKII has been believed to act as a structural protein as well as an enzyme during synaptic plasticity. In this review, we summarized studies in CaMKII field and provide an insight into how enzymatic and structural functions of CaMKII cooperate with each other for long-term potentiation (LTP) in neurons. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".

DGKι regulates presynaptic release during mGluR-dependent LTD.

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (∼2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.

Interleukin-22 promotes epithelial cell transformation and breast tumorigenesis via MAP3K8 activation.

Interleukin-22 (IL-22), one of the cytokines secreted by T-helper 17 (Th17) cells, binds to a class II cytokine receptor containing an IL-22 receptor 1 (IL-22R1) and IL-10R2 and influences a variety of immune reactions. IL-22 has also been shown to modulate cell cycle and proliferation mediators such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but little is known about the underlying molecular mechanisms of IL-22 in tumorigenesis. In this paper, we propose that IL-22 has a crucial role to play in controlling epithelial cell proliferation and tumorigenesis in the breast. IL-22 increased MAP3K8 phosphorylation through IL-22R1, followed by the induction of MEK-ERK, JNK-c-Jun, and STAT3 signaling pathways. Furthermore, IL-22-IL-22R1 signaling pathway activated activator protein-1 and HER2 promoter activity. In addition, Pin1 was identified as a key positive regulator for the phosphorylation-dependent MEK, c-Jun and STAT3 activity induced by IL-22. Pin1(-/-) mouse embryonic fibroblasts (MEF) exhibited significantly a decrease in IL-22-induced MEK1/2, c-Jun, and STAT3 phosphorylation compared with Pin1(+/+) MEF. In addition, a knockdown of Pin1 prevented phosphorylation induced by IL-22. The in vivo chorioallantoic membrane assay also showed that IL-22 increased tumor formation of JB6 Cl41 cells. Moreover, the knockdown of MAP3K8 and Pin1 attenuated tumorigenicity of MCF7 cells. Consistent with these observations, IL-22 levels positively correlate with MAP3K8 and Pin1 expression in human breast cancer. Overall, our findings point to a critical role for the IL-22-induced MAP3K8 signaling pathway in promoting cancer-associated inflammation in the tumor microenvironment.

Activation of sodium-dependent glutamate transporters regulates the morphological aspects of oligodendrocyte maturation via signaling through calcium/calmodulin-dependent kinase IIß's actin-binding/-stabilizing domain.

Signaling via the major excitatory amino acid glutamate has been implicated in the regulation of various aspects of the biology of oligodendrocytes, the myelinating cells of the central nervous system (CNS). In this respect, cells of the oligodendrocyte lineage have been described to express a variety of glutamate-responsive transmembrane proteins including sodium-dependent glutamate transporters. The latter have been well characterized to mediate glutamate clearance from the extracellular space. However, there is increasing evidence that they also mediate glutamate-induced intracellular signaling events. Our data presented here show that the activation of oligodendrocyte expressed sodium-dependent glutamate transporters, in particular GLT-1 and GLAST, promotes the morphological aspects of oligodendrocyte maturation. This effect was found to be associated with a transient increase in intracellular calcium levels and a transient phosphorylation event at the serine (S)(371) site of the calcium sensor calcium/calmodulin-dependent kinase type IIß (CaMKIIß). The potential regulatory S(371) site is located within CaMKIIß's previously defined actin-binding/-stabilizing domain, and phosphorylation events within this domain were identified in our studies as a requirement for sodium-dependent glutamate transporter-mediated promotion of oligodendrocyte maturation. Furthermore, our data provide good evidence for a role of these phosphorylation events in mediating detachment of CaMKIIß from filamentous (F)-actin, and hence allowing a remodeling of the oligodendrocyte's actin cytoskeleton. Taken together with our recent findings, which demonstrated a crucial role of CaMKIIß in regulating CNS myelination in vivo, our data strongly suggest that a sodium-dependent glutamate transporter-CaMKIIß-actin cytoskeleton axis plays an important role in the regulation of oligodendrocyte maturation and CNS myelination.

Synaptic removal of diacylglycerol by DGKzeta and PSD-95 regulates dendritic spine maintenance.

Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKzeta is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD-95. Overexpression of DGKzeta in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD-95 binding. Conversely, DGKzeta knockdown reduces spine density. Mice deficient in DGKzeta expression show reduced spine density and excitatory synaptic transmission. Time-lapse imaging indicates that DGKzeta is required for spine maintenance but not formation. We propose that PSD-95 targets DGKzeta to synaptic DAG-producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.

Inhibitory effects of a new 1H-pyrrolo[3,2-c]pyridine derivative, KIST101029, on activator protein-1 activity and neoplastic cell transformation induced by insulin-like growth factor-1.

Diarylureas and diarylamides derivatives are reported to have antitumor activity. Encouraged by the interesting antiproliferative activity of diarylurea and diarylamide derivatives, we synthesized a new series of diarylureas and diarylamides containing pyrrolo[3,2-c]pyridine scaffold. In this study, we demonstrate that a N-(3-(4-benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-4-morpholino-3-(trifluoromethyl)benzamide, KIST101029, inhibits neoplastic cell transformation induced by insulin-like growth factor 1 (IGF-1) in mouse epidermal JB6 Cl41 cells. The KIST101029 compound inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase kinases (MEK), c-jun N-terminal kinases (JNK), and mechanistic target of rapamycin (mTOR) signaling pathways induced by IGF-1 in JB6 Cl41 cells, resulting in the inhibition of c-fos and c-jun transcriptional activity. In addition, the KIST101029 inhibited the associated activator protein-1 (AP-1) transactivation activity and cell transformation induced by IGF-1 in JB6 Cl41 cells. Consistent with these observations, in vivo chorioallantoic membrane assay also showed that the KIST101029 inhibited IGF-1-induced tumorigenicity of JB6 Cl41 cells. Importantly, KIST101029 suppressed the colony formation of A375 cells in soft agar. Taken together, these results indicate that a KIST101029 might exert chemopreventive effects through the inhibition of phosphorylation of MAPK and mTOR signaling pathway.

The Effect of Denosumab in Elderly Patients Regarding Bone Density and Fracture Risk.

BACKGROUND: With an aging population, the importance of treating and diagnosing osteoporosis is increasing. Osteoporosis, previously known as a resorptive change primarily related to endocrinological mechanisms, is also being approached as a phenomenon of senile change. Denosumab is gaining popularity among osteoporosis medications due to its ability to increase bone mineral density (BMD) and the economic benefit arising from the 6-month cycle. In line with previous literature, this study aimed to examine the BMD-augmenting effect of denosumab through which it reduces fracture risk in individuals aged over 80 years. METHODS: We reviewed patients who received denosumab between 2018 and 2022 with a minimum clinical observation period of 12 months. BMD was measured every 12 months, and patients were classified per their period of denosumab use. Fracture risk was evaluated using the fracture risk assessment tool (FRAX) and fracture incidence during the observation period were assessed. RESULTS: Among 155 patients, a significant increase in BMD was observed at 3 sites: the lumbar spine, femoral neck, and total hip (p<0.001, p<0.001, and p=0.001, respectively). The patients were divided according to the length of clinical follow-up they received, and similar results were found in all subgroups. Fracture risk assessment was performed using FRAX and the incidence of fracture events during follow-up. FRAX significantly decreased in all subgroups except those who received 24 months of follow-up (p=0.003, p=0.41, p=0.001 in the 12, 24, and ≥36 months groups, respectively). CONCLUSIONS: Denosumab use resulted in long-term BMD increase and reduced fracture risk in individuals aged 80 and above.

α1-Adrenergic receptor-PKC-Pyk2-Src signaling boosts L-type Ca2+ channel CaV1.2 activity and long-term potentiation in rodents.

The cellular mechanisms mediating norepinephrine (NE) functions in brain to result in behaviors are unknown. We identified the L-type Ca2+ channel (LTCC) CaV1.2 as a principal target for Gq-coupled α1-adrenergic receptors (ARs). α1AR signaling increased LTCC activity in hippocampal neurons. This regulation required protein kinase C (PKC)-mediated activation of the tyrosine kinases Pyk2 and, downstream, Src. Pyk2 and Src were associated with CaV1.2. In model neuroendocrine PC12 cells, stimulation of PKC induced tyrosine phosphorylation of CaV1.2, a modification abrogated by inhibition of Pyk2 and Src. Upregulation of LTCC activity by α1AR and formation of a signaling complex with PKC, Pyk2, and Src suggests that CaV1.2 is a central conduit for signaling by NE. Indeed, a form of hippocampal long-term potentiation (LTP) in young mice requires both the LTCC and α1AR stimulation. Inhibition of Pyk2 and Src blocked this LTP, indicating that enhancement of CaV1.2 activity via α1AR-Pyk2-Src signaling regulates synaptic strength.

Regulation of hippocampal long-term potentiation and long-term depression by diacylglycerol kinase ζ.

Diacylglycerol (DAG) is an important signaling molecule at neuronal synapses. Generation of synaptic DAG is triggered by the activation of diverse surface receptors including N-methyl-D-aspartate (NMDA) receptors and metabotropic glutamate receptors. The action of DAG is terminated by enzymatic conversion of DAG to phosphatidic acid (PA) by DAG kinases (DGKs). DGKζ, one of many mammalian DGKs, is localized to synapses through direct interaction with the postsynaptic scaffolding protein PSD-95, and regulates dendritic spine maintenance by promoting DAG-to-PA conversion. However, a role for DGKζ in the regulation of synaptic plasticity has not been explored. We report here that Schaffer collateral-CA1 pyramidal synapses in the hippocampus of DGKζ-knockout (DGKζ(-/-) ) mice show enhanced long-term potentiation (LTP) and attenuated long-term depression (LTD). The attenuated LTD at DGKζ(-/-) synapses involves both NMDA receptors and metabotropic glutamate receptors. These changes in LTP and LTD were reversed by phospholipase C inhibition, which blocks DAG production. Similar reversals in both LTP and LTD were also induced by inhibition of protein kinase C, which acts downstream of DAG. These results suggest that DGKζ regulates hippocampal LTP and LTD by promoting DAG-to-PA conversion, and establish that phospholipase C and protein kinase C lie upstream and downstream, respectively, of DGKζ-dependent regulation of hippocampal LTP and LTD.

Regulated RalBP1 binding to RalA and PSD-95 controls AMPA receptor endocytosis and LTD.

Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)-dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.

Regulation of synaptic Rac1 activity, long-term potentiation maintenance, and learning and memory by BCR and ABR Rac GTPase-activating proteins.

Rho family small GTPases are important regulators of neuronal development. Defective Rho regulation causes nervous system dysfunctions including mental retardation and Alzheimer's disease. Rac1, a member of the Rho family, regulates dendritic spines and excitatory synapses, but relatively little is known about how synaptic Rac1 is negatively regulated. Breakpoint cluster region (BCR) is a Rac GTPase-activating protein known to form a fusion protein with the c-Abl tyrosine kinase in Philadelphia chromosome-positive chronic myelogenous leukemia. Despite the fact that BCR mRNAs are abundantly expressed in the brain, the neural functions of BCR protein have remained obscure. We report here that BCR and its close relative active BCR-related (ABR) localize at excitatory synapses and directly interact with PSD-95, an abundant postsynaptic scaffolding protein. Mice deficient for BCR or ABR show enhanced basal Rac1 activity but only a small increase in spine density. Importantly, mice lacking BCR or ABR exhibit a marked decrease in the maintenance, but not induction, of long-term potentiation, and show impaired spatial and object recognition memory. These results suggest that BCR and ABR have novel roles in the regulation of synaptic Rac1 signaling, synaptic plasticity, and learning and memory, and that excessive Rac1 activity negatively affects synaptic and cognitive functions.

Sensitive label-free imaging of brain samples using FxClear-based tissue clearing technique.

Optical clearing has emerged as a powerful tool for volume imaging. Although volume imaging with immunostaining have been successful in many protocols, yet obtaining homogeneously stained thick samples remains challenging. Here, we propose a method for label-free imaging of brain slices by enhancing the regional heterogeneity of the optical properties using the tissue clearing principle. We used FxClear, a method for delipidation of brain tissue, to retain a larger proportion of lipids at the white matter (WM). Furthermore, the embedding media affected the contrasts for the lipid-rich or extracellular matrix-rich areas, depending on their chemical properties. Thus, we tailored clearing conditions for the enhancement of the refractive indices (RIs) differences between gray and WM, or several pathological features. RI differences can be imaged using conventional light microscopy or optical coherence tomography. We propose that our protocol is simple, reliable, and flexible for label-free imaging, easily implementable to routine histology laboratory.

SALM synaptic cell adhesion-like molecules regulate the differentiation of excitatory synapses.

Synaptic cell adhesion molecules (CAMs) are known to play key roles in various aspects of synaptic structures and functions, including early differentiation, maintenance, and plasticity. We herein report the identification of a family of cell adhesion-like molecules termed SALM that interacts with the abundant postsynaptic density (PSD) protein PSD-95. SALM2, a SALM isoform, distributes to excitatory, but not inhibitory, synaptic sites. Overexpression of SALM2 increases the number of excitatory synapses and dendritic spines. Mislocalized expression of SALM2 disrupts excitatory synapses and dendritic spines. Bead-induced direct aggregation of SALM2 results in coclustering of PSD-95 and other postsynaptic proteins, including GKAP and AMPA receptors. Knockdown of SALM2 by RNA interference reduces the number of excitatory synapses and dendritic spines and the frequency, but not amplitude, of miniature excitatory postsynaptic currents. These results suggest that SALM2 is an important regulator of the differentiation of excitatory synapses.