The incidence and influencing factors of in-hospital frailty progression following transcatheter aortic valve implantation.

Objective Patients undergoing transcatheter aortic valve implantation (TAVI) are generally older and frailty is therefore an important clinical issue. The baseline degree of frailty is associated with the prognosis in patients undergoing TAVI; however, the incidence of in-hospital frailty progression and its influencing factors have not yet been elucidated. Methods This observational, single-center study retrospectively evaluated 281 patients who underwent TAVI. The degree of frailty at baseline and discharge was evaluated using the Clinical Frailty Scale (CFS). In-hospital frailty progression was defined as an increase of at least one level in the CFS score at discharge from baseline, and predictors of frailty progression were assessed. Results The median baseline CFS score was 4.0 (interquartile range: 3.0-4.0). In-hospital frailty progression was observed in 49 patients (17.4%). No significant differences were observed in age, sex, comorbidities, or surgical risk scores between patients with and without frailty progression. Patients with frailty progression experienced stroke more frequently during hospitalization than those without (12.2% vs. 1.3%, p = 0.001). A multivariable logistic analysis showed that in-hospital stroke was a significant predictor of frailty progression (odds ratio, 10.7; 95% confidence interval: 2.34-49.2, p = 0.002). Patients with frailty progression had a longer hospital stay than those without frailty progression [7.0 (4.0-17.0) vs. 4.0 (4.0-8.0) days, p = 0.001]. Conclusions In-hospital frailty progression was not uncommon in patients undergoing TAVI. Stroke incidence was a significant influencing factor in frailty progression, whereas baseline comorbidities and surgical risks were not.

Deep-Learning-Based Analysis Reveals a Social Behavior Deficit in Mice Exposed Prenatally to Nicotine.

Cigarette smoking during pregnancy is known to be associated with the incidence of attention-deficit/hyperactive disorder (ADHD). Recent developments in deep learning algorithms enable us to assess the behavioral phenotypes of animal models without cognitive bias during manual analysis. In this study, we established prenatal nicotine exposure (PNE) mice and evaluated their behavioral phenotypes using DeepLabCut and SimBA. We optimized the training parameters of DeepLabCut for pose estimation and succeeded in labeling a single-mouse or two-mouse model with high fidelity during free-moving behavior. We applied the trained network to analyze the behavior of the mice and found that PNE mice exhibited impulsivity and a lessened working memory, which are characteristics of ADHD. PNE mice also showed elevated anxiety and deficits in social interaction, reminiscent of autism spectrum disorder (ASD). We further examined PNE mice by evaluating adult neurogenesis in the hippocampus, which is a pathological hallmark of ASD, and demonstrated that newborn neurons were decreased, specifically in the ventral part of the hippocampus, which is reported to be related to emotional and social behaviors. These results support the hypothesis that PNE is a risk factor for comorbidity with ADHD and ASD in mice.

Impact of proteinuria on long-term prognosis in patients with coronary artery disease.

BACKGROUND: This study aimed to investigate the association between proteinuria and long-term prognosis in patients with coronary artery disease. METHODS: This was a single-center observational study. A total of 1351 patients were identified who underwent percutaneous coronary intervention, and whose urine data were available. Patients were divided into two groups according to the presence (n = 245) or absence (n = 1106) of proteinuria. All-cause and cardiovascular deaths were primarily evaluated. RESULTS: The prevalence rates of hypertension and diabetes were significantly higher, and the baseline estimated glomerular filtration rate (eGFR) was lower in patients with proteinuria than in those without proteinuria. During the median follow-up of 4.1 years (interquartile range, 1.7-6.8 years), the occurrences of all-cause and cardiovascular deaths were significantly higher in patients with proteinuria. Multivariable Cox regression analysis indicated that the presence of proteinuria was a significant predictor of cardiovascular death as well as age, BMI, reduced eGFR, and left ventricular ejection fraction. When stratified into four groups based on eGFR category (eGFR <60 or ≥60 ml/min/1.73 m2) and absence or presence of proteinuria, the incidence rates of all-cause and cardiovascular deaths were highest in patients with proteinuria and eGFR less than 60 ml/min/1.73 m2. Furthermore, the incidence rates of all-cause and cardiovascular deaths were significantly higher in patients with proteinuria among both diabetic and nondiabetic patients. CONCLUSION: Proteinuria is associated with the long-term prognosis, and all-cause and cardiovascular deaths in patients with coronary artery disease, regardless of eGFR and the presence or absence of diabetes mellitus.

Structural Analysis Implicates CASK-Liprin-α2 Interaction in Cerebellar Granular Cell Death in MICPCH Syndrome.

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a neurodevelopmental disorder caused by the deficiency of the X-chromosomal gene CASK. However, the molecular mechanisms by which CASK deficiency causes cerebellar hypoplasia in this syndrome remain elusive. In this study, we used CASK knockout (KO) mice as models for MICPCH syndrome and investigated the effect of CASK mutants. Female CASK heterozygote KO mice replicate the progressive cerebellar hypoplasia observed in MICPCH syndrome. CASK KO cultured cerebellar granule (CG) cells show progressive cell death that can be rescued by co-infection with lentivirus expressing wild-type CASK. Rescue experiments with CASK deletion mutants identify that the CaMK, PDZ, and SH3, but not L27 and guanylate kinase domains of CASK are required for the survival of CG cells. We identify missense mutations in the CaMK domain of CASK derived from human patients that fail to rescue the cell death of cultured CASK KO CG cells. Machine learning-based structural analysis using AlphaFold 2.2 predicts that these mutations disrupt the structure of the binding interface with Liprin-α2. These results suggest that the interaction with Liprin-α2 via the CaMK domain of CASK may be involved in the pathophysiology of cerebellar hypoplasia in MICPCH syndrome.

Clinical implications of the cardio-ankle vascular index before and after transcatheter aortic valve implantation.

BACKGROUND: Arterial stiffness indices are used to assess the material properties of the arterial wall and are associated with cardiovascular events. Aortic stenosis (AS) is commonly caused by degenerative calcification and can be associated with increased arterial stiffness. However, the clinical implications of arterial stiffness indices in AS patients before and after treatment are unknown. METHODS: This single-center observational study enrolled 150 consecutive patients who underwent transcatheter aortic valve implantation (TAVI) for severe AS. The cardio-ankle vascular index (CAVI) was measured before and after TAVI. The patients were divided into two groups according to the CAVI values before and after TAVI: high CAVI group and low CAVI group. Patient and echocardiographic data and clinical outcomes, including cardiac death and hospitalization for heart failure (HF), were compared. RESULTS: The pre- and postprocedural CAVI was 7.90 (6.75-9.30) and 9.65 (8.90-10.65), respectively. In the analyses with preprocedural CAVI, preprocedural echocardiographic aortic valve peak flow velocity was significantly lower in the high CAVI group. No significant differences between the two groups were observed in the occurrence of cardiac death or hospitalization for HF. In the analyses with postprocedural CAVI, B-type natriuretic peptide levels and E / e ' ratio after TAVI were significantly higher in the high CAVI group. The composite of cardiac death and hospitalization occurrence for HF was significantly higher in the high CAVI group. CONCLUSION: CAVI before TAVI is mainly affected by the AS severity, while CAVI after TAVI is associated with left ventricular diastolic dysfunction and late cardiac events, which may reflect arterial stiffness.

Impact of C-Reactive Protein on Long-Term Cardiac Events in Stable Coronary Artery Disease Patients with Chronic Kidney Disease.

AIM: Chronic inflammation is associated with atherosclerosis development. Chronic kidney disease (CKD) is an independent risk factor for cardiovascular events and is associated with chronic inflammation. We aimed to investigate the influence of C-reactive protein (CRP), an important marker of inflammation, on the clinical outcomes of patients with CKD and stable coronary artery disease (CAD) undergoing percutaneous coronary intervention (PCI). METHODS: Among patients with stable CAD and CKD who underwent PCI, 516 patients whose CRP levels were available before the PCI procedure were identified. The patients were divided into two groups according to the CRP levels: those with CRP ≥ 2.0 mg/L (high-CRP group) and those with CRP ＜2.0 mg/L (low-CRP group). The primary endpoint of this study was the occurrence of major adverse cardiac events (MACE), defined as a composite of cardiac death, myocardial infarction, and unplanned revascularization. RESULTS: Overall, the mean age of the patients was 72.5±9.7 years, and 20.7% were female. The median CRP level was 1.43 mg/L (0.6-4.9 mg/L). The median follow-up period was 3.6 years. The occurrence of MACE was significantly higher in the high-CRP group than in the low-CRP group (log-rank p＜0.001). Notably, the incidence rate of cardiac death was significantly higher in the high-CRP group (log-rank p＜0.001). According to the multivariable analysis, CRP level ≥ 2.0 mg/L was found to be a significant predictor of MACE (hazard ratio [HR]: 1.54, 95% confidence interval [CI]: 1.04-2.28, p=0.003), as well as estimated glomerular filtration rate (HR: 0.98, 95% CI: 0.97-0.99, p＜0.01). CONCLUSION: High-CRP levels adversely affect long-term cardiac events in patients with stable CAD and CKD.

Impact of Handgrip Strength on Clinical Outcomes after Percutaneous Coronary Intervention.

AIM: The relationship between handgrip strength (HGS) and clinical outcomes after percutaneous coronary intervention (PCI) has not yet been thoroughly investigated. METHODS: This was a single-center, observational study. A total of 469 patients who underwent PCI and whose periprocedural HGS was measured were included. Patients were divided into two groups: the low HGS group (men, ＜28 kg; women, ＜18 kg) and the high HGS group (men, ≥ 28 kg; women, ≥ 18 kg). The primary outcome was the composite endpoint of all-cause death, myocardial infarction (MI), and heart failure readmission. RESULTS: There were 151 patients in the low HGS group and 318 patients in the high HGS group. The age of patients in the low HGS group was significantly higher (median [interquartile range]: 78 [71-82] vs. 70 [61-75] years, p＜0.001), while the body mass index and serum albumin level were significantly lower (body mass index: 22.5 [20.2-24.3] vs. 24.3 [22.3-26.6] kg/m2, p＜0.001; serum albumin: 3.6 [3.1-3.9] vs. 4.0 [3.7-4.3] g/dL, p＜0.001) than those in the high HGS group. During the median follow-up period of 778 days, the low HGS group had a higher incidence of composite endpoint than the high HGS group (p＜0.001). The low HGS group had a higher risk of all-cause, cardiac, and non-cardiac death (p＜0.001). Multivariable Cox proportional hazards analysis showed that low handgrip strength was an independent predictor for the composite endpoint (hazard ratio 1.80, 95% confidence interval 1.04-3.12, p=0.04). CONCLUSIONS: Low HGS was independently associated with adverse outcomes after PCI.

The ridge line of left pulmonary vein isolation from left atrial appendage can subsequently increase the completion rate of the mitral isthmus block line.

BACKGROUND AND OBJECTIVES: Mitral isthmus (MI) ablation for mitral flutter is technically difficult, and incomplete block line is not uncommon. The objective of this study is to investigate the effect of the ridge line of left pulmonary vein isolation (LPVI) from left atrial appendage (LAA) on completion rate of mitral isthmus (MI) block line and recurrence rate of atrial tachycardia (AT) or atrial flutter (AFL) after the first MI ablation. METHODS: We identified 611 patients who underwent first MI ablation for mitral flutter during the study period. Finally, 559 patients were enrolled and divided into two groups according to the method of ridge line ablation of LPVI (LAA group, n = 467, conventional group, n = 92). Outcome measures were the completion of MI block line by first MI ablation, the recurrence of AT/AFL, and repeat MI ablation after the first MI ablation. RESULTS: The first MI block line completion rate was significantly higher in the LAA group than the conventional group (95% vs. 85%, p < 0.001). The recurrence rate of AT/AFL after 3 months from first MI ablation was significantly lower in the LAA group. The requirement of additional MI ablation tended to be lower in the LAA group. CONCLUSIONS: Our novel approach of ablating LPV-LAA ridge from the LAA side during PVI can increase the success rate of MI block line completion, and reduce the recurrence rate of AT/AFL and the need for additional MI block line ablation. Graphical abstract Ablation of the left pulmonary vein-left atrial appendage ridge from the left atrial appendage side during PVI increased the success rate of mitral isthmus block line completion.

Heterogeneous Carotid Plaque Predicts Cardiovascular Events after Percutaneous Coronary Intervention.

AIM: The relationship between carotid artery ultrasound findings and clinical outcomes in patients who undergo percutaneous coronary intervention (PCI) has not been completely elucidated. METHODS: This single-center retrospective study investigated 691 patients who underwent PCI and carotid ultrasound testing. Maximum carotid intima-media thickness (CIMT) was defined as the greatest CIMT at the maximally thick point among the common carotid artery, carotid bulb, and internal carotid artery. A carotid plaque was defined as vessel wall thickening with a CIMT ≥ 1.5 mm. The characteristics of carotid plaque (heterogeneity, calcification, or irregular/ulcerated surface) were evaluated visually. Patients were divided into those with and without heterogeneous carotid plaque (maximum CIMT ≥ 1.5 mm and heterogeneous texture). The endpoint was the incidence of a major adverse cardiovascular event (MACE) defined as a composite of cardiovascular (CV) death, myocardial infarction, and ischemic stroke. RESULTS: Among 691 patients, 309 were categorized as having a heterogeneous plaque. Patients with heterogeneous plaques were at a higher risk of MACE than those without (p=0.002). A heterogeneous plaque was independently associated with MACE after adjusting for covariates (hazard ratio [HR], 1.71; 95% confidence interval [CI], 1.01-2.90; p=0.046). Calcified or irregular/ulcerated plaques were correlated with a higher incidence of MACE, but both were not independently associated with MACE (HR, 1.35; 95% CI, 0.69-2.64, p=0.38 and HR, 0.98; 95% CI, 0.57-1.69; p=0.95, respectively). CONCLUSION: The presence of a heterogeneous carotid plaque in patients who underwent PCI predicted future CV events. These patients may require more aggressive medical therapy and careful follow-up.

Transcatheter Aortic Valve Replacement for Bicuspid Aortic Stenosis With a Severely Calcified Raphe Using An Under-Sized SAPIEN 3 Valve: Sizing by the Circle Method and Deployment by the Pressure-Regulated Method.

Transcatheter aortic valve replacement (TAVR) for bicuspid aortic valve stenosis is challenging, and the absence of established methods for sizing the bicuspid aortic valve (BAV) complicates TAVR. We report a case of successful TAVR for bicuspid aortic stenosis with a severely calcified raphe. We used an undersized SAPIEN 3 valve, with three safety measures based on assessment of structural characteristics, sizing by the circle method, and deployment of the valve by the pressure-regulated method.

Impact of Albuminuria on the Prognosis After Transcatheter Aortic Valve Implantation.

The impact of preoperative albuminuria on the prognosis after transcatheter aortic valve implantation (TAVI) has not been studied. A total of 228 patients who underwent TAVI for severe aortic stenosis (AS) and for whom preoperative urinary data was available were retrospectively investigated. Patients were divided into two groups according to the urinary albumin-to-creatinine ratio (ACR): high (ACR≥ 30 mg/g) and low (ACR<30 mg/g). The urinary total protein-to-creatinine ratio (PCR) and dipstick proteinuria were also evaluated. The primary outcome was the composite outcome of all-cause death and readmission for heart failure. In total, 117 patients had a high ACR and 111 patients had a low ACR. During the median follow-up period of 467 days, patients with a high ACR had a higher incidence of the primary outcome than those with a low ACR (p<0.001). Patients with a high PCR or positive dipstick proteinuria were also at a higher risk for the primary outcome (p<0.001 and p=0.008, respectively). Multivariable Cox proportional hazards analysis showed a high ACR was independently associated with a primary outcome (hazard ratio, 4.98; 95% confidence interval, 1.84-13.49; p=0.002). In conclusion, preoperative albuminuria is an independent predictor of cardiac events in patients with severe AS undergoing TAVI.

An Epilepsy-Associated Mutation of Salt-Inducible Kinase 1 Increases the Susceptibility to Epileptic Seizures and Interferes with Adrenocorticotropic Hormone Therapy for Infantile Spasms in Mice.

Six mutations in the salt-inducible kinase 1 (SIK1) have been identified in developmental and epileptic encephalopathy (DEE-30) patients, and two of the mutations are nonsense mutations that truncate the C-terminal region of SIK1. In a previous study, we generated SIK1 mutant (SIK1-MT) mice recapitulating the C-terminal truncated mutations using CRISPR/Cas9-mediated genome editing and found an increase in excitatory synaptic transmission and enhancement of neural excitability in neocortical neurons in SIK1-MT mice. NMDA was injected into SIK1-MT males to induce epileptic seizures in the mice. The severity of the NMDA-induced seizures was estimated by the latency and the number of tail flickering and hyperflexion. Activated brain regions were evaluated by immunohistochemistry against c-fos, Iba1, and GFAP. As another epilepsy model, pentylenetetrazol was injected into the adult SIK1 mutant mice. Seizure susceptibility induced by both NMDA and PTZ was enhanced in SIK1-MT mice. Brain regions including the thalamus and hypothalamus were strongly activated in NMDA-induced seizures. The epilepsy-associated mutation of SIK1 canceled the pharmacological effects of the ACTH treatment on NMDA-induced seizures. These results suggest that SIK1 may be involved in the neuropathological mechanisms of NMDA-induced spasms and the pharmacological mechanism of ACTH treatment.

Albuminuria Predicts Short-Term Worsening Renal Function After Transcatheter Aortic Valve Replacement.

Albuminuria is a major risk factor of cardiovascular events, however, the impact of albuminuria on clinical outcomes of patients undergoing transcatheter aortic valve replacement (TAVR) has not been fully investigated. This retrospective study included 206 patients who underwent TAVR for severe aortic stenosis. Patients were divided into two groups according to the preoperative urinary albumin-to-creatinine ratio (ACR): high (ACR ≥ 30 mg/g) and low (ACR < 30 mg/g). The incidence of 1-month worsening renal function (WRF), defined as a decrease in estimated glomerular filtration rate (eGFR) ≥10% from baseline after TAVR, was investigated. Patients with high ACR had acute kidney injury (8.5% vs. 1.0%, p = 0.01) and 1-month WRF (29.2% vs. 12.0%, p = 0.002) more frequently than those with low ACR. High ACR was independently associated with 1-month WRF (odds ratio, 3.72; 95% confidence interval, 1.72-8.08; p < 0.001). Albuminuria can be a useful predictor of deterioration of renal function at various time points after TAVR.

Large interventricular membranous septal aneurysm renders the anatomy unsuitable for transcatheter aortic valve replacement.

An interventricular membranous septal aneurysm, though rare, can coexist with aortic valve stenosis. In this report, we present an unsuitable anatomy for transcatheter aortic valve replacement (TAVR) due to large interventricular membranous septal aneurysm. This case suggests that the feasibility of TAVR would depend on the location and size of the aneurysm and its relationship with the aortic root.

IQSEC3 Deletion Impairs Fear Memory Through Upregulation of Ribosomal S6K1 Signaling in the Hippocampus.

BACKGROUND: IQSEC3, a gephyrin-binding GABAergic (gamma-aminobutyric acidergic) synapse-specific guanine nucleotide exchange factor, was recently reported to regulate activity-dependent GABAergic synapse maturation, but the underlying signaling mechanisms remain incompletely understood. METHODS: We generated mice with conditional knockout (cKO) of Iqsec3 to examine whether altered synaptic inhibition influences hippocampus-dependent fear memory formation. In addition, electrophysiological recordings, immunohistochemistry, and behavioral assays were used to address our question. RESULTS: We found that Iqsec3-cKO induces a specific reduction in GABAergic synapse density, GABAergic synaptic transmission, and maintenance of long-term potentiation in the hippocampal CA1 region. In addition, Iqsec3-cKO mice exhibited impaired fear memory formation. Strikingly, Iqsec3-cKO caused abnormally enhanced activation of ribosomal P70-S6K1-mediated signaling in the hippocampus but not in the cortex. Furthermore, inhibiting upregulated S6K1 signaling by expressing dominant-negative S6K1 in the hippocampal CA1 of Iqsec3-cKO mice completely rescued impaired fear learning and inhibitory synapse density but not deficits in long-term potentiation maintenance. Finally, upregulated S6K1 signaling was rescued by IQSEC3 wild-type, but not by an ARF-GEF (adenosine diphosphate ribosylation factor-guanine nucleotide exchange factor) inactive IQSEC3 mutant. CONCLUSIONS: Our results suggest that IQSEC3-mediated balanced synaptic inhibition in hippocampal CA1 is critical for the proper formation of hippocampus-dependent fear memory.

Comparison of the empirical linear ablation and low voltage area-guided ablation in addition to pulmonary vein isolation in patients with persistent atrial fibrillation: a propensity score-matched analysis.

BACKGROUND: The efficacy of pulmonary vein isolation (PVI) alone is not guaranteed for persistent atrial fibrillation (PeAF), and it is unclear which type of ablation approach should be applied in addition to PVI. This study aimed to compare outcomes and prognosis between empirical linear ablation and low-voltage area (LVA) ablation after PVI for PeAF. METHODS: We enrolled 128 patients with PeAF who were assigned to the linear ablation group (n = 64) and the LVA ablation group (n = 64) using a propensity score-matched model. After PVI and cardioversion, the patients underwent either empirical linear ablation or LVA ablation during sinus rhythm. All patients in the linear ablation group underwent both roof line and mitral valve isthmus (MVI) ablations. An electrical-guided ablation targeting LVA (< 0.5 mV) was performed in the LVA group. When there was no LVA in the LVA group, only PVI was applied. We compared the procedural outcomes and recurrence after ablation between the two groups. RESULTS: The baseline characteristics were well-balanced between the two groups. Fifty patients had LVA (22 and 28 patients in the linear and LVA groups). The roof and MVI lines were completed in 100% and 96.9% of the patients. During the mean follow-up of 279.5 ± 161.3 days, the LVA group had significantly lower recurrence than the linear group (15 patients [23%] vs. 29 patients [45%], p = 0.014). Thirty-five patients were prescribed antiarrhythmic drugs during the follow-up period (linear group, n = 17; LVA group, n = 18); amiodarone and bepridil were administered to most of the patients (15 and 17 patients, respectively). The difference in the prognosis was relevant among the patients with LVA, while this trend was not observed in those without LVA. The LVA ablation group demonstrated significantly lower radiofrequency energy and shorter procedural time compared to the linear ablation group. The recurrence of atrial flutter was more likely to occur in the linear group than in the LVA group (14 [22%] vs. 6 [9.4%], p = 0.052). CONCLUSION: The electrophysiological-guided LVA ablation is more effective than empirical linear ablation in PeAF patients with LVA. Unnecessary empirical linear ablation might have a risk of iatrogenic gap and atrial flutter recurrence.

IQSEC2 Deficiency Results in Abnormal Social Behaviors Relevant to Autism by Affecting Functions of Neural Circuits in the Medial Prefrontal Cortex.

IQSEC2 is a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factor 6 (Arf6), of which protein is exclusively localized to the postsynaptic density of the excitatory synapse. Human genome studies have revealed that the IQSEC2 gene is associated with X-linked neurodevelopmental disorders, such as intellectual disability (ID), epilepsy, and autism. In this study, we examined the behavior and synapse function in IQSEC2 knockout (KO) mice that we generated using CRIPSR/Cas9-mediated genome editing to solve the relevance between IQSEC2 deficiency and the pathophysiology of neurodevelopmental disorders. IQSEC2 KO mice exhibited autistic behaviors, such as overgrooming and social deficits. We identified that up-regulation of c-Fos expression in the medial prefrontal cortex (mPFC) induced by social stimulation was significantly attenuated in IQSEC2 KO mice. Whole cell electrophysiological recording identified that synaptic transmissions mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), N-methyl-D-aspartate receptor (NMDAR), and γ-aminobutyric acid receptor (GABAR) were significantly decreased in pyramidal neurons in layer 5 of the mPFC in IQSEC2 KO mice. Reexpression of IQSEC2 isoform 1 in the mPFC of IQSEC2 KO mice using adeno-associated virus (AAV) rescued both synaptic and social deficits, suggesting that impaired synaptic function in the mPFC is responsible for social deficits in IQSEC2 KO mice.

Risperidone Mitigates Enhanced Excitatory Neuronal Function and Repetitive Behavior Caused by an ASD-Associated Mutation of SIK1.

Six mutations in the salt-inducible kinase 1 (SIK1)-coding gene have been identified in patients with early infantile epileptic encephalopathy (EIEE-30) accompanied by autistic symptoms. Two of the mutations are non-sense mutations that truncate the C-terminal region of SIK1. It has been shown that the C-terminal-truncated form of SIK1 protein affects the subcellular distribution of SIK1 protein, tempting to speculate the relevance to the pathophysiology of the disorders. We generated SIK1-mutant (SIK1-MT) mice recapitulating the C-terminal-truncated mutations using CRISPR/Cas9-mediated genome editing. SIK1-MT protein was distributed in the nucleus and cytoplasm, whereas the distribution of wild-type SIK1 was restricted to the nucleus. We found the disruption of excitatory and inhibitory (E/I) synaptic balance due to an increase in excitatory synaptic transmission and enhancement of neural excitability in the pyramidal neurons in layer 5 of the medial prefrontal cortex in SIK1-MT mice. We also found the increased repetitive behavior and social behavioral deficits in SIK1-MT mice. The risperidone administration attenuated the neural excitability and excitatory synaptic transmission, but the disrupted E/I synaptic balance was unchanged, because it also reduced the inhibitory synaptic transmission. Risperidone also eliminated the repetitive behavior but not social behavioral deficits. These results indicate that risperidone has a role in decreasing neuronal excitability and excitatory synapses, ameliorating repetitive behavior in the SIK1-truncated mice.

Non-microtubule tubulin-based backbone and subordinate components of postsynaptic density lattices.

A purification protocol was developed to identify and analyze the component proteins of a postsynaptic density (PSD) lattice, a core structure of the PSD of excitatory synapses in the central nervous system. "Enriched"- and "lean"-type PSD lattices were purified by synaptic plasma membrane treatment to identify the protein components by comprehensive shotgun mass spectrometry and group them into minimum essential cytoskeleton (MEC) and non-MEC components. Tubulin was found to be a major component of the MEC, with non-microtubule tubulin widely distributed on the purified PSD lattice. The presence of tubulin in and around PSDs was verified by post-embedding immunogold labeling EM of cerebral cortex. Non-MEC proteins included various typical scaffold/adaptor PSD proteins and other class PSD proteins. Thus, this study provides a new PSD lattice model consisting of non-microtubule tubulin-based backbone and various non-MEC proteins. Our findings suggest that tubulin is a key component constructing the backbone and that the associated components are essential for the versatile functions of the PSD.

Inhibition of DNA ligase IV enhances the CRISPR/Cas9-mediated knock-in efficiency in mouse brain neurons.

CRISPR/Cas9-mediated gene knock-in in in vivo neurons using in utero electroporation is a powerful technique, but the knock-in efficiency is generally low. We previously demonstrated that co-transfection with RAD51, a key molecule of the initial step of homology-directed repair (HDR), expression vector increased EGFP knock-in efficiency in the ß-actin site up to 2.5-fold in the pyramidal neurons in layer 2/3 of the somatosensory cortex of mouse brain. To further improve the efficiency, we examined the effect of inhibition of DNA ligase IV (LIG4) that is an essential molecule for non-homologous end joining (NHEJ). Co-transfection with small hairpin RNA for LIG4 (shlig4) expression vector increased the EGFP knock-in efficiency in the ß-actin site up to 3.6-fold compared to the condition without shlig4. RAD51 and shlig4 expression vector co-transfection further increased the knock-in efficiency up to 4.7-fold of the control condition. These results suggest that the inhibition of LIG4 is more effective than RAD51 overexpression, and it enhances the effect of RAD51 overexpression on HDR-mediated gene knock-in in vivo neurons.