Favorable liver and skeletal muscle changes in patients with MASLD and T2DM receiving glucagon-like peptide-1 receptor agonist: A prospective cohort study.

To investigate changes in skeletal muscle mass and fat fraction in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and type 2 diabetes mellitus (T2DM) undergoing treatment with Semaglutide for 6months. This single-arm pilot study included 21 patients with MASLD who received semaglutide for T2DM. Body weight, metabolic parameters, liver enzymes, fibrosis markers, skeletal muscle index (cm2/m2), and fat fraction (%) at the L3 level using the two-point Dixon method on magnetic resonance imaging (MRI), as well as liver steatosis and liver stiffness assessed using MRI-based proton density fat fraction (MRI-PDFF) and MR elastography, respectively, were prospectively examined before and 6 months after semaglutide administration. The mean age of the patients was 53 years and 47.6% were females. The median liver steatosis-fraction (%) and skeletal muscle steatosis-fraction values (%) significantly decreased (22.0 vs 12.0; P = .0014) and (12.8 vs 9.9; P = .0416) at baseline and 6 months, respectively, while maintaining muscle mass during treatment. Semaglutide also dramatically reduced hemoglobin A1c (%) (6.8 vs 5.8, P = .0003), AST (IU/L) (54 vs 26, P < .0001), ALT (IU/L) (80 vs 34, P = .0004), and γ-GTP (IU/L) levels (64 vs 34, P = .0007). Although not statistically significant, Body weight (kg) (79.9 vs 77.4), body mass index (BMI) (kg/m2) (28.9 vs 27.6), and liver stiffness (kPa) (28.9 vs 27.6) showed a decreasing trend. Fibrosis markers such as M2BPGi, type IV collagen, and skeletal muscle area did not differ. Semaglutide demonstrated favorable effects on liver and skeletal muscle steatosis, promoting improved liver function and diabetic status.

SynGAP regulates synaptic plasticity and cognition independently of its catalytic activity.

SynGAP is an abundant synaptic GTPase-activating protein (GAP) critical for synaptic plasticity, learning, memory, and cognition. Mutations in SYNGAP1 in humans result in intellectual disability, autistic-like behaviors, and epilepsy. Heterozygous Syngap1-knockout mice display deficits in synaptic plasticity, learning, and memory and exhibit seizures. It is unclear whether SynGAP imparts structural properties at synapses independently of its GAP activity. Here, we report that inactivating mutations within the GAP domain do not inhibit synaptic plasticity or cause behavioral deficits. Instead, SynGAP modulates synaptic strength by physically competing with the AMPA-receptor-TARP excitatory receptor complex in the formation of molecular condensates with synaptic scaffolding proteins. These results have major implications for developing therapeutic treatments for SYNGAP1-related neurodevelopmental disorders.

Context-dependent hyperactivity in syngap1a and syngap1b zebrafish autism models.

Background and Aims: SYNGAP1 disorder is a prevalent genetic form of Autism Spectrum Disorder and Intellectual Disability (ASD/ID) and is caused by de novo or inherited mutations in one copy of the SYNGAP1 gene. In addition to ASD/ID, SYNGAP1 disorder is associated with comorbid symptoms including treatment-resistant-epilepsy, sleep disturbances, and gastrointestinal distress. Mechanistic links between these diverse symptoms and SYNGAP1 variants remain obscure, therefore, our goal was to generate a zebrafish model in which this range of symptoms can be studied. Methods: We used CRISPR/Cas9 to introduce frameshift mutations in the syngap1a and syngap1b zebrafish duplicates (syngap1ab) and validated these stable models for Syngap1 loss-of-function. Because SYNGAP1 is extensively spliced, we mapped splice variants to the two zebrafish syngap1a and b genes and identified mammalian-like isoforms. We then quantified locomotory behaviors in zebrafish syngap1ab larvae under three conditions that normally evoke different arousal states in wild type larvae: aversive, high-arousal acoustic, medium-arousal dark, and low-arousal light stimuli. Results: We show that CRISPR/Cas9 indels in zebrafish syngap1a and syngap1b produced loss-of-function alleles at RNA and protein levels. Our analyses of zebrafish Syngap1 isoforms showed that, as in mammals, zebrafish Syngap1 N- and C-termini are extensively spliced. We identified a zebrafish syngap1 α1-like variant that maps exclusively to the syngap1b gene. Quantifying locomotor behaviors showed that syngap1ab larvae are hyperactive compared to wild type but to differing degrees depending on the stimulus. Hyperactivity was most pronounced in low arousal settings, with overall movement increasing with the number of mutant syngap1 alleles. Conclusions: Our data support mutations in zebrafish syngap1ab as causal for hyperactivity associated with elevated arousal that is especially pronounced in low-arousal environments.

Mouse models of SYNGAP1-related intellectual disability.

SYNGAP1 is a Ras-GTPase-activating protein highly enriched at excitatory synapses in the brain. De novo loss-of-function mutations in SYNGAP1 are a major cause of genetically defined neurodevelopmental disorders (NDDs). These mutations are highly penetrant and cause SYNGAP1-related intellectual disability (SRID), an NDD characterized by cognitive impairment, social deficits, early-onset seizures, and sleep disturbances. Studies in rodent neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, and heterozygous Syngap1 knockout mice have deficits in synaptic plasticity, learning, and memory and have seizures. However, how specific SYNGAP1 mutations found in humans lead to disease has not been investigated in vivo. To explore this, we utilized the CRISPR-Cas9 system to generate knock-in mouse models with two distinct known causal variants of SRID: one with a frameshift mutation leading to a premature stop codon, SYNGAP1; L813RfsX22, and a second with a single-nucleotide mutation in an intron that creates a cryptic splice acceptor site leading to premature stop codon, SYNGAP1; c.3583-9G>A. While reduction in Syngap1 mRNA varies from 30 to 50% depending on the specific mutation, both models show ~50% reduction in Syngap1 protein, have deficits in synaptic plasticity, and recapitulate key features of SRID including hyperactivity and impaired working memory. These data suggest that half the amount of SYNGAP1 protein is key to the pathogenesis of SRID. These results provide a resource to study SRID and establish a framework for the development of therapeutic strategies for this disorder.

Tumor steatosis and glutamine synthetase expression in patients with advanced hepatocellular carcinoma receiving atezolizumab plus bevacizumab therapy.

AIM: The anti-programmed death-ligand 1 antibody atezolizumab and vascular endothelial growth factor-neutralizing antibody bevacizumab in combination (Atezo + Bev) have become the first-line therapy in advanced hepatocellular carcinoma (HCC). Distinct types of tumor immune microenvironment (TIME) and their associations with specific molecular subclasses and driver gene mutations have been identified in HCC; however, these insights are mainly based on surgically resected early-stage tumors. The current study aimed to reveal the biology and TIME of advanced HCC and their significance in predicting clinical outcomes of Atezo + Bev therapy. METHODS: Thirty-three patients with advanced HCC who were scheduled for treatment with Atezo + Bev therapy were included in this study. Pretreatment tumor biopsy, pre- and posttreatment diffusion-weighted magnetic resonance imaging (MRI) with nine b values (0-1500 s/mm2 ), and other clinicopathologic factors were analyzed. RESULTS: Compared with resectable HCC, advanced HCC was characterized by higher proliferative activity, a higher frequency of Wnt/ß-catenin-activated HCC, and lower lymphocytic infiltration. Prognostically, two metabolism-related factors, histopathologically determined tumor steatosis and/or glutamine synthetase (GS) expression, and MRI-determined tumor steatosis, were the most significant prognostic indicators for progression-free survival (PFS) and overall survival after Atezo + Bev therapy. Furthermore, changes in the pre- and posttreatment true diffusion coefficients on MRI, which might reflect changes in TIME after treatment, were significantly associated with better PFS. CONCLUSIONS: The biology and TIME of HCC were strikingly different in advanced HCC compared with those of surgically resected HCC. Two metabolism-related factors, pathologically determined tumor steatosis and/or GS expression, and MRI-determined tumor steatosis, were found to be the most significant prognostic indicators for Atezo + Bev therapy in advanced HCC.

Mouse models of SYNGAP1 -related intellectual disability.

SYNGAP1 is a Ras-GTPase activating protein highly enriched at excitatory synapses in the brain. De novo loss-of-function mutations in SYNGAP1 are a major cause of genetically defined neurodevelopmental disorders (NDD). These mutations are highly penetrant and cause SYNGAP1 -related intellectual disability (SRID), a NDD characterized by cognitive impairment, social deficits, early-onset seizures, and sleep disturbances (1-5). Studies in rodent neurons have shown that Syngap1 regulates developing excitatory synapse structure and function (6-11), and heterozygous Syngap1 knockout mice have deficits in synaptic plasticity, learning and memory, and have seizures (9, 12-14). However, how specific SYNGAP1 mutations found in humans lead to disease has not been investigated in vivo. To explore this, we utilized the CRISPR-Cas9 system to generate knock-in mouse models with two distinct known causal variants of SRID: one with a frameshift mutation leading to a premature stop codon, SYNGAP1; L813RfsX22, and a second with a single-nucleotide mutation in an intron that creates a cryptic splice acceptor site leading to premature stop codon, SYNGAP1; c.3583-9G>A . While reduction in Syngap1 mRNA varies from 30-50% depending on the specific mutation, both models show ∼50% reduction in Syngap1 protein, have deficits in synaptic plasticity, and recapitulate key features of SRID including hyperactivity and impaired working memory. These data suggest that half the amount of SYNGAP1 protein is key to the pathogenesis of SRID. These results provide a resource to study SRID and establish a framework for the development of therapeutic strategies for this disorder. Significance Statement: SYNGAP1 is a protein enriched at excitatory synapses in the brain that is an important regulator of synapse structure and function. SYNGAP1 mutations cause SYNGAP1 -related intellectual disability (SRID), a neurodevelopmental disorder with cognitive impairment, social deficits, seizures, and sleep disturbances. To explore how SYNGAP1 mutations found in humans lead to disease, we generated the first knock-in mouse models with causal SRID variants: one with a frameshift mutation and a second with an intronic mutation that creates a cryptic splice acceptor site. Both models show decreased Syngap1 mRNA and Syngap1 protein and recapitulate key features of SRID including hyperactivity and impaired working memory. These results provide a resource to study SRID and establish a framework for the development of therapeutic strategies. Highlights: Two mouse models with SYNGAP1 -related intellectual disability (SRID) mutations found in humans were generated: one with a frameshift mutation that results in a premature stop codon and the other with an intronic mutation resulting in a cryptic splice acceptor site and premature stop codon. Both SRID mouse models show 35∼50% reduction in mRNA and ∼50% reduction in Syngap1 protein.Both SRID mouse models display deficits in synaptic plasticity and behavioral phenotypes found in people. RNA-seq confirmed cryptic splice acceptor activity in one SRID mouse model and revealed broad transcriptional changes also identified in Syngap1 +/- mice. Novel SRID mouse models generated here provide a resource and establish a framework for development of future therapeutic intervention.

The feasibility of superparamagnetic iron oxide-enhanced magnetic resonance imaging for assessing liver lesions in patients with contraindications for iodine CT contrast media or gadolinium-based MR contrast media: a retrospective case-control study.

Background: The detection and characterization of liver lesions are problematic in patients with bronchial asthma, renal dysfunction, or a history of allergy to gadolinium-based magnetic resonance contrast media or iodine-computed tomography contrast media because these contrast media cannot be used. Hence, the information on the lesion vascularity cannot be obtained. Therefore, this retrospective case-control study evaluated the feasibility of superparamagnetic iron oxide (SPIO) in patients with one or more of these contraindications who underwent SPIO-enhanced magnetic resonance imaging for the assessment of liver lesions. Methods: Twenty-six patients with a total of 48 lesions were analyzed. SPIO was used in the case of all patients because each patient had at least one reason not to use iodine contrast or gadolinium-based contrast media. Additionally, all patients were subjected to the perfusion study. A total volume of 1.3 mL of SPIO was injected via the cubital vein at a rate of 3 mL per second, followed by 40 mL saline at the same speed. The scanning of the perfusion study was started 4 s after the beginning of superparamagnetic iron oxide injection and scanning took 50 s. Two radiologists independently evaluated whether the lesion was malignant or benign. Receiver operating characteristic analysis (ROC) was performed to determine the additional benefit of the perfusion study. Results: There were no adverse effects associated with SPIO. The area under the curve (AUC) value without perfusion study for observers 1 and 2 were 0.473 (P=0.794, 95% CI: 0.275-0.672) and 0.602 (P=0.305, 95% CI: 0.407-0.798), respectively, whereas the Az values with perfusion study for observers 1 and 2 were 0.782 (P=0.011, 95% CI: 0.565-0.998) and 0.784 (P=0.004, 95% CI: 0.591-0.977), respectively. Az value became significantly better when the perfusion study has added (P=0.001 and 0.012 by observers 1 and 2). Conclusions: SPIO can be used safely in patients with bronchial asthma, renal dysfunction, or a history of contrast media allergy. Furthermore, the diagnostic accuracy of SPIO was acceptable.

Arc weakens synapses by dispersing AMPA receptors from postsynaptic density via modulating PSD phase separation.

In response to stimuli, the immediate early gene product Arc can acutely down-regulate synaptic strength by removing AMPA receptors (AMPARs) from synapses and thus regulate synaptic plasticity. How Arc, a scaffold protein, can specifically facilitate synaptic removal of AMPARs is unknown. We found that Arc directly antagonizes with PSD-95 in binding to TARPs, which are the auxiliary subunits of AMPARs. Arc, in a highly concentration-sensitive manner, acutely disperses TARPs from the postsynaptic density (PSD) condensate formed via phase separation. TARPs with the Ser residue in the "P-S-Y"-motif of its tail phosphorylated are completely refractory from being dispersed by Arc, suggesting that Arc cannot displace AMPARs from PSDs in active synapses. Conversely, strengthening the interaction between Arc and TARPs enhances Arc's capacity in weakening synapses. Thus, Arc can specifically and effectively modulate synaptic AMPAR clustering via modulating PSD phase separation. Our study further suggests that activity-dependent, bi-directional modulation of PSD condensate formation/dispersion represents a general regulatory mechanism for synaptic plasticity.

Endogenous Syngap1 alpha splice forms promote cognitive function and seizure protection.

Loss-of-function variants in SYNGAP1 cause a developmental encephalopathy defined by cognitive impairment, autistic features, and epilepsy. SYNGAP1 splicing leads to expression of distinct functional protein isoforms. Splicing imparts multiple cellular functions of SynGAP proteins through coding of distinct C-terminal motifs. However, it remains unknown how these different splice sequences function in vivo to regulate neuronal function and behavior. Reduced expression of SynGAP-α1/2 C-terminal splice variants in mice caused severe phenotypes, including reduced survival, impaired learning, and reduced seizure latency. In contrast, upregulation of α1/2 expression improved learning and increased seizure latency. Mice expressing α1-specific mutations, which disrupted SynGAP cellular functions without altering protein expression, promoted seizure, disrupted synapse plasticity, and impaired learning. These findings demonstrate that endogenous SynGAP isoforms with α1/2 spliced sequences promote cognitive function and impart seizure protection. Regulation of SynGAP-αexpression or function may be a viable therapeutic strategy to broadly improve cognitive function and mitigate seizure.

Relevance of diffusion-weighted imaging with background body signal suppression for staging, prognosis, morphology, treatment response, and apparent diffusion coefficient in plasma-cell neoplasms: A single-center, retrospective study.

Accurate staging and evaluation of therapeutic effects are important in managing plasma-cell neoplasms. Diffusion-weighted imaging with body signal suppression magnetic resonance imaging (DWIBS-MRI) allows for acquisition of whole-body volumetric data without radiation exposure. This study aimed to investigate the usefulness of DWIBS-MRI in plasma-cell neoplasms. We retrospectively analyzed 29 and 8 Japanese patients with multiple myeloma and monoclonal gammopathy of undetermined significance, respectively, who underwent DWIBS-MRI. We conducted a histogram analysis of apparent diffusion coefficient values. The correlations between each histogram parameter and staging, cell maturation, prognosis, and treatment response were evaluated. We found that the apparent diffusion coefficient values in patients with monoclonal gammopathy of undetermined significance were lower than those in patients with multiple myeloma. Pretreatment apparent diffusion coefficient values of immature myeloma were lower than those of mature myeloma. Moreover, these values decreased in proportion to stage progression in Durie-Salmon classification system but showed no significant correlation with other staging systems or prognosis. Patients were stratified as responder, stable, and non-responder based on the International Myeloma Working Group criteria. The magnitude of changes in apparent diffusion coefficients differed significantly between responders and non-responders (0.154 ± 0.386 ×10-3 mm2/s vs. -0.307 ± 0.424 ×10-3 mm2/s, p = 0.003). Although its usefulness has yet to be established, DWIBS-MRI combined with apparent diffusion coefficient measurement allowed for excellent response evaluation in patients with multiple myeloma. Furthermore, apparent diffusion coefficient analysis using DWIBS-MRI may be useful in predicting cell maturation and total tumor volume.

Magnetic Resonance Imaging of Orbital Solitary Fibrous Tumors: Radiological-Pathological Correlation Analysis.

BACKGROUND: Solitary fibrous tumors (SFTs) are rare and can be misdiagnosed because of their various radiological appearances. PURPOSE: To clarify the characteristic MRI findings of SFTs by analyzing their radiological-pathological correlation. MATERIAL AND METHODS: Nine consecutive patients with SFT who underwent magnetic resonance imaging (MRI) prior to surgery were analyzed. Eight patients underwent contrast-enhanced MRI, and three underwent dynamic MRI. Radiological-pathological correlation analysis, co-occurrence matrix, run-length matrix, and histogram analysis were performed to assess the relationship between pathological findings T1- and T2-weighted images (T1-WI and T2-WI). RESULTS: All nine lesions ranged in size from 20 to 36 mm. Seven lesions were located in the superior portion of the retrobulbar space found outside of the muscle cone, and two lesions in the inferior portion were located within it. No significant correlation was observed between the amount of collagenous tissue and the qualitative evaluation of the signal on T1-WI and T2-WI. Kurtosis on T2-WI was significantly correlated with the amount of collagenous tissue (ρ = -0.97, p < 0.0001) and endothelial cells (ρ = -0.49, p = 0.0479). CONCLUSION: Kurtosis in the histogram analysis on T2WI showed a strong correlation with the amount of collagenous tissue.

Abdominal Organ Enhancement in Dynamic MRI using 1 M Gadobutrol vs 0.5 M Meglumine Gadoterate in Liver of Hemangioma Patients.

BACKGROUND: The utility of gadobutrol (GAD) which is higher r1 value contrast media for evaluating abdominal solid organ have not been fully evaluated. OBJECTIVE: To compare the contrast enhancement of abdominal organs on dynamic MRI using 0.1 mmol/kg 1.0 M GAD or 0.5 M meglumine gadoterate (MG) in patients with a liver hemangioma. METHODS: A phantom study was performed at different concentrations (0.05, 0.1, 0.3, 0.5, 0.7, 0.9, 1.0, 5.0 and 10 mmol/L) of GAD and MG. Sixty-two patients with a liver hemangioma were enrolled. Contrast media was injected at a rate of 2 mL/s followed by 40 mL of saline. Two arterial phases, a portal phase and an equilibrium phase were obtained. One certified radiologist set regions of interest on the abdominal aorta, liver, pancreas, spleen and the liver hemangioma. The relative enhancement ratio (RER) was calculated. RESULTS: In the phantom study the signal intensity of both contrast media was similar at lower concentrations. However, the signal intensity of MG was higher at concentrations of more than 5.0 mmol/L. In the clinical study the RER of the abdominal viscera during the portal and equilibrium phases was higher with GAD. The hemangioma had a higher equilibrium phase enhancement with GAD. The aortic RER was equivalent during all phases and the liver RER during the 2nd arterial phase was higher with GAD. The arterial phase during GAD imaging might have been measured later than was optimal. CONCLUSION: When the same injection protocol was used for an abdominal dynamic MRI, arterial phase imaging was late when GAD was used. The higher T1 relaxation value was significantly higher in the abdominal viscera during the portal and equilibrium phases, while the liver hemangioma also had significantly higher contrast enhancement during the equilibrium phase. CLINICAL TRIAL REGISTRATION NUMBER: 3186.

Evaluation of abdominal hemodynamics through compressed sensing accelerated functional imaging.

PURPOSE: To compare the imaging characteristics of the volumetric-interpolated breath-hold examination (VIBE) using compressed-sensing (CS) acceleration (CS-VIBE) with the conventional sequence relying on parallel imaging to assess the potential use of CS-VIBE as a functional imaging technique for upper abdominal haemodynamics. MATERIALS AND METHODS: Patients (30 men, 27 women) suspected of having a hepatic disease underwent magnetic resonance imaging (MRI) of the liver, including a dynamic contrast-enhanced study. Gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid was used as the contrast agent. MRI data of two multi-phase breath-hold exams were used for intra-individual comparisons. The VIBE and CS-VIBE were performed on different days. Image quality in both sequences was qualitatively assessed by three experienced radiologists. Moreover, the contrast ratio (CR) of the aorta, portal vein, liver and pancreas to muscle tissue were measured as a quantitative assessment. For the CS-VIBE, a five-phase time-intensity curve (TIC) was created to evaluate haemodynamics. The measurement area included the pancreas, common hepatic artery, portal vein and superior mesenteric vein. The ratio of that area to the muscle tissue in the same cross section was used to create the TICs. RESULTS: The qualitative assessment showed that artefacts were significantly different between the VIBE and CS-VIBE sequences. This finding indicated that the conventional VIBE had fewer artefacts. The CR was significantly higher for the CS-VIBE than for the VIBE images in all phases (p < 0.001). An evaluation of haemodynamics compared with those obtained by CT angiography showed almost the same temporal characteristics in the common hepatic artery, portal vein and superior mesenteric vein signals as those in a previous study. CONCLUSION: Compared with the conventional VIBE, the CS-VIBE had significantly higher temporal resolution and higher image contrast. The temporal resolution of the CS-VIBE was sufficient for viewing abdominal haemodynamics. If the remaining limitation of acquisition speed for dynamic MRI can be adequately addressed, we believe that CS-VIBE functional images with high-contrast haemodynamics will be very useful in clinical practise.

Validation study of perfusion parameter in hypervascular hepatocellular carcinoma and focal nodular hyperplasia using dynamic susceptibility magnetic resonance imaging with super-paramagnetic iron oxide: comparison with single level dynamic CT arteriography.

BACKGROUND: Dynamic susceptibility contrast MR imaging (DSC-MRI) offers direct evaluation of neo-vascularity. Ferucarbotran does not accumulate in the interstitial space, instead remaining in the intravascular space during early phase imaging. We investigate tracer kinetic analysis with DSC-MRI with ferucarbotran and single level CT during hepatic arteriography (SL-CTHA) in assessment of hypervascular hepatocellular lesions and evaluate the usefulness of DSC-MRI with ferucarbotran. METHODS: Six patients having hypervascular hepatocellular carcinoma (HCC) and 3 patients having focal nodular hyperplasia (FNH) were included in the study. SL-CTHA was performed with the infusion of 3 mL of contrast media at a rate of 1 mL/s and scanned at a rate of 0.8 second per rotation. DSC-MRI was acquired with the echo-planar method at 1.5T system. A total dose of 1.4 mL (0.5 mol Fe/L) of ferucarbotran was used. Ferucarbotran was injected at a rate of 2 mL/s with 40 mL of physiological saline. Imaging was obtained at a temporal resolution of 1.2 or 0.46 seconds in 5 and 4 patients, respectively. For both CT and MRI modalities, a model-free analysis method was used to derive region of interest-based perfusion parameters. Plasma flow, distribution volume (DV) of contrast agent and estimated mean transit time (EMTT) were estimated. RESULTS: A strong correlation was obtained with plasma flow (r=0.8231, P=0.0064) between DSC-MRI and SL-CTHA. No significant correlation was obtained for DV and EMTT between DSC-MRI and SL-CTHA. All perfusion parameters showed no significant difference between SL-CTHA and DSC-MRI in FNH. On the other hand, in HCC, DV and EMTT showed significant differences (P=0.046 and 0.046), and plasma flow showed no significant difference between DSC-MRI and SL-CTHA. CONCLUSIONS: This pilot study demonstrates the possibility of quantitative analysis of liver tumor using superparamagnetic iron oxide (SPIO)-based agent and highlights the potential for SPIO-based agent in more precisely assessing the perfusion characteristic of hypervascular liver tumors than by using extracellular contrast media.

SynGAP isoforms differentially regulate synaptic plasticity and dendritic development.

SynGAP is a synaptic Ras GTPase-activating protein (GAP) with four C-terminal splice variants: α1, α2, ß, and γ. Although studies have implicated SYNGAP1 in several cognitive disorders, it is not clear which SynGAP isoforms contribute to disease. Here, we demonstrate that SynGAP isoforms exhibit unique spatiotemporal expression patterns and play distinct roles in neuronal and synaptic development in mouse neurons. SynGAP-α1, which undergoes liquid-liquid phase separation with PSD-95, is highly enriched in synapses and is required for LTP. In contrast, SynGAP-ß, which does not bind PSD-95 PDZ domains, is less synaptically targeted and promotes dendritic arborization. A mutation in SynGAP-α1 that disrupts phase separation and synaptic targeting abolishes its ability to regulate plasticity and instead causes it to drive dendritic development like SynGAP-ß. These results demonstrate that distinct intrinsic biochemical properties of SynGAP isoforms determine their function, and individual isoforms may differentially contribute to the pathogenesis of SYNGAP1-related cognitive disorders.

Twenty Years of SynGAP Research: From Synapses to Cognition.

SynGAP is a potent regulator of biochemical signaling in neurons and plays critical roles in neuronal function. It was first identified in 1998, and has since been extensively characterized as a mediator of synaptic plasticity. Because of its involvement in synaptic plasticity, SynGAP has emerged as a critical protein for normal cognitive function. In recent years, mutations in the SYNGAP1 gene have been shown to cause intellectual disability in humans and have been linked to other neurodevelopmental disorders, such as autism spectrum disorders and schizophrenia. While the structure and biochemical function of SynGAP have been well characterized, a unified understanding of the various roles of SynGAP at the synapse and its contributions to neuronal function remains to be achieved. In this review, we summarize and discuss the current understanding of the multifactorial role of SynGAP in regulating neuronal function gathered over the last two decades.

SynGAP splice variants display heterogeneous spatio-temporal expression and subcellular distribution in the developing mammalian brain.

The SynGAP protein is a major regulator of synapse biology and neural circuit function. Genetic variants linked to epilepsy and intellectual disability disrupt synaptic function and neural excitability. SynGAP has been involved in multiple signaling pathways and can regulate small GTPases with very different roles. Yet, the molecular bases behind this pleiotropy are poorly understood. We hypothesize that different SynGAP isoforms will mediate different sets of functions and that deciphering their spatio-temporal expression and subcellular localization will accelerate understanding their multiple functions. Using isoform-specific antibodies recognizing SynGAP in mouse and human samples we found distinctive developmental expression patterns for all SynGAP isoforms in five mouse brain areas. Particularly noticeable was the delayed expression of SynGAP-α1 isoforms, which directly bind to postsynaptic density-95, in cortex and hippocampus during the first 2 weeks of postnatal development. Suggesting that during this period other isoforms would have a more prominent role. Furthermore, we observed subcellular localization differences between isoforms, particularly throughout postnatal development. Consistent with previous reports, SynGAP was enriched in the postsynaptic density in the mature forebrain. However, SynGAP was predominantly found in non-synaptic locations in a period of early postnatal development highly sensitive to SynGAP levels. While, α1 isoforms were always found enriched in the postsynaptic density, α2 isoforms changed from a non-synaptic to a mostly postsynaptic density localization with age and ß isoforms were always found enriched in non-synaptic locations. The differential expression and subcellular distribution of SynGAP isoforms may contribute to isoform-specific regulation of small GTPases, explaining SynGAP pleiotropy.

Low-Dose Perampanel Rescues Cortical Gamma Dysregulation Associated With Parvalbumin Interneuron GluA2 Upregulation in Epileptic Syngap1+/- Mice.

BACKGROUND: Loss-of-function SYNGAP1 mutations cause a neurodevelopmental disorder characterized by intellectual disability and epilepsy. SYNGAP1 is a Ras GTPase-activating protein that underlies the formation and experience-dependent regulation of postsynaptic densities. The mechanisms that contribute to this proposed monogenic cause of intellectual disability and epilepsy remain unresolved. METHODS: We established the phenotype of the epileptogenesis in a Syngap1+/- mouse model using 24-hour video electroencephalography (vEEG)/electromyography recordings at advancing ages. We administered an acute low dose of perampanel, a Food and Drug Administration-approved AMPA receptor (AMPAR) antagonist, during a follow-on 24-hour vEEG to investigate the role of AMPARs in Syngap1 haploinsufficiency. Immunohistochemistry was performed to determine the region- and location-specific differences in the expression of the GluA2 AMPAR subunit. RESULTS: A progressive worsening of the epilepsy with emergence of multiple seizure phenotypes, interictal spike frequency, sleep dysfunction, and hyperactivity was identified in Syngap1+/- mice. Interictal spikes emerged predominantly during non-rapid eye movement sleep in 24-hour vEEG of Syngap1+/- mice. Myoclonic seizures occurred at behavioral-state transitions both in Syngap1+/- mice and during an overnight EEG from a child with SYNGAP1 haploinsufficiency. In Syngap1+/- mice, EEG spectral power analyses identified a significant loss of gamma power modulation during behavioral-state transitions. A significant region-specific increase of GluA2 AMPAR subunit expression in the somas of parvalbumin-positive interneurons was identified. CONCLUSIONS: Acute dosing with perampanel significantly rescued behavioral state-dependent cortical gamma homeostasis, identifying a novel mechanism implicating Ca2+-impermeable AMPARs on parvalbumin-positive interneurons underlying circuit dysfunction in SYNGAP1 haploinsufficiency.

Brain-specific Drp1 regulates postsynaptic endocytosis and dendrite formation independently of mitochondrial division.

Dynamin-related protein 1 (Drp1) divides mitochondria as a mechano-chemical GTPase. However, the function of Drp1 beyond mitochondrial division is largely unknown. Multiple Drp1 isoforms are produced through mRNA splicing. One such isoform, Drp1ABCD, contains all four alternative exons and is specifically expressed in the brain. Here, we studied the function of Drp1ABCD in mouse neurons in both culture and animal systems using isoform-specific knockdown by shRNA and isoform-specific knockout by CRISPR/Cas9. We found that the expression of Drp1ABCD is induced during postnatal brain development. Drp1ABCD is enriched in dendritic spines and regulates postsynaptic clathrin-mediated endocytosis by positioning the endocytic zone at the postsynaptic density, independently of mitochondrial division. Drp1ABCD loss promotes the formation of ectopic dendrites in neurons and enhanced sensorimotor gating behavior in mice. These data reveal that Drp1ABCD controls postsynaptic endocytosis, neuronal morphology and brain function.

Mitochondrial Stasis Reveals p62-Mediated Ubiquitination in Parkin-Independent Mitophagy and Mitigates Nonalcoholic Fatty Liver Disease.

It is unknown what occurs if both mitochondrial division and fusion are completely blocked. Here, we introduced mitochondrial stasis by deleting two dynamin-related GTPases for division (Drp1) and fusion (Opa1) in livers. Mitochondrial stasis rescues liver damage and hypotrophy caused by the single knockout (KO). At the cellular level, mitochondrial stasis re-establishes mitochondrial size and rescues mitophagy defects caused by division deficiency. Using Drp1KO livers, we found that the autophagy adaptor protein p62/sequestosome-1-which is thought to function downstream of ubiquitination-promotes mitochondrial ubiquitination. p62 recruits two subunits of a cullin-RING ubiquitin E3 ligase complex, Keap1 and Rbx1, to mitochondria. Resembling Drp1KO, diet-induced nonalcoholic fatty livers enlarge mitochondria and accumulate mitophagy intermediates. Resembling Drp1Opa1KO, Opa1KO rescues liver damage in this disease model. Our data provide a new concept that mitochondrial stasis leads the spatial dimension of mitochondria to a stationary equilibrium and a new mechanism for mitochondrial ubiquitination in mitophagy.