Modulating Endoplasmic Reticulum Chaperones and Mutant Protein Degradation in GABRG2(Q390X) Associated with Genetic Epilepsy with Febrile Seizures Plus and Dravet Syndrome.

A significant number of patients with genetic epilepsy do not obtain seizure freedom, despite developments in new antiseizure drugs, suggesting a need for novel therapeutic approaches. Many genetic epilepsies are associated with misfolded mutant proteins, including GABRG2(Q390X)-associated Dravet syndrome, which we have previously shown to result in intracellular accumulation of mutant GABAA receptor γ2(Q390X) subunit protein. Thus, a potentially promising therapeutic approach is modulation of proteostasis, such as increasing endoplasmic reticulum (ER)-associated degradation (ERAD). To that end, we have here identified an ERAD-associated E3 ubiquitin ligase, HRD1, among other ubiquitin ligases, as a strong modulator of wildtype and mutant γ2 subunit expression. Overexpressing HRD1 or knockdown of HRD1 dose-dependently reduced the γ2(Q390X) subunit. Additionally, we show that zonisamide (ZNS)-an antiseizure drug reported to upregulate HRD1-reduces seizures in the Gabrg2+/Q390X mouse. We propose that a possible mechanism for this effect is a partial rescue of surface trafficking of GABAA receptors, which are otherwise sequestered in the ER due to the dominant-negative effect of the γ2(Q390X) subunit. Furthermore, this partial rescue was not due to changes in ER chaperones BiP and calnexin, as total expression of these chaperones was unchanged in γ2(Q390X) models. Our results here suggest that leveraging the endogenous ERAD pathway may present a potential method to degrade neurotoxic mutant proteins like the γ2(Q390X) subunit. We also demonstrate a pharmacological means of regulating proteostasis, as ZNS alters protein trafficking, providing further support for the use of proteostasis regulators for the treatment of genetic epilepsies.

γ-Aminobutyric acid transporter and GABAA receptor mechanisms in Slc6a1+/A288V and Slc6a1+/S295L mice associated with developmental and epileptic encephalopathies.

We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 (SLC6A1) in vitro and concluded that a partial or complete loss of γ-aminobutyric acid uptake due to impaired protein trafficking is the primary aetiology. Impairment of γ-aminobutyric acid transporter 1 function could cause compensatory changes in the expression of γ-aminobutyric acid receptors, which, in turn, modify disease pathophysiology and phenotype. Here we used different approaches including radioactive 3H γ-aminobutyric acid uptake in cells and synaptosomes, immunohistochemistry and confocal microscopy as well as brain slice surface protein biotinylation to characterize Slc6a1+/A288V and Slc6a1+/S295L mice, representative of a partial or a complete loss of function of SLC6A1 mutations, respectively. We employed the γ-aminobutyric acid transporter 1-specific inhibitor [3H]tiagabine binding and GABAA receptor subunit-specific radioligand binding to profile the γ-aminobutyric acid transporter 1 and GABAA receptor expression in major brain regions such as cortex, cerebellum, hippocampus and thalamus. We also determined the total and surface expression of γ-aminobutyric acid transporter 1, γ-aminobutyric acid transporter 3 and expression of GABAA receptor in the major brain regions in the knockin mice. We found that γ-aminobutyric acid transporter 1 protein was markedly reduced in cortex, hippocampus, thalamus and cerebellum in both mutant mouse lines. Consistent with the findings of reduced γ-aminobutyric acid uptake for both γ-aminobutyric acid transporter 1(A288V) and γ-aminobutyric acid transporter 1(S295L), both the total and the γ-aminobutyric acid transporter 1-mediated 3H γ-aminobutyric acid reuptake was reduced. We found that γ-aminobutyric acid transporter 3 is only abundantly expressed in the thalamus and there was no compensatory increase of γ-aminobutyric acid transporter 3 in either of the mutant mouse lines. γ-Aminobutyric acid transporter 1 was reduced in both somatic regions and nonsomatic regions in both mouse models, in which a ring-like structure was identified only in the Slc6a1+/A288V mouse, suggesting more γ-aminobutyric acid transporter 1 retention inside endoplasmic reticulum in the Slc6a1+/A288V mouse. The [3H]tiagabine binding was similar in both mouse models despite the difference in γ-aminobutyric acid uptake function and γ-aminobutyric acid transporter 1 protein expression for both mutations. There were no differences in GABAA receptor subtype expression, except for a small increase in the expression of α5 subunits of GABAA receptor in the hippocampus of Slc6a1S295L homozygous mice, suggesting a potential interaction between the expression of this GABAA receptor subtype and the mutant γ-aminobutyric acid transporter 1. The study provides the first comprehensive characterization of the SLC6A1 mutations in vivo in two representative mouse models. Because both γ-aminobutyric acid transporter 1 and GABAA receptors are targets for anti-seizure medications, the findings from this study can help guide tailored treatment options based on the expression and function of γ-aminobutyric acid transporter 1 and GABAA receptor in SLC6A1 mutation-mediated neurodevelopmental and epileptic encephalopathies.

Retrospective analysis of preoperative application of triple-modal pre-rehabilitation on postoperative recovery of colorectal cancer patients.

PURPOSE: To retrospectively analyze the difference between triple-modal pre-rehabilitation and common treatment in patients with colorectal cancer (CRC). METHODS: A total of 145 patients with CRC diagnosed by pathology and admitted to our hospital for surgery between June 2020 and June 2022 were included in the study. All patients were divided into two groups: the triple-modal pre-rehabilitation group (pre-rehabilitation group) and the common treatment group. The triple-modal pre-rehabilitation strategy included exercise (3-5 times per week, with each session lasting more than 50 min), nutritional support, and psychological support. The study was designed to assess the potential of the pre-rehabilitation intervention to accelerate postoperative recovery by assessing the 6-min walk test, nutritional indicators, and HADS score before and after surgery. RESULTS: The pre-rehabilitation intervention did not reduce the duration of initial postoperative recovery or the incidence of postoperative complications, but it did increase the patients' exercise capacity (as determined by the 6-min walk test), with the pre-rehabilitation group performing significantly better than the common group (433.0 (105.0) vs. 389.0 (103.5), P < 0.001). The study also found that triple-modal pre-rehabilitation was beneficial for the early recovery of nutritional status in surgical patients and improved anxiety and depression in patients after surgery, especially in those who had not received neoadjuvant therapy. CONCLUSION: The triple-modal pre-rehabilitation strategy is of significant importance for reducing stress and improving the functional reserve of patients with colorectal cancer (CRC) during the perioperative period. The results of our study provide further support for the integration of the triple-modal pre-rehabilitation strategy into the treatment and care of CRC patients.

4-Phenylbutyrate promoted wild-type γ-aminobutyric acid type A receptor trafficking, reduced endoplasmic reticulum stress, and mitigated seizures in Gabrg2+/Q390X mice associated with Dravet syndrome.

OBJECTIVE: γ-Aminobutyric acid type A (GABAA ) receptor subunit gene mutations are major causes of various epilepsy syndromes, including severe kinds such as Dravet syndrome. Although the GABAA receptor is a major target for antiseizure medications, treating GABAA receptor mutations with receptor channel modulators is ineffective. Here, we determined the effect of a novel treatment with 4-phenylbutyrate (PBA) in Gabrg2+/Q390X knockin mice associated with Dravet syndrome. METHODS: We used biochemistry in conjunction with differential tagging of the wild-type and the mutant alleles, live brain slice surface biotinylation, microsome isolation, patch-clamp whole-cell recordings, and video-monitoring synchronized electroencephalographic (EEG) recordings in Gabrg2+/Q390X mice to determine the effect of PBA in vitro with recombinant GABAA receptors and in vivo with knockin mice. RESULTS: We found that PBA reduced the mutant γ2(Q390X) subunit protein aggregates, enhanced the wild-type GABAA receptor subunits' trafficking, and increased the membrane expression of the wild-type receptors. PBA increased the current amplitude of GABA-evoked current in human embryonic kidney 293T cells and the neurons bearing the γ2(Q390X) subunit protein. PBA also proved to reduce endoplasmic reticulum (ER) stress caused by the mutant γ2(Q390X) subunit protein, as well as mitigating seizures and EEG abnormalities in the Gabrg2+/Q390X mice. SIGNIFICANCE: This research has unveiled a promising and innovative approach for treating epilepsy linked to GABAA receptor mutations through an unconventional antiseizure mechanism. Rather than directly modulating the affected mutant channel, PBA facilitates the folding and transportation of wild-type receptor subunits to the cell membrane and synapse. Combining these findings with our previous study, which demonstrated PBA's efficacy in restoring GABA transporter 1 (encoded by SLC6A1) function, we propose that PBA holds significant potential for a wide range of genetic epilepsies. Its ability to target shared molecular pathways involving mutant protein ER retention and impaired protein membrane trafficking suggests broad application in treating such conditions.

Epilepsy plus blindness in microdeletion of GABRA1 and GABRG2 in mouse and human.

OBJECTIVE: GABAA receptor subunit gene (GABR) mutations are significant causes of epilepsy, including syndromic epilepsy. This report for the first time, describes intractable epilepsy and blindness due to optic atrophy in our patient, who has a microdeletion of the GABRA1 and GABRG2 genes. We then characterized the molecular phenotypes and determined patho-mechanisms underlying the genotype-phenotype correlations in a mouse model who is haploinsufficient for both genes (Gabra1+/-/Gabrg2+/- mouse). METHODS: Electroencephalography was conducted in both human and mice with the same gene loss. GABAA receptor expression was evaluated by biochemical and imaging approaches. Optic nerve atrophy was evaluated with fundus photography in human while electronic microscopy, visual evoked potential and electroretinography recordings were conducted in mice. RESULTS: The patient has bilateral optical nerve atrophy. Mice displayed spontaneous seizures, reduced electroretinography oscillatory potential and reduced GABAA receptor α1, ß2 and γ2 subunit expression in various brain regions. Electronic microscopy showed that mice also had optic nerve degeneration, as indicated by increased G-ratio, the ratio of the inner axonal diameter to the total outer diameter, suggesting impaired myelination of axons. More importantly, we identified that phenobarbital was the most effective anticonvulsant in mice and the patient's seizures were also controlled with phenobarbital after failing multiple anti-seizure drugs. CONCLUSIONS: This study is the first report of haploinsufficiency of two GABR epilepsy genes and visual impairment due to altered axonal myelination and resultant optic nerve atrophy. The study suggests the far-reaching impact of GABR mutations and the translational significance of animal models with the same etiology.

SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis.

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10-3, 95% confidence interval: 1.5-15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).

GABAA Receptor ß3 Subunit Mutation N328D Heterozygous Knock-in Mice Have Lennox-Gastaut Syndrome.

Lennox-Gastaut Syndrome (LGS) is a developmental and epileptic encephalopathy (DEE) characterized by multiple seizure types, electroencephalogram (EEG) patterns, and cognitive decline. Its etiology has a prominent genetic component, including variants in GABRB3 that encodes the GABAA receptor (GABAAR) ß3 subunit. LGS has an unknown pathophysiology, and few animal models are available for studying LGS. The objective of this study was to evaluate Gabrb3+/N328D knock-in mice as a model for LGS. We generated a heterozygous knock-in mouse expressing Gabrb3 (c.A982G, p.N238D), a de novo mutation identified in a patient with LGS. We investigated Gabrb3+/N328D mice for features of LGS. In 2-4-month-old male and female C57BL/J6 wild-type and Gabrb3+/N328D mice, we investigated seizure severity using video-monitored EEG, cognitive impairment using a suite of behavioral tests, and profiled GABAAR subunit expression by Western blot. Gabrb3+/N328D mice showed spontaneous seizures and signs of cognitive impairment, including deficits in spatial learning, memory, and locomotion. Moreover, Gabrb3+/N328D mice showed reduced ß3 subunit expression in the cerebellum, hippocampus, and thalamus. This phenotype of epilepsy and neurological impairment resembles the LGS patient phenotype. We conclude that Gabrb3+/N328D mice provide a good model for investigating the pathophysiology and therapeutic intervention of LGS and DEEs.

MG53 ameliorates nerve injury induced neuropathic pain through the regulation of Nrf2/HO-1 signaling in rats.

Neuropathic pain is one of the most common types of chronic pain, and it arises as a direct consequence of a lesion or disease that affects the somatosensory system. Mitsugumin53 (MG53), which is a member of the TRIM family of proteins and is known as TRIM72, exerts protective effects on muscle, lung, kidney, brain, and other cells or tissues. Recently, increasing evidence has indicated that MG53 plays a vital role in regulating neuroinflammation and oxidative stress. However, the relationship between MG53 and neuropathic pain is unclear. In this study, we aimed to explore the role of MG3 in neuropathic pain after chronic constriction injury (CCI) to the sciatic nerve in rats. To explore the mechanism of MG53 regulating the development of neuropathic pain, the rats was injected (intrathecal injection) of recombinant human MG53 (rhMG53) protein and/or nuclear factor erythroid 2-related factor 2 (Nrf2) siRNA after CCI. Mechanical allodynia or thermal hyperalgesia was assessed by the 50% paw withdrawal threshold (PWT) or the paw withdrawal latency (PWL). The target molecules was detected using western blotting (WB), immunofluorescence (IF), quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), biochemical evaluations, and Dihydroethidium (DHE) staining. The results indicated that the expression level of MG53 in the spinal cord was increased after CCI in rats. Moreover, intrathecal injection with rhMG53 protein notably alleviated CCI-induced mechanical allodynia, thermal hyperalgesia, neuroinflammation，oxidative stress and the increased level of reactive oxygen species (ROS) via activation of the Nrf2/heme oxygenase-1 (HO-1) signaling pathway. However, administration of Nrf2 siRNA abrogated the analgesic, anti-inflammatory and antioxidant effects of rhMG53 in CCI model rats. Our study demonstrated that MG53 improved neuropathic pain, neuroinflammation, and oxidative stress via activation of the Nrf2/HO-1 signaling pathway in the spinal cord of CCI model rats, which suggested that MG53 may serve as a new target for the treatment of neuropathic pain.

Liver Transplantation Outcomes of HBV-, HCV-, and Alcohol-induced Hepatocellular Carcinoma in the United States: Analysis of National Inpatient Samples.

OBJECTIVE: Liver transplantation is a current treatment option for hepatocellular carcinoma (HCC). The United States National Inpatient Sample database was utilized to identify risk factors that influence the outcome of liver transplantation, including locoregional recurrence, distant metastasis, and in-hospital mortality, in HCC patients with concurrent hepatitis B infection, hepatitis C infection, or alcoholic cirrhosis. METHODS: This retrospective cohort study included HCC patients (n=2391) from the National Inpatient Sample database who underwent liver transplantation and were diagnosed with hepatitis B or C virus infection, co-infection with hepatitis B and C, or alcoholic cirrhosis of the liver between 2005 and 2014. Associations between HCC etiology and post-transplant outcomes were examined with multivariate analysis models. RESULTS: Liver cirrhosis was due to alcohol in 10.5% of patients, hepatitis B in 6.6%, hepatitis C in 10.8%, and combined hepatitis B and C infection in 24.3%. Distant metastasis was found in 16.7% of patients infected with hepatitis B and 9% of hepatitis C patients. Local recurrence of HCC was significantly more likely to occur in patients with hepatitis B than in those with alcohol-induced disease. CONCLUSION: After liver transplantation, patients with hepatitis B infection have a higher risk of local recurrence and distant metastasis. Postoperative care and patient tracking are essential for liver transplant patients with hepatitis B infection.

Generation and characterization of iPS cell line (CTGUi001-A) from skin fibroblasts of a patient with Fabry disease.

We have generated an iPSCs line (CTGUi001-A) from dermal fibroblasts of a 16-year-old male Fabry disease patient with a novel GLA gene mutation (c.156C > A) using Sendai virus encoding the four Yamanaka factors OCT4, SOX2, KLF4, and c-MYC. The CTGUi001-A iPSC line displayed typical embryonic stem cell-like morphology, carried the GLA gene mutation, expressed several pluripotent stem cell makers, retained normal karyotype (46, XY) and was capable of forming teratomas containing three germ layers.

Heterozygous GABAA receptor ß3 subunit N110D knock-in mice have epileptic spasms.

OBJECTIVE: Infantile spasms is an epileptic encephalopathy of childhood, and its pathophysiology is largely unknown. We generated a heterozygous knock-in mouse with the human infantile spasms-associated de novo mutation GABRB3 (c.A328G, p.N110D) to investigate its molecular mechanisms and to establish the Gabrb3+/N110D knock-in mouse as a model of infantile spasms syndrome. METHODS: We used electroencephalography (EEG) and video monitoring to characterize seizure types, and a suite of behavioral tests to identify neurological and behavioral impairment in Gabrb3+/N110D knock-in mice. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded from layer V/VI pyramidal neurons in somatosensory cortex, and extracellular multi-unit recordings from the ventral basal nucleus of the thalamus in a horizontal thalamocortical slice were used to assess spontaneous thalamocortical oscillations. RESULTS: The infantile spasms-associated human de novo mutation GABRB3 (c.A328G, p.N110D) caused epileptic spasms early in development and multiple seizure types in adult Gabrb3+/N110D knock-in mice. Signs of neurological impairment, anxiety, hyperactivity, social impairment, and deficits in spatial learning and memory were also observed. Gabrb3+/N110D mice had reduced cortical mIPSCs and increased duration of spontaneous oscillatory firing in the somatosensory thalamocortical circuit. SIGNIFICANCE: The Gabrb3+/N110D knock-in mouse has epileptic spasms, seizures, and other neurological impairments that are consistent with infantile spasms syndrome in patients. Multiple seizure types and abnormal behaviors indicative of neurological impairment both early and late in development suggest that Gabrb3+/N110D mice can be used to study the pathophysiology of infantile spasms. Reduced cortical inhibition and increased duration of thalamocortical oscillatory firing suggest perturbations in thalamocortical circuits.

Variable Expression of GABAA Receptor Subunit Gamma 2 Mutation in a Nuclear Family Displaying Developmental and Encephalopathic Phenotype.

Mutations in GABAA receptor subunit genes (GABRs) are a major etiology for developmental and epileptic encephalopathies (DEEs). This article reports a case of a genetic abnormality in GABRG2 and updates the pathophysiology and treatment development for mutations in DEEs based on recent advances. Mutations in GABRs, especially in GABRA1, GABRB2, GABRB3, and GABRG2, impair GABAergic signaling and are frequently associated with DEEs such as Dravet syndrome and Lennox-Gastaut syndrome, as GABAergic signaling is critical for early brain development. We here present a novel association of a microdeletion of GABRG2 with a diagnosed DEE phenotype. We characterized the clinical phenotype and underlying mechanisms, including molecular genetics, EEGs, and MRI. We then compiled an update of molecular mechanisms of GABR mutations, especially the mutations in GABRB3 and GABRG2 attributed to DEEs. Genetic therapy is also discussed as a new avenue for treatment of DEEs through employing antisense oligonucleotide techniques. There is an urgent need to define treatment targets and explore new treatment paradigms for the DEEs, as early deployment could alleviate long-term disabilities and improve quality of life for patients. This study highlights biomolecular targets for future therapeutic interventions, including via both pharmacological and genetic approaches.

4-Phenylbutyrate restored γ-aminobutyric acid uptake and reduced seizures in SLC6A1 patient variant-bearing cell and mouse models.

We have studied the molecular mechanisms of variants in solute carrier Family 6 Member 1 associated with neurodevelopmental disorders, including various epilepsy syndromes, autism and intellectual disability. Based on functional assays of solute carrier Family 6 Member 1 variants, we conclude that partial or complete loss of γ-amino butyric acid uptake due to reduced membrane γ-amino butyric acid transporter 1 trafficking is the primary aetiology. Importantly, we identified common patterns of the mutant γ-amino butyric acid transporter 1 protein trafficking from biogenesis, oligomerization, glycosylation and translocation to the cell membrane across variants in different cell types such as astrocytes and neurons. We hypothesize that therapeutic approaches to facilitate membrane trafficking would increase γ-amino butyric acid transporter 1 protein membrane expression and function. 4-Phenylbutyrate is a Food and Drug Administration-approved drug for paediatric use and is orally bioavailable. 4-Phenylbutyrate shows promise in the treatment of cystic fibrosis. The common cellular mechanisms shared by the mutant γ-amino butyric acid transporter 1 and cystic fibrosis transmembrane conductance regulator led us to hypothesize that 4-phenylbutyrate could be a potential treatment option for solute carrier Family 6 Member 1 mutations. We examined the impact of 4-phenylbutyrate across a library of variants in cell and knockin mouse models. Because γ-amino butyric acid transporter 1 is expressed in both neurons and astrocytes, and γ-amino butyric acid transporter 1 deficiency in astrocytes has been hypothesized to underlie seizure generation, we tested the effect of 4-phenylbutyrate in both neurons and astrocytes with a focus on astrocytes. We demonstrated existence of the mutant γ-amino butyric acid transporter 1 retaining wildtype γ-amino butyric acid transporter 1, suggesting the mutant protein causes aberrant protein oligomerization and trafficking. 4-Phenylbutyrate increased γ-amino butyric acid uptake in both mouse and human astrocytes and neurons bearing the variants. Importantly, 4-phenylbutyrate alone increased γ-amino butyric acid transporter 1 expression and suppressed spike wave discharges in heterozygous knockin mice. Although the mechanisms of action for 4-phenylbutyrate are still unclear, with multiple possibly being involved, it is likely that 4-phenylbutyrate can facilitate the forward trafficking of the wildtype γ-amino butyric acid transporter 1 regardless of rescuing the mutant γ-amino butyric acid transporter 1, thus increasing γ-amino butyric acid uptake. All patients with solute carrier Family 6 Member 1 variants are heterozygous and carry one wildtype allele, suggesting a great opportunity for treatment development leveraging wildtype protein trafficking. The study opens a novel avenue of treatment development for genetic epilepsy via drug repurposing.

Astrocytic GABA transporter 1 deficit in novel SLC6A1 variants mediated epilepsy: Connected from protein destabilization to seizures in mice and humans.

OBJECTIVE: Mutations in γ-aminobutyric acid (GABA) transporter 1 (GAT-1)-encoding SLC6A1 have been associated with myoclonic atonic epilepsy and other phenotypes. We determined the patho-mechanisms of the mutant GAT-1, in order to identify treatment targets. METHODS: We conducted whole-exome sequencing of patients with myoclonic atonic epilepsy (MAE) and characterized the seizure phenotypes and EEG patterns. We studied the protein stability and structural changes with homology modeling and machine learning tools. We characterized the function and trafficking of the mutant GAT-1 with 3H radioactive GABA uptake assay and confocal microscopy. We utilized different models including a knockin mouse and human astrocytes derived from induced pluripotent stem cells (iPSCs). We focused on astrocytes because of their direct impact of astrocytic GAT-1 in seizures. RESULTS: We identified four novel SLC6A1 variants associated with MAE and 2 to 4 Hz spike-wave discharges as a common EEG feature. Machine learning tools predicted that the variant proteins are destabilized. The variant protein had reduced expression and reduced GABA uptake due to endoplasmic reticular retention. The consistent observation was made in cortical and thalamic astrocytes from variant-knockin mice and human iPSC-derived astrocytes. The Slc6a+/A288V mouse, representative of MAE, had increased 5-7 Hz spike-wave discharges and absence seizures. INTERPRETATION: SLC6A1 variants in various locations of the protein peptides can cause MAE with similar seizure phenotypes and EEG features. Reduced GABA uptake is due to decreased functional GAT-1, which, in thalamic astrocytes, could result in increased extracellular GABA accumulation and enhanced tonic inhibition, leading to seizures and abnormal EEGs.

[Voluntary wheel running exercise regulates microglia polarization in hippocampus through STAT3 signal pathway to inhibit depression-like behavior induced by chronic stress in rats].

This paper was aimed to investigate the effect of voluntary wheel running exercise on depression-like behavior induced by chronic water immersion restraint stress (CWIRS) and the underlying mechanism. Sprague-Dawley (SD) rats received CWIRS to induce depression-like behavior and 4-week voluntary wheel running exercise. Meanwhile, the rats were treated with lipopolysaccharide (LPS) or STAT3 over-expression vector (pcDNA-STAT3) by intracerebroventricular injection. Behavioral tests were used to detect depression-like behavior. ELISA assay was used to detect levels of various inflammatory factors in the rat hippocampus. Western blot was used to detect protein expression levels of ionized calcium binding adaptor molecule 1 (Iba1), inducible nitric oxide synthase (iNOS), arginase 1 (Arg1), phosphorylated STAT3 (p-STAT3) and total STAT3 (t-STAT3). The results showed that, compared with stress group, stress + exercise group exhibited improved depression-like behavior, decreased interleukin-1ß (IL-1ß) and IL-6 levels, increased IL-4 and IL-10 levels, down-regulated Iba-1 and iNOS protein expression levels, up-regulated Arg1 protein expression level, and decreased p-STAT3/t-STAT3 ratio in hippocampal tissue. LPS reversed the improving effect of voluntary wheel running exercise on depression-like behavior in rats, and the over-expression of STAT3 reversed the promoting effects of voluntary wheel running on M2 polarization of microglial cells in rat hippocampus and depression-like behavior. These results suggest that voluntary wheel running ameliorates the depression-like behavior induced by CWIRS in rats, and the mechanism may be related to regulating hippocampal microglia polarization via STAT3 signaling pathway.

Disruption of the Ubiquitin-Proteasome System and Elevated Endoplasmic Reticulum Stress in Epilepsy.

The epilepsies are a broad group of conditions characterized by repeated seizures, and together are one of the most common neurological disorders. Additionally, epilepsy is comorbid with many neurological disorders, including lysosomal storage diseases, syndromic intellectual disability, and autism spectrum disorder. Despite the prevalence, treatments are still unsatisfactory: approximately 30% of epileptic patients do not adequately respond to existing therapeutics, which primarily target ion channels. Therefore, new therapeutic approaches are needed. Disturbed proteostasis is an emerging mechanism in epilepsy, with profound effects on neuronal health and function. Proteostasis, the dynamic balance of protein synthesis and degradation, can be directly disrupted by epilepsy-associated mutations in various components of the ubiquitin-proteasome system (UPS), or impairments can be secondary to seizure activity or misfolded proteins. Endoplasmic reticulum (ER) stress can arise from failed proteostasis and result in neuronal death. In light of this, several treatment modalities that modify components of proteostasis have shown promise in the management of neurological disorders. These include chemical chaperones to assist proper folding of proteins, inhibitors of overly active protein degradation, and enhancers of endogenous proteolytic pathways, such as the UPS. This review summarizes recent work on the pathomechanisms of abnormal protein folding and degradation in epilepsy, as well as treatment developments targeting this area.

[Identification of Cordyceps cicadae and Tolypocladium dujiaolongae based on ITS sequences and chemical pattern recognition method].

Based on ITS sequences, the molecular identification of Cordyceps cicadae and Tolypocladium dujiaolongae was carried out, and high-performance liquid chromatography(HPLC) fingerprint combined with chemical pattern recognition method was established to differentiate C. cicadae from its adulterant T. dujiaolongae. The genomic DNA from 10 batches of C. cicadae and five batches of T. dujiaolongae was extracted, and ITS sequences were amplified by PCR and sequenced. The stable differential sites of these two species were compared and the phylogenetic tree was constructed via MEGA 7.0. HPLC was used to establish the fingerprints of C. cicadae and T. dujiaolongae, and similarity evaluation, cluster analysis(CA), principal component analysis(PCA), and partial least squares discriminant analysis(PLS-DA) were applied to investigate the chemical pattern recognition. The result showed that the sources of these two species were different, and there were 115 stable differential sites in ITS sequences of C. cicadae and T. dujiao-longae. The phylogenetic tree could distinguish them effectively. HPLC fingerprints of 18 batches of C. cicadae and 5 batches of T. dujiaolongae were established. The results of CA, PCA, and PLS-DA were consistent, which could distinguish them well, indicating that there were great differences in chemical components between C. cicadae and T. dujiaolongae. The results of PLS-DA showed that six components such as uridine, guanosine, adenosine, and N~6-(2-hydroxyethyl) adenosine were the main differential markers of the two species. ITS sequences and HPLC fingerprint combined with the chemical pattern recognition method can serve as the identification and differentiation methods for C. cicadae and T. dujiaolongae.

Epileptic Mechanisms Shared by Alzheimer's Disease: Viewed via the Unique Lens of Genetic Epilepsy.

Our recent work on genetic epilepsy (GE) has identified common mechanisms between GE and neurodegenerative diseases including Alzheimer's disease (AD). Although both disorders are seemingly unrelated and occur at opposite ends of the age spectrum, it is likely there are shared mechanisms and studies on GE could provide unique insights into AD pathogenesis. Neurodegenerative diseases are typically late-onset disorders, but the underlying pathology may have already occurred long before the clinical symptoms emerge. Pathophysiology in the early phase of these diseases is understudied but critical for developing mechanism-based treatment. In AD, increased seizure susceptibility and silent epileptiform activity due to disrupted excitatory/inhibitory (E/I) balance has been identified much earlier than cognition deficit. Increased epileptiform activity is likely a main pathology in the early phase that directly contributes to impaired cognition. It is an enormous challenge to model the early phase of pathology with conventional AD mouse models due to the chronic disease course, let alone the complex interplay between subclinical nonconvulsive epileptiform activity, AD pathology, and cognition deficit. We have extensively studied GE, especially with gene mutations that affect the GABA pathway such as mutations in GABAA receptors and GABA transporter 1. We believe that some mouse models developed for studying GE and insights gained from GE could provide unique opportunity to understand AD. These include the pathology in early phase of AD, endoplasmic reticulum (ER) stress, and E/I imbalance as well as the contribution to cognitive deficit. In this review, we will focus on the overlapping mechanisms between GE and AD, the insights from mutations affecting GABAA receptors, and GABA transporter 1. We will detail mechanisms of E/I imbalance and the toxic epileptiform generation in AD, and the complex interplay between ER stress, impaired membrane protein trafficking, and synaptic physiology in both GE and AD.

[Herbal textual research on Rubi Fructus].

Rubi Fructus is a commonly used traditional Chinese medicine. The origin of Rubi Fructus is the dried fruit of Rubus chingii, a plant of the family Rosaceae, according to the 2015 edition of Chinese pharmacopoeia. There are some differences in the plant origin of Rubi Fructus in ancient herbal literature, to trace back its sources, we conducted a textual research on its origin, producing areas, quality evaluation, processing and concocting, properties, tastes and efficacy etc. based on the records of ancient herbal literatures and combined with plant morphology and related investigation. RESULTS:: showed that the variety of Rubi Fructus was more complex among ancient herbal literature, including R. coreanus, R. hirsutus, R. corchorifolius, R. foliolosus and other mixed varieties. Most scholars believe that the R. chingii has not been recorded in ancient herbal literature, while R. chingii was recorded as early as the Ming Dynasty in Compendium of materia medica through our textual research. Ancient Chinese herbs recorded that Rubi Fructus was mostly produced in Hubei, Shandong, Shanxi and Jiangsu provinces, while R. chingii mainly produced in Anhui, Jiangsu, Zhejiang, Jiangxi, Fujian and other provinces nowadays. Also, it was recorded that Rubi Fructus harvested in wheat field during May were the best. Besides, R. chingii with big, full, grain integrate, firm, yellow and green color, sour taste and impurity free possess the best quality in the contemporary. The ancient records of processing and concocting, properties, tastes and efficacy were basically the same as modern ones.These results provide the basis for the correct utilization and further development of Rubi Fructus.

[Systematic analysis on chemical constituents of Mori Cortex, mulberry root bark and its phellem layer based on HPLC-ESI-MS].

In this study, HPLC-ESI-MS and HPLC methods were established to explore the differences in the main chemical components and content of Mori Cortex with(mulberry root bark) and without(Mori Cortex) the phellem layer from both qualitative and quantitative aspects. The HPLC-ESI-MS method was used for quality analysis in positive and negative ion modes, and 33 compounds were identified in mulberry root bark, 22 compounds in Mori Cortex, and 26 compounds in phellem layer; mulberry root bark and Mori Cortex shared 22 components, and mulberry root bark has 11 unique compounds; Mori Cortex and its phellem layer shared 15 components, while Mori Cortex has 7 unique compounds. HPLC method was used to simultaneously determine 7 major constituents, including mulberroside A, chlorogenic acid, dihydromorin, oxyresveratrol, moracin O, kuwanon G, and kuwanon H, and the developed method showed good linearity(r>0.998 9) within the concentration range and the recoveries varied from 99.88% to 103.0%, and the RSD was 1.7%-2.9%. The HPLC results showed that the contents of the 7 compounds have great differences in 13 batches samples, compared with mulberry root bark, the contents of mulberroside A, chlorogenic acid, dihydromorin and moracin O of Mori Cortex were increased, while the contents of oxyresveratrol, kuwanon G and kuwanon H were decreased after peeling process. These results can provide a basis for the rationality and quality control of Mori Cortex required to remove the phellem layer.