Locally Reducing KCC2 Activity in the Hippocampus is Sufficient to Induce Temporal Lobe Epilepsy

Mesial temporal lobe epilepsy (mTLE) is the most common form of epilepsy, believed to arise in part from compromised GABAergic inhibition. The neuronal specific K+/Cl− co-transporter 2 (KCC2) is a critical determinant of the efficacy of GABAergic inhibition and deficits in its activity are observed in mTLE patients and animal models of epilepsy. To test if reductions of KCC2 activity directly contribute to the pathophysiology of mTLE, we locally ablated KCC2 expression in a subset of principal neurons within the adult hippocampus. Deletion of KCC2 resulted in compromised GABAergic inhibition and the development of spontaneous, recurrent generalized seizures. Moreover, local ablation of KCC2 activity resulted in hippocampal sclerosis, a key pathological change seen in mTLE. Collectively, our results demonstrate that local deficits in KCC2 activity within the hippocampus are sufficient to precipitate mTLE

Isolation and Characterization of Multi-Protein Complexes Enriched in the K-Cl Co-transporter 2 From Brain Plasma Membranes

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity

Direct activation of KCC2 arrests benzodiazepine refractory status epilepticus and limits the subsequent neuronal injury in mice

Hyperpolarizing GABAAR currents, the unitary events that underlie synaptic inhibition, are dependent upon efficient Cl− extrusion, a process that is facilitated by the neuronal specific K+/Cl− co-transporter KCC2. Its activity is also a determinant of the anticonvulsant efficacy of the canonical GABAAR-positive allosteric: benzodiazepines (BDZs). Compromised KCC2 activity is implicated in the pathophysiology of status epilepticus (SE), a medical emergency that rapidly becomes refractory to BDZ (BDZ-RSE). Here, we have identified small molecules that directly bind to and activate KCC2, which leads to reduced neuronal Cl− accumulation and excitability. KCC2 activation does not induce any overt effects on behavior but prevents the development of and terminates ongoing BDZ-RSE. In addition, KCC2 activation reduces neuronal cell death following BDZ-RSE. Collectively, these findings demonstrate that KCC2 activation is a promising strategy to terminate BDZ-resistant seizures and limit the associated neuronal injury

Association of PPARγ2 polymorphisms with carcass and meat quality traits in a Pietrain x Jinhua F2 population

The PPARγ2 gene is a key regulator of both proliferation and preadipocyte differentiation in mammals. Herein its genotype and allele frequencies were analyzed using PCR-SSCP in eight pig breeds (N = 416). Two kinds of polymorphisms of the PPARγ2 gene were detected, including a previously reported shift SNP A177G (Met59Val) in exon 1 and a novel silent mutation G876A in exon 5. The results revealed that European pig breeds carry a higher allele A frequency at the A177G locus and a fixed GG genotype at the G876A locus. Allele A at the G876A locus was only found in Jinhua pigs. The association between haplotype (A177G/G876A) and carcass and meat quality traits was analyzed in a Pietrain x Jinhua F2 population (N = 248). The PPARγ2 gene was found to be significantly associated with backfat thickness at the shoulder (p < 0.05), 6–7th ribs (p < 0.01), last rib (p < 0.01), gluteus medius (p <0.05) and ham weight (p < 0.01). Significant effects of different haplotypes on ham weight and backfat thickness at the 6–7th ribs, last rib, and gluteus medius were also observed

The International Deep Brain Stimulation Registry and Database for Gilles de la Tourette Syndrome: How Does It Work?

Tourette Syndrome (TS) is a neuropsychiatric disease characterized by a combination of motor and vocal tics. Deep brain stimulation (DBS), already widely utilized for Parkinson's disease and other movement disorders, is an emerging therapy for select and severe cases of TS that are resistant to medication and behavioral therapy. Over the last two decades, DBS has been used experimentally to manage severe TS cases. The results of case reports and small case series have been variable but in general positive. The reported interventions have, however, been variable, and there remain non-standardized selection criteria, various brain targets, differences in hardware, as well as variability in the programming parameters utilized. DBS centers perform only a handful of TS DBS cases each year, making large-scale outcomes difficult to study and to interpret. These limitations, coupled with the variable effect of surgery, and the overall small numbers of TS patients with DBS worldwide, have delayed regulatory agency approval (e.g., FDA and equivalent agencies around the world). The Tourette Association of America, in response to the worldwide need for a more organized and collaborative effort, launched an international TS DBS registry and database. The main goal of the project has been to share data, uncover best practices, improve outcomes, and to provide critical information to regulatory agencies. The international registry and database has improved the communication and collaboration among TS DBS centers worldwide. In this paper we will review some of the key operation details for the international TS DBS database and registry

Positive association of the hepatic lipase gene polymorphism c.514C > T with estrogen replacement therapy response

<p>Abstract</p> <p>Background</p> <p>Hepatic lipase (HL), an enzyme present in the hepatic sinusoids, is responsible for the lipolysis of lipoproteins. Human HL contains four polymorphic sites: G-250A, T-710C, A-763G, and C-514T single-nucleotide polymorphism (SNPs). The last polymorphism is the focus of the current study. The genotypes associated with the C-514T polymorphism are CC (normal homozygous - W), CT (heterozygous - H), and TT (minor-allele homozygous - M). HL activity is significantly impaired in individuals of the TT and CT genotypes. A total of 58 post-menopausal women were studied. The subjects were hysterectomized women receiving hormone replacement therapy consisting of 0.625 mg of conjugated equine estrogen once a day. The inclusion criteria were menopause of up to three years and normal blood tests, radiographs, cervical-vaginal cytology, and densitometry. DNA was extracted from the buccal and blood cells of all 58 patients using a commercially available kit (GFX^®- Amersham-Pharmacia, USA).</p> <p>Results</p> <p>Statistically significant reductions in triglycerides (t = 2.16; n = 58; p = 0.03) but not in total cholesterol (t = 0.14; n = 58; p = 0.89) were found after treatment. This group of good responders were carriers of the T allele; the CT and TT genotypes were present significantly more frequently than in the group of non-responders (p = 0.02 or p = 0.07, respectively). However, no significant difference in HDL-C (t = 0.94; n = 58; p = 0.35) or LDL-C (t = -0.83; n = 58; p = 0.41) was found in these patients.</p> <p>Conclusions</p> <p>The variation in lipid profile associated with the C-514T polymorphism is significant, and the T allele is associated with the best response to ERT.</p

The Fate of Porous Hydroxyapatite Granules Used in Facial Skeletal Augmentation

Facial appearance is largely determined by the morphology of the underlying skeleton. Hydroxyapatite is one of several materials available to enhance projection of the facial skeleton. This study evaluated the long-term maintenance of augmented bony projection when porous hydroxyapatite granules are used on the facial skeleton. Ten female patients aged 28–58 years were studied following aesthetic augmentation of the facial skeleton at 24 sites using porous hydroxyapatite granules. Postoperative CT scans at 3 months served as the baseline measurement and compared with scans taken at 1 and 2 years, with the thickness of the hydroxyapatite measured in axial and coronal planes. Thickness of original bone plus overlay of hydroxyapatite, thickness of the overlying soft tissue, and the overall projection (bone plus soft tissue) were recorded. It was found that 99.7% of the hydroxyapatite was maintained at 2 years, with no statistical difference (t test) from the baseline measurement. The overall projection (bony and soft tissue) was maintained as there was no evidence of native bone resorption or soft tissue atrophy. Radiographic results confirmed that the use of porous hydroxyapatite granules for enhancement of the facial skeleton is not only a predictable procedure, but maintains full bony projection at 2 years

Relationships of dietary patterns with body composition in older adults differ by gender and PPAR-γ Pro12Ala genotype

Dietary patterns may better capture the multifaceted effects of diet on body composition than individual nutrients or foods. The objective of this study was to investigate the dietary patterns of a cohort of older adults, and examine relationships of dietary patterns with body composition. The influence of a polymorphism in the peroxisome proliferator-activated receptor-γ (PPAR-γ) gene was considered. The Health, Aging and Body Composition (Health ABC) Study is a prospective cohort study of 3,075 older adults. Participants’ body composition and genetic variation were measured in detail. Food intake was assessed with a semi-quantitative food frequency questionnaire (Block Dietary Data Systems, Berkeley, CA), and dietary patterns of 1,809 participants with complete data were derived by cluster analysis. Six clusters were identified, including a ‘Healthy foods’ cluster characterized by higher intake of low-fat dairy products, fruit, whole grains, poultry, fish and vegetables. An interaction was found between dietary patterns and PPAR-γ Pro12Ala genotype in relation to body composition. While Pro/Pro homozygous men and women in the ‘Healthy foods’ cluster did not differ significantly in body composition from those in other clusters, men with the Ala allele in the ‘Healthy foods’ cluster had significantly lower levels of adiposity than those in other clusters. Women with the Ala allele in the ‘Healthy foods’ cluster differed only in right thigh intermuscular fat from those in other clusters. Relationships between diet and body composition in older adults may differ by gender and by genetic factors such as PPAR-γ Pro12Ala genotype