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Glaucoma is one of the most common optic neuropathies, featuring progressive retinal ganglion cell damage and visual field loss (Tham et al., 2014; Xu et al., 2020). Currently, the only effective treatment for this condition is the reduction of intraocular pressure (IOP) (Palmberg, 2001; Heijl et al., 2002). Canaloplasty is a proven bleb-independent surgery with good efficacy and safety profiles in primary open-angle glaucoma (POAG) (Golaszewska et al., 2021). However, early transient postoperative IOP elevation has been reported in up to 30% of cases (Riva et al., 2019), similar to that commonly observed in other internal drainage glaucoma surgeries such as implantation using iStent (0%-21.0%), CyPass (10.8%), and Hydrus (4.8%-6.5%) (Lavia et al., 2017). This complication may be a predictor of poor reserve in the outflow system and is potentially associated with surgical failure. Nonetheless, the exact pathophysiology of glaucoma remains unknown, and studies clarifying the risk factors for postoperative IOP elevation have been scarce.

The Patterns of Visual Field Defects in Primary Angle-Closure Glaucoma Compared to High-Tension Glaucoma and Normal-Tension Glaucoma.

INTRODUCTION: The aim of this study was to compare the patterns of visual field (VF) defects in primary angle-closure glaucoma (PACG) to control groups of eyes with high-tension glaucoma (HTG) and normal-tension glaucoma (NTG). METHODS: Forty-eight eyes with PACG were enrolled, and control eyes with HTG and NTG matched for age, sex, and mean deviation of VF defect were selected. VF tests were performed using the 24-2 program of the Humphrey field analyzer. VF defects were classified into six patterns with the Ocular Hypertension Treatment Study classification system and were categorized into three stages (early, moderate, and advanced). Each hemifield was divided into five regions according to the Glaucoma Hemifield Test (GHT). The mean total deviation (TD) of each GHT region was calculated. RESULTS: Compared with HTG and NTG groups, the partial arcuate VF defects were more common in the PACG group. In the PACG group, the nasal GHT region in the inferior hemifield had the worst mean TD (-8.48 ± 8.62 dB), followed by the arcuate 1 (-7.81 ± 7.91 dB), arcuate 2 (-7.46 ± 7.43 dB), paracentral (-7.19 ± 7.98 dB), and central (-5.14 ± 6.24 dB) regions; the mean TD of the central region was significantly better than those for all other regions (all p < 0.05). A similar trend was observed in the superior hemifield in the PACG group but not the VF hemifields of the HTG and NTG groups. CONCLUSION: Patterns of VF defect in PACG patients differ from those with HTG and NTG. This discrepancy might be due to the differences in the pathogenic mechanisms of glaucomatous optic neuropathy.

Surgical Outcomes of Penetrating Canaloplasty in Patients with Uncontrolled Posner-Schlossman Syndrome: A Prospective Study.

PURPOSE: To evaluate the surgical outcomes in patients with Posner-Schlossman syndrome (PSS) at one year. METHODS: A prospective interventional study in PSS patients with penetrating canaloplasty. Main outcome measure was success rate (6 mmHg≤ intraocular pressure (IOP) ≤ 21 mmHg) with or without medications. RESULTS: Thirteen eyes in 13 patients with PSS underwent complete catheterization. The mean IOP and medications (Meds) were reduced to 16.1 ± 4.8 mmHg on 0.5 ± 1.0 Meds at 12 months. Complete and qualified success rates were 61.5% and 84.6% at 12 months. The postoperative recurrent rate of PSS was 69.2%, the mean peak IOP during attacks and episodes were decreased to 26.7 ± 8.3 mmHg and 1.7 ± 2.0 times respectively. Transient IOP spike (61.5%) and hyphema (38.5%) were the two most common postoperative complications. CONCLUSION: Penetrating canaloplasty achieves a high success rate in PSS without serious complications.

Clinical outcomes of penetrating canaloplasty in patients with traumatic angle recession glaucoma: a prospective interventional case series.

BACKGROUND/AIM: To evaluate the clinical outcomes of penetrating canaloplasty in traumatic angle recession glaucoma at 1 year. METHODS: Patients with angle recession glaucoma underwent penetrating canaloplasty, a new Schlemm's canal-based internal drainage procedure, which creates a direct canal for flow of aqueous humour from the anterior chamber to the ostia of Schlemm's canal via a window created at the corneal scleral bed without use of antimetabolites. Postoperative intraocular pressure (IOP), number of glaucoma medications, and procedure-related complications were evaluated. Success was defined as an IOP ≤21 mm Hg without (complete) or with (qualified) use of glaucoma medication. RESULTS: Forty eyes in 40 patients with angle recession glaucoma underwent successful circumferential catheterisation. The mean patient age was 42±13 years. In patients with penetrating canaloplasty that was deemed to be completely successful, the mean IOP decreased from a preoperative value of 37.8±12.3 mm Hg on 3.3±1.2 anti-glaucoma medications to 18.5±6.4 mm Hg on 1.2±1.4 medications, 14.9±4.6 mm Hg on 0.1±0.5 medications, 15.7±5.4 mm Hg on 0.1±0.4 medications and 14.8±3.6 mm Hg on 0.1±0.5 medications at 1, 3, 6 and 12 months postoperatively (p<0.05). Complete success was achieved in 35/40 eyes (87.5%) at 6 months and in 34/38 (89.5%) at 12 months. Hyphema (18/40, 45.0%) and transient IOP elevation (≥30 mm Hg, 9/40, 22.5%) were the most common postoperative complications. CONCLUSION: Penetrating canaloplasty significantly reduces IOP and has a high success rate in angle recession glaucoma. TRIAL REGISTRATION NUMBER: ChiCTR1900020511.

Outcomes of Penetrating Canaloplasty in Childhood Glaucoma.

PRCIS: Penetrating canaloplasty was safe and effective for the management of refractory childhood glaucoma. PURPOSE: The purpose of this paper is to report the safety and efficacy of penetrating canaloplasty for the management of childhood glaucoma. MATERIALS AND METHODS: This single-center prospective interventional case series enrolled 50 eyes of 32 patients with childhood glaucoma between July 2017 and July 2020. Penetrating canaloplasty to create direct communication between the anterior chamber and tensioning suture-dilated the Schlemm canal was performed through a trabecular meshwork ostium in the affected eyes. Preoperative and postoperative intraocular pressure (IOP), number of glaucoma medications, and procedure-related complications were recorded. Qualified and complete success was defined as IOP≥5 and ≤21 mm Hg with or without glaucoma medications, respectively. RESULTS: A total of 43 eyes (86.0%) achieved 360 degrees catheterization of Schlemm canal and completed penetrating canaloplasty. The median age at surgery was 5 years (range, 10 d-17 y, mean 6.20±5.46 y). The mean IOP decreased from 33.11±10.89 mm Hg on 2 medications (median, range: 0-5) before surgery to 13.46±4.71 mm Hg on 0 medications (median, range:0-2) postoperatively at 12 months ( P <0.001). Complete and qualified success rates were 81.08% and 89.19%, respectively, at the 12-month follow-up. Hyphema (6/43, 13.95%) and a transient shallow anterior chamber with hypotony (5/43, 11.63%) were the most commonly observed early complications. CONCLUSION: Penetrating canaloplasty demonstrated good safety and efficacy in eyes with childhood glaucoma, and may be considered an option for managing refractory childhood glaucoma.

A new bleb-independent surgery namely penetrating canaloplasty for corticosteroid-induced glaucoma: a prospective case series.

AIM: To report the outcomes of penetrating canaloplasty for corticosteroid-induced glaucoma in a case series. METHODS: Penetrating canaloplasty is a bleb-independent filtering surgery unifying canaloplasty and trabeculectomy. In this study, the surgery was performed to restore the natural outflow through surgically expanded Schlemm's canal and generated trabeculum ostium. A total of 10 eyes of 8 patients were treated with penetrating canaloplasty for corticosteroid-induced glaucoma. Intraocular pressure (IOP) and the number of glaucoma medications at postoperative 3, 6, 12, 18, 24, 36, and 48mo were documented as primary endpoint. Complications after the surgery were recorded as secondary endpoint. RESULTS: Penetrating canaloplasty was accomplished successfully for all 10 eyes, with a mean follow-up of 20.4±13.0mo (range 6-48mo). The mean preoperative IOP and number of anti-glaucoma medications were 45.1±6.5 mm Hg and 3.3±0.5 respectively. The mean post-operative IOP at 3, 6, 12, 18, 24, 36, and 48mo were 15.8±6.0, 14.7±3.3, 15.3±2.0, 15.6±2.6, 17.5±1.8, 16.5±4.9, and 14.0 mm Hg. The number of anti-glaucoma medications at these time points were all 0. This surgery failed to control the IOP in 1 eye at 1mo after surgery. Hyphaema occurred in 3 eyes on the first day after surgery. Postoperative transient IOP increasing was encountered with in two eyes from 1wk to 1mo after surgery. Choroidal detachment developed in one eye but responded well to conservative treatment. CONCLUSION: Penetrating canaloplasty is effective for corticosteroid-induced glaucoma without serious complications, making it a viable or preferred alternative option.

Achieving Inner Aqueous Drain in Glaucoma Secondary to Iridocorneal Endothelial Syndrome: One Year Results of Penetrating Canaloplasty.

PURPOSES: To report the efficacy of a bleb-independent penetrating canaloplasty in the management of glaucoma secondary to iridocorneal endothelial syndrome (GS-ICE). DESIGN: Prospective, non-comparative clinical study. METHODS: Penetrating canaloplasty was performed on 35 eyes from 35 patients with GS-ICE and medically uncontrolled intraocular pressure (IOP) between January 2018 and April 2020. Patients were followed up at 1 week, months 1, 3, 6, 12 postoperatively, and semi-annually thereafter. The IOP, anti-glaucoma medication, and surgery-related complications were recorded. Surgical success was defined as IOP ≥ 5 mmHg and ≤ 21 mmHg without (complete success) or with/without (qualified success) IOP-lowering medication. RESULTS: A total of 29 eyes (82.9%) had 360° catheterization and successfully received penetrating canaloplasty. Of these eyes, 24 (82.8%) achieved qualified success and 22 (75.9%) achieved qualified success at 12 months after surgery. The mean IOP decreased from 39.5 ± 11.8 mmHg on 2.9 ± 1.0 medications before surgery to 16.6 ± 5.3 mmHg (P < .001) on 0.2 ± 0.6 medications (P < .001) at 12 months postoperatively, respectively. Hyphema (37.9%), transient hypotony (34.5%), and transient postoperative IOP elevation (≥ 30 mmHg, 17.9%) were the most commonly observed early complications at the 1 week and 1 month visits. From 1 month and beyond, all treated eyes showed no obvious bleb at the operation quadrant. CONCLUSIONS: Penetrating canaloplasty rescued the inner aqueous outflow in ICE eyes and demonstrated acceptable success in IOP control with few complications, providing a new option for the management of GS-ICE.

Evidence of nutrient translocation in response to smoke exposure by the East African ant acacia, Vachellia drepanolobium.

Fire is a major selective force on arid grassland communities, favoring traits such as the smoke-induced seed germination response seen in a wide variety of plant species. However, little is known about the relevance of smoke as a cue for plants beyond the seedling stage.We exposed a fire-adapted savanna tree, Vachellia (=Acacia) drepanolobium, to smoke and compared nutrient concentrations in leaf and root tissues to unexposed controls. Experiments were performed on three age cohorts: 2-year-old, 9-month-old, and 3-month-old plants.For the 2-year-old plants exposed to smoke, carbon and nitrogen concentrations were lower in the leaves and higher in the roots than controls. Less pronounced trends were found for boron and magnesium.In contrast, smoke-exposed 3-month-old plants had lower root nitrogen concentrations than controls. No significant differences were found in the 9-month-old plants, and no significant shifts in other nutrient concentrations were observed between plant tissues for any of the three age cohorts. Synthesis: Our findings are consistent with smoke-induced translocation of nutrients from leaves to roots in 2-year-old V. drepanolobium. This could represent a novel form of fire adaptation, with variation over the course of plant development. The translocation differences between age cohorts highlight the need to investigate smoke response in older plants of other species. Accounting for this adaptation could better inform our understanding of savanna community structure and nutrient flows under fire regimes altered by anthropogenic land use and climate change.

Phylogenomics and diversification drivers of the Eastern Asian - Eastern North American disjunct Podophylloideae.

Evolutionary and biogeographic processes determine species richness patterns of vascular plants between Eastern Asia (EA) and Eastern North America (ENA). However, the strikingly higher species richness of EA relative to ENA remains poorly understood from this perspective. Here, we studied the relative importance of biogeographical, evolutionary and ecological factors underlying differences in species richness between EA and ENA in Podophylloideae (Berberidaceae, Ranunculales; in total 10 spp. in EA vs. 2 spp. in ENA). Based on large-scale transcriptome data, our phylogenomic analyses strongly supported Podophylloideae and its two multi-species genera, i.e. Dysosma (EA) and Diphylleia (EA/ENA), as monophyletic groups. Sinopodophyllum hexandrum (EA) was identified as sister to the remainder of Podophylloideae. Dysosma (7 spp.) was recovered as sister to Podophyllum peltatum (ENA), forming an EA-ENA disjunct pair with a strong bias of species diversity in the EA counterpart. Our biogeographic analyses support the 'out-of-Tibet' hypothesis, suggesting that Podophylloideae started to diversify in the Himalaya-Hengduan Mountains (Mid-Miocene) and migrated eastward (since the Late Miocene) into Central-eastern China, Japan, and ENA (only P. peltatum and Diphylleia cymosa). Overall, we conclude that the striking species diversity anomaly between EA and ENA in Podophylloideae may be explained by a combination of (1) a longer period of time available to accumulate species in EA; and (2) a greater diversification rate in EA, which might have been promoted by greater physiographic and environmental heterogeneity in this region.

Intraocular asymmetry of visual field defects in primary angle-closure glaucoma, high-tension glaucoma, and normal-tension glaucoma in a Chinese population.

Direct comparison data on spatial patterns of visual field (VF) defects among primary angle-closure glaucoma (PACG), high-tension glaucoma (HTG) and normal-tension glaucoma (NTG) are not available. We aimed to compare the intraocular asymmetry of VF loss among patients with PACG, NTG and HTG across different severity levels. A total of 162 eyes of 114 patients with PACG, 111 eyes of 74 patients with HTG and 148 eyes of 102 patients with NTG were included. VF defects were categorized into 3 stages (early, moderate, and advanced), and each hemifield was divided into 5 regions according to the Glaucoma hemifield test (GHT). The mean total deviation (TD) of each GHT region was calculated. In the early stage, the paracentral, peripheral arcuate 1 and peripheral arcuate 2 regions in the superior hemifield in the NTG group had significantly worse mean TDs than their corresponding regions in the inferior hemifield. In the advanced stage, the central region in the superior hemifield in the PACG group had a significantly worse mean TD than that in the inferior hemifield. There was no significant difference in the mean TD for any of the five regions between hemifields across all severity levels in the in the HTG group. The superior hemifield was affected more severely than the inferior hemifield in all three subtypes of primary glaucoma. This asymmetric tendency was more pronounced in NTG than in PACG and HTG.

Genomic insights on the contribution of balancing selection and local adaptation to the long-term survival of a widespread living fossil tree, Cercidiphyllum japonicum.

'Living fossils' are testimonies of long-term sustained ecological success, but how demographic history and natural selection contributed to their survival, resilience, and persistence in the face of Quaternary climate fluctuations remains unclear. To better understand the interplay between demographic history and selection in shaping genomic diversity and evolution of such organisms, we assembled the whole genome of Cercidiphyllum japonicum, a widespread East Asian Tertiary relict tree, and resequenced 99 individuals of C. japonicum and its sister species, Cercidiphyllum magnificum (Central Japan). We dated this speciation event to the mid-Miocene, and the intraspecific lineage divergence of C. japonicum (China vs Japan) to the Early Pliocene. Throughout climatic upheavals of the late Tertiary/Quaternary, population bottlenecks greatly reduced the genetic diversity of C. japonicum. However, this polymorphism loss was likely counteracted by, first, long-term balancing selection at multiple chromosomal and heterozygous gene regions, potentially reflecting overdominance, and, second, selective sweeps at stress response and growth-related genes likely involved in local adaptation. Our findings contribute to a better understanding of how living fossils have survived climatic upheaval and maintained an extensive geographic range; that is, both types of selection could be major factors contributing to the species' survival, resilience, and persistence.

Evolutionary insights from comparative transcriptome and transcriptome-wide coalescence analyses in Tetrastigma hemsleyanum.

BACKGROUND: Tetrastigma hemsleyanum is of great medicinal importance and used as a model system to address the evolutionary history of warm-temperate evergreen (WTE) forest biomes in East Asia over Neogene time scales. However, further studies on the neutral and adaptive divergence processes of T. hemsleyanum are currently impeded by a lack of genomic resources. In this study, we de novo assembled and annotated a reference transcriptome for two cpDNA lineages (Central-South-East vs. Southwest) of T. hemsleyanum. We further used comparative genomic and multilocus coalescent approaches to investigate the tempo and mode of lineage diversification in T. hemsleyanum. RESULTS: A total of 52,838 and 65,197 unigenes with an N50 of 1,667 and 1,841 bp for Central-South-East (CSE) and Southwest (SW) lineages, respectively, were recovered, and 6,692 putative orthologs were identified between the two lineages. Estimation of Ka/Ks ratios for these orthologs revealed that ten genes had Ka/Ks values significantly greater than 0.5 (P < 0.05), whereas 2,099 (Ka/Ks < 0.5, P < 0.05) were inferred to be under purifying selection. Based on three bioinformatic strategies, we identified a total of 1,018 single-copy nuclear genes (SCNGs) from the orthologs. We successfully designed eight nuclear gene primer pairs with high intraspecific variation (e.g. hT = 0.923, πT = 1.68×10-3), when surveyed across a subset of T. hemsleyanum individuals. Concordant with the previous cpDNA data, the haplotype networks constructed for most nuclear gene loci clearly identified the two lineages. A multilocus coalescence analysis suggested that the separation between the two lineages appears to have occurred during the mid-Pliocene. Despite their ancient divergence, both lineages experienced expansion at rather localized scales and have continued to exchange genes at a low rate. CONCLUSIONS: This study demonstrated the utility of transcriptome sequencing as a basis for SCNG development in non-model species and the advantages of integrating multiple nuclear loci for phylogeographic and phylogenetic studies.

Phylogenomic and Comparative Analyses of Complete Plastomes of Croomia and Stemona (Stemonaceae).

The monocot genus Croomia (Stemonaceae) comprises three herbaceous perennial species that exhibit EA (Eastern Asian)⁻ENA (Eastern North American) disjunct distribution. However, due to the lack of effective genomic resources, its evolutionary history is still weakly resolved. In the present study, we conducted comparative analysis of the complete chloroplast (cp) genomes of three Croomia species and two Stemona species. These five cp genomes proved highly similar in overall size (154,407⁻155,261 bp), structure, gene order and content. All five cp genomes contained the same 114 unique genes consisting of 80 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Gene content, gene order, AT content and IR/SC boundary structures were almost the same among the five Stemonaceae cp genomes, except that the Stemona cp genome was found to contain an inversion in cemA and petA. The lengths of five genomes varied due to contraction/expansion of the IR/SC borders. A/T mononucleotides were the richest Simple Sequence Repeats (SSRs). A total of 46, 48, 47, 61 and 60 repeats were identified in C. japonica, C. heterosepala, C. pauciflora, S. japonica and S. mairei, respectively. A comparison of pairwise sequence divergence values across all introns and intergenic spacers revealed that the ndhF⁻rpl32, psbM⁻trnD and trnS⁻trnG regions are the fastest-evolving regions. These regions are therefore likely to be the best choices for molecular evolutionary and systematic studies at low taxonomic levels in Stemonaceae. Phylogenetic analyses of the complete cp genomes and 78 protein-coding genes strongly supported the monophyly of Croomia. Two Asian species were identified as sisters that likely diverged in the Early Pleistocene (1.62 Mya, 95% HPD: 1.125⁻2.251 Mya), whereas the divergence of C. pauciflora dated back to the Late Miocene (4.77 Mya, 95% HPD: 3.626⁻6.162 Mya). The availability of these cp genomes will provide valuable genetic resources for further population genetics and phylogeographic studies on Croomia.

Plastome organization, genome-based phylogeny and evolution of plastid genes in Podophylloideae (Berberidaceae).

Species of Podophylloideae (Berberidaceae, Ranunculales) are of great pharmacogenetic importance and represent the classic biogeographic disjunction between eastern Asia (EA; 10 ssp.) and eastern North America (ENA; 2 ssp.). However, previous molecular studies of this group suffered from low phylogenetic resolution and/or insufficient marker variability. This study is the first to report whole-plastome sequence data for all 12 species of Podophylloideae (14 individuals) and a close relative, Achlys triphylla. These 15 plastomes proved highly similar in overall size (156,240-157,370 bp), structure, gene order and content, also when compared to other Ranunculales, but also revealed some structural variations caused by the expansion or contraction of the inverted repeats (IRs) into or out of adjacent single-copy regions. Our phylogenomic analysis, based on 63 plastome-derived protein-coding genes (CDS), supported the monophyly of Podophylloideae and its two major genera (EA: Dysosma, EA/ENA: Diphylleia), with Podophyllum peltatum L. (ENA) being more closely related to Diphylleia than to the group's earliest diverging species, Sinopodophyllum hexandrum (EA). Furthermore, within this subfamily/dataset, matK was identified as the fastest evolving gene, which proved to be under positive selection especially in more recently derived, lower-elevation lineages of Dysosma, possibly reflecting an adaptive response to novel environmental (i.e. subtropical compared to higher-elevation/alpine) conditions. Finally, several highly variable noncoding regions were identified in the plastomes of Podophylloideae and Ranunculales. These highly variable loci should be the best choices for future phylogenetic, phylogeographic, and population-level genetic studies. Overall, our results demonstrate the power of plastid phylogenomics to improve phylogenetic resolution, and contribute to a better understanding of plastid gene evolution in Podophylloideae.

Volume Reduction in Enlarged Kidneys in Autosomal Dominant Polycystic Kidney Disease (ADPKD) Prior to Renal Transplant with Transcatheter Arterial Embolization (TAE): A Systematic Review and Meta-Analysis.

PURPOSE: Symptomatically enlarged kidneys observed in autosomal dominant polycystic kidney disease (ADPKD) patients can lead to compression symptoms and contraindications to renal transplantation. Surgical nephrectomy can be utilized to increase space in the abdomen prior to renal transplantation; however, not all individuals are appropriate candidates for this procedure. Transcatheter arterial embolization (TAE) of the renal arteries can provide a noninvasive way to reduce renal volume in ADPKD. MATERIALS AND METHODS: We performed a systematic literature review on the usage of TAE to reduce renal volume prior to kidney transplantation and to relief compression symptoms in ADPKD. PubMed, Web of Science, and Cochrane Library were searched for articles focused on the usage of TAE to reduce renal volume in symptomatic enlarged kidneys. Renal volume data were compiled, and meta-analysis was performed with three or more studies. RESULTS: Six papers satisfied the inclusion and exclusion criteria. Significant renal volume reduction was observed by 12 months in all studies. Success of TAE was measured with three clinical outcomes: removal of contraindication for renal transplant, relief of compression symptoms, and pulmonary function test. Proportionality meta-analysis of three studies measuring relief of compression symptoms showed no significant differences in heterogeneity (p = 0.4543). CONCLUSION: Current studies conclude that TAE is an effective and minimally invasive option for reduction in renal volume in order to optimize patient outcome for renal transplantation and for relief of compression symptoms. Further prospective studies involving increased sample size and multiple centers should be pursued to establish evidence-based guidelines.

The complete chloroplast genome of Vancouveria planipetala, with implication for the phylogeny of Ranunculales.

Vancouveria planipetala (Berberidaceae) is a perennial herb which has high ornamental and ecological values. In this study, we assembled the complete chloroplast (cp) genome of V. planipetala. The whole cp genome of V. planipetala is 156,871 bp in length, comprising a pair of inverted repeat (IR) regions (25,888 bp) separated by a large single copy (LSC) region (88,321 bp) and a small single copy (SSC) region (16,772 bp). The cp genome contains 114 unique genes, including 80 protein-coding genes, 30 tRNA, and four rRNA genes, with 17 genes duplicated in IRs. Phylogenetic analyses showed that Papaveraceae is the basal group of Ranunculales.

A kainate receptor subunit promotes the recycling of the neuron-specific K+-Cl- co-transporter KCC2 in hippocampal neurons.

Synaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K+-Cl- co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an ∼40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (EGABA). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.

Beneficial Effects of Metformin and/or Salicylate on Palmitate- or TNFα-Induced Neuroinflammatory Marker and Neuropeptide Gene Regulation in Immortalized NPY/AgRP Neurons.

Neuropeptide Y (NPY)/Agouti-related peptide (AgRP)-expressing neurons in the hypothalamus induce feeding and decrease energy expenditure. With consumption of a diet high in fat, there is an increase in circulating saturated free fatty acids, including palmitate, leading to the development of neuroinflammation and secretion of cytokines, such as TNFα, and in turn activation of the canonical IKKß/NFκB cascade. We describe a model of palmitate- and TNFα-induced neuroinflammation in a functionally characterized, immortalized NPY/AgRP-expressing cell model, mHypoE-46, to study whether the anti-diabetic metformin alone or in combination with the anti-inflammatory agent salicylate can ameliorate these detrimental effects. Treatment with palmitate increased mRNA expression of feeding peptides Npy and Agrp, and inflammatory cytokines Tnfa and Il-6, whereas treatment with TNFα increased mRNA expression of Npy, Nfkb, Ikba, Tnfa, and Il-6. The effects of metformin and/or sodium salicylate on these genes were assessed. Metformin increased phosphorylation of AMPK and S6K, while sodium salicylate increased phospho-AMPK and decreased phospho-S6K, but neither had any effect on phospho-ERK, -JNK or -p38 in the mHypoE-46 NPY/AgRP neurons. Furthermore, we utilized a pre-treatment and/or co-treatment paradigm to model potential clinical regimens. We determined co-treatment with metformin or sodium salicylate alone was successful in alleviating changes observed in feeding peptide mRNA regulation, whereas a preventative pre-treatment with metformin and sodium salicylate together was able to alleviate palmitate- and TNFα-induced induction of NPY and/or AgRP mRNA levels. These results highlight important differences in reactive versus preventative treatments on palmitate- and TNFα-induced neuroinflammation in NPY/AgRP neurons.

Divergent Regulation of ER and Kiss Genes by 17ß-Estradiol in Hypothalamic ARC Versus AVPV Models.

Kisspeptin (Kiss) and G-protein-coupled receptor (Gpr)54 have emerged as key regulators of reproduction. 17ß-estradiol (E2)-mediated regulation of these neurons is nuclei specific, where anteroventral periventricular (AVPV) Kiss neurons are positively regulated by E2, whereas arcuate nucleus (ARC) neurons are inhibited. We have generated immortalized Kiss cell lines from male and female adult-derived murine hypothalamic primary culture, as well as cell lines from microdissected AVPV and ARC from female Kiss-green fluorescent protein (GFP) mice. All exhibit endogenous Kiss-1 expression, estrogen receptors (ER)s (ERα, ERß, and Gpr30), as well as known markers of AVPV Kiss neurons in the mHypoA-50 and mHypoA-Kiss/GFP-4, vs markers of ARC Kiss neurons in the mHypoA-55 and the mHypoA-Kiss/GFP-3 lines. There was an increase in Kiss-1 mRNA expression at 24 hours in the AVPV lines and a repression of Kiss-1 mRNA at 4 hours in the ARC lines. An E2-mediated decrease in ERα mRNA expression at 24 hours in the AVPV cell lines was detected, and a significant decrease in Gpr30, ERα, and ERß mRNA levels at 4 hours in the ARC cell lines was evident. ER agonists and antagonists determined the specific ERs responsible for mediating changes in gene expression. In the AVPV, ERα is required but not ERß or GPR30, vs the ARC Kiss-expressing cell lines that require GPR30, and either ERα and/or ERß. We determined cAMP response element-binding protein 1 was necessary for the down-regulation of Kiss-1 mRNA expression using small interfering RNA knockdown in the ARC cell model. These studies elucidate some of the molecular events involved in the differential E2-mediated regulation of unique and specific Kiss neuronal models.

Cellular insulin resistance disrupts hypothalamic mHypoA-POMC/GFP neuronal signaling pathways.

POMC neurons play a central role in the maintenance of whole-body energy homeostasis. This balance requires proper regulation of POMC neurons by metabolic hormones, such as insulin. However, the heterogeneous cellular population of the intact hypothalamus presents challenges for examining the molecular mechanisms underlying the potent anorexigenic effects of POMC neurons, and there is currently a complete lack of mature POMC neuronal cell models for study. To this end, we have generated novel, immortalized, adult-derived POMC-expressing/α-MSH-secreting cell models, mHypoA-POMC/GFP lines 1-4, representing the fluorescence-activated cell-sorted POMC population from primary POMC-eGFP mouse hypothalamus. The presence of Pomc mRNA in these cell lines was confirmed, and α-MSH was detected via immunofluorescence. α-MSH secretion in the mHypoA-POMC/GFP-1 was found to increase in response to 10  ng/ml ciliary neurotrophic factor (CNTF) or 10  nM insulin as determined by enzyme immunoassay. Further experiments using the mHypoA-POMC/GFP-1 cell line revealed that 10  ng/ml CNTF increases Pomc mRNA at 1 and 2  h after treatment, whereas insulin elicited an increase in Pomc mRNA level and decreases in insulin receptor (Insr (Ir)) mRNA level at 4  h. Furthermore, the activation of IR-mediated downstream second messengers was examined by western blot analysis, following the induction of cellular insulin resistance, which resulted in a loss of insulin-mediated regulation of Pomc and Ir mRNAs. The development of these immortalized neurons will be invaluable for the elucidation of the cellular and molecular mechanisms that underlie POMC neuronal function under normal and perturbed physiological conditions.