Long-Term Continuous Corticosterone Treatment Decreases VEGF Receptor-2 Expression in Frontal Cortex

Objective: Stress and increased glucocorticoid levels are associated with many neuropsychiatric disorders including schizophrenia and depression. Recently, the role of vascular endothelial factor receptor-2 (VEGFR2/Flk1) signaling has been implicated in stress-mediated neuroplasticity. However, the mechanism of regulation of VEGF/Flk1 signaling under longterm continuous glucocorticoid exposure has not been elucidated. Material and Methods: We examined the possible effects of long-term continuous glucocorticoid exposure on VEGF/Flk1 signaling in cultured cortical neurons in vitro, mouse frontal cortex in vivo, and in post mortem human prefrontal cortex of both control and schizophrenia subjects. Results: We found that long-term continuous exposure to corticosterone (CORT, a natural glucocorticoid) reduced Flk1 protein levels both in vitro and in vivo. CORT treatment resulted in alterations in signaling molecules downstream to Flk1 such as PTEN, Akt and mTOR. We demonstrated that CORT-induced changes in Flk1 levels are mediated through glucocorticoid receptor (GR) and calcium. A significant reduction in Flk1-GR interaction was observed following CORT exposure. Interestingly, VEGF levels were increased in cortex, but decreased in serum following CORT treatment. Moreover, significant reductions in Flk1 and GR protein levels were found in postmortem prefrontal cortex samples from schizophrenia subjects. Conclusions: The alterations in VEGF/Flk1 signaling following long-term continuous CORT exposure represents a molecula

Common Variants at 9p21 and 8q22 Are Associated with Increased Susceptibility to Optic Nerve Degeneration in Glaucoma

Optic nerve degeneration caused by glaucoma is a leading cause of blindness worldwide. Patients affected by the normal-pressure form of glaucoma are more likely to harbor risk alleles for glaucoma-related optic nerve disease. We have performed a meta-analysis of two independent genome-wide association studies for primary open angle glaucoma (POAG) followed by a normal-pressure glaucoma (NPG, defined by intraocular pressure (IOP) less than 22 mmHg) subgroup analysis. The single-nucleotide polymorphisms that showed the most significant associations were tested for association with a second form of glaucoma, exfoliation-syndrome glaucoma. The overall meta-analysis of the GLAUGEN and NEIGHBOR dataset results (3,146 cases and 3,487 controls) identified significant associations between two loci and POAG: the CDKN2BAS region on 9p21 (rs2157719 [G], OR = 0.69 [95%CI 0.63–0.75], p = 1.86×10−18), and the SIX1/SIX6 region on chromosome 14q23 (rs10483727 [A], OR = 1.32 [95%CI 1.21–1.43], p = 3.87×10−11). In sub-group analysis two loci were significantly associated with NPG: 9p21 containing the CDKN2BAS gene (rs2157719 [G], OR = 0.58 [95% CI 0.50–0.67], p = 1.17×10−12) and a probable regulatory region on 8q22 (rs284489 [G], OR = 0.62 [95% CI 0.53–0.72], p = 8.88×10−10). Both NPG loci were also nominally associated with a second type of glaucoma, exfoliation syndrome glaucoma (rs2157719 [G], OR = 0.59 [95% CI 0.41–0.87], p = 0.004 and rs284489 [G], OR = 0.76 [95% CI 0.54–1.06], p = 0.021), suggesting that these loci might contribute more generally to optic nerve degeneration in glaucoma. Because both loci influence transforming growth factor beta (TGF-beta) signaling, we performed a genomic pathway analysis that showed an association between the TGF-beta pathway and NPG (permuted p = 0.009). These results suggest that neuro-protective therapies targeting TGF-beta signaling could be effective for multiple forms of glaucoma

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

PKBγ/AKT3 loss-of-function causes learning and memory deficits and deregulation of AKT/mTORC2 signaling: Relevance for schizophrenia

<div><p>Psychiatric genetic studies have identified genome-wide significant loci for schizophrenia. The AKT3/1q44 locus is a principal risk region and gene-network analyses identify AKT3 polymorphisms as a constituent of several neurobiological pathways relevant to psychiatric risk; the neurobiological mechanisms remain unknown. AKT3 shows prenatal enrichment during human neocortical development and recurrent copy number variations involving the 1q43-44 locus are associated with cortical malformations and intellectual disability, implicating an essential role in early brain development. Here, we investigated the role of AKT3 as it relates to aspects of learning and memory and behavioral function, relevant to schizophrenia and cognitive disability, utilizing a novel murine model of Akt3 genetic deficiency. Akt3 heterozygous (Akt3^-/+) or null mice (Akt3^-/-) were assessed in a comprehensive test battery. Brain biochemical studies were conducted to assess the impact of Akt3 deficiency on cortical Akt/mTOR signaling. Akt3^-/+ and Akt3^-/- mice exhibited selective deficits of temporal order discrimination and spatial memory, tasks critically dependent on intact prefrontal-hippocampal circuitry, but showed normal prepulse inhibition, fear conditioned learning, memory for novel objects and social function. Akt3 loss-of-function, reduced brain size and dramatically impaired cortical Akt Ser⁴⁷³ activation in an allele-dose dependent manner. Such changes were observed in the absence of altered Akt1 or Akt2 protein expression. Concomitant reduction of the mTORC2 complex proteins, Rictor and Sin1 identifies a potential mechanism. Our findings provide novel insight into the neurodevelopmental role of Akt3, identify a non-redundant role for Akt3 in the development of prefrontal cortical-mediated cognitive function and show that Akt3 is potentially the dominant regulator of AKT/mTOR signaling in brain.</p></div

Demographic data for postmortem samples.

<p>Demographic data for postmortem samples.</p

Chronic CORT-induced Flk1 regulation is mediated through calcium.

<p>(<i>A</i>) Calcium chelator BAPTA-AM blocked CORT (CORT)-induced reduction in Flk1 protein levels. BAPTA-AM (50 µM) was applied 30 min before CORT (1 µM) treatment to cultured neurons at DIV 5. Cell lysates were collected at 48 h after CORT treatment and Flk1 protein levels were determined by western blot analysis. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 5) expressed as fold change in Flk1 protein levels as compared to CON. *<i>P</i><0.01 versus CON; #<i>P</i><0.01 versus CORT (Bonferroni's test). (<i>B</i>) Chronic CORT treatment increases NCS-1 protein levels in neurons. CORT (CORT; 1 µM) was applied to mouse primary cortical neurons at DIV 5. NCS-1 protein levels were determined by western blotting analysis at 48 h following CORT treatment. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 5) expressed as fold change in NCS-1 protein levels as compared to CON. *<i>P</i><0.01 (Bonferroni's test). (C) Chronic CORT treatment increases NCS-1 protein levels in mouse frontal cortex. NCS-1 protein levels in frontal cortex of mice treated with CORT (5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were determined by western blot analysis. Data represent mean±SE (<i>n</i> = 6) expressed as fold change in NCS-1 protein levels as compared to CON. β-actin is the loading control.*<i>P</i><0.05 (Bonferroni's test).</p

Long-term Continuous CORT treatment increases VEGF protein levels <i>in vitro</i> and <i>in vivo</i>.

<p>(<i>A</i>) CORT (CORT; 1 µM) was applied to mouse primary cortical neurons at DIV 5. VEGF protein levels were determined by western blotting analysis at 48 hand 72 h following CORT treatment. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 6) expressed as fold change in VEGF protein levels as compared to CON. *<i>P</i><0.05 (Bonferroni's test). (<i>B</i>) VEGF protein levels in frontal cortex of mice treated with CORT (5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were determined by western blot analysis. Data represent mean±SE (<i>n</i> = 5) expressed as fold change in VEGF protein levels as compared to CON. *<i>P</i><0.01 (t test). <i>(C)</i> VEGF protein levels in serum samples collected from mice treated with CORT (CORT; 5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were analysed by ELISA. Data represent mean±SE (<i>n</i> = 5–6) expressed as fold change in VEGF protein levels as compared to CON. *<i>P</i><0.01 (t test).</p

Proposed model showing the effects of corticosterone on VEGF/Flk1 signaling pathway in mouse frontal cortex.

<p>The signaling events induced by corticosterone (CORT) are mediated through the Glucocorticoid receptor (GR). The reduction in Flk1 levels following long-term continuous CORT exposure results in the activation of PTEN, but inhibition of Akt and mTOR phosphorylation. The effects of CORT on Flk1 are mediated through calcium (Ca²⁺). CORT exposure results in increased levels of VEGF in cortex. The role of VEGF in Flk1 regulation (or vice versa) under CORT exposure remains unknown. Solid arrows represent activation, whereas dashed arrows represent inhibition of the pathways.</p

PI3K signaling is not involved in long-term continuous CORT-induced increases in VEGF protein levels.

<p>(<i>A</i>) VEGF protein levels were determined by western blotting analysis in neuronal cell lysates treated with LY294002 (LY; 20 µM) for 48 h. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 6) expressed as fold change in VEGF protein levels as compared to CON. *<i>P</i><0.05 (Bonferroni's test). (<i>B</i>) Pretreatment with LY did not prevent CORT-induced induction in VEGF protein levels in neurons. Cortical neurons at DIV 5 were treated with LY (20 µM) for 30 min followed by CORT (1 µM) exposure for 48 h. VEGF protein levels were determined in cell lysates by western blot analysis. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 6) expressed as fold change in VEGF protein levels as compared to CON. *<i>P</i><0.05 (Bonferroni's test).</p