Interplay between Thyroid Hormones and Stearoyl-CoA Desaturase 1 in the Regulation of Lipid Metabolism in the Heart

Stearoyl-CoA desaturase 1 (SCD1), an enzyme that is involved in the biosynthesis of monounsaturated fatty acids, induces the reprogramming of cardiomyocyte metabolism. Thyroid hormones (THs) activate both lipolysis and lipogenesis. Many genes that are involved in lipid metabolism, including Scd1, are regulated by THs. The present study used SCD1 knockout (SCD1^−/−) mice to test the hypothesis that THs are important factors that mediate the anti-steatotic effect of SCD1 downregulation in the heart. SCD1 deficiency decreased plasma levels of thyroid-stimulating hormone and thyroxine and the expression of genes that regulate intracellular TH levels (i.e., Slc16a2 and Dio1-3) in cardiomyocytes. Both hypothyroidism and SCD1 deficiency affected genomic and non-genomic TH pathways in the heart. SCD1 deficiency is known to protect mice from genetic- or diet-induced obesity and decrease lipid content in the heart. Interestingly, hypothyroidism increased body adiposity and triglyceride and diacylglycerol levels in the heart in SCD1^−/− mice. The accumulation of triglycerides in cardiomyocytes in SCD1^−/− hypothyroid mice was caused by the activation of lipogenesis, which likely exceeded the upregulation of lipolysis and fatty acid oxidation. Lipid accumulation was also observed in the heart in wildtype hypothyroid mice compared with wildtype control mice, but this process was related to a reduction of triglyceride lipolysis and fatty acid oxidation. We also found that simultaneous SCD1 and deiodinase inhibition increased triglyceride content in HL-1 cardiomyocytes, and this process was related to the downregulation of lipolysis. Altogether, the present results suggest that THs are an important part of the mechanism of SCD1 in cardiac lipid utilization and may be involved in the upregulation of energetic metabolism that is associated with SCD1 deficiency.     

Altered mRNA and Protein Expression of Monocarboxylate Transporter MCT1 in the Cerebral Cortex and Cerebellum of Prion Protein Knockout Mice

The effect of a cellular prion protein (PrP^c) deficiency on neuroenergetics was primarily analyzed via surveying the expression of genes specifically involved in lactate/pyruvate metabolism, such as monocarboxylate transporters (MCT1, MCT2, MCT4). The aim of the present study was to elucidate a potential involvement of PrP^c in the regulation of energy metabolism in different brain regions. By using quantitative real-time polymerase chain reaction (qRT-PCR), we observed a marked reduction in MCT1 mRNA expression in the cortex of symptomatic Zürich I Prnp^−/− mice, as compared to their wild-type (WT) counterparts. MCT1 downregulation in the cortex was accompanied with significantly decreased expression of the MCT1 functional interplayer, the Na⁺/K⁺ ATPase α2 subunit. Conversely, the MCT1 mRNA level was significantly raised in the cerebellum of Prnp^−/− vs. WT control group, without a substantial change in the Na⁺/K⁺ ATPase α2 subunit expression. To validate the observed mRNA findings, we confirmed the observed change in MCT1 mRNA expression level in the cortex at the protein level. MCT4, highly expressed in tissues that rely on glycolysis as an energy source, exhibited a significant reduction in the hippocampus of Prnp^−/− vs. WT mice. The present study demonstrates that a lack of PrP^c leads to altered MCT1 and MCT4 mRNA/protein expression in different brain regions of Prnp^−/− vs. WT mice. Our findings provide evidence that PrP^c might affect the monocarboxylate intercellular transport, which needs to be confirmed in further studies.     

The Amino Acid Transporter Mct10/Tat1 Is Important to Maintain the TSH Receptor at Its Canonical Basolateral Localization and Assures Regular Turnover of Thyroid Follicle Cells in Male Mice

Cathepsin K-mediated thyroglobulin proteolysis contributes to thyroid hormone (TH) liberation, while TH transporters like Mct8 and Mct10 ensure TH release from thyroid follicles into the blood circulation. Thus, thyroid stimulating hormone (TSH) released upon TH demand binds to TSH receptors of thyrocytes, where it triggers Gα_q-mediated short-term effects like cathepsin-mediated thyroglobulin utilization, and Gα_s-mediated long-term signaling responses like thyroglobulin biosynthesis and thyrocyte proliferation. As reported recently, mice lacking Mct8 and Mct10 on a cathepsin K-deficient background exhibit excessive thyroglobulin proteolysis hinting towards altered TSH receptor signaling. Indeed, a combination of canonical basolateral and non-canonical vesicular TSH receptor localization was observed in Ctsk^−/−/Mct8^−/y/Mct10^−/− mice, which implies prolonged Gα_s-mediated signaling since endo-lysosomal down-regulation of the TSH receptor was not detected. Inspection of single knockout genotypes revealed that the TSH receptor localizes basolaterally in Ctsk^−/− and Mct8^−/y mice, whereas its localization is restricted to vesicles in Mct10^−/− thyrocytes. The additional lack of cathepsin K reverses this effect, because Ctsk^−/−/Mct10^−/− mice display TSH receptors basolaterally, thereby indicating that cathepsin K and Mct10 contribute to TSH receptor homeostasis by maintaining its canonical localization in thyrocytes. Moreover, Mct10^−/− mice displayed reduced numbers of dead thyrocytes, while their thyroid gland morphology was comparable to wild-type controls. In contrast, Mct8^−/y, Mct8^−/y/Mct10^−/−, and Ctsk^−/−/Mct8^−/y/Mct10^−/− mice showed enlarged thyroid follicles and increased cell death, indicating that Mct8 deficiency results in altered thyroid morphology. We conclude that vesicular TSH receptor localization does not result in different thyroid tissue architecture; however, Mct10 deficiency possibly modulates TSH receptor signaling for regulating thyrocyte survival.     

Comparative Proteome Research in a Zebrafish Model for Vanishing White Matter Disease

Vanishing white matter (VWM) disease is a genetic leukodystrophy leading to severe neurological disease and early death. VWM is caused by bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (EIF2B). Previous studies have attempted to investigate the molecular mechanism of VWN by constructing models for each subunit of EIF2B that causes VWM disease. The underlying molecular mechanisms of the way in which mutations in EIF2B3 result in VWM are largely unknown. Based on our recent results, we generated an eif2b3 knockout (eif2b3^−/−) zebrafish model and performed quantitative proteomic analysis between the wild-type (WT) and eif2b3^−/− zebrafish, and identified 25 differentially expressed proteins. Four proteins were significantly upregulated, and 21 proteins were significantly downregulated in eif2b3^−/− zebrafish compared to WT. Lon protease and the neutral amino acid transporter SLC1A4 were significantly increased in eif2b3^−/− zebrafish, and crystallin proteins were significantly decreased. The differential expression of proteins was confirmed by the evaluation of mRNA levels in eif2b3^−/− zebrafish, using whole-mount in situ hybridization analysis. This study identified proteins which candidates as key regulators of the progression of VWN disease, using quantitative proteomic analysis in the first EIF2B3 animal model of VWN disease.     

GCase and LIMP2 Abnormalities in the Liver of Niemann Pick Type C Mice

The lysosomal storage disease Niemann–Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1^−/− mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1^−/− liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1^−/− liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1^−/− liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1^−/− hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.     

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh^Δex5−6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh^Δex5−6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh^Δex5−6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh^Δex5−6 mutant larvae in a context-dependent manner. We expect the sgsh^Δex5−6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.     

Transport Turnover Rates for Human OCT2 and MATE1 Expressed in Chinese Hamster Ovary Cells

MATE1 (multidrug and toxin extruder 1) and OCT2 (organic cation transporter 2) play critical roles in organic cation excretion by the human kidney. The transporter turnover rate (TOR) is relevant to understanding both their transport mechanisms and interpreting the in vitro–in vivo extrapolation (IVIVE) required for physiologically-based pharmacokinetic (PBPK) modeling. Here, we use a quantitative western blot method to determine TORs for MATE1 and OCT2 proteins expressed in CHO cells. MATE1 and OCT2, each with a C-terminal V-5 epitope tag, were cell surface biotinylated and the amount of cell surface MATE1 and OCT2 protein was quantified by western analysis, using standard curves for the V5 epitope. Cell surface MATE1 and OCT2 protein represented 25% and 24%, respectively, of the total expression of these proteins in CHO cells. The number of cell surface transporters was ~55 fmol cm⁻² for MATE1 and ~510 fmol cm⁻² for OCT2. Dividing these values into the different J_max values for transport of MPP, metformin, and atenolol mediated by MATE1 and OCT2 resulted in calculated TOR values (±SE, n = 4) of 84.0 ± 22.0 s⁻¹ and 2.9 ± 0.6 s⁻¹; metformin, 461.0 ± 121.0 s⁻¹ and 12.6 ± 2.4 s⁻¹; atenolol, 118.0 ± 31.0 s⁻¹, respectively. These values are consistent with the TOR values determined for a variety of exchangers (NHEs), cotransporters (SGLTs, Lac permease), and uniporters (GLUTs, ENTs).     

Deficiency of Thyroid Hormone Reduces Voltage-Gated Na+ Currents as Well as Expression of Na+/K+-ATPase in the Mouse Hippocampus

Mice lacking functional thyroid follicular cells, Pax8^−/− mice, die early postnatally, making them suitable models for extreme hypothyroidism. We have previously obtained evidence in postnatal rat neurons, that a down-regulation of Na⁺-current density could explain the reduced excitability of the nervous system in hypothyroidism. If such a mechanism underlies the development of coma and death in severe hypothyroidism, Pax8^−/− mice should show deficits in the expression of Na⁺ currents and potentially also in the expression of Na⁺/K⁺-ATPases, which are necessary to maintain low intracellular Na⁺ levels. We thus compared Na⁺ current densities in postnatal mice using the patch-clamp technique in the whole-cell configuration as well as the expression of three alpha and two beta-subunits of the Na⁺/K⁺-ATPase in wild type versus Pax8^−/− mice. Whereas the Na⁺ current density in hippocampal neurons from wild type mice was upregulated within the first postnatal week, the Na⁺ current density remained at a very low level in hippocampal neurons from Pax8^−/− mice. Pax8^−/− mice also showed significantly decreased protein expression levels of the catalytic α1 and α3 subunits of the Na⁺/K⁺-ATPase as well as decreased levels of the β2 isoform, with no changes in the α2 and β1 subunits.     

Microcirculatory Function during Endotoxemia—A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 μg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3^−/− compared to Nos2^−/− mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2^−/− mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3^−/− or Nos2^−/−/Nos3^−/− mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2^−/− mice), as this beneficial effect was absent in Nos3^−/− or Nos2^−/−/Nos3^−/− mice.     

Aryl Hydrocarbon Receptor Repressor and TiPARP (ARTD14) Use Similar,but also Distinct Mechanisms to Repress Aryl Hydrocarbon Receptor Signaling

The aryl hydrocarbon receptor (AHR) regulates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The AHR repressor (AHRR) is an AHR target gene and functions as a ligand-induced repressor of AHR; however, its mechanism of inhibition is controversial. Recently, we reported that TCDD-inducible poly (ADP-ribose) polymerase (TiPARP; ARTD14) also acts as a repressor of AHR, representing a new player in the mechanism of AHR action. Here we compared the ability of AHRR- and TiPARP-mediated inhibition of AHR activity. TCDD increased AHRR mRNA levels and recruitment of AHRR to cytochrome P450 1A1 (CYP1A1) in MCF7 cells. Knockdown of TiPARP, but not AHRR, increased TCDD-induced CYP1A1 mRNA and AHR protein levels. Similarly, immortalized TiPARP^−/− mouse embryonic fibroblasts (MEFs) and AHRR^−/− MEFs exhibited enhanced AHR transactivation. However, unlike TiPARP^−/− MEFs, AHRR^−/− MEFs did not exhibit increased AHR protein levels. Overexpression of TiPARP in AHRR^−/− MEFs or AHRRΔ8, the active isoform of AHRR, in TiPARP^−/− MEFs reduced TCDD-induced CYP1A1 mRNA levels, suggesting that they independently repress AHR. GFP-AHRRΔ8 and GFP-TiPARP expressed as small diffuse nuclear foci in MCF7 and HuH7 cells. GFP-AHRRΔ8_Δ1-49, which lacks its putative nuclear localization signal, localized to both the nucleus and the cytoplasm, while the GFP-AHRRΔ8_Δ1-100 mutant localized predominantly in large cytoplasmic foci. Neither GFP-AHRRΔ8_Δ1-49 nor GFP-AHRRΔ8_Δ1-100 repressed AHR. Taken together, AHRR and TiPARP repress AHR transactivation by similar, but also different mechanisms.     

Nitro-Oleic Acid (NO2-OA) Improves Systolic Function in Dilated Cardiomyopathy by Attenuating Myocardial Fibrosis

Nitro-oleic acid (NO₂-OA), a nitric oxide (NO)- and nitrite (NO₂⁻)-derived electrophilic fatty acid metabolite, displays anti-inflammatory and anti-fibrotic signaling actions and therapeutic benefit in murine models of ischemia-reperfusion, atrial fibrillation, and pulmonary hypertension. Muscle LIM protein-deficient mice (Mlp^−/−) develop dilated cardiomyopathy (DCM), characterized by impaired left ventricular function and increased ventricular fibrosis at the age of 8 weeks. This study investigated the effects of NO₂-OA on cardiac function in Mlp^−/− mice both in vivo and in vitro. Mlp^−/− mice were treated with NO₂-OA or vehicle for 4 weeks via subcutaneous osmotic minipumps. Wildtype (WT) littermates treated with vehicle served as controls. Mlp^−/− mice exhibited enhanced TGFβ signalling, fibrosis and severely reduced left ventricular systolic function. NO₂-OA treatment attenuated interstitial myocardial fibrosis and substantially improved left ventricular systolic function in Mlp^−/− mice. In vitro studies of TGFβ-stimulated primary cardiac fibroblasts further revealed that the anti-fibrotic effects of NO₂-OA rely on its capability to attenuate fibroblast to myofibroblast transdifferentiation by inhibiting phosphorylation of TGFβ downstream targets. In conclusion, we demonstrate a substantial therapeutic benefit of NO₂-OA in a murine model of DCM, mediated by interfering with endogenously activated TGFβ signaling.     

BMP3 Affects Cortical and Trabecular Long Bone Development in Mice

Bone morphogenetic proteins (BMPs) have a major role in tissue development. BMP3 is synthesized in osteocytes and mature osteoblasts and has an antagonistic effect on other BMPs in bone tissue. The main aim of this study was to fully characterize cortical bone and trabecular bone of long bones in both male and female Bmp3^−/− mice. To investigate the effect of Bmp3 from birth to maturity, we compared Bmp3^−/− mice with wild-type littermates at the following stages of postnatal development: 1 day (P0), 2 weeks (P14), 8 weeks and 16 weeks of age. Bmp3 deletion was confirmed using X-gal staining in P0 animals. Cartilage and bone tissue were examined in P14 animals using Alcian Blue/Alizarin Red staining. Detailed long bone analysis was performed in 8-week-old and 16-week-old animals using micro-CT. The Bmp3 reporter signal was localized in bone tissue, hair follicles, and lungs. Bone mineralization at 2 weeks of age was increased in long bones of Bmp3^−/− mice. Bmp3 deletion was shown to affect the skeleton until adulthood, where increased cortical and trabecular bone parameters were found in young and adult mice of both sexes, while delayed mineralization of the epiphyseal growth plate was found in adult Bmp3^−/− mice.     

The Dipeptidyl Peptidase-4 Inhibitor Sitagliptin Protects against Dyslipidemia-Related Kidney Injury in Apolipoprotein E Knockout Mice

The goal of this study was to investigate the possible protective effects of sitagliptin against dyslipidemia-related kidney injury in apolipoprotein E knockout (apoE^−/−) mice. Eight-week-old male apoE^−/− mice were randomized to receive either a high fat diet (HFD, apoE^−/− group) or HFD mixed with sitagliptin (sita + apoE^−/− group) for 16 weeks. A control group of age- and gender-matched C57BL/6J mice were fed a HFD. The apoE^−/− group exhibited increases in body weight and serum lipid levels in addition to high-density lipoprotein, and increases in 24-h urinary 8-hydroxy-2-deoxyguanosine and albuminuria excretion. Decreased insulin sensitivity was also observed in the apoE^−/− group. These mice additionally contained enlargements of the glomerular mesangial matrix area, lipid deposition area, and renal interstitium collagen area. The apoE^−/− group also demonstrated down-regulation of phosphorylated AMP-activated protein kinase (AMPK), increases in renal mRNA expression of transforming growth factor-beta 1 (TGF-β1) and fibronectin (FN), and increased protein expression of Akt, TGF-β1, FN and p38/ERK mitogen-activated protein kinase (MAPK). Sitagliptin treatment successfully ameliorated all the deleterious effects of dyslipidemia tested. To our knowledge, this is the first time that sitagliptin has been shown to reverse the renal dysfunction and structural damage induced by dyslipidemia in apoE^−/− mice. Our results suggest that the renoprotective mechanism of sitagliptin may be due to a reduction in Akt levels, a restoration of AMPK activity, and inhibition of TGF-β1, FN, and p38/ERK MAPK signaling pathways.     

Substance P Activates the Wnt Signal Transduction Pathway and Enhances the Differentiation of Mouse Preosteoblastic MC3T3-E1 Cells

Recent experiments have explored the impact of Wnt/β-catenin signaling and Substance P (SP) on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10⁻¹⁰ to 10⁻⁸ M). To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1) antagonists and Dickkopf-1 (DKK1) and gene expression levels of Wnt/β-catenin signaling pathway components, as well as osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin, and Runx2), were measured using quantitative polymerase chain reaction (PCR). Furthermore, protein levels of Wnt/β-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on β-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10⁻⁹ to 10⁻⁸ M) significantly up-regulated the expressions of osteoblastic genes. SP (10⁻⁸ M) also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1), as well as c-myc and β-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10⁻⁸ M) promoted the transfer of β-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/β-catenin signaling pathway.     

Collagen Family and Other Matrix Remodeling Proteins Identified by Bioinformatics Analysis as Hub Genes Involved in Gastric Cancer Progression and Prognosis

Gastric cancer has remained in the top five cancers for over ten years, both in terms of incidence and mortality due to the shortage of biomarkers for disease follow-up and effective therapies. Aiming to fill this gap, we performed a bioinformatics assessment on our data and two additional GEO microarray profiles, followed by a deep analysis of the 40 differentially expressed genes identified. PPI network analysis and MCODE plug-in pointed out nine upregulated hub genes coding for proteins from the collagen family (COL12A1, COL5A2, and COL10A1) or involved in the assembly (BGN) or degradation of collagens (CTHRC1), and also associated with cell adhesion (THBS2 and SPP1) and extracellular matrix degradation (FAP, SULF1). Those genes were highly upregulated at the mRNA and protein level, the increase being correlated with pathological T stages. The high expression of BGN (p = 8 × 10⁻¹²), THBS2 (p = 1.2 × 10⁻⁶), CTHRC1 (p = 1.1 × 10⁻⁴), SULF1 (p = 3.8 × 10⁻⁴), COL5A1 (p = 1.3 × 10⁻⁴), COL10A1 (p = 5.7 × 10⁻⁴), COL12A1 (p = 2 × 10⁻³) correlated with poor overall survival and an immune infiltrate based especially on immunosuppressive M2 macrophages (p-value range 4.82 × 10⁻⁷–1.63 × 10⁻¹³). Our results emphasize that these genes could be candidate biomarkers for GC progression and prognosis and new therapeutic targets.     

Dysfunctional cGMP Signaling Leads to Age-Related Retinal Vascular Alterations and Astrocyte Remodeling in Mice

The nitric oxide–guanylyl cyclase-1–cyclic guanylate monophosphate (NO–GC-1–cGMP) pathway is integral to the control of vascular tone and morphology. Mice lacking the alpha catalytic domain of guanylate cyclase (GC1^−/−) develop retinal ganglion cell (RGC) degeneration with age, with only modest fluctuations in intraocular pressure (IOP). Increasing the bioavailability of cGMP in GC1^−/− mice prevents neurodegeneration independently of IOP, suggesting alternative mechanisms of retinal neurodegeneration. In continuation to these studies, we explored the hypothesis that dysfunctional cGMP signaling leads to changes in the neurovascular unit that may contribute to RGC degeneration. We assessed retinal vasculature and astrocyte morphology in young and aged GC1^−/− and wild type mice. GC1^−/− mice exhibit increased peripheral retinal vessel dilation and shorter retinal vessel branching with increasing age compared to Wt mice. Astrocyte cell morphology is aberrant, and glial fibrillary acidic protein (GFAP) density is increased in young and aged GC1^−/− mice, with areas of dense astrocyte matting around blood vessels. Our results suggest that proper cGMP signaling is essential to retinal vessel morphology with increasing age. Vascular changed are preceded by alterations in astrocyte morphology which may together contribute to retinal neurodegeneration and loss of visual acuity observed in GC1^−/− mice.     

The Lrat−/− Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa

Purpose: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle. Methods: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat^−/− and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays. Results: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat^−/− animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat^−/− animals. Lrat^−/− animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat^−/− animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals. Conclusions: Our Lrat^−/− rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.