Transplanted human cones incorporate into the retina and function in a murine cone degeneration model.

Once human photoreceptors die, they do not regenerate, thus, photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation, and synaptic connectivity to the host will be critical in advancing this technology for use in clinical practice. Unlike the unstructured grafts of prior cell-suspension transplantations into end-stage degeneration models, we describe the extensive incorporation of induced pluripotent stem cell (iPSC) retinal organoid-derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extended throughout the graft, even forming a series of adherens junctions between mouse and human cells, reminiscent of an outer limiting membrane. Donor-host interaction appeared to promote polarization as well as the development of morphological features critical for light detection, namely the formation of inner and well-stacked outer segments oriented toward the retinal pigment epithelium. Putative synapse formation and graft function were evident at both structural and electrophysiological levels. Overall, these results show that human photoreceptors interacted readily with a partially degenerated retina. Moreover, incorporation into the host retina appeared to be beneficial to graft maturation, polarization, and function.

Evidence for endogenous exchange of cytoplasmic material between a subset of cone and rod photoreceptors within the adult mammalian retina via direct cell-cell connections.

Photoreceptor cell transplantation into the mouse retina has been shown to result in the transfer of cytoplasmic material between donor and host photoreceptors. Recently it has been found that this inter-photoreceptor material transfer process is likely to be mediated by nanotube-like structures connecting donor and host photoreceptors. By leveraging cone-specific reporter mice and super-resolution microscopy we provide evidence for the transfer of cytoplasmic material also from endogenous cones to endogenous rod photoreceptors and the existence of nanotube-like cell-cell connections possibly mediating this process in the adult mouse retina, together with preliminary data indicating that horizontal material transfer may also occur in the human retina.

Label-free imaging flow cytometry for analysis and sorting of enzymatically dissociated tissues.

Biomedical research relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic activated cell sorting. Labelling processes potentially alter the cells' properties and should be avoided, especially when purifying cells for clinical applications. A promising alternative is the label-free identification of cells based on physical properties. Sorting real-time deformability cytometry (soRT-DC) is a microfluidic technique for label-free analysis and sorting of single cells. In soRT-FDC, bright-field images of cells are analyzed by a deep neural net (DNN) to obtain a sorting decision, but sorting was so far only demonstrated for blood cells which show clear morphological differences and are naturally in suspension. Most cells, however, grow in tissues, requiring dissociation before cell sorting which is associated with challenges including changes in morphology, or presence of aggregates. Here, we introduce methods to improve robustness of analysis and sorting of single cells from nervous tissue and provide DNNs which can distinguish visually similar cells. We employ the DNN for image-based sorting to enrich photoreceptor cells from dissociated retina for transplantation into the mouse eye.

Osteoblastic glucocorticoid signaling exacerbates high-fat-diet- induced bone loss and obesity.

Chronic high-fat diet (HFD) consumption not only promotes obesity and insulin resistance, but also causes bone loss through mechanisms that are not well understood. Here, we fed wild-type CD-1 mice either chow or a HFD (43% of energy from fat) for 18 weeks; HFD-fed mice exhibited decreased trabecular volume (-28%) and cortical thickness (-14%) compared to chow-fed mice. In HFD-fed mice, bone loss was due to reduced bone formation and mineral apposition, without obvious effects on bone resorption. HFD feeding also increased skeletal expression of sclerostin and caused deterioration of the osteocyte lacunocanalicular network (LCN). In mice fed HFD, skeletal glucocorticoid signaling was activated relative to chow-fed mice, independent of serum corticosterone concentrations. We therefore examined whether skeletal glucocorticoid signaling was necessary for HFD-induced bone loss, using transgenic mice lacking glucocorticoid signaling in osteoblasts and osteocytes (HSD2OB/OCY-tg mice). In HSD2OB/OCY-tg mice, bone formation and mineral apposition rates were not suppressed by HFD, and bone loss was significantly attenuated. Interestingly, in HSD2OB/OCY-tg mice fed HFD, both Wnt signaling (less sclerostin induction, increased ß-catenin expression) and glucose uptake were significantly increased, relative to diet- and genotype-matched controls. The osteocyte LCN remained intact in HFD-fed HSD2OB/OCY-tg mice. When fed a HFD, HSD2OB/OCY-tg mice also increased their energy expenditure and were protected against obesity, insulin resistance, and dyslipidemia. Therefore, glucocorticoid signaling in osteoblasts and osteocytes contributes to the suppression of bone formation in HFD-fed mice. Skeletal glucocorticoid signaling is also an important determinant of glucose uptake in bone, which influences the whole-body metabolic response to HFD.

Skeletal glucocorticoid signalling determines leptin resistance and obesity in aging mice.

OBJECTIVE: Aging and chronic glucocorticoid excess share a number of critical features, including the development of central obesity, insulin resistance and osteoporosis. Previous studies have shown that skeletal glucocorticoid signalling increases with aging and that osteoblasts mediate the detrimental skeletal and metabolic effects of chronic glucocorticoid excess. Here, we investigated whether endogenous glucocorticoid action in the skeleton contributes to metabolic dysfunction during normal aging. METHODS: Mice lacking glucocorticoid signalling in osteoblasts and osteocytes (HSD2OB/OCY-tg mice) and their wild-type littermates were studied until 3, 6, 12 and 18 months of age. Body composition, adipose tissue morphology, skeletal gene expression and glucose/insulin tolerance were assessed at each timepoint. Leptin sensitivity was assessed by arcuate nucleus STAT3 phosphorylation and inhibition of feeding following leptin administration. Tissue-specific glucose uptake and adipose tissue oxygen consumption rate were also measured. RESULTS: As they aged, wild-type mice became obese and insulin-resistant. In contrast, HSD2OB/OCY-tg mice remained lean and insulin-sensitive during aging. Obesity in wild-type mice was due to leptin resistance, evidenced by an impaired ability of exogenous leptin to suppress food intake and phosphorylate hypothalamic STAT3, from 6 months of age onwards. In contrast, HSD2OB/OCY-tg mice remained leptin-sensitive throughout the study. Compared to HSD2OB/OCY-tg mice, leptin-resistant wild-type mice displayed attenuated sympathetic outflow, with reduced tyrosine hydroxylase expression in both the hypothalamus and thermogenic adipose tissues. Adipose tissue oxygen consumption rate declined progressively in aging wild-type mice but was maintained in HSD2OB/OCY-tg mice. At 18 months of age, adipose tissue glucose uptake was increased 3.7-fold in HSD2OB/OCY-tg mice, compared to wild-type mice. CONCLUSIONS: Skeletal glucocorticoid signalling is critical for the development of leptin resistance, obesity and insulin resistance during aging. These findings underscore the skeleton's importance in the regulation of body weight and implicate osteoblastic/osteocytic glucocorticoid signalling in the aetiology of aging-related obesity and metabolic disease.

Crocetin Prevents RPE Cells from Oxidative Stress through Protection of Cellular Metabolic Function and Activation of ERK1/2.

Age-related macular degeneration (AMD) is a leading cause for visual impairment in aging populations with limited established therapeutic interventions available. Oxidative stress plays an essential role in the pathogenesis of AMD, damaging the retinal pigment epithelium (RPE), which is essential for the function and maintenance of the light-sensing photoreceptors. This study aimed to evaluate the effects of crocetin, one of the main components of Saffron, on an in vitro RPE model of tert-butyl hydroperoxide (TBHP) induced oxidative stress using ARPE19 cells. The effects of crocetin were assessed using lactate de-hydrogenase (LDH) and ATP assays, as well as immunocytochemistry for cell morphology, junctional integrity, and nuclear morphology. The mechanism of crocetin action was determined via assessment of energy production pathways, including mitochondrial respiration and glycolysis in real-time as well as investigation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and distribution. Our results show that crocetin pre-treatment protects ARPE19 cells from TBHP-induced LDH release, intracellular ATP depletion, nuclear condensation, and disturbance of junctional integrity and cytoskeleton. The protective effect of crocetin is mediated via the preservation of energy production pathways and activation of ERK1/2 in the first minutes of TBHP exposure to potentiate survival pathways. The combined data suggest that a natural antioxidant, such as crocetin, represents a promising candidate to prevent oxidative stress in RPE cells and might halt or delay disease progression in AMD.

Correction: Karimi, P., et al. Crocetin Prevents RPE Cells from Oxidative Stress through Protection of Cellular Metabolic Function and Activation of ERK1/2. Int. J. Mol. Sci. 2020, 21, 2949.

The authors wish to make the following correction to Figure 6B of this article [...].

Restoration of visual function by transplantation of optogenetically engineered photoreceptors.

A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). Here, for an RPE-independent treatment approach, we introduce a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplant them into blind mice lacking the photoreceptor layer. These optogenetically-transformed photoreceptors are light responsive and their transplantation leads to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generate cone photoreceptors from human induced pluripotent stem cells, expressing the chloride pump Jaws. After transplantation into blind mice, we observe light-driven responses at the photoreceptor and ganglion cell levels. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics.

Androgens sensitise mice to glucocorticoid-induced insulin resistance and fat accumulation.

AIMS/HYPOTHESIS: Chronic glucocorticoid therapy causes insulin resistance, dyslipidaemia, abnormal fat accumulation, loss of muscle mass and osteoporosis. Here we describe a hitherto unknown sexual dimorphism in the metabolic response to chronic glucocorticoid exposure in mice. This led us to investigate whether glucocorticoid-induced insulin resistance and obesity were dependent on sex hormones. METHODS: Male and female CD1 mice were treated for 4 weeks with supraphysiological doses (~250 µg/day) of corticosterone, the main glucocorticoid in rodents, or equivalent volume of vehicle (drinking water without corticosterone). To investigate the effects of sex hormones, a separate group of mice were either orchidectomised or ovariectomised prior to corticosterone treatment, with or without dihydrotestosterone replacement. Body composition was determined before and after corticosterone treatment, and insulin tolerance was assessed after 7 and 28 days of treatment. Adipocyte morphology was assessed in white and brown adipose tissues by immunohistochemistry, and fasting serum concentrations of NEFA, triacylglycerols, total cholesterol and free glycerol were measured using colorimetric assays. Obesity- and diabetes-related hormones were measured using multiplex assays, and RNA and protein expression in adipose tissues were measured by RT-PCR and immunoblotting, respectively. RESULTS: Chronic corticosterone treatment led to insulin resistance, fasting hyperinsulinaemia, increased adiposity and dyslipidaemia in male, but not female mice. In males, orchidectomy improved baseline insulin sensitivity and attenuated corticosterone-induced insulin resistance, but did not prevent fat accumulation. In androgen-deficient mice (orchidectomised males, and intact and ovariectomised females) treated with dihydrotestosterone, corticosterone treatment led to insulin resistance and dyslipidaemia. In brown adipose tissue, androgens were required for corticosterone-induced intracellular lipid accumulation ('whitening'), and dihydrotestosterone specifically exacerbated corticosterone-induced accumulation of white adipose tissue by increasing adipocyte hypertrophy. Androgens also suppressed circulating adiponectin concentrations, but corticosterone-induced insulin resistance did not involve additional suppression of adiponectin levels. In white adipose tissue, androgens were required for induction of the glucocorticoid target gene Gilz (also known as Tsc22d3) by corticosterone. CONCLUSIONS/INTERPRETATION: In mice, androgens potentiate the development of insulin resistance, fat accumulation and brown adipose tissue whitening following chronic glucocorticoid treatment.

Metabolic and skeletal homeostasis are maintained in full locus GPRC6A knockout mice.

The G protein-coupled receptor class C, group 6, subtype A (GPRC6A) is suggested to have a physiological function in glucose and bone metabolism, although the precise role lacks consensus due to varying findings in different knockout (KO) mouse models and inconsistent findings on the role of osteocalcin, a proposed GPRC6A agonist. We have further characterized a full locus GPRC6A KO model with respect to energy metabolism, including a long-term high-dose glucocorticoid metabolic challenge. Additionally, we analyzed the microarchitecture of tibiae from young, middle-aged and aged GPRC6A KO mice and wildtype (WT) littermates. Compared to WT, vehicle-treated KO mice presented with normal body composition, unaltered insulin sensitivity and basal serum insulin and glucose levels. Corticosterone (CS) treatment resulted in insulin resistance, abnormal fat accrual, loss of lean mass and suppression of serum osteocalcin levels in both genotypes. Interestingly, serum osteocalcin and skeletal osteocalcin mRNA levels were significantly lower in vehicle-treated GPRC6A KO mice compared to WT animals. However, WT and KO age groups did not differ in long bone mass and structure assessed by micro-computed tomography. We conclude that GPRC6A is not involved in glucose metabolism under normal physiological conditions, nor does it mediate glucocorticoid-induced dysmetabolism in mice. Moreover, GPRC6A does not appear to possess a direct, non-compensable role in long bone microarchitecture under standard conditions.

Transplantation of photoreceptors into the degenerative retina: Current state and future perspectives.

The mammalian retina displays no intrinsic regenerative capacities, therefore retinal degenerative diseases such as age-related macular degeneration (AMD) or retinitis pigmentosa (RP) result in a permanent loss of the light-sensing photoreceptor cells. The degeneration of photoreceptors leads to vision impairment and, in later stages, complete blindness. Several therapeutic strategies have been developed to slow down or prevent further retinal degeneration, however a definitive cure i.e. replacement of the lost photoreceptors, has not yet been established. Cell-based treatment approaches, by means of photoreceptor transplantation, have been studied in pre-clinical animal models over the last three decades. The introduction of pluripotent stem cell-derived retinal organoids represents, in principle, an unlimited source for the generation of transplantable human photoreceptors. However, safety, immunological and reproducibility-related issues regarding the use of such cells still need to be solved. Moreover, the recent finding of cytoplasmic material transfer between donor and host photoreceptors demands reinterpretation of several former transplantation studies. At the same time, material transfer between healthy donor and dysfunctional patient photoreceptors also offers a potential alternative strategy for therapeutic intervention. In this review we discuss the history and current state of photoreceptor transplantation, the techniques used to assess rescue of visual function, the prerequisites for effective transplantation as well as the main roadblocks, including safety and immune response to the graft, that need to be overcome for successful clinical translation of photoreceptor transplantation approaches.

Chronic Mild Stress Causes Bone Loss via an Osteoblast-Specific Glucocorticoid-Dependent Mechanism.

Chronic stress and depression are associated with alterations in the hypothalamic-pituitary-adrenal signaling cascade and considered a risk factor for bone loss and fractures. However, the mechanisms underlying the association between stress and poor bone health are unclear. Using a transgenic (tg) mouse model in which glucocorticoid signaling is selectively disrupted in mature osteoblasts and osteocytes [11ß-hydroxysteroid-dehydrogenase type 2 (HSD2)OB-tg mice], the present study examines the impact of chronic stress on skeletal metabolism and structure. Eight-week-old male and female HSD2OB-tg mice and their wild-type (WT) littermates were exposed to chronic mild stress (CMS) for the duration of 4 weeks. At the endpoint, L3 vertebrae and tibiae were analyzed by micro-computed tomography and histomorphometry, and bone turnover was measured biochemically. Compared with nonstressed controls, exposure to CMS caused an approximately threefold increase in serum corticosterone concentrations in WT and HSD2OB-tg mice of both genders. Compared with controls, CMS resulted in loss of vertebral trabecular bone mass in male WT mice but not in male HSD2OB-tg littermates. Furthermore, both tibial cortical area and area fraction were reduced in stressed WT but not in stressed HSD2OB-tg male mice. Osteoclast activity and bone resorption marker were increased in WT males following CMS, features absent in HSD2OB-tg males. Interestingly, CMS had little effect on vertebral and long-bone structural parameters in female mice. We conclude that in male mice, bone loss during CMS is mediated via enhanced glucocorticoid signaling in osteoblasts (and osteocytes) and subsequent activation of osteoclasts. Female mice appear resistant to the skeletal effects of CMS.

Continuous corticosterone delivery via the drinking water or pellet implantation: A comparative study in mice.

In order to investigate the effects of glucocorticoid excess in rodent models, reliable methods of continuous glucocorticoid delivery are essential. The current study compares two methods of corticosterone (CS) delivery in regards to their ability to induce typical adverse outcomes such as fat accrual, insulin resistance, sarcopenia and bone loss. Eight-week-old mice received CS for 4weeks either via the drinking water (25-100µgCS/mL) or through weekly surgical implantation of slow release pellets containing 1.5mg CS. Both methods induced abnormal fat mass accrual, inhibited lean mass accretion and bone expansion, suppressed serum osteocalcin levels and induced severe insulin resistance. There was a clear dose dependant relationship between the CS concentrations in the drinking water and the severity of the phenotype, with a concentration of 50µg CS/mL drinking water most closely matching the metabolic changes induced by weekly pellet implantations. In contrast to pellets, however, delivery of CS via the drinking water resulted in a consistent diurnal exposure pattern, closely mimicking the kinetics of clinical glucocorticoid therapy. In addition, the method is safe, inexpensive, easily adjustable, non-invasive and avoids operative stress to the animals. Our data demonstrate that delivery of CS via the drinking water has advantages over weekly implantations of slow-release pellets. A dose of 50µg CS/mL drinking water is appropriate for the investigation of chronic glucocorticoid excess in mice.

Reactivity and Speciation of Anti-Diabetic Vanadium Complexes in Whole Blood and Its Components: The Important Role of Red Blood Cells.

Reactions with blood components are crucial for controlling the antidiabetic, anticancer, and other biological activities of V(V) and V(IV) complexes. Despite extensive studies of V(V) and V(IV) reactions with the major blood proteins (albumin and transferrin), reactions with whole blood and red blood cells (RBC) have been studied rarely. A detailed speciation study of Na3[V(V)O4] (A), K4[V(IV)2O2(citr)2]·6H2O (B; citr = citrato(4-)); [V(IV)O(ma)2] (C; ma = maltolato(-)), and (NH4)[V(V)(O)2(dipic)] (D; dipic = pyridine-2,6-dicarboxylato(2-)) in whole rat blood, freshly isolated rat plasma, and commercial bovine serum using X-ray absorption near-edge structure (XANES) spectroscopy is reported. The latter two compounds are potential oral antidiabetic drugs, and the former two are likely to represent their typical decomposition products in gastrointestinal media. XANES spectral speciation was performed by principal component analysis and multiple linear regression techniques, and the distribution of V between RBC and plasma fractions was measured by electrothermal atomic absorption spectroscopy. Reactions of A, C, or D with whole blood (1.0 mM V, 1-6 h at 310 K) led to accumulation of ∼50% of total V in the RBC fraction (∼10% in the case of B), which indicated that RBC act as V carriers to peripheral organs. The spectra of V products in RBC were independent of the initial V complex, and were best fitted by a combination of V(IV)-carbohydrate (2-hydroxyacid moieties) and/or citrate (65-85%) and V(V)-protein (15-35%) models. The presence of RBC created a more reducing environment in the plasma fraction of whole blood compared with those in isolated plasma or serum, as shown by the differences in distribution of V(IV) and V(V) species in the reaction products of A-D in these media. At physiologically relevant V concentrations (<50 µM), this role of RBC may promote the formation of V(III)-transferrin as a major V carrier in the blood plasma. The results reported herein have broad implications for the roles of RBC in the transport and speciation of metal pro-drugs that have broad applications across medicine.

Glucocorticoids and bone: local effects and systemic implications.

Glucocorticoids (GCs) are highly effective in the treatment of inflammatory and autoimmune conditions but their therapeutic use is limited by numerous adverse effects. Recent insights into the mechanisms of action of both endogenous and exogenous GCs on bone cells have unlocked new approaches to the development of effective strategies for the prevention and treatment of GC-induced osteoporosis. Furthermore, topical studies in rodents indicate that the osteoblast-derived peptide, osteocalcin, plays a central role in the pathogenesis of GC-induced diabetes and obesity. These exciting findings mechanistically link the detrimental effects of GCs on bone and energy metabolism. In this article we review the physiology and pathophysiology of GC action on bone cells, and discuss current and emerging concepts regarding the molecular mechanisms underlying adverse effects of GCs such as osteoporosis and diabetes.

Characterisation of fibroblast-like synoviocytes from a murine model of joint inflammation.

INTRODUCTION: Fibroblast-like synoviocytes (FLS) play a central role in defining the stromal environment in inflammatory joint diseases. Despite a growing use of FLS isolated from murine inflammatory models, a detailed characterisation of these cells has not been performed. METHODS: In this study, FLS were isolated from inflamed joints of mice expressing both the T cell receptor transgene KRN and the MHC class II molecule Ag7 (K/BxN mice) and their purity in culture determined by immunofluorescence and real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Basal expression of proinflammatory genes was determined by real-time RT-PCR. Secreted interleukin 6 (IL-6) was measured by enzyme-linked immunosorbent assay (ELISA), and its regulation by tumor necrosis factor-alpha (TNF-α and corticosterone (the major glucocorticoid in rodents) measured relative to other mesenchymal cell populations. RESULTS: Purity of FLS culture was identified by positive expression of fibronectin, prolyl 4-hydroxylase, cluster of differentiation 90.2 (CD90.2) and 248 (CD248) in greater than 98% of the population. Cultured FLS were able to migrate and invade through matrigel, a process enhanced in the presence of TNF-α. FLS isolated from K/BxN mice possessed significantly greater basal expression of the inflammatory markers IL-6, chemokine ligand 2 (CCL-2) and vascular cell adhesion molecule 1 (VCAM-1) when compared to FLS isolated from non-inflamed tissue (IL-6, 3.6 fold; CCL-2, 11.2 fold; VCAM-1, 9 fold; P<0.05). This elevated expression was abrogated in the presence of corticosterone at 100 nmol/l. TNF-α significantly increased expression of all inflammatory markers to a much greater degree in K/BxN FLS relative to other mesenchymal cell lines (K/BxN; IL-6, 40.8 fold; CCL-2, 1343.2 fold; VCAM-1, 17.8 fold; ICAM-1, 13.8 fold; P<0.05), with secreted IL-6 mirroring these results (K/BxN; con, 169±29.7 versus TNF-α, 923±378.8 pg/ml/1×105 cells; P<0.05). Dose response experiments confirmed effective concentrations between 10 and 100 nmol/l for corticosterone and 1 and 10 ng/ml for TNF-α, whilst inflammatory gene expression in FLS was shown to be stable between passages four and seven. CONCLUSIONS: This study has established a well characterised set of key inflammatory genes for in vitro FLS culture, isolated from K/BxN mice and non-inflamed wild-type controls. Their response to both pro- and anti-inflammatory signalling has been assessed and shown to strongly resemble that which is seen in human FLS culture. Additionally, this study provides guidelines for the effective characterisation, duration and treatment of murine FLS culture.

Osteoblasts mediate the adverse effects of glucocorticoids on fuel metabolism.

Long-term glucocorticoid treatment is associated with numerous adverse outcomes, including weight gain, insulin resistance, and diabetes; however, the pathogenesis of these side effects remains obscure. Glucocorticoids also suppress osteoblast function, including osteocalcin synthesis. Osteocalcin is an osteoblast-specific peptide that is reported to be involved in normal murine fuel metabolism. We now demonstrate that osteoblasts play a pivotal role in the pathogenesis of glucocorticoid-induced dysmetabolism. Osteoblast-targeted disruption of glucocorticoid signaling significantly attenuated the suppression of osteocalcin synthesis and prevented the development of insulin resistance, glucose intolerance, and abnormal weight gain in corticosterone-treated mice. Nearly identical effects were observed in glucocorticoid-treated animals following heterotopic (hepatic) expression of both carboxylated and uncarboxylated osteocalcin through gene therapy, which additionally led to a reduction in hepatic lipid deposition and improved phosphorylation of the insulin receptor. These data suggest that the effects of exogenous high-dose glucocorticoids on insulin target tissues and systemic energy metabolism are mediated, at least in part, through the skeleton.