Search

BACKGROUND This study systematically investigated circulating and retinal tissue lipid determinants of human diabetic retinopathy (DR) to identify underlying lipid alterations associated with severity of DR. METHODS Retinal tissues were retrieved from postmortem human eyes, including 19 individuals without diabetes, 20 with diabetes but without DR, and 20 with diabetes and DR, for lipidomic study. In a parallel study, serum samples from 28 American Indians with type 2 diabetes from the Gila River Indian Community, including 12 without DR, 7 with mild nonproliferative DR (NPDR), and 9 with moderate NPDR, were selected. A mass-spectrometry–based lipidomic platform was used to measure serum and tissue lipids. RESULTS In the postmortem retinas, we found a graded decrease of long-chain acylcarnitines and longer-chain fatty acid ester of hydroxyl fatty acids, diacylglycerols, triacylglycerols, phosphatidylcholines, and ceramide(NS) in central retina from individuals with no diabetes to those with diabetes with DR. The American Indians’ sera also exhibited a graded decrease in circulating long-chain acylcarnitines and a graded increase in the intermediate-length saturated and monounsaturated triacylglycerols from no DR to moderate NPDR. CONCLUSION These findings suggest diminished synthesis of complex lipids and impaired mitochondrial β-oxidation of fatty acids in retinal DR, with parallel changes in circulating lipids. TRIAL REGISTRATION ClinicalTrials.gov NCT00340678. FUNDING This work was supported by NIH grants R24 DK082841, K08DK106523, R03DK121941, P30DK089503, P30DK081943, P30DK020572, P30 EY007003; The Thomas Beatson Foundation; and JDRF Center for Excellence (5-COE-2019-861-S-B).

International audience; Michel Jouvet proposed in 1959 that REM sleep is a paradoxical state since it was characterized by the association of a cortical activation similar to wakefulness (W) with muscle atonia. Recently, we showed using cFos as a marker of activity that cortical activation during paradoxical sleep (PS) was limited to a few limbic cortical structures in contrast to W during which all cortices were strongly activated. However, we were not able to demonstrate whether the same neurons are activated during PS and W and to rule out that the activation observed was not linked with stress induced by the flowerpot method of PS deprivation. In the present study, we answered to these two questions by combining tdTomato and cFos immunostaining in the innovative TRAP2 transgenic mice exposed one week apart to two periods of W (W-W mice), PS rebound (PSR-PSR) or a period of W followed by a period of PSR (W-PSR mice). Using such method, we showed that different neurons are activated during W and PSR in the anterior cingulate (ACA) and rostral and caudal retrosplenial (rRSP and cRSP) cortices as well as the claustrum (CLA) previously shown to contain a large number of activated neurons after PSR. Further, the distribution of the neurons during PSR in the rRSP and cRSP was limited to the superficial layers while it was widespread across all layers during W. Our results clearly show at the cellular level that PS and W are two completely different states in term of neocortical activation.

Diabetes adversely affects multiple organs, including the kidney, eye and nerve, leading to diabetic kidney disease, diabetic retinopathy and diabetic neuropathy, respectively. In both type 1 and type 2 diabetes, tissue damage is organ specific and is secondary to a combination of multiple metabolic insults. Hyperglycaemia, dyslipidaemia and hypertension combine with the duration and type of diabetes to define the distinct pathophysiology underlying diabetic kidney disease, diabetic retinopathy and diabetic neuropathy. Only recently have the commonalities and differences in the metabolic basis of these tissue-specific complications, particularly those involving local and systemic lipids, been systematically examined. This review focuses on recent progress made using preclinical models and human-based approaches towards understanding how bioenergetics and metabolomic profiles contribute to diabetic kidney disease, diabetic retinopathy and diabetic neuropathy. This new understanding of the biology of complication-prone tissues highlights the need for organ-specific interventions in the treatment of diabetic complications.

The chaperone and anti-apoptotic activity of α-crystallins (αA- and αB-) and their derivatives has received increasing attention due to their tremendous potential in preventing cell death. While originally known and described for their role in the lens, the upregulation of these proteins in cells and animal models of neurodegenerative diseases highlighted their involvement in adaptive protective responses to neurodegeneration associated stress. However, several studies also suggest that chronic neurodegenerative conditions are associated with progressive loss of function of these proteins. Thus, while external supplementation of α-crystallin shows promise, their potential as a protein-based therapeutic for the treatment of chronic neurodegenerative diseases remains ambiguous. The current review aims at assessing the current literature supporting the anti-apoptotic potential of αA- and αB-crystallins and its potential involvement in retinal neurodegenerative diseases. The review further extends into potentially modulating the chaperone and the anti-apoptotic function of α-crystallins and the use of such functionally enhanced proteins for promoting neuronal viability in retinal neurodegenerative disease.

Purpose: We have previously demonstrated that HspB4/αA-crystallin, a molecular chaperone, plays an important intrinsic neuroprotective role during diabetes, by its phosphorylation on residue 148. We also reported that HspB4/αA-crystallin is highly expressed by glial cells. There is a growing interest in the potential causative role of low-grade inflammation in diabetic retinopathy pathophysiology and retinal Müller glial cells’ (MGCs’) participation in the inflammatory response. MGCs indeed play a central role in retinal homeostasis via secreting various cytokines and other mediators. Hence, this study was carried out to delineate and understand the regulatory function of HspB4/αA-crystallin in the inflammatory response associated with metabolic stresses. Methods: Primary MGCs were isolated from knockout HspB4/αA-crystallin mice. These primary cells were then transfected with plasmids encoding either wild-type (WT), phosphomimetic (T148D), or non-phosphorylatable mutants (T148A) of HspB4/αA-crystallin. The cells were exposed to multiple metabolic stresses including serum starvation (SS) or high glucose with TNF-alpha (HG + T) before being further evaluated for the expression of inflammatory markers by qPCR. The total protein expression along with subcellular localization of NF-kB and the NLRP3 component was assessed by Western blot. Results: Elevated levels of IL-6, IL-1β, MCP-1, and IL-18 in SS were significantly diminished in MGCs overexpressing WT and further in T148D as compared to EV. The HG + T-induced increase in these inflammatory markers was also dampened by WT and even more significantly by T148D overexpression, whereas T148A was ineffective in either stress. Further analysis revealed that overexpression of WT or the T148D, also led to a significant reduction of Nlrp3, Asc, and caspase-1 transcript expression in serum-deprived MGCs and nearly abolished the NF-kB induction in HG + T diabetes-like stress. This mechanistic effect was further evaluated at the protein level and confirmed the stress-dependent regulation of NLRP3 and NF-kB by αA-crystallin. Conclusions: The data gathered in this study demonstrate the central regulatory role of HspB4/αA-crystallin and its modulation by phosphorylation on T148 in retinal MGCs. For the first time, this study demonstrates that HspB4/αA-crystallin can dampen the stress-induced expression of pro-inflammatory cytokines through the modulation of multiple key inflammatory pathways, therefore, suggesting its potential as a therapeutic target for the modulation of chronic neuroinflammation.

Study Objectives Determine whether in the hippocampus and the supramammillary nucleus (SuM) the same neurons are reactivated when mice are exposed 1 week apart to two periods of wakefulness (W-W), paradoxical sleep rebound (PSR-PSR) or a period of W followed by a period of PSR (W-PSR) Methods We combined the innovative TRAP2 mice method in which neurons expressing cFos permanently express tdTomato after tamoxifen injection with cFos immunohistochemistry. Results We found out that a large number of tdTomato+ and cFos+ cells are localized in the dentate gyrus (DG) after PSR and W while CA1 and CA3 contained both types of neurons only after W. The number of cFos+ cells in the infrapyramidal but not the suprapyramidal blade of the DG was positively correlated with the amount of PS. In addition, we did not find double-labeled cells in the DG whatever the group of mice. In contrast, a high percentage of CA1 neurons were double-labeled in W-W mice. Finally, in the supramammillary nucleus, a large number of cells were double-labeled in W-W, PSR-PSR but not in W-PSR mice. Conclusions Altogether, our results are the first to show that different neurons are activated during W and PS in the supramammillary nucleus and the hippocampus. Further, we showed for the first time that granule cells of the infrapyramidal blade of the DG are activated during PS but not during W. Further experiments are now needed to determine whether these granule cells belong to memory engrams inducing memory reactivation during PS.

The prevalence of diabetes has been rising steadily in the past half-century, along with the burden of its associated complications, including diabetic retinopathy (DR). DR is currently the most common cause of vision loss in working-age adults in the United States. Historically, DR has been diagnosed and classified clinically based on what is visible by fundoscopy; that is vasculature alterations. However, recent technological advances have confirmed pathology of the neuroretina prior to any detectable vascular changes. These, coupled with molecular studies, and the positive impact of anti-inflammatory therapeutics in DR patients have highlighted the central involvement of the innate immune system. Reminiscent of the systemic impact of diabetes, immune dysregulation has become increasingly identified as a key element of the pathophysiology of DR by interfering with normal homeostatic systems. This review uses the growing body of literature across various model systems to demonstrate the clear involvement of all three pillars of the immune system: immune-competent cells, mediators, and the complement system. It also demonstrates how the relative contribution of each of these requires more extensive analysis, including in human tissues over the continuum of disease progression. Finally, although this review demonstrates how the complex interactions of the immune system pose many more questions than answers, the intimately connected nature of the three pillars of the immune system may also point to possible new targets to reverse or even halt reverse retinopathy.

In mammals including humans, the convention defining rapid-eye-movement (REM) sleep — also called paradoxical sleep — states that it is mainly characterized by a low voltage fast electrocortical rhythms, rapid eye movements and a muscle atonia occasionally accompanied by brief distal twitches. When facing sleep pathologies, all these criteria are not always met and identifying REM sleep becomes thus a challenge. Here we review such criteria and report the adjustments proposed to identify and measure REM sleep in animal models of sleep pathologies such as REM sleep Behaviour Disorder and narcolepsy type 1. We also argue that video-recordings synchronized to multi-parametric polysomnography are mandatory to recognize REM sleep and differentiate it from dissociated states in animal models of sleep disorders.

The retina is one of the most metabolically active tissues, yet the processes that control retinal metabolism remains poorly understood. The mTOR complex (mTORC) that drives protein and lipid biogenesis and autophagy has been studied extensively in regards to retinal development and responses to optic nerve injury but the processes that regulate homeostasis in the adult retina have not been determined. We previously demonstrated that normal adult retina has high rates of protein synthesis compared to skeletal muscle, associated with high levels of mechanistic target of rapamycin (mTOR), a kinase that forms multi-subunit complexes that sense and integrate diverse environmental cues to control cell and tissue physiology. This study was undertaken to: 1) quantify expression of mTOR complex 1 (mTORC1)- and mTORC2-specific partner proteins in normal adult rat retina, brain and liver; and 2) to localize these components in normal human, rat, and mouse retinas. Immunoblotting and immunoprecipitation studies revealed greater expression of raptor (exclusive to mTORC1) and rictor (exclusive for mTORC2) in normal rat retina relative to liver or brain, as well as the activating mTORC components, pSIN1 and pPRAS40. By contrast, liver exhibits greater amounts of the mTORC inhibitor, DEPTOR. Immunolocalization studies for all three species showed that mTOR, raptor, and rictor, as well as most other known components of mTORC1 and mTORC2, were primarily localized in the inner retina with mTORC1 primarily in retinal ganglion cells (RGCs) and mTORC2 primarily in glial cells. In addition, phosphorylated ribosomal protein S6, a direct target of the mTORC1 substrate ribosomal protein S6 kinase beta-1 (S6K1), was readily detectable in RGCs, indicating active mTORC1 signaling, and was preserved in human donor eyes. Collectively, this study demonstrates that the inner retina expresses high levels of mTORC1 and mTORC2 and possesses active mTORC1 signaling that may provide cell- and tissue-specific regulation of homeostatic activity. These findings help to define the physiology of the inner retina, which is key for understanding the pathophysiology of optic neuropathies, glaucoma and diabetic retinopathy.

AIMS/HYPOTHESIS: In diabetic macular oedema (DMO), blood components passing through the disrupted blood-retinal barrier cause neuroinflammation, but the mechanism by which autoantibodies induce neuroglial dysfunction is unknown. The aim of this study was to identify a novel autoantibody and to evaluate its pathological effects on clinically relevant photoreceptor injuries. METHODS: Biochemical purification and subsequent peptide fingerprinting were applied to identify autoantigens. The titres of autoantibodies in DMO sera were quantified and their associations with clinical variables were evaluated. Two animal models (i.e. passive transfer of autoantibodies and active immunisation) were characterised with respect to autoimmune mechanisms underlying photoreceptor injuries. RESULTS: After screening serum IgG from individuals with DMO, fumarase, a Krebs cycle enzyme expressed in inner segments, was identified as an autoantigen. Serum levels of anti-fumarase IgG in participants with DMO were higher than those in diabetic participants without DMO (p

We review here classical and recent knowledge on the state of the cortex during paradoxical (REM) sleep (PS). Recent data indicate that only a few limbic cortical structures including the anterior cingulate, retrosplenial and medial entorhinal cortices and the dentate gyrus are strongly activated during PS. In contrast, most of the other cortices including the somatosensory ones are rather deactivated during PS. Further, recent results suggest that tonic activation of limbic cortical neurons during PS is due to projections from glutamate neurons of the claustrum and GABA/glutamate neurons of the supramammillary nucleus while their pacing with theta is induced by projections from GABAergic neurons of the medial septum. The limbic structures activated during PS have all been implicated in spatial memory and it is therefore likely that such activation is crucial for memory consolidation.

The role of gamma-amino butyric acid (GABA) in sleep induction and maintenance is well accepted since most insomnia treatments target GABAa receptors. However, the population(s) of GABAergic neurons involved in the beneficial effect of GABA on sleep remains to be identified. This is not an easy task since GABAergic neurons are widely distributed in all brain structures. A recently growing number of populations of GABAergic neurons have been involved in sleep control. We first review here possible candidates for inducing non-rapid eye movement (NREM) sleep including the GABAergic neurons of the ventrolateral preoptic area, the parafacial zone in the brainstem, the nucleus accumbens and the cortex. We also discuss the role of several populations of GABAergic neurons in rapid eye movement (REM) sleep control. Indeed, it is well accepted that muscle atonia occurring during REM sleep is due to a GABA/glycinergic hyperpolarization of motoneurons. Recent evidence strongly suggests that these neurons are located in the ventral medullary reticular formation. It has also recently been shown that neurons containing the neuropeptide melanin concentrating hormone and GABA located in the lateral hypothalamic area control REM sleep expression. Finally, a population of REM-off GABAergic neurons located in the ventrolateral periaqueductal gray has been shown to gate REM sleep by inhibiting glutamatergic neurons located in the sublaterodorsal tegmental nucleus. In summary, recent data clearly indicate that multiple populations of GABAergic neurons located throughout the brain from the cortex to the medulla oblongata control NREM and REM sleep.

Crystallin proteins are the most prominent protein of the lens and have been increasingly shown to play critical roles in other tissues, especially the retina. Members of all 3 sub-families of crystallins, alpha-, beta- and gamma-crystallins have been reported in the retina during diabetes, traumatic injury and other retinal diseases. While their specific role in the retina is still unclear and may vary, beta-crystallin proteins have been shown to play a critical role in ganglion cell survival following trauma. We recently reported the correlation between a gene conversion in the betaB2-crystallin gene and a phenotype of familial congenital cataract. Interestingly, in half of the patients, this phenotype was associated with glaucoma. Taken together, these data suggested that the mutations we recently reported could have an impact on the role of betaB2-crystallin in both lens epithelial cells and retinal neurons. Consistent with this hypothesis, we show in the current study that the gene conversion leading to an amino acid conversion lead to a loss of solubility and a change of subcellular localization of betaB2-crystallin in both cell types. While the overall observations were similar in both cell types, there were some important nuances between them, suggesting different roles and regulation of betaB2-crystallin in lens cells versus retinal neurons. The data reported in this study strongly support a significant role of betaB2-crystallin in both lenticular and retinal ocular tissues and warrant further analysis of its regulation and its impact not only in cataract formation but also in retinal neurodegenerative diseases.

International audience

BACKGROUND: In this study, we identified the lipidomic predictors of early type 2 diabetic kidney disease (DKD) progression, which are currently undefined. METHODS: This longitudinal study included 92 American Indians with type 2 diabetes. Serum lipids (406 from 18 classes) were quantified using mass spectrometry from baseline samples when iothalamate-based glomerular filtration rate (GFR) was at least 90 mL/min. Affymetrix GeneChip Array was used to measure renal transcript expression. DKD progression was defined as at least 40% decline in GFR during follow-up. RESULTS: Participants had a mean age of 45 ± 9 years and median urine albumin/creatinine ratio of 43 (interquartile range 11–144). The 32 progressors had significantly higher relative abundance of polyunsaturated triacylglycerols (TAGs) and a lower abundance of C16–C20 acylcarnitines (ACs) (P < 0.001). In a Cox regression model, the main effect terms of unsaturated free fatty acids and phosphatidylethanolamines and the interaction terms of C16–C20 ACs and short-low-double-bond TAGs by categories of albuminuria independently predicted DKD progression. Renal expression of acetyl-CoA carboxylase–encoding gene (ACACA) correlated with serum diacylglycerols in the glomerular compartment (r = 0.36, and P = 0.006) and with low-double-bond TAGs in the tubulointerstitial compartment (r = 0.52, and P < 0.001). CONCLUSION: Collectively, the findings reveal a previously unrecognized link between lipid markers of impaired mitochondrial β-oxidation and enhanced lipogenesis and DKD progression in individuals with preserved GFR. Renal acetyl-CoA carboxylase activation accompanies these lipidomic changes and suggests that it may be the underlying mechanism linking lipid abnormalities to DKD progression. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00340678. FUNDING: NIH R24DK082841, K08DK106523, R03DK121941, P30DK089503, P30DK081943, and P30DK020572.

International audience; The cFos immunostaining allowed the identification of multiple populations of neu- rons involved in the generation of paradoxical sleep. We adopted the transgenic (tar- geted recombination in active populations) mouse model, which following injection of tamoxifen, allows expression of Cre-dependent reporter constructs (i.e., mCherry) in neurons expressing cFos during waking or paradoxical sleep hypersomnia follow- ing automatic paradoxical sleep deprivation. Three groups of mice were subjected to two periods of waking, one period of waking and one of paradoxical sleep hypersom- nia, or two periods of paradoxical sleep hypersomnia. A high percentage of double- labelled neurons was observed in the lateral hypothalamic area and zona incerta of two periods of waking and two periods of paradoxical sleep hypersomnia in mice, but not in those of one period of waking and one of paradoxical sleep hypersom- nia in animals. Melanin-concentrating hormone neurons in the lateral hypothalamic area and Lhx6+ cells in the zona incerta constituted 5.7 ± 1.5% and 8.8 ± 2.3% of all mCherry+ cells and 20.6 ± 4.8% and 24.6 ± 5.9% of all cFos+ neurons in two periods of paradoxical sleep hypersomnia in animals. In addition, melanin-concentrating hor- mone cells as well as Lhx6+ neurons rarely expressed mCherry (or cFos) in the wak- ing condition, in contrast to orexin neurons, which constituted approximately 30% of mCherry+ and cFos+ neurons. Our results validate the TRAP methodology and open the way to use it for identifying the neurons activated during waking and para- doxical sleep hypersomnia. Furthermore, they indicate for the first time that Lhx6+ neurons in the zona incerta, like melanin-concentrating hormone cells in the lateral hypothalamic area, are activated during paradoxical sleep hypersomnia but not dur- ing waking. These results indicate that Lhx6+ neurons might play a role in the control of paradoxical sleep, like the melanin-concentrating hormone cells.

This chapter presents hypotheses on the mechanisms responsible for the succession of the three vigilance states, namely waking, nonrapid eye movement (non-REM) (slow-wave sleep-SWS), and REM sleep (paradoxical sleep-PS). It can be proposed that waking is induced by the activity of multiple waking systems, including the serotonergic, noradrenergic, cholinergic, and hypocretin systems. At the onset of sleep, the SWS-active neurons are activated by the circadian clock localized in the suprachiasmatic nucleus and a hypnogenic factor, adenosine, which progressively accumulates in the brain during waking. A number of studies support the hypothesis that SWS results from the activation of GABAergic neurons localized in the ventrolateral preoptic nucleus. However, additional GABAergic systems have been described, localized in the parafacial, accumbens, and reticular thalamic nuclei, and these are also presented. In addition, the chapter discusses the fact that a large body of data strongly suggests that the switch from SWS to PS is due to the interaction of multiple populations of glutamatergic and GABAergic neurons localized in the posterior hypothalamus and the brainstem.

This chapter presents hypotheses on the mechanisms responsible for the succession of the three vigilance states, namely waking, nonrapid eye movement (non-REM) (slow-wave sleep-SWS), and REM sleep (paradoxical sleep-PS). It can be proposed that waking is induced by the activity of multiple waking systems, including the serotonergic, noradrenergic, cholinergic, and hypocretin systems. At the onset of sleep, the SWS-active neurons are activated by the circadian clock localized in the suprachiasmatic nucleus and a hypnogenic factor, adenosine, which progressively accumulates in the brain during waking. A number of studies support the hypothesis that SWS results from the activation of GABAergic neurons localized in the ventrolateral preoptic nucleus. However, additional GABAergic systems have been described, localized in the parafacial, accumbens, and reticular thalamic nuclei, and these are also presented. In addition, the chapter discusses the fact that a large body of data strongly suggests that the switch from SWS to PS is due to the interaction of multiple populations of glutamatergic and GABAergic neurons localized in the posterior hypothalamus and the brainstem.

The retinal insulin receptor (IR) exhibits basal kinase activity equivalent to that of the liver of fed animals, but unlike the liver, does not fluctuate with feeding and fasting; it also declines rapidly after the onset of insulin-deficient diabetes. The ligand(s) that determine basal IR activity in the retina has not been identified. Using a highly sensitive insulin assay, we found that retinal insulin concentrations remain constant in fed versus fasted rats and in diabetic versus control rats; vitreous fluid insulin levels were undetectable. Neutralizing antibodies against insulin-like growth factor 2 (IGF-2), but not insulin-like growth factor 1 (IGF-1) or insulin, decreased IR kinase activity in normal rat retinas, and depletion of IGF-2 from serum specifically reduced IR phosphorylation in retinal cells. Immunoprecipitation studies demonstrated that IGF-2 induced greater phosphorylation of the retinal IR than the IGF-1 receptor. Retinal IGF-2 mRNA content was 10-fold higher in adults than pups and orders of magnitude higher than in liver. Diabetes reduced retinal IGF-2, but not IGF-1 or IR, mRNA levels, and reduced IGF-2 and IGF-1 content in vitreous fluid. Finally, intravitreal administration of IGF-2 (mature and pro-forms) increased retinal IR and Akt kinase activity in diabetic rats. Collectively, these data reveal that IGF-2 is the primary ligand that defines basal retinal IR activity and suggest that reduced ocular IGF-2 may contribute to reduced IR activity in response to diabetes. These findings may have importance for understanding the regulation of metabolic and prosurvival signaling in the retina.

The fetal environment plays a decisive role in modifying the risk for developing diabetes later in life. Developing novel methodology for noninvasive imaging of β-cell development in vivo under the controlled physiological conditions of the host can serve to understand how this environment affects β-cell growth and differentiation. A number of culture models have been designed for pancreatic rudiment but none match the complexity of the in utero or even normal physiological environment. Speier et al. recently developed a platform of noninvasive in vivo imaging of pancreatic islets using the anterior chamber of the eye where islets get vascularized, grow and respond to physiological changes. The same methodology was adapted for the study of pancreatic development. E13.0, still undifferentiated rudiments with fluorescent lineage tracing were implanted in the AC of the eye, allowing the longitudinal study of their growth and differentiation. Within 48 h the anlages get vascularized and grow but their mesenchyme displays a selective growth advantage. The resulting imbalance leads to alteration in the differentiation pattern of the progenitors. Reducing the mesenchyme to its bare minimum before implantation allows the restoration of a proper balance and a development that mimics the normal pancreatic development. These groundbreaking observations demonstrate that the anterior chamber of the eye provides a good system for noninvasive in vivo fluorescence imaging of the developing pancreas under the physiology of the host and can have important implications for designing strategies to prevent or reverse the deleterious effects of hyperglycemia on altering β-cell function later in life.

Despite decades of research, there is a persistent debate regarding the localization of GABA/glycine neurons responsible for hyperpolarizing somatic motoneurons during paradoxical (or REM) sleep (PS), resulting in the loss of muscle tone during this sleep state. Combining complementary neuroanatomical approaches in rats, we first show that these inhibitory neurons are localized within the ventromedial medulla (vmM) rather than within the spinal cord. We then demonstrate their functional role in PS expression through local injections of adeno-associated virus carrying specific short-hairpin RNA in order to chronically impair inhibitory neurotransmission from vmM. After such selective genetic inactivation, rats display PS without atonia associated with abnormal and violent motor activity, concomitant with a small reduction of daily PS quantity. These symptoms closely mimic human REM sleep behavior disorder (RBD), a prodromal parasomnia of synucleinopathies. Our findings demonstrate the crucial role of GABA/glycine inhibitory vmM neurons in muscle atonia during PS and highlight a candidate brain region that can be susceptible to α-synuclein-dependent degeneration in RBD patients., Loss of muscle tone is a distinguishing feature of paradoxical or REM sleep (PS) and is disrupted in REM sleep behavior disorder. Here the authors report that GABA/glycine inhibitory neurons in the ventromedial medulla are essential for producing PS muscle atonia without affecting PS quantity.

Purpose: The addition of short chain acyl groups to lysines is emerging as key post-translational modifications (PTM) linking metabolite levels to metabolic enzyme activities. Malonyl-lysine (Kmal) targets and inhibits glycolysis and beta-oxidation enzymes. Recently, formation of Kmal was suggested to act as a sink for mitochondrial malate, preventing its inhibition of succinate dehydrogenase in the electron transport chain (ETC). Using the db/db T2DM mouse model, we recently found that diabetes increases retinal glycolysis and beta-oxidation metabolite levels, whereas malate was the only TCA cycle metabolite significantly increased. Metabolic flux measurement with 13C-labeled glucose also demonstrated increased enrichment of labeled malate. We therefore compared the relative levels of Kmal-modified proteins in retinas from diabetic mice as well as in postmortem retinas from diabetic patients to determine if this PTM is altered by diabetes. Methods: Immunofluorescence (IF) with antibodies specific to acetyl-lysine, to succinyl-lysine and to Kmal was used to probe retinal sections from BKS-db/db mice and BKS-db/+ controls (n=9/group) at 24 weeks of age. Kmal modifications were also examined in mouse retinas from mice made diabetic with streptozotocin (STZ) and controls (n=6/group) and a limited number of retinal sections from postmortem eyes of diabetic (n=4) and control (n=2) patients. Antibodies to markers of retinal ganglion and glial cells were used to co-localize Kmal-modified proteins. Results: Only Kmal IF intensity was significantly reduced by approximately 30% in both db/db and STZ diabetic mouse retinas. Kmal IF exhibited a 25% reduction in human diabetic retinas. Kmal-modified proteins were concentrated in the ganglion cell layer and largely coincided with Müller glia endfeet and/or astrocytes. Conclusions: The results suggest that protein malonylation in glial cells is diminished in the diabetic retina, which may increase glycolysis and beta-oxidation while inhibiting the ETC. Disclosure S.F. Abcouwer: None. S. Shanmugam: None. H.M. Hager: None. C. Lin: None. P.E. Fort: None. T.W. Gardner: Research Support; Self; Novo Nordisk A/S. K. Sas: None. S. Pennathur: None.

Purpose Loss of retinal capillary endothelial cells and pericytes through apoptosis is an early event in diabetic retinopathy (DR). Inflammatory pathways play a role in early DR, yet the biochemical mechanisms are poorly understood. In this study, we investigated the role of indoleamine 2,3-dioxygenase (IDO), an inflammatory cytokine-inducible enzyme, on retinal endothelial apoptosis and capillary degeneration in the diabetic retina. Methods IDO was detected in human and mouse retinas by immunohistochemistry or Western blotting. Interferon-γ (IFN-γ) levels were measured by ELISA. IDO levels were measured in human retinal capillary endothelial cells (HREC) cultured in the presence of IFN-γ ± 25 mM D-glucose. Reactive oxygen species (ROS) were measured using CM-H2DCFDA dye and apoptosis was measured by cleaved caspase-3. The role of IDO in DR was determined in IDO knockout (IDO-/-) mice with streptozotocin-induced diabetes. Results The IDO and IFN-γ levels were higher in human diabetic retinas with retinopathy relative to nondiabetic retinas. Immunohistochemical data showed that IDO is present in capillary endothelial cells. IFN-γ upregulated the IDO and ROS levels in HREC. The blockade of either IDO or kynurenine monooxygenase led to inhibition of ROS in HREC. Apoptosis through this pathway was inhibited by an ROS scavenger, TEMPOL. Capillary degeneration was significantly reduced in diabetic IDO-/- mice compared to diabetic wild-type mice. Conclusions The results suggest that the kynurenine pathway plays an important role in the inflammatory damage in the diabetic retina and could be a new therapeutic target for the treatment of DR.

In the present chapter, hypotheses on the mechanisms responsible for the genesis of the three vigilance states, namely, waking, non-rapid eye movement (non-REM) also called slow-wave sleep (SWS), and REM sleep also called paradoxical sleep (PS), are presented. A huge number of studies first indicate that waking is induced by the activation of multiple waking systems, including the serotonergic, noradrenergic, cholinergic, and hypocretin systems. At the onset of sleep, the SWS-active neurons would be activated by the circadian clock localized in the suprachiasmatic nucleus and a hypnogenic factor, adenosine, which progressively accumulates in the brain during waking. A number of studies support the hypothesis that SWS results from the activation of GABAergic neurons localized in the ventrolateral preoptic nucleus (VLPO). However, new GABAergic systems recently described localized in the parafacial, accumbens, and reticular thalamic nuclei will be also presented. In addition, we will show that a large body of data strongly suggests that the switch from SWS to PS is due to the interaction of multiple populations of glutamatergic and GABAergic neurons localized in the posterior hypothalamus and the brainstem.

In the present chapter, hypotheses on the mechanisms responsible for the genesis of the three vigilance states, namely, waking, non-rapid eye movement (non-REM) also called slow-wave sleep (SWS), and REM sleep also called paradoxical sleep (PS), are presented. A huge number of studies first indicate that waking is induced by the activation of multiple waking systems, including the serotonergic, noradrenergic, cholinergic, and hypocretin systems. At the onset of sleep, the SWS-active neurons would be activated by the circadian clock localized in the suprachiasmatic nucleus and a hypnogenic factor, adenosine, which progressively accumulates in the brain during waking. A number of studies support the hypothesis that SWS results from the activation of GABAergic neurons localized in the ventrolateral preoptic nucleus (VLPO). However, new GABAergic systems recently described localized in the parafacial, accumbens, and reticular thalamic nuclei will be also presented. In addition, we will show that a large body of data strongly suggests that the switch from SWS to PS is due to the interaction of multiple populations of glutamatergic and GABAergic neurons localized in the posterior hypothalamus and the brainstem.

Neurodegeneration is a central aspect of the early stages of diabetic retinopathy, the primary ocular complication associated with diabetes. While progress has been made to improve the vascular perturbations associated with diabetic retinopathy, there are still no treatment options to counteract the neuroretinal degeneration associated with diabetes. Our previous work suggested that the molecular chaperones α-crystallins could be involved in the pathophysiology of diabetic retinopathy; however, the role and regulation of α-crystallins remained unknown. In the present study, we demonstrated the neuroprotective role of αA-crystallin during diabetes and its regulation by its phosphorylation on residue 148. We further characterized the dual role of αA-crystallin in neurons and glia, its essential role for neuronal survival, and its direct dependence on phosphorylation on this residue. These findings support further evaluation of αA-crystallin as a treatment option to promote neuron survival in diabetic retinopathy and neurodegenerative diseases in general.

Retinoblastoma (RB) is the most common primary intraocular cancer in children, and the third most common cancer overall in infants. No molecular-targeted therapy for this lethal tumor exists. Since the tumor suppressor RB1, whose genetic inactivation underlies RB, is upstream of the epigenetic regulator EZH2, a pharmacologic target for many solid tumors, we reasoned that EZH2 might regulate human RB tumorigenesis. Histologic and immunohistochemical analyses were performed using an EZH2 antibody in sections from 43 samples of primary, formalin-fixed, paraffin-embedded human RB tissue, cryopreserved mouse retina, and in whole cell lysates from human RB cell lines (Y79 and WERI-Rb1), primary human fetal retinal pigment epithelium (RPE) and fetal and adult retina, mouse retina and embryonic stem (ES) cells. Although enriched during fetal human retinal development, EZH2 protein was not present in the normal postnatal retina. However, EZH2 was detected in all 43 analyzed human RB specimens, indicating that EZH2 is a fetal protein expressed in postnatal human RB. EZH2 expression marked single RB cell invasion into the optic nerve, a site of invasion whose involvement may influence the decision for systemic chemotherapy. To assess the role of EZH2 in RB cell survival, human RB and primary RPE cells were treated with two EZH2 inhibitors (EZH2i), GSK126 and SAH-EZH2 (SAH). EZH2i impaired intracellular adenosine triphosphate (ATP) production, an indicator of cell viability, in a time and dose-dependent manner, but did not affect primary human fetal RPE. Thus, aberrant expression of a histone methyltransferase protein is a feature of human RB. This is the first time this mechanism has been implicated for an eye, adnexal, or orbital tumor. The specificity of EZH2i toward human RB cells, but not RPE, warrants further in vivo testing in animal models of RB, especially those EZH2i currently in clinical trials for solid tumors and lymphoma.

The cognitive role of melanin-concentrating hormone (MCH) neurons, a neuronal population located in the mammalian postero-lateral hypothalamus sending projections to all cortical areas, remains poorly understood. Mainly activated during paradoxical sleep (PS), MCH neurons have been implicated in sleep regulation. The genetic deletion of the only known MCH receptor in rodent leads to an impairment of hippocampal dependent forms of memory and to an alteration of hippocampal long-term synaptic plasticity. By using MCH/ataxin3 mice, a genetic model characterized by a selective deletion of MCH neurons in the adult, we investigated the role of MCH neurons in hippocampal synaptic plasticity and hippocampal-dependent forms of memory. MCH/ataxin3 mice exhibited a deficit in the early part of both long-term potentiation and depression in the CA1 area of the hippocampus. Post-tetanic potentiation (PTP) was diminished while synaptic depression induced by repetitive stimulation was enhanced suggesting an alteration of pre-synaptic forms of short-term plasticity in these mice. Behaviorally, MCH/ataxin3 mice spent more time and showed a higher level of hesitation as compared to their controls in performing a short-term memory T-maze task, displayed retardation in acquiring a reference memory task in a Morris water maze, and showed a habituation deficit in an open field task. Deletion of MCH neurons could thus alter spatial short-term memory by impairing short-term plasticity in the hippocampus. Altogether, these findings could provide a cellular mechanism by which PS may facilitate memory encoding. Via MCH neuron activation, PS could prepare the day's learning by increasing and modulating short-term synaptic plasticity in the hippocampus.

Acta Pharmacologica Sinica (2018), doi:10.1038/aps.2017.168, published online 28 December 2017. After publication of the article, it has been brought to our attenetion that there is a discrepancy between the publication date on the pdf and online formats. The date on the pdf is 28 December 2017 and online is 28 December 2018. The correct publication date is the one on the pdf, 28 December 2017.

Study Objectives Experimental studies over the last 15 years established a role in sleep of the tuberal hypothalamic neurons that express melanin-concentrating hormone (MCH). Controversies still remain regarding their actual contribution to both slow-wave sleep (SWS) and paradoxical sleep (PS also known as REM sleep) or PS alone. Methods To address this point, we compared effects of chemogenetic activation and inhibition of MCH neurons on SWS and PS amounts and EEG rhythmic activities in transgenic Pmch-cre mice. Results In agreement with recently reported optogenetic data, the activation of MCH neurons invariably facilitates PS onset and maintenance. Our chemogenetic experiments further disclose that the ultradian rhythm of SWS is also notably related to the activity of MCH neurons. We observed that the mean duration of SWS episodes is significantly extended when MCH neurons are inhibited. Conversely, when they were excited, SWS bouts were drastically shortened and depicted substantial changes in δ rhythmic activities in electroencephalographic recording likely reflecting a deeper SWS. Conclusions According to these original findings, we propose that when MCH neurons are physiologically recruited, SWS depth is increased and the extinction of SWS episodes is accelerated, two joint physiological processes strengthening the probability for natural SWS to PS transition and likely facilitating PS onset.

REM (paradoxical) sleep is a state characterized by rapid eye movements, EEG activation, and muscle atonia. REM sleep behavior disorder (RBD) is a parasomnia characterized by loss of muscle atonia during REM sleep. Cataplexy, a key symptom of narcolepsy, is a striking sudden episode of muscle weakness comparable to REM sleep atonia triggered by emotions during wakefulness. This chapter presents recent results on the neuronal network responsible for REM sleep and explores hypotheses explaining RBD and cataplexy. RBD could be due to a specific degeneration of glutamatergic neurons responsible for muscle atonia, localized in the pontine sublaterodorsal tegmental nucleus (SLD) or the glycinergic/GABAergic premotoneurons localized in the ventral medullary reticular nuclei. Cataplexy in narcoleptics could be due to activation during waking of SLD neurons. In normal conditions, activation of SLD neurons would be blocked by simultaneous excitation by hypocretins of REM sleep-off GABAergic neurons localized in the ventrolateral periaqueductal gray.

International audience; We recently demonstrated that granule cells located in the dorsal dentate gyrus (dDG) are activated by neurons located in the lateral supramammillary nucleus (SumL) during paradoxical sleep (PS) hypersomnia. To determine whether these neurons are glutamatergic and/or GABAergic, we combined FOS immunostaining with in situ hybridization of vesicular glutamate transporter 2 (vGLUT2, a marker of glutamatergic neurons) or that of the vesicular GABA transporter (vGAT, a marker of GABAergic neurons) mRNA in rats displaying PS hyper-somnia (PSR). We found that 84 and 76 % of the FOS? SumL neurons in PSR rats expressed vGLUT2 and vGAT mRNA, respectively. Then, we examined vGLUT2 and FOS immunostaining in the dorsal and ventral DG of PSR rats with a neurochemical lesion of the Sum. In PSR-lesioned animals but not in sham animals, nearly all vGLUT2? fibers and FOS? neurons disappeared in the dDG, but not in the ventral DG (vDG). To identify the pathway (s) responsible (s) for the activation of the vDG during PS hypersomnia, we combined Fluorogold (FG) injection in the vDG of PSR rats with FOS staining. We found a large number of neurons FOS-FG?, specifically in the medial entorhinal cortex (ENTm). Altogether, our results suggest that SumL neurons with a unique dual glutamatergic and GABAergic phenotype are responsible for the activation of the dDG during PS hypersomnia, while vDG granule neurons are activated by ENTm cortical neurons. These results suggest differential mechanisms and functions for the activation of the dDG and the vDG granule cells during PS.

Diabetes mellitus (DM) has detrimental effects on every ocular tissue but most greatly impacts the retina. These effects can be moderated with glycemic control and blood pressure regulation, but complications such as diabetic retinopathy (DR) may still occur. DR can result in vision loss due to several mechanisms, such as retinal detachment and macular ischemia. This chapter explains the current classification and treatment of DR as well as other ocular complications of DM, such as neurotrophic keratitis, neovascular glaucoma, and cataract.

Poorly controlled diabetes has long been known as a catabolic disorder with profound loss of muscle and fat body mass resulting from a simultaneous reduction in protein synthesis and enhanced protein degradation. By contrast, retinal structure is largely maintained during diabetes despite reduced Akt activity and increased rate of cell death. Therefore, we hypothesized that retinal protein turnover is regulated differently than in other insulin-sensitive tissues, such as skeletal muscle. Ins2Akita diabetic mice and streptozotocin-induced diabetic rats exhibited marked reductions in retinal protein synthesis matched by a concomitant reduction in retinal protein degradation associated with preserved retinal mass and protein content. The reduction in protein synthesis depended on both hyperglycemia and insulin deficiency, but protein degradation was only reversed by normalization of hyperglycemia. The reduction in protein synthesis was associated with diminished protein translation efficiency but, surprisingly, not with reduced activity of the mTORC1/S6K1/4E-BP1 pathway. Instead, diabetes induced a specific reduction of mTORC2 complex activity. These findings reveal distinctive responses of diabetes-induced retinal protein turnover compared with muscle and liver that may provide a new means to ameliorate diabetic retinopathy.

Aim: Most Duchenne muscular dystrophy patients and the mdxCv3 mouse strain, lacking expression of both dystrophins Dp260 and Dp71, show a high attenuation of the dark-adapted electroretinogram (ERG) b-wave amplitude, whereas mice lacking the expression of Dp260 show normal b-wave amplitude. Here, we completed our assessment of whether the sole absence of Dp71 affects the ERG. Methods: Ganzfeld ERGs were performed on dark-adapted Dp71-null mice and littermates. Scotopic flash ERGs were recorded at light intensities from 3.10−5 to 1 cd.s/m2. Oscillatory potentials (OPs) were extracted at 1 cd.s/m2. Photopic flash ERGs were recorded at 10 cd.s/m2 after light adaptation. Results: Dp71-null mice showed a slight but significant reduction in b-wave amplitudes, normal a-wave amplitudes and nonaffected implicit times of the scotopic ERGs. No changes were observed in the amplitudes and implicit times of the OPs and the photopic ERGs. Conclusions: Our results demonstrate that together both Dp71 and Dp260 are required for the generation of the ERG b-wave in mice.

In the recently published consensus-based care recommendations for adults with myotonic dystrophy type 1 (DM1),1 visual symptoms caused by retinal dysfunction are not considered despite several studies reporting frequent abnormal electroretinograms (ERGs) in patients with DM1.2–4