RESUMEN
Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.
Asunto(s)
Isquemia Encefálica , Péptidos de Penetración Celular/farmacología , Ferroptosis/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hemorragias Intracraneales , Neuronas , Fosfolípido Hidroperóxido Glutatión Peroxidasa/biosíntesis , Selenio/farmacología , Accidente Cerebrovascular , Transcripción Genética/efectos de los fármacos , Animales , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Hemorragias Intracraneales/tratamiento farmacológico , Hemorragias Intracraneales/metabolismo , Hemorragias Intracraneales/patología , Masculino , Ratones , Neuronas/metabolismo , Neuronas/patología , Factor de Transcripción Sp1/metabolismo , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Factor de Transcripción AP-2/metabolismoRESUMEN
Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.
Asunto(s)
Quimiocina CX3CL1/metabolismo , Retinopatía Diabética/patología , Microglía/fisiología , Retina/patología , Animales , Bencimidazoles/farmacología , Compuestos de Bifenilo/farmacología , Quimiocina CX3CL1/farmacología , Retinopatía Diabética/inducido químicamente , Retinopatía Diabética/metabolismo , Perfilación de la Expresión Génica , Ratones , Microglía/metabolismo , Neuronas/fisiología , Pericitos/patología , Ratas , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/genética , Retina/metabolismo , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/patología , Transducción de Señal/efectos de los fármacos , Estreptozocina/farmacología , Tetrazoles/farmacología , Vasoconstricción/efectos de los fármacosRESUMEN
Genetic disorders which present during development make treatment strategies particularly challenging because there is a need to disentangle primary pathophysiology from downstream dysfunction caused at key developmental stages. To provide a deeper insight into this question, we studied a mouse model of X-linked juvenile retinoschisis (XLRS), an early-onset inherited condition caused by mutations in the Rs1 gene encoding retinoschisin (RS1) and characterized by cystic retinal lesions and early visual deficits. Using an unbiased approach in expressing the fast intracellular calcium indicator GCaMP6f in neuronal, glial, and vascular cells of the retina of RS1-deficient male mice, we found that initial cyst formation is paralleled by the appearance of aberrant spontaneous neuro-glial signals as early as postnatal day 15, when eyes normally open. These presented as glutamate-driven wavelets of neuronal activity and sporadic radial bursts of activity by Müller glia, spanning all retinal layers and disrupting light-induced signaling. This study confers a role to RS1 beyond its function as an adhesion molecule, identifies an early onset for dysfunction in the course of disease, establishing a potential window for disease diagnosis and therapeutic intervention.Significance StatementDevelopmental disorders make it difficult to distinguish pathophysiology due to ongoing disease from pathophysiology due to disrupted development. Here, we investigated a mouse model for X-linked retinoschisis (XLRS), a well-defined monogenic degenerative disease caused by mutations in the Rs1 gene, which codes for the protein retinoschisin. We evaluated the spontaneous activity of explanted retinas lacking retinoschisin at key stages of development using the unbiased approach of ubiquitously expressing GCaMP6f in all retinal neurons, vasculature and glia. In mice lacking RS1, we found an array of novel phenotypes which present around eye-opening, are linked to glutamatergic neurotransmission, and affect visual processing. These data identify novel pathophysiology linked to RS1, and define a window where treatments might be best targeted.
RESUMEN
X-linked juvenile retinoschisis (XLRS) is an early-onset inherited condition that affects primarily males and is characterized by cystic lesions of the inner retina, decreased visual acuity and contrast sensitivity and a selective reduction of the electroretinogram (ERG) b-wave. Although XLRS is genetically heterogeneous, all mouse models developed to date involve engineered or spontaneous null mutations. In the present study, we have studied three new Rs1 mutant mouse models: (1) a knockout with inserted lacZ reporter gene; (2) a C59S point mutant substitution and (3) an R141C point mutant substitution. Mice were studied from postnatal day (P15) to 28 weeks by spectral domain optical coherence tomography and ERG. Retinas of P21-22 mice were examined using biochemistry, single cell electrophysiology of retinal ganglion cells (RGCs) and by immunohistochemistry. Each model developed intraretinal schisis and reductions in the ERG that were greater for the b-wave than the a-wave. The phenotype of the C59S mutant appeared less severe than the other mutants by ERG at adult ages. RGC electrophysiology demonstrated elevated activity in the absence of a visual stimulus and reduced signal-to-noise ratios in response to light stimuli. Immunohistochemical analysis documented early abnormalities in all cells of the outer retina. Together, these results provide significant insight into the early events of XLRS pathophysiology, from phenotype differences between disease-causing variants to common mechanistic events that may play critical roles in disease presentation and progression.
Asunto(s)
Genes Ligados a X , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Fenotipo , Retinosquisis/genética , Retinosquisis/patología , Animales , Biomarcadores , Moléculas de Adhesión Celular/genética , Modelos Animales de Enfermedad , Electrorretinografía , Proteínas del Ojo/genética , Estudios de Asociación Genética/métodos , Inmunohistoquímica , Ratones , Mutación , Estimulación Luminosa , Retinosquisis/diagnóstico , Índice de Severidad de la Enfermedad , Tomografía de Coherencia ÓpticaRESUMEN
Diabetic retinopathy (DR) is a frequent complication of diabetes mellitus and an increasingly common cause of visual impairment. Blood vessel damage occurs as the disease progresses, leading to ischemia, neovascularization, blood-retina barrier (BRB) failure and eventual blindness. Although detection and treatment strategies have improved considerably over the past years, there is room for a better understanding of the pathophysiology of the diabetic retina. Indeed, it has been increasingly realized that DR is in fact a disease of the retina's neurovascular unit (NVU), the multi-cellular framework underlying functional hyperemia, coupling neuronal computations to blood flow. The accumulating evidence reveals that both neurochemical (synapses) and electrical (gap junctions) means of communications between retinal cells are affected at the onset of hyperglycemia, warranting a global assessment of cellular interactions and their role in DR. This is further supported by the recent data showing down-regulation of connexin 43 gap junctions along the vascular relay from capillary to feeding arteriole as one of the earliest indicators of experimental DR, with rippling consequences to the anatomical and physiological integrity of the retina. Here, recent advancements in our knowledge of mechanisms controlling the retinal neurovascular unit will be assessed, along with their implications for future treatment and diagnosis of DR.
Asunto(s)
Retinopatía Diabética/fisiopatología , Pericitos/fisiología , Neuronas Retinianas/fisiología , Animales , Barrera Hematorretinal , Retinopatía Diabética/metabolismo , Humanos , Receptores Colinérgicos/fisiología , Receptores Dopaminérgicos/fisiología , Receptores de Neurotransmisores/fisiología , Flujo Sanguíneo Regional/fisiología , Vasos Retinianos/fisiopatologíaRESUMEN
The nervous system demands an adequate oxygen and metabolite exchange, making pericytes (PCs), the only vasoactive cells on the capillaries, essential to neural function. Loss of PCs is a hallmark of multiple diseases, including diabetes, Alzheimer's, amyotrophic lateral sclerosis (ALS) and Parkinson's. Platelet-derived growth factor receptors (PDGFRs) have been shown to be critical to PC function and survival. However, how PDGFR-mediated PC activity affects vascular homeostasis is not fully understood. Here, we tested the hypothesis that imatinib, a chemotherapeutic agent and a potent PDGFR inhibitor, alters PC distribution and thus induces vascular atrophy. We performed a morphometric analysis of the vascular elements in sham control and imatinib-treated NG2-DsRed mice. Vascular morphology and the integrity of the blood-retina barrier (BRB) were evaluated using blood albumin labeling. We found that imatinib decreased the number of PCs and blood vessel (BV) coverage in all retinal vascular layers; this was accompanied by a shrinkage of BV diameters. Surprisingly, the total length of capillaries was not altered, suggesting a preferential effect of imatinib on PCs. Furthermore, blood-retina barrier disruption was not evident. In conclusion, our data suggest that imatinib could help in treating neurovascular diseases and serve as a model for PC loss, without BRB disruption.
Asunto(s)
Barrera Hematorretinal/efectos de los fármacos , Mesilato de Imatinib/farmacología , Pericitos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Animales , Barrera Hematorretinal/citología , Ratones , Ratones Endogámicos C57BL , Pericitos/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
Adequate blood flow is essential to brain function, and its disruption is an early indicator in diseases, such as stroke and diabetes. However, the mechanisms contributing to this impairment remain unclear. To address this gap, in the diabetic and nondiabetic male mouse retina, we combined an unbiased longitudinal assessment of vasomotor activity along a genetically defined vascular network with pharmacological and immunohistochemical analyses of pericytes, the capillary vasomotor elements. In nondiabetic retina, focal stimulation of a pericyte produced a robust vasomotor response, which propagated along the blood vessel with increasing stimulus. In contrast, the magnitude, dynamic range, a measure of fine vascular diameter control, and propagation of vasomotor response were diminished in diabetic retinas from streptozotocin-treated mice. These functional changes were linked to several mechanisms. We found that density of pericytes and their sensitivity to stimulation were reduced in diabetes. The impaired response propagation from the stimulation site was associated with lower expression of connexin43, a major known gap junction unit in vascular cells. Indeed, selective block of gap junctions significantly reduced propagation but not initiation of vasomotor response in the nondiabetic retina. Our data establish the mechanisms for fine local regulation of capillary diameter by pericytes and a role for gap junctions in vascular network interactions. We show how disruption of this balance contributes to impaired vasomotor control in diabetes.SIGNIFICANCE STATEMENT Identification of mechanisms governing capillary blood flow in the CNS and how they are altered in disease provides novel insight into early states of neurological dysfunction. Here, we present physiological and anatomical evidence that both intact pericyte function as well as gap junction-mediated signaling across the vascular network are essential for proper capillary diameter control and vasomotor function. Changes to capillary blood flow precede other anatomical and functional hallmarks of diabetes establishing a significant window for prevention and treatment.
Asunto(s)
Conexina 43/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Uniones Comunicantes/metabolismo , Pericitos/metabolismo , Vasos Retinianos/metabolismo , Animales , Velocidad del Flujo Sanguíneo/fisiología , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/patología , Uniones Comunicantes/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Vasos Retinianos/patologíaRESUMEN
In retinal degenerative disease (RD), the diminished light signal from dying photoreceptors has been considered the sole cause of visual impairment. Recent studies show a 10-fold increase in spontaneous activity in the RD network, challenging this paradigm. This aberrant activity forms a new barrier for the light signal, and not only exacerbates the loss of vision, but also may stand in the way of visual restoration. This activity originates in AII amacrine cells and relies on excessive activation of gap junctions. However, it remains unclear whether aberrant activity affects central visual processing and what mechanisms lead to this excessive activation of gap junctions. By combining genetic manipulation with electrophysiological recordings of light-induced activity in both living mice and isolated wholemount retina, we demonstrate that aberrant activity extends along retinotectal projections to alter activity in higher brain centers. Next, to selectively eliminate Cx36-containing gap junctions, which are the primary type expressed by AII amacrine cells, we crossed rd10 mice, a slow-degenerating model of RD, with Cx36 knockout mice. We found that retinal aberrant activity was reduced in the rd10/Cx36KO mice compared to rd10 controls, a direct evidence for involvement of Cx36-containing gap junctions in generating aberrant activity in RD. These data provide an essential support for future experiments to determine if selectively targeting these gap junctions could be a valid strategy for reducing aberrant activity and restoring light responses in RD.
Asunto(s)
Conexinas/genética , ADN/genética , Uniones Comunicantes/genética , Mutación , Degeneración Retiniana/genética , Células Fotorreceptoras Retinianas Bastones/metabolismo , Visión Ocular , Células Amacrinas/metabolismo , Células Amacrinas/patología , Animales , Conexinas/metabolismo , Análisis Mutacional de ADN , Femenino , Uniones Comunicantes/metabolismo , Genotipo , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Degeneración Retiniana/metabolismo , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/patología , Proteína delta-6 de Union ComunicanteRESUMEN
Retinal degeneration leads to progressive photoreceptor cell death, resulting in vision loss. Subsequently, inner retinal neurons develop aberrant synaptic activity, compounding visual impairment. In retinal ganglion cells, light responses driven by surviving photoreceptors are obscured by elevated levels of aberrant spiking activity. Here, we demonstrate in rd10 mice that targeting disruptive neuronal circuitry with a gap junction antagonist can significantly reduce excessive spiking. This treatment increases the sensitivity of the degenerated retina to light stimuli driven by residual photoreceptors. Additionally, this enhances signal transmission from inner retinal neurons to ganglion cells, potentially allowing the retinal network to preserve the fidelity of signals either from prosthetic electronic devices, or from cells optogenetically modified to transduce light. Thus, targeting maladaptive changes to the retina allows for treatments to use existing neuronal tissue to restore light sensitivity, and to augment existing strategies to replace lost photoreceptors.
Asunto(s)
Uniones Comunicantes/efectos de los fármacos , Fototransducción/efectos de los fármacos , Degeneración Retiniana/genética , Potenciales de Acción/efectos de los fármacos , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Potenciales Postsinápticos Excitadores , Uniones Comunicantes/fisiología , Técnicas In Vitro , Luz , Fototransducción/genética , Ácido Meclofenámico/farmacología , Ácido Meclofenámico/uso terapéutico , Ratones , Ratones Transgénicos , Células Fotorreceptoras/fisiología , Degeneración Retiniana/tratamiento farmacológico , Células Ganglionares de la Retina/fisiología , Visión Ocular/efectos de los fármacos , Visión Ocular/genéticaRESUMEN
We previously reported in adult mice that visuomotor experience during monocular deprivation (MD) augmented enhancement of visual-cortex-dependent behavior through the non-deprived eye (NDE) during deprivation, and enabled enhanced function to persist after MD. We investigated the physiological substrates of this experience-enabled form of adult cortical plasticity by measuring visual behavior and visually evoked potentials (VEPs) in binocular visual cortex of the same mice before, during, and after MD. MD on its own potentiated VEPs contralateral to the NDE during MD and shifted ocular dominance (OD) in favor of the NDE in both hemispheres. Whereas we expected visuomotor experience during MD to augment these effects, instead enhanced responses contralateral to the NDE, and the OD shift ipsilateral to the NDE were attenuated. However, in the same animals, we measured NMDA receptor-dependent VEP potentiation ipsilateral to the NDE during MD, which persisted after MD. The results indicate that visuomotor experience during adult MD leads to enduring enhancement of behavioral function, not simply by amplifying MD-induced changes in cortical OD, but through an independent process of increasing NDE drive in ipsilateral visual cortex. Because the plasticity is resident in the mature visual cortex and selectively effects gain of visual behavior through experiential means, it may have the therapeutic potential to target and non-invasively treat eye- or visual-field-specific cortical impairment.
Asunto(s)
Potenciales Evocados Visuales/fisiología , Plasticidad Neuronal/fisiología , Desempeño Psicomotor/fisiología , Visión Monocular/fisiología , Corteza Visual/fisiología , Factores de Edad , Animales , Predominio Ocular/fisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Lateralidad Funcional/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Piperazinas/farmacología , Privación Sensorial/fisiología , Umbral Sensorial/fisiología , Campos Visuales/fisiología , Vías Visuales/fisiologíaRESUMEN
Intermittent fasting (IF) is a diet with salutary effects on cognitive aging, Alzheimer's disease (AD), and stroke. IF restricts a number of nutrient components, including glucose. 2-deoxyglucose (2-DG), a glucose analog, can be used to mimic glucose restriction. 2-DG induced transcription of the pro-plasticity factor, Bdnf, in the brain without ketosis. Accordingly, 2-DG enhanced memory in an AD model (5xFAD) and functional recovery in an ischemic stroke model. 2-DG increased Bdnf transcription via reduced N-linked glycosylation, consequent ER stress, and activity of ATF4 at an enhancer of the Bdnf gene, as well as other regulatory regions of plasticity/regeneration (e.g., Creb5, Cdc42bpa, Ppp3cc, and Atf3) genes. These findings demonstrate an unrecognized role for N-linked glycosylation as an adaptive sensor to reduced glucose availability. They further demonstrate that ER stress induced by 2-DG can, in the absence of ketosis, lead to the transcription of genes involved in plasticity and cognitive resilience as well as proteostasis.
Asunto(s)
Enfermedad de Alzheimer , Cetosis , Accidente Cerebrovascular , Humanos , Desoxiglucosa/farmacología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Glucosa/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismoRESUMEN
Anterior Uveitis (AU) is the inflammation of the anterior part of the eye, the iris and ciliary body and is strongly associated with HLA-B*27. We report AU exome sequencing results from eight independent cohorts consisting of 3,850 cases and 916,549 controls. We identify common genome-wide significant loci in HLA-B (OR = 3.37, p = 1.03e-196) and ERAP1 (OR = 0.86, p = 1.1e-08), and find IPMK (OR = 9.4, p = 4.42e-09) and IDO2 (OR = 3.61, p = 6.16e-08) as genome-wide significant genes based on the burden of rare coding variants. Dividing the cohort into HLA-B*27 positive and negative individuals, we find ERAP1 haplotype is strongly protective only for B*27-positive AU (OR = 0.73, p = 5.2e-10). Investigation of B*27-negative AU identifies a common signal near HLA-DPB1 (rs3117230, OR = 1.26, p = 2.7e-08), risk genes IPMK and IDO2, and several additional candidate risk genes, including ADGFR5, STXBP2, and ACHE. Taken together, we decipher the genetics underlying B*27-positive and -negative AU and identify rare and common genetic signals for both subtypes of disease.
Asunto(s)
Uveítis Anterior , Humanos , Uveítis Anterior/genética , Inflamación/genética , Haplotipos , Genes MHC Clase I , Antígenos HLA-B/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Aminopeptidasas/genética , Antígenos de Histocompatibilidad MenorRESUMEN
The visual system is highly sensitive to dynamic features in the visual scene. However, it is not known how or where this enhanced sensitivity first occurs. We investigated this phenomenon by studying interactions between excitatory and inhibitory synapses in the second synaptic layer of the mouse retina. We found that these interactions showed activity-dependent changes that enhanced signaling of dynamic stimuli. Excitatory signaling from cone bipolar cells to ganglion cells exhibited strong synaptic depression, attributable to reduced glutamate release from bipolar cells. This depression was relieved by amacrine cell inhibitory feedback that activated presynaptic GABA(C) receptors. We found that the balance between excitation and feedback inhibition depended on stimulus frequency; at short interstimulus intervals, excitation was enhanced, attributable to reduced inhibitory feedback. This dynamic interplay may enrich visual processing by enhancing retinal responses to closely spaced temporal events, representing rapid changes in the visual environment.
Asunto(s)
Inhibición Neural/fisiología , Dinámicas no Lineales , Retina/citología , Sinapsis/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Benzodiazepinas/farmacología , Estimulación Eléctrica/métodos , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Técnicas In Vitro , Ratones , Inhibición Neural/efectos de los fármacos , Técnicas de Placa-Clamp , Ácidos Fosfínicos/farmacología , Estimulación Luminosa/métodos , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , Piridinas/farmacología , Células Bipolares de la Retina/efectos de los fármacos , Células Bipolares de la Retina/fisiología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/fisiología , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Endothelial cells (ECs) are key players in the development and maintenance of the vascular tree, the establishment of the blood-brain barrier and control of blood flow. Disruption in ECs is an early and active component of vascular pathogenesis. However, our ability to selectively target ECs in the CNS for identification and manipulation is limited. Here, in the mouse retina, a tractable model of the CNS, we utilized a recently developed AAV-BR1 system to identify distinct classes of ECs along the vascular tree using a GFP reporter. We then developed an inducible EC-specific ectopic Connexin 43 (Cx43) expression system using AAV-BR1-CAG-DIO-Cx43-P2A-DsRed2 in combination with a mouse line carrying inducible CreERT2 in ECs. We targeted Cx43 because its loss has been implicated in microvascular impairment in numerous diseases such as diabetic retinopathy and vascular edema. GFP-labeled ECs were numerous, evenly distributed along the vascular tree and their morphology was polarized with respect to the direction of blood flow. After tamoxifen induction, ectopic Cx43 was specifically expressed in ECs. Similarly to endogenous Cx43, ectopic Cx43 was localized at the membrane contacts of ECs and it did not affect tight junction proteins. The ability to enhance gap junctions in ECs provides a precise and potentially powerful tool to treat microcirculation deficits, an early pathology in numerous diseases.
Asunto(s)
Conexina 43 , Retinopatía Diabética , Animales , Conexina 43/genética , Conexina 43/metabolismo , Células Endoteliales , Uniones Comunicantes/metabolismo , Ratones , RetinaRESUMEN
Abnormalities in brain glucose metabolism and accumulation of abnormal protein deposits called plaques and tangles are neuropathological hallmarks of Alzheimer's disease (AD), but their relationship to disease pathogenesis and to each other remains unclear. Here we show that succinylation, a metabolism-associated post-translational protein modification (PTM), provides a potential link between abnormal metabolism and AD pathology. We quantified the lysine succinylomes and proteomes from brains of individuals with AD, and healthy controls. In AD, succinylation of multiple mitochondrial proteins declined, and succinylation of small number of cytosolic proteins increased. The largest increases occurred at critical sites of amyloid precursor protein (APP) and microtubule-associated tau. We show that in vitro, succinylation of APP disrupted its normal proteolytic processing thereby promoting Aß accumulation and plaque formation and that succinylation of tau promoted its aggregation to tangles and impaired microtubule assembly. In transgenic mouse models of AD, elevated succinylation associated with soluble and insoluble APP derivatives and tau. These findings indicate that a metabolism-linked PTM may be associated with AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Placa Amiloide/metabolismo , Procesamiento Proteico-Postraduccional , Ácido Succínico/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Animales , Autopsia , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Placa Amiloide/genética , Placa Amiloide/patología , Agregado de Proteínas , Proteolisis , Proteoma/genética , Proteoma/metabolismo , Proteínas tau/genéticaRESUMEN
Pericytes are a unique class of mural cells essential for angiogenesis, maintenance of the vasculature and are key players in microvascular pathology. However, their diversity and specific roles are poorly understood, limiting our insight into vascular physiology and the ability to develop effective therapies. Here, in the mouse retina, a tractable model of the CNS, we evaluated distinct classes of mural cells along the vascular tree for both structural characterization and physiological manipulation of blood flow. To accomplish this, we first tested three inducible mural cell-specific mouse lines using a sensitive Ai14 reporter and tamoxifen application either by a systemic injection, or by local administration in the form of eye drops. The specificity and pattern of cre activation varied significantly across the three lines, under either the PDGFRß or NG2 promoter (Pdgfrß-CreRha, Pdgfrß-CreCsln, and Cspg4-Cre). In particular, a mouse line with Cre under the NG2 promoter resulted in sparse TdTomato labeling of mural cells, allowing for an unambiguous characterization of anatomical features of individual sphincter cells and capillary pericytes. Furthermore, in one PDGFRß line, we found that focal eye drop application of tamoxifen led to an exclusive Cre-activation in pericytes, without affecting arterial mural cells. We then used this approach to boost capillary blood flow by selective expression of Halorhodopsin, a highly precise hyperpolarizing optogenetic actuator. The ability to exclusively target capillary pericytes may prove a precise and potentially powerful tool to treat microcirculation deficits, a common pathology in numerous diseases.
Asunto(s)
Velocidad del Flujo Sanguíneo/fisiología , Capilares/fisiología , Pericitos/fisiología , Flujo Sanguíneo Regional/fisiología , Retina/fisiología , Administración Oftálmica , Animales , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Capilares/química , Capilares/efectos de los fármacos , Ratones , Ratones Transgénicos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Pericitos/química , Pericitos/efectos de los fármacos , Flujo Sanguíneo Regional/efectos de los fármacos , Retina/química , Retina/citología , Retina/efectos de los fármacos , Tamoxifeno/administración & dosificaciónRESUMEN
Sensory information is thought to be modulated by presynaptic inhibition. Although this form of inhibition is a well-studied phenomenon, it is still unclear what role it plays in shaping sensory signals in intact circuits. By visually stimulating the retinas of transgenic mice lacking GABAc receptor-mediated presynaptic inhibition, we found that this inhibition regulated the dynamic range of ganglion cell (GC) output to the brain. Presynaptic inhibition acted differentially upon two major retinal pathways; its elimination affected GC responses to increments, but not decrements, in light intensity across the visual scene. The GC dynamic response ranges were different because presynaptic inhibition limited glutamate release from ON, but not OFF, bipolar cells, which modulate the extent of glutamate spillover and activation of perisynaptic NMDA receptors at ON GCs. Our results establish a role for presynaptic inhibitory control of spillover in determining sensory output in the CNS.
Asunto(s)
Potenciales de Acción/fisiología , Inhibición Neural/fisiología , Neuronas Aferentes/fisiología , Terminales Presinápticos/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Antagonistas del GABA/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Técnicas de Cultivo de Órganos , Estimulación Luminosa/métodos , Terminales Presinápticos/efectos de los fármacos , Receptores de GABA/fisiologíaRESUMEN
Axon terminals from the two eyes initially overlap in the dorsal-lateral geniculate nucleus (dLGN) but subsequently refine to occupy nonoverlapping territories. Retinal activity is required to establish and maintain this segregation. We show that despite the presence of retinal activity, segregated projections desegregate when the structure of activity is altered. Early in development, spontaneous retinal activity in the no b-wave (nob) mouse is indistinguishable from that of wild-type mice, and eye-specific segregation proceeds normally. But, around eye-opening, spontaneous and visually evoked activity in nob retinas become abnormal, coincident with a failure to preserve precise eye-specific territories. Dark-rearing studies suggest that altered visual experience is not responsible. Transgenic rescue of the mutated protein (nyctalopin) within nob retinal interneurons, without rescuing expression in either retinal projection neurons or their postsynaptic targets in the dLGN, restores spontaneous retinal activity patterns and prevents desegregation. Thus, normally structured spontaneous retinal activity stabilizes newly refined retinogeniculate circuitry.
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Tipificación del Cuerpo/fisiología , Mapeo Encefálico , Cuerpos Geniculados/crecimiento & desarrollo , Retina/crecimiento & desarrollo , Vías Visuales/crecimiento & desarrollo , Animales , Procesamiento de Imagen Asistido por Computador , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación , Proteoglicanos/genética , Células Ganglionares de la Retina/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Functional hyperemia, or the matching of blood flow with activity, directs oxygen and nutrients to regionally firing neurons. The mechanisms responsible for this spatial accuracy remain unclear but are critical for brain function and establish the diagnostic resolution of BOLD-fMRI. Here, we described a mosaic of pericytes, the vasomotor capillary cells in the living retina. We then tested whether this net of pericytes and surrounding neuroglia predicted a connectivity map in response to sensory stimuli. Surprisingly, we found that these connections were not only selective across cell types, but also highly asymmetric spatially. First, pericytes connected predominantly to other neighboring pericytes and endothelial cells, and less to arteriolar smooth muscle cells, and not to surrounding neurons or glia. Second, focal, but not global stimulation evoked a directional vasomotor response by strengthening connections along the feeding vascular branch. This activity required local NO signaling and occurred by means of direct coupling via gap junctions. By contrast, bath application of NO or diabetes, a common microvascular pathology, not only weakened the vascular signaling but also abolished its directionality. We conclude that the exclusivity of neurovascular interactions may thus establish spatial accuracy of blood delivery with the precision of the neuronal receptive field size, and is disrupted early in diabetes.
RESUMEN
Purpose: Disruption in blood supply to active retinal circuits is the earliest hallmark of diabetic retinopathy (DR) and has been primarily attributed to vascular deficiency. However, accumulating evidence supports an early role for a disrupted neuronal function in blood flow impairment. Here, we tested the hypothesis that selectively stimulating cholinergic neurons could restore neurovascular signaling to preserve the capillary circulation in DR. Methods: We used wild type (wt) and choline acetyltransferase promoter (ChAT)-channelrhodopsin-2 (ChR2) mice expressing ChR2 exclusively in cholinergic cells. Mice were made diabetic by streptozotocin (STZ) injections. Two to 3 months after the last STZ injection, the rate of capillary blood flow was measured in vivo within each retinal vascular layer using high speed two-photon imaging. Measurements were done at baseline and following ChR2-driven activation of retinal cholinergic interneurons, the sole source of the vasodilating neurotransmitter acetylcholine. After recordings, retinas were collected and assessed for physiological and structural features. Results: In retinal explants from ChAT-ChR2 mice, we found that channelrhodopsin2 was selectively expressed in all cholinergic amacrine cells. Its direct activation by blue light led to dilation of adjacent retinal capillaries. In living diabetic ChAT-ChR2 animals, basal capillary blood flow was significantly higher than in diabetic mice without channelrhodopsin. However, optogenetic stimulation with blue light did not result in flickering light-induced functional hyperemia, suggesting a necessity for a concerted neurovascular interaction. Conclusions: These findings provide direct support to the utility and efficacy of an optogenetic approach for targeting selective retinal circuits to treat DR and its complications.