Alteraciones de la barrera hematoencefálica y de la sustancia blanca en un modelo experimental de Acidemia Glutárica tipo I

Tribunal: Dra. Mónica Brauer, Dra. Giselle Prunell y Dra. Alejandra Kun

A nuclear fluorescent dye identifies pericytes at the neurovascular unit

Perivascular pericytes are key regulators of the blood–brain barrier, vascular development, and cerebral blood flow. Deciphering pericyte roles in health and disease requires cellular tracking; yet, pericyte identification remains challenging. A previous study reported that the far-red fluorophore TO-PRO-3 (642/661), usually employed as a nuclear dye in fixed tissue, was selectively captured by live pericytes from the subventricular zone. Herein, we validated TO-PRO-3 as a specific pericyte tracer in the nervous system (NS). Living pericytes from ex vivo murine hippocampus, cortex, spinal cord, and retina robustly incorporated TO-PRO-3. Classical pericyte immunomarkers such as chondroitin sulphate proteoglycan neuron-glial antigen 2 (NG2) and platelet-derived growth factor receptor beta antigen (PDGFrβ) and the new pericyte dye NeuroTrace 500/525 confirmed cellular specificity of dye uptake. The TO-PRO-3 signal enabled quantification of pericytes density and morphometry; likewise, TO-PRO-3 labeling allowed visualization of pericytes associated with other components of the neurovascular unit. A subset of TO-PRO-3 stained cells expressed the contractile protein α–SMA, indicative of their ability to control the capillary diameter. Uptake of TO-PRO-3 was independent of connexin/pannexin channels but was highly sensitive to temperature and showed saturation, suggesting that a yet unidentified protein-mediated active transport sustained dye incorporation. We conclude that TO-PRO-3 labeling provides a reliable and simple tool for the bioimaging of pericytes in the murine NS microvasculature.Comisión Sectorial de Investigación Científica. Proyecto de Investigación y Desarrollo CSIC I+D 2014.Agencia Nacional de Investigación e Innovación FCE_1_2017_1_13610

Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS

Microglia and reactive astrocytes accumulate in the spinal cord of rats expressing the Amyotrophic lateral sclerosis (ALS)-linked SOD1ᴳ⁹³ᴬ mutation. We previously reported that the rapid progression of paralysis in ALS rats is associated with the appearance of prolifer- ative astrocyte-like cells that surround motor neurons. These cells, designated as Aberrant Astrocytes (AbA cells) because of their atypical astrocytic phenotype, exhibit high toxicity to motor neurons. However, the cellular origin of AbA cells remains unknown. Because AbA cells are labeled with the proliferation marker Ki67, we analyzed the phenotypic makers of proliferating glial cells that surround motor neurons by immunohistochemistry. The number of Ki67⁺AbA cells sharply increased in symptomatic rats, displaying large cell bodies with processes embracing motor neurons. Most were co-labeled with astrocytic marker GFAP concurrently with the microglial markers Iba1 and CD163. Cultures of spinal cord prepared from symptomatic SOD1ᴳ⁹³ᴬ rats yielded large numbers of microglia expressing Iba1, CD11b, and CD68. Cells sorted for CD11b expression by flow cytometry transformed into AbA cells within two weeks. During these two weeks, the expression of microglial markers largely disappeared, while GFAP and S100β expression increased. The phenotypic transition to AbA cells was stimulated by forskolin. These findings provide evidence for a subpopulation of proliferating microglial cells in SOD1ᴳ⁹³ᴬ rats that undergo a phenotypic transition into AbA cells after onset of paralysis that may promote the fulminant disease progression. These cells could be a therapeutic target for slowing paralysis progression in ALS

Neuroprotective effects of violacein in a model of inherited amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of motor neurons and muscle atrophy, with defective neuron-glia interplay and emergence of aberrant glial phenotypes having a role in disease pathology. Here, we have studied if the pigment violacein with several reported protective/antiproliferative properties may control highly neurotoxic astrocytes (AbAs) obtained from spinal cord cultures of symptomatic hSOD1G93A rats, and if it could be neuroprotective in this ALS experimental model. At concentrations lower than those reported as protective, violacein selectively killed aberrant astrocytes. Treatment of hSOD1G93A rats with doses equivalent to the concentrations that killed AbAs caused a marginally significant delay in survival, partially preserved the body weight and soleus muscle mass and improved the integrity of the neuromuscular junction. Reduced motor neuron death and glial reactivity was also found and likely related to decreased inflammation and matrix metalloproteinase-2 and -9. Thus, in spite that new experimental designs aimed at extending the lifespan of hSOD1G93A rats are needed, improvements observed upon violacein treatment suggest a significant therapeutic potential that deserves further studies.IIBCE (MEC), PEDECIBA y ANII

The application of artificial gravity in medicine and space

Gravity plays a crucial role in physiology. The lack of gravity, like in long duration spaceflight missions, cause pathologies in e.g., the musculoskeletal system, cardiovascular deconditioning, immune system deprivation or brain abnormalities, to just mention a few. The application of artificial gravity through short-arm human centrifugation (SAHC) has been studied as a possible countermeasure to treat spaceflight deconditioning. However, hypergravity protocols applied by using SAHC have also been used to treat different, ground-based pathologies. Such gravitational therapies have been applied in Uruguay for more than four decades now. The aim of this overview is to summarize the most important findings about the effects of gravitational therapy in different, mainly vascular based pathologies according to the experience in the Gravitational Therapy Center and to discuss the current research in the field of hypergravity applications in medicine but also as multisystem countermeasure for near weightlessness pathologies. New insight is needed on the use of hypergravity in medicine and space research and application

Astrocyte Dysfunction in Developmental Neurometabolic Diseases.

International audienceAstrocytes play crucial roles in maintaining brain homeostasis and in orchestrating neural development, all through tightly coordinated steps that cooperate to maintain the balance needed for normal development. Here, we review the alterations in astrocyte functions that contribute to a variety of developmental neurometabolic disorders and provide additional data on the predominant role of astrocyte dysfunction in the neurometabolic neurodegenerative disease glutaric acidemia type I. Finally, we describe some of the therapeutical approaches directed to neurometabolic diseases and discuss if astrocytes can be possible therapeutic targets for treating these disorders

Informe final del proyecto: Canales de panexina 1 acoplan la interfaz neuro-vascular en pericitos cerebrales

En la interface neurovascular, los pericitos cerebrales contráctiles ajustan el diámetro capilar para acoplar el flujo sanguíneo local a las demandas metabólicas neuronales, fenómeno conocido como acoplamiento neurovascular: sus mecanismos moleculares y su regulación permanecen aún poco explorados. Reportes recientes implican a canales de membrana de gran poro formados por panexina1-Panx1 (panexones) en la regulación de las interacciones neuro-gliovasculares y del tono muscular y flujo sanguíneo de los grandes vasos. Los panexones proporcionan una vía para transferir iones/moléculas (ingreso de Ca2+; liberación de ATP) entre los medios intracelular y extracelular. Demostramos aquí que los pericitos pericapilares cerebrales expresan panexones funcionales capaces de mediar un intercambio molecular con el microentorno cerebral en ratones despiertos (in vivo), rodajas agudas (ex vivo) y cultivos primarios (in vitro). En condiciones control, dicho intercambio es mantenido por la secreción basal de ATP endógeno y receptores purinérgicos ionotrópicos P2X7(P2X7R) y metabotrópicos P2Y6(P2Y6R). El neurotransmisor glutamato, los receptores neuronales glutamatérgicos NMDA/AMPA, la actividad neuronal inducida por inhibición de receptores GABAA, la descarga epiléptica, y los vasodilatadores acetil-colina y adenosina, disminuyeron la interacción pericito-fluido cerebral por cierre de panexones pericitarios; la PGE2 inhibió panexones solamente en presencia de noradrenalina. La deprivación sensorial, los vasoconstrictores ATP, noradrenalina, endotelina-1 y angiotensina-II y los P2Y6R incrementaron dicha interacción por apertura de panexones. En pericitos cerebrales cultivados, la Panx1 amplificó el aumento del Ca2+ intracelular inducido por ATP exógeno. Nuestros resultados sugieren que los panexones pericitarios sensan y responden a la actividad neuronal y los niveles extracelulares de neurotransmisores y moléculas vasoactivas mediante cambios en su actividad; su estado funcional modula el Ca2+ intrapericitario y consecuentemente el tono contráctil de los pericitos. La panexina1 pericitaria, estratégicamente ubicada en la interfaz sangre/cerebro, surge como un nuevo mecanismo para regular el flujo sanguíneo microvascular y el intercambio a través de la barrera hemato-encefálica.Agencia Nacional de Investigación e Innovació