Adult aberrant astrocytes submitted to late passage cultivation lost differentiation markers and decreased their pro-inflammatory profile.

In amyotrophic lateral sclerosis (ALS), astrocytes are considered key players in some non-cell non-neuronal autonomous mechanisms that underlie motor neuron death. However, it is unknown how much of these deleterious features were permanently acquired. To assess this point, we evaluated if the most remarkable features of neurotoxic aberrant glial phenotypes (AbAs) isolated from paralytic rats of the ALS model G93A Cu/Zn superoxide dismutase 1 (SOD1) could remain upon long lasting cultivation. Real time PCR, immunolabelling and zymography analysis showed that upon many passages, AbAs preserved the cell proliferation capacity, mitochondrial function and response to different compounds that inhibit some key astrocyte functions but decreased the expression of parameters associated to cell lineage, homeostasis and inflammation. As these results are contrary to the sustained inflammatory status observed along disease progression in SOD1G93A rats, we propose that the most AbAs remarkable features related to homeostasis and neurotoxicity were not permanently acquired and might depend on the signaling coming from the injuring microenvironment present in the degenerating spinal cord of terminal rats.

Morphological evidence of the protective effects of a synthetic chalcone against the striatal myelin damage induced by glutaric acid.

Ultrastructural features of striatal white matter and cells in an in vivo model of glutaric acidemia type I created by intracerebral injection of glutaric acid (GA) were analyzed by transmission electron microscopy and immunohistochemistry. To test if the white matter damage observed in this model could be prevented, we administered the synthetic chemopreventive molecule CH38 ((E)-3-(4-methylthiophenyl)-1-phenyl-2-propen-1-one) to newborn rats, previous to an intracerebroventricular injection of GA. The study was done when striatal myelination was incipient and when it was already established (at 12 and 45 days post-injection [DPI], respectively). Results obtained indicate that that the ultrastructure of astrocytes and neurons did not appear significantly affected by the GA bolus. Instead, in oligodendrocytes, the most prominent GA-dependent injury defects included endoplasmic reticulum (ER) stress and nuclear envelope swelling at 12 DPI. Altered and reduced immunoreactivities against heavy neurofilament (NF), proteolipid protein (PLP), and myelin-associated glycoprotein (MAG) together with axonal bundle fragmentation and decreased myelin were also found at both ages analyzed. CH38 by itself did not affect striatal cells or axonal packages. However, the group of rats that received CH38 before GA did not show evidence neither of ER stress nor nuclear envelope dilation in oligodendrocytes, and axonal bundles appeared less fragmented. In this group, labeling of NF and PLP was similar to the controls. These results suggest that the CH38 molecule is a candidate drug to prevent or decrease the neural damage elicited by a pathological increase of GA in the brain. Optimization of the treatments and identification of the mechanisms underlying CH38 protective effects will open new therapeutic windows to protect myelin, which is a vulnerable target of numerous nervous system diseases.

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.

Gestational and Lactational Iron Deficiency Anemia Impairs Myelination and the Neurovascular Unit in Infant Rats.

Iron deficiency anemia is a prevalent health problem among pregnant women and infants, particularly in the developing countries that causes brain development deficits and poor cognitive outcomes. Since tissue iron depletion may impair myelination and trigger cellular hypoxic signaling affecting blood vessels, we studied myelination and the neurovascular unit (NVU) in infant rats born to mothers fed with an iron deficient (ID) or control diet from embryonic day 5 till weaning. Blood samples and brains of rat pups at postnatal day (PND) 14 and 30 were analyzed. PND 14 ID rats had severe microcytic hypochromic anemia that was almost reversed at PND 30 although hypomyelination and astrocyte immature phenotype in the corpus callosum were significant at that age. In CA1 hippocampal region, PND 14 and PND 30 ID rats showed significant reduced expression of the receptor ß of the platelet-derived growth factor localized in pericytes and associated to aquaporin 4 (AQP4) immunopositive capillaries. Shorter AQP4 + capillaries and reduced AQP4 expression were also evidenced in PND 14 and PND 30 ID rats. In addition, pericyte membrane permeability through large-pore channels was transiently increased in ID rats at PND 14 but not at PND 30, while the blood-brain barrier permeability was not affected. Remarkably, transient increased pericyte permeability found in PND 14 ID rats was not directly related to iron depletion, suggesting the involvement of other iron deficiency anemia-induced mechanisms. In summary, severe ID during gestation and lactation produces persistent hypomyelination and significantly affects hippocampal pericytes and astrocytes in the NVU which may trigger impaired neurovascular function.

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.

Pericyte Mapping in Cerebral Slices with the Far-red Fluorophore TO-PRO-3.

This protocol describes a method for high-resolution confocal imaging of pericytes with the far-red fluorophore TO-PROTM-3 Iodide 642/661 in cerebral slices of murine. Identification of pericytes with TO-PRO-3 is a short time-consuming, high cost-effective and robust technique to label pericytes with no need for immunostaining or generation of reporter mice. Since the TO-PRO-3 stain resists immunofluorescence, and lacks spectral overlap, the probe is well suited for multiple labelling. Our procedures also combine TO-PRO-3-staining of pericytes with fluorescent markers for astrocytes and vessels in brain slices. These approaches should enable the assessment of pericyte biology in gliovascular unit.

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 (PDGFrß) 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.

Glutaric Acid Affects Pericyte Contractility and Migration: Possible Implications for GA-I Pathogenesis.

Glutaric acidemia I (GA-I) is an inherited neurometabolic childhood disease characterized by bilateral striatal neurodegeneration upon brain accumulation of millimolar concentrations of glutaric acid (GA) and related metabolites. Vascular dysfunction, including abnormal cerebral blood flow and blood-brain barrier damage, is an early pathological feature in GA-I, although the affected cellular targets and underlying mechanisms remain unknown. In the present study, we have assessed the effects of GA on capillary pericyte contractility in cerebral cortical slices and pericyte cultures, as well as on the survival, proliferation, and migration of cultured pericytes. GA induced a significant reduction in capillary diameter at distances up to ~ 10 µm from the center of pericyte somata. However, GA did not affect the contractility of cultured pericytes, suggesting that the response elicited in slices may involve GA evoking pericyte contraction by acting on other cellular components of the neurovascular unit. Moreover, GA indirectly inhibited migration of cultured pericytes, an effect that was dependent on soluble glial factors since it was observed upon application of conditioned media from GA-treated astrocytes (CM-GA), but not upon direct GA addition to the medium. Remarkably, CM-GA showed increased expression of cytokines and growth factors that might mediate the effects of increased GA levels not only on pericyte migration but also on vascular permeability and angiogenesis. These data suggest that some effects elicited by GA might be produced by altering astrocyte-pericyte communication, rather than directly acting on pericytes. Importantly, GA-evoked alteration of capillary pericyte contractility may account for the reduced cerebral blood flow observed in GA-I patients.

Long Lasting High Lysine Diet Aggravates White Matter Injury in Glutaryl-CoA Dehydrogenase Deficient (Gcdh-/-) Mice.

Glutaric acidemia type I (GA-I) is a neurometabolic disease caused by deficient activity of glutaryl-CoA dehydrogenase (GCDH) that results in accumulation of metabolites derived from lysine (Lys), hydroxylysine, and tryptophan catabolism. GA-I patients typically develop encephalopatic crises with striatal degeneration and progressive white matter defects. However, late onset patients as well as Gcdh-/- mice only suffer diffuse myelinopathy, suggesting that neuronal death and white matter defects are different pathophysiological events. To test this hypothesis, striatal myelin was studied in Gcdh-/- mice fed from 30 days of age during up to 60 days with a diet containing normal or moderately increased amounts of Lys (2.8%), which ensure sustained elevated levels of GA-I metabolites. Gcdh-/- mice fed with 2.8% Lys diet showed a significant decrease in striatal-myelinated areas and progressive vacuolation of white matter tracts, as compared with animals fed with normal diet. Myelin pathology increased with the time of exposure to high Lys diet and was also detected in 90-day old Gcdh-/- mice fed with normal diet, suggesting that dietary Lys accelerated the undergoing white matter damage. Gcdh-/- mice fed with 2.8% Lys diet also showed increased GRP78/BiP immunoreactivity in oligodendrocytes and neurons, denoting ER stress. However, the striatal and cortical neuronal density was unchanged with respect to normal diet. Thus, myelin damage seen in Gcdh-/- mice fed with 2.8% Lys seems to be mediated by a long-term increased levels of GA-I metabolites having deleterious effects in myelinating oligodendrocytes over neurons.

Ultrastructural features of aberrant glial cells isolated from the spinal cord of paralytic rats expressing the amyotrophic lateral sclerosis-linked SOD1G93A mutation.

In the rat model of amyotrophic lateral sclerosis expressing the G93A superoxide dismutase-1 mutation, motor neuron death and rapid paralysis progression are associated with the emergence of a population of aberrant glial cells (AbAs) that proliferate in the degenerating spinal cord. Targeting of AbAs with anti-neoplasic drugs reduced paralysis progression, suggesting a pathogenic potential contribution of these cells accelerating paralysis progression. In the present study, analyze the cellular and ultrastructural features of AbAs following their isolation and establishment in culture during several passages. We found that AbAs exhibit permanent loss of contact inhibition, absence of intermediate filaments and abundance of microtubules, together with an important production of extracellular matrix components. Remarkably, AbAs also exhibited exacerbated ER stress together with a significant abundance of lipid droplets, as well as autophagic and secretory vesicles, all characteristic features of cellular stress and inflammatory activation. Taken together, the present data show AbA cells as a unique aberrant phenotype for a glial cell that might explain their pathogenic and neurotoxic effects.

Astrocyte Dysfunction in Developmental Neurometabolic Diseases.

Astrocytes 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.

Striatal neuronal death mediated by astrocytes from the Gcdh-/- mouse model of glutaric acidemia type I.

Glutaric acidemia type I (GA-I) is an inherited neurometabolic childhood disorder caused by defective activity of glutaryl CoA dehydrogenase (GCDH) which disturb lysine (Lys) and tryptophan catabolism leading to neurotoxic accumulation of glutaric acid (GA) and related metabolites. However, it remains unknown whether GA toxicity is due to direct effects on vulnerable neurons or mediated by GA-intoxicated astrocytes that fail to support neuron function and survival. As damaged astrocytes can also contribute to sustain high GA levels, we explored the ability of Gcdh-/- mouse astrocytes to produce GA and induce neuronal death when challenged with Lys. Upon Lys treatment, Gcdh-/- astrocytes synthetized and released GA and 3-hydroxyglutaric acid (3HGA). Lys and GA treatments also increased oxidative stress and proliferation in Gcdh-/- astrocytes, both prevented by antioxidants. Pretreatment with Lys also caused Gcdh-/- astrocytes to induce extensive death of striatal and cortical neurons when compared with milder effect in WT astrocytes. Antioxidants abrogated the neuronal death induced by astrocytes exposed to Lys or GA. In contrast, Lys or GA direct exposure on Gcdh-/- or WT striatal neurons cultured in the absence of astrocytes was not toxic, indicating that neuronal death is mediated by astrocytes. In summary, GCDH-defective astrocytes actively contribute to produce and accumulate GA and 3HGA when Lys catabolism is stressed. In turn, astrocytic GA production induces a neurotoxic phenotype that kills striatal and cortical neurons by an oxidative stress-dependent mechanism. Targeting astrocytes in GA-I may prompt the development of new antioxidant-based therapeutical approaches.

Increased blood-brain barrier permeability and alterations in perivascular astrocytes and pericytes induced by intracisternal glutaric acid.

BACKGROUND: Glutaric acid (GA) is a dicarboxylic acid that accumulates in millimolar concentrations in glutaric acidemia I (GA-I), an inherited neurometabolic childhood disease characterized by extensive neurodegeneration. Vascular dysfunction is a common and early pathological feature in GA-I, although the underlying mechanisms remain unknown. In the present study, we have used a previously-validated rat model of GA-I to determine the effect of GA on the blood- brain barrier (BBB) and the neurovascular unit. METHODS: Newborn rat pups received a single injection of GA (1 µmol/g) or vehicle into the cisterna magna. BBB permeability was analyzed at 14 and 30 days post injection (DPI) by assessing Evans blue (EB) and immunoglobulin G (IgG) extravasation. Blood vessels and microglia were labeled with tomato lectin. Characterization of EB positive cells was made by double labeling with antibodies to astrocyte and neuronal markers. Immunohistochemistry against aquaporin 4 (AQP4), ß receptor of the platelet derived growth factor (PDGFRß) and laminin was used to recognize astrocyte endfeet, pericytes and basal lamina. Zonula occludens 1 (ZO-1) and occludin striatal expression was assessed by Western blotting. RESULTS: Perinatal intracisternal GA administration caused an increased extravasation of free EB, but not of IgG, into the striatal parenchyma at 14 and 30 DPI. EB extravasated through the BBB was internalized exclusively into neurons. GA-injected animals did not show significant changes in the area of small blood vessels in the striatum, but at 30 DPI there was a significant decrease in AQP4, PDGFRß and laminin positive areas associated with small blood vessels. Occludin and ZO-1 expression in the striatal tissue was unchanged in all conditions analyzed. CONCLUSIONS: The present study shows a previously-unknown effect of a perinatal administration of a single intracisternal GA injection on BBB permeability and on key components of the neurovascular unit. The results suggest BBB leakage is a pathogenic mechanism and a potential therapeutic target for patients with GA-I.

White matter injury induced by perinatal exposure to glutaric acid.

Glutaric acid (GA) is a neurotoxic metabolite that accumulates in the CNS of patients with glutaric acidemia-I (GA-I), a neurometabolic disease caused by deficient activity of glutaryl-CoA dehydrogenase. Most GA-I patients display characteristic CNS lesions, mainly in the gray and white matter of basal ganglia and cerebral cortex. Neurons and astrocytes are believed to be vulnerable to millimolar concentrations of GA. However, little is known about the effects of GA on oligodendrocytes (OL) and the myelination process in the postnatal brain. Here, we show that a single intracerebroventricular administration of GA to rat neonatal pups induced a selective and long-lasting myelination failure in the striatum but no deleterious effect in the myelination of the corpus callosum. At 45 days post-GA injection, the myelinated area of striatal axonal bundles was decreased by 35 %, and the expression of myelin basic protein and myelin-associated glycoprotein (MAG) reduced by 25 and 60 %, respectively. This was accompanied by long lasting cytopathology features in MAG and CC-1-expressing OLs, which was confirmed by transmission electron microscopy. Remarkably, GA did not induce acute loss of pre-OLs in the striatum as assessed by NG2 or PDGFRα immunohistochemistry, suggesting an indirect and progressive mechanism for OL damage. In accordance, GA-induced white matter injury was restricted to the striatum and associated to GA-induced astrocytosis and neuronal loss. In conclusion, the current evidence indicates a pathogenic mechanism by which GA can permanently affect myelin status.

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 SOD1 (G93A) mutation. We previously reported that the rapid progression of paralysis in ALS rats is associated with the appearance of proliferative 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 SOD1 (G93A) 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 S100ß expression increased. The phenotypic transition to AbA cells was stimulated by forskolin. These findings provide evidence for a subpopulation of proliferating microglial cells in SOD1 (G93A) 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.