Silvia Olivera-Bravo

Phenotypically aberrant astrocytes that promote motoneuron damage in a model of inherited amyotrophic lateral sclerosis

Motoneuron loss and reactive astrocytosis are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a paralytic neurodegenerative disease that can be triggered by mutations in Cu-Zn superoxide dismutase (SOD1). Dysfunctional astrocytes contribute to ALS pathogenesis, inducing motoneuron damage and accelerating disease progression. However, it is unknown whether ALS progression is associated with the appearance of a specific astrocytic phenotype with neurotoxic potential. Here, we report the isolation of astrocytes with aberrant phenotype (referred as “AbA cells”) from primary spinal cord cultures of symptomatic rats expressing the SOD1G93A mutation. Isolation was based on AbA cells’ marked proliferative capacity and lack of replicative senescence, which allowed oligoclonal cell expansion for 1 y. AbA cells displayed astrocytic markers including glial fibrillary acidic protein, S100β protein, glutamine synthase, and connexin 43 but lacked glutamate transporter 1 and the glial progenitor marker NG2 glycoprotein. Notably, AbA cells secreted soluble factors that induced motoneuron death with a 10-fold higher potency than neonatal SOD1G93A astrocytes. AbA-like aberrant astrocytes expressing S100β and connexin 43 but lacking NG2 were identified in nearby motoneurons, and their number increased sharply after disease onset. Thus, AbA cells appear to be an as-yet unknown astrocyte population arising during ALS progression with unprecedented proliferative and neurotoxic capacity and may be potential cellular targets for slowing ALS progression.

Neonatal Astrocyte Damage Is Sufficient to Trigger Progressive Striatal Degeneration in a Rat Model of Glutaric Acidemia-I

Background We have investigated whether an acute metabolic damage to astrocytes during the neonatal period may critically disrupt subsequent brain development, leading to neurodevelopmental disorders. Astrocytes are vulnerable to glutaric acid (GA), a dicarboxylic acid that accumulates in millimolar concentrations in Glutaric Acidemia I (GA-I), an inherited neurometabolic childhood disease characterized by degeneration of striatal neurons. While GA induces astrocyte mitochondrial dysfunction, oxidative stress and subsequent increased proliferation, it is presently unknown whether such astrocytic dysfunction is sufficient to trigger striatal neuronal loss. Methodology/Principal Findings A single intracerebroventricular dose of GA was administered to rat pups at postnatal day 0 (P0) to induce an acute, transient rise of GA levels in the central nervous system (CNS). GA administration potently elicited proliferation of astrocytes expressing S100β followed by GFAP astrocytosis and nitrotyrosine staining lasting until P45. Remarkably, GA did not induce acute neuronal loss assessed by FluoroJade C and NeuN cell count. Instead, neuronal death appeared several days after GA treatment and progressively increased until P45, suggesting a delayed onset of striatal degeneration. The axonal bundles perforating the striatum were disorganized following GA administration. In cell cultures, GA did not affect survival of either striatal astrocytes or neurons, even at high concentrations. However, astrocytes activated by a short exposure to GA caused neuronal death through the production of soluble factors. Iron porphyrin antioxidants prevented GA-induced astrocyte proliferation and striatal degeneration in vivo, as well as astrocyte-mediated neuronal loss in vitro. Conclusions/Significance Taken together, these results indicate that a transient metabolic insult with GA induces long lasting phenotypic changes in astrocytes that cause them to promote striatal neuronal death. Pharmacological protection of astrocytes with antioxidants during encephalopatic crisis may prevent astrocyte dysfunction and the ineluctable progression of disease in children with GA-I.

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 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 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.

Disruption of brain redox homeostasis in glutaryl-CoA dehydrogenase deficient mice treated with high dietary lysine supplementation.

Deficiency of glutaryl-CoA dehydrogenase (GCDH) activity or glutaric aciduria type I (GA I) is an inherited neurometabolic disorder biochemically characterized by predominant accumulation of glutaric acid and 3-hydroxyglutaric acid in the brain and other tissues. Affected patients usually present acute striatum necrosis during encephalopathic crises triggered by metabolic stress situations, as well as chronic leukodystrophy and delayed myelination. Considering that the mechanisms underlying the brain injury in this disease are not yet fully established, in the present study we investigated important parameters of oxidative stress in the brain (cerebral cortex, striatum and hippocampus), liver and heart of 30-day-old GCDH deficient knockout (Gcdh(-/-)) and wild type (WT) mice submitted to a normal lysine (Lys) (0.9% Lys), or high Lys diets (2.8% or 4.7% Lys) for 60 h. It was observed that the dietary supplementation of 2.8% and 4.7% Lys elicited noticeable oxidative stress, as verified by an increase of malondialdehyde concentrations (lipid oxidative damage) and 2-7-dihydrodichlorofluorescein (DCFH) oxidation (free radical production), as well as a decrease of reduced glutathione levels and alteration of various antioxidant enzyme activities (antioxidant defenses) in the cerebral cortex and the striatum, but not in the hippocampus, the liver and the heart of Gcdh(-/-) mice, as compared to WT mice receiving the same diets. Furthermore, alterations of oxidative stress parameters in the cerebral cortex and striatum were more accentuated in symptomatic, as compared to asymptomatic Gcdh(-/-) mice exposed to 4.7% Lys overload. Histopathological studies performed in the cerebral cortex and striatum of these animals exposed to high dietary Lys revealed increased expression of oxidative stress markers despite the absence of significant structural damage. The results indicate that a disruption of redox homeostasis in the cerebral cortex and striatum of young Gcdh(-/-) mice exposed to increased Lys diet may possibly represent an important pathomechanism of brain injury in GA I patients under metabolic stress.

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

BackgroundGlutaric 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.MethodsNewborn 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), β receptor of the platelet derived growth factor (PDGFRβ) 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.ResultsPerinatal 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, PDGFRβ and laminin positive areas associated with small blood vessels. Occludin and ZO-1 expression in the striatal tissue was unchanged in all conditions analyzed.ConclusionsThe 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.

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.

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.

Telencephalic-olfactory bulb ventricle wall organization in Austrolebias charrua: Cytoarchitecture, proliferation dynamics, neurogenesis and migration

Adult neurogenesis participates in fish olfaction sensitivity in response to environmental challenges. Therefore, we investigated if several populations of stem/progenitor cells that are retained in the olfactory bulbs (OB) may constitute different neurogenic niches that support growth and functional demands. By electron microscopy and combination cell proliferation and lineage markers, we found that the telencephalic ventricle wall (VW) at OB level of Austrolebias charrua fish presents three neurogenic niches (transitional 1, medial 2 and ventral 3). The main cellular types described in other vertebrate neurogenic niches were identified (transient amplifying cells, stem cells and migrating neuroblasts). However, elongated vimentin/BLBP+ radial glia were the predominant cells in transitional and ventral zones. Use of halogenated thymidine analogs chloro- and iodo-deoxyuridine administered at different experimental times showed that both regions have the highest cell proliferation and migration rates. Zone 1 migration was toward the OB and telencephalon, whereas in zone 3, migration was directed toward the OB rostral portion constituting the equivalent of the mammal rostral migratory band. Medial zone (MZ) has fewer proliferating non-migrant cells that are the putative stem cells as indicated by short and long proliferation assays as well as cell lineage markers. Sparse migration observed suggests MZ may collaborate with VW growth. Scanning electron microscopy evidenced that the whole VW has only monociliated cells with remarkable differences in cilium length among regions. In OB there are monociliated cells with dwarf cilium whereas ventral telencephalon shows long cilium. Summarizing, we identified three neurogenic niches that might serve different functional purposes.

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.

A role of astrocytes in mediating postnatal neurodegeneration in Glutaric acidemia-type 1

Astrocytes are crucial for postnatal development of neuronal networks, axon myelination and neurovascular structures. Defects in astrocyte generation or maturation are associated with severe neurological developmental disorders. Glutaric acidemia type I (GAI), an inherited neurometabolic disorder characterized by accumulation of glutaric (GA) and 3‐hydroxyglutaric acids, shows a paradigmatic postnatal neuropathology characterized by massive degeneration of neurons in the striatum. While the disorder is caused by genetic mutations on glutaryl‐CoA dehydrogenase, the neurological defects usually start months after birth. Pathogenesis of GAI has remained largely unknown, and specifically, it is unclear how accumulation of GAI metabolites may result in neurodegeneration. Recent evidence supports a GAI model involving primary defective astrocyte maturation leading to a co‐morbid spectrum of neurologic symptoms similar to those of patients. Astrocytes are vulnerable to GAI metabolites, but instead of dying, they follow long‐lasting phenotypic changes leading to striatal neuron degeneration as well as defective myelination and blood brain barrier maturation. Here, we summarized recent findings on the pathogenic role of GA‐damaged astrocytes in GAI and discuss if astrocyte dysfunction may be a target of therapeutic interventions.