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

GA induced astrocytic mitochondrial depolarization and increased oxidative stress.

<p>Confluent striatal cultured astrocytes were submitted to GA (5 mM, 24 h) and then mitochondrial potential and oxidative status were assessed immediately. <b>A:</b> Representative images of the effects of GA on mitochondrial potential (measured by the probe JC1), glutathione levels (assessed by monochlorobimane), and oxidative activity analyzed with carboxy-H2DFFDA. Note that GA-treated astrocytes have decreased mitochondrial potential and cellular glutathione as evidenced by green mitochondria and less blue fluorescence, respectively. In bottom images, green spots surrounding the DAPI stained nuclei in GA-treated cells denote increased oxidative stress. Calibration: 20 µm in upper and mid panels, and 10 µm in the bottom one. <b>B:</b> Quantitation of GA effects and counteracting antioxidant actions. Charts show percent values of JC1 ratiometric fluorescence, monochlorobimane (MCB) emission and green carboxy-H2DFFDA fluorescence in GA-treated astrocytes alone or pre-incubated with the antioxidants FeTCPP (FeT, 20 µM), apocynin (APO, 1 mM), or FeTMPyP (FeM, 2 µM). Note that antioxidants abrogated GA decreasing effects on mitochondrial potential and glutathione levels, as well as the increased oxidative stress. Control conditions were indicated with dotted black lines. Asterisks indicate statistical significance at p<0.05.</p

Delayed degeneration of striatal neurons following icv GA administration.

<p>P0 pups were treated with GA or vehicle and processed at different times to evaluate Fluoro JadeC (FJC) positive degenerating neurons, neuron number, nitrotyrosine immunoreactivity and phosphorylated neurofilament (PNF) areas. <b>A:</b> Representative microphotographs of the striatal parenchyma at P21 showing that GA treatment caused increased FJC and nitrotyrosine labeling, and both decreased number of NeuN positive neurons and PNF areas. Calibration: 20 µm in upper and mid panels, and 100 µm in the bottom one. Inset: scheme of striatal area analyzed. <b>B:</b> Time course of FJC labeling in striatal sections of GA-injected animals as compared to controls. Note the sharp increase in degenerating FJC positive cells at P21 and P45. <b>C:</b> Time course of nitrotyrosine labeling in striatal sections of GA-injected animals as compared to controls. Note the sharp increase in nitrotyrosine immunoreactivity at all ages but peaking at P21. <b>D:</b> Time course of number of striatal NeuN positive neurons showing a significant decrease at P21 and P45 in GA-injected animals as compared to controls. <b>E:</b> Relative area of PNF bundles at different times after GA-treatment. Note the significant diminution evidenced at P5 and the progressive decrease until P45. Dotted lines in charts B, C and D indicate respective control values. All data are the mean ± SEM. Statistical significance at p<0.05 (*).</p

Astrocyte activation by GA was sufficient to induce neuronal death through soluble factors.

<p>The neurotoxic activity of conditioned media (CM) of astrocytes treated with GA (CM-GA) was tested on cultured E18 striatal neurons 5 DIV after plating. <b>A:</b> Representative MAP-2 immunostaining of striatal neurons upon treatment with CM from control (CM-C), or GA-treated (CM-GA) or FeTCPP/GA-treated astrocytes (CM-FeT/GA). Note decreased neuronal number and neurite growth caused by CM-GA and the maintenance of both neuronal survival and phenotype in the CM-FeT/GA condition. Scale bar: 50 µm. <b>B:</b> Counting of striatal neurons maintained in culture for 24 h with CM-C or increasing dilutions of CM-GA or vehicle. Neuronal number increased when treated with CM-C (first column of the chart). Conversely a concentration dependent neuronal loss was caused by CM-GA when compared with vehicle (taken as 100%, dotted line). *: p<0.05. <b>C:</b> FeTCPP applied immediately before GA abolished the toxicity of CM-GA (1∶5 dilution) preserving both the neuronal number and morphology. Control was indicated by the dotted line. All data is the mean ± SEM. ∧: p<0.01; *: p<0.05.</p

Increased gliogenesis following icv GA administration.

<p>P0 pups were treated with GA or vehicle and immediately injected with a single dose of BrdU ip to label dividing cells. <b>A:</b> Panoramic BrdU immunohistochemistry of the striatal regions lining ventricles showing an increased number of dark BrdU labeled nuclei in both striatal parenchyma and subventricular areas (dotted white lines indicate the limit between both areas) of GA-injected animals. In controls, injected with vehicle, BrdU positive nuclei were mainly restricted to subventricular regions. Inset schematizes areas imaged. Calibration bar: 75 µm. <b>B:</b> The number of BrdU labeled cells in the striatal parenchyma of GA-injected animals related to respective controls remained elevated until P45. All values were significantly higher than controls (taken as 100% and indicated by a dotted line). <b>C:</b> Astroglial phenotype of BrdU+ cells in GA-injected animals evidenced by a double labeling for both BrdU and S100β that were dominant at P5, and a progressive BrdU-GFAP double immunoreactivity at P21 and P45. In all conditions, the number of BrdU positive cells that displayed astroglial phenotype was significantly higher than controls. All data is expressed as mean ± SEM. Asterisks indicate statistical significance related to respective controls at p<0.05).</p

GA did not induce neuronal death in absence of astrocytes.

<p>Isolated striatal neuronal cultures were employed to investigate the mechanism of GA-induced neurotoxicity. <b>A:</b> Phase contrast of E18 striatal neurons at 5 DIV exposed to vehicle or 5 mM GA (pH 7.4, 24 h). Note that GA neither modified the morphology (as shown in respective insets) nor the number of MAP2 positive neurons as indicated in the right chart. Scale bars: 80 µm (images), 40 µm (insets). <b>B:</b> Astrocyte monolayers pretreated with vehicle or GA 5 mM for 24 h were used as feeding layer to cultured E18 striatal neurons. Note the decrease in the number of neurons and that surviving neurons exhibited swollen bodies and sparse neurites. Extensive washings before neuronal seeding discarded a direct effect of GA on striatal neurons. Scale bars: 20 µm. <b>C:</b> FeTCPP applied immediately before GA prevented toxic effects of GA-treated astrocytes as shown by the preserved neuronal number and morphology of neurons co-cultured on GA-treated astrocyte monolayers. Symbol ∧ indicates statistical significance at p<0.01 and NS: means no statistical signification.</p

GA induced long lasting astrocytosis in the striatum.

<p>P0 rat pups were injected icv with GA or vehicle and processed from 5 (P5) until 45 (P45) days later for immunohistochemistry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020831#s4" target="_blank">Materials and Methods</a>. <b>A:</b> Representative S100β (red) and GFAP (green) immunofluorescences of the striatal parenchyma at P5, P21 and P45 evidencing an increased number of astrocytes with nuclear S100β and typical GFAP stains in GA-treated pups (right) when compared to controls (vehicle, left). Some GFAP astrocytes were also positive to S100β (white arrow heads). Box in the inset shows striatal areas analyzed. Scale bar: 25 µm. <b>B:</b> Increased number of astrocytes after the icv GA injection evidenced by 2–3 folds rises in S100β positive cells at P5 and elevated number of GFAP positive cells until P45. All values were significantly higher (p<0.05) than respective controls (taken as 100% and indicated with the dotted line). All data is expressed as mean ± SEM.</p