Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials

Recent studies suggest that the toxicity of familial amyotrophic lateral sclerosis mutant Cu, Zn superoxide dismutase (SOD1) arises from its selective recruitment to mitochondria. Here we demonstrate that each of 12 different familial ALS-mutant SOD1s with widely differing biophysical properties are associated with mitochondria of motoneuronal cells to a much greater extent than wild-type SOD1, and that this effect may depend on the oxidation of Cys residues. We demonstrate further that mutant SOD1 proteins associated with the mitochondria tend to form cross-linked oligomers and that their presence causes a shift in the redox state of these organelles and results in impairment of respiratory complexes. The observation that such a diverse set of mutant SOD1 proteins behave so similarly in mitochondria of motoneuronal cells and so differently from wild-type SOD1 suggests that this behavior may explain the toxicity of ALS-mutant SOD1 proteins, which causes motor neurons to die

A multidisciplinary approach unravels early and persistent effects of X-ray exposure at the onset of prenatal neurogenesis

Background: In humans, in utero exposure to ionising radiation results in an increased prevalence of neurological aberrations, such as small head size, mental retardation and decreased IQ levels. Yet, the association between early damaging events and long-term neuronal anomalies remains largely elusive. Methods: Mice were exposed to different X-ray doses, ranging between 0.0 and 1.0 Gy, at embryonic days (E) 10, 11 or 12 and subjected to behavioural tests at 12 weeks of age. Underlying mechanisms of irradiation at E11 were further unravelled using magnetic resonance imaging (MRI) and spectroscopy, diffusion tensor imaging, gene expression profiling, histology and immunohistochemistry. Results: Irradiation at the onset of neurogenesis elicited behavioural changes in young adult mice, dependent on the timing of exposure. As locomotor behaviour and hippocampal-dependent spatial learning and memory were most particularly affected after irradiation at E11 with 1.0 Gy, this condition was used for further mechanistic analyses, focusing on the cerebral cortex and hippocampus. A classical p53-mediated apoptotic response was found shortly after exposure. Strikingly, in the neocortex, the majority of apoptotic and microglial cells were residing in the outer layer at 24 h after irradiation, suggesting cell death occurrence in differentiating neurons rather than proliferating cells. Furthermore, total brain volume, cortical thickness and ventricle size were decreased in the irradiated embryos. At 40 weeks of age, MRI showed that the ventricles were enlarged whereas N-acetyl aspartate concentrations and functional anisotropy were reduced in the cortex of the irradiated animals, indicating a decrease in neuronal cell number and persistent neuroinflammation. Finally, in the hippocampus, we revealed a reduction in general neurogenic proliferation and in the amount of Sox2-positive precursors after radiation exposure, although only at a juvenile age. Conclusions: Our findings provide evidence for a radiation-induced disruption of mouse brain development, resulting in behavioural differences. We propose that alterations in cortical morphology and juvenile hippocampal neurogenesis might both contribute to the observed aberrant behaviour. Furthermore, our results challenge the generally assumed view of a higher radiosensitivity in dividing cells. Overall, this study offers new insights into irradiation-dependent effects in the embryonic brain, of relevance for the neurodevelopmental and radiobiological field

Astroglial Inhibition of NF-κB Does Not Ameliorate Disease Onset and Progression in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Motor neuron death in amyotrophic lateral sclerosis (ALS) is considered a “non-cell autonomous” process, with astrocytes playing a critical role in disease progression. Glial cells are activated early in transgenic mice expressing mutant SOD1, suggesting that neuroinflammation has a relevant role in the cascade of events that trigger the death of motor neurons. An inflammatory cascade including COX2 expression, secretion of cytokines and release of NO from astrocytes may descend from activation of a NF-κB-mediated pathway observed in astrocytes from ALS patients and in experimental models. We have attempted rescue of transgenic mutant SOD1 mice through the inhibition of the NF-κB pathway selectively in astrocytes. Here we show that despite efficient inhibition of this major pathway, double transgenic mice expressing the mutant SOD1G93A ubiquitously and the dominant negative form of IκBα (IκBαAA) in astrocytes under control of the GFAP promoter show no benefit in terms of onset and progression of disease. Our data indicate that motor neuron death in ALS cannot be prevented by inhibition of a single inflammatory pathway because alternative pathways are activated in the presence of a persistent toxic stimulus

Bcl2a1 serves as a switch in death of motor neurons in amyotrophic lateral sclerosis

Several studies have indicated that apoptotic pathways are responsible for the loss of motor neurons that constitutes the hallmark of adult-onset neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study we demonstrate that motoneuronal death induced by the expression of mutant Cu,Zn superoxide dismutase (SOD1) typical of familial ALS is mediated by Bcl2a1, a protein belonging to the Bcl2 family. Bcl2a1 is the only member of this family to be up-regulated selectively in spinal motor neurons of mice transgenic for G93A-SOD1 already at the asymptomatic stage. Bcl2a1 over-expression is restricted to motor neurons, it is not depending on the genetic background of mice and it is not due to a general activation of NF-kB-regulated genes. Over-expression of Bcl2a1 is protective against death of neuronal cells induced by expression of G93A-SOD1 but is detrimental upon stimulation of those cells with tumor necrosis factor. Since this factor is known to contribute to the complex molecular cross-talk among different cell types taking place in the pathogenesis of ALS, we propose that the increase in Bcl2a1 is a triggering event in the progressive loss of motor neurons occurring in this disease

Inhibition of p65 nuclear translocation in spinal cord primary cultures of astrocytes from GFAP-IκBαAA and double transgenic GFAP-IκBαAA/SOD1^G93A mice after treatment with LPS (10 µg/ml for 20 min).

<p>(A) Immunofluorescent labeling of spinal cord primary astrocytes from non transgenic (-), transgenic GFAP-I<b>κ</b>BαAA (TgA), SOD1^G93A (G93A) and double transgenic GFAP-I<b>κ</b>BαAA/SOD1^G93A (double Tg) mice using antibodies against p65 and GFAP. Scale bar 20 µm. (B) Immunofluorescent labeling of primary motorneurons from mice as in A using antibodies against p65 and SMI32. Scale bar 20 µm; (C) Western Blot analysis of protein extracts for spinal cord primary astrocytes from mice as in (A) using antibodies against COX2 and β-actin, after stimulation with LPS 10 µg/ml for 16 h.</p

Selective expression of IκBαAA in astrocytes causes a weakly delayed microgliosis and astrocytosis only at the onset of disease (120 days).

<p>(A) Immunofluorescence against GFAP (green) and Cd11b (red) in sections of lumbar spinal cord from 100 days (pre-onset), 120 days (onset) and 140 days (symptomatic) old double transgenic mice GFAP-I<b>κ</b>BαAA/SOD1^G93A (a,b,c) and SOD1^G93A mice (d,e,f). Scale bar 100 µm. (B). Schematic representation of counting results in (A). Data are expressed as percentage (mean+/−SEM) of activated microglia (a) or astrocytes (b) from 3 different animals from the three different disease stages: pre-onset (PO); onset (O); symptomatic (S). The number of cells recorded from SOD1^G93A mice at the symptomatic stage was considered 100%. *p<0.05. (C) GFAP and IBA1 protein levels were evaluated by Western blot on 20 µg of proteins/sample. Blots were probed for β-actin as a control.</p

Inhibition of NF-κB activity in astrocytes from GFAP-IκBαAA mice.

<p>(A) EMSA of nuclear protein extracts (5 µg) from primary astrocytes and microglia from brain and spinal cord of transgenic mice line A (Tg-A) and non transgenic mice (-) stimulated or unstimulated for 20 min with recombinant TNFα (10 ng/ml) before analysis. For NF-<b>κ</b>B consensus probe see underlined sequence below. (B) Quantification of the experiments in (A) performed with spinal cord astrocytes and microglia by densitometric assay. Results obtained from non transgenic stimulated cells were defined as 100%. Results are expressed as the means +/− SEM of three independent experiments. *P<0,01. Effect of 3 h LPS treatment (10 µg/ml) on different NF-<b>κ</b>B controlled gene in primary astrocytes from non transgenic (-) or GFAP-I<b>κ</b>BαAA (TgA) transgenic mice. (C) Western blot analysis on protein extract using antibodies against COX2 and β-actin. (D) semi-quantitave PCR for iNOS, IL-1β, TNFα, Fas mRNA expression. Results obtained with cells from stimulated non transgenic mice are defined as 100% and compared with their corresponding results from stimulated transgenic samples. Data are normalized to β-actin. *p<0.05 **p<0.01. Results represent the mean +/−SEM of three groups. (E) Representative semi-quantitave PCR performed on cDNA from total RNA extracted from astrocytes primary cultures from non transgenic (-) and transgenic (Tg-A) mice untreated and treated with LPS 10 µg/ml.</p

Effects of LPS stimulation in GFAP-IκBαAA and GFAP-IκBαAA/SOD1^G93A mice.

<p>(A) Effect of intraperitoneally LPS injection on survival of 10 weeks old non transgenic (-) and transgenic (Tg-A and Tg-D) mice. N = 20 mice/group (10 males and 10 females). (B) Effect of 3 h LPS treatment on iNOS, IL-1β, TNFα, Fas mRNA expression in tissues as brain or spinal cord from GFAP-IκBα-AA mice as determined by semi-quantitave PCR. Results obtained with tissues from stimulated non transgenic mice are defined as 100% and compared with their corresponding results from stimulated transgenic samples. Data are normalized to β-actin. *p<0.05 **p<0.01. Results represent the mean +/−SEM of three animals/group. (C) Western blot analysis on spinal cord protein extract from non transgenic (-) and GFAP-I<b>κ</b>BαAA transgenic mice (TgA) using antibodies against TRL2, TRL4 and β-actin.</p