Thomas Brännström

Toxicity of familial ALS-linked SOD1 mutants from selective recruitment to spinal mitochondria.

One cause of amyotrophic lateral sclerosis (ALS) is mutation in ubiquitously expressed copper/zinc superoxide dismutase (SOD1), but the mechanism of toxicity to motor neurons is unknown. Multiple disease-causing mutants, but not wild-type SOD1, are now demonstrated to be recruited to mitochondria, but only in affected tissues. This is independent of the copper chaperone for SOD1 and dismutase activity. Highly preferential association with spinal cord mitochondria is seen in human ALS for a mutant SOD1 that accumulates only to trace cytoplasmic levels. Despite variable proportions that are successfully imported, nearly constant amounts of SOD1 mutants and covalently damaged adducts of them accumulate as apparent import intermediates and/or are tightly aggregated or crosslinked onto integral membrane components on the cytoplasmic face of those mitochondria. These findings implicate damage from action of spinal cord-specific factors that recruit mutant SOD1 to spinal mitochondria as the basis for their selective toxicity in ALS.

The homeodomain LIM protein Isl-1 is expressed in subsets of neurons and endocrine cells in the adult rat

We have used immunocytochemical methods to localize the homeodomain LIM protein Isl-1 in the adult rat. Isl-1 immunoreactivity is expressed in polypeptide hormone-producing cells of the endocrine system, in neurons of the peripheral nervous system, and in a subset of brain nuclei. Isl-1 is also expressed in a subset of motoneurons in the spinal cord and brain stem, but not in regions of the central nervous system involved in sensory function or in neocortical areas. The pattern of expression of Isl-1 suggests that this gene may be involved in the specification and maintenance of differentiated phenotypical properties of these cells.

Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative syndrome hallmarked by adult-onset loss of motor neurons. We performed exome sequencing of 252 familial ALS (fALS) and 827 control individuals. Gene-based rare variant analysis identified an exome-wide significant enrichment of eight loss-of-function (LoF) mutations in TBK1 (encoding TANK-binding kinase 1) in 13 fALS pedigrees. No enrichment of LoF mutations was observed in a targeted mutation screen of 1,010 sporadic ALS and 650 additional control individuals. Linkage analysis in four families gave an aggregate LOD score of 4.6. In vitro experiments confirmed the loss of expression of TBK1 LoF mutant alleles, or loss of interaction of the C-terminal TBK1 coiled-coil domain (CCD2) mutants with the TBK1 adaptor protein optineurin, which has been shown to be involved in ALS pathogenesis. We conclude that haploinsufficiency of TBK1 causes ALS and fronto-temporal dementia.

Cortical Neurogenesis in Adult Rats After Transient Middle Cerebral Artery Occlusion

Background and Purpose This study explored the possible occurrence of newly generated nerve cells in the ischemic cortex of adult rats after middle cerebral artery occlusion and reperfusion. Methods Nine- to 10-week-old male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion by the monofilament method. Rats received repeated intraperitoneal injections of the cell proliferation-specific marker 5-bromodeoxyuridine (BrdU) after stroke induction. Brain sections were processed for immunohistochemistry with an avidin-biotin complex-alkaline phosphatase and/or -peroxidase method. Brain sections processed with double-immunofluorescent staining were further scanned by confocal microscopy. Results Interspersed among the predominantly newly formed glial cells, some cells were double labeled by BrdU and 1 of the neuron-specific markers, Map-2, &bgr;-tubulin III, and Neu N, at 30 and 60 days after stroke onset. These cells were randomly distributed throughout cortical layers II through VI, occurring with highest density in the ischemic boundary zone. Three-dimensional confocal analyses of BrdU and the neuron-specific marker Neu N confirmed their colocalization within the same cortical cells. Conclusions This study suggests that new neurons can be generated in the cerebral cortex of adult rats after transient focal cerebral ischemia. Cortical neurogenesis may be a potential pathway for brain repair after stroke.

Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients

Mutations in CuZn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) and are found in 6% of ALS patients. Non-native and aggregation-prone forms of mutant SOD1s are thought to trigger the disease. Two sets of novel antibodies, raised in rabbits and chicken, against peptides spaced along the human SOD1 sequence, were by enzyme-linked immunosorbent assay and an immunocapture method shown to be specific for denatured SOD1. These were used to examine SOD1 in spinal cords of ALS patients lacking mutations in the enzyme. Small granular SOD1-immunoreactive inclusions were found in spinal motoneurons of all 37 sporadic and familial ALS patients studied, but only sparsely in 3 of 28 neurodegenerative and 2 of 19 non-neurological control patients. The granular inclusions were by confocal microscopy found to partly colocalize with markers for lysosomes but not with inclusions containing TAR DNA binding protein-43, ubiquitin or markers for endoplasmic reticulum, autophagosomes or mitochondria. Granular inclusions were also found in carriers of SOD1 mutations and in spinobulbar muscular atrophy (SBMA) patients and they were the major type of inclusion detected in ALS patients homozygous for the wild type-like D90A mutation. The findings suggest that SOD1 may be involved in ALS pathogenesis in patients lacking mutations in the enzyme.

Cortical Neurogenesis in Adult Rats after Reversible Photothrombotic Stroke

Neurogenesis occurs throughout life in the dentate gyrus of hippocampus and subventricular zone, but this phenomenon has rarely been observed in other brain regions of adult mammals. The aim of the current study was to investigate the cell proliferation process in the ischemically challenged region-at-risk after focal cerebral ischemia in the adult rat brain. A reversible photothrombotic ring stroke model was used, which features sustained hypoperfusion followed by late spontaneous reperfusion and a remarkable morphologic tissue recovery in the anatomically well defined somatosensory cortical region-at-risk. Twelve-week-old male Wistar rats received repeated intraperitoneal injections of the cell proliferation specific marker 5-bromodeoxyuridine (BrdU) after stroke induction. Immunocytochemistry of coronal brain sections revealed that the majority of BrdU-positive cells were of glial, macrophage, and endothelial origin, whereas 3% to 6% of the BrdU-positive cells were double-labeled by BrdU and the neuron-specific marker Map-2 at 7 and 100 days after stroke onset in the region-at-risk. They were distributed randomly in cortical layers II-VI. Three-dimensional confocal analyses of BrdU and the neuronal-specific marker Neu N by double immunofluorescence confirmed their colocalization within the same cells at 72 hours and 30 days after stroke induction. This study suggests that, as a potential pathway for brain repair, new neurons can be generated in the cerebral cortex of adult rats after sublethal focal cerebral ischemia.

Soluble misfolded subfractions of mutant superoxide dismutase-1s are enriched in spinal cords throughout life in murine ALS models

Mutants of superoxide dismutase-1 (SOD1) cause ALS by an unidentified cytotoxic mechanism. We have previously shown that the stable SOD1 mutants D90A and G93A are abundant and show the highest levels in liver and kidney in transgenic murine ALS models, whereas the unstable G85R and G127X mutants are scarce but enriched in the CNS. These data indicated that minute amounts of misfolded SOD1 enriched in the motor areas might exert the ALS-causing cytotoxicity. A hydrophobic interaction chromatography (HIC) protocol was developed with the aim to determine the abundance of soluble misfolded SOD1 in tissues in vivo. Most G85R and G127X mutant SOD1s bound in the assay, but only minute subfractions of the D90A and G93A mutants. The absolute levels of HIC-binding SOD1 were, however, similar and broadly inversely related to lifespans in the models. They were generally enriched in the susceptible spinal cord. The HIC-binding SOD1 was composed of disulfide-reduced subunits lacking metal ions and also subunits that apparently carried nonnative intrasubunit disulfide bonds. The levels were high from birth until death and were comparable to the amounts of SOD1 that become sequestered in aggregates in the terminal stage. The HIC-binding SOD1 species ranged from monomeric to trimeric in size. These species form a least common denominator amongst SOD1 mutants with widely different molecular characteristics and might be involved in the cytotoxicity that causes ALS.

Histochemical detection of 4-hydroxynonenal protein in Alzheimer amyloid.

The presence of lipid peroxidation product in amyloid deposits from seven patients with Alzheimer disease and nine with non-Alzheimer disease was examined immunohistochemically by means of an affinity purified anti-HNE antibody to hydroxynonenal (HNE), a marker of lipid peroxidation. A positive reaction was found in amyloid deposits in all the specimens examined: most of the perivascular areas (89%) where amyloid deposition was confirmed by Congo red staining, showed immunoreactivity with the antibody in the specimens of Alzheimer disease. Twenty-one percent of senile plaques which were also stained by Congo red staining reacted with this antibody. Several perivascular cells were also stained by anti-HNE antibody. In other neurons both in Alzheimer and non-Alzheimer disease patients, only a few percent reacted with this antibody and no statistical difference was observed between them. These results verify that lipid peroxidation via free radical injury occurs in amyloid deposits in Alzheimer amyloid. Since HNE has been identified as a cytotoxic metabolite of free radical injury, amyloid deposits in the tissue may exhibit a toxic effect during the generation process of HNE.

Epidermal growth factor receptor family (EGFR, ErbB2–4) in gliomas and meningiomas

Overexpression of epidermal growth factor receptor (EGFR, ErbB1) correlates with enhanced malignant potential of many human tumor types including glioblastoma multiforme. The significance of EGFR expression in meningiomas is, however, unclear. Reports regarding the other EGFR family members, ErbB2–4, in brain tumors are sparse. In this study, the expression of the EGFR family members was analyzed in relation to various parameters for the clinical importance of these receptors in 44 gliomas and 26 meningiomas. In gliomas, quantitative real-time reverse transcription (RT)-PCR revealed the highest EGFR mRNA expression in high-grade gliomas, while ErbB2 and ErbB3 mRNA were detected only in a few high-grade gliomas. In contrast, ErbB4 expression was most pronounced in low-grade gliomas. Immunohistochemistry showed significantly higher EGFR protein expression in high-grade gliomas compared to low-grade gliomas (P=0.004). ErbB2 protein expression was mainly seen in high-grade gliomas. ErbB3 protein expression was low in all gliomas analyzed. ErbB4 protein expression was significantly higher in low-grade gliomas than in high-grade gliomas (P=0.007). In meningiomas, quantitative real-time RT-PCR revealed expression of EGFR, ErbB2, and ErbB4 mRNA in the majority of the tumors. ErbB3 was detected in only one of the meningiomas analyzed. Immunohistochemistry demonstrated high ErbB2 protein expression in meningiomas. An intriguing observation in astrocytomas and oligodendrogliomas grade II, was a significantly decreased overall survival for patients with high EGFR protein expression (P=0.04). The high ErbB4 expression in low-grade compared to high-grade gliomas might suggest that ErbB4 acts as a suppressor of malignant transformation in brain tumors, which is in line with previous studies in other tumor types.

Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis

The most common cause of amyotrophic lateral sclerosis (ALS) is mutations in superoxide dismutase-1 (SOD1). Since there is evidence for the involvement of non-neuronal cells in ALS, we searched for signs of SOD1 abnormalities focusing on glia. Spinal cords from nine ALS patients carrying SOD1 mutations, 51 patients with sporadic or familial ALS who lacked such mutations, and 46 controls were examined by immunohistochemistry. A set of anti-peptide antibodies with specificity for misfolded SOD1 species was used. Misfolded SOD1 in the form of granular aggregates was regularly detected in the nuclei of ventral horn astrocytes, microglia, and oligodendrocytes in ALS patients carrying or lacking SOD1 mutations. There was negligible staining in neurodegenerative and non-neurological controls. Misfolded SOD1 appeared occasionally also in nuclei of motoneurons of ALS patients. The results suggest that misfolded SOD1 present in glial and motoneuron nuclei may generally be involved in ALS pathogenesis.