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.

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.

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.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease afflicting the voluntary motor system. More than 100 different mutations in the ubiquitously expressed enzyme superoxide dismutase-1 (SOD1) have been associated with the disease. To search for the nature of the cytotoxicity of mutant SOD1s, amounts, enzymic activities and structural properties of the protein as well as the CNS histopathology were examined in multiple transgenic murine models. In order to generate the ALS phenotype within the short lifespan of the mouse, more than 20-fold increased rates of synthesis of mutant SOD1s appear to be required. The organs of transgenic mice expressing human wild-type SOD1 or either of the G93A and D90A mutant proteins showed high steady-state protein levels. The major proportion of these SOD1s in the CNS were inactive due to insufficient Cu charging and all contained subfractions with a reduced C57-C146 intrasubunit disulphide bond. Both G85R and the truncated G127insTGGG mutant showed low steady-state protein levels, lacked enzyme activity and had no C57-C146 disulphide bond. These mutants were also enriched in the CNS relative to other organs, suggesting inefficient recognition and degradation of misfolded disulphide-reduced SOD1 in susceptible tissues. In end-stage disease, despite 35-fold differences in levels of mutant SOD1s, similar amounts of detergent-resistant aggregates accumulated in the spinal cord. Small granular as well as larger more diffuse human SOD1 (hSOD1)-inclusions developed in all strains, the latter more pronounced in those with high hSOD1 levels. Widespread vacuolizations were seen in the strains with high levels of hSOD1 but not those with low, suggesting these alterations to be artefacts related to high hSOD1 levels and not to the ALS-causing cytotoxicity. The findings suggest that the motoneuron degeneration could be due to long-term exposure to misfolded aggregation-prone disulphide-reduced SOD1, which constitutes minute subfractions of the stable mutants and larger proportions of the unstable mutants.

Mutant forms of superoxide dismutase-1 (SOD1) cause amyotrophic lateral sclerosis (ALS) by an unknown noxious mechanism. Using an antibody against a novel epitope in the G127insTGGG mutation, mutant SOD1 was studied for the first time in spinal cord and brain of an ALS patient. The level was below 0.5% of the SOD1 level in controls. In corresponding transgenic mice the content of mutant SOD1 was also low, although it was enriched in spinal cord and brain compared with other tissues. In the mice the misfolded mutant SOD1 aggregated rapidly and 20% occurred in steady state as detergent-soluble protoaggregates. The misfolded SOD1 and the protoaggregates form, from birth until death, a potentially noxious burden that may induce the motor neuron injury. Detergent-resistant aggregates, as well as inclusions of mutant SOD1 in motor neurons and astrocytes, accumulated in spinal cord ventral horns of the patient and mice with terminal disease. The inclusions and aggregates may serve as terminal markers of long-term assault by misfolded SOD1 and protoaggregates.

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Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflammation-related neurodegeneration. Here, we provide insights into S100A9 specific mechanisms of action in Alzheimer’s disease (AD). Due to its inherent amyloidogenicity S100A9 contributes to amyloid plaque formation together with Aβ. In traumatic brain injury (TBI) S100A9 itself rapidly forms amyloid plaques, which were reactive with oligomer-specific antibodies, but not with Aβ and amyloid fibrillar antibodies. They may serve as precursor-plaques for AD, implicating TBI as an AD risk factor. S100A9 was observed in some hippocampal and cortical neurons in TBI, AD and non-demented aging. In vitro S100A9 forms neurotoxic linear and annular amyloids resembling Aβ protofilaments. S100A9 amyloid cytotoxicity and native S100A9 pro-inflammatory signaling can be mitigated by its co-aggregation with Aβ, which results in a variety of micron-scale amyloid complexes. NMR and molecular docking demonstrated transient interactions between native S100A9 and Aβ. Thus, abundantly present in AD brain pro-inflammatory S100A9, possessing also intrinsic amyloidogenic properties and ability to modulate Aβ aggregation, can serve as a link between the AD amyloid and neuroinflammatory cascades and as a prospective therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-013-1208-4) contains supplementary material, which is available to authorized users.

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.disulfide bond ͉ motor neuron ͉ neurodegeneration A myotrophic lateral sclerosis (ALS) is characterized by motor neuron degeneration, resulting in progressive paralysis, and death from respiratory failure. Approximately 10% of ALS cases are familial (1) and in some of these the disease is linked to mutations in the CuZn-superoxide dismutase (SOD1) gene (2). Overall, Ϸ6% of all cases with ALS show SOD1 mutations, and more than 100 different such mutations have been identified (3). The mutations confer a cytotoxic gain of function to the enzyme (4, 5). SOD1 is ubiquitously expressed, and in several organs at higher levels than in the motor areas in the CNS (6, 7). Both the nature of the cytotoxicity, and the reasons for the particular susceptibility of some parts of the CNS remain unexplained.It is likely that the different mutant SOD1s cause ALS by essentially the same mechanism. Still, the levels of different mutant SOD1s in the human CNS differ by more than 200-fold (7). Similar large differences are observed in the murine ALS models. In spinal cords from mice expressing the D90A, G93A, G85R and G127insTGGG (G127X) mutant human SOD1s (hSOD1s), the levels are 20, 14, 0.9, and 0.45 times as large, respectively, as the levels of the endogenous murine SOD1 (mSOD1) (8). In the high-level models, D90A and G93A, most hSOD1 lacks enzymatic activity owing to Cu-deficiency, and significant proportions lack the stabilizing intrasubunit disulfide bond. The G85R and G127X hSOD1s lack both enzymatic activity and the disulfide bond. Whereas the liver and kidney contain the highest lev...