Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase.

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

Structures of the G85R variant of SOD1 in familial amyotrophic lateral sclerosis.

Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.

Neurofilament protein aggregation in a cell line model system.

Protein aggregates are associated with many diseases and even aggregates of proteins that have no role in disease are inherently toxic to both neuronal and non-neuronal cells. We have developed a model system to explore the mechanism of protein aggregation using a mouse muscle cell line expressing chimeric neurofilament (NF) proteins, a constituent of the protein aggregates in ALS, Lewy body dementia, and Charcot-Marie-Tooth disease. Formation of protein aggregates in these cells leads to reduced cell viability and activated caspases. Aggregates contained both chimeric NF proteins and ubiquitin by immunolocalization and were predominately cytosolic when proteins were expressed at low levels or for shorter periods of time but were present in the nucleus when expression levels increased. This system represents a flexible, new tool to decipher the molecular mechanism of protein aggregation and the contributions of aggregation to cell toxicity.

The single neurofilament subunit of lamprey may need another element for filament assembly.

Regenerating axon tips in transected lamprey spinal cord contain dense accumulations of neurofilaments (NFs), suggesting that NFs may play a role in the mechanism of axonal regeneration. Compared with heteropolymeric assemblies of NF triplet proteins in mammals, NF in lampreys has been thought to contain only a single subunit (NF180). This would imply that NF180 self-assembles, which would be important for manipulating its expression in studies of axonal regeneration. In order to study the possible role of NF in process outgrowth and to determine whether NF180 can self-assemble, its gene was transfected into mammalian and fish cell lines that either contain or lack vimentin. In transfected NIH3T3 cells, NF180 was poorly phosphorylated and its expression did not alter the length or number of cell processes. Nor did it appear to form typical intermediate filaments, suggesting that it may not self-assemble. NF180 also did not form typical filaments in SW13cl cells that either possessed or lacked vimentin, nor in transfected fish cells that were cultured at 18 degrees C. In vitro, NF180 could not self-assemble but interacted with NF-L to interrupt its self-assembly. When cotransfected with rat NF-L into SW13c1.2vim(-) cells, NF180 did form thick, rod-like filamentous structures on immunofluorescence. More typical NFs were observed when NF180 was cotransfected with both NF-L and NF-M. Thus, NF180 cannot self-assemble but appears to require one or more additional elements for incorporation into NFs.

Heparin and other glycosaminoglycans stimulate the formation of amyloid fibrils from alpha-synuclein in vitro.

Parkinson's disease is the second most common neurodegenerative disease and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of alpha-synuclein have been implicated as a causative factor in the disease. Glycosaminoglycans (GAGs) are routinely found associated with amyloid deposits in most amyloidosis diseases, and there is evidence to support an active role of GAGs in amyloid fibril formation in some cases. In contrast to the extracellular amyloid deposits, the alpha-synuclein deposits in Lewy body diseases are intracellular, and thus it is less clear whether GAGs may be involved. To determine whether the presence of GAGs does affect the fibrillation of alpha-synuclein, the kinetics of fibril formation were investigated in the presence of a number of GAGs and other charged polymers. Certain GAGs (heparin, heparan sulfate) and other highly sulfated polymers (dextran sulfate) were found to significantly stimulate the formation of alpha-synuclein fibrils. Interestingly, the interaction of GAGs with alpha-synuclein is quite specific, since some GAGs, e.g., keratan sulfate, had negligible effect. Heparin not only increased the rate of fibrillation but also apparently increased the yield of fibrils. The molar ratio of heparin to alpha-synuclein and the incorporation of fluorescein-labeled heparin into the fibrils demonstrate that the heparin is integrated into the fibrils and is not just a catalyst for fibrillation. The apparent dissociation constant for heparin in stimulating alpha-synuclein fibrillation was 0.19 microM, indicating a strong affinity. Similar effects of heparin were observed with the A53T and A30P mutants of alpha-synuclein. Since there is some evidence that Lewy bodies may contain GAGs, these observations may be very relevant in the context of the etiology of Parkinson's disease.