C44 THE ROLE OF THE ER IN MOTOR NEURON DEGENERATION
ATKIN J1,2
1La Trobe Institute for Molecular Science, La Trobe University, Australia, 2Florey Neuroscience Institute, Melbourne University, Melbourne, Australia, 3Centre for Neuroscience, Melbourne University, Melbourne, Australia
Email address for correspondence: [email protected]
Keywords: ER stress, unfolded protein response, protein disulphide isomerase
There is now substantial evidence for abnormalities to the endoplasmic reticulum (ER) and Golgi apparatus in diseases affecting motor neurons. In ALS, ER stress is emerging as important cellular pathway to cell death. The ER is primarily responsible for the folding, post-translational modification and trafficking of transmembrane and secretory proteins. ER stress is triggered when misfolded proteins accumulate within the ER. This induces the unfolded protein response (UPR): specific signalling pathways which aim to restore cellular homeostasis. However prolonged UPR induces apoptotic cell death. Many cellular insults lead to increased protein misfolding within the ER, including disturbances of the ER-Golgi vesicular transport pathway.
In this presentation I will review the increasing evidence implicating ER stress in the pathophysiology of ALS. Our laboratory previously demonstrated that the UPR is induced in lumbar spinal cords from transgenic SOD1G93A mice and sporadic ALS patients. Other studies have also shown that genetic manipulation of ER stress in SOD1 mice alters disease onset and progression. Furthermore, activation of the UPR is one of the earliest pathological events in affected motor neurons of SOD1G93A mice, and it is specific to those cells which degenerate first. Mutations to other proteins which modulate ER stress, such as VAPB and VCP, further implicate the UPR in ALS. More recently we demonstrated that ER stress is triggered by mutant TDP-43 and mutant FUS, thus implicating the UPR in both sporadic and other familial forms of disease.
The objective of our most recent work has been directed towards understanding the mechanisms by which the UPR is induced in ALS. Most of the proteins linked to ALS, including SOD1, are not usually associated with the ER, so it remains unclear how ER stress is triggered. However our most recent studies suggest that disruption to dynein-mediated protein trafficking between the ER and Golgi apparatus triggers ER stress in ALS. Disruption to ER-Golgi transport can also account for several other mechanisms implicated in neurodegeneration in ALS, including inhibition of axonal transport and fragmentation of the Golgi apparatus. Together these findings therefore implicate ER stress as an upstream mechanism in disease, and they provide a single mechanism to link the UPR to several other events previously described in ALS.
C45 DIFFERENT STRUCTURES OF SOD1 AGGREGATES CORRELATE WITH DIFFERENT DISEASE PHENOTYPES
MARKLUND S1, ANDERSEN PM2, BRÄNNSTRÖM T1, ZETTERSTRÖM P1, BERGH J1, OLIVEBERG m3, GRAFFMO K1, FORSBERG K1
1Department of Medical Biosciences, Umeå University, Umeå, Sweden, 2Department of Pharmcology and Clinical Neuroscience, Umeå University, Umeå, Sweden, 3Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
Email address for correspondence: [email protected]
Keywords: aggregate, misfolded, prionoid
Background: Inclusions containing aggregated SOD1 are hallmarks of ALS in humans and transgenic models carrying mutant SOD1s (1), but are also seen in ALS patients lacking such mutations (2). This suggests that misfolded aggregation-prone monomeric, oligomeric and/or polymeric aggregated SOD1 species are critically involved in ALS pathogenesis.
Objectives: To map the structure of SOD1 aggregates in mice carrying human SOD1 mutants. From the results deduce nature of associations in oligomeric species and the processes involved in polymerization of SOD1 monomers, with the final goal of developing aggregation inhibitors.
Methods: Aggregates in serial dilutions of extracts of spinal cords and brains from terminal mice were captured on cellulose acetate filters in a 96-well dot-blot apparatus. Eight rabbit antibodies versus human SOD1 peptides covering > 90% of the sequence were used for development of the filters similar to Western immunoblots. The antibodies react only with disordered SOD1 segments, and lack reactivity with ordered and native SOD1. In ordered and fibrous aggregates, the antibodies should lack reactivity with a β-strand backbone and react freely with the rest of the protein forming disordered fringes.
Results: The in vivo formed SOD1 aggregates are found to be highly ordered, with nearly equal antibody reactivity patterns (called type A) in the G93A, G85R, G127insTGGG models as well as in aged wild-type SOD1 transgenic mice. Many more aggregates were found in spinal cord than in brain, and none in liver and muscle. Remarkably, a distinct pattern (type B) was detected in D90A mice. SOD1 aggregates formed in vitro under a variety of conditions showed some resemblances with the in vivo aggregates, but were more heterogeneous. Aggregates from ventral horn from a patient carrying the G127insTGGG mutation were similar to those in corresponding mice.
Discussion: The in vivo crowding and proteostasis network (chaperones, transporters, redox regulators and degradation systems) shape highly ordered terminal aggregates, different from those formed in vitro. The antibody reactivity patterns suggest that some β-strands in native SOD1 are retained in the core fibrils, perhaps interacting via domain swaps. While most SOD1 variants seem to form ‘type A’ aggregates, D90A SOD1 formed different ‘type B’ aggregates. The disease phenotype provoked by this mutation is also deviant with very slow progression, sensory symptoms and bladder dysfunction, pointing at the possibility that different SOD1 ‘prionoid’ species cause different diseases.
Conclusions: Highly ordered SOD1 aggregates are formed in vivo, differing between mutants. Correlations with disease phenotypes are found pointing at the possibility that ALS could be a SOD1 ‘prionoid’ disease.
References
- Kato S, Takikawa M, Nakashima K, et al. ALS 2000;1:163–84.
- Forsberg K, Jonsson PA, Andersen PM, et al. PLoS ONE 2010;5(7):e11552.
C46 GLUTATHIONYLATION PROMOTES AGGREGATION OF SUPEROXIDE DISMUTASE IN ALS
REDLER R, WILCOX K, PROCTOR E, FEE L, CAPLOW M, DOKHOLYAN N
University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Email address for correspondence: [email protected]
Keywords: glutathionylation, SOD1 aggregation, oxidative stress
Background: Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that non-genetic factor(s) impact ALS etiology and disease progression.
Objectives: Our objective was to examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T.
Methods: We used size-exclusion chromatography for separation of modified SOD1 species from the unmodified ones, and to determine the dissociation constant Kd of wild type and the mutants. We use surface plasmon resonance to determine the kinetic rate constants for SOD1 dissociation. We use computational modeling to determine the structural implications of the SOD1 glutathionylation.
Results: Glutathionylation results in profound destabilization of SOD1WT dimers, increasing the equilibrium dissociation constant Kd to ∼10-20 μM, comparable to that of the aggressive A4V mutant. SOD1A4V is further destabilized by glutathionylation, experiencing an approximately 30-fold increase in Kd. Dissociation kinetics of glutathionylated SOD1WT and SOD1A4V are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1I112T has a modestly increased dissociation rate but no change in Kd when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface.
Discussion: The link between SOD1 mutations, protein aggregation, and FALS is not fully understood, but there are multiple reports showing that dimer dissociation is an early event during SOD1 aggregation. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. Our finding that modifications can significantly facilitate SOD1 dimer dissociation (up to 1,000x fold) suggests a possible link between the normal characteristics of SOD1 and its role in FALS.
Conclusions: Protein glutathionylation is associated with redox regulation. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.
C47 COMMON MOLECULAR PROFILE OF MISFOLDED SOD1 IN VARIOUS ALS MURINE MODELS
ZETTERSTRÖM P1, BRÄNNSTRÖM T1, ANDERSEN PM2, MARKLUND SL1
1Umeå University, Medical Biosciences, Umeå, Sweden, 2Umeå University, Pharmacology and Clinical Neuroscience, Umeå, Sweden
Email address for correspondence: [email protected]
Keywords: misfolded SOD1, oligomers, crosslink
Background: There should be one common mechanism by which the over 160 known mutations in SOD1 cause ALS. The only protein form common to all mutant SOD1s is a misfolded SOD1 species since variants exists (including truncated forms) that are incapable of folding to the native structure. Small amounts of misfolded SOD1 are a common denominator among murine ALS models expressing widely different forms of mutant SOD1s (1). Evidence for the involvement of misfolded SOD1 in sporadic ALS is emerging with SOD1 positive inclusions found in all types of ALS (2). We have developed a sensitive ELISA to measure low levels of misfolded SOD1 (misELISA) by the use of antibodies specific for misfolded SOD1 (3).
Objectives: To examine in detail the misfolded SOD1 protein in spinal cords from five strains of transgenic mice carrying different SOD1 variants. Mice expressing stable forms of human SOD1 (wt-hSOD1, G93A and D90A) are compared to mice expressing unstable variants (G85R and G127insTGGG)
Methods: Spinal cord extracts were made from fresh non-frozen tissues of pre-symptomatic mice (around 100 days old, n = 3 for each strain). The extracts were separated by size exclusion chromatography (SEC) and eluted fractions analyzed for total SOD1 by Western immunoblots and for misfolded SOD1 by misELISA. Parts of eluted fractions from some SECs were crosslinked to reveal any protein-protein interactions including oligomerizations.
Results: The same pattern of misfolded SOD1 was found in mice expressing widely different SOD1 variants. In all mice examined, the misfolded SOD1 was eluted from SEC as two main peaks with higher apparent molecular weight than the normal SOD1 dimer. The molecular weights of these peaks were estimated to be 60 kDa and 200 kDa by comparison with calibrators of known molecular weight. By crosslinking, the misfolded SOD1 in the 60 kDa peak was shown to be monomeric while the SOD1 in the 200 kDa peak was composed of at least two oligomeric forms or bound to other cellular components.
Discussion: The main form of misfolded SOD1 in different transgenic mice is monomeric but with a significantly enlarged hydrodynamic radius which results in early SEC elution. The misfolded SOD1 with higher molecular weight are likely bound to chaperones but oligomeric forms may also exist. Misfolded soluble SOD1 is highly likely to be a precursor to large and smaller aggregates, the latter recently shown to facilitate spread of cytotoxicity in a prionoid manner.
References
- Zetterstrom P, Stewart HG, Bergemalm D, et al. Proc Natl Acad Sci USA. 2007;104:14157–62.
- Forsberg K, Jonsson PA, Andersen PM, et al. PLoS One 2010;5:e11552.
- Zetterström P, Andersen PM, Brännström T et al. J Neurochem 2011;117:91–9.
C48 A SEEDED AGGREGATION OF TDP-43 REPRODUCES THE INTRACELLULAR FORMATION OF SARKOSYL-INSOLUBLE INCLUSIONS
FURUKAWA Y1,2, KANEKO K2, WATANABE S2, YAMANAKA K2, NUKINA N2
1Keio University, Yokohama, Kanagawa, Japan, 2RIKEN, Brain Science Institute, Wako, Saitama, Japan
Email address for correspondence: [email protected]
Keywords: TDP-43, seeding, protein fibril
Background: A DNA/RNA-binding protein, TDP-43, was identified as a constituent of inclusions in most cases of sporadic ALS. Under pathological conditions, TDP-43 proteins form aggregates that are resistant to be solubilized by a detergent, sarkosyl. Simple overexpression of TDP-43 and its variants in cells have been reported to induce the formation of inclusions, but no cultured cell models are currently available in which sarkosyl-insoluble TDP-43 aggregates are reproduced.
Pathological TDP-43 aggregates are known to possess amyloid-like fibrillar morphologies. In general, formation of amyloid-like aggregates is accelerated by a seeding reaction, in which a pre-formed fibril functions as a structural template upon the recruitment of soluble protein into insoluble fibrils. While a seeded aggregation of pathogenic proteins has been proposed in several other neurodegenerative diseases, it remains unknown whether a seeding mechanism describes formation of sarkosyl-insoluble TDP-43 aggregates in cells.
Objectives: To understand a molecular mechanism of intracellular TDP-43 aggregation, we have first characterized TDP-43 fibrils in vitro and then examined the seeding ability of those in vitro fibrils to trigger the formation of sarkosyl-insoluble TDP-43 aggregates in cells.
Methods and results: Overexpression of TDP-43 in E. coli has led to the formation of inclusion bodies, which can be resolubilized with guanidine hydrochloride. Dilution of the guanidine hydrochloride concentration made it possible to refold our in vitro TDP-43; indeed, a filter-binding assay showed that refolded TDP-43 preferentially recognizes TG repeats of single-stranded DNA. These refolded and functional TDP-43 proteins were found to form sarkosyl-insoluble aggregates by constant agitation at 37 oC overnight. In addition, fibrillar morphologies of in vitro TDP-43 aggregates were confirmed by an electron microscopy.
TDP-43 fibrils in vitro were then transduced into HEK293T cells by using the BioPORTER protein delivery reagent. A TDP-43 variant expressed in a cell is C-terminally fused with an HA tag (TDP-43-HA), which discriminates intracellularly expressed TDP-43-HA from exogenously added TDP-43 fibrils. When in vitro TDP-43 fibrils were transduced into cells, we have found that intracellular TDP-43-HA forms sarkosyl-insoluble aggregates.
Discussion and conclusions: TDP-43 fibrils were found to exhibit a seeding activity in vitro and in vivo, which would reproduce the pathological formation of Sarkosyl-insoluble TDP-43 inclusions in the cell. It remains controversial whether the aggregation of TDP-43 is a cause or a result of the disease; however, as recently proposed in the other neurodegenerative diseases, a seeding activity of TDP-43 proteins may contribute to the propagation of pathological changes with the progression of diseases.