Intracellular amyloid formation in muscle cells of Aβ-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification

Alicia N Minniti 2 Daniela L Rebolledo 2 Paula M Grez 2 Ricardo Fadic 1 Rebeca Aldunate 2 Irene Volitakis 0 Robert A Cherny 0 Carlos Opazo 4 Colin Masters 0 Ashley I Bush 0 3 Nibaldo C Inestrosa 2 0 Oxidation Disorders Laboratory, Mental Health Research Institute of Victoria and Department of Pathology, University of Melbourne , Parkville, Victoria 3052 , Australia 1 Departamento de Neurologia, Facultad de Medicina, Pontificia Universidad Catolica de Chile , Santiago , Chile 2 Centro de Regulacion Celular y Patologia "Joaquin V. Luco" (CRCP), MIFAB, Centro de Envejecimiento y Regeneracion (CARE), Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile , Alameda 340, 8331010 Santiago , Chile 3 Department of Psychiatry, Harvard Medical School, Massachusetts General Hospital , Charlestown, Massachusetts , USA 4 Laboratorio de Neurobiometales, Departamento de Fisiologia, Facultad de Ciencias Biologicas, Universidad de Concepcion , Chile Background: The amyloid -peptide is a ubiquitous peptide, which is prone to aggregate forming soluble toxic oligomers and insoluble less-toxic aggregates. The intrinsic and external/ environmental factors that determine A aggregation in vivo are poorly understood, as well as the cellular meaning of this process itself. Genetic data as well as cell biological and biochemical evidence strongly support the hypothesis that A is a major player in the onset and development of Alzheimer's disease. In addition, it is also known that A is involved in Inclusion Body Myositis, a common myopathy of the elderly in which the peptide accumulates intracellularly. Results: In the present work, we found that intracellular A aggregation in muscle cells of Caenorhabditis elegans overexpressing A peptide is affected by two single amino acid substitutions, E22G (Arctic) and V18A (NIC). Both variations show decrease intracellular amyloidogenesis compared to wild type A. We show that intracellular amyloid aggregation of wild type A is accelerated by Cu2+ and diminished by copper chelators. Moreover, we demonstrate through toxicity and behavioral assays that A-transgenic worms display a higher tolerance to Cu2+ toxic effects and that this resistance may be linked to the formation of amyloid aggregates. Conclusion: Our data show that intracellular A amyloid aggregates may trap excess of free Cu2+ buffering its cytotoxic effects and that accelerated intracellular A aggregation may be part of a cell protective mechanism. - Background Aberrant protein aggregation is a common feature of lateonset amyloidogenic diseases, such as Alzheimer's disease (AD) and Inclusion Body Myositis (IBM) [1]. A peptide of 3943 mer derived from the amyloid precursor protein (APP), the amyloid -peptide (A), is the main constituent of senile plaques (SPs) in AD [2], and it is also one of the hallmarks of IBM, a common myopathy characterized by the presence of intracellular amyloid aggregates in skeletal muscle cells [1,3]. IBM patients show progressive muscle weakness, compromised muscle innervation [4] and muscle fibre degeneration [5]. Besides the presence of senile plaque-like inclusions, most molecules known to be involved in IBM are also present in AD [6]. Dominant mutations in the APP gene are associated with rare cases of familial AD, in which brain and vascular amyloid deposits are formed earlier than in sporadic cases [7]. One type of familial AD is linked to a point mutation in the A peptide (E22G, Arctic) that accelerates aggregation of A into protofibrils and fibrils in vitro [8-12]. Some transgenic mice carrying the Arctic mutation show increased plaque formation but normal learning and memory compared to strains carrying the wild type A [13]. Opposite to the Arctic variant, in vitro studies demonstrated that the NIC mutation (V18A), which is predicted to favour the helix conformation over the sheet conformation, shows decreased aggregation [14]. Although APP and A have been implicated in several processes in vitro and in vivo, such as neuronal development and cell survival, the in vivo functions of APP and A remain unclear [7,15,16]. Moreover, the inactivation of the complete APP gene family has shown that APP is necessary for neurodevelopment and cell adhesion [17]. In addition, neither the in vivo mechanisms of amyloid formation nor the factors required for this process have been properly established. It is also unknown if the formation of insoluble amyloid aggregates is instrumental for the onset of these amyloidogenic diseases, or if the formation of amyloid structures is the end product of the cell protective machinery. SPs are structured as metal-enriched aggregates that accumulate Cu2+, Fe3+, and Zn2+ [18-20]. In agreement with this, the aggregation state of A-peptide is increased by Cu2+ or Zn2+ [21] and reduced by metal chelators such as clioquinol [22-24]. However, the role of copper as deleterious or beneficial in the context of amyloidogenic diseases is very controversial. For instance, in a transgenic APP23 mouse model, supplementation of the diet with bioavailable copper restored normal levels of SOD-1 activity and decreased the production of soluble A [25]. From these studies, the structural plasticity of the SP is becoming evident in agreement with previous structural data [26]. In light of this evidence we postulate that the formation of the SP might be triggered by fluctuations on the levels of transition metals, and could be a part of a protective homeostatic mechanism gone off-course. However, it is unknown if these metals are accumulated in the intracellular amyloid aggregates observed in IBM and if the aggregation state of A is modulated by transition metals in this intracellular muscle environment. Interestingly, IBM patients do not develop dementia and AD patients do not have the muscle weakness characteristic of IBM, which indicates that these diseases may be triggered by independent mechanisms [1]. Caenorhabditis elegans has an APP homologue but this protein lacks a region equivalent to the A-peptide [27]. However, several studies carried out in A overexpressing C. elegans [6] have established that the formation of amyloid deposits [28] could induce oxidative stress [29], stress response [30,31] and up- or down-regulation of different genes [32]. However, the toxic A-species responsible for these effects detected in A-transgenic worms have not been thoroughly identified, but they are likely to be a specific type of A oligomers rather than mature A amyloid aggregates [33-35]. It is important to mention that the paralysis phenotype is only observed in A expressing C. elegans in a dominant rol-6 background (strains carrying a mutation in a collagen gene) [32]. In fact, the great majority of IBM patients show muscle weakness that translates in a lower quality of life but not paralysis or decreased longevity [36]. In the present work, we evaluated intrinsic and extrinsic factors on the aggregation of intracellular A pe (...truncated)