ABSTRACT
Alpha-Synuclein (alpha-Syn) accounts, as a major component of Lewy bodies (LB), for the filamentous deposits in many cases of neurodegenerative diseases. Yet, little is known about the molecular mechanisms of neuronal loss in these diseases. The correlation between alpha-Syn oligomerization/aggregation and pathologies raises the key question of which molecular form of alpha-Syn (i.e. monomeric alpha-Syn, protofibrils or mature fibrils) represents the damage-inducing culprit in the scenario of synucleinopathies. We show that human alpha-Syn protofibrils (PFs) are potent activators of parallel proinflammatory signalling pathways (p38 and ERK1/2 MAP kinases and NF-kappaB) in microglial cells in vitro. Furthermore, stereotactic injection of alpha-Syn PFs into the substantia nigra of adult rats leads to a profound activation of microglia and adjacent neuronal cell loss, which can be attenuated by the MAP kinase inhibitor semapimod. We propose that the neurodegenerative process of alpha-synucleinopathies involves microglial activation through alpha-Syn released or extruded from cells with pathogenic alpha-Syn metabolism. Compounds that inhibit the MAPK/NF-kappaB pathways might be a promising pharmacological strategy for the treatment of the inflammatory component of synucleinopathies including PD.
Subject(s)
Hydrazones/pharmacology , Microglia/drug effects , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Neurons/drug effects , Protein Kinase Inhibitors/pharmacology , alpha-Synuclein/metabolism , Animals , Animals, Newborn , Cell Death/drug effects , Cells, Cultured , Coculture Techniques , Humans , Male , Microglia/enzymology , Microglia/pathology , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Neurons/enzymology , Neurons/pathology , Rats , Rats, Wistar , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Time Factors , Transfection , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
alpha-Synuclein (alpha-SYN) is deposited in intraneuronal cytoplasmic inclusions (Lewy bodies, LBs) characteristic for Parkinson's disease (PD) and LB dementias. alpha-SYN forms LB-like fibrils in vitro, in contrast to its homologue beta-SYN. Here we have investigated the solubility of SYNs in human LB diseases and in transgenic mice expressing human wild-type and PD-associated mutant [A30P]alpha-SYN driven by the brain neuron-specific promoter, Thy1. Distinct alpha-SYN species were detected in the detergent-insoluble fractions from brains of patients with PD, dementia with LBs, and neurodegeneration with brain iron accumulation type 1 (formerly known as Hallervorden-Spatz disease). Using the same extraction method, detergent-insolubility of human alpha-SYN was observed in brains of transgenic mice. In contrast, neither endogenous mouse alpha-SYN nor beta-SYN were detected in detergent-insoluble fractions from transgenic mouse brains. The nonamyloidogenic beta-SYN was incapable of forming insoluble fibrils because amino acids 73 to 83 in the central region of alpha-SYN are absent in beta-SYN. In conclusion, the specific accumulation of detergent-insoluble alpha-SYN in transgenic mice recapitulates a pivotal feature of human LB diseases.
Subject(s)
Lewy Body Disease/metabolism , Nerve Tissue Proteins/metabolism , Amino Acids/genetics , Animals , Binding Sites/genetics , Blotting, Western , Brain/metabolism , Brain/pathology , Detergents , Disease Models, Animal , Humans , Lewy Body Disease/genetics , Lewy Body Disease/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/genetics , Solubility , Subcellular Fractions , Synaptosomes/metabolism , Synucleins , alpha-SynucleinABSTRACT
The mitochondrial ADP/ATP carrier (AAC) is generally believed to function as a homodimer (Wt. Wt). It remains unknown whether the two monomers possess two independent but fully anticooperative channels or they form a single central channel for nucleotide transport. Here we generated fusion proteins consisting of two tandem covalent-linked AAC monomers and studied the kinetics of ADP/ATP transport in reconstituted proteoliposomes. Functional 64-kDa fusion proteins Wt-Wt and Wt-R294A (wild-type AAC linked to a mutant having low ATP transport activity) were expressed in mitochondria of yeast transformants. Compared to homodimer Wt. Wt, the fusion protein Wt-Wt retained the transport activity and selectivity of ADP versus ATP. The strongly divergent selectivities of Wt and R294A were partially propagated in the Wt-R294A fusion protein, suggesting a limited cooperativity during solute translocation. The rates of ADP or ATP transport were significantly higher than those predicted by the two-channel model. Fusion proteins for Wt-R204L (Wt linked to an inactive mutant) and R204L-Wt were not expressed in aerobically grown yeast cells, which contained plasmid rearrangements that regenerated the fully active 32-kDa homodimer Wt. Wt, suggesting that these fusion proteins are inactive in ADP/ATP transport. These results favor a single binding center gated pore model [Klingenberg, M. (1991) in A Study of Enzymes, Vol. 2: pp. 367-388] in which two AAC subunits cooperate for a coordinated ADP/ATP exchange through a single channel.
Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Mitochondrial ADP, ATP Translocases/chemistry , Mitochondrial ADP, ATP Translocases/metabolism , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Biological Transport , Blotting, Western , Dimerization , Intracellular Membranes/chemistry , Intracellular Membranes/metabolism , Kinetics , Mitochondria/chemistry , Mitochondria/metabolism , Mitochondrial ADP, ATP Translocases/genetics , Molecular Weight , Neurospora crassa/enzymology , Neurospora crassa/genetics , Protein Conformation , Proteolipids/chemistry , Proteolipids/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/geneticsABSTRACT
Previously, the role of residues in the ADP/ATP carrier (AAC) from Saccharomyces cerevisiae has been studied by mutagenesis, but the dependence of mitochondrial biogenesis on functional AAC impedes segregation of the mutational effects on transport and biogenesis. Unlike other mitochondrial carriers, expression of the AAC from yeast or mammalians in Escherichia coli encountered difficulties because of disparate codon usage. Here we introduce the AAC from Neurospora crassa in E. coli, where it is accumulated in inclusion bodies and establish the reconstitution conditions. AAC expressed with heat shock vector gave higher activity than with pET-3a. Transport activity was absolutely dependent on cardiolipin. The 10 single mutations of intrahelical positive residues and of the matrix repeat (+X+) motif resulted in lower activity, except of R245A. R143A had decreased sensitivity toward carboxyatractylate. The ATP-linked exchange is generally more affected than ADP exchange. This reflects a charge network that propagates positive charge defects to ATP(4-) more strongly than to ADP(3-) transport. Comparison to the homologous mutants of yeast AAC2 permits attribution of the roles of these residues more to ADP/ATP transport or to AAC import into mitochondria.
