Accumulation and clearance of α‐synuclein aggregates demonstrated by time‐lapse imaging

Abstract

1Authors share senior authorship.Abbreviations used: 3MA, 3-methyladenine; ALLN, N-acetyl-leucylleucyl-norleucinal; EGFP, enhanced green fluorescent protein; PD, Parkinson's disease; WT, wildtype; DC, C-terminally truncated a-synuclein consisting of amino acids 1-108. AbstractAggregates of a-synuclein are the pathological hallmark of sporadic Parkinson's disease (PD), and mutations in the a-synuclein gene underlie familial forms of the disease. To characterize the formation of a-synuclein aggregates in living cells, we developed a new strategy to visualize a-synuclein by fluorescence microscopy: a-synuclein was tagged with a six amino acid PDZ binding motif and co-expressed with the corresponding PDZ domain fused to enhanced green fluorescent protein (EGFP). In contrast to the traditional approach of a-synuclein-EGFP fusion proteins, this technique provided several-fold higher sensitivity; this allowed us to compare a-synuclein variants and perform time-lapse imaging. A C-terminally truncated a-synuclein variant showed the highest prevalence of aggregates and toxicity, consistent with stabilization of the a-synuclein monomer by its C-terminus. Timelapse imaging illustrated how cells form and accumulate aggregates of a-synuclein. A substantial number of cells also reduced their aggregate load, primarily through formation of an aggresome, which could itself be cleared from the cell. The molecular chaperone Hsp70 not only prevented the formation of aggregates, but also increased their reduction and clearance, underlining the therapeutic potential of similar strategies. In contrast to earlier assumptions build-up, reduction and clearance of a-synuclein aggregation thus appear a highly dynamic process. Keywords: aggregation, aggresomes, molecular chaperones, time-lapse imaging, a-synuclein. J. Neurochem. (2008) 106, 529-540. JOURNAL OF NEUROCHEMISTRY | 2008 | 106 | 529-540 doi: 10.1111/j.1471-4159.2008 Next to WT and the two disease-related mutations A30P and A53T, we also used a C-terminally deleted variant, which had shown increased aggregation in vitro (Bertoncini et al. 2005b). Furthermore, we wanted to characterize the in vivo effects of the molecular chaperone Hsp70, which has been shown to reduce the formation of a-synuclein fibrils observed in vitro (Dedmon et al. 2005) and the prevalence of aggregates in vivo (McLean et al. 2002;Klucken et al. 2004). Experimental procedures CloningFor the fusion protein of PDZ domain and EGFP (PDZ-EGFP), the PDZ1 domain of S-SCAM (accession NP_446073, a gift from the lab of Nils Brose, MPI-em, Göttingen) was cloned into pEGFP-N1, resulting in PDZ-EGFP (Fw: 5¢-ATTAGATCTATGGGAACATTC-CTCAGCACCAC-3¢; Rev: 5¢-ATTACCGGTTCCTCCGGATATGT-GCCAT CTAGTTGG-3¢). The corresponding PDZ binding motif (amio acids HSTTRV, from neuroligin-1, accession NP_446320) was added to the C-termini of a-synuclein variants by PCR (Fw: 5¢-CTCAAGCTTATGGATGTATTCATG-3¢; Rev: 5¢-ATGG-ATATCTCACACTCTGGTGGTGCTGTGGGCTTCAGGTTCG-3¢; DCRev: 5¢-GTTGATATCTCACACTCTGGTGGTGCTGTGTGG-GGCTCCTTCTTC-3¢) and PDZ-EGFP was subclo...

14-3-3 targets chaperone-associated misfolded proteins to aggresomes

“…It is known that overexpression of a-Syn leads to formation of a specific type of inclusions termed aggresome, which is uniquely located in the vicinity of the centrosome and assembled by retrograde transport of dynein motors along the microtubule network (Opazo et al, 2008;Tanaka et al, 2004). Consistently, these perinuclear structures were enriched with vimentin, a known aggresome marker that displayed homogeneous distribution in cells without aggresome formation (Fig.…”

Kinetic properties of ASC protein aggregation in epithelial cells

“…In addition, since specks highly resemble perinuclear structures called aggresomes (Opazo et al, 2008), we examined ASC specks for the expression of several markers of aggresome formation. Aggresomes are large perinuclear collections of misfolded proteins that dynamically form and are subsequently dismantled by lysosomes during autophagy (Opazo et al, 2008).…”

The Hsp70 Chaperone System in Parkinson’s Disease

“…Following the discovery that Hsp70 can abrogate the neurotoxicity of abnormal polyglutamine proteins (Warrick et al, 1999), it was shown that Hsp70 can also prevent dopaminergic neuronal loss associated with αSyn in a Drosophila PD model (Auluck et al, 2002). Numerous studies that followed have reported that over-expression of Hsp70 is able to reduce αSyn aggregation and/or toxicity in various cellular models Klucken et al, 2004b;McLean et al, 2004;Opazo et al, 2008;Outeiro et al, 2008;Zhou et al, 2004). In particular, McLean and co-workers (Outeiro et al, 2008) have found that Hsp70 rescues αSyn-linked toxicity by promoting the cellular clearance of αSyn oligomers, rather than monomers.…”

“…In particular, McLean and co-workers (Outeiro et al, 2008) have found that Hsp70 rescues αSyn-linked toxicity by promoting the cellular clearance of αSyn oligomers, rather than monomers. Another study (Opazo et al, 2008) found that Hsp70 manages αSyn intracellular aggregation by increasing the clearing of aggregates primarily through the aggresome, and the subsequent removal of small aggregates and aggresomes from the cytosol. An interesting study by McLean and colleagues has recently shown that Hsp70 can also inhibit the formation of extracellular αSyn oligomers and rescue the cytotoxicity produced by such secreted oligomers, in a cellular model.…”