Abstract
A major hallmark of Parkinson’s disease (PD) is the presence of Lewy bodies (LBs) in certain neuronal tissues. LBs are protein-rich inclusions, in which α-synuclein (α-syn) is the most abundant protein. Since these inclusions are not present in healthy individuals, despite the high concentration of α-syn in neurons, it is important to investigate whether natural control mechanisms are present to efficiently suppress α-syn aggregation. Here, we demonstrate that a CRISPR/Cas9-mediated knockout (KO) of a DnaJ protein, DNAJB6, in HEK293T cells expressing α-syn, causes a massive increase in α-syn aggregation. Upon DNAJB6 re-introduction into these DNAJB6-KO HEK293T-α-syn cells, aggregation is reduced to the level of the parental cells. We then show that the suppression of α-syn aggregation is dependent on the J-domain of DNAJB6, as the catalytically inactive protein, which carries the H31Q mutation, does not suppress aggregation, when re-introduced into DNAJB6-KO cells. We further demonstrate, that the suppression of α-syn aggregation is dependent on the molecular chaperone Hsp70, which is consistent with the well-known function of J-domains of transferring unfolded and misfolded proteins to Hsp70. These data identify a natural control strategy to suppress α-syn aggregation and suggest potential therapeutic approaches to prevent or treat PD and related disorders.
Highlights
A major hallmark of Parkinson’s disease (PD) is the presence of Lewy bodies (LBs) in certain neuronal tissues
In order to assess if DNAJB6 can act as an endogenous suppressor of α-syn aggregation, we designed RNA guides to target the DNAJB6 gene and used the CRISPR/Cas[9] system[21] for disruption of endogenous expression of the DNAJB6 gene in HEK293T cells
As seen by the western blot stained for DNAJB6, there are 2 splice forms of the protein resulting in a longer isoform (DNAJB6a) and a shorter one (DNAJB6b) (Fig. 1A)
Summary
A major hallmark of Parkinson’s disease (PD) is the presence of Lewy bodies (LBs) in certain neuronal tissues. That the suppression of α-syn aggregation is dependent on the molecular chaperone Hsp[70], which is consistent with the well-known function of J-domains of transferring unfolded and misfolded proteins to Hsp[70] These data identify a natural control strategy to suppress α-syn aggregation and suggest potential therapeutic approaches to prevent or treat PD and related disorders. The cardinal motor symptoms of PD are primarily associated with the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the brain[1,2,3,4] This neurodegenerative process correlates with the formation of large protein-rich cytoplasmic inclusions, known as Lewy bodies (LBs), in which aggregated α-synuclein (α-syn) is the main protein component[2,3,4,5]. DNAJB6 is able to inhibit the formation of amyloid aggregates of proteins and peptides, such as Aβ17 and polyQ18–20, when overexpressed in cell www.nature.com/scientificreports/
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