Research articleDNAJC13 p.Asn855Ser, implicated in familial parkinsonism, alters membrane dynamics of sorting nexin 1
Introduction
Parkinson’s disease (PD) is a chronically progressive, irreversible neurodegenerative disorder characterized by the loss of dopaminergic neurons of the Substantia Nigra pars compacta (SNpc) and proteinaceous inclusions rich in α-synuclein (Lewy bodies; LB). The etiology is challenging to define in most sporadic patients as it is multifactorial. However, highly-penetrant, albeit rare mutations have implicated select cellular pathways in parkinsonism [1]. Of note, mutations in LRRK2, SNCA and VPS35 all result in an autosomal dominant late-onset syndrome that most closely resembles idiopathic PD [[2], [3], [4], [5]]. These proteins functionally converge on synaptic function/transmission and endo-lysosomal trafficking [[6], [7], [8]]. In 2014 DNAJC13 (coding receptor-mediated endocytosis-8; RME-8) was linked to late-onset autosomal dominant parkinsonism in a multi-incident Mennonite kindred with post-mortem SNpc neuronal loss and Lewy body (LB) pathology [3,9]. Although the underlying mechanism for disease is unknown, prior studies on DNAJC13 p.Asn855Ser describe impaired recycling of membrane-associated receptors and α-synuclein accumulation [3,10].
RME-8 is a ˜254 kDa protein consisting of 2243 amino acids that localizes to early endosomal compartments via an N-terminal PI(3)P targeting motif (1–453 aa) [11]. The central J domain (1301–1366 aa) binds the co-chaperone Heat shock cognate-70 (Hsc70) [12], whereas the remainder interacts with the BAR (Bin-Amphiphysin-Rvs)-domain containing sorting nexin 1 (SNX1) protein and the FAM21 subunit of the WASH complex (composed of WASH1, strumpellin, KIAA1033, FAM21 and CCDC53 proteins) [13,14]. Loss-of-function produced by depletion of RME-8 in immortalized cell lines compromises the organization of the endosomal network, induces extensive SNX1-positive tubule formation/stabilization and impairs sorting of retromer-dependent cargos [[11], [12], [13],15].
Collectively, SNX1 and RME-8 likely cooperate to partition endocytosed membrane and associated cargos into alternate pathways for: 1) recycling, via retromer complexes, versus; 2) endosomal retention via ESCRT (Endosomal Sorting Complexes Required for Transport) for subsequent lysosomal autophagy degradation [16].
To study the effects of DNAJC13p.Asn855Ser mutant gene dysfunction in a physiological context, and to help define molecular mechanisms for disease, we have established a Dnajc13 p.Asn855Ser knock-in (DKI) mouse model. The DNAJC13 linkage assignment has been debated [[17], [18], [19]] and now biologic investigations must resolve the issue. Here, we show that endogenous expression of Rme-8 p.Asn855Ser in mature neuronal cultures [Days in Vitro 21 (DIV21)] parallels the transient depletion of wild-type RME-8 and results in an increased percentage of GFP-SNX1 positive tubular structures. Notably, increased tubular formation observed in DKI cultures is not a consequence of differences in GFP-SNX1 puncta density or area, nor is the association of Rme-8 with SNX1, retromer or WASH complexes overtly perturbed. Rather Dnajc13p.Asn855Ser expression induces a dominant-negative gain-of-function.
Section snippets
Mouse model generation
Conditional knock-in mice (cDKI) were generated by TaconicArtemis GmbH (Cologne, Germany) using a gene targeting approach in C57BL/6N Tac embryo stem cells. Genetic engineering targeted the Dnajc13 locus on murine chromosome 9 (104,151,282-104,262,930 bp reverse strand, GRCm38.p4); Ensemble genomic reference sequence ENSMUSG00000032560; NCBI mRNA reference sequence NM_001163026.1. The targeting vector contained: 1) a 5’ long homology arm encompassing exons 21 and 22 of the wild-type murine
Dnajc13 p.Asn855Ser knock-in mice
To examine the effects of the DNAJC13 p.Asn855Ser mutation at physiological levels, and avoid random insertion and overexpression, C57BL/6J mice were engineered to express Dnajc13 exon 24 c.2564A > G (NM_001163026.1; p.Asn855Ser) (Fig. 1A); PCR-based genotyping of genomic DNA was used to confirm the indicated genotypes (Fig. 1B). Wild-type (WT) and mutant allele-specific Dnajc13 mRNA expression was equimolar and unaltered by gene targeting in DKI animals (Fig. 1C; p = 0.77) and successful
Discussion
In this study, we explored the effect of Dnajc13 p.Asn855Ser expression on the membrane dynamics of SNX1 in primary cortical neurons using a novel DKI mouse model. The expression levels of Rme-8 were consistent between WT and DKI cultures, as were the steady-state levels of known interacting partners such as retromer and WASH complexes subunits. The recruitment of the retromer and WASH complexes to endosomal membranes is unaffected by RME-8 expression (and is unaltered in DKI cultures)
Competing interests
The authors declare no competing interests.
Funding
The work was only made possible with funds from the Canada Excellence Research Chair, British Columbia Leadership Endowment funds and the Canadian Foundation for Innovation (MF).
Author contributions
JF conceptualization, project design and administration, investigation, data curation, formal analysis, writing-original draft, editing
JDF investigation, formal analysis, writing-original draft, editing
EG investigation, formal analysis, editing
CK investigation; performed imaging in Fig. 4A.
LNM investigation; performed imaging in Fig. 4A
LPC colony management and cell culture, editing
IT investigation, formal analysis, editing
AJM investigation; performed imaging in Fig. 4A
MJF identified DNAJC13
Acknowledgements
We sincerely appreciate the efforts of staff at the Centre of Disease Modelling, University of British Columbia, for animal husbandry. We thank Dr. Lucia Tapia for her help in testing RME-8 antibodies.
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