Elsevier

Neuroscience Letters

Volume 706, 27 July 2019, Pages 114-122
Neuroscience Letters

Research article
DNAJC13 p.Asn855Ser, implicated in familial parkinsonism, alters membrane dynamics of sorting nexin 1

https://doi.org/10.1016/j.neulet.2019.04.043Get rights and content

Highlights

  • Parkinson’s disease causing mutation (DNAJC13 p.Asn855Ser) does not alter DNAJC13/RME-8 levels.

  • SNX1 membrane dynamics are altered in a DNAJC13 p.Asn855Ser knock-in (DKI) mouse model.

  • DNAJC13 p.Asn855Ser does not disrupt RME-8 binding to SNX1 or Retromer-WASH complexes.

Abstract

DNAJC13 (RME-8) is a core co-chaperone that facilitates membrane recycling and cargo sorting of endocytosed proteins. DNAJ/Hsp40 (heat shock protein 40) proteins are highly conserved throughout evolution and mediate the folding of nascent proteins, and the unfolding, refolding or degradation of misfolded proteins while assisting in associated-membrane translocation. DNAJC13 is one of five DNAJ ‘C’ class chaperone variants implicated in monogenic parkinsonism. Here we examine the effect of the DNAJC13 disease-linked mutation (p.Asn855Ser) on its interacting partners, focusing on sorting nexin 1 (SNX1) membrane dynamics in primary cortical neurons derived from a novel Dnajc13 p.Asn855Ser knock-in (DKI) mouse model. Dnajc13 p.Asn855Ser mutant and wild type protein expression were equivalent in mature heterozygous cultures (DIV21). While SNX1-positive puncta density, area, and WASH-retromer assembly were comparable between cultures derived from DKI and wild type littermates, the formation of SNX1-enriched tubules in DKI neuronal cultures was significantly increased. Thus, Dnajc13 p.Asn855Ser disrupts SNX1 membrane-tubulation and trafficking, analogous to results from RME-8 depletion studies. The data suggest the mutation confers a dominant-negative gain-of-function in RME-8. Implications for the pathogenesis of Parkinson’s disease are discussed.

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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