VPS13A is a multitasking protein at the crossroads between organelle communication and protein homeostasis

University of Groningen

VPS13A is a multitasking protein at the crossroads between organelle communication and protein homeostasis

Yeshaw, Wondwossen Melaku

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VPS13A IS A MULTITASKING PROTEIN AT THE CROSSROADS BETWEEN ORGANELLE COMMUNICATION

AND PROTEIN HOMEOSTASIS

Wondwossen Melaku Yeshaw

The research described in this thesis was conducted at the Department of Cell Biology, University Medical Center Groningen, the Netherlands. Printing of this thesis was financially supported by University of Groningen, University Medical Center Groningen and Graduate School of Medical Sciences, Groningen.

ISBN: 978-94-034-0767-8 ISBN: 978-94-034-0768-5 (Ebook)

Copyright©2018 W.M. Yeshaw. All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means without prior written permission of the author.

Cover and layout design by Thesisexpert.nl

Printed by Gildeprint, Enschede, the Netherlands

VPS13A IS A MULTITASKING PROTEIN AT THE CROSSROADS BETWEEN ORGANELLE COMMUNICATION

AND PROTEIN HOMEOSTASIS

PhD thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus Prof. E. Sterken

and in accordance with the decision by the College of Deans.

This thesis will be defended in public on Monday 18 June 2018 at 11.00 hours

by

Wondwossen Melaku Yeshaw

born on 29 May 1986

in Haik, Ethiopia

Supervisor Prof. O.C.M. Sibon

Assessment Committee

Prof. E.A.A. Nollen

Prof. J.W. Jonker

Prof. S.M.A. Lens

TABLE OF CONTENTS

Chapter 1 Introduction and aim of the thesis 7

Chapter 2 Drosophila Vps13 is required for protein homeostasis in the brain 21 Chapter 3 Drosophila Vps13 mutants show overgrowth of larval neuromuscular Junctions 49

Chapter 4 Human VPS13A is associated with multiple organelles and required for lipid droplet homeostasis 65

Chapter 5 Summarizing discussion and perspectives 105

Appendices Nederlandse samenvatting 123

Acknowledgements 127

Curriculum vitae 131

List of publications 133

CHAPTER 1

Introduction and aim of the thesis

INTRODUCTION

Chorea-Acanthocytosis

Chorea-Acanthocytosis (ChAc) (MIM 200150) is a rare autosomal recessive neurodegenerative disorder and member of a family of neurological disorders broadly known as neuroacanthocytosis (NA) syndromes

. NA involves neurological abnormalities coupled with the presence of abnormally spiked red blood cells (acanthocytes) in the peripheral blood circulation

. NA syndromes are broadly classified into two categories; the “core” NA syndromes and NA with lipoprotein disorders

. The “core” NA group consists of ChAc, McLeod syndrome (MLS), Huntington’s disease-like 2 (HDL-2) and pantothenate kinase associated neurodegeneration (PKAN); all of which display degeneration of basal ganglia and acanthocytosis

. ChAc is characterized by progressive adult onset involuntary movements, behavioral and cognitive changes, oral dystonia and occasional seizures

. Increased creatine kinase levels and a 7-50%

acanthocytosis in blood circulation are common features of ChAc

. Causative mutations for the onset of ChAc are mapped on the Vacuolar Protein Sorting 13A (VPS13A) gene

. In most patients, these mutations lead to reduction or absence of detectable protein levels in red blood cells

and hence, Western blotting for VPS13A is used as a diagnostic tool in clinical setups

.

The main cause of red blood cell abnormalities and neurodegeneration in ChAc is largely unknown. In this chapter, we will describe a general background of VPS13 family proteins with emphasis on VPS13A. Domain architecture, subcellular localizations and functions of VPS13A will be discussed in the context of various ChAc model organisms.

The human VPS13 family proteins

The human VPS13 family consists of four ubiquitously expressed proteins (VPS13A, VPS13B, VPS13C and VPS13D) that share similarity with yeast Vps13

. Mutations in all human VPS13 genes are associated with the onset of neurological and developmental disorders. VPS13A, VPS13B, VPS13C and VPS13D are linked to the onsets of ChAc, Cohen syndrome, Parkinson’s disease and septic shock mortality respectively

. The VP13A gene spans 73 exons and is located on chromosome 9q21. There are two splicing variants of VPS13A (variant 1a and variant 1b). Variant 1a consists of exons 1-68 and 70-73 whereas variant 1b contains only exons 1-69

. Mutations in ChAc patients can be found distributed randomly throughout the VPS13A gene and so far there are no potential hotspots identified

.

VPS13B is mutated in patients with Cohen syndrome

. Cohen syndrome is a rare autosomal recessive

disorder characterized by obesity, motor clumsiness, microcephaly, mental retardation, neutropenia,

facial, oral and ocular abnormalities

. VPS13B is located on chromosome 8q22 and widely expressed

in a variety of human tissues and unlike VPS13A, its expression in adult brain is marginally low

. VPS13B is

required to maintain Golgi integrity and proper protein glycosylation

. Although it was initially predicted

to contain 10 transmembrane domains

, subcellular fractionation study shows that VPS13B is a peripheral

membrane protein localized at the Golgi where it interacts with Rab6 to regulate neurite outgrowth with a 1

mechanism that remains to be determined

.

VPS13C is more similar to VPS13A compared to other VPS13 family proteins

. VPS13C is located on chromosome 15q22 where truncating mutations and polymorphisms are causally linked to Parkinson’s disease

. In addition, mutations and single polynucleotide polymorphisms (SNPs) of VPS13C are associated with the risk of type 2 diabetes

. VPS13C is localized at the mitochondrial membrane and its absence aggravates mitochondrial fragmentation and clearance

.

VPS13D is a ubiquitin binding protein that regulates mitochondrial size and clearance both in Drosophila and human cultured cells

. Furthermore, a VPS13D gene variant is associated with increased septic shock mortality and overproduction of interleukin-6 (IL-6) in patients’ plasma and cultured cells

. Recent molecular autopsy analysis identified VPS13D gene mutation as one of the genes linked to early embryonic mortality

.

All of the human VPS13 family proteins share conserved N- and C-terminal domains

. Nonetheless, the diversity of diseases associated with different human VPS13 family proteins predict that each protein may function in different cellular pathways. Indeed, not all human VPS13 family proteins have similar subcellular localization patterns. VPS13B is localized to the Golgi complex while VPS13C is localized to mitochondria and lipid droplets (LDs)

. VPS13D, on the other hand, colocalizes with the lysosomal protein, LAMP1

. The biggest issues to be solved in VPS13A research are to define the localization of the protein in mammalian cells

and to identify functional domains of the VPS13A protein.

Domains of VPS13A

Sequence alignment studies identify multiple domains of VPS13A. The known domains of VPS13A include Chorein, two phenylalanines in an acidic tract (FFAT), short root transcription factor-binding domain (SHR- BD), aberrant pollen transcription 1 (APT1), ATG-C terminal domain (ATG-C) and pleckstrin homology (PH) domain (Figure 1)

.

Figure 1. Schematic representations of VPS13A and ATG2. Known domains of both proteins are labelled and similar domains are color-coded. FFAT (two phenylalanines in acidic tract), SHR-BD (short root transcription factor-binding domain), APT1 (aberrant

pollen transcription 1), ATG-C (ATG-C terminal domain) and PH (pleckstrin homology), ATG2-CAD (cysteine-alanine-aspartic acid

triad). CLR (C-terminal localization region)

.

The Chorein domain is an evolutionarily conserved domain with an unknown function

. The FFAT is a short stretch of amino acids commonly present in lipid transfer proteins with properties of building membrane contact sites with ER

. The APT1 domain was first identified in maize APT1 protein. APT1 colocalizes with a Golgi marker protein in tobacco pollen tubes and mutations in APT1 protein lead to defective pollen tube germination and transmission

. In vitro, Vps13 APT1 fragments bind specifically to PtdIns3p

. In the primary structure of VPS13A, the APT1 domain is located between SHR-BD and ATG-C domains

. SHR-BD is a highly conserved domain that was previously known as domain of unknown function 1162 (DUF1162)

. SHR-BD is present in vacuolar protein sorting (At5g24740) of A. thaliana. At5g24740 is also known as SHRUBBY and mutation in this gene leads to an aberrant root growth in Arabidopsis

. The SHR-BD fragment of Vps13 binds to a variety of phosphoinositides as well as to lysophosphatidic acid and phosphatidic acid

. Interestingly, the SHR-BD-APT1 fragment binds specifically to PtdIns3p unlike the SHR-BD alone, indicating that APT1 determines the specificity of lipid binding

. VPS13 also contains a PH domain and two ATG-C domains that are conserved in both yeast and human

.

A PH domain is composed of approximately 100 amino acids

and is considered as one of the most common domains in the human proteome. PH domain containing proteins are known for their affinities to phosphoinositides; specifically to those with a pair of adjacent phosphate groups such as (PtdIns(4,5) P2 and (PtdIns(3,4,5)P3

.

Additionally, VPS13A has two ATG-C domains that show homology with the C-terminal region of ATG2A

. There is a 25% identity between the C-terminal regions of VPS13A (aa 2939-3025) and ATG2A (aa 1830- 1916)(Figure 1)

. ATG2 proteins have a membrane binding ability and are essential for autophagy and LD distribution

. Similarly, VPS13A is also required to maintain proper autophagic flux

.

Cellular functions of VPS13A

Most of our current understanding about the cellular functions of Vps13 is derived from studies in

yeast

. Vps13 was first identified in a genetic screen for mutants displaying impaired delivery of

carboxypeptidase Y (CPY) to the vacuole

. Carboxypeptidase Y is a vacuolar protease synthesized in the

endoplasmic reticulum (ER) as pro-CPY. Pro-CPY is transported to the Golgi complex where glycosylation

occurs and subsequently delivered to the vacuole where modification to the active form takes place

.

Mutants that fail to transport CPY to the vacuolar compartment secrete pro-CPY to the periplasm and

ultimately to the extracellular medium

. By screening for secretion defects, together with morphological

examinations, 41 Vps mutant strains were identified. These mutants are grouped into six classes based on

their vacuolar morphology

. The different classes of Vps mutants and the description of their vacuolar

morphology is summarized in table 1.

1

Table 1. Classification of Vps mutants based on their vacuolar morphology. All Vps mutants secrete CPY at various degrees59.

Green circles (Vacuoles), small blue circles (fragmented vacuole like structures), orange circles (pre-vacuolar or class E compartment).

Class Vps mutant Characteristic A Vps8, Vps10,Vps13,Vps29

Vps30, Vps35,Vps38, Vps44 Vps46

Normal vacuolar morphology with 1-3 large vacuoles per cell.

B Vps5, Vps17,Vps39,Vps41 Vps43

Large number (20-40) of small and fragmented vacuolar like compartments.

C Vps11, Vps16,Vps18,Vps33 Severe defect of vacuole assembly. These mutants barely show vacuoles, but instead accumulate small fragmented vesicles.

D Vps3, Vps6,Vps9,Vps15 Vps19, Vps21, Vps34,Vps45

One large vacuole in the parent cell, which fails to be acidified and to segregate to budding daughter cells.

E Vps2, Vps4,Vps20,Vps22 Vps24, Vps25, Vps27,Vps28 Vps31,Vps32, Vps36, Vps37

Possess a different population of vesicles (prevacoular endosome like compartment) that contain proteins from both late Golgi and vacuole.

F Vps1, Vps26 Large central vacuole surrounded by

small fragments without any observable segregation defects.

As a member of class-A Vps mutants, Vps13 mutants possess morphologically normal vacuoles

. Further characterization revealed that Vps13 is a peripheral membrane protein involved in the transport of membrane bound proteins between the trans-Golgi network (TGN) and pre-vacuolar compartment (PVC)

or from endosome to vacuole

. In vps13 mutant cells, there is an increased secretion of insulin and pro-CPY

. In control cells pro-CPY is actively sorted from late Golgi to vacuole by the sorting receptor Vps10

. In Vps13 mutant strains however, Vps10 is mislocalized and rapidly degraded which accounts for an apparent extracellular secretion of pro-CPY

. Severe impairment in the production of viable spores is also an apparent phenotype of Vps13 mutants

.

At the earliest phase of sporulation, Vps13 is diffusely distributed throughout the cytoplasm. Whereas later

in meiosis, it is localized at the prospore membrane

. Compared to wild type strains, Vps13 mutants have

a few very small prospores that often fail to encapsulate nuclei

.

The cellular functions of VPS13 proteins are intricately broad. Studies in several model organisms revealed that VPS13A plays an array of conserved roles to maintain protein homeostasis, phosphoinositide metabolism, actin cytoskeleton, membrane contact sites and LD homeostasis.

Knock-out of one of the six Dictyostelium VPS13 genes (VPS13F) delays intracellular destruction of phagocytic cargo attributed to failure in sensing bacterial folate without affecting phagosome maturation

. Similarly, Tetrahymena VPS13A (TtVPS13A) decorates the phagosome membrane and is required for efficient clearance of phagocytic cargo

. In cultured insect cells, Vps13 depletion delays endocytic processing

. Another Dictyostelium VPS13 (TipC), was identified in a screen for mutations affecting tip formation

, similar to a phenotype that is observed in autophagy mutants

. tipc

cells accumulate ubiquitinated protein aggregates accompanied by a decreased number of GFP-LC3 and GFP-ATG18 puncta. In mammalian cells, depletion of VPS13A raises the number of GFP-LC3 puncta but decreases liberation of free GFP indicative for a slow autophagic flux

.

Both endocytic and autophagic degradation pathways are highly regulated by phosphoinositides

. Interestingly, synthetic genetic screens revealed that Vps13 mutants show similar sets of genetic interactions with Vps30 and Vps38

. Vps30 and Vps38 are the components of yeast complex I and complex II phosphatidylinositol 3-phosphate kinase (PI3K) complexes, respectively

. A plausible importance of Vps13 in phosphoinositide metabolism is further established as Vps13 directly binds to an array of phosphoinositides

. In addition, lipids -such as phosphatidic acid (PA), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4p) are reduced at the prospore membrane of Vps13 mutants

. Phosphoinositides regulate a multiplicity of cellular processes including actin polymerization and their mis-regulation is linked to a variety of human diseases

. Of importance, impaired actin polymerization is apparent in ChAc patient cells, VPS13A depleted cultured cells and Vps13 mutant yeast cells. In different organisms, VPS13A forms a complex with actin

.

Vps13 is localized at multiple membrane contact sites (MCS)

. MCSs regulate lipid distribution and maintain proper lipid gradients across membranes of different organelles

. The type and abundance of lipid species determines the subcellular localization of MCS proteins

. A number of proteins involved in MCSs are identified through bioinformatics, imaging, biochemical and synthetic biology screens

. ER occupies the largest intracellular space in eukaryotic cells and is essential for the biosynthesis of proteins and lipids and the ER regulates cellular calcium homeostasis. It is therefore not surprising that most organelles communicate with the ER

. Organelle communication is not limited to the ER and it is now clear that MCSs are established between LDs and mitochondria

, peroxisomes and mitochondria

, lysosomes and peroxisomes

, LDs and endosomes

, nucleus and vacuoles

and mitochondria and vacuoles

.

Vps13 is recruited to ER-Mitochondrial Encounter Structure (ERMES), vacuole and mitochondria patch

(vCLAMP) and NVJ (nuclear vacuole junction) depending on metabolic growth conditions

. ERMES

mutants are synthetically lethal when combined with Vps13 loss of function

. When harboring a

dominant point mutation (Vps13-D716H), Vps13 is able to restore growth defects of ERMES mutants

suggesting that Vps13 and ERMES are functionally redundant

. The mammalian ERMES counterpart

has yet to be identified and the role of VPS13A in organelle communication is unknown.

AIM AND OUTLINE OF THE THESIS 1

The overall aim of our research was to uncover the cellular functions of VPS13A in health and disease. The biggest hurdles to study the biology of VPS13A were the absence of reliable genetic model systems and limited biochemical and labelling tools. This is mainly attributed to the absence of antibodies that would detect endogenous VPS13A protein in immunolabelling experiments and partly because of the inherently big size of the protein which in turn makes cloning and overexpression difficult. Indeed, Vps13 is the fifth largest protein in the yeast proteome

. We initially characterized a Drosophila model of ChAc with an aim to investigate phenotypic consequences of VPS13A-loss of function at the organismal level. We next aimed to identify the localization and interaction partners of VPS13A in cultured human cell lines. Through combined applications molecular biology, biochemistry and cellular imaging, we uncovered previously unknown functions of VPS13A in membrane contacts and LD homeostasis.

Chapter 2: Drosophila Vps13 is required for protein homeostasis in the brain.

Reports about Vps13 function are mainly derived from studies in unicellular eukaryotes such as Saccharomyces cerevisiae and Tetrahymena thermophile. The availability of multicellular models to study ChAc is limited and there is an obvious demand to generate and validate genetic ChAc models. Although VPS13A mutant mouse models that recapitulate some of the ChAc phenotype were generated

, it later became clear that the phenotypes were not merely caused by VPS13A loss of function but rather dependent on genetic backgrounds

. In this chapter, we aimed to characterize an isogenic Vps13 mutant Drosophila line and showed that Vps13 deficient flies have motor impairments, shorter lifespan, neurodegeneration and accumulation of ubiquitinated protein aggregates. Some of these phenotypes were reverted by ubiquitous expression of human VPS13A in Vps13 deficient lines.

Chapter 3: Drosophila Vps13 mutants show overgrowth of larval neuromuscular junctions.

Impaired synaptic communication and plasticity have been previously implicated in many neurodegenerative diseases

. The neuromuscular junction (NMJ) is a specialized type of synapse that regulates muscle movement by controlling output of neuronal signals

. Cytoskeletal integrity of the NMJ not only determines synaptic architecture but also the quality of neuronal impulses

. Although, VPS13A depletion leads to mis-stabilized actin cytoskeleton

and abnormal bleb formation at neurite terminals of cultured cells

, it is unclear whether defective neuro-synaptic architecture contributes to neurodegeneration in ChAc patients or the Drosophila model. In this chapter, we investigated and the NMJ anatomy of Vps13 mutants that were validated in chapter 2. Body wall muscles of Vps13 mutants were equally developed as their wild type counterparts. Nonetheless, Vps13 mutant larvae were highly mobile.

Our data also indicate that Vps13 loss of function is associated with a large increase in number of boutons

that are smaller in size compared to wild type controls.

Chapter 4: Human VPS13A is associated with multiple organelles and required for lipid droplet homeostasis

In chapter 2, we described that Drosophila Vps13 co-fractionates with endosomal proteins. However, the subcellular localization of mammalian VPS13A and interaction partners were unresolved for a long time. In this chapter, we provide evidence that VPS13A is localized at the ER-mitochondria interface and directly binds to the ER resident protein, VAP-A, through a specific motif. We also show that the VPS13 C-terminal part acts as a mitochondrial localization signal. When cellular lipid is surplus, VPS13A shifts from its reticular arrangement to LDs where it halts LD mobility. Moreover, we find that, upon VPS13A loss of function, LDs accumulate in both cultured cells and Drosophila brain. We also discuss that improper organelle communication and LD handling could contribute to the onset and progression of ChAc.

Chapter 5: Summarizing discussion

Our research highlights a conserved function of VPS13A to control proper protein homeostasis, neuronal

growth, organelle communication and LD dynamics. Initially reported as a class A Vps family in yeast,

Vps13 later emerged as a protein with multiplicity of cellular functions ranging from intracellular transport,

prospore formation, mitochondrial clearance and MCSs. In this chapter, we summarize and discuss

the available literature in the VPS13 field and propose a model in which VPS13A is not limited to a single

subcellular compartment but it is associated of with multiple organelles dependent on cellular lipid

content.

REFERENCES 1

1. Rubio, J. P. et al. Chorea-Acanthocytosis: Genetic Linkage to Chromosome 9q21. Am. J. Hum. Genet. 61, 899–908 (1997).

2. Rampoldi, L. et al. A conserved sorting-associated protein is mutant in chorea-acanthocytosis. Nat. Genet. 28, 119–20 (2001).

3. Walker, R. H., Jung, H. H. & Danek, A. in Handbook of clinical neurology 100, 141–151 (2011).

4. Walker, R. H. Untangling the Thorns: Advances in the Neuroacanthocytosis Syndromes. J. Mov. Disord. 8, 41–54 (2015).

5. Jung, H. H., Danek, A. & Walker, R. H. Neuroacanthocytosis Syndromes. Orphanet J. Rare Dis. 6, 68 (2011).

6. Prohaska, R. et al. Brain, blood, and iron: perspectives on the roles of erythrocytes and iron in neurodegeneration.

Neurobiol. Dis. 46, 607–24 (2012).

7. Velayos Baeza, A. et al. Chorea-Acanthocytosis.

GeneReviews® (2002). at <http://www.ncbi.nlm.nih.gov/

pubmed/20301561>

8. Peikert, K., Danek, A. & Hermann, A. Current state of knowledge in Chorea-Acanthocytosis as core Neuroacanthocytosis syndrome. Eur. J. Med. Genet. (2017).

doi:10.1016/j.ejmg.2017.12.007

9. Dobson-Stone, C. et al. Mutational spectrum of the CHAC gene in patients with chorea-acanthocytosis. Eur. J. Hum.

Genet. 10, 773–81 (2002).

10. Dobson-Stone, C. et al. Chorein detection for the diagnosis of chorea-acanthocytosis. Ann. Neurol. 56, 299–302 (2004).

11. Lupo, F. et al. A new molecular link between defective autophagy and erythroid abnormalities in chorea- acanthocytosis. Blood 128, 2976–2987 (2016).

12. Velayos-Baeza, A., Vettori, A., Copley, R. R., Dobson-Stone, C. & Monaco, A. P. Analysis of the human VPS13 gene family.

Genomics 84, 536–49 (2004).

13. Kolehmainen, J. et al. Cohen syndrome is caused by mutations in a novel gene, COH1, encoding a transmembrane protein with a presumed role in vesicle- mediated sorting and intracellular protein transport. Am. J.

Hum. Genet. 72, 1359–69 (2003).

14. Lesage, S. et al. Loss of VPS13C Function in Autosomal- Recessive Parkinsonism Causes Mitochondrial Dysfunction and Increases PINK1/Parkin-Dependent Mitophagy. Am. J.

Hum. Genet. 98, 500–13 (2016).

15. Shamseldin, H. E. et al. Molecular autopsy in maternal–fetal medicine. Genet. Med. (2017). doi:10.1038/gim.2017.111 16. Cohen, M. M., Hall, B. D., Smith, D. W., Graham, C. B. &

Lampert, K. J. A new syndrome with hypotonia, obesity, mental deficiency, and facial, oral, ocular, and limb anomalies. J. Pediatr. 83, 280–4 (1973).

17. Kivitie-Kallio, S. & Norio, R. Cohen syndrome: essential features, natural history, and heterogeneity. Am. J. Med.

Genet. 102, 125–35 (2001).

18. Seifert, W. et al. Mutational spectrum of COH1 and clinical heterogeneity in Cohen syndrome. J. Med. Genet. 43, e22–

e22 (2006).

19. Seifert, W. et al. Expanded mutational spectrum in Cohen syndrome, tissue expression, and transcript variants of COH1. Hum. Mutat. 30, E404-20 (2009).

20. Seifert, W. et al. Cohen syndrome-associated protein, COH1, is a novel, giant Golgi matrix protein required for Golgi integrity. J. Biol. Chem. 286, 37665–75 (2011).

21. Duplomb, L. et al. Cohen syndrome is associated with major glycosylation defects. Hum. Mol. Genet. 23, 2391–2399 (2014).

22. Seifert, W. et al. Cohen syndrome-associated protein COH1 physically and functionally interacts with the small GTPase RAB6 at the Golgi complex and directs neurite outgrowth. J.

Biol. Chem. 290, 3349–58 (2015).

23. Schormair, B. et al. Diagnostic exome sequencing in early- onset Parkinson’s disease confirms VPS13C as a rare cause of autosomal-recessive Parkinson’s disease. Clin. Genet. (2017).

doi:10.1111/cge.13124

24. Chen, C.-M. et al. Association of GCH1 and MIR4697 , but not SIPA1L2 and VPS13C polymorphisms, with Parkinson’s disease in Taiwan. Neurobiol. Aging 39, 221.e1-221.e5 (2016).

25. Safaralizadeh, T. et al. SIPA1L2 , MIR4697 , GCH1 and VPS13C loci and risk of Parkinson’s diseases in Iranian population: A case-control study. J. Neurol. Sci. 369, 1–4 (2016).

26. Grarup, N. et al. The diabetogenic VPS13C/C2CD4A/

C2CD4B rs7172432 variant impairs glucose-stimulated insulin response in 5,722 non-diabetic Danish individuals.

Diabetologia 54, 789–794 (2011).

27. Holstein, J. et al. Genetic variants in GCKR, GIPR, ADCY5 and VPS13C and the risk of severe sulfonylurea-induced hypoglycaemia in patients with type 2 diabetes. Exp. Clin.

Endocrinol. Diabetes 121, 54–57 (2012).

28. Strawbridge, R. J. et al. Genome-Wide Association Identifies Nine Common Variants Associated With Fasting Proinsulin Levels and Provides New Insights Into the Pathophysiology of Type 2 Diabetes. Diabetes 60, 2624–2634 (2011).

29. Windholz, J. et al. Effects of Genetic Variants in ADCY5, GIPR, GCKR and VPS13C on Early Impairment of Glucose and Insulin Metabolism in Children. PLoS One 6, e22101 (2011).

30. Anding, A. L. et al. Vps13D Encodes a Ubiquitin-Binding Protein that Is Required for the Regulation of Mitochondrial Size and Clearance. Curr. Biol. 28, 287–295.e6 (2018).

31. Nakada, T. et al. VPS13D Gene Variant Is Associated with Altered IL-6 Production and Mortality in Septic Shock. J.

Innate Immun. 7, 545–553 (2015).

32. Ramseyer, V. D., Kimler, V. A. & Granneman, J. G. Vacuolar protein sorting 13C is a novel lipid droplet protein that inhibits lipolysis in brown adipocytes. Mol. Metab. (2017).

doi:10.1016/j.molmet.2017.10.014

33. Pappas, S. S. et al. Eighth International Chorea- Acanthocytosis Symposium: Summary of Workshop Discussion and Action Points. Tremor Other Hyperkinet.

Mov. (N. Y). 7, 428 (2017).

34. Mikitova, V. & Levine, T. P. Analysis of the Key Elements of FFAT-Like Motifs Identifies New Proteins That Potentially Bind VAP on the ER, Including Two AKAPs and FAPP2. PLoS

One 7, e30455 (2012).

35. Rzepnikowska, W. et al. Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate.

Hum. Mol. Genet. 26, 1497–1510 (2017).

36. Xu, Z. & Dooner, H. K. The maize aberrant pollen transmission 1 gene is a SABRE/KIP homolog required for pollen tube growth. Genetics 172, 1251–61 (2006).

37. Rzepnikowska, W. et al. Yeast and other lower eukaryotic organisms for studies of Vps13 proteins in health and disease.

Traffic 18, 711–719 (2017).

38. Chowdhury, S. et al. Structural analyses reveal that the ATG2A-WIPI4 complex functions as a membrane tether for autophagosome biogenesis. bioRxiv 180315 (2017).

doi:10.1101/180315

39. Tamura, N. et al. Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets.

FEBS Lett. (2017). doi:10.1002/1873-3468.12901

40. Koizumi, K. & Gallagher, K. L. Identification of SHRUBBY, a SHORT-ROOT and SCARECROW interacting protein that controls root growth and radial patterning. Development 140, 1292–300 (2013).

41. Fidler, D. R. et al. Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains. Traffic 17, 1214–1226 (2016).

42. Muñoz-Braceras, S., Calvo, R. & Escalante, R. TipC and the chorea-acanthocytosis protein VPS13A regulate autophagy in Dictyostelium and human HeLa cells. Autophagy 11, 918–

27 (2015).

43. Scheffzek, K. & Welti, S. Pleckstrin homology (PH) like domains - versatile modules in protein-protein interaction platforms. FEBS Lett. 586, 2662–73 (2012).

44. Lemmon, M. A. Pleckstrin homology (PH) domains and phosphoinositides. Biochem. Soc. Symp. 74, 81 (2007).

45. Velikkakath, A. K. G., Nishimura, T., Oita, E., Ishihara, N. &

Mizushima, N. Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets. Mol. Biol. Cell 23, 896–909 (2012).

46. Brickner, J. H. & Fuller, R. S. SOI1 encodes a novel, conserved protein that promotes TGN-endosomal cycling of Kex2p and other membrane proteins by modulating the function of two TGN localization signals. J. Cell Biol. 139, 23–36 (1997).

47. Nakanishi, H., Suda, Y. & Neiman, A. M. Erv14 family cargo receptors are necessary for ER exit during sporulation in Saccharomyces cerevisiae. J. Cell Sci. 120, 908–16 (2007).

48. Park, J.-S. & Neiman, A. M. VPS13 regulates membrane morphogenesis during sporulation in Saccharomyces cerevisiae. J. Cell Sci. 125, 3004–11 (2012).

49. Lang, A. B., Peter, A. T. J., Walter, P. & Kornmann, B. ER- mitochondrial junctions can be bypassed by dominant mutations in the endosomal protein Vps13. J. Cell Biol. 210, 883–90 (2015).

50. De, M. et al. The Vps13p–Cdc31p complex is directly required for TGN late endosome transport and TGN homotypic fusion. J. Cell Biol. jcb.201606078 (2017). doi:10.1083/

jcb.201606078

51. John Peter, A. T. et al. Vps13-Mcp1 interact at vacuole–

mitochondria interfaces and bypass ER–mitochondria contact sites. J. Cell Biol. 216, 3219–3229 (2017).

52. Dalton, L. E., Bean, B. D. M., Davey, M. & Conibear, E.

Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes. Mol. Biol. Cell 28, 1539–1550 (2017).

53. Xue, Y. et al. Endoplasmic reticulum–mitochondria junction is required for iron homeostasis. J. Biol. Chem. 292, 13197–

13204 (2017).

54. Park, J.-S. et al. Yeast Vps13 promotes mitochondrial function and is localized at membrane contact sites. Mol. Biol. Cell 27, 2435–49 (2016).

55. Bankaitis, V. A., Johnson, L. M. & Emr, S. D. Isolation of yeast mutants defective in protein targeting to the vacuole. Proc.

Natl. Acad. Sci. U. S. A. 83, 9075–9 (1986).

56. Shiba, Yoichiro Ichikawa, Kimihisa Serizawa, Nobufusa Yoshikawa, H. No Title. J. Ferment. Bioeng. 86, 545–549 (1998).

57. Klionsky, D. J. & Emr, S. D. A new class of lysosomal/vacuolar protein sorting signals. J. Biol. Chem. 265, 5349–52 (1990).

58. Tabuchi, M. et al. Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe. J.

Bacteriol. 179, 4179–89 (1997).

59. Raymond, C. K., Howald-Stevenson, I., Vater, C. A. & Stevens, T. H. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol. Biol. Cell 3, 1389–

402 (1992).

60. Rothman, J. H. & Stevens, T. H. Protein sorting in yeast:

mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell 47, 1041–51 (1986).

61. Redding, K., Brickner, J. H., Marschall, L. G., Nichols, J. W. &

Fuller, R. S. Allele-specific suppression of a defective trans- Golgi network (TGN) localization signal in Kex2p identifies three genes involved in localization of TGN transmembrane proteins. Mol. Cell. Biol. 16, 6208–17 (1996).

62. Luo, W. j & Chang, A. Novel genes involved in endosomal traffic in yeast revealed by suppression of a targeting- defective plasma membrane ATPase mutant. J. Cell Biol. 138, 731–46 (1997).

63. Zhang, B., Chang, A., Kjeldsen, T. B. & Arvan, P. Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex. J. Cell Biol. 153, 1187–98 (2001).

64. Marcusson, E. G., Horazdovsky, B. F., Cereghino, J. L., Gharakhanian, E. & Emr, S. D. The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene.

Cell 77, 579–86 (1994).

65. Leiba, J. et al. Vps13F links bacterial recognition and intracellular killing in Dictyostelium. Cell. Microbiol. 19, e12722 (2017).

66. Samaranayake, H. S., Cowan, A. E. & Klobutcher, L. A.

Vacuolar Protein Sorting Protein 13A, TtVPS13A, Localizes to the Tetrahymena thermophila Phagosome Membrane and Is Required for Efficient Phagocytosis. Eukaryot. Cell 10, 1207–1218 (2011).

67. Korolchuk, V. I. et al. Drosophila Vps35 function is necessary

1

for normal endocytic trafficking and actin cytoskeleton organisation. J. Cell Sci. 120, 4367–76 (2007).

68. Stege, J. T., Laub, M. T. & Loomis, W. F. tip genes act in parallel pathways of earlyDictyostelium development. Dev. Genet.

25, 64–77 (1999).

69. Mesquita, A. et al. Autophagy in Dictyostelium : Mechanisms, regulation and disease in a simple biomedical model.

Autophagy 13, 24–40 (2017).

70. Balla, T. Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation. Physiol. Rev. 93, 1019–1137 (2013).

71. Shi, Y., Azab, A. N., Thompson, M. N. & Greenberg, M. L.

Inositol phosphates and phosphoinositides in health and disease. Subcell. Biochem. 39, 265–92 (2006).

72. Idevall-Hagren, O. & De Camilli, P. Detection and manipulation of phosphoinositides. Biochim. Biophys. Acta - Mol. Cell Biol. Lipids 1851, 736–745 (2015).

73. Costanzo, M. et al. The Genetic Landscape of a Cell. Science (80-. ). 327, 425–431 (2010).

74. Hoppins, S. et al. A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria. J. Cell Biol.

195, 323–340 (2011).

75. Kihara, A., Noda, T., Ishihara, N. & Ohsumi, Y. Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. J. Cell Biol. 152, 519–30 (2001).

76. Vanhaesebroeck, B., Guillermet-Guibert, J., Graupera, M. &

Bilanges, B. The emerging mechanisms of isoform-specific PI3K signalling. Nat. Rev. Mol. Cell Biol. 11, 329–341 (2010).

77. Vicinanza, M., D’Angelo, G., Di Campli, A. & De Matteis, M. A.

Phosphoinositides as regulators of membrane trafficking in health and disease. Cell. Mol. Life Sci. 65, 2833–2841 (2008).

78. Wen, P. J., Osborne, S. L. & Meunier, F. A. Dynamic control of neuroexocytosis by phosphoinositides in health and disease. Prog. Lipid Res. 50, 52–61 (2011).

79. Shiokawa, N. et al. Chorein, the protein responsible for chorea-acanthocytosis, interacts with β-adducin and β-actin. Biochem. Biophys. Res. Commun. 441, 96–101 (2013).

80. Toulmay, A. & Prinz, W. A. Lipid transfer and signaling at organelle contact sites: the tip of the iceberg. Curr. Opin.

Cell Biol. 23, 458–63 (2011).

81. Raiborg, C. et al. Repeated ER–endosome contacts promote endosome translocation and neurite outgrowth. Nature 520, 234–238 (2015).

82. Mesmin, B. et al. A four-step cycle driven by PI(4)P hydrolysis directs sterol/PI(4)P exchange by the ER-Golgi tether OSBP.

Cell 155, 830–43 (2013).

83. Kornmann, B. et al. An ER-Mitochondria Tethering Complex Revealed by a Synthetic Biology Screen. Science (80-. ). 325, 477–481 (2009).

84. Lahiri, S. et al. A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria. PLoS Biol. 12, e1001969 (2014).

85. Gatta, A. T. et al. A new family of StART domain proteins at

membrane contact sites has a role in ER-PM sterol transport.

Elife 4, (2015).

86. Hönscher, C. et al. Cellular Metabolism Regulates Contact Sites between Vacuoles and Mitochondria. Dev. Cell 30, 86–94 (2014).

87. Elbaz-Alon, Y. et al. A Dynamic Interface between Vacuoles and Mitochondria in Yeast. Dev. Cell 30, 95–102 (2014).

88. Rocha, N. et al. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning. J. Cell Biol. 185, 1209–25 (2009).

89. Hua, R. et al. VAPs and ACBD5 tether peroxisomes to the ER for peroxisome maintenance and lipid homeostasis. J. Cell Biol. jcb.201608128 (2017). doi:10.1083/jcb.201608128 90. Costello, J. L. et al. ACBD5 and VAPB mediate membrane

associations between peroxisomes and the ER. J. Cell Biol.

jcb.201607055 (2017). doi:10.1083/jcb.201607055

91. Giordano, F. et al. PI(4,5)P(2)-dependent and Ca(2+)- regulated ER-PM interactions mediated by the extended synaptotagmins. Cell 153, 1494–509 (2013).

92. Galmes, R. et al. ORP5/ORP8 localize to endoplasmic reticulum–mitochondria contacts and are involved in mitochondrial function. EMBO Rep. 17, 800–810 (2016).

93. Saheki, Y. et al. Control of plasma membrane lipid homeostasis by the extended synaptotagmins. Nat. Cell Biol.

18, 504–515 (2016).

94. Salo, V. T. et al. Seipin regulates ER-lipid droplet contacts and cargo delivery. EMBO J. e201695170 (2016). doi:10.15252/

embj.201695170

95. Stoica, R. et al. ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/

FTD-associated TDP-43. Nat. Commun. 5, 3996 (2014).

96. Herms, A. et al. AMPK activation promotes lipid droplet dispersion on detyrosinated microtubules to increase mitochondrial fatty acid oxidation. Nat. Commun. 6, 7176 (2015).

97. Fan, J., Li, X., Issop, L., Culty, M. & Papadopoulos, V. ACBD2/

ECI2-Mediated Peroxisome-Mitochondria Interactions in Leydig Cell Steroid Biosynthesis. Mol. Endocrinol. 30, 763–82 (2016).

98. Fransen, M., Lismont, C. & Walton, P. The Peroxisome- Mitochondria Connection: How and Why? Int. J. Mol. Sci. 18, 1126 (2017).

99. Chu, B.-B. et al. Cholesterol Transport through Lysosome- Peroxisome Membrane Contacts. Cell 161, 291–306 (2015).

100. Guimaraes, S. C. et al. Peroxisomes, lipid droplets, and endoplasmic reticulum &quot;hitchhike&quot; on motile early endosomes. J. Cell Biol. 211, 945–54 (2015).

101. Pan, X. et al. Nucleus-vacuole junctions in Saccharomyces cerevisiae are formed through the direct interaction of Vac8p with Nvj1p. Mol. Biol. Cell 11, 2445–57 (2000).

102. Tomemori, Y. et al. A gene-targeted mouse model for chorea-acanthocytosis. J. Neurochem. 92, 759–766 (2005).

103. Sakimoto, H., Nakamura, M., Nagata, O., Yokoyama, I. & Sano, A. Phenotypic abnormalities in a chorea-acanthocytosis mouse model are modulated by strain background.

Biochem. Biophys. Res. Commun. 472, 118–124 (2016).

104. Bae, J. R. & Kim, S. H. Synapses in neurodegenerative

diseases. BMB Rep. 50, 237–246 (2017).

105. Zhou, L. et al. Tau association with synaptic vesicles causes presynaptic dysfunction. Nat. Commun. 8, 15295 (2017).

106. Milnerwood, A. J. & Raymond, L. A. Early synaptic pathophysiology in neurodegeneration: insights from Huntington’s disease. Trends Neurosci. 33, 513–523 (2010).

107. Wu, H., Xiong, W. C. & Mei, L. To build a synapse:

signaling pathways in neuromuscular junction assembly.

Development 137, 1017–33 (2010).

108. Dobbins, G. C., Zhang, B., Xiong, W. C. & Mei, L. The Role of the Cytoskeleton in Neuromuscular Junction Formation. J.

Mol. Neurosci. 30, 115–118 (2006).

109. Nelson, J. C., Stavoe, A. K. H. & Colón-Ramos, D. A. The actin cytoskeleton in presynaptic assembly. Cell Adh. Migr. 7, 379–87 (2013).

110. Kapitein, L. C. & Hoogenraad, C. C. Building the Neuronal Microtubule Cytoskeleton. Neuron 87, 492–506 (2015).

111. Foller, M. et al. Chorein-sensitive polymerization of cortical actin and suicidal cell death in chorea-acanthocytosis.

FASEB J. 26, 1526–1534 (2012).

112. Schmidt, E.-M. et al. Chorein sensitivity of cytoskeletal organization and degranulation of platelets. FASEB J. 27, 2799–2806 (2013).

113. Alesutan, I. et al. Chorein Sensitivity of Actin Polymerization, Cell Shape and Mechanical Stiffness of Vascular Endothelial Cells. Cell. Physiol. Biochem. 32, 728–742 (2013).

114. Park, J.-S., Halegoua, S., Kishida, S. & Neiman, A. M. A Conserved Function in Phosphatidylinositol Metabolism for Mammalian Vps13 Family Proteins. PLoS One 10, e0124836 (2015).

1

CHAPTER 2

Drosophila Vps13 is required for protein homeostasis in the brain.

Jan J. Vonk

, Wondwossen M. Yeshaw

, Francesco Pinto

, Anita I.E. Faber

, Liza L. Lahaye

, , Bart Kanon

, Marianne van der Zwaag

, Antonio Velayos-Baeza

, Raimundo Freire

, Sven C. van Ijzendoorn

, Nicola A. Grzeschik

, Ody C.M. Sibon

1

Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.

2

Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom. 3Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Ofra s/n, La Laguna, Tenerife, Spain.

*

these authors contributed equally

PLoS One. 2017 Jan 20;12(1):e0170106. doi: 10.1371/journal.pone.0170106.

ABSTRACT

Chorea-Acanthocytosis (ChAc, MIM 200150) is a rare, neurodegenerative disorder characterized by

progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the

Vacuolar Protein Sorting 13A (VPS13A) gene, which is conserved from yeast to human. The consequences

of VPS13A dysfunction in the nervous system are still largely unspecified. In order to study the

consequences of VPS13A protein dysfunction in the ageing central nervous system, we characterized a

Drosophila melanogaster Vps13 mutant line. The Drosophila Vps13 gene encoded a protein of similar size

as human VPS13A. Our data suggest that Vps13 is a peripheral membrane protein located to endosomal

membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age

associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated

ubiquitylated proteins. Levels of Ref(2)P, the Drosophila orthologue of p62, were increased and protein

aggregates accumulated in the central nervous system. Overexpression of the human VPS13A protein

in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of

human VPS13A and Drosophila Vps13. Our results demonstrate that Vps13 is essential to maintain protein

homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate

the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for

potential therapeutic targets for ChAc.

INTRODUCTION

Chorea-Acanthocytosis (ChAc) is a rare neurodegenerative disorder characterized by chorea, orofacial dyskinesia and psychiatric symptoms including tics (reviewed in

). In addition to the neurological symptoms, spiky red blood cells (acanthocytes) are often observed. ChAc is a recessively inherited disease caused by mutations in the VPS13A gene, hereafter called HsVPS13A

. These mutations mostly lead to absence or reduced levels of the HsVPS13A (or also called chorein) protein

. Symptoms manifest on average at the age of 32

. The pathophysiology of ChAc is largely unknown and it is not clear why HsVPS13A loss of function leads to the symptoms presenting in ChAc patients. HsVPS13A is evolutionarily conserved and orthologues are present in various organisms such as Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Tetrahymena thermophila, Dyctiostelium discoidenum and Saccharomyces cerevisiae

.

HsVPS13A belongs to the VPS13 family of proteins, which in humans consists of four members, VPS13A to D.

All members have an N-terminal chorein domain of unknown function. Besides HsVPS13A other members of this family are also associated with medical conditions. VPS13B mutations cause Cohen syndrome, a developmental disorder characterized by mental retardation, microcephaly and facial dysmorphisms

. VPS13B has been reported to be a Rab6 effector that controls Golgi integrity

. VPS13C mutations have recently been described to cause autosomal-recessive early-onset Parkinson’s disease, probably by alteration of mitochondrial morphology and function

. The VPS13C protein has also been suggested to play a role in adipogenesis

. Additionally, a number of genetic studies have found an association of VPS13C with glucose and insulin metabolism

, and of VPS13D with altered interleukin 6 production

. Knowledge about the cellular function of the Vps13 protein family members mainly comes from investigations in S. cerevisiae where a single VPS13 gene encodes a peripheral membrane protein

, Vps13, which is involved in the trafficking of multiple proteins from the trans-Golgi network to the pre-vacuolar compartment

. Vps13 is also required for the formation of the prospore membrane by controlling the levels of phosphatidylinositol-4-phosphate

. Recently, it has been demonstrated that Vps13 is important for mitochondrial integrity and at least some functions of Vps13 are redundant with functions of ERMES, a protein complex that tethers the endoplasmic reticulum and the mitochondria

. Although ERMES plays an important role in yeast, so far no counterpart has been identified in metazoans.

In various organisms Vps13 function has been linked to lysosomal degradation pathways. In the ciliate Tetrahymena thermophila TtVPS13A is required for phagocytosis

and in Dictyostelium discoideum TipC, the HsVPS13A Dictyostelium orthologue, plays a role in autophagic degradation

. A role for HsVPS13A in autophagy has also been supported by experiments performed in human epitheloid cervix carcinoma cells, where knock down of HsVps13A leads to an impairment of the autophagic flux

.

Studies to understand a possible function of VPS13A in the brain are limited. Vps13A knockout mice show

recapitulation of some of the patient’s characteristics such as acanthocytic red blood cells and an altered

gait at an older age. Additionally, gliosis and TUNEL positive cells are present in the brain of these mice

.

However, it is reported that the severity and penetrance of neurological phenotypes in mouse models of

2

ChAc are variable or absent depending on the genetic background of the strains

. Therefore, additional animal models are required to identify genetic modifiers and to further understand the role of VPS13A in an ageing brain.

To further study the cellular function of VPS13A in an aging, multicellular model organism with a complex central nervous system we used Drosophila melanogaster. We established a Drosophila model for ChAc which showed a reduced life span, decreased climbing ability and age-associated neurodegeneration.

Additionally it showed sensitivity to proteotoxic stress and impaired protein homeostasis. The phenotypes of Vps13 mutant flies were rescued by overexpression of the Human VPS13A protein, indicating a functional conservation of Drosophila Vps13 and HsVPS13A. Drosophila Vps13 mutants will be valuable for further detailed studies to investigate the role of VPS13A in brain tissue and to screen for possible therapeutic strategies.

RESULTS

Characterization of Drosophila Vps13 mutant flies

ChAc is caused by mutations in the VPS13A gene

, which lead to absence or reduced levels of HsVPS13A

protein

. The Drosophila genome encodes for three predicted Vps13 proteins, orthologues to human

VPS13A, B and D; in this study we focused on the structural orthologue of HsVPS13A, further referred to

as Vps13

. The Exelixis Drosophila fly line Vps13

carries a PiggyBac transposable element in an intronic

region of the Vps13 gene (Figure 1A)

. Flies heterozygous for this mutation (Vps13

) did not show any

mutant phenotype; homozygous mutants (Vps13

) were viable and were investigated further. Analysis

by qPCR showed lower levels of Vps13 mRNA in homozygous Vps13 mutant flies (Figure 1B). Polyclonal

antibodies were raised against two different epitopes of the Vps13 protein (Figure 1A). Both antibodies

recognized a band in extracts from control fly heads (Figure 1C,D), which migrated with the same mobility

as the human protein in samples derived from HEK293 cells and detected with a HsVPS13A-specific

antibody (Figure 1F). Vps13 was highly enriched in samples from fly heads compared to samples from

whole flies (Figure 1E), suggesting that Vps13 is enriched in the Drosophila central nervous system. In

homozygous Vps13 mutant flies full length Vps13 protein levels were below the detection limit, visualized

using Western blot analysis using the antibody against the C-terminal domain (Figure 1C). The antibody

directed against the N-terminal part of the protein, recognized a truncated Vps13 product in extracts

of homozygous mutants, consistent with the presence of the Piggybac element insertion, indicating

that the antibodies are specific, that the expression of full length Vps13 is strongly decreased and a

truncated Vps13 product is present in mutant flies (Figure 1C-E). Exact excision of the PiggyBac element

in 3 independent lines resulted in recovery of the expression of a full length Vps13 protein in fly heads

(Figure 1F, Supplementary Figure 2A). The excision lines were used as controls in further studies. These

results indicate that the Vps13 mutant line is a suitable tool to study the function of Vps13 in Drosophila.

Control Vps13 Vps13 NT

Tubulin 2R:43D7 - 43E1 1 kb

PBac{PB}Vps13[c03628]

RNA

Vps13

Vps13 #62 Vps13 NT

Vps13 antibodies

β-actin Vps13 #62

Control Vps13

Control Vps13Excision line 1Excision line 2Excision line 3Hek293 cells Vps13 #62

Tubulin

HsVPS13A

A

B C D

E F

0.0 0.2 0.4 0.6 0.8 1.0

Relative vps13 mRNA level

Control Vps13 Protein

Vps13 #62

Tubulin

Control headsControl whole flies

~360 kDa

~360 kDa 170 kDa

~360 kDa ~360 kDa

42 kDa

50 kDa

50 kDa 50 kDa

Fig 1. Vps13

encodes for a truncated Vps13 protein.

(A) Schematic representation of the Vps13 gene and the genomic localization, RNA and protein is depicted. The epitopes of the polyclonal Vps13 antibodies (Vps13 NT and Vps13 #62) are indicated.(B) Relative levels of Vps13 mRNA in control and Vps13 mutant flies were determined by Q-PCR. Mean and SEM (n=2) are plotted. (C) Western blot analysis of Vps13 protein in control and Vps13 mutant fly heads using the Vps13 #62 antibody. β-Actin was used as a loading control. (D) Western blot analysis of the level of Vps13 protein in control and Vps13 mutant fly head extracts analyzed with the Vps13 NT antibody. α-tubulin was used as a loading control.

(E) Lysates of the heads of control flies and whole control flies were analyzed for Vps13 levels. α-tubulin was used as a loading control.

(F) Lysates of the heads of control flies, Vps13 mutant flies and three excision lines were analyzed for Vps13 levels. Human VPS13A

was detected in samples of Hek293 cells. Drosophila samples and human samples were run on the same gel, separated by a lane

containing the molecular weight standards, after transfer of the membrane, the marker lane was split to detect human and Drosophila

VPS13 separately using species specific antibodies. The marker lane was used to align the blots after antibody detection. α-tubulin

was used as a loading control.

2

Control Vps13 Vps13 NT

Tubulin 2R:43D7 - 43E1 1 kb

PBac{PB}Vps13[c03628]

RNA

Vps13

Vps13 #62 Vps13 NT

Vps13 antibodies

β-actin Vps13 #62

Control Vps13

Control Vps13 Excision line 1Excision line 2Excision line 3 Hek293 cells Vps13 #62

Tubulin

HsVPS13A

A

B C D

E F

0.0 0.2 0.4 0.6 0.8 1.0

Relative vps13 mRNA level

Control Vps13 Protein

Vps13 #62

Tubulin

Control headsControl whole flies

~360 kDa

~360 kDa 170 kDa

~360 kDa ~360 kDa

42 kDa

50 kDa

50 kDa 50 kDa

Fig 1. Vps13

encodes for a truncated Vps13 protein.

(A) Schematic representation of the Vps13 gene and the genomic localization, RNA and protein is depicted. The epitopes of the polyclonal Vps13 antibodies (Vps13 NT and Vps13 #62) are indicated.(B) Relative levels of Vps13 mRNA in control and Vps13 mutant flies were determined by Q-PCR. Mean and SEM (n=2) are plotted. (C) Western blot analysis of Vps13 protein in control and Vps13 mutant fly heads using the Vps13 #62 antibody. β-Actin was used as a loading control. (D) Western blot analysis of the level of Vps13 protein in control and Vps13 mutant fly head extracts analyzed with the Vps13 NT antibody. α-tubulin was used as a loading control.

(E) Lysates of the heads of control flies and whole control flies were analyzed for Vps13 levels. α-tubulin was used as a loading control.

(F) Lysates of the heads of control flies, Vps13 mutant flies and three excision lines were analyzed for Vps13 levels. Human VPS13A was detected in samples of Hek293 cells. Drosophila samples and human samples were run on the same gel, separated by a lane containing the molecular weight standards, after transfer of the membrane, the marker lane was split to detect human and Drosophila VPS13 separately using species specific antibodies. The marker lane was used to align the blots after antibody detection. α-tubulin was used as a loading control.

Vps13 co-fractionates with Rab7 and Rab5

We aimed to determine the subcellular localization of Vps13 in brain tissue; however, the antibodies that were generated against Vps13 failed to show a specific staining using immunolabeling. We therefore followed a cell fractionation approach to determine the subcellular localization of Vps13. We found that Vps13 was mainly, but not exclusively, present in the isolated membrane fraction (Figure 2A). To determine whether Vps13 is a peripheral or integral membrane protein, the membranes were treated with a variety of buffers to extract proteins as previously described

. High salt buffer could not remove Vps13 from the membrane fraction, while high pH and high concentration of urea did (Figure 2B). This shows that Vps13 has characteristics similar to a peripheral membrane protein, such as Golgi Matrix protein 130 kDa (GM130)

, but different from an integral membrane protein like Epidermal Growth Factor Receptor (EGFR), both of them were used as controls in these experiments (Figure 2B). The membrane fraction was separated on a sucrose gradient and the distribution of Vps13 was determined in relation to marker proteins for various organelles. The distribution of Vps13 was different compared to the distribution of markers for Golgi (GM130), lysosomes (Lamp1) and mitochondria (ATP5A) (Figure 2C and E). Vps13 was mainly present in fractions 12 to 16 in which also Rab5 and Rab7, Rab-GTPases involved in the regulation of endosomal trafficking, were present. Rab5 is mainly present on early endosomes and Rab7 is enriched on late endosomes

. To study this further, Rab7 positive membranes from fraction 14 were immuno-isolated and Vps13 was shown to be present in these samples (Figure 2D). Furthermore, Rab7, but not Rab5 was enriched in membranes immuno-isolated with Vps13 antibodies (Figure 2D). Together, these data suggest that Vps13 is a peripheral membrane protein associated with Rab7 positive membranes.

Vps13 mutant flies have a decreased life span and show age dependent neurodegeneration After validation of the Drosophila Vps13 mutant and characterizing its subcellular localization, we investigated the physiological consequences of impaired Vsp13 function. Characteristics of several Drosophila models for neurodegenerative diseases are a decreased life span, impaired locomotor function and the presence of brain vacuoles

. As a control an isogenic fly line (w

) and 3 independent precise excision lines were used. Homozygous Vps13 mutant flies showed a decreased life span compared to isogenic controls and the excision lines (Figure 3A-C, Supplementary Table 1). 75% of the mutant flies died between 16 and 20 days of age while control flies showed a more gradual decline (Figure 3B). Young Vps13 mutant flies showed climbing capabilities comparable to controls, however around day 17 the climbing ability of Vps13 mutant flies was decreased (Figure 3D, Supplementary movie 1).

To further investigate neurodegenerative features in Vps13 mutants, brain sections were analyzed by light

microscopy and an increase in vacuoles was observed in brains of 20 day old flies while they were absent

in brains from isogenic controls (Figure 3E). Vacuoles in Vps13 mutant flies were (among other regions)

present in the central complex, known for its function in locomotor control (Figure 3F)

. The impaired

locomotor function upon ageing, shortened life span and the presence of large vacuoles in the brain of

Vps13 mutant flies are all characteristics of neurodegeneration in Drosophila

.

PNS Cytosol Membrane

GAPDH Vps13 #62

EGFR GM130

GAPDH Vps13 #62

EGFR

PNS CytosolMembraneSol. controlInsol. controlSol. 1M KClInsol. 1M KClSol. Na

2CO^3, pH 1 1

Sol. 6M UreaInsol. 6M Urea Insol. Na

2CO^3, pH 1 1

Vps13 lysate

Rab7

Rab5 GM130 Lamp1 ATP5A

Vps13 #62 2 4 6 8 10 12 14 16 18 20 22 Fractions:

A B

C

Input Control IgGRab7 Vps13 Rab5

Rab7 Vps13 #62

Rab5

ATP5A

D

Fraction

% of total

E

~360 kDa ~360 kDa

~360 kDa

~360 kDa 37 kDa

170 kD

37 kDa 170 kDa 130 kDa

23 kDa

24 kDa

130 kDa

120 kDa

60 kDa

23 kDa

24 kDa

60 kDa

2 4 6 8 10 12 14 16 18 20 22

0 10 20 30 40 50

Vps13 Rab7 Rab5 GM130 Lamp1 Atp5a

Fig 2. Vps13 co-fractionates with Rab7 and Rab5

(A) Western blot analysis of control fly head samples fractionated into a cytosolic and membrane fraction from postnuclear supernatant

(PNS). EGFR was used as a membrane marker and GAPDH as a cytosolic marker. (B) Membrane fractions from control fly heads treated

with 1 M KCl, Na

CO

pH 11 or 6 M urea were centrifuged to separate the soluble and insoluble (membrane containing) fractions. The

level of Vps13 was determined in these fractions. Markers for peripheral membrane proteins (GM130), integral membrane proteins

(EGFR) and the cytosolic proteins (GAPDH) were used. The “Vps13 lysate” lane contains a lysate derived from Vps13 homozygous

mutant fly heads, as expected no Vps13 is detected, demonstrating the specificity of the antibody against Vps13. (C) Membranes from

control fly heads were fractionated on a sucrose gradient. Western blot analysis was performed to analyze the distribution of Vps13 in

relation to markers associated with membranes of various organelles: Rab7 (late endosomes), Rab5 (early endosomes), GM130 (golgi),

Lamp1 (lysosomes) and ATP5A (mitochondria). (D) Immunoisolation of membranes from fraction 14 of the sucrose gradient using

Vps13 NT, Rab7 and Rab5 antibodies. (E) Quantification of the sucrose gradient fractionation of Fig 2C.

2

A

D C

B

0 20 40 60

0 20 40 60 80 100

Age (days)

Survival (%)

Control Vps13

0 20 40 60 80

0 20 40 60 80 100

Age (days)

Survival (%)

Vps13 Excision line 1 Excision line 2 Excision line 3

0 - 4 4 - 8

8 - 12 12 - 16

16 - 20 20 - 24

24 - 28 28 - 32

32 - 36 36 - 40

40 - 44 44 - 48

48 - 52 52 - 56

56 - 60 0.0

0.2 0.4 0.6 0.8

Days

Fraction of flies died

Control Vps13

4 days old

17 days old 0

20 40 60 80 100

**

*

Climbers (%)

Control Vps13

E

F

Vps13 Vps13

Fig 3. Vps13 mutant flies show a decreased life span, age dependent impairment of locomotor function and neurodegeneration.

(A) Life span analysis of isogenic control and Vps13 mutant flies. (B) The fraction of dead flies of total flies used,observed within the

indicated time intervals. (C) Life span curve of Vps13 mutant flies and three excision lines. (D) Climbing behavior was analyzed by

determining the percentage of isogenic control and Vps13 mutant flies (4 and 17 days old) able to climb 5 cm against gravity within 15

seconds. Mean and SEM are plotted (n=5). For statistical analysis a two-tailed students T-test was used. P<0.001 is ***. (E) Fly heads (20

day old) of control and homozygous Vps13 mutant flies were fixed, dehydrated and embedded in epon. Sections, visualizing a cross

section of the complete brain, were stained with toluidine blue. The scale bar indicates 50 µm.(F) Higher magnification images of the

boxed area’s in Fig E. The central complex is denoted with a dotted line. The scale bar indicates 25 µm.