Vacuolar Storage Disease in Lagotto Romagnolo Dogs

Department of Veterinary Biosciences Section of Veterinary Pathology Faculty of Veterinary Medicine University of Helsinki Finland

VACUOLAR STORAGE DISEASE IN LAGOTTO ROMAGNOLO DOGS

PATHOLOGY, AUTOPHAGY AND EXTRACELLULAR VESICLES

Pernilla Syrjä

ACADEMIC DISSERTATION to be presented for public examination with the permission of the Faculty of Veterinary Medicine of the University of Helsinki, in the Forest Sciences Building, Hall 108 (LSB3) on the 4th of November, 2020 at 12 o’clock.

Helsinki 2020

Doctoral Programme in Clinical Veterinary Medicine CVM The Faculty of Veterinary Medicine uses the Urkund system (plagiarism recognition) to examine all doctoral dissertations

Study director: Professor Antti Sukura, Faculty of Veterinary Medicine, University of Helsinki Supervisor: Professor Eeva-Liisa Eskelinen, Institute of Biomedicine, University of Turku

Pre-examiners: Professor Achim Gruber Freie Universität Berlin, Department of Veterinary Pathology, Germany Professor Horst Posthaus Universität Bern, Department of Infectious Diseases and Pathobiology, Switzerland

Opponent: Privatdozentin Frauke Seehusen University of Zurich UZH, Institute of Veterinary Pathology, Switzerland

Front cover: Lagottos Mimmi and Charlie, of which one carries the ATG4D variant. Picture courtesy of Tere Jääskeläinen.

Unigrafia Helsinki 2020 DSHealth thesis series 71/2020 ISSN 2342-3161 ISBN 978-951-51-6678-4 (nid) ISBN 978-951-51-6679-1 (PDF)

ABSTRACT

This thesis aimed at describing the pathology of a novel progressive neurologic disease in dogs of the Lagotto Romagnolo (LR) breed, at testing the hypothesis of altered autophagy in affected dogs, derived from genetic investigations, and at testing the hypothesis of a lysosomal storage disease affecting the dogs, derived from the histopathologic changes. Autophagy is a degradative cellular process, responsible for the turnover of damaged organelles and clearance of protein aggregates, that is disturbed in several neurodegenerative diseases in man. Morphologically, altered autophagy commonly leads to neuronal proteinaceous inclusions, as well as spheroid formation, in histology. Occasionally, neuronal vacuoles have been linked to altered autophagy. Cytoplasmic vacuolation is histologically very typical for lysosomal storage diseases, a group of inherited diseases characterized by defective lysosomal degradation, which often affect the nervous system. Compensatory rerouting of cargo during defective autophagy and lysosomal degradation may affect the content and release of extracellular vesicles (EVs) from cells. Post mortem examination of 13 affected LRs confirmed a distinct histopathological phenotype, consisting of intracellular vacuole storage in neurons, axonal damage and neuronal loss. Secretory epithelial cells and selected mesenchymal cells were also affected by vacuolation. In order to link the histopathologic findings to altered autophagy, immunohistochemical staining of affected organs, along with age- and breed matched control tissue, was performed. Markers for autophagosomal, lysosomal and endosomal membranous antigens, as well as for autophagy receptors and cargo were used. For obtaining more specific results regarding the subcellular morphological changes, electron microscopy and immunoelectron microscopy were performed, using the same membranous markers. Autophagy was functionally monitored by the LC3 I/II conversion assay and by quantifying autophagosomes with immunofluorescence in cultured fibroblasts from affected and control dogs. The screening for a lysosomal storage disease included urine analysis to detect excessive oligosaccharide or glucosaminoglycan excretion. Histochemical staining and electron microscopy were used to examine the affected tissues for lysosomally stored material. In addition, the activity of three lysosomal enzymes were measured in fibroblasts from affected dogs and controls, as well as in culture medium, in order to identify alterations in lysosomal function. The size, amount and cargo of EVs released from fibroblasts of affected dogs were compared to those released from control cells, in order to detect changes in EV release or content that could support or contradict either hypothesis. Nano-particle tracking analysis of EVs, as well as mass

spectrometry and functional enrichment analysis of the EV proteomes were performed. Both autophagosomal markers and autophagic cargo accumulated within the lesions of the nervous system in affected dogs. The stored vacuoles in affected tissues represented hybrid organelles of the autophagic and endolysosomal pathways, displaying membranous markers of both pathways. Fibroblasts showed changes in basal, but not starvation-induced autophagy, as both basal LC3II levels and basal number of LC3 positive spots were significantly elevated. In addition, the basal EV release was significantly increased and the basal EV proteome enriched in substrates of basal autophagy in the cells of affected dogs in comparison to control cells. These findings suggest altered basal autophagy as a pathogenetic mechanism in this novel disease. Lysosomal storage of a specific material or a lysosomal enzyme deficiency were not detected. A novel candidate gene for neurodegeneration, ATG4D, was discovered by the genetic groups in the collaborative research where this thesis was part. In aspects of comparative pathology, altered basal autophagy was uncovered as an additional etiology and morphological differential diagnosis for cytoplasmic vacuolation reminiscent of lysosomal storage diseases in dogs, and functionally linked to the ATG4D variant through this thesis. The importance of basal autophagy in cells was also hereby shown, as disease occurred despite starvation-induced autophagy being functional in the dogs. For veterinary medicine, the characterization of this novel inherited progressive neurodegenerative disease provides a broader scientific knowledge as basis for veterinarians regarding prognostication of affected dogs, and for LR owners regarding breeding.

SAMMANFATTNING

Den här avhandlingen beskriver de vävnads förändringarna hos hundar av rasen Lagotto Romagnolo (LR) som lider av en tidigare okänd, fortskridande neurologisk sjukdom. Avhandlingens syfte är att undersöka om sjukdomen uppkommer pga störd autofagi, vilket den genetiska bakgrunded tyder på, eller om sjukdomen är en lysosomal inlagringssjukdom, vilket vävnadsförändringarna indikerar. Autofagi är en återvinningsprocess i cellen som bland annat ansvarar för att skadade organeller förnyas och proteinansamlingar löses upp. Störd autofagi är en central sjukdomsmekanism i flere neurodegenerativa sjukdomar hos människan. Störd autofagi leder ofta till proteinaggregat och/eller vakuoler i nervcellerna samt till svullna nervutskott, aksonala sferoider. Vakuoler i nervcellerna är ett typiskt histologiskt fynd också vid lysosomala inlagringssjukdomar, en grupp ärftliga sjukdomar som ofta skadar nervsystemet och där cellernas lysosomer inte fungerar normalt. Både störd autofagi och störd lysosomal nedbrytning i cellerna kan leda till att ämnen i stället utsöndras ur cellen som extracellulära vesiklar (EV); små signalbärare som potentiellt kan användas inom diagnostik av störningarna i dessa cellfunktioner. Obduktionsfynd hos 13 neurologiskt sjuka LR hundar bekräftade distinkta histopatologiska förändringar in vävnaden; upplagring av vakuoler i nervcellerna, axonskador och neuronförlust. Körtelepitelceller och vissa mesenkymala celler uppvisade också cytoplasmiska vakuoler hos beträffade LR hundar. I syfte att påvisa störd autofagi färgades vävnadsförändringarna, och oförändrad kontrollvävnad från friska LR i samma ålder, med markörer för autofagosomala, lysosomala och endosomala membranprotein, samt för autofagireceptorer och -last. För att mer specifikt undersöka förändringar i cellernas inre användes elektronmikroskopi och immunelektronmikroskopi med respektive markörer. Autofagi undersöktes funktionellt i bindvävsceller från sjuka och friska LR hundar genom uppföljning av LC3I/II konvertering och genom att kvantifiera bindvävscellernas autofagosomer med hjälp av immunofluorescens. För att påvisa en eventuell lysosomal inlagringssjukdom mättes oligosackarid- och glukosaminoglykan mängden i hundarnas urin. Histokemiska färgningar och elektronmikroskopi användes för att påvisa upplagrat material i cellernas vakuoler. Enzymaktiviteten hos tre lysosomala enzym mättes i och utanför bindvävsceller från beträffade hundar och kontrol hundar för att hitta störningar i enzymfunktionen. EVs storlek, mängd och innehåll jämfördes sjuka och kontrol bindvävsceller emellan genom uppföljning av nano-partiklars rörelse (nano-

particle tracking analysis) samt mass-spektrometri och funktionell analys av protein som speciallt anrikades i EV från sjuka LR hundar. Både autofagosomala markörer och autofagilast samlades i vävnadsförändringarna hos sjuka LR hundar. Bindvävscellerna från beträffade hundar uppvisade förändrad basal autofagi, men oförändrad svält- inducerad autofagi. Dessutom utsöndrades signifikant mera EV från de sjuka hundarnas celler och förmert substrat för basal autofagi kunde påvisas i dessa EV. Dessa fynd framför störd basal autofagi som en patogenetisk mekanism bakom den nya, beskrivna sjukdomen. Lysosomal upplagring av ett specifikt substrat eller en bristande lysosomal enzymfunktion kunde inte påvisas. De lagrade vakuolerna representerar hybridorganeller som uppstått mellan cellernas autofagi- och endolysosomala vesikelflöden. En ny kandidatgen för nervcellsdegeneration, ATG4D, upptäcktes av de genetiska forskningsgrupperna inom forskningssamarbetet där den här avhandlingen var delaktig. För komparativ patologi framför avhandlingen störd basal autofagi som en alternativ orsak till vävnadsförändringar som histologiskt påminner om lysosomal upplagringssjukdom, samt verifierar bristande autofagi hos LR hundar med genvarianten i ATG4D. En grundläggande relevans för basal autofagi i specifika celler blir också tydlig genom avhandlingen, eftersom hundarna är symptomatiska trots att svält- inducerad autofagi fungerar normalt. Inom veterinärmedicin ger avhandlingen, och de kliniska studierna inom forskningssamarbetet, en bredare vetenskaplig basis för veterinärer och LR ägare gällande vårdbeslut, prognosbedömning och rasens avel.

CONTENTS

Abstract Sammanfattning Table of Contents List of original publications Author’s contribution Abbreviations

1 Introduction………………………………………………………………………….. 14 2 Review of the literature ...... 16

2.1 Autophagy ...... 16

2.1.1 Autophagy outlined ...... 16

2.1.2 The core autophagic molecular machinery ...... 18

2.1.2.1 The LC3 conjugation system……………………………………..18 2.1.2.2 ATG4….……………………………………………………….20

2.1.3 Basal versus induced autophagy ...... 20

2.1.4 Autophagy in neurodegeneration ...... 21

2.2 The endolysosomal system ...... 23

2.2.1 Endolysosomal vesicular pathways outlined ...... 23

2.2.2 Lysosomal storage diseases ...... 24

2.3 Extracellular vesicles ...... 25

2.3.1 Composition, release mechanisms and function ...... 25

2.3.2 EVs in neurodegeneration ...... 27

2.4 Crosstalk between cellular vesicular pathways ...... 27

2.5 Comparative neuropathology of canine and human genetic disorders linked to autophagy alterations ...... 29

2.6 The Lagotto Romagnolo breed………………………………………………..32

3 Aims of the studies………………………………………………………………….34

4 Materials and methods……………………………………………………………35

4.1 Ethical statement ...... 35

4.2 Study cohorts ...... 35

4.3 Biochemical screening………………………………………………………… 36

4.3.1 Urine analysis ...... 36

4.3.2 Enzyme activity ...... 36

4.4 Post mortem examination ...... 37

4.4.1 Histopathology ...... 37

4.4.2 Transmission electron microscopy ...... 37

4.4.3 Immunohistochemistry ...... 37

4.4.4 Immunoelectron microscopy ...... 38

4.5 Monitoring autophagy ...... 39

4.5.1 Cell culture experiments and Western Blot ...... 39

4.5.2 Immunofluorescence ...... 40

4.6 Extracellular vesicles ...... 41

4.6.1 Cell culture, vesicle isolation and quality control ...... 41

4.6.2 Nanoparticle tracking analysis and vesicle quantification ...... 42

4.6.3 Mass-spectrometry and proteomic analysis ...... 43

4.7 Statistical analysis ...... 44

5 Results…………………………………………………………………………………..45

5.1 The histopathologic phenotype of LRs with the ATG4D variant comprises neurodegeneration and intracellular vacuolar storage ...... 45

5.2 Basal autophagy is altered in LRs with the ATG4D variant ...... 48

5.3 The histological lesions are associated with accumulation of autophagic cargo and autophagic membranes…………………………………………………….50

5.4 Lysosomal storage or a lysosomal enzyme deficiency were not detected in the affected dogs ...... 51

5.5 Cells with the ATG4D variant release substrates of basal autophagy via EVs ...... 54

6 Discussion…………………………………………………………………………….56

6.1 A novel neurodegenerative disease in LRs ...... 56

6.2 ATG4D as a candidate gene for neurodegeneration ...... 57

6.3 Altered autophagy as a histopathologic differential diagnosis for lysosomal storage diseases ...... 58

6.4 Disturbed basal autophagy can cause disease despite induced autophagy is functional ...... 60

6.5 EV-content reflects the disease process in cells with the ATG4D variant 62

6.6 Limitations of the study ...... 63

6.7 Future aspects ...... 64

7 Conclusions……….…………………………………………………………………65

8 Acknowledgements

9 References

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following publications:

I Kyöstilä K, Syrjä P, Jagannathan V, Chandrasekar G, Jokinen TS, Seppälä EH, Becker D, Drögemüller M, Dietschi E, Drögemüller C, Lang J, Steffen F, Rohdin C, Jäderlund KH, Lappalainen AK, Hahn K, Wohlsein P, Baumgärtner W, Henke D, Oevermann A, Kere J, Lohi H, Leeb T. A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease. PLoS Genet. 2015 Apr 15;11(4):1005169

II Syrjä P, Anwar T, Jokinen T, Kyöstilä K, Jäderlund KH, Cozzi F, Rohdin C, Hahn K, Wohlsein P, Baumgärtner W, Henke D, Oevermann A, Sukura A, Leeb T, Lohi H, Eskelinen EL. Basal Autophagy Is Altered in Lagotto Romagnolo Dogs with an ATG4D Mutation. Vet Pathol. 2017 Nov;54(6):953-963.

III Syrjä P, Palviainen M. Jokinen T, Kyöstilä K, Lohi H, Roosje P, Anderegg L, Leeb T, Sukura A, Eskelinen EL. Altered basal autophagy affects extracellular vesicle release in cells of Lagotto Romagnolo dogs with a variant ATG4D. Accepted for publication in Vet Pathol. 27.08.2020 DOI: 10.1177/0300985820959243

The publications are referred to in the text by their roman numerals.

AUTHOR’S CONTRIBUTION

I Post mortem examination of affected Finnish and Norwegian dogs, including planning and performing histopathology, immunohistochemistry and electron microscopy of affected tissues. Writing the canine pathology part in the manuscript, contributing to the discussion. Kaisa Kyöstilä organized and performed the genotyping of Finnish LR sample cohorts under supervision of Professors Hannes Lohi and Tosso Leeb. Kyöstilä performed the RNA-level experiments and had the main responsibility in combining the results for publication.

II Planning and performing the histopathological, immunohistochemical and electron microscopical examinations of extra neuronal tissues. Planning of cell experiments for autophagy monitoring under supervision by Eeva-Liisa Eskelinen, cell culture for the LC3 conversion assay, performing the immunofluorescence study. Tahira Anwar from Eeva-Liisa Eskelinen’s group performed the Western blots. Statistical evaluation of results, writing the manuscript.

III Planning and performing the immunoelectron microscopy. Cell culture, isolation and electron microscopy of extracellular vesicles. Functional analysis and interpretation of the proteomic data from extracellular vesicels. Nanoparticle tracking analysis and mass spectrometry were performed by the core facilities of the University of Helsinki. Writing the manuscript.

ABBREVIATIONS

AD Alzheimer’s disease ATG autophagy related gene ATG4D autophagy-related cystein proteinase 4D BFJE benign familial juvenile epilepsy CCT chaperonin-containing T-complex CNS central nervous system DMEM Dulbecco’s Modified Eagle Medium DRG dorsal root ganglium EBSS Earl's balanced salt solution EM electron microscopy ER endoplasmic reticulum ESCRT endosomal sorting complexes for transport EV extracellular vesicle FBS fetal bovine serum GABARAP γ-amino-butyric acid receptor-associated protein GABARAPL GABARAP-like protein GAG glycosaminoglycan GAP GTPase activator protein GFAP glial fibrillary acidic protein HD Huntington's disease HE hematoxylin-eosin HSP hereditary spastic paraparesis IEM immunoelectron microscopy IF immunofluorescence IHC immunohistochemistry ILV intra-luminal vesicle LAMP2 lysosomal-associated membrane protein 2 LC3 microtubule associated protein 1A/B light chain 3 LGI2 Leucine-Rich, Glioma Inactivated 2 LR Lagotto Romagnolo LSD lysosomal storage disease MFN2 mitofusin2 MRI magnetic resonance imaging mTORC1 mechanistic target of rapamycin complex 1 MVB multivesicular body MVP major vault protein NAD neuroaxonal dystrophy NCL neuronal ceroid lipofuscinosis NTA nanoparticle-tracking-analysis PAS periodic-acid-Schiff’s PD Parkinson’s disease

PE phosphatidyletanolamine PNS peripheral nervous system PSM peptide spectrum match RAB24 Ras-related protein Rab-24 RCD regulated cell death SDS sodium dodecyl sulfate SQSTM1/p62 sequestosome-1/p62 TCP-1 T-complex protein 1 s TECPR2 tectonin beta-propeller repeat-containing protein 2 TEM transmission electron microscopy TFR2 transferrin receptor 2 ULK1 Unc-51-like kinase 1 V-ATPase vacuolar H+-adenosine triphosphatase WB Western blot VPS11 vacuolar protein sorting 11 gene

1 INTRODUCTION

The study of spontaneous genetic diseases in animals can provide insights into the function of genes and, consequently, their role in disease processes. These studies have have enabled the discovery of novel disease-associated candidate genes across species and revealed conserved pathogenetic mechanisms common to man and animals. The process of disease-linked genetic discovery works in two directions ; firstly, a hypothesis regarding the genetic cause for a novel sporadic disease may arise from discovery of a genetic variant strongly linked to the disease phenotype. Hereby functional and morphological studies of the disease-associated pathology, linking the gene defect to the lesions, serve as support for causality. Secondly, a hypothesis regarding which cellular function is disturbed, hence which genes may be altered in a specific disease, can be based on the typical disease-associated morphological changes and derived from comparative pathology. Tissue-based studies and confirmation of the diagnosis largely relies on autopsy material in research regarding neurodegenerative disease. Hereby the dog with its similar disease-mechanisms and a shorter lifespan, has proven a valuable animal model and research in this field has intensified after the sequence and structure of the canine genome was revealed (Jagannathan, Drogemuller, Leeb, & Dog Biomedical Variant Database Consortium, (DBVDC), 2019; Lindblad-Toh et al., 2005). Generally, neurodegenerative diseases in man can have a genetic cause and occur at an early age, or be multifactorial with genetic risk-factors and affect the elderly (Dickson Dennis W., Weller Roy O., 2011; Goodarzi et al., 2019). The number of known neurodegenerative diseases with an overlapping pathology (Schutt et al., 2016) and similar genetic background occurring in man and dog is increasing (Chambers et al., 2018; Fyfe et al., 2011; Hahn et al., 2015; Lucot et al., 2018; Ogawa et al., 2015; Walvoort, Dormans, & van den Ingh, T S, 1985). Several neurodegenerative diseases are proteinopathies, where excessive, unfolded, missfolded or disease-related protein(s) aggregate in neurons, leading to neuronal dysfunction and degeneration (Dickson Dennis W., Weller Roy O., 2011; Menzies, Fleming, & Rubinsztein, 2015). Two mechanisms counteract intraneuronal protein accumulation ; autophagy and proteasomal degradation (Amm, Sommer, & Wolf, 2014; Boland et al., 2018; Korolchuk, Menzies, & Rubinsztein, 2010; Mizushima, Levine, Cuervo, & Klionsky, 2008), and neurons also release vesicles containing proteins into the extracellular space (Minakaki et al., 2018; Schneider & Simons, 2013). Consequently, research regarding the cause and pathomechanisms of novel neurodegenerative diseases with protein accumulation commonly focus on functional disturbances in these pathways, and on genes encoding the proteins involved therein. Notably, there are also important differences between canine and human neurodegenerative disease, despite partially overlapping phenotypes

and genetic background (Chambers et al., 2018; Fyfe et al., 2011; Hahn et al., 2015; Wiedmer et al., 2015) . During the course of this thesis work, Lagotto Romagnolo dogs (LRs) with a progressive neurological disease, comprising ataxia, episodes of nystagmus and behavioral changes, were found to have a common histopathological phenotype, not previously described in the breed (I). The main histopathological findings were indicative of a lysosomal storage disease (I, II). Genetical analyses by the research groups of Professor Hannes Lohi in Helsinki and Professor Tosso Leeb in Bern, however, revealed a missense variant in the autophagy-related gene 4D (ATG4D) associating with the disease (I). The aim of this thesis was to describe the pathology of this novel vacuolar storage disease, and to test both the hypothesis derived from the genetic findings, suggesting that autophagy is altered in the affected dogs, and the hypothesis derived from the morphological changes, indicating this being a lysosomal storage disease.

2 REVIEW OF THE LITERATURE

AUTOPHAGY

Autophagy, a term derived from the Greek words for auto i.e. self, and phagein i.e. eating, is a vesicular degradative process for intracellular turnover of damaged or redundant organelles, and for clearance of aggregate prone, long- lived proteins (Mizushima et al., 2008; Xie & Klionsky, 2007). It was first described mainly as a digestive pathway, providing quality control and recyclable material, as part of cellular homeostasis (Tsukada & Ohsumi, 1993). The detailed autophagic process has only more recently been revealed and its conserved functions studied broadly in yeast and mammals (Nakatogawa, Suzuki, Kamada, & Ohsumi, 2009, Mizushima et al., 2008). In 2016, Yoshinori Ohsumi was awarded the Nobel prize in Physiology or Medicine for his work on the molecular mechanisms of autophagy, emphasizing the role of autophagy as a fundamentally important cellular process.

2.1.1 AUTOPHAGY OUTLINED There are several known forms of autophagy (Fig.1), and autophagy can also be categorized as selective or non-selective regarding the targeted material (Galluzzi et al., 2017; Mizushima et al., 2008). Macroautophagy is the best characterized form and starts with the formation of a membrane structure, the isolation membrane, that sequesters the damaged organelle or cytosolic protein cargo to be degraded. The isolation membrane closes around the targeted cytoplasmic material to form the autophagosome, a cargo-containing cytoplasmic vesicle, limited by a double membrane (Galluzzi et al., 2017; Xie & Klionsky, 2007). For degradation of autophagic cargo, the process requires lysosomal enzymes. To enable this, the outer autophagosomal membrane fuses with the lysosome, and lysosomal enzymes permeabilize and digest the inner autophagosomal membrane along with the autophagosomal content. This step comprises the formation of the autolysosome; a single-membrane- bound vesicle containing various, partially degraded content (Eskelinen, 2005; Xie & Klionsky, 2007). In this thesis, the term autophagy refers to macroautophagy (Fig. 1 A). Autophagy substrates can also be directly engulfed by an invagination of the lysosomal or late endosomal membrane, which closes into an intraluminal vesicle and is degraded via microautophagy (Fig. 1 B) (Li, W. W., Li, & Bao, 2012). Endosomal microautophagy differs from lysosomal microautophagy, as endosomal microautophagy depends on multiple endosomal sorting complexes for transport (ESCRT) (Galluzzi et al., 2017; Sahu et al., 2011). The third form of autophagic cargo delivery into the lysosome is chaperone- mediated autophagy (Fig. 1 C), which requires cargo-recognition by

cytoplasmic chaperones, consequent binding of the chaperone to receptors at the lysosomal membrane and transmembrane delivery of unfolded cargo via lysosome-associated membrane protein 2, isoform A (LAMP2A) into the lysosomal lumen (Galluzzi et al., 2017; Mizushima et al., 2008). During the final phase of autophagy, autolysosomes release nutrients into the cytoplasm, and lysosomes reform from them through autophagic lysosome reformation (Chen & Yu, 2013). Autolysosomes also release their degraded cargo into the extracellular space after fusing with the plasma membrane (Medina et al., 2011; Sirois et al., 2012).

Figure 1 The different forms on autophagy outlined: A. Macroautophagy: the isolationmambrane forms next to the substrate, encloses it into the double membrane bound autophagosome, which fuses with the lysosome to form the single membrane bound autolysosome. B. Microautophagy: cytosolic and nuclear proteins, lipid droplets, peroxisomes and damaged mitochondria are taken up by the lysosome or the late endosome via membrane invagination. In endosomal microautophagy, the process is dependent on the ESCRT proteins. C. Chaperone-mediated autophagy: cytosolic soluble protein, containing a specific amino-acid sequence code, are recognized by chaperones and translocated via the LAMP2A translocation complex directly into the lysosomal lumen for degradation.

2.1.2 THE CORE AUTOPHAGIC MOLECULAR MACHINERY Autophagy-related genes (ATG) encode the proteins involved in the autophagic process (Mizushima, 2019; Nakatogawa et al., 2009; Tsukada & Ohsumi, 1993). There are more than 40 known ATG genes, however, approximately 20 ATG genes encode the core proteins involved in autophagy (Mizushima, 2019). The relevance of different ATG proteins for the autophagic process however varies, as mice lacking ATG5 or ATG7 died as neonates (Komatsu et al., 2005a; Kuma et al., 2004), whereas only a mild atactic phenotype was described in mice lacking ATG4B (Read, Savelieva, Baker, Hansen, & Vogel, 2011). Especially yeast Atg8, and one of its mammalian homologues, microtubule-associated protein 1 light chain 3 (LC3), which is part of the autophagosomal membrane, have been used for monitoring autophagy in cells and tissues (Mizushima & Yoshimori, 2007; Wild, McEwan, & Dikic, 2014). The six mammalian forms of ATG8 proteins include LC3A, B and C along with the γ-amino-butyric acid receptor-associated protein (GABARAP) and GABARAP-like protein 1 and 2 (GABARAPL1 and GABARAPL2) (Mizushima, 2019; Wild et al., 2014). Mammalian ATG4 also has four isoforms, A, B, C and D. The ATG4 and LC3/GABARAP isoform expression patterns provide additional organ- and tissue specific modulation of autophagy (Fernandez & Lopez-Otin, 2015a; Li, M. et al., 2011; Nemos et al., 2003).

2.1.2.1 The LC3 conjugation system LC3 belongs functionally to the ATG8 conjugation system, which is one of two ubiquitin-like conjugation systems functioning during the biogenesis of autophagosomes (Nakatogawa, 2013). During LC3-conjugation, the ATG4- encoded cysteine peptidase ATG4 first cleaves a 22 amino acid peptide from the C-terminus of pro-LC3, revealing a glycine-residue, and conformationally changing pro-LC3 into LC3I (Fig. 2). This enables activation of LC3I by ATG7 and covalent binding of the lipid phosphatidyletanoleamine (PE) to LC3I by ATG3, reversibly integrating lipidated LC3II into the autophagosomal membrane (Kabeya et al., 2000). During or after the fusion step between autophagosome and the lysosome, LC3II is delipidated and released by ATG4 from the outer autophagosomal limiting membrane back into the cytosol as LC3I (Fig.2) (Kauffman et al., 2018; Nakatogawa, Ishii, Asai, & Ohsumi, 2012). The inner limiting membrane of the autophagosome, and LC3II bound to it, are degraded by the lysosomal enzymes along with the autophagosomal content.

Figure 2 The functions of ATG4 in LC3 conjugation. After cleavage of proLC3 by ATG4 to reveal a glycine (G) at the c-terminal, LC3I is activated by ATG7, requiring ATP Thereafter, LC3I forms a thioester with ATG3 and is eventually conjugated to phosphatidylethanolamine (PE) and integrated into the autophagosomal membrane. At or after fusio with the lysosome, ATG4 deconjugates LC3II from the membrane PE, releasing it into the cytosol as LC3I.

Lipidated, autophagosomal-bound LC3II can be distinguished from cytosolic LC3I based on the mobility in SDS-PAGE gels, and the amount of LC3II and number of LC3II positive structures correlate with the quantity of autophagosomes in a cell (Mizushima & Yoshimori, 2007; Mizushima, Yoshimori, & Levine, 2010). It does not, however, reveal if autophagosomes are increased due to increased autophagosome formation, or due to disturbed maturation or diminished clearance of the autophagosomes (Mizushima & Yoshimori, 2007; Mizushima et al., 2010). LC3II can also bind to Golgi- derived (Gomes-da-Silva et al., 2019), endosomal or phagosomal membranes (Florey, Kim, Sandoval, Haynes, & Overholtzer, 2011; Kondylis et al., 2013; Sanjuan et al., 2007), which further complicates the interpretation of LC3 lipidation data (Jacquin et al., 2017; Jacquin, Fletcher, & Florey, 2019). Autophagy can also be selective with respect to the cytoplasmic cargo. Several molecules, characterized by a cargo-binding domain and an ATG8/LC3 interacting domain, serve as receptors for autophagic cargo. Some receptors recognize ubiquitin on a potential target, routing ubiquinated cargo into autophagy. Other receptors bind directly to a specific target protein, causing its sequestration into the autophagosome (Khaminets, Behl, & Dikic, 2016). SQSTM1/p62 is one of the ubiquitin-binding autophagy receptors that is widely used as an indicator of autophagic activity (Pankiv et al., 2007).

2.1.2.2 ATG4 The Atg4 cystein peptidase primes Atg8 for PE-conjugation and integration into the autophagosomal membrane in yeast, as well as delipidates Atg8 after autophagosomal fusion with the vacuole (Nakatogawa et al., 2012). Atg4 has four mammalian isoforms, ATG4A, B, C and D (Fernandez & Lopez-Otin, 2015b). Functional redundancy and differences in substrate affinity among these isoforms are only being revealed (Li et al., 2011). Systematic genetic depletion of ATG4 paralogues in human cells have indicated ATG4B as the functionally most relevant, as its loss severely affected autophagy (Agrotis, A., Pengo, Burden, & Ketteler, 2019). The defect was incomplete, however, and rescued by the residual priming activity of the other three isoforms. Autolysosomes formed despite a total lack of ATG4-activity, indicating that ATG4 is not functionally required for autophagy, but rather has a regulatory role, especially in counteracting erroneous binding of ATG8 homologues to other membranes (Agrotis, Alexander et al., 2019; Nakatogawa et al., 2012). Enzyme kinetics and activation status may cause functional differences between the ATG4 isoforms, as fast priming of LC3 in early autophagosome biogenesis is performed by ATG4B, whereas all four isoforms can regulate the slow delipidation of LC3II (Kaufman 2018). Reactive oxygen species can reversibly inhibit the delipidating activity of ATG4A and ATG4B during starvation-induced autophagy, ensuring that LC3II stays conjugated to the forming autophagosome (Scherz-Shouval et al., 2007; Zheng et al., 2020). Little is known on the physiological functions of the ATG4D isoform. Interestingly, caspase cleavage increases the substrate affinity of ATG4D (Betin & Lane, 2009b).

2.1.3 BASAL VERSUS INDUCED AUTOPHAGY Autophagy is constitutively active in cells at low levels, assuring cellular homeostasis through renewal of proteins and organelles. This basal autophagy prevents cellular build-up of residual material, that forms during normal cellular metabolism. Basal autophagy is especially important in postmitotic cells such as neurons (Hara et al., 2006) and cardiomyocytes (Mizushima et al., 2008), accounting for almost 50% of the protein turnover in cells (Zhang, T., Shen, Qu, & Ghaemmaghami, 2016). Under nutrient-rich conditions the mechanistic target of rapamycin complex 1 (mTORC1) phosphorylates Unc- 51-like kinases (ULK1, the homologue of yeast Atg1) and downregulates autophagy (Komatsu et al., 2005b; Mizushima et al., 2008; Mizushima, 2019). Under stressful conditions, such as during amino-acid deprivation, oxidative stress, ischemia or hypoxia, autophagy is induced in order to provide nutrients and to assure enhanced degradation of excessive damaged or aggregating cytosolic material (Mizushima et al., 2008; Xie & Klionsky, 2007). Hereby mTORC1 is inactivaed, ULK1 activated and autophagy induced. Recent studies suggest that basal and induced autophagy are not simply the same process executed at different intensity. Specific substrates of basal

autophagy have been described, such as sialyloligosaccharides and subunits of the proteasome (Seino et al., 2013; Zhang et al., 2016), and molecules affecting basal but not induced autophagy have been identified. ATG12-ATG3 interactions outside their role in autophagosomal biogenesis are required for efficient basal, but not induced autophagy (Murrow, Malhotra, & Debnath, 2015). Further, the activity and stability of ATG4B is specifically regulated during basal conditions (Kuang et al., 2012). The end phase of autophagy is also differently regulated during basal conditions and starvation, as RAB24 is required for the clearance of autophagic compartments during basal autophagy, whereas degradation in autolysosomes formed during short starvation is unaffected by RAB24 silencing (Yla-Anttila et al., 2015). Secondary signaling molecules can have opposite effects on basal and starvation-induced autophagy, as reactive oxygen species are required for starvation-induced autophagy (Scherz-Shouval et al., 2007) but can inhibit basal autophagy (Underwood et al., 2010).

2.1.4 AUTOPHAGY IN NEURODEGENERATION Dysfunctional basal autophagy causes cellular degeneration in neurons, even without other disease-causing mutations or stressors (Hara et al., 2006; Komatsu et al., 2005). Accumulation of misfolded, unfolded or mutant protein(s) within neurons due to altered autophagic degradation, is a central feature in neurodegeneration and loss (Dickson Dennis W., Weller Roy O., 2011; Lie & Nixon, 2019; Menzies et al., 2015; Zatyka, Sarkar, & Barrett, 2020). This is rarely solely due to primary accumulation of disease specific proteins that stress functional autophagy, but also due to direct interaction of the soluble form of the protein with autophagic proteins, inhibiting effective autophagy (Menzies et al., 2015). As examples, Huntingtin, aggregating in Huntington’s disease (HD), interferes with autophagic cargo recognition (Martinez-Vicente et al., 2010). The protein Ataxin3, which contains extended polyglutamine tracts in spinocerebellar ataxia 3, loses its ability to protect Beclin 1, initiator of autophagy, from proteasomal degradation (Ashkenazi et al., 2017). Axonal function and structure are susceptible to autophagic alterations, since autophagosomes forming distally within the axon need to reach the neural soma by retrograde intra-axonal transport to spatially fuse with lysosomes (Cheng, Zhou, Lin, Cai, & Sheng, 2015). Intra-axonal autophagosome and autolysosome accumulation with spheroid formation and axonal degeneration are consequently a main feature of several neuroaxonal dystrophies (NAD) and axonopathies linked to mutations in genes involved in autophagy or vesicular transport in the axon (Cheng et al., 2015; Colecchia et al., 2018; Wang, Y., Song, & Song, 2018). Recently, functions for ATG proteins in regulating axonal microtubule stability, were discovered (Negrete-Hurtado et al., 2020). These functions were independent of the role of the proteins in

degradative autophagy, indicating that dysfunctional ATGs can damage he axonal structure despite functional autophagy. Autophagy has a complex role in neurodegeneration, since it may be a beneficial response to protein accumulation and cellular nutrient stress, but lethal if overactive. In fact, inhibiting autophagy decreased neuronal death in models of hypoxic-excitotoxic stress (Ginet et al., 2014; Liu & Levine, 2015). Autophagy is also involved in regulating neuronal excitability via degradation of internalized receptors postsynaptically, which again links autophagy dysfunction to neuronal loss through excitotoxicity (Lieberman et al., 2020). Genetic neurodegenerative diseases, pathogenetically linked to defective autophagy, are numerous and are continously being reviewed (Menzies et al., 2015; van Beek, Klionsky, & Reggiori, 2018; Zatyka et al., 2020). Variants that alter autophagy and lead to neurodegenerative disease have been described in genes encoding proteins functioning at various stages including, but not limited to, proteins that regulate autophagy induction, function as autophagy receptors, act in formation and/or closure of the autophagosome, coordinate autolysosomal fusion and maturation, affect lysosomal degradation, enable autophagic lysosomal reformation and facilitate axonal transport of autophagic structures. Some examples are outlined in Table 1.

Table 1. Examples of genetic neurodegenerative diseases affecting different stages of autophagy. Summarized from Lie & Nixon, 2019; Menzies et al., 2015; van Beek et al., 2018; Zatyka et al., 2020. Dickson Dennis, W. 2011.

Altered Affected gene/ proposed Disease Pathology autophagy pathogenesis phase Induction dysregulation of mTORC1 Tuberous sclerosis Purkinje cell loss, due to TSC1, TSC2 (TSC) tumors mutations ND5 mutations linked to Leigh syndrome vacuolation, gliosis failure of autophagy in brain stem and induction during ATP basal ganglia depletion Cargo SQSTM1 mutations familial amyotrophic motor neuron recognition hamper p62 function lateral sclerosis degeneration with p62 inclusions mutated genes encoding autosomal recessive dopaminergic altered PINK1 and/or Parkinson’s Disease neuronal loss, parkin decrease mitophagy α-synuclein accumulation Autophagosome mutated WDR45- encoded β-propeller protein- spheroid formation biogenesis protein interaction with associated ATG9 neurodegeneration

mutations in EPM2A or Lafora disease accumulation of EPM2B with reduced polyglucosan laforin or malin levels bodies mutations in ATG5, hereditary childhood ataxia impairing ATG12-ATG5 ataxia binding Autophagosomal impaired amphisome Charcot-Marie-Tooth peripheral maturation formation due to RAB7 type2 axonopathy mutations mutations in the ectopic P- VICI syndrome Purkinje cell loss, granules autophagy cerebral atrophy, protein 5 (EPG5) gene callosal hypoplasia causes abnormal or agenesia autolysosomes Autolysosomal Presenilin-1 (PSEN-1) early-onset familial giant neuritic degradation mutations lead to Alzheimer’s disease swellings increased lysosomal pH Lysosomal mutations in genes Hereditary spastic axonal damage with reformation encoding spastizin paraplegia accumulating (ZFYVE26) and spatacsin autolysosomes (SPG11) decrease lysosome numbers

THE ENDOLYSOSOMAL SYSTEM

The lysosome is a membrane-bound organelle, named according to the lytic acidic hydrolases it contains. Several degradative processes of the cell, including phagocytosis, endocytosis and autophagy, rely on the digestive capacity of lysosomal enzymes (Ballabio & Bonifacino, 2020; Florey et al., 2011; Lie & Nixon, 2019; Sanjuan et al., 2007). Fusion with lysosomes also provides the capacity to acidify the lumen of autophagosomes, phagosomes and endosomes, to regulate the vesicular membrane permeability for releasing content into the cytosol (Abu-Remaileh et al., 2017) or into the extracellular space through exocytosis (Medina et al., 2011). While autophagy degrades and turns over cytosolic proteins and organelles, the endolysosomal system’s main substrates are extracellular, endocytosed and phagocytosed material, as well as plasma membrane-derived proteins and receptors.

2.2.1 ENDOLYSOSOMAL VESICULAR PATHWAYS OUTLINED

Cells internalize extracellular molecules through inward budding of the plasma membrane and formation of endocytic cytosolic vesicles, the early

endosomes. Homotypic fusion and fission events during endosomal maturation enable recycling of membrane and receptors back to the plasma membrane via recycling endosomes, whereas both membrane and content can be further sorted within the endosomal compartment to form multivesicular bodies (MVBs) (Fader & Colombo, 2009; Lin et al., 2019; Xu, Camfield, & Gorski, 2018). Transferrin receptors (TFRs) are an example of receptors cycling from endosomes back to the plasma membrane, after internalization and release of the substrate; iron, into the endosomal vesicle (Fader & Colombo, 2009). A single outer membrane characterizes the MVB, surrounding several smaller vesicles, intra-luminal vesicles (ILVs) that form through inward budding into the MVB lumen. The MVB has a central role in the endolysosomal pathway, as it may release the intraluminal vesicles into the extracellular space as exosomes (Jeppesen et al., 2019), or fuse with the lysosome for content degradation (Xu et al., 2018). Before fusion with the lysosome or the plasma membrane, MVBs can receive autophagic cargo via fusion with autophagosomes, forming the amphisomes (Eskelinen, 2005; Lin et al., 2019). The lysosome contains hydrolytic enzymes, such as proteases, peptidases, lipidases, sulfatases, galactosideases that are functional in an acidic milieu. Most lysosomal enzymes are targeted to the lysosome through a mannose-6- phosphate tag (Coutinho, Prata, & Alves, 2012). Mannose-6-phosphate is recognized by specific receptors in the Golgi area, and the receptor-enzyme complex is then targeted to the endo-lysosomal system. In addition to its degradative capacity deriving from the lumen, the lysosomal membrane contains several important functional molecules. The vacuolar H+-adenosine triphosphatase (V-ATPase) proton pump ensures the acidic luminal pH (Mindell, 2012). This proton gradient is also required for transporter molecules regulating efflux of metabolites from the lysosomal lumen (Abu- Remaileh et al., 2017). One membranous glycoprotein widely used as a marker for the organelle is LAMP2 (Abu-Remaileh et al., 2017; Ogawa et al., 2015; Sirois et al., 2012) .

2.2.2 LYSOSOMAL STORAGE DISEASES

Lysosomal storage diseases (LSD) are a group of hereditary disorders characterized by failure of lysosome function, with consequent intracellular accumulation of undegraded lysosomal substrates, cellular degeneration and loss (Schultz, Tecedor, Chang, & Davidson, 2011; Seranova et al., 2017; Tancini et al., 2019). The primary genetic defect in LSDs may cause deficiency or functional loss of a lysosomal enzyme (Schultz et al., 2011; Walvoort et al., 1985), interfere with membranous transport and/or acidification of the lysosome (Hortnagel et al., 2016; Schultz et al., 2011) or affect the targeting of lysosomal enzymes to the lysosome (Boonen, van Meel, Oorschot, Klumperman, & Kornfeld, 2011). Mutations in lysosomal membrane proteins,

including LAMP2, also cause disease (Nishino et al., 2000). LSDs typically have an early onset, are progressive, and histologically characterized by the accumulation of enlarged secondary lysosomes or autolysosomes, visible in light microscopy and containing storage material (Boonen et al., 2011; Mink, Augustine, Adams, Marshall, & Kwon, 2013; Schultz et al., 2011). Morphological and/or biochemical analysis of the stored material can provide initial insight to the pathogenesis and the enzymatic defect. The storage does however not always reflect a specific degradative enzyme deficit, as it may be due to decreased efflux from the lysosome (Schultz et al., 2011) or reflect overall decreased lysosomal degradative function and therefore relate to the metabolism of a specific cell type rather than to lack of a specific enzyme (Boonen et al., 2011). In several LSDs, the disease pathogenesis is unclear, even when the causative gene defect and the specific storage material are known (Adams et al., 2019; Mink et al., 2013) .

EXTRACELLULAR VESICLES

As a consequence of intracellular degradative processes, and as part of intercellular communication, cells release extracellular vesicles (EVs) of various size and diverse intracellular origin (Thery et al., 2018). The content of EVs may derive directly from the cytoplasm, or from the endolysosomal or the autophagic pathway (Jeppesen et al., 2019; Tancini et al., 2019). EV analysis has emerged as a non-invasive method to gain information about intracellular processes and disease states (Ichii et al., 2017; Minakaki et al., 2018; Tancini et al., 2019). EVs have been isolated from various body fluids, including serum and plasma (Aguilera-Rojas, Badewien-Rentzsch, Plendl, Kohn, & Einspanier, 2018; Brady et al., 2018), central spinal fluid (Grapp et al., 2013), urine (Ichii et al., 2017), and from cell culture media (Brady et al., 2018). The general morphology and the content of EVs appear comparable between species (Lawson, Kovacs, Finding, Ulfelder, & Luis-Fuentes, 2017).

2.3.1 COMPOSITION, RELEASE MECHANISMS AND FUNCTION

EVs comprise membrane-bound vesicles of different size and cellular origin, co-isolating with free cytosolic and nuclear nanoparticles (Table 2). The classification of EVs is constantly evolving and recently related also to the type and cytosolic origin of the EV content (Jeppesen et al., 2019). The main classification consensus is based on vesicle size and release mechanism (Thery et al., 2018).

Table 2. Summary of EVs and co-isolating particles described in body fluid and culture medium of human cells (Jeppesen et al., 2019; Thery et al., 2018; Zhang, H. et al., 2018) .

Type Size Release Subcellular origin Origin of limiting approximate mechanism of the content membrane

Extracellular vesicles Microvesicles 150-1000 nm budding cytosol plasma membrane Large up to 10 μm blebbing cytosol of neoplastic plasma membrane oncosome cells Exosomes 40-150 nm ILVs released endocytosis and endosomal from MVBs cytosol Apoptotic up to 1,5 μm apoptosis nuclear and plasma membrane bodies cytosolic with phosphatidyl serin Co-isolating particles Exomeres 35 nm not known derived from not known, partially specific pathways membrane bound (coagulation, glycolysis, mTOR signalling) Microvesicles 40-100 nm budding cytosol plasma membrane Autophagic 40-1000nm not known autophagic cargo autophagic pathway EVs Amphisomal co-isolates amphisome, nuclear; histones, not membrane derived with small but not ILV, DNA bound nanoparticles EVs, <100 derived nm Non-vesicular co-isolates transmembrane enzymes, cytosolic not membrane material with small release proteins, tubulin bound EVs, <100 nm

The diversity of EVs is well depicted by the description of non-classical exosomes, which are of a similar size and origin as exosomes, but do not display the protein markers typical for exosomes (CD9, CD83 or CD61), and vesicles within the size range of exosomes but limited by plasma membrane (Jeppesen et al., 2019). EVs may contain messenger-RNA (mRNA), microRNA (miRNA) and non-coding RNA, DNA (Asada et al., 2019; Jeppesen et al., 2019), protein complexes including receptors (Grapp et al., 2013; Schneider & Simons, 2013), and lipids (Zhang et al., 2018).

2.3.2 EVS IN NEURODEGENERATION Intracellular protein accumulation plays a central role in the pathogenesis of several neurodegenerative diseases and EV release can counteract this intracellular stress in neurons (Minakaki et al., 2018; You & Ikezu, 2019). EVs may also contain biomarkers of neurodegeneration, aiding the diagnosis of these diseases (You & Ikezu, 2019). Together these properties have made the role of EVs in neurodegeneration a new field of intense research. Increased EV release can reduce neuronal loss, synaptic malfunction and immune activation, which all contribute to neurodegeneration (You & Ikezu, 2019). In addition to a protective and regulatory role of EVs in the diseased nervous system, EVs are one way of spreading disease-associated proteins from neuron to neuron. Toxic and/or disease-specific proteins have been detected in neuronal EVs (Minakaki et al., 2018; Schneider & Simons, 2013). Synaptic activity facilitates EV release in the nervous system. Thus, EV release and uptake by neighbouring cells enables spreading of the proteins and other EV cargos along neuronal connections (Dickson Dennis W., Weller Roy O., 2011; Schneider & Simons, 2013).

CROSSTALK BETWEEN CELLULAR VESICULAR PATHWAYS

There is intense interaction between the autophagic and the endocytic pathways and their alterations affect EV release (Fig. 3). Crosstalk occurs as vesicular fusion and fission between the autophagic and the endocytic compartments at the level of the amphisome and MVB (Fader & Colombo, 2009; Lin et al., 2019; Xu et al., 2018), that both contribute to the formation of EVs (Jeppesen et al., 2019). Consequently, a block in lysosomal function, as occurs in lysosomal storage diseases, may cause secondary accumulation of upstream autophagic compartments in the cell (Walvoort et al., 1985) (Seranova et al., 2017; Yim & Mizushima, 2020). Hereby EV release serves as one compensatory route for cargo disposal from MVBs and amphisomes (Minakaki et al., 2018; Tancini et al., 2019).

Figure 3 Overview of the macroautophagy (blue) and endosomal (green) vesicular pathways in the cell, with lysosomal degradation (purple) and EV formation as alternative endpoints. MVB: multivesicular body, TFR2: transferrin receptor 2, LC3: micro-tubule-associated protein 1 light chain 3, PE: phosphatidylethanolamine, LAMP2: lysosome-associated membrane protein 2

In addition to direct vesicular interaction, shared regulatory mechanisms for lysosomal function, autophagy and EV formation have been described. Upon inactivation of mTORC1 during starvation, autophagy related genes and lysosomal genes are activated by the same transcription factor (Ballabio & Bonifacino, 2020; Medina et al., 2011; Yim & Mizushima, 2020). mTORC1 inactivation during starvation also induces autophagy, as described earlier. Under nutrient-rich conditions, the activated mTORC1 is linked to the lysosomal membrane (Yim & Mizushima, 2020). After starvation-induced downregulation of mTORC1 activity with consequent induction of autophagy, during the final stages of autophagy mTORC1 returns to the active form on the lysosome, ensuring the efflux of amino acids from the lysosome and reformation of lysosomes after completed autophagic degradation (Abu-

Remaileh et al., 2017; Chen & Yu, 2013; Yim & Mizushima, 2020). EV release may also have different roles or be differently regulated in basal as opposed to starvation-induced autophagy. Exosome release is upregulated during autophagy impairment but decreased in murine cells when blocking specifically basal autophagy (Murrow et al., 2015).

COMPARATIVE NEUROPATHOLOGY OF CANINE AND HUMAN GENETIC DISORDERS LINKED TO AUTOPHAGY ALTERATIONS

Morphologically, histopathological findings in canine genetic neurodegenerative diseases linked to the endosomal or autophagy-lysosomal system include axonal damage with spheroid formation (Agler et al., 2014; Cork, Troncoso, Price, Stanley, & Griffin, 1983; Fyfe et al., 2011; Hahn et al., 2015; Lucot et al., 2018; Ogawa et al., 2015; Tsuboi et al., 2017), vacuolar changes in the neuronal soma (Mhlanga-Mutangadura et al., 2016; Wiedmer et al., 2015) and intraneuronal accumulations (Agler et al., 2014; Chambers et al., 2018; Hahn et al., 2015; Tsuboi et al., 2017; Walvoort et al., 1985). Intra-axonal autophagosome accumulation with spheroid formation and axonal degeneration are the main features of neuroaxonal dystrophy (NAD) in the Spanish Water Dog and hereditary spastic paraparesis (HSP) in man, both caused by mutations in tectonin beta-propeller repeat-containing protein 2 (TECPR2) (Hahn et al., 2015; Oz-Levi et al., 2012). The exact function of TECPR2 in autophagy is not clear, however, autophagy is impaired in fibroblasts from individuals with HSP due to TECPR2 mutations (Oz-Levi et al., 2012) and TECPR2 is assists in cargo sorting at ER exit sites together with lipidated LC3C (Zatyka et al., 2020). Intra-axonal autophagosome and autolysosome accumulation was also described in Old English Sheepdogs and Gordon setters with a defect in the RAB24-gene (Agler et al., 2014). Widespread axonal spheroid formation is also the main change in cross-bred Schnautzer-Beagle laboratory dogs with fetal-onset NAD linked to mitofusin 2 (MFN2) mutations (Fyfe et al., 2010; Fyfe et al., 2011), whereas the human phenotype in Charcot-Marie-tooth 2A with MFN2 mutations comprises a peripheral axonopathy with altered axonal mitochondrial transport (Misko, Jiang, Wegorzewska, Milbrandt, & Baloh, 2010). MFN2 is expressed in the outer mitochondrial membrane and the ER, mainly functioninig in tethering events between mitochondria and ER, and in linking mitochondria to the microtubule-associated motors of axonal transport (Misko et al., 2010). During starvation, MFN2 participates in providing mitochondrial-derived membrane lipids to the forming autophagosome, functionally linking the protein to autophagy (Hailey et al., 2010). Demyelination and NAD with young adult onset and a predilection to sensory pathways in Rottweiler dogs was described in the 1980s (Cork et al., 1983), but was only recently associated with a missense mutation in the vacuolar protein sorting 11 gene (VPS11)(Lucot et

al., 2018). The human phenotype of neurodegeneration linked to mutations in VPS11 has an infantile onset and has not been thoroughly characterized, however described as reminiscent of lysosomal storage (Hortnagel et al., 2016) and hypomyelination. VPS11 is important for fusion of the late endosomal compartments with the vacuole/lysosome and facilitates autophagy (Zhang et al., 2016). A vacuolated cell morphology has been described in autophagy- dependent cell death, one form of regulated cell death (RCD) caused by excessive autophagy (Liu & Levine, 2015). Per definition, autophagy-dependent death is independent of other RCDs, such as apoptosis and necrosis, and dependent on autophagy related genes, thus can be inhibited by genetic or pharmacologic inhibition hereof (Galluzzi et al., 2018). The vacuolation is due to the accumulation of autophagosomes, autolysosomes and other dilated organelles, such as Golgi or ER in autophagy-dependent cell death. Neuronal accumulation of autophagic vacuoles occurs in several neurodegenerative diseases in man, where impaired autophagy is a central pathogenetic mechanism, including cortical neurons in Alzheimers disease (AD) (Nixon et al., 2005) and spinal motor neurons in amyotrophic laterals sclerosis (Sasaki, 2011). Neuronal vacuolation visible in light microscopy is described in both Alaskan Huskies and Black Russian terriers with mutations in RAB3GAP1, though neuronal loss in conjunction with the vacuolation was only described in the Huskies (Mhlanga-Mutangadura et al., 2016; Wiedmer et al., 2015). RAB3GAP1 codes a GTPase activator protein (GAP) that enhances the GTPase activity of RAB3 and has been shown to affect both basal and induced autophagy (Spang et al., 2014). RAB3GAP1 mutations are linked to neurodegenerative congenital disease in man, the Warburg micro syndrome and the Martsolf syndrome, with phenotypes partially overlapping the canine disease (Handley et al., 2013). Vacuolation and loss of cerebellar Purkinje cells is also mentioned among the findings in Old English Sheepdogs and Gordon Setters with canine hereditary ataxia associated with a point mutation in another Rab-encoding gene, RAB24 (Agler et al., 2014). RAB24 has been shown to act in basal autophagy during the clearance of autophagic compartments (Yla-Anttila et al., 2015). RAB24 alterations has been associated with fatty liver (Seitz et al., 2019) and cancer (Yla-Anttila & Eskelinen, 2018), but not with neurogenerative disease, in man. Accumulation of ubiquitin-positive structures (Agler et al., 2014) and p62 (Chambers et al., 2018; Ogawa et al., 2015) have been described within the neuronal soma or dendrites of dogs with genetic neurodegenerative disease, similar to human neurodegenerative proteinopathies. Coarse material within the neuronal soma was also described, but not further specified, in the Spanish Water dogs affected by NAD due to a TECPR2 variant (Hahn et al., 2015). Canine Lafora disease, with intradendritic accumulation of several proteins (laforin, alfa/beta synuclein, ubiquitin, LC3, heat-shock protein 70 and p62) in the Purkinje cells as polyglucosan bodies, is comparable to Lafora disease

in man regarding neuropathology (Chambers et al., 2018; Dickson Dennis W., Weller Roy O., 2011) . In addition to the canine neurodegenerative diseases where the altered gene is directly coding proteins involved in the autophagic or endolysosomal pathways, secondary alterations in autophagic flux with LC3 accumulation have also been described in dogs. Papillon dogs with NAD and intra-axonal LC3 accumulation due to a mutation in the phospholipase A2 group VI gene (PLA2G6) (Tsuboi et al., 2017; Uchida, 2017) model human infantile NAD without peripheral nervous system involvement or iron accumulation. Welsh Corgi Pembroke dogs, with a mutation in the superoxide dismutase gene SOD 1, have a higher risk of developing degenerative myelopathy with spinal neuron loss, spinal accumulation of LC3 and autophagic cargo receptors within diseased neurites (Ogawa et al., 2015), reminiscent of the inclusions seen in lower motor neurons in sporadic amyotrophic lateral sclerosis in man (Sasaki, 2011). As discussed above, similar clinical features, morphological changes and genetic background between several canine and human degenerative neurological diseases exist. However, important differences have also been reported, some outlined in Table 3.

Table 3. Differences in the neuropathology of canine disease in comparison to human disease with a common genetic background.

Altered Canine Human References gene histopathology histopathology spheroid spheroid formation Oz-Levi et al. 2013, TECPR2 formation, neuronal in CNS Hahn et al. 2015 iron accumulation demyelination, hypomyelination, Hörtnagel et al VPS11 spheroid formation in lysosomal 2016, Lucot et al. 2018 CNS accumulations Handley et al. peripheral 2013, Mhlanga- CNS neuronal RAB3GAP1 axonopathy, CNS Mutangadura et al. vacuolation lesions not described 2015, Wiedmer et al. 2015 Fyfe et al. 2010, spheroid formation peripheral MFN2 Misko et al. 2010 in CNS axonopathy

Despite the dog being a valuable, spontaneous animal model for human neurodegenerative disease (Ambrosini et al., 2019), the overlap is limited between the histopathology of canine disease and important neurodegenerative diseases in man, such as Alzheimer’s disease (AD),

Parkinson’s disease (PD) and Huntington’s disease. The amyloid pathology in canine cognitive dysfunction is reminiscent of early histopathological lesions in AD, comprising extracellular diffuse β-amyloid deposits. Mature amyloid plaques or tau-pathology, hallmarks of AD in man, have not been described in dogs (Schutt et al., 2016; Smolek et al., 2016). The spontaneous canine model for PD, canine multiple system atrophy in Kerry Blue Terriers and Chinese Crested dogs, has neuroanatomical overlap regarding areas of neural loss, however, not synucleinopathy (deLahunta & Averill, 1976; O'Brien et al., 2005). The role of autophagy in canine cognitive dysfunction or canine multiple systems atrophy has not been studied further. Studies on alterations within the autophagy-lysosomal system in dogs with neuronal ceroid lipofuscinosis (NCL) are lacking, despite similar genetic background and disease morphology (Farias et al., 2011; Guo et al., 2019; Schmutz et al., 2019) and the proven role of autophagy in the pathogenesis of human NCL (Adams et al., 2019; Seranova et al., 2017).

THE LAGOTTO ROMAGNOLO BREED

The Lagotto Romagnolo dog breed originates in the area of Comacchia at the Italian east coast, approximately 100 km south of Venice, and was originally a swimming, retrieving breed. As the swamps in the coastal areas dried out, the Lagotto Romagnolo was increasingly used to seek truffles in the Romagna area of North-East Italy. The breed is mentioned in the Italian literature already in the early 1900th century as Lago´t or Lagotto, the retrieving dog; “cane di riporto”, and as the truffle dog; “cane da tartufo” (Morri, 1840). The Federation Cynologique Internationale (FCI) accepted the breed on a provisional basis in 1995, and 10 years later, on a definitive basis. The LR belongs to FCI group no8 of Retrievers, Flushing dogs and Water dogs as breed number 298 (www.fci.be). It is a small or mid-sized dog with height (ideal height: 46 cm males, 43 cm females) and length being in the same dimensions, giving the dog a proportionate body shape (Fig. 4). The LR is easy to train, has an excellent sense of smell and is described as a keen, undemanding and affectionate pet (www.fci.be/en/nomenclature/ROMAGNA-WATER-DOG- 298). The LR is an increasingly popular breed in Finland, with 322 new registered dogs in 2015 and 355 in 2019, equaling an 8.6% increase in this currently 35th most popular breed. Of the around 510 000 registered dogs in Finland in 2019, 5 404 were LRs (www.kennelliitto.fi). Since 2008, heritable disorders such as joint- and eye disease are monitored in LRs as a prerequisite for registration of a pup and the breeding strategy program of the Finnish LR Club recommends a coefficient of inbreeding below 6.25% within four to five generations (https://www.lagottoromagnolo.fi/kasvatus/pevisa-ja-jto). The reported coefficient of inbreeding, this being the probability of inheriting

identical copies of an allele from both parents due to a common ancestor, is clearly lower than recommendations in the breed with an average of 1.69% from 2006-2016 (https://www.lagottoromagnolo.fi/kasvatus/pevisa-ja-jto). A strong lack of heterozygosity is not evident, as the median heterozygosity was 35.5% in the LR breed, in comparison to 34.3% in other purebred dogs and 43.2% in mixed breed (www.os.genoscoper.com. retrieved 1.9 2020). Despite this, inherited diseases occur in the breed, possibly due to a relatively narrow genetic background in the LRs breed, relatively few dogs being used in breeding and incomplete pedigree records.

Figure 4 A four-year-old male healthy Lagotto Romagnolo dog, Fonzie (Mandaraban Rock Around the Clock). Owner and photography by Marjo Kullberg, courtesy of Tere Jääskeläinen

3 AIMS OF THE STUDY

This study aimed at testing the following hypotheses :

1. Dogs of the Lagotto Romagnolo breed suffer from a novel neurodegenerative disease with a distinct histopathologic phenotype. 2. Autophagy is altered in the affected dogs, which leads to the histological lesions. 3. The disease is not linked to a lysosomal enzyme deficiency.

The specific aims, in order to confirm or reject each of the hypotheses, were :

x To characterize the histopathological phenotype of the disease, enabling genetic analysis of the diseased cohort in comparison to healthy LRs and dogs of other breeds (I) x To monitor basal and induced autophagy, and release of EVs, in cells of affected dogs in comparison to cells of controls (II, III) x To link in vitro alterations in autophagy to the histopathological phenotype of affected LRs (I, II, III) x To exclude any known LSD with similar phenotype in the affected LRs (II)

4 MATERIALS AND METHODS

ETHICAL STATEMENT

The LRs included in this study were all examined with the consent of the owner, as privately-owned pets. The dogs were euthanized at the owner’s request, due to progressive neurological signs. The post mortem examination and sampling were approved by the Cantonal Committee for animal experiments (I and III, canton Bern; permit 23/10) and the Animal Ethics Committee at the state provincial office of southern Finland (I, II and III; ESAVI/6054/ 04.10.03/2012).

STUDY COHORTS

The affected and control LRs used in this thesis are presented in Tables 4 and 5. All affected LRs were genetically confirmed homozygous for the ATG4D variant. Controls 1-4 and 6 were homozygous wild-type for the ATG4D variant and controls 5 and 7 heterozygous, clinical healthy carriers of the variant.

Table 4. ATG4Dmut/mutLRs with the histopathological phenotype confirmed by post mortem examination (PM) or skin biopsy (B).

Case Nationality Gender Age at sampling Disease Sample type Study duration 1 FI male 2 y 5 mo 10 mo PM I, II 2 FI male 2 y 4 mo 15 mo PM I, II 3 FI female 1 y 8 mo 16 mo PM I 4 FI female 8 y 4 y PM I 5 FI female 8 y 5 y PM, B I, II, III* 6 FI male 8 y 5 y PM, B II, III* 7 FI female 7 y 3 y PM, B II 8 CH female 2 y 9 mo 3 mo PM I 9 CH female 7 y unknown PM, B III 10 SE male 2 y 3 mo PM I 11 SE male 6 y 8 mo 5 y PM I 12 NO female 4 y 2,5 y PM, B I, II 13 ITA male 8 y not specifed PM, B II 14 FR female 8 y no signs B I, II 15 USA female 2 y 6 mo B I, II *skin biopsy for dermal fibroblasts at the age of 5 years, PM: post mortem, B: skin biopsy

Table 5. Finnish LRs used as controls.

Control Gender Age Sample type Used for Disease Study

1 female 8 y PM histology, IHC Idiopathic II epilepsy 2 male 5 y PM histology, IHC hemangio- I, II sarcoma 3 female 4 y B WB, IF mammary II tumor 4 female 5 y B WB, IF skin tumor II 5 female 4 y B EV studies ovario- III hysterectomy 6 female 5 y B EV studies mammary III tumor 7 male 8 y B EV studies skin tumor III

BIOCHEMICAL SCREENING

4.3.1 URINE ANALYSIS Urine samples of cases 1 and 5 were examined by thin-layer chromatography stained with resorcinol, in order to identify the excreted oligosaccharides (Professor Martin Renlund, Department of Pediatrics, Helsinki University Central Hospital). Urine samples of two clinically healthy dogs were included as canine controls. Samples of a healthy human and a human patient with Salla disease and excretion of sialic acid in the urine were included as negative and positive laboratory controls for the sugar staining. The glycosaminoglycan concentration (GAG) was determined as described by de Jong (de Jong, Wevers, & Liebrand-van Sambeek, 1992), based on the color reaction with dimethylmethylene blue. The urinary GAG to creatinine ratio was used to compensate for the concentration differences in the native urine samples.

4.3.2 ENZYME ACTIVITY Dermal fibroblasts from cases 5, 6 and 12 (Table 3), and controls 3 and 4 (Table 4) were cultured as described for autophagy monitoring and the enzyme activity levels of three lysosomal enzymes : arylsulfatase A (ARYLS-A), beta- galactosidase (b-Gal), and glycosylasparaginase (GA) were measured within the lysed cells. The GA activity in the culture medium of the affected and control cells was also measured to test for mistargeting of the enzyme to the

extracellular milieu. Briefly, the formation of p-nitrocatechol from p- nitrocatechol sulfate by ARYLS-A, 4-methylumbelliferone from 4- methylumbelliferyl-beta-D-galactoside by b-Gal, and 7-amido-4- methylcoumarin from L-aspartic acid β-7-amido-4-methylcoumarin by GA, was followed using a kinetic measurement, by spectrophotometry for ARYLS- A and microtiter plate spectrofluorometry for b-Gal and GA. The enzyme activity was expressed as nanomoles of substrate hydrolyzed/minute/mg protein in the sample preparation.

POST MORTEM EXAMINATION

Autopsy was performed in 13 LRs during the study (case Nos. 1-13). Samples of the major internal organs, endocrine glands, eyes, inner ear, skin, central nervous system (CNS) and peripheral nerves were formalin fixed, paraffin embedded and processed for routine histology. Only the brain and a skin biopsy were examined from one of these LRs (case 13). Skin biopsies from the dorsal flank area of four LRs (cases 5, 6, 14, 15) were formalin-fixed, paraffin- embedded, and processed for routine histology.

4.4.1 HISTOPATHOLOGY The formalin-fixed material was sectioned and stained with hematoxylin- eosin (HE), periodic-acid-Schiff’s reaction (PAS) and treated with diastase for glycogen digestion (PAS-diastase). Fresh-frozen tissue samples were prepared from pancreas and skin of cases 2 and 3, as well as control 1, and stained with PAS and Oil-red-O for glycoproteins and lipids, respectively.

4.4.2 TRANSMISSION ELECTRON MICROSCOPY Transmission electron microscopy (TEM) samples from the cerebellar cortex and thalamus of case 2, mandibular salivary gland and pancreas of cases 2 and 12, and skin of cases 2, 5 and 12 were fixed in 2.5% glutaraldehyde, post fixed with 1 % osmium tetroxide, stained with 8 % uranyl acetate in 0.69 % maleic acid and embedded in epoxy resin along with respective tissues from control dog number 1. Ultrathin sections, mounted on copper grids, were stained with Reynolds lead citrate and viewed with a Jeol JEM-1400 (Jeol Ltd., Tokyo, Japan) electron microscope, with a Gatan Orius SC 1000B bottom mounted CCD-camera (Gatan Inc., USA), at 80 KV.

4.4.3 IMMUNOHISTOCHEMISTRY Antibodies used for immunohistochemical staining (IHC) on formalin-fixed, paraffin embedded material are summarized in Table 6. Antigens were

retrieved with 0.01 M citrate buffer of pH6 and heat (99°C for 20 minutes). The UltraVision Detection System HRP/DAB kit (Thermo Fisher Scientific Inc.), reacting with mouse- and rabbit primary antibodies, using an enzyme- labelled polymer-linked secondary antibody and DAB as chromogen, was used for revealing the label. Sections from affected dogs, stained without the primary antibody, were used as negative controls. Sections of control cases 1 and 2 were used for assessing unspecific label of CNS.

Table 6. Primary antibodies used with different methods in this thesis.

Antigen Specific Type and Reactivity* Method antibody clonality LAMP2 3144, LSBio mouse monoclonal Human, Dog IHC, IEM, IF LC3I and II ab48394, rabbit polyclonal Human, Mouse, Rat, IHC, IEM, IF Abcam Zebrafish, Syrian hamster, Bacteria LC3I and II L7543 Sigma- rabbit polyclonal Human, Mouse, Rat WB Aldrich ATG4D SAB1301447, rabbit polyclonal Mouse IHC, IEM Sigma-Aldrich GFAP MCA1909, mouse monoclonal Human IHC BioRad TFR2 PA5-42732 rabbit polyclonal Bovine, Dog, Horse, IEM ThermoFisher Guinea pig, Human, Mouse, Rabbit, Rat p62 P0067, Sigma- rabbit polyclonal Human, Mouse, Rat IHC Aldrich (affinity isolated) Ubiquitin ab7780 Abcam rabbit polyclonal Mouse, Rat, Human, IHC African green monkey β-actin PA5-16914 rabbit polyclonal Human, Mouse, Dog, WB Thermo Fisher Sheep Unspecific ab37415, rabbit polyclonal non-specific, IgG IEM Abcam isotype-control

GFAP: glial fibrillary acidic protein, * according to manufacturer

4.4.4 IMMUNOELECTRON MICROSCOPY For immunoelectron microscopy (IEM), tissue samples of pancreas and the dorsal root ganglion (DRG) from one affected dog (case 5) were collected within 1 h after euthanasia. Tissues were fixed in 4% paraformaldehyde for 2 hours, transferred to 2% paraformaldehyde and embedded into Lowicryl K4M. Immunolabeling was performed on thin sections, using 5% bovine serum albumin (BSA Sigma-Aldrich®), 1% fetal bovine serum (FBS 10500056

Gibco®) containing NaPO4 buffer, pH 7.4, and 0.5% fish skin gelatin (G7765, Sigma-Aldrich® CAS 9000-70-8) as blocking agents. The grids were then incubated with primary antibodies against LAMP2, LC3, ATG4D and TFR2 (Table 6) at room temperature for 2 h. For evaluating the density of unspecific staining, control sections were incubated with a rabbit-isotype control antibody (Table 6) as well as without primary antibody. All sections were incubated with either protein-A-conjugated 10-nm gold or gold-conjugated goat-anti mouse secondary antibody for 20 minutes. After washing, a 5 min post-fixation with 5% glutaraldehyde was used, and the sections were then post-stained with uranyl acetate and lead citrate. ImageJ software (Fiji, by Wayne Rasband, National Institute of Mental Health, Bethesda, Maryland, USA) was used for determining the gold labelling density of vacuolar membranes and of non-membranous areas in the cells. Considering the size of the primary and secondary antibodies, gold particles within 15 nm from a membrane were counted as membranous label. A minimum of 50 gold particles, or all gold particles in 10 fields of view at 10 000x magnification, were counted for each tissue, antibody and control. The total non-membranous and membranous areas viewed in each sample were also measured. The specific labelling density, D(s), was determined for each antibody, as the total membranous gold density D(+) (dots/μm2), minus the unspecific membranous labelling density D(-) noted in control sections stained either without a primary antibody, or with the isotype control antibody. The specificity of the gold label regarding antibodies binding both to a protein on the vacuolar membrane and free in the cytosol (LC3II and ATG4D) was evaluated using the chi-squared test. Hereby the non- membranous versus membranous label distribution of LC3 and ATg4D stained sections was compared to the membranous versus non-membranous label distribution in the isotype stained control sections.

MONITORING AUTOPHAGY

4.5.1 CELL CULTURE EXPERIMENTS AND WESTERN BLOT Fibroblasts from two cases (5 and 12), and two controls (3 and 4) were cultured as primary cells in high glucose Dulbecco’s Modified Eagle Medium (DMEM 41965-039 GibcoTM) supplemented with 10% fetal bovine serum (FBS 10500056 GibcoTM) and penicillin-streptomycin (PenStrep 15140122 GibcoTM). During 2nd or 3rd passage, the fibroblasts were seeded with equal densities on Petri dishes and glass cover slips. When semi-confluent, the cells were incubated in fresh medium for 2 h, and subsequently subjected to a 2 h starvation in Earl´s Balanced Salt Solution (EBSS 24010043 GibcoTM) with, and without, 0.4 μM lysosomal H+ATPase inhibitor Bafilomycin A1 (Bafilomycin A1 BioViotica NSC381866). Control samples were subjected to a 2 h starvation with 0.4% DMSO in EBSS.

The following samples were harvested: 2 h under basal conditions with and without Bafilomycin A1, 2-h starvation, with and without Bafilomycin A1. Cells were lysed with RIPA buffer containing 0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 10% NP-40, 2.5% deoxycholic acid, 10 mM EDTA (R0278 Sigma) and a protease inhibitor cocktail (SigmaFAST™, S8830). The cell lysates were subjected to Western blotting (WB) with LC3 antibody (Table 6). Beta actin was used as loading control. The signals were visualized and quantified using enhanced chemiluminescence. The WB experiments were conducted as two technical replicates of two case/control pairs, representing biological replicates.

4.5.2 IMMUNOFLUORESCENCE Fibroblasts from two affected (5 and 12) and two control LRs (3 and 4) were grown on coverslips and starved for 2 h or left untreated. The cells were fixed with cold methanol at -20 °C for 5 minutes and stained for immunofluorescence with primary antibodies against LC3 and LAMP2 (Table 6). Secondary antibodies conjugated to Alexa Fluor 594 (A11037, Thermo Fisher) for LC3 and Alexa Fluor 488 (A11029, Thermo Fischer) for LAMP2 were used. Nuclei were visualized with 4, 5-diamidina-2-phenylindole (1 mg/mL DAPI, 62248, Thermo Fisher). The stained coverslips were mounted onto microscope slides and viewed with a Zeiss Axio Imager A2 fluorescence microscope. Images were captured with a Zeiss AxioCam MRc5 CCD camera, set at 40 ms and 90% exposure intensity. The number of LC3 positive spots was counted in 50 cells from each sample, using Cell Profiler software. Briefly, the count for number of cells was based on the DAPI signal, cytoplasmic area defined by the LAMP2 signal from lysosomes, and number of LC3-positive spots were accepted only if overlapping with cytoplasm (Fig. 5).

Figure 5 Detecting LC3-positive (red) spots in cells (green outline) with Cellprofiler Software. Cells exceeding the pictures border were discarded (2, 5, 7, 8). The analyzed pictures were acquired with a Zeiss AxioCam MRc5, set at 40ms and 90% exposure intensity in an Imager A2 fluorescence microscope.

EXTRACELLULAR VESICLES

4.6.1 CELL CULTURE, VESICLE ISOLATION AND QUALITY CONTROL Fibroblasts from three affected (5, 6, 9) and three control LRs (5, 6, 7) were seeded at equal cell density into two 175 cm2 flasks in high glucose DMEM, containing 10% FBS and PenStrep. After 48 h, the cells were washed trice with PBS and further incubated in 25 ml DMEM containing 10% EV-free fetal bovine serum (EV core facility, University of Helsinki) and PenStrep. At semiconfluent growth, after 72 h of culture and a visual control of cellular integrity, the medium from affected and control cells was decantered into sterile Falcon tubes. After three washes with PBS, affected and control cells were subjected to a 2 h starvation in EBSS. The conditioned EBSS was harvested after the starvation. The total harvested medium (50ml) from each C for 30ﹾexperiment was cooled immediately and centrifuged at 2500 g, 4

C before furtherﹾ minutes. The supernatants with EVs were frozen at -80 processing. EVs were then pelleted through ultracentrifugation at 110 000g for 2 h, washed and resuspended in 1 ml buffer and further ultracentrifuged at 110 000 g for 90 minutes. The final EV pellet was resuspended in 100 μl buffer. One aliquot from each dog was viewed in negative TEM as quality control. For this, EVs were loaded on 200 mesh pioloform- and carbon-coated glow-discharged copper grids. Fixation with 2% PFA (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M NaPO4 buffer (pH 7.0) was used and grids were then stained with 2% neutral uranyl acetate, embedded into a methyl cellulose uranyl acetate mixture (1.8/0.4%) and viewed with TEM, using a Tecnai 12 (FEI Company, Eindhoven, The Netherlands) operating at 80 kV. Images were taken with Gatan Orius SC 1000B CCD-camera (Gatan Inc., USA). Proteins from category 1, 2 and 3 detailed in Table 3 in the Minimal Information for Studies of Extracellular Vesicles 2018 were used as reference for protein based EV characterization and quality control (Thery et al., 2018).

4.6.2 NANOPARTICLE TRACKING ANALYSIS AND VESICLE QUANTIFICATION The concentration and size-distribution of EVs were determined using nanoparticle -tracking -analysis (NTA). In brief, NTA is based on detecting the scattering of laser light, caused by particles randomly moving in suspension. A microscope is used to visualize the scattered light, which is recorded over time. The movement of particles in suspension is dependent on their size, which can be calculated by computer software. NTA provides a size distribution curve of particles in suspension, along with the particle concentration in the NTA aliquot (Fig. 6). From this analysis, the EV concentration in the original sample can be calculated. Comparisons were made between the mean concentration of EVs and their size-distribution in media from affected and control fibroblasts under basal conditions (FM=full media) and starvation (ST=starvation). The mean EV concentration was determined as an average of five measurements using NTA. The EV size distribution in affected and control samples was compared using mean percentiles of vesicles less than 100 nm, 150 nm and 250 nm in diameter.

Figure 6 Example of the NTA result, based on five repeated measurements from one EV sample.

4.6.3 MASS-SPECTROMETRY AND PROTEOMIC ANALYSIS The total proteome of EVs harvested from 50 ml media of affected and control fibroblasts during basal conditions, and after a 2 h starvation, were analyzed by mass-spectrometry (MS). The EVs were trypsinized, and peptides were washed and sorted with a C18 liquid chromatography column according to hydrophobicity. A mass spectrometer sorted the peptides according to mass/charge and the 10 most abundant precursor peaks of the MS1 spectrum were used for further analysis. The fragmentation spectrum was filtered at a false discovery rate (i e., the portion of false positive spectrum matches allowed in the peptide’s further analyzed) was set at 5 %. Peptides were identified based on peptide spectrum matches to canine proteins annotated in the Uniprot data base (https://www.uniprot.org/proteomes/UP000002254) using the Proteome Discoverer TM software of ThermoFisher. Unreviewed canine proteins and the ATG4D variant c.1288G>A were included in the search. Proteins identified based on two or more specific peptides and with two or more peptide spectrum matches (PSM) were included in further analysis.

The genes encoding proteins that were 1) common to 2 or more affects, respectively two or more controls, 2) only present in EVs of affects and 3) only present in EVs of control cells, were determined by Venn diagrams. The subcellular origin, molecular function, biological process and interactions of the proteomes specified above were mapped using FunRich Software for functional enrichment and interaction network analysis of genes and proteins (http://www.funrich.org/).

STATISTICAL ANALYSIS

SPSS® IBM software was used for statistical testing and differences with p< 0.05 considered statistically significant. The two-tailed t-test was used when comparing differences between affected and controls if results showed a normal distribution (Western blotting). Nonparametric tests comparing two samples (Mann-Whitney-U test) or all four samples (Kruskall-Wallis test) were used when the distribution of the results was skewed (immunofluorescence: number of spots/cell). Cross tabulation and the Chi Square test were used for comparison of the distribution of a categorical variable (IEM: membranous dot vs nonmembranous dot). The linear mixed model method for dependent samples was used when the mean was based on repeated measurements (EVs: concentration, size distribution).

5 RESULTS

The age distribution at elected euthanasia (Fig. 7) showed that affected LRs were generally either euthanized at below 3 years of age (5/13) or coped with the disease until the age of 7 years or more (6/13), regardless of country of origin.

Figure 7 The age and gender of individual affected LRs (female=red dot, male= blue dot) at euthanasia showing 5 of 13 dogs being euthanized before 3 years-of-age, whilst 6 lived up to 7 years-of-age.

THE HISTOPATHOLOGIC PHENOTYPE OF LRS WITH THE ATG4D VARIANT COMPRISES NEURODEGENERATION AND INTRACELLULAR VACUOLAR STORAGE

During post mortem examination, cerebellar atrophy was evident as thinning of the cortical grey matter and broadening of cerebellar cortical sulci in the visual macroscopic examination of the brain in two dogs (cases 6 and 9). Histologically neurons, secreting epithelial cells, and specific mesenchymal cells were distended by clear cytoplasmic clear vacuoles (Fig. 8 A-F) in the HE stain. This change affected neurons in both the CNS and the PNS, epithelial cells of several endo- and exocrine glands (parathyroid gland, prostate, adrenal cortical cells, thyroidal follicular cells, exocrine pancreatic cells,

dermal sweat glands, mammary and para-anal glands, salivary glands and their secretory ducts) as well as mesenchymal cells involved in transcytosis or unconventional secretion, such as vascular endothelial cells, pericytes, and glomerular podocytes. Also macrophages and plasma cells were affected. Mainly small vesicles, 1-2 μm in diameter, were present in secretory epithelial cells (Fig. 8 E and F), whilst neurons and mesenchymal cells contained also larger vacuoles, exceeding 40 μm in diameter (Fig. 8 A, C, D). The neuronal vacuolation was widely distributed within the CNS, and evident throughout the neuroaxis from the cerebral cortex to motor neurons and dorsal root ganglia of the spinal cord in dogs with a prolonged disease. The severity of the vacuolation, however, varied greatly even between neurons, even of the same affected area (front page). Consistently severely affected areas included the cerebellar cortex, specifically the Purkinje cells, and the deep cerebellar nuclei as well as the vestibular nuclei. Multifocal neuronal loss was evident as cell bodies lacking within the Purkinje cell layer and consequent granular cell loss in dogs with a prolonged disease course. Consistently vacuolated were also neurons of the red nucleus, the lateral and medial geniculate nuclei of the thalamus. The cerebral cortex, caudate nucleus and specific hindbrain nuclei, such as the oculomotor and hypoglossal nuclei, were less severely affected even in dogs with a prolonged disease history. Dorsal root ganglia and the coeliac ganglion were severely affected, whereas cell bodies of the parasympatic intramural nerve plexi in visceral organs were less altered. Vacuolation was also evident in the cytoplasm of choroid plexus cells and cells of the subcommisural organ. Axonal spheroids were numerous, both in the dorsal funiculus of the spinal cord and the white matter of the cerebellum, as well as in the cerebellar cortical granular cell layer, the cerebellar nuclei and nuclei of the medulla oblongata. Both homogenously eosinophilic spheroids and spheroids containing coarse granular material were present (Fig. 8 B). In areas of spheroid formation and neuronal loss, reactive gliosis was evident as both astrocytosis ; an increase in GFAP- positive cells in IHC, and also as an increase GFAP-negative glia cells. The axonal changes were limited to the CNS. Only the nervous system showed loss of cells, as extraneural organs displaying cytoplasmic vacuolar storage were not affected by scarring or atrophy (Fig. 8 E and F). Small amounts of granular, intracellular, eosinophilic material were present in the smooth muscle cells of the skin (m. erector pili), the tunica muscularis of the gastrointestinal wall and the urinary bladder. The sweat gland vacuolization was evident in all skin biopsies, except one (case 5), examined from live LRs cases. To conclude, the histopathological phenotype of affected dogs was indicative of a lysosomal storage disease.

Figure 8 Histological findings in the nervous system and extra neural organs in LRs with the ATG4D variant (modified from I and II). A. Cerebellar cortex: Purkinje cells (arrows) contain clear cytoplasmic vacuoles. HE B. Medulla oblongata: numerous axonal spheroids, some with a granular texture (arrow) are present. HE. C. Arteriole: the pericytes of arteriolar walls, enwrapped by basement membrane (arrow) are distended by large clear vacuoles. PAS. D. Lymph node: plasma cells and macrophages are severely vacuolated. HE. E. Mandibular salivary gland: acinar cells and the epithelial cells of intralobular ducts show fine vacuolation. HE. F. Pancreas: pancreatic acinar cells are severely vacuolized, without an inflammatory or fibrotic tissue response. HE. Modified from study II, Figs 4 and 6.

BASAL AUTOPHAGY IS ALTERED IN LRS WITH THE ATG4D VARIANT

When monitoring autophagic flux in fibroblasts from affected LRs using LC3I/LC3II Western blot, basal LC3II levels were elevated compared to LC3II levels in control cells (Fig. 9 A). Increased LC3II levels can be due to increased synthesis of autophagosomes, with normal formation of autolysosomes and lysosomal degradation, representing an upregulated autophagic flux (Mizushima & Yoshimori, 2007; Mizushima et al., 2010). Alternatively, LC3II levels can increase if autolysosome formation, lysosomal LC3II degradation and normal turnover of LC3II from the autolysosome back to cytosolic LC3I is blocked (Galluzzi et al., 2017; Mizushima et al., 2010). Hereby autophagic flux is decreased in cells and further inhibition of lysosomal function does not significantly increase LC3II levels, as this later part of the autophagic process is already compromised. When disabling lysosomal degradation by adding Bafilomycin A1 during basal conditions, LC3II increased less in affected cells than in controls. This indicates that basal lysosomal degradation of autophagic cargo is partly disabled in the affected cells, and that the basal LC3II increase in the affected cells was not due to upregulation of autophagic flux. During starvation-induced autophagy, the LC3II levels increased equally in control and affected cells, and a comparable further increase was noted in both samples after lysosomal inhibition by Bafilomycin A1 during starvation. Thus, starvation-induced autophagy and lysosomal degradation of autophagosomes and cargo is not altered in cells with the ATG4D variant. The difference in LC3II levels during basal conditions was significant when quantified against the loading control (II, Fig. 1).

Figure 9 Monitoring autophagy in cells from affected LRs. A.) WB detecting LC3I and LC3II in fibroblasts from control (lanes 1-4) and affected (lanes 5-8) LRs at basal conditions (DMEM), at basal conditions during lysosomal inhibition (DMEM+Baf.) and after 2h starvation (EBSS) as well as 2h starvation and lysosomal inhibition (EBSS +Baf.). LC3II is almost absent in control cells during basal conditions (lane marked in green), whereas cells from the affected LRs have a clear LC3II band at basal conditions (lane marked in red). B.) IF of LC3-positive spots (red) in cells from affected LRs and control LRs at basal conditions (DMEM) and after 2h of starvation (EBSS 2h). Fibroblasts with the ATG4D variant show an increased number of spots during basal conditions, in comparison to control cells. Modified from study II, Fig.1 and 2.B

When using IF to visualize and quantify autophagosomes in fibroblasts from affected LRs, significantly more LC3-positive spots were noted under basal conditions in affected, than in control cells (Fig. 9 B: A vs C). In agreement with results from the WB, there was a milder increase in the number of LC3-positive spots in affected cells after lysosomal inhibition under basal conditions. However, the change in number of LC3-positive spots induced by starvation in affected cells did not differ from control cells (Fig. 8 B: B vs D). The difference in the median number of LC3 positive spots under basal conditions was significant, as was the difference in the median increase in number of LC3-positive spots after lysosomal inhibition (II Fig.2 g-i). Taken together, the WB and IF findings indicate that basal autophagy is dysfunctional in cells with the ATG4D variant, while starvation-induced autophagy functions normally. Since lysosomal inhibition during basal conditions caused a milder increase in both LC3II level and number of LC3- positive spots in affected cells, it is likely that the later phases of autophagy, involving autolysosome formation and/or lysosomal degradation, are altered.

THE HISTOLOGICAL LESIONS ARE ASSOCIATED WITH ACCUMULATION OF AUTOPHAGIC CARGO AND AUTOPHAGIC MEMBRANES

Accumulation of autophagic cargo and autophagic membranes was detected by IHC within the lesions and organs of affected LRs (Fig. 10 A-F), supporting the results from WB and IF experiments and the hypothesis of altered autophagy. The results were most consistent regarding the axonal spheroids, as they were positive for LC3 as well as for ubiquitin and p62, markers of protein aggregates destined for autophagy. The axonal spheroids contained numerous subcellular vacuoles, with variable content and delined by multiple membranes, consistent with autophagosome /autolysosome accumulation on EM (Fig. 10 G, H).

Figure 10 A-F, IHC of CNS and extraneural tissues with markers for autophagic cargo (ubiquitin, p62), autophagosomal membranes (LC3) and ATG4D. G-H, EM of axonal spheroids. A: IHC Ubiquitin, medulla oblongata: axonal spheroids contain coarse ubiquitinated material (arrows). B: IHC ATG4D, cerebellar cortex: the homogenous axonal spheroids show weak ATG4D positivity (arrow). C: IHC LC3, medulla oblongata: axonal spheroids are diffusely LC3 positive. D: IHC LC3, kidney: the vacuolated glomerular podocytes are LC3 positive. Inset: LC3 IHC kidney of control dog. E: IHC p62, cerebellar cortex: spheroids contain also coarse p62 positive material (arrows). F: IHC p62, smooth muscle: there is granular p62 positivity in smooth muscle. G and H: EM, axonal spheroid: the axon is distended by vacuolar profiles (arrowheads) lined by double or multiple membranes consistent with autophagosomal/autolysosomal structures. Higher magnification in panel H shows that the accumulated vacuoles resemble autolysosomes. GCL: granular cell layer. WM: white matter. ML: molecular layer. PCL: Purkinje cell layer. Modified from study I Fig.4 and II Fig. 8.

LYSOSOMAL STORAGE OR A LYSOSOMAL ENZYME DEFICIENCY WERE NOT DETECTED IN THE AFFECTED DOGS

As the morphological phenotype provided the differential diagnosis of LSD, investigations for evidence supporting this alternate hypothesis were performed. Histochemical staining of formalin fixed, and of frozen samples, for glycogen, glycoprotein and lipofuscin (PAS) as well as for glycolipids (oil-red- O) failed to reveal specific content within the intracellular vacuoles in affected tissues. The cytoplasmic vacuoles of neurons and extraneural cells were lined by a single membrane (Fig. 11 A and B) in TEM. They appeared empty or contained sparse floccular or membranous material (Fig. 11 A and B). These

features indicated that the vacuoles were not autophagosomes, as autophagosomes are lined by double membranes and contain engulfed material. Occasionally, inward budding, fusion and tethering occurred between the vacuoles. The vacuoles did not contain a specific, stored material visible on TEM.

Figure 11 TEM, IHC and IEM of the cytoplasmic vacuoles. A.TEM pancreas: the cytoplasmic vacuoles are limited by a single membrane, and often appear to be fusing with each other. B. TEM, sweat gland acinar cell, 5000x: the vacuolar content is sparse, comprised of loose floccular material and small vesicular profiles. C. IHC LAMP2, cerebellar cortex: faint membranous LAMP2 positivity in neuronal somal vacuoles (arrows). Inset: LAMP2 IHC of control neuron. D. IHC LC3, cerebellar cortex: increased cytoplasmic and coarse perivacuolar LC3 positivity in neurons (arrow). Inset: LC3 IHC of control neuron. E and F: IEM, gold labelled LC3 antibody (E) in dorsal root ganglium and LAMP2 (F) antibody in pancreas bind to the vacuolar membrane (arrows). Modified from study I Fig.4 and study III Fig.2.

Weak membranous LAMP2 and LC3 positivity was noted in the neuronal vacuoles (Fig. 11 C and D), but the IHC result was not clear enough to definitely characterize the nature of the limiting membrane of the cytoplasmic vacuoles, especially in several extraneural organs. IEM increased the specificity of the staining, and a positive membranous LAMP2 and LC3 label were confirmed in neurons and pancreas (Fig. 11 E and F). Transferrin receptor-2 and ATG4D were also detected on the membrane limiting the cytoplasmic vacuoles of neuronal and pancreatic acinar cells by IEM, with a comparable specific labeling density as the LC3 signal. The urine analysis of affected LRs displayed an oligosaccharide pattern similar to that of canine controls, with no excessive excretion of a specific sugar. No excretion of excessive glucosaminoglycanes was detected in the urine, as the average GAG/Crea ratio of affected LRs (2,3) was comparable to that of controls (2,1). No marked intracellular decrease or extracellular increase in the activity of selected lysosomal enzymes was detected in the affected LRs, as would be expected in primary LSD (Fig. 12).

Lysosomal enzyme activity 100 90 80 70 60 50 Average control 40 Average affected 30 20 10 0 Beta-gal. Arylsulf. Glycosylasp. Ic Glycosylasp. Ec nmol/min/mg nmol/mg/min pmol/min/mg pmol/mg/min

Figure 12 The enzyme activity of selected lysosomal enzymes in fibroblasts of affected LRs (red) in comparison to control LRs (blue). The activity of beta-galactosidase and arylsulfatase-A was mildly increased within cells of affected LRs, however the difference was less than two-fold. The activity of glycosyl-asparaginase in cells (Ic) and culture medium (Ec) from affected cells did not exceed that of the controls.

Summarizing the results, the variant ATG4D is linked to disturbed basal autophagy in cells, with accumulation of autolysosome-like vacuoles within the axons and spheroid formation in the CNS. In addition, hybrid vacuoles derived from the autophagy-lysosomal-endosomal pathways accumulate within the soma of various cell types. No specific storage material is evident in the vacuoles and the biochemical screen results do not support a LSD in affected LRs.

CELLS WITH THE ATG4D VARIANT RELEASE SUBSTRATES OF BASAL AUTOPHAGY VIA EVS

There was no significant difference in the morphology (Fig. 13A) or size distribution of EVs released from fibroblasts of affected LRs, when compared to those released from control fibroblasts. Notably, fibroblasts carrying the ATG4D variant released significantly more EVs during basal culturing conditions than control cells (Fig. 13B). This indicates that the various EV release mechanisms are functional in cells with the ATG4D variant, and that the vacuolar accumulation is not due to blocked extracellular release of EVs.

A B EV concentration in medium 10 8 6 4

Mean E10/ml 2 0 DMEM EBSS 2h Control Affected

Figure 13 EV morphology and concentration in medium conditioned by fibroblasts with the ATG4D variant. A: the EVs from affected cells showed a round and cupped shape, comparable to EVs of control cells (inset). EM of EVs, negative staining. Modified from III Fig. 3. B: fibroblasts from affected LRs released significantly more EVs during basal conditions than control cells. *P< 0.05

The proteins of EVs released under basal conditions were further analyzed regarding purity, cellular compartment of origin and molecular function. Altogether 80% of the proteins identified in control EVs and 78% of those in EVs from affected cells have previously been associated with the exosomal fraction, including proteins from categories 1 and 2 in Table 3 of the guidelines for EV purity assessment (Thery et al., 2018). Non-EV derived, co-isolating proteins (category 3 , Table 3, MISEV 2018) were identified at lower levels than exosomal proteins, and with comparable frequency, in all samples, indicating that the sample quality was comparable between affected and control cells. During basal conditions, the EV proteome of affected cells showed qualitative differences in comparison to the EV proteome of control cells when annotated according to function and subcellular origin (Fig. 14). There was an increased amount of cytosolic, ER and mitochondrial-derived proteins in the EVs of affected cells, corresponding to chaperones and heat-shock proteins from these organelles. Chaperones unique to EVs of affected cells included

calreticulin, calnexin, heat-shock protein family A, T-complex protein 1 (TCP1) subunits, chaperonin containing TCP1 (CCT) and endoplasmin. p62 was also identified in EVs of affected but not control cells, however based on only one unique peptide in two samples, and three unique peptides in one sample. Proteins unique to control EVs, involved in vesicular dymanics and lacking in EVs from affected cells, included Rab-GTPases Rab1A and Rab5C. Wild-type or mutant ATG4D was not detected in the EV proteomes of affected of control cells.

A. B.

* * * *

Figuregure 1144 Comparison of the cellular compartment of origin (A) and the molecular function (B) of the proteproteinsi in basal EVs of affected cells and control cells identified by MS. The mapping is basedsed on ththe known functions of genes coding the identified proteins using FunRich Software. The EVEVs of f affected cells are enriched in proteins from the ER, mitochondria and cytosol *, whilst depleted on cytoskeletal and nucelar proteins*. When mapped according to function, more chaperones, proteins with heat shock activity or extracellular matrix proteins were identified in the EV proteome of affected cells, compared to controls. Modified from study III Fig.4.

6 DISCUSSION

This thesis work describes a novel neurodegenerative disease of LRs, regarding the histopathology, alterations in the autophagy-endolysosomal system and EVs of cells from affected dogs.

A NOVEL NEURODEGENERATIVE DISEASE IN LRS

Several neurologic diseases, which affect the cerebellum and clinically cause ataxia, are known in the LR breed. The benign familial juvenile epilepsy (BFJE) of LRs was the first spontaneously regressing genetic epilepsy described in dogs (Jokinen, Metsahonkala et al., 2007), where interictal ataxia occurs in the most severe cases. BFJE is caused by a change in the Leucine- Rich, Glioma Inactivated 2 (LGI2) gene, that prevents the secretion of the LGI2 protein from neurons (Seppala et al., 2011) rendering them prone to electric instability. The disease remits spontaneously when LGI2 expression is downregulated naturally, at the age of approximately four months in LRs (Seppala et al., 2011). The histologic lesions of LRs with BFJE were restricted to Purkinje cells in the cerebellar cortex (Jokinen et al., 2007). Two other forms of cerebellar cortical degeneration also affect the LRs, one targeting the Purkinje cells, and another targeting the granular cell layer. The causes of these progressive disorders are undefined (Jokinen, Rusbridge et al., 2007). The vacuolar storage disease described in this thesis provides a fourth differential diagnosis for ataxic young LRs, however, with a later age of onset, and importantly, a prolonged disease course without remission. It is unclear whether the two-weighted age distribution of dogs at euthanasia is solely due to the owners’ perception of the dogs’ quality of life, or whether it reflects disease biology. Interestingly, the predominant ages at euthanasia coincide with the change in metabolism from a growing dog into the basal state of a full-grown young adult, and with the start of aging. The efficiency of autophagy decreases with age (Mizushima et al., 2008; Rubinsztein, Mariño, & Kroemer, 2011), and canine age-related cognitive changes are known to occur from 6 years onwards (Snigdha, de Rivera, Milgram, & Cotman, 2016). It is possible that a further, age-related, decrease in autophagic function could exaggerate the clinical signs in elder LRs with disturbed basal autophagy. Despite the occurrence of histological lesions in several extra neural organs, the clinical signs in this disease are neurological. Other organs known to depend on efficient autophagy, such as the heart and liver (Mizushima et al., 2008), showed only mild histological changes that did not translate into clinical signs. One possibility is that induced autophagy, caused by changes in nutrient and glucose levels during normal canine life, compensate the deficits

in basal autophagy more efficiently in extraneural tissues than in the brain in LRs. Interestingly, changes in autophagy in the brain in response to fasting are region specific. The cerebellum, which was constantly severely affected in diseased LRs, showed no change in LC3 or p62 levels in response to 12-h fasting in mice (Nikoletopoulou, Sidiropoulou, Kallergi, Dalezios, & Tavernarakis, 2017). In comparison, autophagic flux was increased in the cerebral cortex and hypothalamus, while decreasing to sub-basal levels in the murine hippocampus after 12-h fasting (Nikoletopoulou et al., 2017). As the weight was not recorded at onset of signs in this study, a possible correlation between body condition and severity or age at onset of signs could not be examined.

ATG4D AS A CANDIDATE GENE FOR NEURODEGENERATION

Variants in the ATG4D gene had not been linked to neurologic disease in man or animals at the time of the first publication. The ATG4D gene is expressed throughout the CNS with a strong expression in the adult cerebellum, based on murine studies (Magdaleno et al., 2006). The ATG4D transcript was also present in the cerebellum of healthy and affected LRs (I). ATG4D has laso functionally been linked to the nervous system. Firstly, the priming and delipidation activity of ATG4D was increased towards GABARAPL1 after caspase cleavage (Betin & Lane, 2009) and secondly, GABARAPL1 of the LC3 homologues is predominantly expressed in the nervous system in man (Nemos et al., 2003). No ATG4D-deficient mouse models are available, but the Atg4b- knockout mice are vital and histological findings include ubiquitin-containing material within the neuropil of the deep cerebellar nuclei, without apparent neurodegeneration (Read et al., 2011). Atg4b-knockout mice show a systemic decrease in autophagy, mild functional neurologic motor deficit and impaired sense of balance (Marino et al., 2010; Read et al., 2011). At a cellular level, the link between disturbed ATG4D function and neurodegeneration is yet to be clarified. A regulatory function of ATG4D during oxidative stress at the beginning of apoptosis has been proposed. ATG4D contains a caspase cleavage site, in contrast to ATG4B and A, and a mitochondrial target motif absent in the other ATG4 paralogues (Fernandez & Lopez-Otin, 2015). Primarily, caspase-3 cleavage of ATG4D leads to increased autophagy as a cytoprotective response via the increased processing of GABARAPLI (Betin & Lane, 2009a). Under sustained pro-apoptotic signals and prolonged casepase-3 activation, however, ATG4D may translocate to mitochondria and induce apoptosis via mitochondrial stress, as seen during overexpression of the caspase-cleaved ATG4D form in cells (Betin & Lane, 2009). Increased ATG4D expression under oxidative stress has been described in cardiomyocytes in models of human tissue ischemia (Singh et al., 2014).

A role for ATG4D mutations in spontaneous disease has mainly been described in neoplasia. ATG4D amplifications and deletions are present several forms of cancer in humans and expression is downregulated in breast carcinoma (Zhang, X. et al., 2016), uterine fibroids (Andaloussi et al., 2017), acute myeloid leukemia (Jin et al., 2018) and colorectal cancer (Gil et al., 2018). ATG4D has been interpreted as a tumor suppressor gene based on these results. However, initial ATG4D suppression is indirectly blocked in a set of gliomas, where ATG4D upregulation correlates with decreased apoptosis, increased proliferation and unfavorable prognosis (Fu et al., 2017). Upregulation of ATG4D is also described in pulmonary squamous cell carcinoma (Lebovitz et al., 2015). This inconsistency may reflect the tumor specific carcinogenesis and the regulatory role of ATG4D regarding apoptosis and oxidative stress. Increased ATG4D expression may also have a beneficial or protective role, as in ageing, shown in centenarians (Xiao et al., 2018). In chronic inflammatory bowel disease in man, variants in the ATG4D gene were protective when correlated with granuloma formation and prognosis. The effect of the variants on autophagy or ATG4D at the protein level in the lesions was however not investigated (Brinar et al., 2012). Genetic investigations by collaborators showed an unaltered ATG4D transcription in the cerebellum of affected LRs (I). The ATG4D protein was detected within the axonal spheroids and at the limiting membrane of the cytoplasmic vacuoles, indicating alteration mainly in protein function. The results of the in vitro studies, altered basal autophagy in cells of affected LRs, support this. Taken together, the histological and in vitro results strongly link the gene variant and altered autophagy to the disease phenotype in LRs.

ALTERED BASAL AUTOPHAGY AS A HISTOPATHOLOGIC DIFFERENTIAL DIAGNOSIS FOR LYSOSOMAL STORAGE DISEASES

Intracellular vacuolation has long been considered the histologic hallmark of lysosomal storage diseases (Cantile & Youssef, 2016). Due to the specific biochemical composition of neurons, and neurons being post mitotic cells, the CNS is a major target organ in several LSDs (Cantile & Youssef, 2016; Schultz et al., 2011; Seranova et al., 2017). Morphologically, the vacuolar storage disease in LRs does not challenge this concept, as the vacuoles accumulating in LRs are positive for markers of the vesicular pathways fusing with lysosomes to acquire degradative enzymes. However, the disease in LRs provides evidence that the etiology of this morphologic change can be upstream of actual lysosomal degradation; in autophagy, MVB- and amphisome formation and function. It is known that autophagy is secondarily affected in several LSDs as the final degradative step, lysosomal degradation, is dysfunctional (Schultz et al., 2011; Seranova et al., 2017). Therefore, it was

important to distinguish between secondary autophagic alterations due to a potential primary LDS and novel, primary autophagic disease, with a morphology mimicking LSDs in the diseased LRs. Several findings in this study support the interpretation that lysosomal degradation is functional in LRs with vacuolar storage. Firstly, no specific content could be revealed in the vacuoles by histochemical staining of fixed or frozen sections for lipid, glycolipids, glycoproteins and glycogen, which accumulate in most LSDs. Secondly, specific content could not be detected in the vacuoles when viewed at the ultrastructural level. TEM is a widely used first-step screening method in diagnosing lysosomal storage, since storage of a specific material may directly provide clues on which enzyme activity is lacking. Stored material in mucopolysaccharidosis, gangliosidosis and lipofuscinosis, as examples, have a typical ultrastructure of zebra bodies, concentric membranous stacks and granular, non-membrane-bound cytoplasmic material, respectively (Cantile & Youssef, 2016). Urine analysis is another screening method for increased excretion of specific substrates that accumulate due to defective degradation. Water- soluble sugars that accumulate in some LSDs may disperse from the cells during fixation and therefore go undetected in histochemical investigations (Bolfa et al., 2019; Cantile & Youssef, 2016). The ultrastructural findings and target organs of oligosaccharide accumulation described in feline α- and canine β-mannosidosis overlap with those of affected LRs (Cantile & Youssef, 2016). The stored sugar was however excreted into the urine in dogs affected by β-mannosidosis and the disease progressed to elected euthanasia around 8 months-of-age (Jolly et al., 2019) or 1 year (Bolfa et al., 2019). Oligosaccharides and sialic acid patterns of affected LRs were similar to those of control urine and affected LRs managed with the disease until 7 or 8 years of age. Another indicator of a sustained lysosomal function was the even slightly elevated activity of selected lysosomal enzymes in cells from affected LRs, as the activity of a defective enzyme is commonly decreased in LSDs (Bolfa et al., 2019; Cantile & Youssef, 2016). The slight activity increase in cells of affected LRs, can be due to the accumulation of vacuoles with partially lysosomal content, as lysosomal membrane proteins were detected on the accumulated vacuoles by IHC and IEM. The histopathology and ultrastructural changes in affected LRs also mimicked Inclusion-cell disease (I-cell disease) or mucolipidosis type II, as described in man and cats (Boonen et al., 2011; Mazrier et al., 2003). In I-cell disease, lysosomal enzymes are erroneously secreted into the extracellular space due to a dysfunctional mannose-6- phosphate pathway. The ratio of extra- and intracellular activities of the lysosomal enzyme glycosyl-asparaginase, which is delivered to the lysosome via the mannose-6-phosphate tag, was therefore studied in cells of affected LRs. An increase in the extracellular activity of the enzyme, as described in I- cell disease (Boonen et al., 2011; Mazrier et al., 2003; Wang, P., Mazrier, Caverly Rae, Raj, & Giger, 2018) was not detected in the affected LR dogs.

The degradative capacity of the lysosome towards autophagy substrates can additionally be evaluated as part of the LC3 starvation essay for monitoring autophagy if a lysosomal inhibitor, such as Bafilomycin A1, is added to the medium. An increase in LC3II level after Bafilomycin A1 application indicates that autophagosomes fuse with lysosomes and LC3II is turned over normally, i.e., either released from the outer autophagosomal membrane as LC3I or degraded by lysosomal enzymes inside the autolysosome (Galluzzi et al., 2017; Mizushima et al., 2010) under normal conditions without the drug. The LC3II level in Western blot and the number of LC3-positive spots in immunofluorescence did not differ when adding Bafilomycin 1A to starved affected cells in comparison to control cells. This suggests that lysosomal degradation is not altered during starvation in cells from affected LRs. Increased release of EVs has been described as a pathway for extracellular release of accumulating substrates in LSDs (Tancini et al., 2019) Thus, it is possible that a quantitative assessment of the EV proteome could have detected accumulation of a specific protein in the EVs of affected cells. The proteomic analyses were however normalized towards the number of cells releasing them, not the EV amount. Since the released EV amount was much higher in affected cells, conclusions regarding the quantitative changes between the EV proteomes of affected cells in comparison to controls cannot be made based on these studies. The vast majority of the EV proteome in both affected cells (78%) and control cells (80%) comprised diverse proteins that have previously been shown to locate to exosomes.

DISTURBED BASAL AUTOPHAGY CAN CAUSE DISEASE DESPITE INDUCED AUTOPHAGY IS FUNCTIONAL

Under basal conditions, the LC3II level were increased in cells of affected LRs. In addition, increased granular LC3 staining was detected in neurons, axons, and smooth muscle cells in tissues from affected dogs by IHC. When inducing autophagy by starvation in fibroblasts, the LC3II level and the number of LC3- positive spots were similar in control and affected cells. Based on these findings, basal autophagy, but not starvation-induced autophagy, is dysfunctional in LRs with the vacuolar storage disease. Cellular models of autophagy have indeed shown that basal autophagy can be altered and lead to morphological changes in the cell, despite induced autophagy is functional (Murrow et al., 2015; Yla-Anttila et al., 2015). The vacuolar storage disease in LRs represents a spontaneous canine disease modelling disturbed basal autophagy. In cells with the ATG4D variant, both LC3II levels and the number of LC3 positive spots increased less than in controls when lysosomal function was inhibited during basal autophagy. This finding indicates that the increased LC3II level at basal state is not due to upregulated autophagosome formation

in cells from affected LRs, but rather due to altered autolysosome formation or LC3II turnover from the autolysosomal membrane. LC3II increases the intrinsic tendency for tethering and polymerization of membranes (Florey et al., 2011) and ATG4 peptidases counteract this (Agrotis, A. et al., 2019; Nakatogawa et al., 2012). Assuming that ATG4D is involved in releasing LC3II from the autophagosomal membrane, decreased function of the enzyme could cause prolonged and/or excessive association of LC3II on the autolysosome during basal autophagy. When assessed by IEM, ATG4D was indeed located to the vacuolar membrane in DRG and pancreas, in contrast to its diffuse cytoplasmic localization described elsewhere (Betin & Lane, 2009). Recently, a putative functional link between ATG4 and specifically basal autophagy was revealed. When preprocessed LC3I, resistant to ATG4- mediated deconjucation, was expressed in human cells lacking ATG4 activity, LC3 was covalently bound to other targets than PE, namely ATG3 (Agrotis et al., 2019). Interestingly, when ATG4-mediated deconjugation was not functional, LC3 linked to ATG3 at the same location, Lys 243, as ATG12 (Agrotis et al., 2019). The ATG3-ATG12 complex is a prerequisite for functional basal autophagy: murine fibroblasts lacking the ATG3-ATG12 complex accumulate enlarged MVBs, and have decreased basal, but functional starvation-induced, autophagy (Murrow et al., 2015). These findings are reminiscent of the morphological changes noted in cells of affected LRs. At the molecular level, it is possible that deficient ATG4D-mediated deconjugation of LC3 could lead to an increase in ATG3-LC3 complexes and a decrease in ATG3- ATG12 complexes, which could disturb basal autophagy. Further studies are needed to clarify why basal, but not induced, autophagy is altered in LRs. One possibility is that ATG4D functions mostly during nutrient rich conditions. Another possibility is that the autophagic machinery and ATG4D are involved in basal functions other than autophagic degradation in the target organs of this disease. A few studies link the function of ATG4, and specifically ATG4D, to unconventional secretion and release of intracellular vacuoles. Autophagic vacuoles, and ATG4D as the sole ATG4 isoform, were secreted from murine endothelial cells during starvation and caspase-3 activation (Sirois et al., 2012). In endothelial cells lacking caspase- 3, autophagic vacuoles were retained within the cytoplasm as vacuolization visible on TEM (Sirois et al., 2012). In maturing human erythroblasts, autophagy normally clears the cell from organelles. When ATG4D function was downregulated by a dominant negative ATG4D mutant in these cells, enlarged amphisomes, morphologically comparable to those observed in affected cells of LRs, were retained within the cells (Betin, Singleton, Parsons, Anstee, & Lane, 2013). The cytoplasmic vacuolation in LRs with the ATG4D variant, especially in cells involved in transcytosis or secretion, thus resembles the findings in functional studies linking ATG4D to release of autophagic vacuoles in vitro and in murine models. Neither the variant, nor the native ATG4D protein however, was detected in the proteomes of EVs released from affected or control fibroblasts in this study.

EV-CONTENT REFLECTS THE DISEASE PROCESS IN CELLS WITH THE ATG4D VARIANT

Significantly more EVs were released from fibroblasts of affected LRs during basal conditions, when compared to fibroblasts of control LRs. This is in agreement with previous reports describing increased EV release from cellular models during inhibition of autophagic-lysosomal function (Eitan, Suire, Zhang, & Mattson, 2016; Xu et al., 2018). During starvation, the difference in released EV amount between affected and control cells disappeared. The EV size spectrum was not significantly different from controls in fibroblasts from affected LRs, indicating that the ATG4D-variant does not alter the general EV profile. In comparison to previous reports of EV release from normal canine fibroblasts, the EV concentration released from LR control fibroblasts was similar (Aguilera-Rojas et al., 2018). The specific content of EVs released from cells of affected LRs, not present in the EV proteome of controls, included ER- and mitochondria-derived chaperones and heat-shock proteins, assisting in protein folding. Among them were several subunits of the chaperonin-containing T-complex/CCTs. CCTs have been identified as substrates of basal, but not induced, autophagy in human fibroblasts (Zhang et al., 2016). Furthermore, inhibition of autophagy has been linked to an increased secretion of its substrates via EVs in other studies (Eitan, Suire, Zhang, & Mattson, 2016; Xu et al., 2018) and when autophagic-lysosomal degradation was decreased in neuronal cells, the autophagy substrate α-synuclein was increasingly released via EVs (Minakaki et al., 2018). Our findings suggest that cells with mutant ATG4D can redirect substrates of basal autophagy into EVs. Two small GTPases, Rab1A and Rab5C, were observed in EVs of control cells, but not detected in those released from cells of affected dogs. It is known that small GTPases, circulating between an inactive GDP state and the active GTP form, are core regulators of intracytoplasmic membrane dynamics, and involved in autophago-lysosomal and endosomal pathways (Spang et al., 2014; Yla-Anttila & Eskelinen, 2018). It is tempting to speculate that the two GTPases may be retained intracellularly within the membrane traffic between vacuoles in affected cells. Alternatively, these two GTPases may not be expressed in the ATG4D mutant cells.

LIMITATIONS OF THE STUDY

This study was based on investigating a spontaneous genetic canine disease with alterations in basal autophagy. Fibroblasts used for the in vitro essays were not immortalized. The possibilities for repeating the experiments and changing experimental set up were therefore limited, since cells were used only up to the 3rd passage. The purpose of using low passage cells was to avoid changes in expression profiles that occur when removing cells from their natural microenvironment. The use of study subjects with a naturally occurring disease also limited the number of affected dogs and biological replicates available for further investigation of the disease process. A small number of study subjects may lead to type II statistical errors, accepting false negative results as true. The insights into the molecular mechanisms of autophagy pathways and the methods for monitoring autophagy are continuously evolving. Despite the LC3 assay still being a common method for assessing autophagy induction, it was recently described that pro-LC3 moves similarly to LC3II in SDS-PAGE gels (Agrotis et al., 2019). In theory, an increased LC3II band could be due to decreased preprocessing of pro-LC3 by ATG4. This would, however, interfere with autophagosome formation as preprocessing of pro-LC3 occurs before integration into the autophagosomal membrane. Thus, defects in preprocessing of pro-LC3 should be detectable in the immunofluorescence staining as a decreased in vesicular LC3 staining. The results showed the opposite, an increase of vesicular LC3 in the affected cells. In addition, in LRs, the phenotype showed excessive vacuole formation, with LC3 label on the vacuolar membrane consistent with formation of LC3II (Kabeya et al., 2000). In addition, the role of ATG4D is proposed to be in delipidation, as prepriming of pro-LC3 is mainly the task of ATG4B in mammalian cells (Kauffman et al., 2018). It is also reported that pro-LC3 is only transiently expressed in cells and immediately transformed into LC3I (Wild et al., 2014). One limitation in this study is the fact that the phenotype of vacuolation was most pronounced in epithelial cells and neurons, not in fibroblasts, which were used in the functional studies. Culture of epithelial cells was not attempted in this study. The methods available to study intracellular processes and subcellular structures in fixed material are limited compared to experiments that can be done with cell cultures. IEM was performed on neurons and epithelial cells, in order to evaluate cells with the morphological phenotype and links tissue IHC findings to IF results from the fibroblast studies. On the other hand, this discrepancy shows that even cells that do not show histological changes may be affected functionally in this disease. The EV results in this study are based solely on findings in vitro and it cannot be extrapolated that similar EV changes occur at organismal level in the affected dogs.

FUTURE ASPECTS

Assays testing specifically the enzyme activity of ATG4 have now been described (Li, M., Fu, Yang, & Yin, 2017). This methodology could confirm whether the missense variant affects the enzyme activity of ATG4D. Furthermore, the effect of starvation-induced autophagy on the ATG4D expression could be monitored at the RNA and protein level in fibroblasts from affected and control dogs, to further investigate if ATG4D expression changes or not between basal and starvation-induced autophagy. Quantitative and qualitative comparison of EVs in serum and plasma of affected and control LRs could be performed, to examine if the changes in the EV proteome of fibroblasts from affected LRs can be detected also in the blood. The changes noted in basal autophagy in cells from affected LRs in this study, including the increased LC3II in Western blot, the increased number of LC3- positive spots in IF and the quantitative change in EVs, were not detected during starvation-induced autophagy. It would be interesting to test if inducing autophagy at an organismal level, e.g. via calorie-restriction, would influence clinical signs in the affected LRs. If so, further investigations regarding a possible medical treatment of the disease, with agents inducing autophagy, could be considered.

7 CONCLUSIONS

x A neurodegenerative disease comprising vacuolation and loss of neurons, as well as axonal spheroid formation, is linked to a variant ATG4D in LRs, supporting the first hypothesis. Variants in the ATG4D gene have not been linked to neurologic disease previously, thus the genetic groups in the collaborative research where this thesis was part discovered a novel candidate gene for neurodegeneration. Several extra neural cell types are affected by cytoplasmic vacuolation in LRs with the ATG4D variant. This does not translate into clinical signs.

x In agreement with the second hypothesis, autophagic cargo, autophagic cargo-receptors and autophagic membrane protein accumulate in the histologic lesions, linking the histopathological phenotype to disturbed autophagy. Cell experiments specify the alteration to basal autophagy. The functional importance of basal autophagy in cells hereby becomes evident, as disease occurred despite starvation-induced autophagy being functional in the affected LRs.

x In aspects of comparative pathology, altered basal autophagy was verified as an etiologic and morphologic differential diagnosis for cytoplasmic vacuolation reminiscent of lysosomal storage diseases in dogs. The accumulating hybrid organelles derive from the autophagic and the endolysosomal pathways.

x Results contradicting the third hypothesis and supporting the existence of a known lysosomal storage disease in the affected LRs, were not uncovered during this study.

x For veterinary medicine, characterization of this novel inherited progressive neurological disease of LRs provides additional knowledge as basis for prognostic judgement by veterinary clinicians and for pedigree planning by LR breeders.

ACKNOWLEDGEMENTS

This thesis work was carried out at the Section for Veterinary Pathology and Parasitology, Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, with the help of core services at the Viikki Campus, including the Electron Microscopy Unit and the EV Core. The studies, and periods of education in and focus on research, were partially supported by grants from the CVM-doctoral school, the Finnish Veterinary Foundation, the Swedish Cultural Foundation in Finland and the Finnish Foundation of Veterinary Research, which is gratefully acknowledged. I thank my study director, supervisor and long-time chief of everyday work, Professor Antti Sukura, for guidance in this project, patience with my winding road towards academic competence and for allowing me to invest a decade into promoting the profession of veterinary pathologists before focusing on a PhD. This project would have finished after the first publication, had it not been for the invaluable help provided by my second supervisor, Professor Eeva-Liisa Eskelinen. Thank You Eeva-Liisa, for your interest in this canine disease, for your expert supervision of the autophagy studies and your thorough review of the text. My thesis committee members, Professors Johanna Arola and Tomi Taira, are acknowledged for practical advice on how to master this task. I sincerely thank my opponent, Privatdozentin Frauke Seehusen, for accepting the invitation to examine this work and join a virtual oral defense. “Walk regularly among giants” they say. I am fortunate to have had this chance in two fields through this thesis: canine genetics with Professor Hannes Lohi’s Group and veterinary neuropathology with the competence of mentors and colleagues in Hannover and Bern. Being deeply anchored in practical and diagnostic veterinary pathology, it has been a privilege for me to experience ambitious, high-class research through collaboration with these expert scientists. I especially thank veterinary neurologist Tarja Pääkkönen for the cooperation over the years and for continuously providing me with excellently examined neurological cases, a prerequisite for successful neuropathology. Thank You Kaisa Kyöstilä for being my grass root contact in canine genetics and for all your help with the ATG4D- and other projects around the canine cerebellum. I warmly acknowledge Tahira Anwar´s hands on help with blots and dots, and your sincere engagement in my project despite having an own PhD to attend to. Mari Palviainen is gratefully acknowledged for guiding me regarding studies on EVs. My sincere thank you to all my colleagues and coworkers around the coffee table at the Section for Veterinary Pathology, University of Helsinki; your collegiality is immense and all the collective efforts and adjustments made in your routines, to enable that I could complete this thesis, are warmly acknowledged. I thank my colleagues Susanne Kilpinen and Katja Selby, for

your friendship and discussions in- and outside this project, and all the Lagotto-owners who contribute to the study. Finally, it is not only about what you do, but also about who you are. I am part of a supportive, large family with sisters, parents, Erin, Aron and Rami around me. This not only gives me examples of pursuing goals and leading a purposive life, but has also keeps the ordinary going and regularly brings everything, including this PhD project, into perspective. For this, my foundation, I am most thankful.

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