In vitro and in vivo characterization of pathomechanisms of inherited neurodegenerative disorders in dogs: Nebent.

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ISBN 978-3-86345-248-3

Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH 35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375

E-Mail: info@dvg.de · Internet: www.dvg.de

Kerstin Caroline Hahn Hannover 2014

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Bibliografische Informationen der Deutschen Bibliothek Die Deutsche Bibliothek verzeichnet diese Publikation in der

Deutschen Nationalbibliografie;

Detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar.

1. Auflage 2015

Printed in Germany

ISBN 978-3-86345-248-3

Verlag: DVG Service GmbH Friedrichstraße 17

35392 Gießen 0641/24466 info@dvg.de www.dvg.de

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University of Veterinary Medicine Hannover Department of Pathology

Center for Systems Neuroscience

In vitro and in vivo characterization of pathomechanisms of inherited neurodegenerative disorders in dogs

Thesis

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY (PhD)

awarded by the University of Veterinary Medicine Hannover

by

Kerstin Caroline Hahn Völklingen

Hannover, Germany 2014

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Supervisor: Prof. Dr. Wolfgang Baumgärtner, Ph.D.

Supervision Group: Prof. Dr. Wolfgang Baumgärtner, Ph.D.

Prof. Dr. Peter Claus Prof. Dr. Herbert Hildebrandt

1^st Evaluation: Prof. Dr. Wolfgang Baumgärtner, Ph.D.

Department of Pathology,

University of Veterinary Medicine Hanover Prof. Dr. Peter Claus

Institute of Neuroanatomy, Hannover Medical School Prof. Dr. Herbert Hildebrandt Department of Cellular Chemistry, Hannover Medical School

2^nd Evaluation: Prof. Dr. Tosso Leeb Institute of Genetics

Vetsuisse Faculty, University of Bern

Date of final exam: 13.03.2015

Kerstin Caroline Hahn was supported by the Department of Pathology, University of Veterinary Medicine Hannover and the Foundation of German Business (Stiftung der Deutschen Wirtschaft).

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Parts of the thesis have been published:

Gerhauser I, Hahn K, Baumgärtner W, Wewetzer K. 2012.

Culturing adult canine sensory neurons to optimise neural repair.

Vet Rec 170:102.

Parts of the thesis have been presented at congresses:

Hahn K, Rhodin C, Jagannathan V, Wohlsein P, Baumgärtner W, Grandon R, Jäderlund KH, Drögemüller C.

Tectonin beta-propeller repeat-containing protein 2 (TECPR2) missense mutation associated with neuroaxonal dystrophy in Perros de Agua Espanol.

Second joint European Congress of the ESVP, ECVP and ESTP; Berlin, 2014.

Tectonin beta-propeller repeat-containing protein 2 (TECPR2) missense mutation associated with neuroaxonal dystrophy in Perros de Agua Espanol.

Second International Workshop of Veterinary Neuroscience; Hannover, 2014.

Neuroaxonale Dystrophie beim Spanischen Wasserhund infolge einer Mutation im Tectonin beta-propeller repeat containing protein 2 (TECPR2) Gen

57. Jahrestagung der Fachgruppe Pathologie der Deutschen Veterinärmedizinischen Gesellschaft. Fulda 2014. Tierärztliche Praxis Kleintiere: A15-A25.

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To my parents and my

brother Christian

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There is only one thing that makes a dream impossible to achieve: the fear of failure.

(Paulo Coelho, The Alchemist)

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Contents I

Abbreviation list

AD: Alzheimers disease

ALR: autophagic lysosome reformation ALS: amyotrophic lateral sclerosis AMPK: AMP-activated protein kinase AP: adaptor protein (AP)

ARHGEF16: rho guanine nucleotide exchange factor 16 (also termed Nbr) ATG: autophagy-related genes in mammals

Atg: autophagy-related genes in yeast ATP: adenosine triphosphate

ATP13A2: lysosomal type 5 P-type ATPase encoding gene BDNF: brain-derived neurotrophic factor

BECN1: beclin-1

BNIP3L: BCL2/adenovirus E1B 19kDa interacting protein 3-like (also termed Nix) BPAN: beta-propeller associated neurodegeneration

BSA: bovine serum albumin

C19orf12: chromosome 19 open reading frame 12 Ca²⁺: calcium

CLEAR: coordinated lysosomal expression and regulation (CLEAR) consensus sequence CNPase: 2’,3’-cyclic nucleotide 3’-phosphohydrolase

CNS: central nervous system COPI: coatomer complex I

CSF1R: colony stimulating factor 1 receptor encoding gene DME: Dulbecco’s modified Eagle

DMEM: Dulbecco’s modified Eagle medium dps: days post seeding

DRG: dorsal root ganglia

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IV Abbreviation list

EGF: epidermal growth factor EGR2: early growth response 2 ER: endoplasmatic reticulum ERAD: ER-associated degradation

ESCRT: endosomal sorting complex required for transport FCS: fetal calf serum

FGF2: fibroblast growth factor 2

FIG: figure

FYCO1: FYVE (phenylalanine, tyrosine, valine, glutamic acid) and coiled-coil domain containing protein 1

GAN: gigaxonin encoding gene GAP43: growth associated protein 43 GBA: glucocerebrosidase encoding gene GFAP: glial fibrillary acidic protein

GGAs: Golgi-localized γ-ear-containing ADP ribosylation factor-binding proteins GLB1: acid β galactosidase

GM1: GM1-ganglioside GS: glutamine synthetase GSK-3: glycogen synthase kinase-3 HBSS: Hank's Balanced Salt Solution HD: Huntingtons disease HDAC6: histone deacetylase 6

HDLS: hereditary neuroaxonal leukodystophy with spheroids HE: haematoxylin/eosin

HOPS: homotypic vacuole fusion and vacuole protein sorting (HOPS) complex HSP: human hereditary spastic paraparesis/ paraplegia

Hsp70: heat shock cognate protein 70

Iba1: ionized calcium-binding adapter molecule 1

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Abbreviation list V

KFERQ: amino acid motiv: lysine (K), phenylalanine (F), glutamic acid (E), arginine (R), Glutamine (Q)

LAMP: lysosomal-associated membrane protein LC3: microtubule-associated protein-light chain 3 LFB-CV: Luxol fast blue-cresyl violet

LIR: LC3-interacting regions LL: large light (neurons)

LRRK2: leucine-rich repeat kinase 2 encoding gene LSD: lysosomal storage disorders

MAP2: microtubule-associated protein 2 MFN2: mitofusin 2

ml: milliliter

MPAN: mitochondrial membrane protein associated neurodegeneration MPRs: mannose-6-phosphate receptors

mTORC1: mammalian or mechanistic target of rapamycin complex 1 MVB: multivesicular body

NAD: neuroaxonal dystrophy

NBIA: neurodegeneration with brain iron accumulation NBR: ARHGEF16 homolog in Drosophila melanogaster NDP52: nuclear dot protein 52

ng: nano-gram

NGF: nerve growth factor Nix: also termed BNIP3L

nm: nano meter

nNF: non-phosphorylated neurofilament OCT: optimum cutting temperature OECs: olfactory ensheathing cells OPTN: optineurin

p53: tumor protein p53

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VI Abbreviation list

p62: also termed SQSTM1

p75^NTR: low affinity neurotrophin receptor PANK2: pantothenate kinase 2 encoding gene PARK2: Parkin encoding gene

PARK6 : PINK1 encoding gene

Parkin: Parkin RBR E3 ubiquitin protein ligase PD: Parkinson’s disease

PE: phosphatidylethanolamine pH: potentia Hydrogenii

PI3KC3: class III phosphoinositide 3-kinase (also termed Vps34) PINK1: PTEN induced putative kinase 1

PIP3: phosphatidylinositol 3-phosphate

PKAN: pantothenate kinase-associated neurodegeneration PLA2G6: phospholipase A2, group VI encoding gene PLAN: PLA2G6-associated neurodegeneration

PLOSL: Polycystic Lipomembranous Osteodysplasia with Sclerosing Leukoencephalopathy

pNF: phosphorylated neurofilament PNS: peripheral nervous system

POLD : pigmentary orthochromatic leukodystrophy Rab: Ras-related proteins in brain GTPase RabGAPs: Rab GTPase-activating proteins ROS: reactive oxygen species

Rubicon: RUN domain cysteine rich domain containing, beclin-1 interacting protein S100: S100-protein

SA-GLB1: senescence-associated β-galactosidase SD: small dark (neurons)

SGCs: satellite glial cells Sirt1: sirtulin 1

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Abbreviation list VII

SNARE: N-ethylmaleimide-sensitive-factor attachment receptor protein SNCA: synuclein encoding gene

SOX2: sex-determining region Y-box 2 SPG11: spastic paraplegia 11 or spatacsin

SPG15: zinc finger FYVE domain-containing protein 26 or spastizin SPG49: tectonin beta-propeller repeat-containing protein 2 (TECPR2) SPG60: WD repeat-containing protein 48

SQSTM1: sequestosome 1(also termed p62) SV: synaptic vesicles

Tau1: Tau 1-protein

TECPR2: tectonin beta-propeller repeat-containing protein 2 or SPG49 TFEB: transcription factor EB

TREM2: triggering receptor expressed on myeloid cells 2 encoding gene Trk: tyrosine kinase

TYROBP: TYRO protein tyrosine kinase binding protein encoding gene ULK: Unc51-like kinase

UPR: unfolded protein response UPS: ubiquitin proteasome system V-ATPase: vacuolar-type H+-ATPase VCP: p97/valosin-containing protein

WDR: WD (tryptophan, aspartatic acid) repeat domain-containing protein WIPI: WD (tryptophan, aspartic acid) repeat domain phosphoinositide-interacting

protein

ZKSCAN3: zinc finger with KRAB and SCAN domains 3 βIII tubulin: neuronal class III β tubulin

μm: micro meter

μM: micro mole

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General introduction 1

1 General introduction

1.1 The dog as a translational model for human inherited neurodegenerative diseases During recent years, the relevance of the dog as a translational large animal model for human neurodegenerative conditions including lysosomal storage disorders (LSD), amyotrophic lateral sclerosis (ALS), Alzheimer´s disease (AD), epilepsy, spinal cord injury, but also physiological ageing has significantly increased (Katz et al., 2005; Hytönen et al., 2012;

Bock et al., 2013; Head, 2013; Morgan et al., 2013; Potschka et al., 2013). All these naturally occurring conditions in humans and dogs are modulated or even determined by genetic factors (Ball et al., 1982; Platt et al., 2012; Tanzi, 2012; Browne et al., 2014; Busch et al., 2014; Deelen et al., 2014). Consequently, understanding the genetic basis and the corresponding pathogenetic mechanisms of neurodegenerative diseases in animals and humans is essential to develop therapeutic approaches. Since the beginning of the twentieth century, the foremost model for laboratory studies in mammals has been the mouse (Paigen, 1995; Karlsson and Lindblad-Toh, 2008; Webster et al., 2014). However, the mouse has several restrictions as a model for complex human disease such as AD, ALS, and spinal cord injury, which was highlighted by the limited therapeutic success compared to promising preclinical data based on studies in rodent models (Benatar, 2007; Tator et al., 2012;

Cavanaugh et al., 2014). The reasons for this frequent observation are generally unknown, but morphological, physiological, and genetic differences might partly account for difficulties to extrapolate data from murine models to humans. In contrast, structure and organization of the canine and human central nervous system (CNS) is similar to a large extent (Techangamsuwan et al., 2008; Omar et al., 2011; Wewetzer et al., 2011). Furthermore, the dog genome is less diverged from the human than the mouse genome (Lindblad-Toh et al., 2005; Karlsson and Lindblad-Toh; 2008). The successful treatment of inherited blindness in dogs by gene therapy demonstrates that the canine model provides a useful approach to test novel therapies in vitro and in vivo (Bennicelli et al., 2008). Furthermore, the identification of causative loci in dogs can identify genes and pathways that help to understand and modulate the pathogenesis of human diseases.

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2 General introduction

1.2 Dorsal root ganglia cultures as an in vitro model in neuroscience

The paravertebral located dorsal roots of the spinal cord contain sensory ganglia. These dorsal root ganglia (DRGs) are composed of afferent, pseudounipolar neurons, ensheathing satellite glial cells (SGCs), and connective tissue cells (Hanani, 2005). DRG neurons transmit autonomic and sensomotoric signals from the periphery to the CNS. According to ultrastructural properties, DRG neurons were classified into two main subtypes termed as

“large light” (LL) and “small dark” neurons (SD; Lawson, 1992). LL neurons give rise to Aα fibers and Aβ fibers (myelinated, fast conducting, nociceptive or non-nociceptive). Aγ fibers (thinly myelinated, slow conducting, nociceptive) arise from the smaller population of LL neurons, with a diameter similar to SD neurons, whereas C type fibers (non-myelinated, slow conducting, nociceptive) originate from SD neurons (Harper and Lawson, 1985; Ruscheweyh et al., 2007). However, due to overlapping sizes of the neuronal cell body and also differences in sensory quality this classification just reflects tendencies (Ruscheweyh et al., 2007). SGCs form a sheath around the DRG neurons, control their microenvironment, and carry receptors for numerous neuroactive agents. Moreover, they communicate with neighboring cells including DRG neurons. Consequently, SGCs represent an essential component of signal processing and transmission within the DRG and functionally substitute the lacking blood-brain barrier in sensory ganglia (Hanani, 2005; Krames, 2014).

DRG neurons from neonatal and adult rodents, chicken, pigs, and primates can easily be accessed in order to cultivate them in vitro (Bray et al., 1978; Li, 1998; Roggenkamp et al., 2012; Ramesh et al., 2013). Consequently, DRG cultures represent a widely used model to study the pathogenesis and underlying molecular mechanisms of pathogen-host interactions, neuropathic pain, and its pharmacological modulation (Ramesh et al., 2013;

Biggs et al., 2014; Liu et al., 2014; Krames, 2014). Additionally, DRG in vitro systems enable the characterization of neuron-glia interactions, axonal growth and their modulation by various types of neurotrophins and neuropharmacological compounds (Sondell et al., 1999;

Zhao et al., 2006; Päiväläinen et al., 2008). Neonatal murine and adult DRGs from rats represent a potential source of stem/progenitor cells, which might originate from the SGCs in adults (Namaka et al., 2001; Li et al., 2007). In vitro these cells can differentiate into glia,

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smooth muscle cells, and neurons and seem to be involved in the recovery of neuronal numbers in the DRG after peripheral nerve injury (Groves et al., 2003; Li et al., 2007). These DRG inherent precursors might represent interesting candidates for gene therapy and/or homologous cell transplantation assays as a treatment option for neurodegenerative conditions.

Furthermore DRGs are affected in lysosomal storage disorders such as GM1-gangliosidosis or Tay-Sachs disease (Abe et al., 1985; Bieber et al., 1986). Ganglioside accumulations are also induced in DRG neurons after application of compounds such as chloroquine or suramin that selectively accumulate in the lysosomes (Klinghardt et al., 1981; Gill and Windebank, 1998).

Additionally, many other disorders with potential mitochondrial or cytoskeletal impairments including ALS, AD, Parkinson's disease (PD), and diabetic and giant axonal neuropathy affect DRGs (Tshala-Katumbay et al., 2005; Sasaki et al., 2007; Figueroa-Romero et al., 2008;

Sábado et al., 2014). Neuroaxonal dystrophy (NAD) in aged sympathetic ganglia, manifesting as swollen, dystrophic, preterminal axons compressing or displacing the perikarya represents also a common finding in humans and animals, whose pathogenesis is unknown (Schmidt et al., 1990).

Consequently, DRGs may represent a valuable in vitro system to study the pathogenetic mechanisms of lysosomal, mitochondrial, and/or age-associated neurodegenerative conditions as well as therapeutically options.

1.3 GM1-ganglioside in ageing, age-associated neurodegenerative diseases and GM1- gangliosidosis

Gangliosides represent sialic acid-containing glycosphingolipids found in cellular membranes (Leeden et al., 1998). The highest ganglioside concentrations are present in neurons, in which they account for 10 % of the total lipid content (Ledeen, 1978). GM1-ganglioside (GM1) represents the most commonly used ganglioside in brain-related research and seems to influence cellular ageing, age-related neurodegenerative conditions and is involved in the pathogenesis of lysosomal storage disorders as GM1-gangliosidosis (McJarrow et al., 2009;

Pernber et al., 2012; Wu et al., 2012; Regier and Tifft, 2013).

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The brain ganglioside content changes in an age-related manner. In the fetal brain of humans and rodents GM1 accumulates during synaptogenesis and early stages of myelination (Irwin et al., 1980; Skaper et al., 1989). In humans, ageing is accompanied by a decrease in brain GM1 content, whereas GM1 levels increase with ageing in the rodent brain (Aydin et al., 2000). Species-specific and age-associated differences in GM1 degradation pathways might account for the differences in GM1 content in the aged rodent and human brain. For instance, mice possess an alternative GM1 asialo degradation pathway in contrast to humans and dogs (Suzuki et al., 1988; Hahn et al., 1997). The relevance of this pathway in murine GM1 degradation and its age-associated alterations are not known. However, differences in the GM1 content between rodent and human brains should be considered in the extrapolation of experimental results.

GM1 levels are also determined by its lysosomal degradation rate and depend on the activity of the enzyme acid β-galactosidase (GLB1). The GLB1 activity detectable at suboptimal pH 6.0 was defined as senescence-associated β-galactosidase (SA-GLB1) and is a widely used marker for neuronal senescence (Dimri et al., 1995; Geng et al., 2010). SA-GLB1 seems to represent the accumulation of GLB1 in lysosomes, which might explain its activity also at suboptimal pH conditions (Lee et al., 2006). Consequently, the age-associated variations in GM1 metabolism might reflect impairments of the endosomal/autophagy pathway.

The age-associated decline of GM1 in the human brain parallels the age-dependent synaptic loss and is discussed as a factor involved in the pathogenesis of several age-related neurodegenerative conditions including AD and PD (Pernber et al., 2012; Wu et al., 2012).

This hypothesis is supported by the successful application of GM1 to improve motoric and cognitive skills in these diseases but also in patients with brain lesions due to vascular disorders and peripheral neurotoxicity associated with chemotherapeutical agents (Battistin et al., 1985; Zhu et al., 2013). Furthermore, age-associated alterations in GM1 distribution at presynaptic neuritic terminals were discussed in AD patients to promote amyloid β-protein fibrillogenesis (Yamamoto et al., 2007).

The lysosomal storage disorder GM1-gangliosidosis represents a pathological condition of increased neuronal GM1 content, which clinically manifests in neurological symptoms. This autosomal recessive inherited disease is described in humans, dogs, and several animal

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species and results from a deficiency of the lysosomal GM1 degrading enzyme GLB1. The progression of the disease depends on the residual GLB1 activity (Regier and Tifft, 2013).

However, the molecular mechanisms involved in disease pathogenesis are still incompletely understood (Brunetti-Pierri and Scaglia, 2008). Neuronal apoptosis, endoplasmatic reticulum (ER) stress, abnormal axoplasmic transport resulting in myelin deficiency, disturbed neuronal–oligodendroglial interactions, and mitochondrial dysfunction have been proposed to play a role in GM1- gangliosidosis (Kaye et al., 1992; Folkerth, 1999; Tessitore et al., 2004;

van der Voorn et al., 2004; d'Azzo et al., 2006; Brunetti-Pierri and Scaglia, 2008; Takamura et al., 2008). Consequently, despite the accumulations of other substrates in GM1- gangliosidosis, the study of GM1 modulatory effects on neurons, glia cells, and cellular senescence might reveal new therapeutic approaches for the different types of GM1- gangliosidosis.

To address the question how changes in GM1 metabolism result in neurotropic effects or neuropathology, numerous in vitro and in vivo studies were performed. GM1 was demonstrated to promote neurite outgrowth, arborization, as well as neuronal differentiation in vitro and neuronal repair in vivo (Ferrari et al., 1983; Leon et al., 1984;

Wang et al., 1995). This effect seems to depend on structural synaptic alterations, modulations of Ca²⁺ influx, potentiation of receptor-mediated neurotrophin signaling, and/

or regulation of receptor trafficking (Cuello et al., 1989; Di Patre et al., 1989;

Hadjiconstantinou et al., 1992; Fong et al., 1995; Ando et al., 1998; Wu et al., 2007; Suzuki et al., 2011; Prendergast et al., 2014). Furthermore, GM1 might function as a receptor or co- receptor and/or might modulate receptor-ligand interactions as demonstrated for fibroblast growth factor 2 (FGF2) signaling (Rusnati et al., 1999; Rusnati et al., 2002; Chinnapen et al., 2012). FGF2 neurotrophic effects were partly considered as secondary and astrocytes, oligodendrocytes as well as microglia may represent the primary mode of FGF2/GM1 action (Perkins and Cain 1995). In Schwann cells, GM1 was described to reduce cell proliferation and to promote a phenotype with extremely elongated processes (Sobue et al., 1988). In addition, GM1-mediated NGF production by Schwann cells was reported (Ohi et al., 1990). In general, the effects of external added GM1 on glia cells and glia cell differentiation were not characterized in detail.

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However, the impact of GM1 on the metabolism of neurons and glial cells and its role in age- related neurodegeneration is complex. In this regard, the detailed characterization of GM1

mediated effects in different physiological and pathological conditions provides the basic for successful therapeutic applications of GM1 and its saver and more potent semisynthetic derivatives.

1.4 Autophagy in mammalian cells 1.4.1 Overview and subtypes of autophagy

Autophagy (from the Greek, “auto” oneself, “phagy” to eat) defines a primarily degradative pathway that takes place in all eukaryotic cells (Feng et al., 2014). The term “autophagy” was defined by the Nobel laureate Christian de Duve in 1963, based on his discovery of lysosomes (De Duve et al., 1955). Autophagy implies the delivery of cytoplasmic cargo to the lysosome and its subsequent degradation to generate macromolecular building blocks and energy under stress conditions, to remove superfluous and damaged organelles, to adapt to changing nutrient conditions, and to maintain cellular homeostasis (Levine and Kroemer, 2008; Feng et al., 2014). Autophagy and the endocytic compartment are structurally and regulatory closely connected forming the lysosomal network (Nixon, 2013).

Under non-stress conditions, low levels of autophagy (basal autophagy) perform essential housekeeping and quality control functions preserving cellular homeostasis, whereas under stress conditions the autophagic flux is upregulated (activated autophagy; Glick et al., 2010).

Autophagy is classified according to the mode of cargo delivery to the lysosome into macroautophagy, microautophagy, and chaperone-mediated autophagy (Nixon, 2013). Both micro- and macroautophagy can be selective or non-selective (Shintani and Klionsky, 2004).

In macroautophagy, the double-membrane-delimited autophagosome sequesters parts of the cytoplasm and then fuses directly with the lysosome or after preceeding fusion events with late endosomes (Klionsky, 2005; Figure 1.4.2).

Microautophagy refers to the direct engulfment of cytoplasm by the endosome or lysosome.

In microautophagy, the lysosomal/vacuolar membrane is randomly invaginated and differentiates to the autophagic tubes enclosing portions of the cytosol. Subsequent vesicle

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formation at the top of the tube incorporates the cytosolic components into the endosome or lysosome (Li, et al., 2012). The formation of multivesicular bodies (MVB) also determined as late endosomes are suggested as one type of microautophagy in mammalian cells (Sahu et al., 2011).

Chaperone-mediated autophagy implies the recognition of proteins with a KFERQ or a KFERQ-like motif by the heat shock cognate protein 70 (Hsp70) and the subsequent binding of the protein-chaperon complex to the lysosomal-associated membrane protein (LAMP) 2A mediating the transfer into the lysosomal lumen (Agarraberes et al., 1997).

Non-selective autophagy is used for the turnover of dispensable cytoplasm under starvation conditions, whereas selective autophagy specifically targets damaged or superfluous organelles (Feng et al., 2014).

A further subclassification of selective autophagy refers to the substrate for lysosomal degradation and implies e.g. mitophagy (Lemasters, 2014), lipophagy (Zechner and Madeo, 2009), aggrephagy of protein aggregations (Hyttinen et al., 2014), ribophagy (Kristensen et al., 2008; Baltanás et al., 2011), reticulophagy (Rubio et al., 2012), pexophagy of peroxisomes (Jiang et al., 2014a), crinophagy of secretory granules (Glaumann, 1989), lysophagy (Hung et al., 2013), heterophagy of exogenous proteins (Ohshita et al., 1986), and xenophagy of infectious agents (Alexander and Leib, 2008; Pujol et al., 2009).

1.4.2 The molecular mechanisms of macroautophagy

The molecular understanding of autophagy in mammals is largely based on genetic studies and the definition of autophagy-related genes in yeast (Atg), enabling the identification of mammalian homologs (ATG; Tsukada and Ohsumi, 1993, Klionsky, 2003). The first step in macroautophagy implies the formation of a membranous structure, termed phagophore.

Phagophore formation is initiated from different membranous cellular origins, as discussed later. The phagophore elongates and encloses the substrate for degradation and fuses to a double-membrane vesicular structure, the autophagosme. The autophagosome subsequently fuses directly with lysosomes and forms the autophagolysosome. An alternative way, coupling autophagy and the endocytic compartment comprises the

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formation of autophagosome-late endosome hybrid organelles termed amphisomes that subsequently fuse with the lysosome (Gordon and Seglen, 1988; Figure 1.4.2).

Figure 1.4.2: Overview of macroautophagy

The molecular degradation products, released from the autophagolysosome represent a regulatory component of the autophagy controlling complex mammalian target of rapamycin complex-1 (mTORC1). Inhibition of mTORC1 results in signaling complex translocation, autophagy induction and formation of the phagophore. The extending membrane encloses dispensable cytoplasm, protein aggregates or organelles for degradation. The autophagosome represents a double membrane-structure, generated by the closure of the inner and outer bilayers of the elongating phagophore. Autophagosomes fuse predominantly with late endosomes to hybrid- organelles termed amphisomes. The amphisome fuses subsequently with the lysosome, where hydrolytic degradation occurs. The direct autophagosome-lysosome fusion is considered as a less frequently event (Modified from Nixon, 2013).

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1.4.3 Autophagosome formation

The majority of the proteins encoded by the ATG genes are involved in the autophagosome formation process (Figure 1.4.3). The membrane sources for phagophore nucleation and expansion is still a matter of discussion. Cell imaging studies suggested the endoplasmatic reticulum (ER; Hayashi-Nishino et al., 2009), the Golgi apparatus (Yen et al., 2010), the plasma membrane (Ravikumar et al., 2010), recycling endosomes (Puri et al., 2013), mitochondria (Hailey et al., 2010), and ER-mitochondria contact sites (Hamasaki et al., 2013) as potential origins of pre-autophagosomal structures.

On the molecular level, autophagosome generation is primarily regulated by different cellular stress signals, including lowered concentrations of essential amino acids, adenosine triphosphate (ATP), growth factors, hypoxia, occurrence of protein aggregates, and ER stress (Kroemer et al., 2010). These signals mediate mammalian (or mechanistic) target of rapamycin complex-1 (mTORC1) inhibition or AMP-activated protein kinase (AMPK) activation and trigger the Unc51-like kinase (ULK) complex (Kim et al., 2011).

Phosphorylation of ULK1 mediates the translocation of a multiprotein complex containing beclin-1 (BECN1) and class III phosphoinositide 3-kinase (PI3KC3 or Vps34) from the cytoskeleton to the phagophore (Fimia et al., 2007; Suzuki et al., 2007; Di Bartolomeo et al., 2010). The subsequent expansion of the phagophore is mediated by PI3KC3 activity that phosphorylates phosphatidylinositol to generate phosphatidylinositol 3-phosphate (PIP3).

PIP3 binds to two proteins namely WD repeat domain phosphoinositide-interacting (WIPI)1 and WIPI2 (Proikas-Cezanne et al., 2004; Polson et al., 2010). Both were suggested to regulate the formation of the ATG5-ATG12-ATG16L complex, essential for further phagophore maturation and elongation (Fujita et al., 2008). The ATG5–ATG12–ATG16L complex decorates pre-phagophore structures and phagophores but dissociates from completed autophagosomes (Mizushima et al., 2003; Zavodszky et al., 2013). Microtubule- associated protein-light chain 3 (LC3) is cleaved by the involvement of mammalian Atg4 homologs to form cytoplasmic LC3-I, which is subsequently activated by ATG7, transferred to ATG3, and conjugated to phosphatidylethanolamine (PE) forming LC3-II (Tanida et al., 2001, 2002; Hemelaar et al., 2003; Zavodszky et al., 2013). This transfer reaction is supported by

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the ATG5–ATG12–ATG16L complex, which may also determine the site of the production of LC3–PE and therefore the phagophore formation site (Fujita et al., 2008).

Both, the mammalian Atg 4 homologs and ATG9 are involved in autophagosome formation and maturation but the distinct function in the mammalian autophagy pathway is only poorly defined. Humans and mice possess ATG4A, ATG4B, ATG4C, and ATG4D, all functioning as cysteine proteases, which are suggested to interact with the seven different mammalian Atg8 homologs , with broad diversity in the catalytic efficiency among different ATG4-ATG8 pairs (Mariño et al., 2003; Li et al., 2011). The function of the different ATG4 subtypes in LC3 lipidation and redistribution has still to be defined in detail.

ATG9 represents a dynamic component that is concentrated under basal conditions in the juxtannuclear soma, associated to the trans-Golgi network and/or recycling endosomes or late endosomes. Autophagy induction results in ATG9 redistribution to peripheral endosomal membranes and is suggested to co-localize with phagophores (Orsi et al., 2012;

Young et al., 2006). It is discussed, that ATG9 vesicles interact dynamically with phagophores and autophagosomes without finally becoming incorporated into them (Orsi et al., 2012;

Zavodszky et al., 2013). WIPI2 is involved in removing and recycling ATG9 from the association to phagophores (Orsi et al., 2012).

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Figure 1.4.3: Autophagy induction and autophagosome biogenesis.

Autophagy is initiated via mammalian target of rapamycin complex-1 (mTORC1) inhibition or AMP-activated protein kinase (AMPK) activation. These regulatory instances catalyze the phosphorylation of Unc51-like kinase1 (ULK1) and activation of the ULK complex. This complex activates the class III phosphoinositide 3-kinase (PI3KC3) complex and mediates its subsequent relocation to the phagophore formation membrane. Vps34 generates phosphatidylinositol 3 phosphate (PI3P) that binds to WD repeat domain phosphoinositide- interacting proteins (WIPIs) and catalyzes the first of two ubiqutination-like reactions. The first reaction implies the ATG7 and ATG10-mediated formation of the ATG-5-ATG12-ATG16L complex. The attachment of this complex on the phagophore induces the microtubule-associated protein-light chain 3 (LC3) lipidation cascade.

The resulting LC3 II (LC3 bound to phosphatidylethanolamine; PE) facilitates the closure of the phagophore.

(Modified from Nixon, 2013).

1.4.4 Fusion of autophagosomes with lysosomes or endosomes

Autophagosomes fuse either directly with lysosomes or secondary after preceding fusion with early or predominantly late endosomes (Klionsky et al., 2012). The primary generation of autophagolysosomes occurs accentuated in the juxtanuclear region, where lysosomes are concentrated near the microtubule-organizing centre (Lee et al., 2011). In neurons, a high

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proportion of autophagosomes fuse with late endosomes during the transport along axons or dendrites towards the soma (Lee et al., 2011; Nixon, 2013).

The detailed mechanisms controlling the fusion of completed autophagosomes with endosomes and lysosomes are not completely understood (Shen and Mizushima, 2014).

Most studies in mammalian cells focused on the fusion of late endosomes with lysosomes, whereas autophagosome- or amphisome-lysosome fusion is less characterized. The endosomal sorting complex required for transport (ESCRT) is suggested to be essential for delivery of the fusion machinery to lysosomes or autophagosomes (Metcalf and Isaacs, 2010). Tethering of lysosomes to autophagosomes is mediated by the Ras-related proteins in brain GTPase (Rab) 7 and the homotypic vacuole fusion and vacuole protein sorting (HOPS) complex (Pawelec et al., 2010; Jiang et al., 2014b). Rab7 and LC3 form a complex with the FYVE and coiled-coil domain containing protein 1 (FYCO1), and promote the trafficking of autophagosomes towards the lysosome (Pankiv et al., 2010; Weidberg et al., 2011; Deegan et al., 2013). Furthermore, HOPS complex interactions with soluble N- ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins e.g. Syntaxin 17 participate in autophagosome-lysosome fusion (Itakura and Mizushima, 2013; Jiang et al., 2014b). Lysosomal membrane components as vacuolar-type H+-ATPase (V-ATPase) complex and the lysosomal-associated membrane protein (LAMP) 1 as well as Rubicon were also characterized as positive or negative regulators of autophagosome- and also endosome- lysosome fusion and could interact with the ESCRT pathway (Yamamoto et al., 1998;

Matsunaga et al., 2009; Metcalf and Isaacs, 2010). Other proteins suggested to mediate autophagosome fusion with endosomes or lysosomes include LAMP2 (Saftig et al., 2008), Rab11 (Fader et al., 2008), histone deacetylase 6 (HDAC6; Lee et al., 2010), the ubiquitin- binding proteins ubiquilin (N'Diaye et al., 2009), and p97/valosin-containing protein (VCP; Ju et al., 2009; Metcalf and Isaacs, 2010). Additionally, deletion of the dynein-dynactin complex results in autophagosome accumulation, defining the intact microtubule-based transport as a crucial event in autophagosome-lysosome fusion (Kimura et al., 2008). The fusion event is also influenced by the lipid content of autophagosomes (Koga et al., 2010) and the lysosomal pH, independently of V-ATPase activity (Kawai et al., 2007).

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Many of these proteins including Rab7, ESCRT complex, and SNARE proteins not only mediate autophagosome fusion with lysosomes, but are also involved in endosome- lysosome fusion, for which several models were currently discussed (Luzio et al., 2007). The principles may be equally considered for autophagosome/amphisome-lysosome fusion. The maturation model suggests the gradual formation of lysosomes from late endosomes by adding lysosomal molecules while removing endosomal components (Roederer et al., 1987;

Murphy, 1991). The vesicular hypothesis implies vesicles budding from the late endosome delivering the content to the lysosome (Thilo et al., 1995). The “kiss and run” model suggests a transient fusion of late endosome and lysosome and an exchange of contents (kiss) followed by a separation of the two organelles (run; Storrie and Desjardins, 1996; Duclos et al., 2003). The forth model hypothesizes, that endosomes and lysosomes fuse to a hybrid organelle containing lysosome and late endosome components and subsequent lysosome recycling by the selective removal of late endosome constituents (Luzio et al., 2000).

However, the relevance of these models for autophagosome/amphisome-lysosome fusion has to be clarified in experimental studies.

1.4.5 Reformation of lysosomes from autophagolysosomes

The restoration of lysosomes from hybrid organelles is essential to maintain the proper function of the endosomal, autophagosomal, and lysosomal network and maintainance of cellular homeeostasis. This process implies on the one hand recycling of lysosomal components from autophagolysosomes as well as de novo protein synthesis of lysosomal membrane proteins and lysosomal hydrolases.

Autophagic lysosome reformation (ALR) is suggested to be regulated by mTORC1 through Rab7 and requires an intact microtubule network. ALR starts with the budding of LAMP1 positive tubules from autophagosomes and subsequent segregation of vesicles as proto- lysosomal structures (Yu et al., 2010). Clathrin participates in ALR and is directed to autophagolysosomes via phosphatidylinositol-4,5-bisphosphate (Rong et al., 2012).

Prolonged starvation decreases autophagy and promotes ALR. This is mediated by mTORC1 reactivation due to lysosomal amino acid release as a result of autophagic degradation (Yu et

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al., 2010). This process deserves as a self-regulatory feedback mechanism ensuring the recycling of lysosome membranes and the restoration of lysosome number (Yu et al., 2010).

Newly synthesized acid hydrolases are tagged with mannose-6-phosphate in the cis-Golgi compartment and subsequently bind to mannose-6-phosphate receptors (MPRs) in the trans-Golgi network (Rouillé et al., 2000). Clathrin, adaptor protein (AP) complex components, and Golgi-localized γ-ear-containing ADP ribosylation factor-binding proteins (GGAs) mediate the vesicular transfer to endosomes. The MPRs dissociate and recycle to the trans-Golgi compartment whereas the hydrolases were transferred to the lysosome (Dell'Angelica et al., 2000; Hirst et al., 2001). The transfer of de novo synthesized lysosomal membrane proteins occurs directly via late endosomes or indirectly via the plasma membrane. AP3 is considered as a regulatory component in both pathways (Ihrke et al., 2004).

1.4.6 Transcriptional regulation of lysosomal network proteins

The transcription of lysosomal and autophagy-associated genes is regulated by a signaling axis between mTORC1, the transcription factor EB (TFEB), the transcription factor zinc finger with KRAB and SCAN domains 3 (ZKSCAN3), as well as the lysosome and lysosome- associated complexes.

Autophagy inducing conditions such as nutrient starvation, metabolic and lysosomal stress mediate an inhibition of the negative regulator of autophagy mTORC1. This results in the dephosphorylation of TFEB and its translocation from the cytoplasm to the nucleus (Peña- Llopis and Brugarolas, 2011). Subsequent binding of TFEB to a coordinated lysosomal expression and regulation (CLEAR) consensus sequence activates de novo gene transcription of lysosomal network proteins (Sardiello et al., 2009; Settembre and Ballabio, 2011; Martina et al., 2012; Settembre et al., 2013). ZKSCAN3 functions as a suppressive factor of autophagy-associated gene transcription that is cytoplasmatically sequestered during starvation conditions (Chauhan et al., 2013). Similarly, the autophagy is negatively regulated via lysosomal amino acids that activate mTORC1 via V-ATPase and Ragulator and subsequent inhibition of TFEB (Sancak et al., 2010; Zoncu et al., 2011).

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1.4.7 Selective autophagy

In response to starvation, autophagy comprises non-selective and mainly selective degradative processes. In conditions of amino acid deprivation, cytosolic proteins are primary autophagy targets, whereas proteins linked to various complexes and organelles are degraded later (Kristensen et al., 2008). Receptors for selective autophagy are characterized by the presence of one or multiple LC3-interacting regions (LIR), which interact with the autophagosome membrane-bound LC3 family members (Birgisdottir et al., 2013). Cargo binding occurs via one or multiple cargo binding domains except for adaptors that are transmembrane proteins and consequently directly linked to their cargo (Kirkin et al., 2009).

The selectivity of adaptor proteins for their respective cargo is mediated by distinct types of cargo ubiquitination patterns. The adaptor molecule undergoes degradation together with the ubiquitinated cargo (Schreiber and Peter, 2014).

Several proteins including Sequestosome 1 (SQSTM1 or p62), Rho Guanine Nucleotide Exchange Factor 16 (ARHGEF16 or Nbr), BCL2/Adenovirus E1B 19kDa Interacting Protein 3- Like (BNIP3L or Nix), Nuclear Dot Protein 52 (NDP52), and optineurin (OPTN), but also ATG4B and ULK1 were identified as LIR containing selective autophagy receptors (Schreiber and Peter, 2014). p62 and/or ARHGEF16 are involved in the selective degradation of various substrates as aggregated protein complexes, mitophagy and xenophagy (Bjørkøy et al., 2005;

Zheng et al., 2009). Nix and the Parkinson disease-associated proteins PTEN induced putative kinase 1 (PINK1) and the Parkin RBR E3 Ubiquitin Protein Ligase (Parkin) mediate mitophagy (Ding et al., 2010). NDP52 and OPTN have been described as important mediators for the targeting of invasive pathogens to the autophagosome (Thurston et al., 2009; Wild et al., 2011).

However, for several forms of selective autophagy, the adaptor molecules or the possible involvement of known adaptors are not defined yet.

1.4.8 Autophagy modulating factors and signaling mechanisms

Autophagy is a highly sensitive process induced by almost every stressful condition affecting cellular homeostasis (Kroemer et al., 2010). Changes in molecular concentrations of amino

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acids, ATP, and oxygen levels are related to the cellular balance of anabolic and catabolic processes reflecting the cells or the bodies nutrient state (Russell et al., 2014). The lysosome is considered as the key side of amino acid sensing (Zoncu et al., 2011). Coupling of autophagy to ATP and oxygen levels is related to complex mechanisms involving the mitochondrial production of reactive oxygen species (ROS), mitochondrial and ER Ca²⁺

homeostasis, and the unfolding protein response (Høyer-Hansen et al., 2007; Moore et al., 2011; Chang et al., 2012; Filomeni et al., 2014).

These factors modulate the activity of mTORC1, AMPK and sirtulin1 (Sirt1), a nutrient- sensing deacetylase (Lee et al., 2008; Russell et al., 2014). Furthermore, starvation induces lipophagy, an alternative, catabolic pathway to hydrolytic enzyme and lipase-mediated degradation of lipid droplet-associated triglycerides and cholesterol (Liu and Czaja, 2013).

Lipophagy represents a compensatory mechanism to adapt to nutrient deprivation by generating energy from increased oxidation of free fatty acids, but is also important to handle conditions of lipid excess, that might otherwise result in cytotoxic effects (Singh and Cuervo, 2012). Sphingolipids, as components of the plasma membrane and internal membrane systems including autophagosomes and lysosomes and their metabolism display another autophagy modulating factor (Hamer et al., 2012; Li et al., 2014). Ceramide, the central molecule in sphingolipid metabolism, regulates autophagy in a cytoprotective manner but also induces autophagy-mediated cell death (Daido et al., 2004; Spassieva et al., 2009). However, underlying mechanisms and the influences of dysregulations in sphingolipid metabolism on autophagosomal, endosomal, and lysosomal membrane properties, fusion events, and cytoskeletal transport were not studied in detail.

These considerations underline the relevance of autophagy as a sentinel and executive pathway to maintain cellular homeostasis and the coupling to mitochondrial and ER metabolism.

1.4.9 Autophagy, ER stress, unfolded protein response and ER-associated degradation Protein folding occurs in the rough ER and is mediated by chaperones (Gotoh et al., 2011).

Glucose deprivation, ER Ca²⁺ release, and hypoxia result in the accumulation of misfolded

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proteins in the ER and induce the unfolded protein response (UPR), but also inhibit mTORC1 and induce autophagy (Wouters and Koritzinsky, 2008; Mekahli et al., 2011; de la Cadena et al., 2014). The UPR serves as a mechanism to sustain cell viability by attenuating protein synthesis and restoring cellular homeostasis. UPR coupled processes activate transcription factors, which regulate the expression of genes encoding chaperones, components of the ER-associated degradation (ERAD) system, and proteins associated with autophagy (Ding et al., 2007; Ron et al., 2011). ERAD implies the cytosolic degradation of misfolded ER proteins by the ubiquitin proteasome system (UPS) (Vembar and Brodsky, 2008). The UPS serves in parallel to autophagy as a mechanism to remove incorrectly folded ER proteins (Vembar and Brodsky, 2008). These considerations underline the coupling of ER stress, UPR, ERAD, the UPS and autophagy.

1.4.10 Autophagy and the ubiquitin proteasome system

Autophagy and the UPS degrade ubiquitinated cargo and therefore regulate cellular toxicity due to misfolded or aggregated proteins, especially implicated in the pathogenesis of neurodegenerative diseases. Proteasomal protein degradation serves on the one hand as a basic mechanism providing amino acids, but also controls cell homeostasis by targeted degradation of regulatory proteins or processing of protein precursors (Palombella et al., 1994). The UPS and autophagy are suggested to fulfill roughly different tasks in protein catabolism with proteasomal degradation of short lived proteins and autophagy-mediated degradation of long lived, endocytosed, or aggregated proteins (Fuertes et al., 2003; Metcalf and Isaacs, 2010). Despite these distinct functions, autophagy and the UPS represent a communicating network, with one pathway dependent on the other, but not able to compensate completely the impairment of one system.

Several studies revealed that proteasome inhibition results in upregulation of macroautophagy (Korolchuk et al., 2009). This triggering of autophagy may be related to the accumulation of proteins and to the decay in cellular amino acid concentrations. However, autophagy is not sufficient to compensate the lack of amino acids normally provided by proteasomal degradation, whereas the accumulation of proteins is tolerated by the cell

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(Suraweera et al., 2012). Additionally, the proteasomal impairment might result in the accumulation of autophagy triggering regulatory proteins such as p53 (Tavernarakis et al., 2008; Zhang et al., 2009).

Furthermore, inhibition of autophagy results in reduced proteasomal degradation of proteins. It has been suggested that the delay in proteasomal protein degradation depends on the cargo adaptor p62. This selective autophagy adaptor protein is stabilized upon inhibition of autophagy and sequestrates other autophagy adaptors as well as ubiquitinated substrates. This results in a delayed delivery of polyubiquitinated substrates to the proteasome. Additionally, accumulation of p62 might compete with the deubiquitinating components of the regulatory subunit of the proteasome complex and result in decreased feeding of substrates into the proteasome catalytic core (Korolchuk et al., 2009).

The UPS and autophagy are also directly linked on the molecular level by proteins as ubiquilin, functioning in autophagosome fusion and as a shuttle factor that regulates the translocation of proteolytic substrates to the proteasome (Ko et al., 2004; N'Diaye et al., 2009). Other aspects of autophagy and UPS interactions include the degradation of the catalytic core of the proteasome in conditions of nutrient deprivation through macroautophagy (Cuervo et al., 1995). Furthermore, UPS and autophagy-associated genes were co-regulated at the transcriptional level (Zhao et al., 2007; Schreiber and Peter, 2014).

1.4.11 The lysosomal network: coupling of endocytosis and autophagy

The convergence of autophagy and the endosomal pathway involves multiple steps including the formation of amphisomes, the endosomal transmission of extracellular signals to the autophagy triggering machinery, the generation of phagophores, and the transfer of lysosomal components from the ER-Golgi network to lysosomes. Furthermore, multiple regulatory molecules modulate different steps of both, autophagy and endocytosis.

Endocytotic cargoes as ligand receptor complexes were internalized from the plasma membrane, undergo fusion events with further endocytic vesicles and form tubulovesicular compartments termed early endosomes. In these structures receptors such as the low density lipoprotein receptor dissociate from their ligands, whereas other receptors including

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the epidermal growth factor (EGF) receptor remain associated with their binding partners (Luzio et al., 2007). The dissociated receptors can be returned to the plasma membrane via recycling endosomes, representing structures that separate from the primary endocytosed vesicles. Cargos for further degradation such as ligands or receptor ligand complexes remain in the vesicular elements of the early endosome (Luzio et al., 2007). After further fusion events, early endosomes mature to late endosomes, also termed multivesicular bodies. Late endosomes fuse directly with lysosomes or more frequently with autophagosomes to form amphisomes. These amphisomes further fuse with lysosomes to generate the autophagolysosomes (Nixon, 2013).

Internalized receptors including the EGF and insulin receptors activate signaling pathways such as the PI3K/Akt pathway, which are involved in the regulation of autophagy (Han et al., 2011, Chan et al., 2012). Consequently, the endocytic pathway serves as a mechanism transferring extracellular growth or nutrition state signals to the autophagy machinery.

It is suggested, that phagophores originate from different membrane sources including the plasma membrane, the ER, but also endosomes (Axe et al., 2008; Ravikumar et al., 2010;

Longatti and Tooze, 2012). Several molecules mediating phagophore formation perform also functions in the endocytic system. For example SNARE proteins were suggested to be involved in endocytosis, fusion events of early endosomes and the generation of phagophores from the plasma membrane (Moreau et al., 2011; Wu et al., 2014).

Additionally, SNAREs mediate autophagosome-lysosome and endosome-lysosome fusion characterizing them as molecules involved in multiple steps of endocytosis and autophagy (Luzio et al., 2007; Itakura and Mizushima, 2013). Further molecules regulating both endocytosis and autophagy include coat proteins such as coatomer complex I (COPI), Rab GTPases (Rab 5, Rab7, and Rab11) and their specific inhibitors Rab GTPase-activating proteins (RabGAPs), as well as BECN1 complex regulating molecules such as RUN domain cysteine rich domain containing, beclin-1 interacting protein (Rubicon; Ravikumar et al., 2008; Razi et al., 2009; Tabata et al., 2010; Zeigerer et al., 2012; Lamb et al., 2013).

The lysosome represents the final degradative compartment of autophagy and the endocytic pathway. The maintenance of lysosomal degradation depends on endosomal delivery of

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lysosomal hydrolyases, the acidifying machinery (V-ATPase), as well as the transporters and permeases to the lysosome (Singh and Cuervo, 2011; Lamb et al., 2013).

This active communication between the endocytic and autophagic degradative compartments and the numerous regulatory mechanisms shared between these pathways emphasizes the use of the term ‘lysosomal network’ and underlines that impairments of autophagy and the endocytic system should be regarded as a unity in the pathogenesis of autophagy-associated disease conditions.

1.4.12 Impairments of the lysosomal network in inherited neurodegenerative diseases Impairments of the lysosomal network were implicated in the pathogenesis of numerous neurodegenerative diseases such as AD, PD, Huntington’s disease (HD), ALS, LSD, various types of neuroaxonal dystrophies, and neuroaxonal dystrophy (NAD) -related conditions.

Neuronal loss, accumulation of autophagic vesicles and/or the occurrence of misfolded proteins or peptide aggregations represent pathological features found in all of the above mentioned diseases, but also in physiological ageing (Bethlem and Den Hartog Jager, 1960;

Mukaetova-Ladinska et al., 2000; Yu et al., 2005; Settembre et al., 2008; Yao et al., 2009;

Song et al., 2012; Fink, 2013; Nixon, 2013; Levi and Finazzi, 2014; Martin et al., 2014).

Lysosomal storage disorders are predominantly caused by primary lysosomal dysfunction. In other neurodegenerative conditions, the role of autophagy as a primary or secondary mechanism and the relevance of specific steps remain to be defined. Furthermore, especially AD and PD represent “classical” old age-associated neurodegenerative conditions, whose progression might be promoted by the decline in neuronal autophagy and mitochondrial impairments (Navarro and Boveris, 2004; He et al., 2013).

Disorders of the autophagy machinery frequently involve the CNS. Neurons as extremely specialized, postmitotic cells with a high-energy demand seem especially vulnerable to disturbances of the autophagy machinery (Lee et al., 2011). In neurodegenerative conditions, accumulations of autophagic vesicles were found accentuated at synapses, but also axons. The axonal involvement might be related to the long distance that autophagic vacuoles must travel to reach lysosomes, which are concentrated mainly in the neuronal

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soma (Lee et al., 2011; Nixon, 2013). Synapses represent regions of high-energy demand and protein turnover and contain abundant mitochondria and polyribosomes, which makes them more susceptible to the consequences of dysfunctional autophagy (Son et al., 2012).

Likewise, synaptic pathology is accompanied by abnormal accumulation of autophagosomes in the hippocampus of young AD model mice (Sanchez-Varo et al., 2012). In G2019S- leucine- rich repeat kinase 2 (LRRK2) transgenic mice, an animal model for PD, autophagosomes appear in synaptic terminals in the cerebral cortex of old mice (Ramonet et al., 2011; Yang et al., 2013). In addition, synaptic alterations, accumulations of autophagic vesicles in neurites and synapses, and mitochondrial dysfunctions are detected before the occurrence of neuritic plaques or Lewy bodies in AD and PD, respectively (Yu et al., 2005; Yao et al., 2009;

Hattingen et al., 2009). These accumulations might result from the disruption of the neurite transport machinery, impairment of autophagic fusion, and also an excessive autophagy induction. The latter is controversially discussed in HD but also ALS pathogenesis (Nixon et al., 2005; Ma et al., 2010; Yang et al., 2013; Martin et al., 2014).

Autophagy-related factors considered to modulate AD pathogenesis include autophagosomal impairment due to accumulation of amyloid-precursor-protein or tau aggregates, reduced expression of BECN1, destabilization of lysosomal membranes, disturbances of lysosomal acidification, abnormal up-regulation of Rab5, and excessive endocytosis as well as mitochondrial alterations and ROS generation (Cataldo et al., 2000; Ji et al., 2006; Cataldo et al., 2008; Pickford et al., 2008; Lee et al., 2011; Pinho et al., 2014).

The recessive inherited PD types caused by mutations in Parkin (PARK2) or PINK1 (PARK6) are clearly associated with disturbances of mitophagy (Kitada et al., 1998; Valente et al., 2004; Gasser, 2005; Kawajiri et al., 2010). The dominant synuclein (SNCA) and LRRK2 mutation-associated forms of PD seem to be induced by disturbances of macroautophagy and chaperon-mediated autophagy, but the precise mechanism is not defined yet (Yu et al., 2009; Winslow et al., 2010; Manzoni et al., 2013; Tanik et al., 2013). Further Parkinson- related genes such as glucocerebrosidase (GBA) and lysosomal type 5 P-type ATPase (ATP13A2) are also associated with lysosomal impairments (Ramirez et al., 2006; Mazzulli et al., 2011; Pan and Yue, 2014).

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HD, caused by a repetitive DNA sequence in the huntingtin gene, seems to result from complex disturbances of autophagy (Duyao et al., 1993; Kegel et al., 2000; Martin et al., 2014). Wild-type huntingtin is suggested to function as an autophagy adaptor, to mediate trafficking of autophagosomes along microtubules, and to modulate mitophagy (Ravikumar et al., 2005; Kalvari et al., 2014; Rotblat et al., 2014). Consequently, mutant huntingtin- associated disturbances of autophagy involve the formation of toxic protein aggregates sequestering mTOR, ongoing with autophagy upregulation and excessive formation of autophagosomes as well as disruption of autophagosome motility and subsequent impairment of autophagosome-lysosome fusion (Ravikumar et al., 2004; Martinez-Vicente et al., 2010; Roscic et al., 2011; Wong and Holzbaur, 2014).

In familial and sporadic ALS, numerous underlying genetic mutations were identified many of them associated with primary or secondary functions in the autophagy pathway (Guo et al., 2010; Iguchi et al., 2013). Impairments of autophagy including increased numbers of autophagosomes occur early in the disease together with numerous accumulations of aberrant proteins in affected neurons (Guo et al., 2010; Zhang et al., 2011). In addition, ALS- associated mutations in the dynein and dynactin gene indicate that disturbances of lysosome or autophagosome transport play a major role in ALS pathogenesis (Moughamian and Holzbaur, 2012 a, b).

LSD are among the first diseases linking lysosomal dysfunctions and neurodegeneration (Klein and Futerman, 2013). LSD are caused by defects in lysosomal enzymes (e.g. acid β- galactosidase in GM1–gangliosidosis, β-glucocerebrosidase in Gaucher disease), lysosomal enzyme trafficking (e.g. N-acetyl glucosamine phosphoryl transferase α/β in mucolipidosis type II), soluble non-enzymatic lysosomal proteins (e.g. cholesterol binding NPC in Niemann- Pick disease type C2), and lysosomal membrane proteins (e.g. LAMP2 in Danon disease;

Suzuki and Chen, 1968; Tsuji et al., 1987; Canfield et al., 1998; Nishino et al., 2000; Park et al., 2003; Platt et al., 2012). Furthermore, secondary effects such as impairments of lysosome reformation, failure of endo- and autolysosomal clearance, accumulation of protein aggregates, and deficient mitophagy can increase cargo storage in autophagic vesicles (Tessitore et al., 2009; Goldman und Krise, 2010).

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In NAD and NAD-related conditions such as human hereditary spastic paraparesis (HSP) mutations in genes encoding autophagy-associated proteins including WD repeat domain- containing protein 45 (WDR45), spatacsin (SPG11), zinc finger FYVE domain-containing protein 26 (Spastizin or SPG15), tectonin beta-propeller repeat-containing protein 2 (TECPR2 or SPG49), and WD repeat-containing protein 48 (WDR48 or SPG60) were characterized (Haack et al., 2012; Oz-Levi et al., 2012; Khundadze et al., 2013; Chang et al., 2014; Novarino et al., 2014; Vantaggiato et al., 2014). Spatacsin and Spastizin mutations are associated with impairment of autophagosome maturation, accumulation of immature autophagosomes, and autophagic lysosome reformation (ALR; Chang et al., 2014;

Vantaggiato et al., 2014). WDR45 is suggested to regulate autophagosome formation, whereas the deubiquitinating enzyme WDR48 is associated with the endosomal/lysosomal compartment and interacts with mTOR regulating proteins (Park et al., 2002; Lu et al., 2011;

Gangula and Maddika, 2013; Saitsu et al., 2013). TECPR2 is suggested to interact with the mammalian Atg8 homologs and to function as a positive regulator of autophagosome accumulation (Behrends et al., 2010; Oz-Levi et al., 2012).

All these findings underline the relevance of autophagy in ageing and neurodegeneration and suggest this pathway as a potential therapeutic target. In addition, they might partially explain the influence of nutrition and other environmental factors on the progress of degenerative CNS diseases. However, the association of autophagy and senescence as well as the dependence of neuronal subpopulations on autophagic pathways is far from understood provoking future studies.

1.5 Human hereditary spastic paraparesis and neuroaxonal dystrophies in humans and dogs

Human hereditary spastic paraparesis (HSP) and neuroaxonal dystrophies represent a group of heterogeneous neurodegenerative conditions with clinical and pathological overlapping features (Vaher et al., 2001, Fink, 2013; Schneider et al., 2013). Furthermore, neuroaxonal dystrophy (NAD) and HSP-associated mutations affect similar pathways.