Vacuolar Storage Disease in Lagotto Romagnolo Dogs : Pathology, autophagy and extracellular vesicles

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. Histologically, altered autophagy commonly leads to neuronal proteinaceous inclusions as well as spheroid formation. Occasionally, neuronal vacuoles have been linked to altered autophagy. Cytoplasmic vacuolation is histologically more 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, affected organs and age- as well as breed matched control tissue, were immunohistochemically stained with markers for autophagosomal, lysosomal and endosomal membranous antigens, autophagy receptors and autophagic cargo. Electron microscopy and immunoelectron microscopy with the same membranous markers were used in order to obtain more specific results regarding the subcellular morphological changes. Autophagy was functionally monitored by the LC3 I/II conversion assay and by quantifying autophagosomes with immunofluorescence in cultured fibroblasts from affected dogs in comparison to control. 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. The genetic groups in the collaborative research where this thesis was part discovered a novel candidate gene for neurodegeneration, ATG4D. 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.

Avhandlingen beskriver vävnadsförändringarna hos Lagotto Romagnolo (LR) hundar, med en genvariant i ATG4D, som lider av en ny, fortskridande neurologisk sjukdom. Avhandlingens syfte är att undersöka om sjukdomen uppkommer till följd av störd autofagi eller om sjukdomen är en lysosomal inlagringssjukdom. Störd autofagi är en central sjukdomsmekanism i flera neurodegenerativa sjukdomar hos människan, därför är det av intresse att undersöka sjukdomsmekanismer och genetiken bakom liknande sjukdomar hos hundar. Lysosomala inlagringssjukdomar är också ärftliga, beträffar både människor och djur och skadar ofta nervsystemet. Både störd autofagi och störd lysosomal nedbrytning kan leda till att ämnen i stället frigörs som extracellulära vesiklar (EV); små signalbärare som potentiellt kan användas inom diagnostik av störningarna i dessa cellfunktioner. Obduktionsfynden hos 13 beträffade LR hundar bekräftade en enhetlig sjukdomsbild på cellnivå; upplagring av vakuoler i nervcellerna, skador i nervutskotten och nervcellsförlust. Körtelepitelceller och vissa mesenkymala celler uppvisade också cytoplasmiska vakuoler. I immunhistokemiska specialfärgningar syntes ansamling av autofagosomala och lysosomala membranprotein, autofagireceptorer och –last i vävnadsskadorna som tecken på störd autofagi. Med hjälp av elektronmikroskopi och immunelektronmikroskopi kunde delvis samma protein påvisas i de upplagrade vakuolernas membran. Bindvävsceller från beträffade hundar uppvisade förändrad basal autofagi, men oförändrad svält-inducerad autofagi, när autofagi-flödet undersöktes funktionellt i cellkultur. Motsvarade fynd syntes som signifikant flera autofagosomer i bindvävsceller från sjuka LRs under basala förhållanden. Dessutom utsöndrades signifikant flera EV ur de sjuka hundarnas celler under basala förhållanden och förmerat substrat för basal autofagi kunde påvisas i dessa EV. Sammanfattande tyder fynden på störd basal autofagi som en patogenetisk mekanism i sjukdomen. Lysosomal upplagring kunde inte påvisas hos de sjuka hundarna när urin, tre lysosomala enzyms aktivitet och vakuolernas innehåll undersöktes. En ny kandidatgen för nervcellsdegeneration, ATG4D, upptäcktes av genetikerna inom forskningssamarbetet där den här avhandlingen verifierade bristande autofagi hos LR hundar med genvarianten. Avhandlingens bidrag till jämförande patologi är att påvisa störd basal autofagi som en alternativ orsak till vävnadsförändringar som påminner om lysosomal upplagring. En grundläggande relevans för basal autofagi i specifika celler blir också tydlig genom avhandlingen då hundarna har symptom 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.