Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10 -136 ) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

Neurodegenerative disorders affect millions of people worldwide. We describe a novel neurodegenerative disease in a canine model, characterized by progressive cerebellar ataxia and cellular vacuolization. Our genetic analyses identified a single nucleotide change in the autophagy-related ATG4D gene in affected dogs. The ATG4D gene has not been linked to inherited diseases before. The autophagy-lysosome pathway plays an important role in degrading and recycling different cellular components. Disturbed autophagy has been reported in several different diseases but mutations in core autophagy components are rare. Histological analyses of affected canine brain tissues revealed altered autophagic flux, and a knockdown of the gene in the zebrafish model caused marked neurodevelopmental alterations and neurodegeneration. Our findings identify a new disease-causing pathway and implicate the ATG4D protease as an important mediator for neuronal homeostasis. Furthermore, our study establishes a large animal model to investigate the role of ATG4D in autophagy and to test possible treatment options.

Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: A genetic test will be available later from Genoscoper Ltd, which is partly owned by HL.

Funding: This study was partly supported by a grant from the Albert-Heim-Foundation ( http://www.albert-heim-stiftung.ch/ ) to TL, by grants to HL from the Academy of Finland ( http://www.aka.fi/en-GB/A/ ), ERCStG (260997) ( http://erc.europa.eu/funding-and-grants ), the Sigrid Juselius Foundation ( http://www.sigridjuselius.fi/foundation ), Biocentrum Helsinki ( http://www.helsinki.fi/biocentrum/ ) and The Jane and Aatos Erkko Foundation ( http://www.jaes.fi/en/ ), by a grant from the Biomedicum Helsinki Foundation ( http://www.biomedicum.com/ ) to KK, by grants to JK from the Swedish Research Council ( http://www.vr.se/ ) and Swedish Brain Foundation ( http://www.hjarnfonden.se/ ), and by a postdoctoral grant to GC from the Swedish Brain Foundation ( http://www.hjarnfonden.se/ ). TL received a Humboldt Research Award from the Alexander von Humboldt Foundation ( http://www.humboldt-foundation.de ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2015 Kyöstilä et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Inherited neurological diseases also occur in the domestic dog (Canis lupus familiaris), and many are caused by mutations in the same genes as corresponding human conditions [ 14 ]. In the present work, we report a novel inherited neurodegenerative condition in the Lagotto Romagnolo (LR) dog breed, and describe its clinical and pathological characteristics and the likely genetic cause. The affected dogs present with progressive cerebellar ataxia, and histological findings reveal intracellular vacuolization, altered neural autophagy and neurodegeneration. Our results suggest that the disorder is caused by a recessive missense mutation in the ATG4D gene, which encodes an autophagy-related proteinase [ 15 ].

The LSDs form a family of around 50 inherited metabolic diseases characterized by accumulation of macromolecules within intracellular vacuoles of the endosomal-autophagic-lysosomal pathways [ 8 , 9 ]. LSDs can be subgrouped on the basis of the stored material, ranging from carbohydrates (e.g. mucopolysaccharidoses) to different types of lipids (e.g. sphingolipidoses) and proteins, or a combination of these [ 9 ]. Although the disease usually involves multiple organs, central nervous system (CNS) dysfunction and neurodegeneration are present in the majority of LSDs [ 8 – 10 ]. The classical causative mutations disrupt lysosomal enzymes, leading to accumulation of their unprocessed substrates within lysosomes [ 8 , 10 ]. However, there are LSDs that differ from this classical example. Dysfunction of other types of proteins important for lysosomal function, such as the lysosomal membrane protein LAMP2 [ 11 ], can also cause LSD. Furthermore, the pathological changes in some LSDs appear to result rather from defects in intracellular membrane trafficking than in processing of the lysosomal substrates, and many show signs of altered autophagic flow [ 12 ]. In fact, the involvement of the autophagy pathways in several LSDs has prompted a suggestion that LSDs could in part be seen as autophagy disorders [ 13 ].

The intracellular homeostasis of neurons, especially in the cerebellar Purkinje cells, is easily disturbed by dysfunction in degradative processes and accumulation of different cellular materials [ 1 ]. The autophagy-lysosome pathway [ 2 ] and the ubiquitin-proteasome system [ 3 ] are two major cellular degradation pathways. The autophagy (or self-eating) process is particularly important in the degradation of organelles and long-lived proteins, whereas the proteasome complex targets more short-lived proteins [ 4 , 5 ]. Macroautophagy (usually referred to simply as autophagy) is an evolutionary conserved intracellular process, in which proteins and organelles are sequestered within double-membrane autophagosomes and delivered to the lysosome for degradation. This recycling process is orchestrated by several different autophagy related (ATG) proteins in order to maintain proper cellular homeostasis under both basal state and stressful conditions, such as nutrient deprivation [ 2 ]. The ubiquitin-proteasome system and the autophagy-lysosome pathway are interlinked [ 4 ], and their dysfunction has been implicated in various detrimental neurodegenerative disorders, such as inherited ataxias, Alzheimer and Parkinson disease, and the lysosomal storage disorders (LSDs) [ 6 , 7 ].

Results

Clinical characterization reveals progressive ataxia with occasional nystagmus and behavioral abnormalities A particular neurodegenerative disease was first recognized in three LRs that presented with progressive neurological signs. The three affected dogs comprised two full siblings and a distantly related dog. Similar clinical history, clinical examination findings and corresponding histological changes in post mortem pathological examination in all three dogs indicated a shared disease etiology. During the course of the genetic study, we performed a detailed neurological examination in altogether 16 affected LR dogs, and another six LRs were reported by their owners to suffer from comparable neurological signs. The typical clinical presentation in affected dogs was progressive ataxia (S1 Video), and many of the dog owners reported that their dogs had been a bit clumsy even before they noticed obvious ataxia. Ten of the 22 affected dogs had episodes of abnormal eye movements (nystagmus) and this was the first clinical sign noticed by the owners of seven affected dogs. Later in the course of the disease, the owners of seven affected LRs reported behavioral changes, such as restlessness, depression and aggression towards people or other dogs. The age at onset of clinical signs varied considerably between the 22 dogs; the first clinical signs were noticed at the mean age of 23 months, ranging from 4 months to 4 years. The rate of progression of clinical signs to a point where euthanasia had to be considered also varied from months to years. The neurological examination in 16 affected LRs revealed a mild to severe cerebellar ataxia in all examined dogs. The majority of dogs had normal paw positioning responses when postural reactions were tested but showed delayed onset of correction in hopping reactions. Spinal reflexes were normal except for decreased or absent patellar reflexes in five dogs. Menace reaction was decreased in eight dogs, and exaggerated in one dog. Positional nystagmus was visible in four dogs during the neurological examination. Magnetic resonance imaging of the brain was performed in 11 affected dogs. The principal findings included signs of mild atrophy of the cerebellum in nine dogs and of the forebrain in six dogs. In five dogs, lateral ventricles were enlarged. A small corpus callosum was detected in three affected dogs when compared to age matched LRs. In two affected dogs, the brain imaging was unremarkable.

Pathological findings indicate disturbed autophagy and vesicular trafficking We performed pathological examination on seven LRs that were euthanized due to progressive neurological signs. Atrophy of the cerebellum was clearly visible on macroscopic examination in two of the examined dogs. Histological examination revealed widespread swelling and clear vacuolization of the neuronal cytoplasm, diffusely affecting the central and peripheral nervous system. The cytoplasmic vacuolization varied from fine vesiculation to large confluent vacuoles (Fig 1A). The cerebellar cortex was consistently affected (Fig 1B), showing marked progressive Purkinje cell loss and granular cell depletion, especially in dogs with a prolonged clinical course or more severe clinical signs (Fig 1C and 1D) The deep cerebellar nuclei, nucleus ruber, nucleus vestibularis and the lateral and medial geniculate nuclei also showed consistent, severe changes. The lesions were milder in the cerebral cortex, the basal ganglia and in specific nuclei, such as the oculomotor and hypoglossal nucleus. Disturbed axonal transport was evident as numerous morphologically diverse axonal spheroids (Fig 1E) in the cerebellar white matter, in the thalamic, brainstem and cerebellar nuclei as well as in the dorsal funiculus of the spinal cord. The spheroids were accompanied by mild to moderate astrocytosis of the cerebellar and brainstem white matter, indicated by an increase in glial fibrillary acidic protein (GFAP)-positive cells. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Histological findings in neurons and pancreas. (A) Swelling of neurons in the vestibular nucleus due to fine vesiculation (arrows) and clear vacuolization (arrowhead) of the cytoplasm. HE, scale bar 100 μm. (B) Clear cytoplasmic vacuolization (arrows) in cerebellar cortical Purkinje cells. HE, scale bar 100 μm. (C) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells (arrows) and a dense granular cell layer. HE, scale bar 100 μm. (D) Marked neuronal loss is present the cerebellar cortex of an affected dog. The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain (arrow). HE, scale bar 100 μm. (E) Axonal spheroids of varying quality were seen in the white matter (arrows) of cerebellum and brainstem. HE, scale bar 100 μm. (F) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells. HE, scale bar 100 μm. (G) Purkinje cell with numerous single-membrane bound, cytoplasmic vacuoles tethering to each other (arrows, inset). Electronmicrograph, scale bar 2 μm. Inset: scale bar 1 μm. (H) Axonal spheroid containing aggregated degenerated mitochondria, occasional double-membrane-bound autophagosomes (arrows) and free electron dense material, compressed by a peripheral clear vacuolar space. Electronmicrograph, scale bar 0.5 μm. Abbreviations: n, nucleus; ml, molecular layer; pl, Purkinje cell layer; gl, granular cell layer. https://doi.org/10.1371/journal.pgen.1005169.g001 In addition to the findings in neural tissue, we detected hypertrophy and vesicular vacuolization of the cytoplasm in several secretory epithelial cells, including the cells of the choroid plexus and the subcommisural organ. Outside of the nervous system, vacuolization affected pancreatic acinar cells (Fig 1F), the parathyroid gland, adrenal cortical cells, the prostate, the salivary glands, the mammary gland and bronchial epithelial cells. Furthermore, vacuolization and granular aggregates were present in cells of mesodermal origin, as seen in smooth muscle cells, in the vascular tunica media and occasionally in endothelial cells, but also in cells with macrophage morphology in lymphoid tissue, pulmonary alveoli and in GFAP-negative glial cells scattered along the interface of Purkinje and granular cell layers and throughout the cerebellar white matter. Furthermore, fine vesicular vacuolization of the cytoplasm was seen in the apocrine sweat glands in skin biopsies of three live dogs that suffered from corresponding clinical signs. This vacuolization was comparable to that present in the secretory epithelia of the necropsied dogs. The content of the neuronal vacuoles did not stain in hematoxylin-eosin (HE) staining and was periodic-acid-Schiff´s (PAS) negative, which suggests that glycogen, glycoprotein, glycolipid or lipofuscin did not accumulate within the vacuoles. Electron microscopy sections of Purkinje cells showed single membrane bound cytoplasmic vacuoles of varying size that either appeared empty or contained very few small membranous profiles or lucent floccular material (Fig 1G). The vacuoles tethered and formed contact sites reminiscent of hemifusion (Fig 1G inset). The axonal swellings contained peripherally coalescing clear vacuoles that compressed degenerated mitochondria, occasional double-membrane-bound autophagosomes, and free electron dense aggregated material (Fig 1H).

Bioinformatic analysis of the ATG4D missense variant The mammalian ATG4D protein belongs to the ATG4 family of cysteine proteinases, together with ATG4A, B and C [15]. The c.1288G>A variant is located in the last exon of the canine ATG4D gene (Fig 3B). At the protein level, the missense variant is predicted to cause an alanine to threonine amino acid change, p.A430T. The main functional domain of the ATG4D protein, the C54 peptidase domain, is located at the center of the protein body. The amino-terminal region of the ATG4D protein is suggested to contain a PEST sequence, a caspase site and a cryptic mitochondrial target sequence, and the carboxy-terminus holds a putative Bcl-2 homology-3 (BH3) domain [16, 17]. The alanine at position 430 does not reside in any of the known domains but is centered between the peptidase domain and the BH3 motif near the carboxy-terminus (Fig 3C). The position is moderately conserved in evolution as is seen in an alignment of 41 different vertebrate species (Fig 3D). A large majority (37 out of 41) of the species has either an alanine or valine at the position, both of which are non-polar, hydrophobic amino acids, whereas four of the investigated species possess a serine residue. Alignment of all four ATG4 paralogs from human and dog revealed valine residues at the corresponding positions in ATG4A, ATG4B, and ATG4C (Fig 3E). We used the PredictSNP program to provide a consensus pathogenicity estimate from several independent prediction algorithms on the functional effect of the p.A430T change [18]. The alanine to threonine change was estimated to be neutral with 85% confidence, which would suggest that the variant does not severely disrupt the protein but may still have an effect on its function.

Analysis of the ATG4D transcript We then examined whether the ATG4D gene is expressed in the affected tissue and if the mutation has an effect on mRNA splicing or mRNA expression levels. We sequenced the ATG4D transcript using RNA samples extracted from the cerebellar cortex of two affected, two carrier and two wild-type LRs. The obtained sequences were in accordance with the reference (XM_542069.3), and the c.1288G>A variant was present in the transcripts of the two affected and two carrier dogs. We did not identify splicing defects or changes in transcript levels. Both transcript alleles were present at comparable levels in the heterozygous carrier dogs (S2 Fig). These results suggest that the canine phenotype is caused by a dysfunction at the protein level and not by any change on the transcript level.

Histological analyses reveal altered autophagy pathway in the affected neurons We next used immunohistochemistry (IHC) to examine the nature of the neuropathological changes in more detail. For this purpose, we used antibodies produced against ATG4D, ubiquitin, the autophagosome membrane marker LC3B, the lysosome membrane marker LAMP2 and the autophagic cargo marker p62, which binds ubiquinated material destined for autophagy. The axonal spheroids showed strong diffuse immunoreactivity for LC3B (Fig 4A), and the granular core was immunoreactive for ubiquitin (Fig 4B) and p62 (Fig 4C), indicating disturbed autophagy in the neurites. Within the cerebellar granular cell layer and cerebellar white matter, the ATG4D protein was detected within the finely granular swollen axons (Fig 4D). Some vacuoles in the neuronal soma were positive for the lysosomal marker LAMP2 (Fig 4F). The ultrastructure of these single membrane bound vacuoles was consistent with distended secondary lysosomes or autolysosomes, containing digested material. Some vacuoles, however, remained unstained with all antibodies used (Fig 4F). Coarse LC3B positivity was present in the perinuclear area in several Purkinje cells, indicating induction of autophagy or blockage of the autophagic flow in the cerebellum of affected dogs (Fig 4E). Although the cause and origin of the neuronal vacuoles remains to be investigated in more detail, these results indicate alterations in the autophagy pathway in neurons of the affected dogs. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Immunohistochemistry indicates disturbed autophagic flow in neurons. (A) Axonal spheroids stain diffusely positive for LC3B. IHC LC3B, scale bar 100 μm. (B,C) The granular cores of the spheroids are positive for (B) ubiquitin (arrow) and (C) p62. IHC ubiquitin and p62, scale bars 20 and 100 μm, respectively. (D) Smooth axonal swellings in the cerebellar cortex contain ATG4D. Inset: control. IHC ATG4D, scale bar 100 μm. (E) Affected neurons show increased perinuclear granular LC3B positivity. Inset: control. IHC LC3B, scale bar 100 μm. (F) Neuronal vacuoles are partially LAMP2 positive (arrow) and partially negative (arrow head). Inset: control. IHC LAMP2, scale bar 20 μm. https://doi.org/10.1371/journal.pgen.1005169.g004