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Comparative Study
. 2015 Nov 10;10(11):e0141824.
doi: 10.1371/journal.pone.0141824. eCollection 2015.

TECPR2 Associated Neuroaxonal Dystrophy in Spanish Water Dogs

Kerstin Hahn  1   2 , Cecilia Rohdin  3   4 , Vidhya Jagannathan  5 , Peter Wohlsein  1 , Wolfgang Baumgärtner  1   2 , Frauke Seehusen  1 , Ingo Spitzbarth  1 , Rodrigo Grandon  6 , Cord Drögemüller  5 , Karin Hultin Jäderlund  7
Affiliations
Comparative Study

TECPR2 Associated Neuroaxonal Dystrophy in Spanish Water Dogs

Kerstin Hahn et al. PLoS One. .

Abstract

Clinical, pathological and genetic examination revealed an as yet uncharacterized juvenile-onset neuroaxonal dystrophy (NAD) in Spanish water dogs. Affected dogs presented with various neurological deficits including gait abnormalities and behavioral deficits. Histopathology demonstrated spheroid formation accentuated in the grey matter of the cerebral hemispheres, the cerebellum, the brain stem and in the sensory pathways of the spinal cord. Iron accumulation was absent. Ultrastructurally spheroids contained predominantly closely packed vesicles with a double-layered membrane, which were characterized as autophagosomes using immunohistochemistry. The family history of the four affected dogs suggested an autosomal recessive inheritance. SNP genotyping showed a single genomic region of extended homozygosity of 4.5 Mb in the four cases on CFA 8. Linkage analysis revealed a maximal parametric LOD score of 2.5 at this region. By whole genome re-sequencing of one affected dog, a perfectly associated, single, non-synonymous coding variant in the canine tectonin beta-propeller repeat-containing protein 2 (TECPR2) gene affecting a highly conserved region was detected (c.4009C>T or p.R1337W). This canine NAD form displays etiologic parallels to an inherited TECPR2 associated type of human hereditary spastic paraparesis (HSP). In contrast to the canine NAD, the spinal cord lesions in most types of human HSP involve the sensory and the motor pathways. Furthermore, the canine NAD form reveals similarities to cases of human NAD defined by widespread spheroid formation without iron accumulation in the basal ganglia. Thus TECPR2 should also be considered as candidate gene for human NAD. Immunohistochemistry and the ultrastructural findings further support the assumption, that TECPR2 regulates autophagosome accumulation in the autophagic pathways. Consequently, this report provides the first genetic characterization of juvenile canine NAD, describes the histopathological features associated with the TECPR2 mutation and provides evidence to emphasize the association between failure of autophagy and neurodegeneration.

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Conflict of interest statement

Competing Interests: VJ and CD are affiliated at the Institute of Genetics, University of Bern, Switzerland, which currently offers genotyping for the causative mutation as a diagnostic genetic test. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Histology of NAD in Spanish water dogs.
(A, B) Histology of the brain stem (cuneate nucleus) stained with hematoxylin and eosin revealed numerous large granular axonal swellings (spheroids; arrow). Note the hypereosinophilic central target-like core structure of distinct spheroids (A). Single neurons displayed an accumulation of a finely or coarse granular, intensely eosinophilic material associated with the soma (arrow) displacing the Nissl substance. A high proportion of neurons adjacent to affected areas displayed a normal morphology with equally distributed Nissl substance (arrowhead, B).
Fig 2
Fig 2. Pedigree of the collected Spanish water dogs with NAD.
Note the inbreeding loops and the likely common ancestors appearing 8 to 9 generations ago. Only for the numbered animals DNA was available. Affected animals are shown with black symbols; genotyped carriers of the causative mutation are indicated with half-filled symbols; females are shown as circles and males as squares.
Fig 3
Fig 3. TECPR2 domain structure and p.R1337W mutation associated with NAD in Spanish water dogs.
(A) TECPR2 possesses three N-terminal WD (tryptophan-aspartic-acid dipeptide) repeats (red), a polylysine tract (green), and six C-terminal tectonin beta-propeller repeat (TECPR) domains (blue). (B) The arginine at position 1337 that is substituted by a tryptophan residue (red) is located in the sixth TECPR domain. Note that the mutation affects a conserved amino acid residue in all known TECPR2 orthologs. Highly conserved residues are marked in green.
Fig 4
Fig 4. Transmission electron microscopy of spinal cord spheroids compared to a normally structured axon.
Ultrastructurally, spheroids lacked myelin sheaths and contained closely packed accumulations of membrane-bound vacuolar structures. High numbers of vacuoles were rimmed by a double layered membrane separated by an electron-lucent cleft and defined as autophagosomes (insert; A). The axons of a healthy, age matched Beagle dog is surrounded by a thick myelin sheath (arrowhead). Isolated vacuolar structures, interpreted as mitochondria (arrow) are present between neurofilaments (B). (A) Bar: overview: 1 μm; insert 0.5 μm. (B) Bar: 2.3 μm.
Fig 5
Fig 5. Expression of autophagosome, lysosome and late endosome associated proteins in the spinal cord white matter of an NAD affected Spanish water dog (A-D) and an age matched control Beagle dog (E-H).
(A) In the affected Spanish water dog, immunohistochemistry using a LC3B specific antibody demonstrated an autophagosome accumulation within spheroids. Only few LAMP2 positive lysosomes (B) and RAB7 stained late endosomes (C) are present within spheroids. No TECPR2 accumulations are found within spheroids (D). In diseased Spanish water dogs, LC3B, LAMP2 and TECPR2 are strongly expressed in glial cells and axons without spheroid formation. In the age matched healthy Beagle dog, LC3B (E), LAMP2 (F) and TECPR2 (H) positive staining is found in axons and glial cells. RAB 7 (G) positive staining is restricted to glial cells. Bar: 20 μm. A-H: Nomarski differential interference-contrast optic.
Fig 6
Fig 6. Distribution of cervical and thoracic spinal cord spheroids in Spanish water dogs with NAD compared to mostly affected areas in human hereditary spastic paraparesis (HSP).
In NAD affected Spanish water dogs (left), spheroids and neuronal loss were restricted to the sensory, ascending pathways localized to the grey matter of the spinal cord dorsal horn and single large spheroids were detected within the cuneate and gracile fasciculus. This might explain the clinical signs as gait disturbances, proprioceptive deficits, decreased spinal reflexes and urinary incontinence. In other human forms of NAD, HSP and Pla2g6 knock-out mice (right), spinal cord spheroid formation is accentuated in the sensory pathways including the gracile fasciculus as well as the corticospinal tracts. Furthermore, also the descending motor pathways including the ventral horns as well as the descending pyramidal tracts are affected. Note that spinal cord histology of TECPR2 associated HSP in humans is unknown. Ascending, sensory pathways (red; transmission of sensory signals from the periphery (red arrow) via dorsal horn (DH) towards the brain): Dorsal funiculus composing of gracile fasciculus (GF) and cuneate fasciculus (CF); Spinocerebellar tracts with: dorsal spinocerebellar tract (DST) and ventral spinocerebellar tract (VST); Descending, motor pathways (blue; signal transmission via the ventral horn (VH) neurons towards the muscles; blue arrow): Pyramidal tracts with lateral corticospinal tract (LCT) and ventral corticospinal tract (VCT).
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