Histology of NAD in Spanish water dogs.

<p>(<b>A</b>, <b>B</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 (<b>A</b>). 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>B</b>).</p

Association of the <i>TECPR2</i> missense mutation with the NAD phenotype in Spanish water dogs.

<p>Association of the <i>TECPR2</i> missense mutation with the NAD phenotype in Spanish water dogs.</p

Pedigree of the collected Spanish water dogs with NAD.

<p>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.</p

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).

<p>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 <i>Pla2g6</i> 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).</p

TECPR2 domain structure and p.R1337W mutation associated with NAD in Spanish water dogs.

<p>(<b>A</b>) 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>B</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.</p

Histological findings in neurons and pancreas.

<p>(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.</p

Muscle and peripheral nerve pathology in Alaskan Malamutes with polyneuropathy and a mutation in NDRG1.

<p>Representative cranial tibial muscle and peroneal nerve biopsy transverse sections from a two year old female Alaskan Malamute (a,b) and a three year old female Alaskan Malamute (c,d) with histopathological findings consistent with polyneuropathy. Large and small groups of atrophic fibers were present with variable severity and fatty infiltration (a,c. H&E stain). A moderate to marked depletion of myelinated fibers was evident in resin embedded nerve biopsy sections (b,d. Toluidine blue stain). Nerve fiber loss resulted from chronic axonal degeneration (arrows in b,d).</p

Multiple alignment of mammalian NDRG1 proteins.

<p>Alignment of NDRG1 protein sequences from five mammalian species: Homo sapiens, Pan troglodytes, Canis lupus, Bos taurus and Mus musculus shows a sequence similarity of 94.7%. Residue 98 (gly, marked with a frame) which is substituted in Alaskan Malamutes with the G>T mutation, is conserved in the five species.</p

Variants detected by whole genome re-sequencing of an affected Lagotto Romagnolo.

<p>^aThe sequences were compared to the reference genome (CanFam 3.1) from a Boxer. Only those variants that were homozygous in the affected Lagotto Romagnolo are reported.</p><p>Variants detected by whole genome re-sequencing of an affected Lagotto Romagnolo.</p

Histological findings in neurons and pancreas.

<p>(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.</p