The participating cells in lesions.

<p>(A) Prominent astrocytic reaction as evidenced by anti-GFAP antibody (GFAP, green; Dapi, blue). Astrocytic reaction, engulfing the lesion; a few astrocytic feet penetrated the lesion (<i>white asterisk</i>). (B) Microglial activation, immunolabelled by anti Iba1, reproduced similar topography as the astrocytic reaction by surrounding the lesion, and few microglial cell processes infiltrated the lesion (<i>white asterisk</i>) (Iba1, green; Dapi, blue). Concurrently, astrocytic and microglial cell activations are present not only around the lesions but also elsewhere. (C) Immunostaining of anti-oligodendrocyte specific protein (anti-OSP), a cell membrane oligodendrocyte marker (OSP, green; Dapi, blue. Big or confluent lesions (<i>white arrows</i>), where the centre is occupied by many OSP-positive and intricate processes of different shapes and sizes, which presumably depend on the plane section. (D) Double immunostaining showed that the two markers–cytoplasmic (MBP) (red) and membrane (OSP) (green)–of oligodendrocytes were present together in the demyelinating lesions and the centre of the plaque was double-immunostained (<i>white arrows</i>). (E) Anti-actin immunostaining (red), Dapi (blue), actin protein aggregates were also accumulated in the centre (<i>arrow</i>) of the plaque and in the cytoplasm of some surrounding cells (<i>white arrows</i>). (F) Double immunostaining of actin (red) and MBP (green) Dapi (blue). Within the core of the plaque with actin immunostaining, note the presence of oligodendrocyte processes (<i>white arrows</i>). Scale bar: (A) and (B) 30 μm, (C) 20 μm, (D) 50 μm, (E) and (F) 20 μm.</p

Genotyping of animals of 7 French breeds for the KIF1C mutation.

<p>Genotyping of animals of 7 French breeds for the KIF1C mutation.</p

Identification of a variant in <i>KIF1C</i> gene in bovine animals affected by progressive ataxia.

<p>(A) Sanger sequence electropherogram traces for the causal mutation in the bovine <i>KIF1C</i> gene done on a wild type (WT), a heterozygous carrier and an affected animal. The G>A substitution affects the last nucleotide of bovine exon 5. Translated amino acids are presented below the genomic sequence. (B) Schematic diagram of coding exons from <i>KIF1C</i> gene in cattle (protein with 1104 amino acids) with the predicted functional domains of the protein, with the position of the mutation indicated (<i>arrow</i>). (C) Based on protein alignment, the affected amino acid is highly conserved in vertebrates and located in a conserved region of the protein. MACMU, <i>Macaca mulatta</i>; FELCA, <i>Felis catus</i>; BOVIN, bovine (<i>Bos taurus</i>); CANLF, <i>Canis lupus familiaris</i>; XENTR, <i>Xenopus tropicalis</i>; DANRE, <i>Danio rerio</i>.</p

Variants detected by whole-genome sequencing of 3 animals, including 2 with progressive ataxia.

<p>Variants detected by whole-genome sequencing of 3 animals, including 2 with progressive ataxia.</p

Association between the ataxia variant and morphology traits in animals of the Charolais breed.

<p>Association between the ataxia variant and morphology traits in animals of the Charolais breed.</p

<i>KIF1C</i> variant affects mRNA expression and leads to a functional knock-out.

<p>(A) Schematic diagram of the <i>KIF1C</i> gene in bovine sequence, located on chromosome 19, with the mutation indicated by an arrow. Primer pairs p1 and p2 (respectively amplifying <i>KIF1C</i> exons 1 to 11 and exons 13 to 20) are shown downstream of the diagram. RT-PCR from WT and affected bovine brains with p1 and p2 primer pairs demonstrated that <i>KIF1C</i> expression is modified in affected animals both in quantity–with mRNA decay–and quality (several transcripts in affected animals). <i>RPL13</i> (ribosomal protein L13) was used as a housekeeping gene. (B) Schematic diagram of <i>KIF1C</i> transcripts in affected bovine. The normal transcript bears the G>A mutation and leads to a mutated protein; the alternative transcript results from defective splicing and leads to exon 5 skipping. (C) Proteins were extracted from brains of WT and affected bovines, and from HeLa cells. Samples were analysed by immunoblotting with antibody against KIF1C proteins. No KIF1C protein was found in affected animals. WT, wild type; Aff, affected.</p

Topographical distribution of lesions in spinal cord.

<p>(A) Cervical spinal cord on paraffin-embedded section and Kluver-Barrera staining. Myelin pallor was diffuse and many tracts, presumably ascending and descending ones, were affected by demyelinating plaques. White matter stacks of various sizes were sparsely distributed. (B) Drawing of white matter lesions one by one, as presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007550#pgen.1007550.s005" target="_blank">S5C Fig</a> (<i>dark arrows</i>), showed that the ventral and lateral tracts harboured the majority of lesions. The dorsal tracts, except the median part, were less affected, but nevertheless contain very few stacks. Scale bar: (A) and (B) 2 mm.</p

Quantification of the length of paranodal section in cerebellar peduncles of non-affected and affected cattle.

<p>(A) Caspr positivity was concentrated in two paranodal compartments on either side of the node of Ranvier. The immunostaining was observed on myelinated fibres of various diameters. (Caspr green, Dapi blue). (B) The lengths of the paranodal region vary slightly; however, in affected white matter this length appeared more variable and greater within and around the demyelinating lesion. (C) Quantitative comparison and graphic representation of these lengths in WT controls, within and outside the lesions. Numbers of quantified paranodal sections in WT controls: 570; in affected cattle: inside lesion 115 paranodes and outside lesion 194 paranodes. Scale bar: (A) and (B) 40 μm.</p

Cytological features of the lesions.

<p>Semi-thin blue toluidine stained section of white matter from spinal cord and cerebellum. (A) Cerebellar white matter less affected region showing a small lesion or “pre-plaque” (<i>arrow</i>) as a hypertrophied cell with histological characteristics reminiscent of an oligodendrocyte. (B) Spinal cord white matter showed a “mature” lesion constituted by amorphous and acellular material engulfing many myelin fibres and cellular debris (<i>arrow</i>). The surrounding nerve fibres were more or less disturbed. Electron micrographs of oligodendrocyte modifications consisting of intracytoplasmic inclusions. (C) Electron micrographs of frontal section of mature demyelinating plaque. The major part of the centre of the lesion (<i>star</i>) is composed of membranous, vesicular structures and fibrillary elements, but no cytological organelles (e.g. mitochondria or endoplasmic reticulum) were identifiable and no membranous binding was observed. Immediately around the centre, there are many myelinic and amyelinic processes, some of which are degenerated. The last “ring” is composed of many surrounding cells (<i>white arrows</i>) and complete the white matter lesion. Some cells are easily recognizable as astrocytes, their cytoplasm containing gliofilament tangles; some others could be histologically reminiscent of oligodendrocyte cells while others are not recognizable in the absence of specific markers. (D) High magnification of the area marked by a star in (C) evidenced myelinic bodies, small vesicles intermingled with membranous processes and fibrillary and amorphous material. These ultrastructural features of lesions were similar irrespective of the studied brain region (cerebellum, spinal cord and internal capsules). Scale bar: (A) and (B) 40 μm, (C) 15 μm, (D) 1.5 μm.</p