A litter of five 12-week-old Norwegian Buhunds (2 males and 3 females) was evaluated because of slowly progressive gait abnormalities, impaired balance, and head tremors in 2 puppies (1 male and 1 female; dogs 1 and 2, respectively). Results of general physical examination were unremarkable for all puppies. Neurologic examination of the 2 affected puppies revealed unremarkable mentation and behavior; broad-base stance in all 4 limbs; persistent, fine head tremors; mild truncal ataxia; and mild hypermetria in all 4 limbs. Results of cranial nerve evaluation were unremarkable except for a bilaterally reduced menace response. Segmental spinal reflexes and muscle bulk and tone were unremarkable. No foci of hyperesthesia were detected on palpation of the head, vertebral column, or appendicular muscles. Given these findings, a cerebellar lesion was suspected. No neurologic abnormalities were detected in the remaining 3 puppies. Six and 10 years previously, 2 other related female Norwegian Buhunds (one 16 weeks old [dog 3] and the other 20 weeks old [dog 4]) had been referred to the same hospital because of similar abnormalities, although clinical signs at that time were slightly more evident than for the later evaluated puppies.
For all 4 puppies, results of CBC and serum biochemical analysis were unremarkable. Magnetic resonance imaging of the brain was performed for dogs 1, 2, and 3 with a 1.5-T unit.a The MRI protocol included dorsal, sagittal, and transverse T2-weighted fast spin echo; transverse T2 fluid-attenuated inversion recovery; and transverse T1-weighted fast spin echo sequences performed before and after contrast mediumb administration. No abnormalities were detected in any sequence.
A CSF sample was collected from the cerebromedullary cistern in dogs 1, 2, and 3. Sample cell counts and total protein concentration were within the reference ranges. Cytologic analysis of the CSF sample revealed no abnormal findings, and PCR assays for Toxoplasma gondii, Neospora caninum, and canine distemper virus yielded negative results. Urinary organic acid screening, enzyme analysis of fibroblasts cultured from skin biopsy specimens, and a brainstem auditory-evoked response test were performed for dogs 1 and 2, and findings were also unremarkable.
All 4 affected puppies were euthanized at the breeder's request via IV injection of pentobarbital sodium.c The brain and spinal cord of each puppy were collected via craniectomy and laminectomy, fixed in neutral-buffered 10% formalin, trimmed, automatically processed, and routinely embedded in paraffin. Multiple transverse sections were obtained from the telencephalon, diencephalon, brainstem, and spinal cord. After transection of the midbrain, longitudinal midline sectioning of the cerebellum and brainstem was performed. One side of the brain underwent sagittal lamellation (3-mm slice thickness; vertical axis), whereas the other half underwent transverse lamellation (3mm slice thickness; 5° caudoventral inclination).
Slides for histologic examination were created from 5-μm-thick tissue sections stained with H&E stain and the Bielschowsky impregnation method for detection of empty baskets.1,2 For dogs 1, 2, and 4, antibody labeling was performed for ITPRI3,d and calbindin-D-28K,4,e which are both markers of Purkinje cell differentiation. The paraffin-embedded samples from dog 3 were unavailable for reprocessing.
Additional neuronal markers used for dogs 1, 2, and 4 included microtubule-associated protein 25,f (for identification of Golgi neurons and stellate, basket, and granule cells) and synaptophysing (panneuronal marker). For identification of velate astrocytes and Bergmann glia, glial fibrillary acidic protein immunohistochemical analysis was performed in singleh-and doublei-labeling mode with a neuronal cell death marker6 or calbindin. Immunolabeling for calbindin, microtubule-associated protein 2, and ITPR1 involved microwave pretreatment of tissue sections in citrate buffer (pH, 6.0). Primary antibody binding was made visible by use of polymer technologyj and diaminobenzidinek or green peroxidase substrate chromogenl as chromogens. Immunohistochemical double labeling was performed as described elsewhere.7 Histologic slides were evaluated by bright-field microscopy, epifluorescence microscopy (excitation wavelength, 488 nm),m or both. The systematic algorithm for detection of abnormalities has been described elsewhere.8
On gross and subgross examination, the architecture of the lobules, sulci, gyri, and folia of the brain was unremarkable in appearance. On sagittal histologic sections, the subarachnoid spaces of the cerebellum appeared mildly enlarged. In dog 4, the underlying molecular and Purkinje cell layer had mild, patchy Bergmann gliosis, whereas lamination, neuronal alignment, and relative layer thickness were unremarkable throughout cerebellar cortex. In dogs 1 and 2, the rostroventral lobules had patchy subpial granule cell retention. The H&E staining revealed multiple dark Purkinje cells throughout the lobules, some of which had positive staining for cell death, in particular at the peripheral tips of the folia. No other degenerative features, dysmorphic Purkinje cells, or axonal changes were evident. However, the amount of ITPR1 and calbindin expression in histologic preparations was abnormal (Figures 1 and 2). Specifically, ITPR1 was absent in multiple Purkinje cells of the paraflocculus and dorsal foliary tips. Lack of staining extended from the soma into the dendritic tree. A similar effect was observed for calbindin expression that left individual cells unstained in the outer dorsal vermis and (in dog 4) up to 25 immunonegative cells in a row in the pyramis and uvula, with the lingula less affected. This finding was milder for dogs 1 and 2. No changes were identified for the other neuronal markers. Likewise, cerebellar white matter and connected fiber tracts had no histologic evidence of change.
Illustrations of cerebellar regions (A and D) and photomicrographs of gray matter sections obtained from those regions (B and C) on postmortem evaluation of a 12-week-old Norwegian Buhund evaluated because of slowly progressive cerebellar ataxia and fine head tremors. This dog was 1 of 4 affected dogs from the same pedigree. The distribution of the affected cerebellar regions is represented in green in panels A (ansiforme lobule and paraflocculus) and D (caudal lobe). The photomicrographs reveal the decreased somatic and dendritic (star) expression of antibody-labeled Purkinje cell (PC) differentiation marker ITPR1 in the affected (C) versus unaffected (B) cerebellar regions. Protein expression is indicated by the brown color of the chromogen diaminobenzidine. Notice that there is no reduction in Purkinje cell density, but ITPR1-negative Purkinje cells appear smaller than ITPR1-positive cells. GCL= Granule cell layer. ML = Molecular layer. H&E stain; bar = 35 μm.
Citation: Journal of the American Veterinary Medical Association 253, 6; 10.2460/javma.253.6.774
Illustrations of cerebellar regions (A and D) and photomicrographs of gray matter sections obtained from those regions (B and C) on postmortem evaluation of the dog described in Figure 1. The distribution of the affected cerebellar regions is represented in red in panels A (paraflocculus and most lateral part of the paravermis) and D (caudal lobe). The photomicrographs reveal extensive loss of calbindin expression in the affected (C) versus unaffected (B) cerebellar regions. Calbindin expression is indicated by the green color of the applied peroxidase substrate chromogen. Glial fibrillary acidic protein, found in astrocytes, is stained brown with the chromogen diaminobenzidine. Notice that there is no reduction in Purkinje cell density, but calbindin-negative Purkinje cells appear smaller than calbindin-positive cells. PC = Purkinje cell. H&E stain; bar = 35 μm. See Figure 1 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 253, 6; 10.2460/javma.253.6.774
Extracerebellar lesions were sparse and featured some hypereosinophilic neurons in the precerebellar nuclei, tegmentum, and spinal cord Rexed laminae VII through IX of the intumescences. The tectum and periaqueductal gray matter were spared, as were the diencephalic nuclei, basal nuclei, and cerebral cortex. No gross abnormalities in any other organ were identified on postmortem examination.
The pedigrees of the affected puppies were provided by the breeder, together with information regarding other relatives affected (Figure 3). The litter siblings of the affected dogs were identified by use of records held by the Kennel Club of the United Kingdom and added to the pedigree. No phenotype information was available for these additional siblings; therefore, whether they had the same clinical signs as the other affected dogs was unknown. For the pedigree analysis, the dogs reported by the breeder as unaffected as well as the dogs with unknown affected status were presumed to be unaffected to allow the mode of inheritance to be inferred. Results of this pedigree analysis suggested an autosomal recessive mode of inheritance.
Diagram of the extended pedigree of the 4 Norwegian Buhunds (black symbols) described in Figure 1. Males are represented by squares and females by circles. Dogs reported by the breeder as having similar clinical signs (slowly progressive ataxia), but for which no clinical information was available, are represented by solid gray symbols. Dogs reported by the breeder as unaffected are represented by white symbols. Dogs confirmed as clinically unaffected by a neurologist are marked with an asterisk. Dogs for which no breeder reported information about their disease status are represented by checkered symbols. In the litters included in this pedigree, 12 dogs were deemed affected and 28 dogs were deemed unaffected.
Citation: Journal of the American Veterinary Medical Association 253, 6; 10.2460/javma.253.6.774
To further explore the hypothesized genetic basis of the observed abnormalities, total RNA was extractedn from cerebellar samples collected from dogs 1 and 2, and genome-wide mRNA sequencing was performed. A kit including RNA fragmentation, first-strand cDNA synthesis, second-strand synthesis, end repair, dA tailing, and PCR amplification modules was used for library preparation.o Between modules, clean-up was performed by use of a purification kit.p,q Reverse transcription of RNA fragments was carried out.r Agarose gel electrophoresis was performed on the resulting products, and size selection of the adaptor ligated library was performed by band excision. The library was purifieds prior to PCR amplification, which involved primers for paired-end multiplexed massively parallel sequencing.t The final library was quantified by quantitative PCRu and sequenced at a genetics research facility.v Paired-end sequencing, with 51-bp reads, was carried out on a partial lane of a massively parallel sequencer.w The reads were aligned9 to the canine reference genome (CanFam3.0).
Candidate genes for ataxia were identified from human and veterinary literature and included the genes CALB1, ITPR1, ATXN1, ATXN2, ATXN3, ATXN7, ATXN10, SPTBN2, CACNA1A, CACNB4, TBP, PLEKHG4, KLHL1, TTBK2, PRKC, FGF14, NOP56, FXN, SACS, SYNE1, CABC1, ANO10, SEL1L, RAB24, GRM1, CAPN1, KCNJ10, and SERAC1.10–12 Visual inspection of the candidate genes was performed to search for single nucleotide polymorphisms and insertions and deletions in the aligned reads.13 Owing to the presumed autosomal recessive mode of inheritance, variants were considered potentially causative only if they were homozygous in both affected dogs and not present in any control mRNA-sequencing data, genomes, or exomes of dogs from different breeds. This method revealed no variants for further evaluation.
Discussion
Hereditary ataxias in dogs can be classified into 5 groups according to the affected structures and clinical signs: cerebellar cortical degeneration, spinocerebellar degeneration, multiple system degeneration, cerebellar ataxia without substantial neurodegeneration, and episodic ataxia.12 Cerebellar cortical degeneration is a neurodegenerative disease of several canine breeds that primarily affects Purkinje cells with secondary changes in the molecular or granular layer.14–19 The granular layer can, more rarely, be primarily affected.20–26 Clinical signs of cerebellar cortical degeneration are due to cerebellar dysfunction and include broad-base stance, ataxia, dysmetria, truncal sway, intention tremors, menace deficits, head tilt, nystagmus, and rarely anisocoria. A breed-specific extent and progression of the disease have been described12 as well as associated causative genetic mutations.27–31
The histopathologic findings in the examined Norwegian Buhunds of the present report suggested defective Purkinje cell differentiation and mild cerebellocortical degeneration, mainly affecting the paraflocculus, dorsal vermis, pyramis, and uvula. A clear discrepancy was evident between the mild degenerative processes affecting the Purkinje cell structure and their evident failure to express specific proteins in certain cerebellar regions.
Calbindin D28K and ITPR1 are proteins involved in cellular calcium homeostasis. Calbindin D28K is an intracellular calcium-binding protein expressed in several organs and throughout the CNS in species ranging from invertebrates to mammals.32,33 Within the CNS, calbindin D28K is expressed predominantly in inhibitory neurons and therefore particularly in Purkinje cells.33,34 Other areas expressing this protein include the dentate gyrus, inferior olivary nucleus, trapezoid body, entorhinal cortex, habenular and mammillary nuclei, and, albeit weakly, spinal dorsal horns.35 Mutant mice carrying a targeted null mutation of the gene for calbindin D28K are clinically severely impaired in tests of motor coordination; however, on histologic examination, Purkinje cell bodies and dendritic trees appear morphologically normal, indicating sublethal dysfunction rather than impairment of Purkinje cell maintenance metabolism.36
The ITPR1 is an intracellular inositol triphosphate–gated calcium channel situated mainly in the smooth endoplasmic reticulum. As with calbindin D28K, ITPR1 is also widely expressed in various tissues within and outside the CNS.37,38 Within the brain, expression of ITPR1 is particularly high in Purkinje cells.37,38 Expression and activation of ITPR1 are involved in Purkinje cell plasticity, synaptogenesis, and synaptic activity, suggesting a primary role of ITPR1 in the pathogenesis of multiple forms of SCAs in humans.39
Spinocerebellar ataxias are a group of autosomal dominant–inherited neurodegenerative disorders in humans characterized by cerebellar ataxia and variably associated with other multisystemic neurologic deficits.10,40 Polyglutamate SCAs (types 1, 2, 3, 6, 7, and 17) represent the most common types in humans and are caused by an expansion of a cytosine-adenine-guanine trinucleotide repeat encoding the amino acid glutamine, which possibly results in a toxic gain of function of the protein encoded by the affected gene.10 Reduction in the expression of calbindin D28K has been associated with neurodegeneration in SCA types 1, 2, 3, and 17 and other polyglutamine diseases.41–45 Histologically, in mice with experimentally induced SCA types 1, 2, and 3, the reduced calbindin D28K expression precedes motor phenotype and morphological changes by several weeks and is associated with only mild depletion of Purkinje cells.41–43 Deletions in gene ITPR1 have been identified in humans with SCA types 15 and 16.46,47 However, other SCAs with causative mutation in genes other than ITPR1 have altered ITPR1 expression, suggesting that the genetic basis of multiple forms of ataxias in humans may ultimately converge on affecting the ITPR1-dependent signaling.38,39,42
A guanine-adenine-adenine repeat expansion in intron 35 of ITPR1 is associated with SCA in Italian Spinone dogs.29 Similar to findings for the Norwegian Buhunds of the present report, histologic examination of Italian Spinone dogs with SCA has revealed no dendritic nor somatic changes in the morphology of Purkinje cells. However, as in the Norwegian Buhunds, expression of ITPR1 and calbindin D-28K is markedly affected. Two affected Norwegian Buhunds of the present report were tested for the Italian Spinone repeat expansion by means of long-range PCR assay,29 and no differences were identified in electrophoretic band patterns, compared with patterns for 2 unaffected Norwegian Buhunds. The mRNA sequencing procedure revealed no other potentially causative genetic variants in the protein coding region of ITPR1 in the Norwegian Buhunds of the present report.
Histologic examination of tissue specimens from the Norwegian Buhunds also revealed mild, sparse extracerebellar changes affecting neurons in pontine and olivary nuclei, tegmentum, and spinal cord Rexed laminae from VI through IX in both intumescences, with sparing of the spinocerebellar tracts. In humans with SCA, the loss of Purkinje cells almost invariably results in retrograde neuronal degeneration in the inferior olivary nuclei.40 Neuronal loss in the pontine nuclei is also associated with some types of SCAs; however, such loss is considered less likely to be due to retrograde degeneration processes.40
Degeneration of olivary and pontine nuclei has been identified in a Labrador Retriever in association with cerebellar cortical degeneration and was suggested to be secondary to the cerebellar lesion.48 The olivary nuclei were reportedly affected in Kerry Blue Terriers and Chinese Crested Dogs with canine multiple system degeneration, which is a hereditary, early-onset, progressive disease characterized by degenerative processes affecting olivopontocerebellar and striatonigral structures.49–51 In the Norwegian Buhunds of the present report, no striatonigral structures were affected; however, a typical progression of the involved structures and related clinical signs has been described for canine multiple system degeneration, and in the early stage of the disease, only Purkinje cells are affected, followed by the olivary nuclei, caudate nuclei, and then substantia nigra.
Degenerative processes affecting the spinal cord gray matter in dogs with Purkinje cell degeneration have been identified in adult Brittany Spaniels with SCA.52 In the Norwegian Buhunds of the present report, only mild degenerative processes were identified in the ventral horns of the spinal cord intumescences. At this stage of the disease, there were no detectable clinical signs associated with these histologic findings, making their clinical relevance unclear; however, we could not exclude the possibility that, with progression of the disease, the changes identified in the spinal gray matter may have become clinically important.
Similar to reported findings for other canine breeds affected by hereditary ataxias,12 the pedigree analysis for the Norwegian Buhund family in the present report revealed a likely autosomal recessive mode of inheritance. Autosomal recessive cerebellar ataxias are another described group of inherited degenerative diseases in humans that affect the cerebellum and various other structures. Genetic mutations have been identified for various types of autosomal recessive cerebellar ataxias, including among the others Friedreich ataxia, autosomal recessive spastic ataxia of Charlevoix-Saguenay, and autosomal recessive cerebella ataxia types 1, 2, and 3.11 No potential causative genetic variants were detected in the evaluated Norwegian Buhunds when considering the candidate genes associated with the aforementioned autosomal recessive disorders. However, the nature of the mRNA sequencing method used meant that only the protein-coding region of these genes could be searched for variants, and any variants in noncoding regions would not have been detected.
The present report represents the first of an early onset, autosomal recessive hereditary form of cerebellar ataxia in a family of Norwegian Buhunds. The histologic findings were characterized by a markedly reduced expression of calbindin D28K and ITPR1 by Purkinje cells with only mild signs of structural degeneration, suggestive of a functional disease related to cellular calcium metabolism. A candidate gene scan involving mRNA sequencing revealed no genetic mutation responsible for the disease among the evaluated genes. The presence of this form of hereditary ataxia should be considered when evaluating young Norwegian Buhunds with clinical signs of cerebellar dysfunction and also when planning breeding strategies for this breed. Unremarkable MRI findings with normal cerebellar size and signal do not exclude the presence of clinically important cerebellar disease.
Acknowledgments
The authors thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (funded by Wellcome Trust grant No. 090532/Z/09/Z and MRC Hub grant No. G090074791070) for the generation of the sequencing data.
Presented in abstract form at the 22nd Symposium of the European Society of Veterinary Neurology and European College of Veterinary Neurology, Bologna, Italy, September 2009.
ABBREVIATIONS
| ITPR1 | Inositol 1,4,5-triphosphate receptor 1 |
| SCA | Spinocerebellar ataxia |
Footnotes
1.5-T SignaEchoSpeed MRI, GE Healthcare, Milwaukee, Wis.
Multihance (gadobendatedimeglumine), Bracco Diagnotics Inc, Milan, Italy.
Animalcare Ltd, York, England.
Anti-ITPR1 (1:200) rabbit polyclonal antibody, Linaris GmbH, Dossenheim, Germany.
Anti-calbindin-D-28K (1:1,000) rabbit/polyclonal antibody, Sigma-Aldrich Corp, St Louis, Mo.
Anti-MAP2 (1:1,000) mouse monoclonal antibody (clone AP18), Kamiya Biomedical Co, Seattle, Wash.
Anti-synaptophysin (1:400) rabbit polyclonal antibody, Synaptic Systems GmbH, Goettingen, Germany.
Anti-GFAP (1:500) rabbit polyclonal antibody, Abcam, Cambridge, England.
FluoroJade-C, AAT Bioquest Inc, Sunnyvale, Calif.
ImmPRESS polymer detection system, Vector Laboratories, Burlingame, Calif.
DAB, Kem-En-Tec Diagnostics, Taastrup, Denmark.
HistoGreen, Linaris, Dossenheim, Germany.
Axiophot Zeiss Inc, Jena, Germany.
RNeasy midi kit, Qiagen, Venlo, Netherlands.
NEBNext mRNA sample prep master mix set 1, New England Biolabs Inc, Ipswich, Mass.
RNeasy mini purification kit, Qiagen, Venlo, Netherlands.
QIAquick PCR purification mini kit, Qiagen, Venlo, Netherlands.
Superscript II reverse transcriptase, Thermo Fisher Scientific, Waltham, Mass.
QIAquick gel extraction kit, Qiagen, Venlo, Netherlands.
Illumina Inc, San Diego, Calif.
Kapa library quantification kit, Kapa Biosystems Inc, Wilmington, Mass.
High-Throughput Genomics Group, Wellcome Trust Centre for Human Genetics, Oxford, England.
Illumina HiSeq2000, Illumina Inc, San Diego, Calif.
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