Immunohistochemical Features of Dystrophic Axons in Papillon Dogs with Neuroaxonal Dystrophy
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Vet Pathol 46:474–483 (2009)
DOI: 10.1354/vp.08-VP-0156-U-FL
Immunohistochemical Features of Dystrophic Axons in Papillon Dogs
with Neuroaxonal Dystrophy
K. NIBE, H. NAKAYAMA, AND K. UCHIDA
Division of the Project for Zoonosis Education and Research, University of Miyazaki, Miyazaki 889-
2199 (KN); and Department of Veterinary Pathology, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657 (HN, KU), Japan
Abstract. The immunohistochemical features of dystrophic axons in brain tissues of Papillon dogs
with neuroaxonal dystrophy (NAD) were examined in comparison with 1 dog with cerebellar cortical
abiotrophy (CCA) and a dog without neurologic signs. Histologically, many dystrophic axons were
observed throughout the central nervous system of all dogs with NAD. These axonal changes were
absent in the dog with CCA and in the control dog. Severe Purkinje cell loss was found in the dog with
CCA, whereas the lesions were milder in all dogs with NAD. Immunohistochemically, the many
dystrophic axons were positive for neurofilaments, tau, a/b-synuclein, HSP70, ubiquitin, synaptophysin,
syntaxin-1, and synaptosomal-associated protein-25 (SNAP-25). A few dystrophic axons were positive
for a-synuclein. In addition, these dystrophic axons, especially in the nucleus gracilis, cuneatus, olivaris,
and spinal tract of the trigeminal nerve, were intensely immunopositive for the 3 calcium-binding
proteins calretinin, calbindin, and parvalbumin. The accumulation of synapse-associated proteins in the
dystrophic axons may indicate dysfunction of the synapse at the presynaptic portion. The accumulation
of a-synuclein in the dystrophic axon and region-specific appearance of calcium-binding protein-positive
spheroids are considered as unique features in NAD of Papillon dogs, providing the key to elucidate the
pathogenesis.
Key words: Calcium-binding proteins; neuroaxonal dystrophy; Papillon dogs; synapse.
Introduction the presence of spheroids and iron accumulation in
the globus pallidus and substantia nigra reticulata.
Canine neuroaxonal dystrophy (NAD) and Similar iron accumulation has never been found in
cerebellar cortical abiotrophy (CCA) are hereditary canine NADs. Previously, the axonal changes in
suspected neurodegenerative diseases with moder- canine NAD are thought to appear through
ate to severe cerebellar atrophy.10,47 Because these 2 abnormal accumulation of neurofilament (NF) as
diseases have similar clinical, neurologic, and a result of distal axonal loss and synaptic abnor-
magnetic resonance imaging (MRI) features, dis- malities.12 Sacre et al.38 described that the axonal
crimination between them during life is very spheroids in canine NAD were located mainly
difficult. Definitive diagnosis of canine NAD or within the sensory systems including the proprio-
CCA is made by complete pathologic examination ceptive and vestibular nuclei, and that these axonal
of the central nervous system (CNS). Recently, we changes appeared to affect the preterminal or
have reported the clinicopathologic features of presynaptic portions. Immunohistochemically, ac-
NAD and CCA in Papillon and Papillon-related cumulation of NF, ubiquitin, Rab-3a, and several
dogs.32 Although NAD and CCA showed similar synaptic proteins, including synaptophysin, synap-
degenerative cerebellar changes, the lesions were tosomal-associated protein 25 (SNAP-25), and
more severe in CCA than in NAD.5,7,33,39,45 synapsin-I, has been observed in the axonal
Furthermore, NAD showed characteristic axonal spheroids of Rottweiler dogs with NAD.43 These
degeneration involving formation of numerous findings were considered to reflect severe disrup-
spheroids throughout the CNS.8,9,11,12,32,38,43 tion of axonal transport in these dystrophic axons.
Human NAD is clinicopathologically classified In addition, several calcium-binding proteins,
into 4 subtypes: infantile, late infantile, juvenile, including calbindin, calretinin, and parvalbumin,
and adult types according to the age at onset. are mainly distributed within the local interneuron
Hallervorden-Spatz syndrome,15,16,24 one of the containing c-aminobutyric acid (GABA) as a
most common infantile NADs, is diagnosed by neurotransmitter.44 Moreover, these calcium-bind-
474
Downloaded from vet.sagepub.com by guest on March 1, 2015Vet Pathol 46:3, 2009 Neuroaxonal Dystrophy in Papillon Dogs 475
ing proteins are known to present in morpholog- visualized with 3,39-diaminobenzidine (Sigma, St. Louis,
ically distinct populations of GABAergic interneu- MO) in Tris-buffered saline. Sections were counter-
ron in monkey and human brains. These proteins stained with hematoxylin or light green.
are used for cyto- and chemo-architectural inves- Results
tigations in several animals.17 Their function are,
although not well defined, suspected to contribute Histologic features
to cytoskeletal organization, cell motility and Spheroid formation was a very characteristic
differentiation, axonal transport, membrane excit- feature in all dogs with NAD, while no spheroid
ability, and the synthesis and release of certain was observed in the brain of the dog with CCA and
neurotransmitters.44 Abnormal accumulation of the control dog. On the other hand, cerebellar
calcium-binding proteins has been demonstrated neuronal cell loss, including Purkinje and granular
in several neurodegenerative disorders, including cells of the dog with CCA, was more severe than
NAD of dogs, cats, and horses.37,42,43 However, the that of all dogs with NAD. Spheroids in NAD were
relationship between axonal degeneration and the scattered throughout the CNS, being especially
accumulation of calcium-binding proteins has not predominant in the dorsal horn of the spinal cord,
been well investigated in canine NAD. nuclei gracilis, cuneatus, and olivaris, and spinal
In the present study, immunohistochemical tract of the trigeminal nerve, and its circumference
analyses using antibodies against several axonal in the brain stem, as described previously.32 In the
components, including NF, tau, synuclein, heat cerebellum of all dogs with NAD, many spheroids
shock proteins (HSPs), several synapse-associated also were found in the white matter and granule cell
proteins, and calcium-binding proteins, were per- layer compared with the cerebrum. They also were
formed to clarify the immunohistochemical fea- densely distributed in the cerebellar nuclei of dog
tures of the spheroids in Papillon dogs with NAD. No. 3. The spheroids varied in size from 20 to
50 mm in diameter and were heterogeneous in
Materials and Methods appearance (Fig. 1). In dog Nos. 2 and 3, severe
Tissue samples and histology lesions, including granular and Purkinje cell loss,
Brain tissues including the cerebrum, cerebellum, were located in the vermis. Berlin blue staining
brain stem, and spinal cords from 5 Papillon dogs, revealed no iron deposits in all brain regions of
including 3 with NAD, 1 with CCA, and 1 necropsied at dogs with NAD and the control dog, including the
4 months of age without any neurologic signs, were globus pallidus and substantia nigra reticulate of
used. The clinical features of these dogs are summarized dog No. 3.
in Table 1. The details have been reported previously.32
All tissue samples were fixed in 10% formalin and Immunohistochemical features
embedded in paraffin. For microscopic study, 6-mm- Control tissues. By immunohistochemistry for
thick paraffin sections were stained with HE. Some NF-L, M, and H, medium- to large-sized neurons
selected sections also were stained with Berlin blue for
in the cerebral cortex and spinal gray matter,
the identification of iron deposits. The globus pallidus
and substantia nigra reticulate of dog No. 3 also were Purkinje cells and cerebellar nucleus, and several
investigated. The spleen tissue with severe hemosiderin nuclei in the brain stem, such as nuclei gracilis,
deposits of a necropsied dog was used as a positive cuneatus, and olivaris, were intensely immunopos-
control. itive. Tau protein diffusely accumulated in the
cytoplasm and their processes of neuronal and glial
Immunohistochemistry cells in almost all brain regions examined. Many
Selected sections from the cerebrum, cerebellum, neuronal and glial cells in the cerebrum, cerebel-
brain stem, and spinal cords were used for immunohis- lum, brain stem, and spinal gray matter were feebly
tochemistry. After antigen retrieval with an autoclave at positive for HSP70. Some HSP70-positive neuronal
121uC, 5 minutes, endogenous peroxidase was blocked and glial cells showed nuclear immunoreactivity.
with 3% hydrogen peroxidase in methanol at room On the other hand, all brain cells were immunoneg-
temperature for 10 minutes. Primary antibodies against ative for ubiquitin. Immunohistochemistry for
for NFs, tau, a-synuclein, a/b-synuclein, HSP70, ubiq-
synapse-associated proteins, including synaptophy-
uitin, synaptophysin, syntaxin-1, SNAP-25, calbindin,
calretinin, and parvalbumin were used. The details of sin, sintaxin-1, and SNAP-25, exhibited similar
these primary antibodies are shown in Table 2. Each staining pattern. These antibodies diffusely labeled
section was incubated with the primary antibody at the neuropile of the cerebral cortex, molecular
37uC, 60 minutes. All sections were incubated with layer, and glomerula cerebellaria in the cerebellum,
Envision polymer reagent (DAKO-Japan, Kyoto, Ja- brain stem, and spinal gray matter. The staining
pan) at 37uC, 30 minutes, and then these section were pattern of both a- and a/b-synuclein was similar to
Downloaded from vet.sagepub.com by guest on March 1, 2015476 Nibe, Nakayama, and Uchida Vet Pathol 46:3, 2009
Table 1. Clinical features of cases investigated in this study.
Dog No. Breeds Gender* Age{ Major Clinical Histories Diagnoses
1 Papillon 3 M 8m 3m: ataxia of hind limb, cerebellar ataxia NAD
Chihuahua (intention tremor, hypermetria), broad-based
stance. 5m: myotonia, ananastasia. 7m:
blindness, deafness, anosmia, disappearance of
swallowing reflex. 8m: euthanasia because of
difficulty of eating.
2 Papillon F 6m 4m: ataxia of hind limb, cerebellar ataxia (head NAD
tremor). 5m: tetraplegia, tongue, and
disappearance of patella reflex, abnormal of
femoral nerve. 6m: euthanasia.
3 Papillon F 9m 3m: ataxia of hind limb, cerebellar ataxia NAD
(intention tremor, hypermetria, head tremor).
4m: ananastasia of hind limb, and then general
paresis, distention of limbs. 9m: death because
of aspiration pneumonia.
4 Papillon M 2y9m 5m: cerebellar ataxia (head tremor, intention CCA
tremor), broad-based stance, truncal ataxia,
and then upper motor neuron sign. 1y6m:
general paralysis. 2y: ananastasia. 2y9m:
difficulty of swallowing, paralysis of left facial
nerve, upper, and then died because of
aspiration pneumonia.
Control Papillon M 1y6m 2m: rhinotracheitis. 4m: hypoglycemia, diarrhea, Peritonitis by
vermination of alimentary tract. perforated
duodenal ulcer
* M 5 male; F 5 female; NAD 5 neuroaxonal dystrophy; CCA 5 cerebellar cortical abiotrophy.
{ m 5 month-old age; y 5 year-old age.
that of synapse-associated proteins. By immuno- were completely negative for calretinin and parval-
histochemistry for calcium-binding proteins, Pur- bumin. The nerve fibers of the medial lemniscus
kinje cells were strongly positive for calbindin, and and trigeminal nerve were immunopositive for
some neurons of the cerebellar nucleus also were calretinin. Parvalbumin immunoreactivity was
weakly positive. On the other hand, Purkinje cells found in the cerebellar peduncle, lateral and medial
Table 2. Antibodies used in the present study.
Antibodies for Clonality* Dilution Source
Cytoskeletal proteins NF-M and -H MAb 1 : 250 Chemicon International,
Temecula, CA
NF-L MAb prediluted DAKO-Japan, Kyoto, Japan
Tau RAS 1 : 100 Sigma, St. Louis, MO
a-Synuclein RAS 1 : 1,000 Chemicon International
a/b-Synuclein MAb 1 : 500 COVANCE, Berkeley, CA
Heat shock proteins Hsp70 MAb 1 : 500 BD, Franklin Lakes, NJ
Ubiquitin MAb 1 : 150 DAKO-Japan
Synapse-associated Synaptophysin MAb 1 : 25 DAKO-Japan
proteins Syntaxin1 MAb 1 : 250 Santa Cruz Biotechnology, Santa
Cruz, CA
SNAP-25 MAb 1 : 500 Chemicon International
Calcium-binding Calretinin MAb 1 : 25 DAKO-Japan
proteins Calbindin MAb 1 : 10 DBS, Pleasanton, CA
Parvalubumin MAb 1 : 500 Chemicon International
* MAb 5 mouse monoclonal antibody; RAS 5 rabbit antiserum.
Downloaded from vet.sagepub.com by guest on March 1, 2015Vet Pathol 46:3, 2009 Neuroaxonal Dystrophy in Papillon Dogs 477
stronger than that in the cerebrum. Ubiquitin
immunoreactivity of survived neuronal and glial
cells of dogs with NAD was very weak compared
with that for HSP70.
Synapse-associated proteins. Synaptophysin la-
beled spheroids in almost all brain regions of dogs
with NAD. The immunoreactivity was same
equivalent to that of NFs. Only a small number
of spheroids in the brain stem and cerebellum were
positive for syntaxin-1 and SNAP25 in dogs with
NAD, except for dog No. 3 (Fig. 2). In dog No. 3,
a large number of spheroids in the cerebellum,
brain stem, and sometimes the cerebrum were
intensely immunopositive for syntaxin-1.
Fig. 1. Cerebellar nucleus; NAD, dog No. 3. The a- and a/b-synuclein. A large number of spher-
spheroids vary in sizes from about 20 to 50 mm and oids in all dogs with NAD also were intensely
show various morphologies, including homogeneous, immunopositive for a/b-synuclein (Fig. 3). Anti-
granular, and concentric appearance. HE stain. Bar 5 body for a-synuclein also labeled dystrophic axons
50 mm. in dogs with NAD, while the number of immuno-
positive spheroids was smaller than those for a/b-
lemniscus, longitudinal fibers of the pons, and synuclein.
medial longitudinal fasciculus in the brain stem,
and some small neurons in the spinal gray matter. Calcium-binding proteins. Calbindin-, calretinin-,
A few neuronal cells in the cerebral cortex were and parvalbumin-immunopositive spheroids were
immunopositive for these calcium-binding proteins, distributed in the limited region of the CNS. In the
while there was no specific distribution pattern of cerebrum of dogs with NAD, the spheroids were
the positive cells. completely negative for these antibodies. On the
The immunohistochemical findings of the dys- other hand, in the cerebellum, many dystrophic
trophic axons of the 3 dogs with NAD are axons in the granule cell layer and white matter
summarized in Table 3. The results for the dog were intensely immunopositive for calbindin
with CCA and the control are not included because (Fig. 4), calretinin, and parvalbumin. Further-
of the absence of spheroids. Briefly, the spheroids more, in dog No. 3, the spheroids in the cerebellar
in almost all CNS regions of all dogs with NAD nucleus also were intensely immunopositive for the
were intensely immunopositive for NF, a/b-synu- proteins. Antibody to calbindin also labeled
clein, HSP70, and ubiquitin. Furthermore, regional morphologically intact Purkinje cells and their
restriction was recognized in the immunoreactivi- processes of dogs with NAD (Fig. 4). In the brain
ties for a-synuclein, syntaxin-1, SNAP-25, calreti- stem, many spheroids in certain nuclei, including
the nucleus gracilis, cuneatus, olivaris, and the
nin, calbindin, and parvalbumin, as detailed below.
spinal tract of the trigeminal nerve, were immuno-
NF and tau. Almost all spheroids in the cere- positive for these 3 antibodies (Fig. 5), while the
brum, cerebellum, brain stem, and spinal cord of intensity was varied in each nucleus. The spheroids
dogs with NAD showed intense immunoreactivity in the nucleus gracilis of dog No. 3 showed very
for NFs. On the other hand, several spheroids were intense immunoreactivity for calretinin, whereas
positive for tau. No specific localization pattern of those in the nucleus cuneatus exhibited weakly or
tau or NFs immunopositive spheroids was recog- no immunoreaction (Fig. 6). The immunoreactivity
nized. of spheroids for parvalbumin was more intense in
the nucleus olivaris and spinal tract of the
Heat shock proteins. Almost all spheroids in the trigeminal nerve than those in nucleus gracilis and
cerebrum, cerebellum, brain stem, and spinal cord cuneatus. In the spinal tract of the trigeminal nerve
of dogs with NAD were intensely positive for both of dog No. 2, some parvalbumin-positive spheroids
HSP70 and ubiquitin. Many surviving neuronal were adjacent to the neuronal cell body (Fig 7).
and glial cells of dogs with NAD also were positive Although spheroid formation was prominent in the
for HSP70. The immunoreactivity of HSP70 in the spinal dorsal horn, only a few spheroids were feebly
cerebellum, brain stem, and spinal cord was positive, and most were negative for calbindin,
Downloaded from vet.sagepub.com by guest on March 1, 2015478 Nibe, Nakayama, and Uchida Vet Pathol 46:3, 2009
Table 3. Results of immunostain.
Immunoreactivity of Spheroids in
Antibodies for Dog 1 Dog 2 Dog 3 Brain Regions
Cytoskeletal proteins NF-M and -H ++ ++ ++ Cerebrum
++ ++ ++ Cerebellum
++ ++ ++ Brain stem
ND* ++ ++ Spinal cord
NF-L ++ ++ ++ Cerebrum
++ ++ ++ Cerebellum
++ ++ ++ Brain stem
ND ++ ++ Spinal cord
Tau - 6 + Cerebrum
6 6 + Cerebellum
6 6 + Brain stem
ND - - Spinal cord
a-Synuclein - 6 - Cerebrum
6 - 6 Cerebellum
+ - 6 Brain stem
ND - - Spinal cord
a/b-Synuclein + ++ ++ Cerebrum
+ + + Cerebellum
++ + 6 Brain stem
ND 6 + Spinal cord
Heat shock proteins Hsp70 ++ ++ ++ Cerebrum
++ ++ ++ Cerebellum
++ ++ ++ Brain stem
ND ++ ++ Spinal cord
Ubiquitin + ++ ++ Cerebrum
++ ++ ++ Cerebellum
++ ++ ++ Brain stem
ND ++ ++ Spinal cord
Synapse-associated proteins Synaptophysin ++ ++ ++ Cerebrum
++ + ++ Cerebellum
++ ++ ++ Brain stem
ND ++ ++ Spinal cord
Syntaxin1 - - 6 Cerebrum
- 6 ++ Cerebellum
+ + ++ Brain stem
ND - 6 Spinal cord
SNAP-25 - - - Cerebrum
- - 6 Cerebellum
- 6 + Brain stem
ND - - Spinal cord
Calcium-binding proteins Calretinin - - - Cerebrum
6 + + Cerebellum
++ ++ ++ Brain stem
ND ++ - Spinal cord
Calbindin - - - Cerebrum
6 ++ ++ Cerebellum
++ ++ ++ Brain stem
ND - - Spinal cord
Parvalubumin - - - Cerebrum
- 6 ++ Cerebellum
++ ++ ++ Brain stem
ND 6 6 Spinal cord
* ND 5 not done; ++ 5 over 50% of spheroids are intensely positive; + 5 50 to 10% of spheroids are intensely positive; 6 5
less 10% of spheroids are immunopositive; - 5 negative.
Downloaded from vet.sagepub.com by guest on March 1, 2015Vet Pathol 46:3, 2009 Neuroaxonal Dystrophy in Papillon Dogs 479
Fig. 2. Cerebellum; NAD, dog No. 2. A spheroid Fig. 3. Cerebellum; NAD, dog No. 1. Many spher-
in the white matter is immunopositive for syntaxin-1 oids immunopositive for a/b-synuclein are evident in the
(arrowhead). The intact molecular layer and glomerula granular cell layer and white matter. Envision polymer
cerebellaria in the granular cell layer also are positive for method. Counterstained by Mayer’s hematoxylin. Bar 5
syntaxin-1. Envision polymer method. Counterstained 50 mm.
by Mayer’s hematoxylin. Bar 5 50 mm.
been suggested to occur in the presynaptic area.38
calretinin, and parvalbumin in all dogs with NAD. Previously, Sisó et al.43 reported that the spheroids
Exceptionally, the spheroids in the dorsal horn of in NAD of Rottweiler dogs were immunopositive
dog No. 2 exhibited intense immunoreaction for for several synapse-associated proteins, including
calretinin. synaptophysin, synapsin-1, SNAP-25, and Rab3a,
together with other molecules such as phosphory-
Discussion
lated NF, ubiquitin, and a-synuclein. In this study,
Axonal degeneration characterized by abundant similar observations were obtained in Papillon dogs
spheroids is a pathologic hallmark of canine NAD. with NAD.
Although abundant iron accumulation also is a Spheroids arise in a variety of circumstances or
characteristic feature of human NAD,24 it has never diseases, and their contents vary in each case.48
been described in canine NAD in either Papillon or Ultrastructually, they are mixture of NFs, tubulo-
Rottweiler dogs.8,11,12,32,43 Even in this study, iron vesicular structures, mitochondria, lysosomes, and
accumulation was not found in all dogs with NAD. membranous bodies.48 This study revealed that
Recently, at least 2 causative genes, PANK2 and NFs were one of the major components of the
PLA2G6, have been identified in human infantile spheroids in canine NAD. The accumulation of
NAD,6,23,30 although alteration of these genes in NFs is considered a rather common property of
canine NAD may not be responsible for the axonal spheroids in several CNS disorders47 and is
pathogenesis because of the difference in the not specific to canine NAD. Tau is a microtubule-
pathologic features. Because the spheroids in binding protein in normal neurons and glial cells,
canine NAD are commonly formed adjacent to which associates with transportation of cytoplas-
the neuronal cell bodies, axonal degeneration has mic proteins and cell organelles. Thus, the local
Downloaded from vet.sagepub.com by guest on March 1, 2015480 Nibe, Nakayama, and Uchida Vet Pathol 46:3, 2009
Fig. 6. Medulla oblongata; NAD, dog No. 3.
Many spheroids in the nucleus gracilis are intensely
positive for calretinin (left), while those in the nucleus
cuneatus exhibit weak (arrowhead) or no (arrow)
immunoreaction (right). Envision polymer method.
Counterstained by Mayer’s hematoxylin. Bar 5 50 mm.
accumulation of tau might indicate the abnormal-
ity of axonal transport. Hsp70, which is one of the
major heat shock proteins and which is expressed
under several stress conditions, protects cells from
various stresses and assists damage repairs.4,22
Fig. 4. Cerebellum; NAD, dog No. 2. A spheroid Ubiquitin exists in cells universally, participating
in the white matter is immunopositive for calbindin in protein modification and being involved in
(arrowhead). Morphologically intact Purkinje cells and various physiologic processes, such as protein
their processes also are positive for calbindin. Envision degradation, DNA repair, translational control,
polymer method. Counterstained by Mayer’s hematox-
ylin. Bar 5 50 mm.
and signal transfer.28,51 The accumulation of these
heat shock proteins within the axonal spheroids in
NAD might be a response directed against
Fig. 5. Medulla oblongata; NAD, dog No. 2.
Many spheroids immunopositive for parvalbumin are Fig. 7. Medulla oblongata; NAD, dog No. 2.
evident in the nucleus of the spinal tract of the Parvalbumin-positive spheroids are sometimes adjacent
trigeminal nerve (arrowhead). Envision polymer meth- to the neuronal cell body (arrowhead). Envision
od. Counterstained by Mayer’s hematoxylin. Bar 5 polymer method. Counterstained by Mayer’s hematox-
50 mm. ylin. Bar 5 50 mm.
Downloaded from vet.sagepub.com by guest on March 1, 2015Vet Pathol 46:3, 2009 Neuroaxonal Dystrophy in Papillon Dogs 481
accumulated proteins within the presynaptic area. the accumulation of a-synuclein in dystrophic
The phenomena also might indicate that the level axons has not been reported in human NAD, the
and/or nature of these deposits resulted from features might be specific for canine NAD.
exceeding the ability of degradation by the protea- With regard to calcium-binding proteins, a large
some system and the lysosomal system. Synapto- number of immunopositive spheroids were found
physin is an integral protein of the synaptic vesicle in the cerebellum, medulla oblongata, but there
membrane, interacting with other synaptic proteins were few spheroids in the cerebrum of dogs with
and participating in exocytosis.20,31 The accumula- NAD. Calcium-binding proteins are widely distrib-
tion of synaptophysin in the dystrophic axons of uted throughout the CNS, and cell-specific expres-
Papillon dogs with NAD may indicate the func- sion has been reported in cats,1 monkeys,3 and
tional obstruction of the synapse at the presynaptic rats,29 although in dogs, there is a report about the
portion. As compared with those positive for abnormal expression on calbindin and parvalbu-
synaptophysin, only a small number of spheroids min in brain tissue under several pathologic
were immunopositive for syntaxin-1 and SNAP25. conditions.44 Hof et al. reported the distribution
Syntaxin-1 is an anchor protein involved in of calcium-binding proteins, including parvalbu-
exocytosis, like SNAP-25, which is a plasma min, calbindin, and calretinin, in the neocortex and
membrane protein located at presynaptic nerve hippocampus of dogs.17,18 Physiologic distribution
terminals.46,50 These differences in location and of these proteins in other parts of canine CNS was
biologic function might reflect the spheroid immu- not been well elucidated. In our study, almost all
noreactivity seen in canine NAD. Synuclein family regions of the cerebrum showed weak and vague
has 3 types of proteins, including a-, b-, and c- immunoreactivity for all calcium-binding proteins
synuclein. This has a highly conserved alpha helical examined. However, in the cerebellum and brain
lipid-binding motif with similarity to the class-A2 stem, many immunopositive spheroids were pre-
lipid-binding domains of the exchangeable apoli- dominantly distributed. Because the phenomena
poproteins. The a- and b-synuclein are cytoplasmic may reflect simply the normal localization of the
proteins found predominantly and abundantly in proteins, further fundamental studies on these
the presynaptic nerve terminals in the brain, molecules in the CNS of dogs will be needed to
primarily near the vesicles. Although the functions elucidate the meaning or roles in canine NAD.
of a- and b-synuclein are not well elucidated, their In humans, dysfunction of the calcium channel
participation in synaptic plasticity has been pro- in CNS is quite important to many neurologic
posed. Abnormal axonal transport or metabolism diseases.21,25 Human SCD is a hereditary disease
of presynaptic portion is thought to be one of the characterized by atrophy of the spinal cord and
causes, but the mechanism remains unclear. cerebellum and is caused by expansion of a CAG
In humans, accumulation of phosphorylated repeat.34 Among the subtypes of human SCDs,
tau2,14,36,53 and synuclein19 proteins is one of the SCA6 is caused by an elongated CAG repeat of the
important factors of several neurodegenerative CACNA1A gene encoding the P/Q-type voltage-
diseases. The terms ‘‘tauopathies’’ and ‘‘synuclei- dependent calcium channel.13 Several murine mod-
nopathies’’ are general terms of diseases that are els with mutations at the a1 subunit of the gene
caused by the accumulation of phosphorylated tau such as groggy rat49,52 are thought to have some
or synuclein proteins. Alzheimer’s disease is one clinical and pathologic similarity with canine
example of a tauopathy.40 A mechanism of NAD. To discuss the relationship between the
neurotoxicity is suggested that hyperphosphory- calcium channel and canine NAD, further molec-
lated and aggregated tau impairs axonal trans- ular investigations, including gene analysis, are
port.27 Likewise, abnormal accumulation of a- necessary.
synuclein is a pathologic feature of ‘‘synucleino-
pathies.’’19,41 Human multiple systems atrophy References
(MSA)26,35 is one of the spinocerebellar degenera-
tions (SCDs), which is one form of ‘‘synucleino- 1 Anelli R, Heckman CJ: The calcium binding
proteins calbindin, parvalbumin, and calretinin have
pathy’’ and is commonly characterized by intracy- specific patterns of expression in the gray matter of
toplasmic accumulation of a-synuclein.19 Although cat spinal cord. J Neurocytol 34:369–385, 2005
intracytoplasmic accumulation of a-synuclein was 2 Arvanitakis Z, Witte RJ, Dickson DW, Tsuboi Y,
not found in the dog with CCA, the molecules Uitti RJ, Slowinski J, Hutton ML, Lin SC, Boeve
accumulated in the spheroids in all dogs with BF, Cheshire WP, Pooley RA, Liss JM, Caviness
NAD. Similar observations have been reported JN, Strongosky AJ, Wszolek ZK: Clinical-patholog-
previously in Rottweiler dogs with NAD.43 Because ic study of biomarkers in FTDP-17 (PPND family
Downloaded from vet.sagepub.com by guest on March 1, 2015482 Nibe, Nakayama, and Uchida Vet Pathol 46:3, 2009
with N279K tau mutation). Parkinsonism Relat 20 Jahn R, Schiebler W, Ouimet C, Greengard P: A
Disord 13:230–239, 2007 38,000-dalton membrane protein (p38) present in
3 Arvidsson U, Ulfhake B, Cullheim S, Ramı́rez V, synaptic vesicles. Proc Natl Acad Sci USA 82:
Shupliakov O, Hökfelt T: Distribution of calbindin 4137–4141, 1985
D28k-like immunoreactivity (LI) in the monkey 21 Jodice C, Mantuano E, Veneziano L, Trettel F,
ventral horn: do Renshaw cells contain calbindin Sabbadini G, Calandriello L, Francia A, Spadaro M,
D28k-LI? J Neurosci 12:718–728, 1992 Pierelli F, Salvi F, Ophoff RA, Frants RR, Frontali
4 Belay HT, Brown IR: Cell death and expression of M: Episodic ataxia type 2 (EA2) and spinocerebellar
heat-shock protein Hsc70 in the hyperthermic rat ataxia type 6 (SCA6) due to CAG repeat expansion
brain. J Neurochem 1:116–119, 2006 in the CACNA1A gene on chromosome 19p. Hum
5 Berry ML, Blas-Machado U: Cerebellar abiotrophy Mol Genet 6:1973–1978, 1997
in a miniature schnauzer. Can Vet J 44:657–659, 22 Kelly S, Yenari MA: Neuroprotection: heat shock
2003 proteins. Curr Med Res Opin 2:55–60, 2002
6 Biancheri R, Rossi A, Alpigiani G, Filocamo M, 23 Khateeb S, Flusser H, Ofir R, Shelef I, Narkis G,
Gandolfo C, Lorini R, Minetti C: Cerebellar atrophy Vardi G, Shorer Z, Levy R, Galil A, Elbedour K,
without cerebellar cortex hyperintensity in infantile Birk OS: PLA2G6 mutation underlies infantile
neuroaxonal dystrophy (INAD) due to PLA2G6 neuroaxonal dystrophy. Am J Hum Genet 79:
mutation. Eur J Paediatr Neurol 11:175–177, 2007 942–948, 2006
7 Bildfell RJ, Mitchell SK, deLahunta A: Cerebellar 24 Koeppen AH, Dickson AC: Iron in the Hallervor-
cortical degeneration in a Labrador retriever. Can den-Spatz syndrome. Pediatr Neurol 25:148–155,
Vet J 36:570–572, 1995 2001
8 Chrisman CL, Cork LC, Gamble DA: Neuroaxonal 25 Koh SH, Kim HT, Kim SH, Lee GY, Kim J, Kim
dystrophy of Rottweiler dogs. J Am Vet Med Assoc MH: Spinocerebellar ataxia type 6 and episodic
184:464–467, 1984 ataxia type 2 in a Korean family. J Korean Med Sci
9 Clark RG, Hartley WJ, Burgess GS, Cameron JS, 16:809–13, 2001
Mitchell G: Suspected inherited cerebellar neuroax- 26 Kulczycki J: Multiple system atrophy. Folia Neuro-
onal dystrophy in collie sheep dogs. NZ Vet J pathol 35:209–213, 1997
30:102–103, 1982
27 Lewis J, McGowan E, Rockwood J, Melrose H,
10 deLahunta A: Abiotrophy in domestic animals:
Nacharaju P, Van Slegtenhorst M, Gwinn-Hardy K,
review. Can J Vet Res 54:65–76, 1990
Paul Murphy M, Baker M, Yu X, Duff K, Hardy J,
11 Evans MG, Mullaney TP, Lowrie CT: Neuroaxonal
Corral A, Lin WL, Yen SH, Dickson DW, Davies P,
dystrophy in a rottweiler pup. J Am Vet Med Assoc
Hutton M: Neurofibrillary tangles, amyotrophy and
192:1560–1562, 1988
progressive motor disturbance in mice expressing
12 Franklin RJ, Jeffery ND, Ramsey IK: Neuroaxonal
mutant (P301L) tau protein. Nat Genet 25:402–405,
dystrophy in a litter of papillon pups. J Small Anim
2000
Pract 36:441–444, 1995
13 Gazulla J, Tintore MA: The P/Q-type voltage- 28 Malik I, Turk J, Mancuso DJ, Montier L, Wohlt-
dependent calcium channel as pharmacological mann M, Wozniak DF, Schmidt RE, Gross RW,
target in spinocerebellar ataxia type 6: gabapentin Kotzbauer PT: Disrupted membrane homeostasis
and pregabalin may be of therapeutic benefit. Med and accumulation of ubiquitinated proteins in a
Hypotheses 68:131–136, 2007 mouse model of infantile neuroaxonal dystrophy
14 Goedert M: Tau protein and neurodegeneration. caused by PLA2G6 mutations. Am J Pathol
Semin Cell Dev Biol 15:45–49, 2004 172:406–416, 2008
15 Gordon N: Pantothenate kinase-associated neuro- 29 Menétrey D, De Pommery J, Thomasset M, Baim-
degeneration (Hallervorden-Spatz syndrome). Eur J bridge KG: Calbindin-D28K (CaBP28k)-like immu-
Paediatr Neurol 6:243–247, 2002 noreactivity in ascending projections. Eur J Neurosci
16 Hayflick SJ: Pantothenate kinase-associated neuro- 4:70–76, 1992
degeneration (formerly Hallervorden-Spatz syn- 30 Morgan NV, Westaway SK, Morton JE, Gregory A,
drome). J Neurol Sci 207:106–107, 2003 Gissen P, Sonek S, Cangul H, Coryell J, Canham N,
17 Hof PR, Bogaert YE, Rosenthal RE, Fiskum G: Nardocci N, Zorzi G, Pasha S, Rodriguez D,
Distribution of neuronal populations containing Desguerre I, Mubaidin A, Bertini E, Trembath
neurofilament protein and calcium-binding proteins RC, Simonati A, Schanen C, Johnson CA, Levinson
in the canine neocortex: regional analysis and cell B, Woods CG, Wilmot B, Kramer P, Gitschier J,
typology. J Chem Neuroanat 11:81–98, 1996 Maher ER, Hayflick SJ: PLA2G6, encoding a
18 Hof PR, Rosenthal RE, Fiskum G: Distribution of phospholipase A2, is mutated in neurodegenerative
neurofilament protein and calcium-binding proteins disorders with high brain iron. Nat Genet
parvalbumin, calbindin, and calretinin in the canine 38:752–754, 2006
hippocampus. J Chem Neuroanat 11:1–12, 1996 31 Navone F, Jahn R, Di Gioia G, Stukenbrok H,
19 Iwatsubo T: Pathological biochemistry of alpha- Greengard P, De Camilli P: Protein p38: an integral
synucleinopathy. Neuropathology 27:474–478, 2007 membrane protein specific for small vesicles of
Downloaded from vet.sagepub.com by guest on March 1, 2015Vet Pathol 46:3, 2009 Neuroaxonal Dystrophy in Papillon Dogs 483
neurons and neuroendocrine cells. J Cell Biol proteins in dystrophic axons. Acta Neuropathol
103:2511–2527, 1986 (Berl.) 102:501–504, 2001
32 Nibe K, Kita C, Morozumi M, Awamura Y, 44 Sisó S, Tort S, Aparici C, Pérez L, Vidal E,
Tamura S, Okuno S, Kobayashi T, Uchida K: Pumarola M: Abnormal neuronal expression of the
Clinicopathological features of canine neuroaxonal calcium-binding proteins, parvalbumin and calbin-
dystrophy and cerebellar cortical abiotrophy in din D-28k, in aged dogs. J Comp Pathol 128:9–14,
Papillon and Papillon-related dogs. J Vet Med Sci 2003
69:1047–1052, 2007 45 Speciale J, deLahunta A: Cerebellar degeneration in
33 Perille AL, Baer K, Joseph RJ, Carrillo JM, Averill DR: a mature Staffordshire terrier. J Am Anim Hosp
Postnatal cerebellar cortical degeneration in Labrador Assoc 39:459–462, 2003
Retriever puppies. Can Vet J 32:619–621, 1991 46 Steegmaier M, Yang B, Yoo JS, Huang B, Shen M,
34 Pulst SM, Santos N, Wang D, Yang H, Huynh D, Yu S, Luo Y, Scheller RH: Three novel proteins of
Velazquez L, Figueroa KP: Spinocerebellar ataxia the syntaxin/SNAP-25 family. J Biol Chem 273:
type 2: polyQ repeat variation in the CACNA1A 34171–34179, 1998
calcium channel modifies age of onset. Brain 47 Summers BA, Cummings JF, deLahunta A: Degen-
128:2297–2303, 2005 erative disease of the central nervous system. In:
35 Quinn N, Wenning G: Multiple system atrophy. Veterinary Neuropathology, ed. Summers BA, Cum-
Curr Opin Neurol 8:323–326, 1995 mings JF, and deLahunta A, pp. 208–350. Mosby-
36 Robert M, Mathuranath PS: Tau and tauopathies. Year Book, St. Louis, MO, 1995
Neurol India 55:11–16, 2007
48 Summers BA, Cummings JF, deLahunta A: Princi-
37 Résibois A, Poncelet L: Purkinje cell neuroaxonal
ples of neuropathology. In: Veterinary Neuropathol-
dystrophy similar to nervous mutant mice phenotype
ogy, ed. Summers BA, Cummings JF, and deLa-
in two sibling kittens. Acta Neuropathol 107:
hunta A, pp. 1–67. Mosby-Year Book, St. Louis,
553–558, 2004
MO, 1995
38 Sacre BJ, Cummings JF, deLahunta A: Neuroaxonal
dystrophy in a Jack Russell terrier pup resembling 49 Takeuchi IK, Aoki E, Takeuchi YK: Age-related
human infantile neuroaxonal dystrophy. Cornell Vet and region-specific increase in number of concentric
83:133–142, 1993 lamellar bodies in axon terminals and presynaptic
39 Sandy JR, Slocombe RE, Mitten RW, Jedwab D: axons in central nervous system of groggy mutant
Cerebellar abiotrophy in a family of Border Collie rat. Acta Neuropathol (Berl) 87:628–634, 1994
dogs. Vet Pathol 39:736–738, 2002 50 Teng FY, Wang Y, Tang BL: The syntaxins.
40 Schindowski K, Belarbi K, Buée L: Neurotrophic Genome Biol 2:1–7, 2001
factors in Alzheimer’s disease: role of axonal 51 Thompson SJ, Loftus LT, Ashley MD, Meller R:
transport. Genes Brain Behav 7:43–56, 2008 Ubiquitin–proteasome system as a modulator of cell
41 Sikorska B, Papierz W, Preusser M, Liberski PP, fate. Curr Opin Pharmacol 8:90–95, 2008
Budka H: Synucleinopathy with features of both 52 Tokuda S, Kuramoto T, Tanaka K, Kaneko S,
multiple system atrophy and dementia with Lewy Takeuchi IK, Sasa M, Serikawa T: The ataxic
bodies. Neuropathol Appl Neurobiol 33:126–129, groggy rat has a missense mutation in the P/Q-type
2007 voltage-gated Ca2+ channel alpha1A subunit gene
42 Sisó S, Ferrer I, Pumarola M: Abnormal synaptic and exhibits absence seizures. Brain Res 1133:
protein expression in two Arabian horses with 168–177, 2007
equine degenerative myeloencephalopathy. Vet J 53 Zarranz JJ, Gómez-Esteban JC, Atarés B, Lezcano
166:238–243, 2003 E, Forcadas M: Tau-predominant-associated pa-
43 Sisó S, Ferrer I, Pumarola M: Juvenile neuroaxonal thology in a sporadic late-onset Hallervorden-Spatz
dystrophy in a Rottweiler: accumulation of synaptic syndrome. Mov Disord 21:107–111, 2006
Request reprints from Dr. K. Uchida, Department of Veterinary Pathology, Graduate School of Agricultural and
Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan). E-mail:
auchidak@mail.ecc.u-tokyo.ac.jp.
Downloaded from vet.sagepub.com by guest on March 1, 2015You can also read
