Immunohistochemical Features of Dystrophic Axons in Papillon Dogs with Neuroaxonal Dystrophy

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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-
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Vet 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

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476                                           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.

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Vet 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,

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478                                               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.

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Vet 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

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480                                          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.

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Vet 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-
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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.

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