NEURONAL INTERMEDIATE FILAMENT ACCUMULATION
IN THE CENTRAL NERVOUS SYSTEM NEURONS OF NUDE RATS (F344/NJcl-rnu/rnu)
Kazufumi KAWASAKO, Masashi Iida, Akio Sekiya, Ryosuke Kobayashi, Naoaki Yamada, Yuki Tomonari, Hiroko Kokoshima, Junko Sato, Yumi Wako, Tetsuro Kurotaki, Takuya Doi, Minoru Tsuchitani, Takeshi Kanno
LSI Medicine Corporation, Ibaraki, Japan

Introduction
Neuronal intermediate filaments are cytoskeletal proteins including
neurofilament (NF) triplets of light, medium and heavy subunits and αinternexin, which are classified into type IV intermediate filaments(IFs).2-4 Accumulation of NF in the neurons has been reported inseveral neurodegenerative human diseases including neuronalintermediate filament inclusion disease (NIFID).1-3 NIFID is one of thefrontotemporal lobar degeneration-fused in sarcoma protein (FTLDFUS),1 and a rare neuropathological human disorder of early onsetwith variable clinical signs including frontotemporal dementia,
pyramidal and extrapyramidal signs.2 Grossly, there is focal atrophy ofthe frontal lobes, and to a less degree, temporal and parietal lobes,
and the caudate is frequently affected.2 Microscopically, all of the typeIV neuronal IFs including α-internexin accumulate excessively as
neuronal cytoplasmic inclusion, which is present throughout the brain
including the spinal cord.2 In addition, neuronal loss and gliosis arealso common findings.2 α-Internexin accumulation is the pathologicalhallmark of human NIFID, which is variably ubiquitinated, but do notcontain tau.1,2 A transgenic mouse model overexpressing rat αinternexin (Tg-mice with rat α-internexin) of human NIFID has beenfound to demonstrate abnormal neuronal IF accumulation and motor
coordination deficits.3 To our knowledge, this is the first description on
spontaneous α-internexin accumulation in the CNS neurons in animals.

Discussion

Results

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Materials and Methods
Nude rat (F344/NJcl-rnu/rnu) were purchased from CLEA Japan,Inc. (Tokyo, Japan). 23 animals aged eight to 18 weeks which showed
no apparent clinical abnormalities and received no prior treatments
were necropsied. Gross examination revealed no abnormalities in any
organ from any animal. Immunohistochemistry (IHC) was performed
using the following primary antibodies.
mouse monoclonal anti-rat α-internexin (GeneTex, Inc., Irvine, California, USA)mouse monoclonal anti-human NFs (Dako, Burlingame, California, USA)mouse monoclonal anti-bovine ubiquitin (LifeSpan Biosciences, Inc., Seattle, Washington, USA)mouse monoclonal anti-human tau (AnaSpec, Inc., Fremont, California, USA)

Figure 1. Thalamus. (a) Pale eosinophilic material accumulation in the neurons. HE. (b) Eosinophilic body (EB) in the degenerative neurons. HE. (c) Central chromatolysis-like change. Nissl. (d) PAS. (e)Schmorl. (f) IHC for ubiquitin. (g) IHC for tau. Figure 2. External pyramidal layer in the cerebral cortex. (a) Swollen dendrites. HE. (b). Higher magnification. Figure 3. Purkinje cell layer. (a) Swollendendrites. HE. (b). Higher magnification. Figure 4. Thalamus. (a) IHC for α-internexin. (b) IHC for NFs. Figure 5. External pyramidal layer in the cerebral cortex. (a) IHC for α-internexin. (b) IHC for NFs.
Figure 6. Purkinje cell layer. (a) IHC for α-internexin. (b) IHC for NFs. Figure 7. Purkinje cell. Filamentous structure in the degenerative neurons. TEM. Figure 8. Purkinje cell. Aggregates of secondary
lysosomes in the neurites. TEM.
These results revealed that the pale eosinophilic materials in the degenerative neurons were accumulations of the neuronal IFs including α-internexin and NFs, and that EB was an aggregate ofubiquitinated materials with lipofuscin-like nature. There were no other histopathological abnormalities including neuronal necrosis or loss, gliosis or microglial proliferation in any area, or torpedo in
Purkinje cell axon. Neuronal changes were observed in three animals out of 23 animals examined (13 %). These three animals were neither littermates nor from the same parents.

α-Internexin is expressed earlier and more abundantly than NFs
throughout the developing CNS neurons.4 As development continues,
the expression levels of α-internexin decrease, whereas those of NFs
α-Internexin is expressed at relatively low levels inincrease.4comparison to NFs, and selective expression is observed in thecerebellar granule cells and the cerebral layer II neurons in humanadult brain.2,4 IHC for α-internexin in the unaffected mature nude rats
revealed selective expression only in the cerebellar granule cells.
Therefore, these results indicated that neuronal α-internexin
accumulation excluding the cerebellar granule cells in mature nude rats
could have pathological significance in human NIFID as well.
In Tg-mice with rat α-internexin, Purkinje cells are most affected byα-internexin overexpression.3 Fusiform swellings of the axons of
Purkinje cells, called torpedoes, are observed in the granular layer in
In contrast, in nude rats, α-internexin
the cerebellar cortex.3
accumulation in the dendrites of Purkinje cells and the pyramidal cells
in the cerebral cortex were observed toward the molecular layer in thecerebellar cortex and the more superficial layer of the cerebral cortex,respectively.
It is suggested that the pattern of α-internexin
accumulation in the dendrites, not in the axons, could be an identifying
feature in nude rats; however, the reason remains unclear.
A motor coordination test using a rotarod demonstrated that Tg-micewith rat α-internexin have a deficit in motor coordination as early as
three month of age.3 It remains unclear whether nude rats without
apparent clinical abnormalities such as tremors or any other overt
symptoms of disorders had presented poor motor coordination. Inaddition, there were no gross abnormalities including focal atrophy ofthe brain or histopathological abnormalities including neuronal loss or
microglial proliferation in any area of the affected animals. Therefore,
the nude rats of this study could not be diagnosed as rat NIFID. On theother hand, α-internexin is a major component of the pathologicalhallmark, and neuronal α-internexin accumulation demonstrates
diagnostic specificity for human NIFID.2 Therefore, it was suggested
that nude rats (F344/NJcl-rnu/rnu) can be used as a spontaneous
animal model for human NIFID.
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