Axonal dystrophy of dorsal root ganglion sensory neurons in a mouse model of Niemann–Pick disease type C
Introduction
Niemann–Pick disease type C (NP-C) is an autosomal recessive disorder of lipid storage metabolism presenting with a variety of neurological symptoms including cerebellar ataxia, dementia, and supranuclear ophthalmoplegia (Pentchev et al., 1995). The defective gene responsible for this disorder is NP-C-1, which causes a disturbance in the trafficking of free cholesterol from lysosome or endosome to other membrane sites Cruz et al., 2000, Davies et al., 2000, Neufield et al., 1999. In the monkey central nervous system (CNS), the NP-C-1 protein has been shown to reside in astrocytic processes adjacent to neural processes at synapses, suggesting its role in regional homeostasis (Patel et al., 1999). However, the cellular mechanisms of neurodegeneration in NP-C are largely unknown.
Neuropathologically, the widespread appearance of swollen neurites (spheroids) and the accumulation of intraneuronal cytoplasmic inclusions with neuronal loss Higashi et al., 1991, Vanier and Suzuki, 1998 are the most characteristic histologic features of the NP-C brain. The distribution of axonal spheroids is not topographically correlated with the sites of neuronal loss that preferentially involve cerebellar Purkinje cells, VPL–VPM thalamic neurons, and GABAergic interneurons Higashi et al., 1993, March et al., 1997, Tanaka et al., 1988, Yamada et al., 2001. On the other hand, recent studies have demonstrated that the degeneration of axons precedes that of neuronal cell body, suggesting the occurrence of dying back processes (Ong et al., 2001). This raises the possibility that spheroid formation may represent a unique form of distal axonopathy in which the terminal portion of axons and presynaptic axon terminals are preferentially affected, which could result in synaptic dysplasia and differential vulnerability of axons depending on their length.
To study on the pathogenetic role of spheroid formation in neurodegeneration in NP-C, we focused on the sites of termination of central processes of dorsal root ganglion (DRG) neurons. The gracile nuclei of the medulla oblongata, one of the principal sites of termination of lengthy myelinated axons derived from large-sized lumbosacral DRG neurons, are known for the development of neuroaxonal dystrophy (NAD) (Jellinger, 1973). NAD, unlike nonspecific axonal swelling, is characterized by unique ultrastructural features such as accumulation of smooth membrane, tubulo-membranous aggregates, and a few neurofilaments (Seitelberger, 1986), and develops as a function of normal aging in rodents as well as humans (Fujisawa and Shiraki, 1978). On the other hand, the cuneate nuclei with shorter length of afferents compared with the gracile are far less prone to develop neuroaxonal dystrophy than the gracile despite both nuclei, collectively known as dorsal column nuclei, having the same physiological functions subserving sensory transmission of tactile and proprioceptive sensations from the upper (cuneate) and the lower (gracile) extremities to the sensory thalamus. In the spinal cord, the substantia gelatinosa of the spinal dorsal horn (lamina II) is the predominant site of termination of small-sized DRG neurons, largely subserving pain and temperature sensation. Unlike dorsal column nuclei, which receive exclusively myelinated fiber afferents, the substantia gelatinosa receives mostly unmyelinated afferents Giuffrida and Rustioni, 1992, Ralston and Ralston, 1979, Rustioni and Sotelo, 1974. Thus, given this inherent propensity for DRG neurons to develop axonal dystrophy and their marked variance in the length and size of central processes, the study of projections of DRG neurons should provide an important clue for the understanding of the mechanism of neurodegeneration in NP-C.
In this study, we employed electron microscopy and morphometric techniques to define structural alterations of the central processes of DRG neurons of Balb/c npcnih, an authentic murine model of human NP-C with identical genetic and chemical defects. We found that the central processes of DRG neurons of the NP-C model mouse are affected in a length-dependent manner; the changes being by far the most dramatic in the gracile nuclei compared to the cuneate and the dorsal horn on the spinal cord, and they were associated with axonal loss and a strikingly early development of axonal dystrophy identical to that seen in the aging gracile nucleus.
Section snippets
Animals
A colony of Balb/c npcnih mutant mice has been maintained and the genotype of each mouse was determined by PCR using genomic DNA isolated from a small piece of the tail as described (Loftus et al., 1997). Thirteen affected mice of 3, 6, and 9 weeks of age (n ≥ 4 each) and 15 age-matched control mice (n ≥ 4) were used in this study. Moreover, to access the effect of aging of the gracile NAD in Balb/c mice, 4- and 5 (n = 1 each)-, 10 (n = 2)-, and 16-month-old mice (n = 2) were also studied.
Light and electron microscopy
Light and electron microscopy of the gracile nuclei of normal aging mice
In the control gracile nuclei of 3, 6, and 9 weeks (n = 4–5) and 5 months (n = 2) of age Balb/c mice, axonal spheroids were not identified. At 11 and 14 months (n = 2 each), the appearance of spheroids was a constant feature in the gracile nucleus, a finding that was more evident at 14 months of age. The dystrophic axons were spherical in shape and exhibited a granular, homogenous, or mixed appearance (Fig. 1a). Ultrastructurally, dystrophic axonal swellings revealed an admixture of
Discussion
It has been well established that the development of axonal swellings (spheroids) is one of the cardinal histopathological features of brains of NP-C involving brains of both human and mice Higashi et al., 1991, Vanier and Suzuki, 1998 along with neuronal storage and neuronal loss, all of which eventually affect the entire CNS. The cerebellum and VPL–VPM thalamus are among the earliest sites of involvement in NP-C, although the underlying mechanism of neurodegeneration is unknown. Recently, it
Acknowledgements
We thank Mr. N. Takeda, Department of Clinical Laboratory research, National Chushin-Matsumoto Hospital, and Ms. K. Suzuki, Research Center for Instrumental Analysis, Shinshu University School of Medicine, for their technical assistance and Prof. S. Ikeda, Department of Medicine, Shinshu University School of Medicine, for his support in the electron microscopic study. We also thank Prof. R.E. Schmidt, Department of Pathology (Neuropathology), Washington University School of Medicine, for his
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