Kinesin and cytoplasmic dynein in spinal spheroids with motor neuron disease

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Abstract

Kinesin and cytoplasmic dynein are two major molecular motors responsible for fast axonal transport. As visualized by immunohistochemistry with monoclonal antibodies, both motors were found to be distributed throughout the cell bodies, dendrites and axons of motor neurons in normal human spinal cords. Large axonal swellings, spheroids, in the spinal cords of patients with motor neuron disease showed massive accumulation of kinesin co-localized with highly phosphorylated neurofilaments. Of 114 spheroids in five spinal cords, 87% were stained heavily with the three anti-kinesin antibodies used in this study. Cytoplasmic dynein was scarce or absent in most of the spheroids. These findings suggest that kinesin selectively accumulates in the spheroids of motor neuron axons, causing disturbance of the machinery for anterograde fast axonal transport in motor neuron disease.

Introduction

The most common human motor neuron disease (MND), amyotrophic lateral sclerosis (ALS), shows a line of evidence of disturbed axonal transport of neurofilament proteins (NFs). In ALS, swollen axons are often seen in the anterior horn of spinal cords, where proximal axons of motor neurons are located. This feature closely resembles the chronic phase of experimentally disturbed axonal transport [19]. Large axonal swellings, spheroids, are characteristic for ALS [3]in contrast to small swellings thought to result from aging [7]. Electron microscopy and immunohistochemistry have shown that the spheroids contain numerous neurofilaments 3, 12, 20that are highly phosphorylated 22, 25. The excessive phosphorylation of NFs in the spheroids has been suggested to result from disturbed transport of NFs.

Transgenic mice experiments overexpressing wild-type NFs indicate that accumulated neurofilaments in cell bodies and proximal axons cause MND-associated abnormalities such as swellings of perikarya and proximal axons 6, 28. Transgenic mice with a modest amount of mouse light-subunit of NF (NF-L) having a point mutation in the tail region showed segregation of neurofilaments from microtubules and massive accumulation of neurofilaments selectively involving motor neurons [15]. In late stages, transgenic mice overexpressing wild-type human heavy-subunit of NF (NF-H) showed a general disturbance of slow and fast axonal transport causing death of spinal motor neurons [5]. Deletions in NF-H have been found in sporadic ALS patients [9].

Microtubule-dependent organelle transport provides fast axonal transport in axons. Microtubules consist of tubulin and microtubule-associated proteins. Kinesin and cytoplasmic dynein are two major molecular motors that power fast axonal transport of organelles in anterograde and retrograde directions, respectively (see review; 23, 27. Breuer [2]used video-enhanced differential interference microscopy to track particle movements in median nerve axons from ALS patients and found disturbed retrograde axonal transport, although electron microscopy has shown only a small membranous organelle accumulation in spinal spheroids [21].

We have examined the distribution of kinesin and cytoplasmic dynein in spinal cords of patients with MND and found that kinesin accumulated selectively in spheroids, co-localizing with highly phosphorylated neurofilaments.

Section snippets

Spinal cords

Five spinal cords of patients with MND and three control spinal cords with non-neurologic disease (Table 1) were obtained 1–6 h after death at autopsy and quickly frozen in dry ice-cooled isopentane or liquid nitrogen without fixation.

Monoclonal antibodies against molecular motors

Anti-kinesin monoclonal antibodies (mAbs) were raised against purified kinesin from chick embryo brain as previously reported [26].

For cytoplasmic dynein, three series of mAbs were employed in this study. The first series was raised against cytoplasmic dynein from

Specificity of mAbs against molecular motors

Anti-chick kinesin heavy-chain mAbs, CKHC 9, CKHC 3, and CKHC 6, were reacted specifically with human kinesin heavy-chain in normal spinal cord (Fig. 1, lanes a, b and c). Anti-cytoplasmic dynein mAbs, 70.2, BDIC 7, and BLD 14.1, were reacted specifically with the intermediate chain of human cytoplasmic dynein (Fig. 1, lanes d, e and f). Purified kinesin and cytoplasmic dynein from human, bovine, rabbit and chick were reacted specifically with these antibodies (data not shown).

Distribution of molecular motors in normal spinal cords

Three

Discussion

We examined the distribution of kinesin and cytoplasmic dynein in normal human spinal cord, where molecular motors were detected throughout neuronal cell bodies, dendrites and axons. Two types of kinesin heavy chains, brain-specific and ubiquitous ones, have been reported in human [17]and in mouse [1]. The staining pattern of mAbs against kinesin in this study is compatible with the distribution of the ubiquitous type of kinesin heavy chain [17]. Cytoplasmic dynein intermediate chains have been

Acknowledgements

We are grateful to Jan Lockman in Duke University and Yuki Watanabe in Akita University for preparations of monoclonal antibodies and Shinobu Fujita in the Mitsubishi-Kagaku Institute of Life Science for an anti-NF antibody (NF-H P+). We are also grateful to the Bryan Alzheimer's Disease Research Center of Duke University Medical School for providing control spinal cords. A part of this work was supported by grants 06670638 and 08670691 (for IT) from the Ministry of Education, Science and

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