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Neurofilaments and orthograde transport are reduced in ventral root axons of transgenic mice that express human SOD1 with a G93A mutation.

Zhang B, Tu P, Abtahian F, Trojanowski JQ, Lee VM - J. Cell Biol. (1997)

Bottom Line: Mice engineered to express a transgene encoding a human Cu/Zn superoxide dismutase (SOD1) with a Gly93 --> Ala (G93A) mutation found in patients who succumb to familial amyotrophic lateral sclerosis (FALS) develop a rapidly progressive and fatal motor neuron disease (MND) similar to amyotrophic lateral sclerosis (ALS).Hallmark ALS lesions such as fragmentation of the Golgi apparatus and neurofilament (NF)-rich inclusions in surviving spinal cord motor neurons as well as the selective degeneration of this population of neurons were also observed in these animals.Quantitative Western blot analyses showed a progressive decrease in the level of NF proteins in the L5 ventral roots of G93A mice and a concomitant reduction in axon caliber with the onset of motor weakness.

View Article: PubMed Central - PubMed

Affiliation: The Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Mice engineered to express a transgene encoding a human Cu/Zn superoxide dismutase (SOD1) with a Gly93 --> Ala (G93A) mutation found in patients who succumb to familial amyotrophic lateral sclerosis (FALS) develop a rapidly progressive and fatal motor neuron disease (MND) similar to amyotrophic lateral sclerosis (ALS). Hallmark ALS lesions such as fragmentation of the Golgi apparatus and neurofilament (NF)-rich inclusions in surviving spinal cord motor neurons as well as the selective degeneration of this population of neurons were also observed in these animals. Since the mechanism whereby mutations in SOD1 lead to MND remains enigmatic, we asked whether NF inclusions in motor neurons compromise axonal transport during the onset and progression of MND in a line of mice that contained approximately 30% fewer copies of the transgene than the original G93A (Gurney et al., 1994). The onset of MND was delayed in these mice compared to the original G93A mice, but they developed the same neuropathologic abnormalities seen in the original G93A mice, albeit at a later time point with fewer vacuoles and more NF inclusions. Quantitative Western blot analyses showed a progressive decrease in the level of NF proteins in the L5 ventral roots of G93A mice and a concomitant reduction in axon caliber with the onset of motor weakness. By approximately 200 d, both fast and slow axonal transports were impaired in the ventral roots of these mice coincidental with the appearance of NF inclusions and vacuoles in the axons and perikarya of vulnerable motor neurons. This is the first demonstration of impaired axonal transport in a mouse model of ALS, and we infer that similar impairments occur in authentic ALS. Based on the temporal correlation of these impairments with the onset of motor weakness and the appearance of NF inclusions and vacuoles in vulnerable motor neurons, the latter lesions may be the proximal cause of motor neuron dysfunction and degeneration in the G93A mice and in FALS patients with SOD1 mutations.

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Schematic diagram  showing the microdissection  of L5 ventral roots and the  microinjection of [35S]methionine into the ventral  horn of the mouse spinal  cord in the experimental paradigm used here to analyze  axonal transport.
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Figure 1: Schematic diagram showing the microdissection of L5 ventral roots and the microinjection of [35S]methionine into the ventral horn of the mouse spinal cord in the experimental paradigm used here to analyze axonal transport.

Mentions: The ventral roots of 24 G93A SOD1 and N1029 transgenic mice as well as age-matched control mice of four different ages (150, 180, 200, and 230 d, n = 3/age group) were harvested after being lethally anesthetized as described above. The L5 segment of the spinal cord was identified after laminectomy of the T13 to L6 vertebral bodies. It was then harvested and the attached ventral root was cut into five consecutive 2-mm-long segments for Western blot analysis (Fig. 1). Each segment was individually homogenized in 50 μl of BUST buffer (0.5% SDS, 8 M urea, 2% β-mercaptoethanol, 0.01% proteinase inhibitor cocktail, and 0.1 M Tris HCl, pH 6.8) as described earlier (Cole et al., 1994; Tu et al., 1995). The solubilized samples were centrifuged in an ultracentrifuge (TL-100; Beckman Instruments, Inc., Fullerton, CA) for 30 min at 25°C, and 8 μl of supernatant from each sample (containing ∼5 μg of protein) was loaded into individual lanes of a 6 or 7.5% polyacrylamide gel and separated by SDS-PAGE. Proteins were then transferred to nitrocellulose membranes for quantitative Western blot analysis as previously described (Cole et al., 1994; Tu et al., 1995).


Neurofilaments and orthograde transport are reduced in ventral root axons of transgenic mice that express human SOD1 with a G93A mutation.

Zhang B, Tu P, Abtahian F, Trojanowski JQ, Lee VM - J. Cell Biol. (1997)

Schematic diagram  showing the microdissection  of L5 ventral roots and the  microinjection of [35S]methionine into the ventral  horn of the mouse spinal  cord in the experimental paradigm used here to analyze  axonal transport.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140205&req=5

Figure 1: Schematic diagram showing the microdissection of L5 ventral roots and the microinjection of [35S]methionine into the ventral horn of the mouse spinal cord in the experimental paradigm used here to analyze axonal transport.
Mentions: The ventral roots of 24 G93A SOD1 and N1029 transgenic mice as well as age-matched control mice of four different ages (150, 180, 200, and 230 d, n = 3/age group) were harvested after being lethally anesthetized as described above. The L5 segment of the spinal cord was identified after laminectomy of the T13 to L6 vertebral bodies. It was then harvested and the attached ventral root was cut into five consecutive 2-mm-long segments for Western blot analysis (Fig. 1). Each segment was individually homogenized in 50 μl of BUST buffer (0.5% SDS, 8 M urea, 2% β-mercaptoethanol, 0.01% proteinase inhibitor cocktail, and 0.1 M Tris HCl, pH 6.8) as described earlier (Cole et al., 1994; Tu et al., 1995). The solubilized samples were centrifuged in an ultracentrifuge (TL-100; Beckman Instruments, Inc., Fullerton, CA) for 30 min at 25°C, and 8 μl of supernatant from each sample (containing ∼5 μg of protein) was loaded into individual lanes of a 6 or 7.5% polyacrylamide gel and separated by SDS-PAGE. Proteins were then transferred to nitrocellulose membranes for quantitative Western blot analysis as previously described (Cole et al., 1994; Tu et al., 1995).

Bottom Line: Mice engineered to express a transgene encoding a human Cu/Zn superoxide dismutase (SOD1) with a Gly93 --> Ala (G93A) mutation found in patients who succumb to familial amyotrophic lateral sclerosis (FALS) develop a rapidly progressive and fatal motor neuron disease (MND) similar to amyotrophic lateral sclerosis (ALS).Hallmark ALS lesions such as fragmentation of the Golgi apparatus and neurofilament (NF)-rich inclusions in surviving spinal cord motor neurons as well as the selective degeneration of this population of neurons were also observed in these animals.Quantitative Western blot analyses showed a progressive decrease in the level of NF proteins in the L5 ventral roots of G93A mice and a concomitant reduction in axon caliber with the onset of motor weakness.

View Article: PubMed Central - PubMed

Affiliation: The Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Mice engineered to express a transgene encoding a human Cu/Zn superoxide dismutase (SOD1) with a Gly93 --> Ala (G93A) mutation found in patients who succumb to familial amyotrophic lateral sclerosis (FALS) develop a rapidly progressive and fatal motor neuron disease (MND) similar to amyotrophic lateral sclerosis (ALS). Hallmark ALS lesions such as fragmentation of the Golgi apparatus and neurofilament (NF)-rich inclusions in surviving spinal cord motor neurons as well as the selective degeneration of this population of neurons were also observed in these animals. Since the mechanism whereby mutations in SOD1 lead to MND remains enigmatic, we asked whether NF inclusions in motor neurons compromise axonal transport during the onset and progression of MND in a line of mice that contained approximately 30% fewer copies of the transgene than the original G93A (Gurney et al., 1994). The onset of MND was delayed in these mice compared to the original G93A mice, but they developed the same neuropathologic abnormalities seen in the original G93A mice, albeit at a later time point with fewer vacuoles and more NF inclusions. Quantitative Western blot analyses showed a progressive decrease in the level of NF proteins in the L5 ventral roots of G93A mice and a concomitant reduction in axon caliber with the onset of motor weakness. By approximately 200 d, both fast and slow axonal transports were impaired in the ventral roots of these mice coincidental with the appearance of NF inclusions and vacuoles in the axons and perikarya of vulnerable motor neurons. This is the first demonstration of impaired axonal transport in a mouse model of ALS, and we infer that similar impairments occur in authentic ALS. Based on the temporal correlation of these impairments with the onset of motor weakness and the appearance of NF inclusions and vacuoles in vulnerable motor neurons, the latter lesions may be the proximal cause of motor neuron dysfunction and degeneration in the G93A mice and in FALS patients with SOD1 mutations.

Show MeSH
Related in: MedlinePlus