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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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SDS-PAGE shows a progressive retardation in fast  transport in the L5 ventral roots of the spinal cord in the G93A  transgenic mice compared to the control (CTR) mice. 12 G93A  and N1029 transgenic mice as well as age-matched control mice  of two different ages (150 and 200 d, n = 3/age group) were killed  3 h after microinjection. Fluorographs show no change in the fast  transport of several proteins (closed triangle, closed square, and  closed circle) in the 150-d-old G93A mice (A and C), but the fast  transport of some proteins is retarded in the 200-d-old G93A  mice (B and D). The graphs in C and D illustrate quantitative  measurements of individual proteins conveyed by fast axonal  transport in pairs of age-matched G93A and CTR mice. The 150-d- old G93A mice fail to show any significant slowing of fast transport (C), but the fast transport of several proteins is retarded in  the 200-d-old G93A mice (D). The symbols in A and B correspond to proteins analyzed in the graphs in C and D.
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Figure 7: SDS-PAGE shows a progressive retardation in fast transport in the L5 ventral roots of the spinal cord in the G93A transgenic mice compared to the control (CTR) mice. 12 G93A and N1029 transgenic mice as well as age-matched control mice of two different ages (150 and 200 d, n = 3/age group) were killed 3 h after microinjection. Fluorographs show no change in the fast transport of several proteins (closed triangle, closed square, and closed circle) in the 150-d-old G93A mice (A and C), but the fast transport of some proteins is retarded in the 200-d-old G93A mice (B and D). The graphs in C and D illustrate quantitative measurements of individual proteins conveyed by fast axonal transport in pairs of age-matched G93A and CTR mice. The 150-d- old G93A mice fail to show any significant slowing of fast transport (C), but the fast transport of several proteins is retarded in the 200-d-old G93A mice (D). The symbols in A and B correspond to proteins analyzed in the graphs in C and D.

Mentions: Since all major cytoskeletal components of slow axonal transport were reduced in G93A mice that developed MND, we asked whether proteins transported in the fast components also are impaired. To do this, we analyzed radiolabeled proteins at 3 h after microinjection of 35S-labeled methionine into the L5 ventral horn of G93A, N1029, and control mice, and we showed that there was no reduction in the intensity of any of the radiolabeled proteins traveling along the ventral roots by fast axonal transport in 150-d-old G93A and control mice (Fig. 7 A). For example, we compared the ratio of three different radiolabeled proteins with molecular mass 100, 43, and 20 kD from G93A mice versus control mice and demonstrated that their movement along all five segments of the ventral roots did not differ significantly (Fig. 7, A and C). In contrast, the fast axonal transport of radiolabeled proteins in 200-d-old G93A mice appeared to be variable when compared to control mice of the same age (Fig. 7 B). For example, the migration of a radiolabeled protein with molecular mass 100 kD (Fig. 7 B) was similar to that of the control, and the transport of a second 43-kD protein was only slightly affected (Fig. 7 B) while the movement of a third 20-kD protein (Fig. 7 B) was reduced by 60% in the ventral root of G95A transgenic mice compared to controls, indicating a selective retardation of specific proteins (Fig. 7, B and D).


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)

SDS-PAGE shows a progressive retardation in fast  transport in the L5 ventral roots of the spinal cord in the G93A  transgenic mice compared to the control (CTR) mice. 12 G93A  and N1029 transgenic mice as well as age-matched control mice  of two different ages (150 and 200 d, n = 3/age group) were killed  3 h after microinjection. Fluorographs show no change in the fast  transport of several proteins (closed triangle, closed square, and  closed circle) in the 150-d-old G93A mice (A and C), but the fast  transport of some proteins is retarded in the 200-d-old G93A  mice (B and D). The graphs in C and D illustrate quantitative  measurements of individual proteins conveyed by fast axonal  transport in pairs of age-matched G93A and CTR mice. The 150-d- old G93A mice fail to show any significant slowing of fast transport (C), but the fast transport of several proteins is retarded in  the 200-d-old G93A mice (D). The symbols in A and B correspond to proteins analyzed in the graphs in C and D.
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Related In: Results  -  Collection

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Figure 7: SDS-PAGE shows a progressive retardation in fast transport in the L5 ventral roots of the spinal cord in the G93A transgenic mice compared to the control (CTR) mice. 12 G93A and N1029 transgenic mice as well as age-matched control mice of two different ages (150 and 200 d, n = 3/age group) were killed 3 h after microinjection. Fluorographs show no change in the fast transport of several proteins (closed triangle, closed square, and closed circle) in the 150-d-old G93A mice (A and C), but the fast transport of some proteins is retarded in the 200-d-old G93A mice (B and D). The graphs in C and D illustrate quantitative measurements of individual proteins conveyed by fast axonal transport in pairs of age-matched G93A and CTR mice. The 150-d- old G93A mice fail to show any significant slowing of fast transport (C), but the fast transport of several proteins is retarded in the 200-d-old G93A mice (D). The symbols in A and B correspond to proteins analyzed in the graphs in C and D.
Mentions: Since all major cytoskeletal components of slow axonal transport were reduced in G93A mice that developed MND, we asked whether proteins transported in the fast components also are impaired. To do this, we analyzed radiolabeled proteins at 3 h after microinjection of 35S-labeled methionine into the L5 ventral horn of G93A, N1029, and control mice, and we showed that there was no reduction in the intensity of any of the radiolabeled proteins traveling along the ventral roots by fast axonal transport in 150-d-old G93A and control mice (Fig. 7 A). For example, we compared the ratio of three different radiolabeled proteins with molecular mass 100, 43, and 20 kD from G93A mice versus control mice and demonstrated that their movement along all five segments of the ventral roots did not differ significantly (Fig. 7, A and C). In contrast, the fast axonal transport of radiolabeled proteins in 200-d-old G93A mice appeared to be variable when compared to control mice of the same age (Fig. 7 B). For example, the migration of a radiolabeled protein with molecular mass 100 kD (Fig. 7 B) was similar to that of the control, and the transport of a second 43-kD protein was only slightly affected (Fig. 7 B) while the movement of a third 20-kD protein (Fig. 7 B) was reduced by 60% in the ventral root of G95A transgenic mice compared to controls, indicating a selective retardation of specific proteins (Fig. 7, B and D).

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