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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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EM photomicrographs show a drastic reduction in the  axonal caliber of the L5 ventral roots of the G93A transgenic  mice by 200 d of age. The ventral roots of the control mouse (A,  C, and E) contain many large myelinated axons that are tightly  packed and evenly distributed in the nerve. Note the relative  abundance of small- and intermediate-sized axons (A, C, and E).  There is no obvious difference in the ventral roots of the G93A  transgenic mice (B) compared to control mice (A) at 150 d. In contrast, the ventral roots of the G93A mouse are mainly composed  of axons of smaller caliber (D and F) compared to age-matched  control mice (C and E). The abundant interaxonal space and the  tangential or longitudinal orientation of axons probably reflects  the collapse of these axons due to impaired transport. Bars: (A) 20  μm; (F) 10 μm.
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Figure 5: EM photomicrographs show a drastic reduction in the axonal caliber of the L5 ventral roots of the G93A transgenic mice by 200 d of age. The ventral roots of the control mouse (A, C, and E) contain many large myelinated axons that are tightly packed and evenly distributed in the nerve. Note the relative abundance of small- and intermediate-sized axons (A, C, and E). There is no obvious difference in the ventral roots of the G93A transgenic mice (B) compared to control mice (A) at 150 d. In contrast, the ventral roots of the G93A mouse are mainly composed of axons of smaller caliber (D and F) compared to age-matched control mice (C and E). The abundant interaxonal space and the tangential or longitudinal orientation of axons probably reflects the collapse of these axons due to impaired transport. Bars: (A) 20 μm; (F) 10 μm.

Mentions: Since NF inclusions are found in proximal axons of the G93A mice and the number of NFs in the ventral roots of these mice are reduced, we asked whether or not the amount of NF subunit proteins was reduced in the G93A mice. To do this, we performed quantitative Western blot analysis on 2-mm segments along the ventral roots of the G93A and N1029 transgenic mice as well as control mice to compare the relative levels of the NF triplet proteins in the ventral root axons (see Fig. 1 for orientation). At 150 d, the levels of NF proteins in the L5 ventral root axons of the G93A mice were similar to N1029 and control mice (Fig. 4, A and E), but there were no NF inclusions or motor neuron degeneration at this time in the G93A mice (data not shown). However, by 180 d, a substantial decrease in NF proteins was detected in the G93A mice compared to age-matched N1029 and the control mice (Fig. 4, B and E). Specifically, a 50% decrease in NFL was first detected in 180-d-old G93A mice (Fig. 4, B and E), and the levels of NFL decreased progressively by 70 and 90% when these mice were 200 (Fig. 5, C and E) and 230 d of age (Fig. 4, D and E), respectively. An obvious decrease in the NFM immunoreactivity also was observed in the ventral root axons of the G93A mice, although it occurred slightly later and to a lesser extent than the decrease in NFL. For example, the levels of NFM were reduced by 50 and 70% when the G93A mice were 200 and 230 d of age, respectively. Similarly, the levels of NFH in the ventral root axons of the G93A mice decreased at 200 and 230 d of age by 90 and 95%, respectively (Fig. 4 E). Since the decrease in the levels of NF proteins was similar in all five consecutive segments of the ventral roots of the G93A mice, this suggests that orthograde transport of NF proteins may be retarded at one or more sites proximal to the ventral root axons studied here. Our analysis also showed that the amount of NF proteins in the ventral root axons of the N1029 transgenic mice was comparable to that seen in the ventral root axons of the control mice at all four ages examined here (data not shown). Thus, both the N1029 transgenic and the wild-type littermates of G93A mice were used as controls and are referred to as such together here.


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)

EM photomicrographs show a drastic reduction in the  axonal caliber of the L5 ventral roots of the G93A transgenic  mice by 200 d of age. The ventral roots of the control mouse (A,  C, and E) contain many large myelinated axons that are tightly  packed and evenly distributed in the nerve. Note the relative  abundance of small- and intermediate-sized axons (A, C, and E).  There is no obvious difference in the ventral roots of the G93A  transgenic mice (B) compared to control mice (A) at 150 d. In contrast, the ventral roots of the G93A mouse are mainly composed  of axons of smaller caliber (D and F) compared to age-matched  control mice (C and E). The abundant interaxonal space and the  tangential or longitudinal orientation of axons probably reflects  the collapse of these axons due to impaired transport. Bars: (A) 20  μm; (F) 10 μm.
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Related In: Results  -  Collection

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Figure 5: EM photomicrographs show a drastic reduction in the axonal caliber of the L5 ventral roots of the G93A transgenic mice by 200 d of age. The ventral roots of the control mouse (A, C, and E) contain many large myelinated axons that are tightly packed and evenly distributed in the nerve. Note the relative abundance of small- and intermediate-sized axons (A, C, and E). There is no obvious difference in the ventral roots of the G93A transgenic mice (B) compared to control mice (A) at 150 d. In contrast, the ventral roots of the G93A mouse are mainly composed of axons of smaller caliber (D and F) compared to age-matched control mice (C and E). The abundant interaxonal space and the tangential or longitudinal orientation of axons probably reflects the collapse of these axons due to impaired transport. Bars: (A) 20 μm; (F) 10 μm.
Mentions: Since NF inclusions are found in proximal axons of the G93A mice and the number of NFs in the ventral roots of these mice are reduced, we asked whether or not the amount of NF subunit proteins was reduced in the G93A mice. To do this, we performed quantitative Western blot analysis on 2-mm segments along the ventral roots of the G93A and N1029 transgenic mice as well as control mice to compare the relative levels of the NF triplet proteins in the ventral root axons (see Fig. 1 for orientation). At 150 d, the levels of NF proteins in the L5 ventral root axons of the G93A mice were similar to N1029 and control mice (Fig. 4, A and E), but there were no NF inclusions or motor neuron degeneration at this time in the G93A mice (data not shown). However, by 180 d, a substantial decrease in NF proteins was detected in the G93A mice compared to age-matched N1029 and the control mice (Fig. 4, B and E). Specifically, a 50% decrease in NFL was first detected in 180-d-old G93A mice (Fig. 4, B and E), and the levels of NFL decreased progressively by 70 and 90% when these mice were 200 (Fig. 5, C and E) and 230 d of age (Fig. 4, D and E), respectively. An obvious decrease in the NFM immunoreactivity also was observed in the ventral root axons of the G93A mice, although it occurred slightly later and to a lesser extent than the decrease in NFL. For example, the levels of NFM were reduced by 50 and 70% when the G93A mice were 200 and 230 d of age, respectively. Similarly, the levels of NFH in the ventral root axons of the G93A mice decreased at 200 and 230 d of age by 90 and 95%, respectively (Fig. 4 E). Since the decrease in the levels of NF proteins was similar in all five consecutive segments of the ventral roots of the G93A mice, this suggests that orthograde transport of NF proteins may be retarded at one or more sites proximal to the ventral root axons studied here. Our analysis also showed that the amount of NF proteins in the ventral root axons of the N1029 transgenic mice was comparable to that seen in the ventral root axons of the control mice at all four ages examined here (data not shown). Thus, both the N1029 transgenic and the wild-type littermates of G93A mice were used as controls and are referred to as such together here.

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