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Impaired axonal transport in motor neurons correlates with clinical prion disease.

Ermolayev V, Cathomen T, Merk J, Friedrich M, Härtig W, Harms GS, Klein MA, Flechsig E - PLoS Pathog. (2009)

Bottom Line: Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally -- after inoculation into the right sciatic nerve.Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods.The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microscopy Group, DFG Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.

ABSTRACT
Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive. There is no evidence of both the functional axonal impairments in vivo and their connection with prion disease. We studied the functional axonal impairments in motor neurons at the onset of clinical prion disease using the combination of tracing as a functional assay for axonal transport with immunohistochemistry experiments. Well-established and novel confocal and ultramicroscopy techniques were used to image and quantify labeled neurons. Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally -- after inoculation into the right sciatic nerve. Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods. Our data connect axonal transport impairments with disease symptoms for different prion strains and inoculation routes and establish further insight on the development of prion pathology in vivo. The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway. These findings suggest novel targets for therapeutic and diagnostic approaches in the early stages of prion disease.

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Reduction of tracer-positive cells in the red nucleus is not attributed to neuronal loss.(A) Co-localization of FB (green) and NeuN (red) analysis in the contralateral and ipsilateral RN of mice upon prion challenge in the sciatic nerve (i.n.) done at the onset of clinical disease (152 dpi). A population of viable NeuN-positive cells is FB-negative (asterisks), which implicates the defects in axonal transport. In other cells NeuN and FB co-localize (arrows, yellow). Scale bars: 50 µm. (B) Distribution histogram of the relative FB to NeuN fluorescence analyzed on z-stacks in the Regions of Interest (ROI). The analysis quantitatively demonstrates that FB fluorescence co-localizes with NeuN in mock controls. 44% of contralateral neurons was below the background threshold and considered FB-negative, and 7%—on the ipsilateral RN. (C) Quantification of NeuN-positive neurons in the sampled mock and i.n. mice revealed no differences between mock and inoculated animals despite the reduction of REx-positive cells. Unpaired t-test was done using GraphPad Prism software. *** – P<0.0001. NS – non-significant.
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ppat-1000558-g003: Reduction of tracer-positive cells in the red nucleus is not attributed to neuronal loss.(A) Co-localization of FB (green) and NeuN (red) analysis in the contralateral and ipsilateral RN of mice upon prion challenge in the sciatic nerve (i.n.) done at the onset of clinical disease (152 dpi). A population of viable NeuN-positive cells is FB-negative (asterisks), which implicates the defects in axonal transport. In other cells NeuN and FB co-localize (arrows, yellow). Scale bars: 50 µm. (B) Distribution histogram of the relative FB to NeuN fluorescence analyzed on z-stacks in the Regions of Interest (ROI). The analysis quantitatively demonstrates that FB fluorescence co-localizes with NeuN in mock controls. 44% of contralateral neurons was below the background threshold and considered FB-negative, and 7%—on the ipsilateral RN. (C) Quantification of NeuN-positive neurons in the sampled mock and i.n. mice revealed no differences between mock and inoculated animals despite the reduction of REx-positive cells. Unpaired t-test was done using GraphPad Prism software. *** – P<0.0001. NS – non-significant.

Mentions: Our system combining unilateral inoculation of prions into the right sciatic nerve and axonal tracer application enabled investigation of axonal defects specifically connected with prion pathogenesis. The ipsilateral side of the RN did not show any significant decrease of tracer-positive neurons and could be used as an internal control. The differences between the ipsilateral site and the contralateral – initially targeted with prions were directly observed on the same z-stack done on serial cryo-sections of i.n. challenged mice but were not detectable in the mock controls. Moreover, on the contralateral side of the RN, we observed a fraction of NeuN-positive neurons, which did not contain FB (Figure 3A, asterisks). In contrast, the ipsilateral RN did not contain such cells but only the cells containing both FB and NeuN (Figure 3A, arrows; for overview, see Figure S2). This observation was confirmed on both visual and computerized co-localization analysis. Due to high background we still observed the fluorescent objects in the vicinity of NeuN-positive cells on the contralateral RN, which were not perfectly co-localizing. In order to perform systematic stereological analysis, we defined Regions of Interest (ROI) according to the NeuN-positive fluorescence in several mock and i.n. samples (Figure S3A) and quantified the FB fluorescence as a per cent of the NeuN fluorescence in z-stacks using available software tools. The analysis of more than 150 cells from 3 i.n. and 4 mock samples revealed practically all the cells in mock controls to contain FB fluorescence over 18% to the level of NeuN signal, which was over the calculated background value of 17%. Fourty-four per cent of the cells in the contralateral RN (21 of 47 totally analyzed) demonstrated the relative FB fluorescence under the background level, and only 7% (3 of 40 analyzed) – in the ipsilateral RN (Figure 3B).


Impaired axonal transport in motor neurons correlates with clinical prion disease.

Ermolayev V, Cathomen T, Merk J, Friedrich M, Härtig W, Harms GS, Klein MA, Flechsig E - PLoS Pathog. (2009)

Reduction of tracer-positive cells in the red nucleus is not attributed to neuronal loss.(A) Co-localization of FB (green) and NeuN (red) analysis in the contralateral and ipsilateral RN of mice upon prion challenge in the sciatic nerve (i.n.) done at the onset of clinical disease (152 dpi). A population of viable NeuN-positive cells is FB-negative (asterisks), which implicates the defects in axonal transport. In other cells NeuN and FB co-localize (arrows, yellow). Scale bars: 50 µm. (B) Distribution histogram of the relative FB to NeuN fluorescence analyzed on z-stacks in the Regions of Interest (ROI). The analysis quantitatively demonstrates that FB fluorescence co-localizes with NeuN in mock controls. 44% of contralateral neurons was below the background threshold and considered FB-negative, and 7%—on the ipsilateral RN. (C) Quantification of NeuN-positive neurons in the sampled mock and i.n. mice revealed no differences between mock and inoculated animals despite the reduction of REx-positive cells. Unpaired t-test was done using GraphPad Prism software. *** – P<0.0001. NS – non-significant.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2723930&req=5

ppat-1000558-g003: Reduction of tracer-positive cells in the red nucleus is not attributed to neuronal loss.(A) Co-localization of FB (green) and NeuN (red) analysis in the contralateral and ipsilateral RN of mice upon prion challenge in the sciatic nerve (i.n.) done at the onset of clinical disease (152 dpi). A population of viable NeuN-positive cells is FB-negative (asterisks), which implicates the defects in axonal transport. In other cells NeuN and FB co-localize (arrows, yellow). Scale bars: 50 µm. (B) Distribution histogram of the relative FB to NeuN fluorescence analyzed on z-stacks in the Regions of Interest (ROI). The analysis quantitatively demonstrates that FB fluorescence co-localizes with NeuN in mock controls. 44% of contralateral neurons was below the background threshold and considered FB-negative, and 7%—on the ipsilateral RN. (C) Quantification of NeuN-positive neurons in the sampled mock and i.n. mice revealed no differences between mock and inoculated animals despite the reduction of REx-positive cells. Unpaired t-test was done using GraphPad Prism software. *** – P<0.0001. NS – non-significant.
Mentions: Our system combining unilateral inoculation of prions into the right sciatic nerve and axonal tracer application enabled investigation of axonal defects specifically connected with prion pathogenesis. The ipsilateral side of the RN did not show any significant decrease of tracer-positive neurons and could be used as an internal control. The differences between the ipsilateral site and the contralateral – initially targeted with prions were directly observed on the same z-stack done on serial cryo-sections of i.n. challenged mice but were not detectable in the mock controls. Moreover, on the contralateral side of the RN, we observed a fraction of NeuN-positive neurons, which did not contain FB (Figure 3A, asterisks). In contrast, the ipsilateral RN did not contain such cells but only the cells containing both FB and NeuN (Figure 3A, arrows; for overview, see Figure S2). This observation was confirmed on both visual and computerized co-localization analysis. Due to high background we still observed the fluorescent objects in the vicinity of NeuN-positive cells on the contralateral RN, which were not perfectly co-localizing. In order to perform systematic stereological analysis, we defined Regions of Interest (ROI) according to the NeuN-positive fluorescence in several mock and i.n. samples (Figure S3A) and quantified the FB fluorescence as a per cent of the NeuN fluorescence in z-stacks using available software tools. The analysis of more than 150 cells from 3 i.n. and 4 mock samples revealed practically all the cells in mock controls to contain FB fluorescence over 18% to the level of NeuN signal, which was over the calculated background value of 17%. Fourty-four per cent of the cells in the contralateral RN (21 of 47 totally analyzed) demonstrated the relative FB fluorescence under the background level, and only 7% (3 of 40 analyzed) – in the ipsilateral RN (Figure 3B).

Bottom Line: Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally -- after inoculation into the right sciatic nerve.Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods.The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microscopy Group, DFG Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.

ABSTRACT
Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive. There is no evidence of both the functional axonal impairments in vivo and their connection with prion disease. We studied the functional axonal impairments in motor neurons at the onset of clinical prion disease using the combination of tracing as a functional assay for axonal transport with immunohistochemistry experiments. Well-established and novel confocal and ultramicroscopy techniques were used to image and quantify labeled neurons. Despite profound differences in the incubation times, 30% to 45% of neurons in the red nucleus of different mouse lines showed axonal transport impairments at the disease onset bilaterally after intracerebral prion inoculation and unilaterally -- after inoculation into the right sciatic nerve. Up to 94% of motor cortex neurons also demonstrated transport defects upon analysis by alternative imaging methods. Our data connect axonal transport impairments with disease symptoms for different prion strains and inoculation routes and establish further insight on the development of prion pathology in vivo. The alterations in localization of the proteins involved in the retrograde axonal transport allow us to propose a mechanism of transport disruption, which involves Rab7-mediated cargo attachment to the dynein-dynactin pathway. These findings suggest novel targets for therapeutic and diagnostic approaches in the early stages of prion disease.

Show MeSH
Related in: MedlinePlus