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Distribution of Misfolded Prion Protein Seeding Activity Alone Does Not Predict Regions of Neurodegeneration

View Article: PubMed Central - PubMed

ABSTRACT

Protein misfolding is common across many neurodegenerative diseases, with misfolded proteins acting as seeds for "prion-like" conversion of normally folded protein to abnormal conformations. A central hypothesis is that misfolded protein accumulation, spread, and distribution are restricted to specific neuronal populations of the central nervous system and thus predict regions of neurodegeneration. We examined this hypothesis using a highly sensitive assay system for detection of misfolded protein seeds in a murine model of prion disease. Misfolded prion protein (PrP) seeds were observed widespread throughout the brain, accumulating in all brain regions examined irrespective of neurodegeneration. Importantly, neither time of exposure nor amount of misfolded protein seeds present determined regions of neurodegeneration. We further demonstrate two distinct microglia responses in prion-infected brains: a novel homeostatic response in all regions and an innate immune response restricted to sites of neurodegeneration. Therefore, accumulation of misfolded prion protein alone does not define targeting of neurodegeneration, which instead results only when misfolded prion protein accompanies a specific innate immune response.

No MeSH data available.


Changes in neuronal markers demonstrate specific neuronal populations targeted to certain brain regions.(a) Tyrosine hydroxylase staining of midbrain neurons. No visible change in staining pattern could be observed in any GSS/101LL animal tested at 240 dpi (n = 6) compared to age- and region-matched NBH/101LL controls (n = 6). A marked loss of staining pattern is observed in the midbrain neurons upon clinical onset (291.1 ± 5.3 dpi), indicative of a loss of tyrosine hydroxylase neurons upon clinical onset of disease. Scale bar = 200 μm. (b) MAP2 staining in brain stem has marked loss of MAP2 cell–associated staining compared to NBH/101LL brain stem age-matched control. Overall, levels of MAP2 staining are visibly lost in the ventral-medial parts of the thalamus compared to region- and age-matched NBH/101LL controls. No change could be observed in the staining pattern of MAP2 in any part of the cortex or cerebellum compared to region- and age-matched NBH/101LL controls. These findings are observed consistently across all animals tested; GSS/101LL (n = 9), NBH/101LL (n = 4). Scale bars = 100 μm. (c) Higher magnification examples of MAP2 neurons lost in the gigantoreticular nuclei of the brain stem but no loss of neurons evidenced in the cortex. (d) Parvalbumin staining of Purkinje cells of the cerebellum at clinical stages of disease in GSS/101LL animals (291.1 ± 5.3 dpi; n = 3) compared to age-matched NBH/101LL controls (n = 3). Scale bars = 100 μm. (e) Neuronal cell counts of substantia nigra (SN) neurons of the midbrain, gigantocellular reticular nuclei (Gi) of the brain stem, and retrosplenial granular region (RSGc) of the cortex from three representative animals. Cells counted based upon the number of cells showing positive staining for either tyrosine hydroxylase (TyHy+) in the SN or MAP2 in the Gi and RSGc. (f) Quantification of MAP2+ staining intensity from three representative animals showing a loss of MAP2 staining in brain stem and the thalamus but no change in the cortex or cerebellum. Quantification of staining was performed using colour deconvolution plug-in of Image-J software.
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pbio.1002579.g004: Changes in neuronal markers demonstrate specific neuronal populations targeted to certain brain regions.(a) Tyrosine hydroxylase staining of midbrain neurons. No visible change in staining pattern could be observed in any GSS/101LL animal tested at 240 dpi (n = 6) compared to age- and region-matched NBH/101LL controls (n = 6). A marked loss of staining pattern is observed in the midbrain neurons upon clinical onset (291.1 ± 5.3 dpi), indicative of a loss of tyrosine hydroxylase neurons upon clinical onset of disease. Scale bar = 200 μm. (b) MAP2 staining in brain stem has marked loss of MAP2 cell–associated staining compared to NBH/101LL brain stem age-matched control. Overall, levels of MAP2 staining are visibly lost in the ventral-medial parts of the thalamus compared to region- and age-matched NBH/101LL controls. No change could be observed in the staining pattern of MAP2 in any part of the cortex or cerebellum compared to region- and age-matched NBH/101LL controls. These findings are observed consistently across all animals tested; GSS/101LL (n = 9), NBH/101LL (n = 4). Scale bars = 100 μm. (c) Higher magnification examples of MAP2 neurons lost in the gigantoreticular nuclei of the brain stem but no loss of neurons evidenced in the cortex. (d) Parvalbumin staining of Purkinje cells of the cerebellum at clinical stages of disease in GSS/101LL animals (291.1 ± 5.3 dpi; n = 3) compared to age-matched NBH/101LL controls (n = 3). Scale bars = 100 μm. (e) Neuronal cell counts of substantia nigra (SN) neurons of the midbrain, gigantocellular reticular nuclei (Gi) of the brain stem, and retrosplenial granular region (RSGc) of the cortex from three representative animals. Cells counted based upon the number of cells showing positive staining for either tyrosine hydroxylase (TyHy+) in the SN or MAP2 in the Gi and RSGc. (f) Quantification of MAP2+ staining intensity from three representative animals showing a loss of MAP2 staining in brain stem and the thalamus but no change in the cortex or cerebellum. Quantification of staining was performed using colour deconvolution plug-in of Image-J software.

Mentions: Midbrain neurons were assessed by their expression pattern of tyrosine hydroxylase, and no change in its staining pattern could be detected until clinical onset of disease (Fig 4A), at which point significant cell loss was observed (Fig 4E). Cell loss was also quantified in specific neuronal populations in the brain stem and cortex and showed substantial cell loss in the brain stem but not in the cortex (Fig 4E). Brain stem, thalamus, cortex, and cerebellum neurons were assessed using antibodies against microtubule associated protein 2 (MAP2), isoforms a+b. No change in MAP2 staining could be observed in cerebellum or cortex neurons at clinical stages of disease (Fig 4B and 4F). Substantial changes in MAP2 staining were observed in brain stem nuclei, such as the gigantocellular reticular nuclei (Fig 4B, 4C and 4F). This encompassed a general loss in precise cell body–associated staining compared to region- and age-matched NBH/101LL controls, which is indicative of neuronal degeneration in GSS/101LL brain stem. A reduction of MAP2 staining associated with dendritic processes was observed in the thalamus at clinical stages of disease, which was particularly prevalent in the ventral-medial parts of the thalamus (Fig 4B and 4F). To further assess specific neuronal populations of the cerebellum, antibodies specific to the calcium-binding protein parvalbumin were used. Parvalbumin is highly expressed in Purkinje and stellate and basket neurons of the cerebellum and has been shown to be lost as a result of pathology in the cerebellum [28]. No change in the pattern of staining or intensity was observed in the GSS/101LL cerebellum compared to uninfected controls, even at clinical stages of disease (Fig 4D).


Distribution of Misfolded Prion Protein Seeding Activity Alone Does Not Predict Regions of Neurodegeneration
Changes in neuronal markers demonstrate specific neuronal populations targeted to certain brain regions.(a) Tyrosine hydroxylase staining of midbrain neurons. No visible change in staining pattern could be observed in any GSS/101LL animal tested at 240 dpi (n = 6) compared to age- and region-matched NBH/101LL controls (n = 6). A marked loss of staining pattern is observed in the midbrain neurons upon clinical onset (291.1 ± 5.3 dpi), indicative of a loss of tyrosine hydroxylase neurons upon clinical onset of disease. Scale bar = 200 μm. (b) MAP2 staining in brain stem has marked loss of MAP2 cell–associated staining compared to NBH/101LL brain stem age-matched control. Overall, levels of MAP2 staining are visibly lost in the ventral-medial parts of the thalamus compared to region- and age-matched NBH/101LL controls. No change could be observed in the staining pattern of MAP2 in any part of the cortex or cerebellum compared to region- and age-matched NBH/101LL controls. These findings are observed consistently across all animals tested; GSS/101LL (n = 9), NBH/101LL (n = 4). Scale bars = 100 μm. (c) Higher magnification examples of MAP2 neurons lost in the gigantoreticular nuclei of the brain stem but no loss of neurons evidenced in the cortex. (d) Parvalbumin staining of Purkinje cells of the cerebellum at clinical stages of disease in GSS/101LL animals (291.1 ± 5.3 dpi; n = 3) compared to age-matched NBH/101LL controls (n = 3). Scale bars = 100 μm. (e) Neuronal cell counts of substantia nigra (SN) neurons of the midbrain, gigantocellular reticular nuclei (Gi) of the brain stem, and retrosplenial granular region (RSGc) of the cortex from three representative animals. Cells counted based upon the number of cells showing positive staining for either tyrosine hydroxylase (TyHy+) in the SN or MAP2 in the Gi and RSGc. (f) Quantification of MAP2+ staining intensity from three representative animals showing a loss of MAP2 staining in brain stem and the thalamus but no change in the cortex or cerebellum. Quantification of staining was performed using colour deconvolution plug-in of Image-J software.
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pbio.1002579.g004: Changes in neuronal markers demonstrate specific neuronal populations targeted to certain brain regions.(a) Tyrosine hydroxylase staining of midbrain neurons. No visible change in staining pattern could be observed in any GSS/101LL animal tested at 240 dpi (n = 6) compared to age- and region-matched NBH/101LL controls (n = 6). A marked loss of staining pattern is observed in the midbrain neurons upon clinical onset (291.1 ± 5.3 dpi), indicative of a loss of tyrosine hydroxylase neurons upon clinical onset of disease. Scale bar = 200 μm. (b) MAP2 staining in brain stem has marked loss of MAP2 cell–associated staining compared to NBH/101LL brain stem age-matched control. Overall, levels of MAP2 staining are visibly lost in the ventral-medial parts of the thalamus compared to region- and age-matched NBH/101LL controls. No change could be observed in the staining pattern of MAP2 in any part of the cortex or cerebellum compared to region- and age-matched NBH/101LL controls. These findings are observed consistently across all animals tested; GSS/101LL (n = 9), NBH/101LL (n = 4). Scale bars = 100 μm. (c) Higher magnification examples of MAP2 neurons lost in the gigantoreticular nuclei of the brain stem but no loss of neurons evidenced in the cortex. (d) Parvalbumin staining of Purkinje cells of the cerebellum at clinical stages of disease in GSS/101LL animals (291.1 ± 5.3 dpi; n = 3) compared to age-matched NBH/101LL controls (n = 3). Scale bars = 100 μm. (e) Neuronal cell counts of substantia nigra (SN) neurons of the midbrain, gigantocellular reticular nuclei (Gi) of the brain stem, and retrosplenial granular region (RSGc) of the cortex from three representative animals. Cells counted based upon the number of cells showing positive staining for either tyrosine hydroxylase (TyHy+) in the SN or MAP2 in the Gi and RSGc. (f) Quantification of MAP2+ staining intensity from three representative animals showing a loss of MAP2 staining in brain stem and the thalamus but no change in the cortex or cerebellum. Quantification of staining was performed using colour deconvolution plug-in of Image-J software.
Mentions: Midbrain neurons were assessed by their expression pattern of tyrosine hydroxylase, and no change in its staining pattern could be detected until clinical onset of disease (Fig 4A), at which point significant cell loss was observed (Fig 4E). Cell loss was also quantified in specific neuronal populations in the brain stem and cortex and showed substantial cell loss in the brain stem but not in the cortex (Fig 4E). Brain stem, thalamus, cortex, and cerebellum neurons were assessed using antibodies against microtubule associated protein 2 (MAP2), isoforms a+b. No change in MAP2 staining could be observed in cerebellum or cortex neurons at clinical stages of disease (Fig 4B and 4F). Substantial changes in MAP2 staining were observed in brain stem nuclei, such as the gigantocellular reticular nuclei (Fig 4B, 4C and 4F). This encompassed a general loss in precise cell body–associated staining compared to region- and age-matched NBH/101LL controls, which is indicative of neuronal degeneration in GSS/101LL brain stem. A reduction of MAP2 staining associated with dendritic processes was observed in the thalamus at clinical stages of disease, which was particularly prevalent in the ventral-medial parts of the thalamus (Fig 4B and 4F). To further assess specific neuronal populations of the cerebellum, antibodies specific to the calcium-binding protein parvalbumin were used. Parvalbumin is highly expressed in Purkinje and stellate and basket neurons of the cerebellum and has been shown to be lost as a result of pathology in the cerebellum [28]. No change in the pattern of staining or intensity was observed in the GSS/101LL cerebellum compared to uninfected controls, even at clinical stages of disease (Fig 4D).

View Article: PubMed Central - PubMed

ABSTRACT

Protein misfolding is common across many neurodegenerative diseases, with misfolded proteins acting as seeds for "prion-like" conversion of normally folded protein to abnormal conformations. A central hypothesis is that misfolded protein accumulation, spread, and distribution are restricted to specific neuronal populations of the central nervous system and thus predict regions of neurodegeneration. We examined this hypothesis using a highly sensitive assay system for detection of misfolded protein seeds in a murine model of prion disease. Misfolded prion protein (PrP) seeds were observed widespread throughout the brain, accumulating in all brain regions examined irrespective of neurodegeneration. Importantly, neither time of exposure nor amount of misfolded protein seeds present determined regions of neurodegeneration. We further demonstrate two distinct microglia responses in prion-infected brains: a novel homeostatic response in all regions and an innate immune response restricted to sites of neurodegeneration. Therefore, accumulation of misfolded prion protein alone does not define targeting of neurodegeneration, which instead results only when misfolded prion protein accompanies a specific innate immune response.

No MeSH data available.