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Analysis of the hippocampal proteome in ME7 prion disease reveals a predominant astrocytic signature and highlights the brain-restricted production of clusterin in chronic neurodegeneration.

Asuni AA, Gray B, Bailey J, Skipp P, Perry VH, O'Connor V - J. Biol. Chem. (2013)

Bottom Line: The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease.Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation.This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.

View Article: PubMed Central - PubMed

Affiliation: From the Centre for Biological Sciences and.

ABSTRACT
Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ(TM)/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.

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Reactive astrocytes in the hippocampus of ME7 animals. Photomicrographs illustrate GFAP expression in NBH and ME7 animals at 21 weeks. Nuclei were counterstained with hematoxylin. GFAP immunoreactivity in astrocytes in the hippocampal layers of NBH animals (see arrowheads in A–D, ×5, ×10, ×20, and ×40 magnifications, respectively) is shown. At this stage, there is minimal GFAP staining in the cortical regions of NBH animals (data not shown). ME7 animals were strongly positive for GFAP, particularly within the swollen astrocytic processes (see arrowheads in E–H, ×5, ×10, ×20, and ×40 magnifications, respectively), and at this stage of the disease, there is strong GFAP staining in the cortex (data not shown). Labeling of hippocampal areas: hippocampal formation (HPF); dentate gyrus (DG); posterior thalamic nucleus (Po); cornu ammonis area 1 (CA1); stratum radiatum (SRad). Brain sections were stained with antibodies against the other astrocyte-associated proteins highlighted in the proteomic analysis, and representative images are shown of the stratum radiatum in NBH and ME7 animals immunostained for Clusterin (I and J), EAAT-2 (K and L), and Prdx6 (M and N) (arrowheads are directed to examples of immunoreactive astrocytes). Scale bar, 20 μm.
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Figure 3: Reactive astrocytes in the hippocampus of ME7 animals. Photomicrographs illustrate GFAP expression in NBH and ME7 animals at 21 weeks. Nuclei were counterstained with hematoxylin. GFAP immunoreactivity in astrocytes in the hippocampal layers of NBH animals (see arrowheads in A–D, ×5, ×10, ×20, and ×40 magnifications, respectively) is shown. At this stage, there is minimal GFAP staining in the cortical regions of NBH animals (data not shown). ME7 animals were strongly positive for GFAP, particularly within the swollen astrocytic processes (see arrowheads in E–H, ×5, ×10, ×20, and ×40 magnifications, respectively), and at this stage of the disease, there is strong GFAP staining in the cortex (data not shown). Labeling of hippocampal areas: hippocampal formation (HPF); dentate gyrus (DG); posterior thalamic nucleus (Po); cornu ammonis area 1 (CA1); stratum radiatum (SRad). Brain sections were stained with antibodies against the other astrocyte-associated proteins highlighted in the proteomic analysis, and representative images are shown of the stratum radiatum in NBH and ME7 animals immunostained for Clusterin (I and J), EAAT-2 (K and L), and Prdx6 (M and N) (arrowheads are directed to examples of immunoreactive astrocytes). Scale bar, 20 μm.

Mentions: GFAP was among the most robustly up-regulated protein based on the relative intensity of a number of peptides independently identified. Furthermore, the intensity of the MS signal in NBH samples also indicated that the protein is among the more abundant proteins profiled even in control samples. Immunocytochemical staining for GFAP in hippocampal sections from NBH and ME7 animals shows the abundance of astrocytes in both cohorts and a markedly increased staining in the ME7 samples (Fig. 3, A–D, compared with E–H). Three of the other candidate proteins that we observed to be up-regulated in ME7 animals included Clusterin, EAAT-2, and Prdx6, which are all known to be predominantly expressed in astrocytes (Fig. 3, I–N) (34–36). Immunoreactivity for these molecules is also observed in astrocytes outside the hippocampus (data not shown). We used double immunocytochemistry of Prdx6 and GFAP to confirm whether this protein is expressed and induced in astrocytes or other cells in ME7 animals. Our data revealed co-localization of Prdx6 and GFAP albeit with weaker immunoreactivity of Prdx6 than GFAP; Prdx6 was expressed in NBH tissue (Fig. 4, A–D) but was markedly increased in the ME7 animals (Fig. 4, E–J). No cells other than astrocytes appear to express Prdx6. ImageJ co-localization is shown in white at the far right, illustrating the pixels having both significant red and green signals (Fig. 4, I–J).


Analysis of the hippocampal proteome in ME7 prion disease reveals a predominant astrocytic signature and highlights the brain-restricted production of clusterin in chronic neurodegeneration.

Asuni AA, Gray B, Bailey J, Skipp P, Perry VH, O'Connor V - J. Biol. Chem. (2013)

Reactive astrocytes in the hippocampus of ME7 animals. Photomicrographs illustrate GFAP expression in NBH and ME7 animals at 21 weeks. Nuclei were counterstained with hematoxylin. GFAP immunoreactivity in astrocytes in the hippocampal layers of NBH animals (see arrowheads in A–D, ×5, ×10, ×20, and ×40 magnifications, respectively) is shown. At this stage, there is minimal GFAP staining in the cortical regions of NBH animals (data not shown). ME7 animals were strongly positive for GFAP, particularly within the swollen astrocytic processes (see arrowheads in E–H, ×5, ×10, ×20, and ×40 magnifications, respectively), and at this stage of the disease, there is strong GFAP staining in the cortex (data not shown). Labeling of hippocampal areas: hippocampal formation (HPF); dentate gyrus (DG); posterior thalamic nucleus (Po); cornu ammonis area 1 (CA1); stratum radiatum (SRad). Brain sections were stained with antibodies against the other astrocyte-associated proteins highlighted in the proteomic analysis, and representative images are shown of the stratum radiatum in NBH and ME7 animals immunostained for Clusterin (I and J), EAAT-2 (K and L), and Prdx6 (M and N) (arrowheads are directed to examples of immunoreactive astrocytes). Scale bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Reactive astrocytes in the hippocampus of ME7 animals. Photomicrographs illustrate GFAP expression in NBH and ME7 animals at 21 weeks. Nuclei were counterstained with hematoxylin. GFAP immunoreactivity in astrocytes in the hippocampal layers of NBH animals (see arrowheads in A–D, ×5, ×10, ×20, and ×40 magnifications, respectively) is shown. At this stage, there is minimal GFAP staining in the cortical regions of NBH animals (data not shown). ME7 animals were strongly positive for GFAP, particularly within the swollen astrocytic processes (see arrowheads in E–H, ×5, ×10, ×20, and ×40 magnifications, respectively), and at this stage of the disease, there is strong GFAP staining in the cortex (data not shown). Labeling of hippocampal areas: hippocampal formation (HPF); dentate gyrus (DG); posterior thalamic nucleus (Po); cornu ammonis area 1 (CA1); stratum radiatum (SRad). Brain sections were stained with antibodies against the other astrocyte-associated proteins highlighted in the proteomic analysis, and representative images are shown of the stratum radiatum in NBH and ME7 animals immunostained for Clusterin (I and J), EAAT-2 (K and L), and Prdx6 (M and N) (arrowheads are directed to examples of immunoreactive astrocytes). Scale bar, 20 μm.
Mentions: GFAP was among the most robustly up-regulated protein based on the relative intensity of a number of peptides independently identified. Furthermore, the intensity of the MS signal in NBH samples also indicated that the protein is among the more abundant proteins profiled even in control samples. Immunocytochemical staining for GFAP in hippocampal sections from NBH and ME7 animals shows the abundance of astrocytes in both cohorts and a markedly increased staining in the ME7 samples (Fig. 3, A–D, compared with E–H). Three of the other candidate proteins that we observed to be up-regulated in ME7 animals included Clusterin, EAAT-2, and Prdx6, which are all known to be predominantly expressed in astrocytes (Fig. 3, I–N) (34–36). Immunoreactivity for these molecules is also observed in astrocytes outside the hippocampus (data not shown). We used double immunocytochemistry of Prdx6 and GFAP to confirm whether this protein is expressed and induced in astrocytes or other cells in ME7 animals. Our data revealed co-localization of Prdx6 and GFAP albeit with weaker immunoreactivity of Prdx6 than GFAP; Prdx6 was expressed in NBH tissue (Fig. 4, A–D) but was markedly increased in the ME7 animals (Fig. 4, E–J). No cells other than astrocytes appear to express Prdx6. ImageJ co-localization is shown in white at the far right, illustrating the pixels having both significant red and green signals (Fig. 4, I–J).

Bottom Line: The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease.Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation.This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.

View Article: PubMed Central - PubMed

Affiliation: From the Centre for Biological Sciences and.

ABSTRACT
Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ(TM)/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.

Show MeSH
Related in: MedlinePlus