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Altered machinery of protein synthesis is region- and stage-dependent and is associated with α-synuclein oligomers in Parkinson's disease.

Garcia-Esparcia P, Hernández-Ortega K, Koneti A, Gil L, Delgado-Morales R, Castaño E, Carmona M, Ferrer I - Acta Neuropathol Commun (2015)

Bottom Line: No modifications were found in the putamen at any time of the study except transient modifications in 28S rRNA and only one RP mRNA at stages 5-6.Altered machinery of protein synthesis is altered in the substantia nigra and cerebral cortex in PD being the frontal cortex area 8 more affected than the angular gyrus and precuneus; in contrast, pathways of protein synthesis are apparently preserved in the putamen.This is associated with the presence of α-synuclein oligomeric species in total homogenates; substantia nigra and frontal cortex are enriched, albeit with different band patterns, in α-synuclein oligomeric species, whereas α-synuclein oligomers are not detected in the putamen.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuropathology, Bellvitge University Hospital, University of Barcelona, Bellvitge Biomedical Research Institute (IDIBELL), Hospitalet de Llobregat; Biomedical Research Center of Neurodegenerative Diseases (CIBERNED), Barcelona, Spain.

ABSTRACT

Introduction: Parkinson's disease (PD) is characterized by the accumulation of abnormal α-synuclein in selected regions of the brain following a gradient of severity with disease progression. Whether this is accompanied by globally altered protein synthesis is poorly documented. The present study was carried out in PD stages 1-6 of Braak and middle-aged (MA) individuals without alterations in brain in the substantia nigra, frontal cortex area 8, angular gyrus, precuneus and putamen.

Results: Reduced mRNA expression of nucleolar proteins nucleolin (NCL), nucleophosmin (NPM1), nucleoplasmin 3 (NPM3) and upstream binding transcription factor (UBF), decreased NPM1 but not NPM3 nucleolar protein immunostaining in remaining neurons; diminished 18S rRNA, 28S rRNA; reduced expression of several mRNAs encoding ribosomal protein (RP) subunits; and altered protein levels of initiation factor eIF3 and elongation factor eEF2 of protein synthesis was found in the substantia nigra in PD along with disease progression. Although many of these changes can be related to neuron loss in the substantia nigra, selective alteration of certain factors indicates variable degree of vulnerability of mRNAs, rRNAs and proteins in degenerating sustantia nigra. NPM1 mRNA and 18S rRNA was increased in the frontal cortex area 8 at stage 5-6; modifications were less marked and region-dependent in the angular gyrus and precuneus. Several RPs were abnormally regulated in the frontal cortex area 8 and precuneus, but only one RP in the angular gyrus, in PD. Altered levels of eIF3 and eIF1, and decrease eEF1A and eEF2 protein levels were observed in the frontal cortex in PD. No modifications were found in the putamen at any time of the study except transient modifications in 28S rRNA and only one RP mRNA at stages 5-6. These observations further indicate marked region-dependent and stage-dependent alterations in the cerebral cortex in PD. Altered solubility and α-synuclein oligomer formation, assessed in total homogenate fractions blotted with anti-α-synuclein oligomer-specific antibody, was demonstrated in the substantia nigra and frontal cortex, but not in the putamen, in PD. Dramatic increase in α-synuclein oligomers was also seen in fluorescent-activated cell sorter (FACS)-isolated nuclei in the frontal cortex in PD.

Conclusions: Altered machinery of protein synthesis is altered in the substantia nigra and cerebral cortex in PD being the frontal cortex area 8 more affected than the angular gyrus and precuneus; in contrast, pathways of protein synthesis are apparently preserved in the putamen. This is associated with the presence of α-synuclein oligomeric species in total homogenates; substantia nigra and frontal cortex are enriched, albeit with different band patterns, in α-synuclein oligomeric species, whereas α-synuclein oligomers are not detected in the putamen.

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Western blotting of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6), Xbox binding protein 1 (XBP1), and phosphorylated inositol requiring kinase 1 (P-IRE-1) in the substantia nigra (a) and frontal cortex area 8 (b) shows significantly increased expression of ATF4 and ATF6 90 kDa at stages 5–6 (p < 0.05), and reduced expression of GRP94 at stages 3–4 and 5–6 (p < 0.01) in the substantia nigra in PD when compared with MA individuals. ATF6f (50 kDa), the cleaved and active form of ATF6, was not identified in any group. In contrast, significant reduction of GRP78 (p < 0.01 at stages 3–4, and p < 0.001 at stages 5–6) with preservation of other reticulum stress markers was found in frontal cortex area 8 in PD. Note that a double band with GRP94, ATF4, and P-IRE-1 antibodies is observed in the substantia nigra but not in the frontal cortex. The expected molecular weight is marked by an arrowhead in SN western blots. β-actin is used as a control of protein loading. AU: arbitrary units; *p < 0.05; **p <0.01
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Fig9: Western blotting of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6), Xbox binding protein 1 (XBP1), and phosphorylated inositol requiring kinase 1 (P-IRE-1) in the substantia nigra (a) and frontal cortex area 8 (b) shows significantly increased expression of ATF4 and ATF6 90 kDa at stages 5–6 (p < 0.05), and reduced expression of GRP94 at stages 3–4 and 5–6 (p < 0.01) in the substantia nigra in PD when compared with MA individuals. ATF6f (50 kDa), the cleaved and active form of ATF6, was not identified in any group. In contrast, significant reduction of GRP78 (p < 0.01 at stages 3–4, and p < 0.001 at stages 5–6) with preservation of other reticulum stress markers was found in frontal cortex area 8 in PD. Note that a double band with GRP94, ATF4, and P-IRE-1 antibodies is observed in the substantia nigra but not in the frontal cortex. The expected molecular weight is marked by an arrowhead in SN western blots. β-actin is used as a control of protein loading. AU: arbitrary units; *p < 0.05; **p <0.01

Mentions: Increased expression levels of ATF4 and ATF6 90 kDa at stages 5–6 (p < 0.05), and reduced GRP94 expression levels at stages 3–4 and 5–6 (p < 0.01), were found in the substantia nigra in PD (Fig. 9a). No ATF6f (50 kDa) was identified in any group (data not shown). Reduced expression levels of GRP78 were observed in frontal cortex area 8 at stages 3–4 and 5–6 (p < 0.01 and p < 0.001, respectively). No changes in the expression of other reticulum stress markers were found in frontal cortex area 8 in PD (Fig. 9b).Fig. 9


Altered machinery of protein synthesis is region- and stage-dependent and is associated with α-synuclein oligomers in Parkinson's disease.

Garcia-Esparcia P, Hernández-Ortega K, Koneti A, Gil L, Delgado-Morales R, Castaño E, Carmona M, Ferrer I - Acta Neuropathol Commun (2015)

Western blotting of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6), Xbox binding protein 1 (XBP1), and phosphorylated inositol requiring kinase 1 (P-IRE-1) in the substantia nigra (a) and frontal cortex area 8 (b) shows significantly increased expression of ATF4 and ATF6 90 kDa at stages 5–6 (p < 0.05), and reduced expression of GRP94 at stages 3–4 and 5–6 (p < 0.01) in the substantia nigra in PD when compared with MA individuals. ATF6f (50 kDa), the cleaved and active form of ATF6, was not identified in any group. In contrast, significant reduction of GRP78 (p < 0.01 at stages 3–4, and p < 0.001 at stages 5–6) with preservation of other reticulum stress markers was found in frontal cortex area 8 in PD. Note that a double band with GRP94, ATF4, and P-IRE-1 antibodies is observed in the substantia nigra but not in the frontal cortex. The expected molecular weight is marked by an arrowhead in SN western blots. β-actin is used as a control of protein loading. AU: arbitrary units; *p < 0.05; **p <0.01
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666041&req=5

Fig9: Western blotting of glucose-regulated protein 78 (GRP78), glucose-regulated protein 94 (GRP94), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6), Xbox binding protein 1 (XBP1), and phosphorylated inositol requiring kinase 1 (P-IRE-1) in the substantia nigra (a) and frontal cortex area 8 (b) shows significantly increased expression of ATF4 and ATF6 90 kDa at stages 5–6 (p < 0.05), and reduced expression of GRP94 at stages 3–4 and 5–6 (p < 0.01) in the substantia nigra in PD when compared with MA individuals. ATF6f (50 kDa), the cleaved and active form of ATF6, was not identified in any group. In contrast, significant reduction of GRP78 (p < 0.01 at stages 3–4, and p < 0.001 at stages 5–6) with preservation of other reticulum stress markers was found in frontal cortex area 8 in PD. Note that a double band with GRP94, ATF4, and P-IRE-1 antibodies is observed in the substantia nigra but not in the frontal cortex. The expected molecular weight is marked by an arrowhead in SN western blots. β-actin is used as a control of protein loading. AU: arbitrary units; *p < 0.05; **p <0.01
Mentions: Increased expression levels of ATF4 and ATF6 90 kDa at stages 5–6 (p < 0.05), and reduced GRP94 expression levels at stages 3–4 and 5–6 (p < 0.01), were found in the substantia nigra in PD (Fig. 9a). No ATF6f (50 kDa) was identified in any group (data not shown). Reduced expression levels of GRP78 were observed in frontal cortex area 8 at stages 3–4 and 5–6 (p < 0.01 and p < 0.001, respectively). No changes in the expression of other reticulum stress markers were found in frontal cortex area 8 in PD (Fig. 9b).Fig. 9

Bottom Line: No modifications were found in the putamen at any time of the study except transient modifications in 28S rRNA and only one RP mRNA at stages 5-6.Altered machinery of protein synthesis is altered in the substantia nigra and cerebral cortex in PD being the frontal cortex area 8 more affected than the angular gyrus and precuneus; in contrast, pathways of protein synthesis are apparently preserved in the putamen.This is associated with the presence of α-synuclein oligomeric species in total homogenates; substantia nigra and frontal cortex are enriched, albeit with different band patterns, in α-synuclein oligomeric species, whereas α-synuclein oligomers are not detected in the putamen.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuropathology, Bellvitge University Hospital, University of Barcelona, Bellvitge Biomedical Research Institute (IDIBELL), Hospitalet de Llobregat; Biomedical Research Center of Neurodegenerative Diseases (CIBERNED), Barcelona, Spain.

ABSTRACT

Introduction: Parkinson's disease (PD) is characterized by the accumulation of abnormal α-synuclein in selected regions of the brain following a gradient of severity with disease progression. Whether this is accompanied by globally altered protein synthesis is poorly documented. The present study was carried out in PD stages 1-6 of Braak and middle-aged (MA) individuals without alterations in brain in the substantia nigra, frontal cortex area 8, angular gyrus, precuneus and putamen.

Results: Reduced mRNA expression of nucleolar proteins nucleolin (NCL), nucleophosmin (NPM1), nucleoplasmin 3 (NPM3) and upstream binding transcription factor (UBF), decreased NPM1 but not NPM3 nucleolar protein immunostaining in remaining neurons; diminished 18S rRNA, 28S rRNA; reduced expression of several mRNAs encoding ribosomal protein (RP) subunits; and altered protein levels of initiation factor eIF3 and elongation factor eEF2 of protein synthesis was found in the substantia nigra in PD along with disease progression. Although many of these changes can be related to neuron loss in the substantia nigra, selective alteration of certain factors indicates variable degree of vulnerability of mRNAs, rRNAs and proteins in degenerating sustantia nigra. NPM1 mRNA and 18S rRNA was increased in the frontal cortex area 8 at stage 5-6; modifications were less marked and region-dependent in the angular gyrus and precuneus. Several RPs were abnormally regulated in the frontal cortex area 8 and precuneus, but only one RP in the angular gyrus, in PD. Altered levels of eIF3 and eIF1, and decrease eEF1A and eEF2 protein levels were observed in the frontal cortex in PD. No modifications were found in the putamen at any time of the study except transient modifications in 28S rRNA and only one RP mRNA at stages 5-6. These observations further indicate marked region-dependent and stage-dependent alterations in the cerebral cortex in PD. Altered solubility and α-synuclein oligomer formation, assessed in total homogenate fractions blotted with anti-α-synuclein oligomer-specific antibody, was demonstrated in the substantia nigra and frontal cortex, but not in the putamen, in PD. Dramatic increase in α-synuclein oligomers was also seen in fluorescent-activated cell sorter (FACS)-isolated nuclei in the frontal cortex in PD.

Conclusions: Altered machinery of protein synthesis is altered in the substantia nigra and cerebral cortex in PD being the frontal cortex area 8 more affected than the angular gyrus and precuneus; in contrast, pathways of protein synthesis are apparently preserved in the putamen. This is associated with the presence of α-synuclein oligomeric species in total homogenates; substantia nigra and frontal cortex are enriched, albeit with different band patterns, in α-synuclein oligomeric species, whereas α-synuclein oligomers are not detected in the putamen.

Show MeSH
Related in: MedlinePlus