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The Mitochondrial Peptidasome, PreP, relation to Alzheimer Disease
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Prof. Elzbieta Glaser)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Amyloid-β (Aβ) is the toxic peptide implicated in the pathogenesis of Alzheimer Disease (AD). Accumulation of Aβ has been shown in brain mitochondria from AD patients and AD mice models. The occurrence of Aβ in the mitochondrial matrix leads to free radical generation and apoptosis in neurons.

In our studies, Aβ was found in brain mitochondria of living patients with plaque pathology. Extracellular Aβ was taken up by neuroblastoma cells and was found in the mitochondria. Moreover, we showed that Aβ40 and Aβ42 are transported into mitochondria via the Translocase of the Outer Membrane, the TOM machinery.

We have identified the mitochondrial Aβ-degrading protease hPreP, in human brain mitochondrial matrix. PreP is a metalloprotease, originally identified as presequence protease, but was also shown to degrade other unstructured peptides including Aβ. Immunoinactivation of PreP in human brain mitochondria revealed PreP to be the protease responsible for Aβ degradation in mitochondria.

Also, we have investigated if genetic variation in the gene encoding hPreP is associated with AD by genotyping 19 single nucleotide polymorphisms (SNPs) in the Swedish population. The study did not show a genetic association between any of the genotyped SNPs and the risk to AD. However, the biochemical analysis of four SNPs selected on the basis of their location within a structural homology model of hPreP revealed a decreased activity compared to wildtype.

Interestingly, the activity of PreP in human brain mitochondrial matrix in AD individuals was significantly lower compared to non-dement aged-matched controls. These findings were also confirmed in brain mitochondrial matrix of AD mouse models. These results suggest that a decreased PreP activity may contribute to Aβ aggregation and accumulation inside mitochondria leading to neuronal death in AD. In summary, our findings show that the degradation of Aβ by hPreP may be of importance in the pathology of AD.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2009. , 64 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-29044ISBN: 978-91-7155-918-0 OAI: oai:DiVA.org:su-29044DiVA: diva2:231469
Public defence
2009-09-25, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 4: In progress.

Available from: 2009-09-03 Created: 2009-08-07 Last updated: 2017-02-22Bibliographically approved
List of papers
1. The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae
Open this publication in new window or tab >>The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae
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2008 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 35, 13145-13150 p.Article in journal (Refereed) Published
Abstract [en]

The amyloid beta-peptide (Abeta) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by Abeta and accumulation of Abeta has been detected in mitochondria. Because Abeta is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial Abeta uptake. Our results from rat mitochondria show that Abeta is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed Abeta association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of Abeta to mitochondrial cristae. A similar distribution pattern of Abeta in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for Abeta in mitochondria and demonstrate both in vitro and in vivo that Abeta is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied Abeta can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of Abeta, a peptide considered to be of major significance in Alzheimer's disease.

Keyword
Alzheimer disease, protein import, human brain biopsies
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Cell Biology
Identifiers
urn:nbn:se:su:diva-14872 (URN)10.1073/pnas.0806192105 (DOI)000259343000093 ()18757748 (PubMedID)
Available from: 2009-01-26 Created: 2009-01-26 Last updated: 2017-12-13Bibliographically approved
2. Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP
Open this publication in new window or tab >>Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP
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2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 39, 29096-29104 p.Article in journal (Refereed) Published
Abstract [en]

Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid beta-protein (Abeta). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Abeta. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu(78) in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Abeta-(1-42), Abeta-(1-40), and Abeta Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 A resolution of AtPreP allowed us to identify Cys(90) and Cys(527) that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Abeta by hPreP may potentially be of importance in the pathology of Alzheimer disease.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-8748 (URN)10.1074/jbc.M602532200 (DOI)16849325 (PubMedID)
Available from: 2007-12-21 Created: 2007-12-21 Last updated: 2017-12-13Bibliographically approved
3. Genetic and biochemical studies of SNPs in the mitochondrial Aβ-degrading protease, hPreP
Open this publication in new window or tab >>Genetic and biochemical studies of SNPs in the mitochondrial Aβ-degrading protease, hPreP
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-29250 (URN)
Available from: 2009-08-18 Created: 2009-08-18 Last updated: 2017-02-22Bibliographically approved
4. Decreased proteolytic activity of the PreP peptidasome in Alzheimer disease brain mitochondria and transgenic models
Open this publication in new window or tab >>Decreased proteolytic activity of the PreP peptidasome in Alzheimer disease brain mitochondria and transgenic models
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-29251 (URN)
Available from: 2009-08-18 Created: 2009-08-18 Last updated: 2017-02-22Bibliographically approved

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