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Mitochondria in Alzheimer's Disease: The Presequence Protease and Mitochondria-Associated ER Membranes
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Elzbieta Glaser)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease (AD) is one of the most prevalent age-related neurodegenerative disorders and the accumulation of the amyloid-β peptide (Aβ) in the temporal lobe has been implicated in the pathology of AD. Synaptic transmission in neuronal cells is a highly energy dependent process, which relies on the presence and proper function of mitochondria. A growing number of studies have analyzed the roles of mitochondria in AD. Interestingly, Aβ accumulation in mitochondria was detected in AD patient brains and in AD mouse models, which was associated with the formation of reactive oxygen species (ROS) and neuronal death. In mitochondria, the only protease capable of clearing Aβ is the Presequence Protease, PreP.

The aim of this thesis was to study the involvement of mitochondria and hPreP in AD. We investigated how the mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), which are involved in the regulation of Ca2+ signaling, phospholipids synthesis and apoptosis, are affected in AD. We observed MAM at synapses and found that these structures are essential for neuronal and astrocytic survival. We detected altered MAM protein levels in AD patient brains and in AD mouse models in early stages of the disease and found that MAM can be functionally modulated by Aβ. We analyzed human PreP (hPreP) activity in brain extracts from AD patients and different factors that can affect hPreP function. Interestingly, we detected low hPreP activity in AD patient brains and in AD mouse models, which were associated with increased ROS levels and lower cytochrome c oxidase activity. This suggested that protein oxidation could contribute to impaired activity. Furthermore, we investigated a potential correlation between 18 single nucleotide polymorphisms (SNPs) in the PITRM1 gene, encoding hPreP, and the risk for developing AD. Even though we could not find any genetic correlation in the Swedish population examined, biochemical analysis of four non-synonymous hPreP-SNPs, selected on the basis of their location in hPreP structure, showed lower hPreP activity. Furthermore, we demonstrated in vivo and in vitro that the hPreP presequence is processed at amino acid 28 by mitochondrial processing peptidase (MPP) and that inefficient processing does not affect the enzymatic activity of hPreP but it decreases the stability of the protein.

Together, these results indicate that MAM dysfunctions, inefficient Aβ clearance in mitochondria by hPreP, hPreP mutations or inefficient processing, may contribute to the development of AD.  

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2013. , 79 p.
Keyword [en]
Alzheimer's Disease, Mitochondria, Mitochondria-associated ER membranes, human Presequence Protease
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-93285ISBN: 978-91-7447-733-7 (print)OAI: oai:DiVA.org:su-93285DiVA: diva2:646630
Public defence
2013-10-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2013-09-19 Created: 2013-09-06 Last updated: 2013-09-11Bibliographically approved
List of papers
1. Modulation of the endoplasmic reticulum-mitochondria interface in Alzheimer's disease and related models
Open this publication in new window or tab >>Modulation of the endoplasmic reticulum-mitochondria interface in Alzheimer's disease and related models
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2013 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 19, 7916-7921 p.Article in journal (Refereed) Published
Abstract [en]

It is well-established that subcompartments of endoplasmic reticulum (ER) are in physical contact with the mitochondria. These lipid raft-like regions of ER are referred to as mitochondria-associated ER membranes (MAMs), and they play an important role in, for example, lipid synthesis, calcium homeostasis, and apoptotic signaling. Perturbation of MAM function has previously been suggested in Alzheimer's disease (AD) as shown in fibroblasts from AD patients and a neuroblastoma cell line containing familial presenilin-2 AD mutation. The effect of AD pathogenesis on the ER-mitochondria interplay in the brain has so far remained unknown. Here, we studied ER-mitochondria contacts in human AD brain and related AD mouse and neuronal cell models. We found uniform distribution of MAM in neurons. Phosphofurin acidic cluster sorting protein-2 and sigma 1 receptor, two MAM-associated proteins, were shown to be essential for neuronal survival, because siRNA knockdown resulted in degeneration. Up-regulated MAM-associated proteins were found in the AD brain and amyloid precursor protein (APP)(Swe/Lon) mouse model, in which up-regulation was observed before the appearance of plaques. By studying an ER-mitochondria bridging complex, inositol-1,4,5-triphosphate receptor-voltage-dependent anion channel, we revealed that nanomolar concentrations of amyloid beta-peptide increased inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevated the number of ER-mitochondria contact points and mitochondrial calcium concentrations. Our data suggest an important role of ER-mitochondria contacts and cross-talk in AD pathology.

Keyword
AD mouse models, hippocampal neurons, human cortical brain tissue
National Category
Natural Sciences Engineering and Technology
Identifiers
urn:nbn:se:su:diva-91531 (URN)10.1073/pnas.1300677110 (DOI)000319327700084 ()
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Note

AuthorCount:17;

Available from: 2013-07-01 Created: 2013-06-28 Last updated: 2017-12-06Bibliographically approved
2. Decreased proteolytic activity of the mitochondrial amyloid-β degrading enzyme, PreP peptidasome, in Alzheimer's disease brain mitochondria
Open this publication in new window or tab >>Decreased proteolytic activity of the mitochondrial amyloid-β degrading enzyme, PreP peptidasome, in Alzheimer's disease brain mitochondria
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2011 (English)In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 27, no 1, 75-87 p.Article in journal (Refereed) Published
Abstract [en]

Accumulation of amyloid-β peptide (Aβ), the neurotoxic peptide implicated in the pathogenesis of Alzheimer's disease (AD), has been shown in brain mitochondria of AD patients and of AD transgenic mouse models. The presence of Aβ in mitochondria leads to free radical generation and neuronal stress. Recently, we identified the presequence protease, PreP, localized in the mitochondrial matrix in mammalian mitochondria as the novel mitochondrial Aβ-degrading enzyme. In the present study, we examined PreP activity in the mitochondrial matrix of the human brain's temporal lobe, an area of the brain highly susceptible to Aβ accumulation and reactive oxygen species (ROS) production. We found significantly lower hPreP activity in AD brains compared with non-AD age-matched controls. By contrast, in the cerebellum, a brain region typically spared from Aβ accumulation, there was no significant difference in hPreP activity when comparing AD samples to non-AD controls. We also found significantly reduced PreP activity in the mitochondrial matrix of AD transgenic mouse brains (Tg mAβPP and Tg mAβPP/ABAD) when compared to non-transgenic aged-matched mice. Furthermore, mitochondrial fractions isolated from AD brains and Tg mAβPP mice had higher levels of 4-hydroxynonenal, an oxidative product, as compared with those from non-AD and nonTg mice. Accordingly, activity of cytochrome c oxidase was significantly reduced in the AD mitochondria. These findings suggest that decreased PreP proteolytic activity, possibly due to enhanced ROS production, contributes to Aβ accumulation in mitochondria leading to the mitochondrial toxicity and neuronal death that is exacerbated in AD. Clearance of mitochondrial Aβ by PreP may thus be of importance in the pathology of AD.

Keyword
Mitochondrial amyloid-β, mitochondrial function, oxidative stress, presequence protease (PreP), proteolysis
National Category
Biochemistry and Molecular Biology Neurosciences
Research subject
Biophysics; Biochemistry
Identifiers
urn:nbn:se:su:diva-70949 (URN)10.3233/JAD-2011-101716 (DOI)000296570400007 ()21750375 (PubMedID)
Note

authorCount :8

Available from: 2012-01-25 Created: 2012-01-25 Last updated: 2017-12-08Bibliographically approved
3. Genetic and biochemical studies of SNPs of the mitochondrial A beta-degrading protease, hPreP
Open this publication in new window or tab >>Genetic and biochemical studies of SNPs of the mitochondrial A beta-degrading protease, hPreP
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2010 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 469, no 2, 204-208 p.Article in journal (Refereed) Published
Abstract [en]

Several studies suggest mitochondrial dysfunction as a possible mechanism underlying the development of Alzheimer disease (AD). There is data showing that amyloid-beta (A beta) peptide is present in AD brain mitochondria. The human presequence protease (hPreP) was recently shown to be the major mitochondrial A beta-degrading enzyme. We investigated if there is an increased susceptibility to AD, which can be attributed to genetic variation in the hPreP gene PITRM1 and if the proteolytic efficiency of recombinant hPreP variants is affected. When a total of 673 AD cases and 649 controls were genotyped for 18 single nucleotide polymorphisms (SNPs), no genetic association between any of the SNPs and the risk for AD was found. In contrast, functional analysis of four non-synonymous SNPs in hPreP revealed a decreased activity compared to wild type hPreP. Using A beta, the presequence of ATP synthase F-1 beta subunit and a fluorescent peptide as substrates, the lowest activity was observed for the hPreP(A525D) variant, corresponding to rs1224893, which displayed only 20-30% of wild type activity. Furthermore, the activity of all variants was restored by the addition of Mg2+, suggesting an important role for this metal during proteolysis. In conclusion, our data suggest that genetic variation in the hPreP gene PITRM1 may potentially contribute to mitochondrial dysfunctions.

Keyword
Amyloid-beta peptide, Alzheimer disease, SNP, Mitochondria, Proteolysis, PreP
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-50536 (URN)10.1016/j.neulet.2009.11.075 (DOI)000274508700006 ()
Note

authorCount :8

Available from: 2010-12-28 Created: 2010-12-28 Last updated: 2017-12-11Bibliographically approved
4. Presequence processing increases the stability of the human Presequence Protease, hPreP
Open this publication in new window or tab >>Presequence processing increases the stability of the human Presequence Protease, hPreP
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Most of the mitochondrial matrix proteins are nuclear encoded, synthesized in the cytoplasm and have to be targeted to the mitochondria. For matrix proteins, this is generally achieved due to the presence of a N-terminal sequence, called presequence. After reaching the mitochondrial matrix, the presequence is cleaved off by the mitochondrial processing peptidase, MPP, giving rise to the mature protein and the presequence. Free presequences are degraded in the mitochondrial matrix by the Presequence Protease, PreP. Previous studies demonstrated that the correct maturation of mitochondrial proteins is important either for stability or catalytic activity of the protein.

In the present study, we estimated the presequence length of the human PreP, hPreP, to be 28 amino acids long, using HEK293T cells and recombinant MPP. Furthermore, we analyzed the activity of the recombinant hPreP precursor and its mature form using two peptides, amyloid-β (1-40) peptide or the synthetic peptide substrate V, and we observed that the proteolytic maturation does not affect hPreP enzymatic activity. However, we detected a significantly lower stability for the hPreP precursor in comparison to the mature form of the enzyme, through pulse-chase experiments using vaccinia virus expression system in mammalian cells. These results show that the mitochondrial processing is required for the hPreP stability.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-93283 (URN)
Available from: 2013-09-06 Created: 2013-09-06 Last updated: 2013-09-10Bibliographically approved

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