Birgitta Wiehager

The amyloid β-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae

The amyloid β-peptide (Aβ) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by Aβ and accumulation of Aβ has been detected in mitochondria. Because Aβ is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial Aβ uptake. Our results from rat mitochondria show that Aβ 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 Aβ association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of Aβ to mitochondrial cristae. A similar distribution pattern of Aβ 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 Aβ in mitochondria and demonstrate both in vitro and in vivo that Aβ is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied Aβ can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of Aβ, a peptide considered to be of major significance in Alzheimers disease.

Modulation of the endoplasmic reticulum–mitochondria interface in Alzheimer’s disease and related models

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 σ1 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 β-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.

Increased β-amyloid release and levels of amyloid precursor protein (APP) in fibroblast cell lines from family members with the Swedish Alzheimer's disease APP670/671 mutation

Cell lines transfected with the Swedish Alzheimers disease amyloid precursor protein APP670/671 mutation release significantly more β‐amyloid than wild‐type cells. Citron et al. [Proc. Natl. Acad. Sci. USA (1994) in press] have recently shown that fibroblasts carrying the APP670/671 mutation also release more β‐amyloid than control cells [1]. The present study confirms a ca. threefold increase in β‐amyloid release from mutation‐bearing fibroblasts. APP mRNA levels did not differ between mutation‐bearing and control cells, although mutation‐bearing fibroblasts contained significantly more APP751/770 than controls. Mild stress decreased β‐amyloid secretion and increased APP751/770 levels in all cell lines. In conclusion, the proportion of APP committed to amyloidogenic processing is increased in fibroblasts from family members with the APP670/671 mutation, and this mutation may also compromise the APP stress response.

Effects of β-Amyloid-(25-35) Peptides on Radioligand Binding to Excitatory Amino Acid Receptors and Voltage-Dependent Calcium Channels: Evidence for a Selective Affinity for the Glutamate and Glycine Recognition Sites of the NMDA Receptor

The neurotoxic fragment corresponding to residues 25-35 of the β-amyloid (Aβ) peptide [Aβ-(25-35)] has been shown to exert effects on (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate ([3H]MK-801) binding to the cation channel of the N-methyl-D-aspartate (NMDA) receptor. In the present study, we investigated whether the amidated and carboxylic acid C-terminated forms of Aβ-(25-35) [Aβ-(25-35-NH2) and Aβ-(25-35-COOH), respectively] exert effects on other excitatory amino acid receptor and cation channel types in rat cortical membranes. Both Aβ-(25-35-NH2) and Aβ-(25-35-COOH) gave statistically significant dose-dependent inhibitions of [3H]glutamate and [3H]glycine binding to the agonist recognition sites of the NMDA receptor. Ten μM Aβ-(25-35-NH2) and Aβ-(25-35-COOH) gave 25% and 20% inhibitions of [3H]glutamate binding and 75% and 70% inhibitions of [3H]glycine binding, respectively. Aβ-(25-35-NH2), but not Aβ-(25-35-COOH), gave a small (ca. 17% at 10 μM) statistically significant increase of [3H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) binding. [3H]kainate binding was not significantly affected by either peptide. Similarly, neither peptide affected either the maximal level or EC50 value for calcium stimulation of [3H]nitrendipine binding. It is concluded that Aβ-(25-35) shows slight affinity for the agonist recognition sites of the NMDA receptor, but not for other excitatory amino acid receptor types or for L-type voltage-dependent calcium channels.

Regionally selective alterations in G protein subunit levels in the Alzheimer's disease brain

In the present study the relative densities of a number of G protein subunits were quantified in membranes prepared from the hippocampus, temporal cortex and angular gyrus of Alzheimers disease and control post-mortem brain by immunoblotting with specific polyclonal antisera against Gs alpha, Gi alpha, Gi alpha-1, G(o) alpha and G beta protein subunits. In addition, basal, Gs-stimulated and Gi-inhibited adenylyl cyclase activities were measured in the same hippocampal membrane samples. Densitometric analysis of the immunoblot data revealed a 58% reduction in the levels of Gi alpha, and a 75% reduction in the levels of Gi alpha-1, in the Alzheimers disease temporal cortex. Gi alpha levels were reduced, by 37% in the angular gyrus of the Alzheimers disease cases. The ratio of large to small molecular weight isoforms of the Gs alpha subunit was significantly increased in both the hippocampus and the angular gyrus of the Alzheimers disease samples when compared to control values, although the difference in individual Gs alpha isoform levels did not attain statistical significance when comparing groups. No statistically significant differences were observed in G(o) alpha or G beta levels when comparing control and Alzheimers disease cases. Gs-stimulated adenylyl cyclase activity was significantly reduced in the Alzheimers disease samples compared to controls, whereas Gi-inhibited adenylyl cyclase activity was unchanged. No significant differences were observed between the control and Alzheimers disease samples for either basal or forskolin stimulated adenylyl cyclase activity. The ratio of hippocampal Gs-stimulated to basal adenylyl cyclase activity correlated significantly with the large to small Gs alpha subunit ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial γ-secretase participates in the metabolism of mitochondria-associated amyloid precursor protein

Intracellular amyloid‐β peptide (Aβ) has been implicated in the pathogenesis of Alzheimers disease (AD). Mitochondria were found to be the target both for amyloid precursor protein (APP) that accumulates in the mitochondrial import channels and for Aβ that interacts with several proteins inside mitochondria and leads to mitochondrial dysfunction. Here, we have studied the role of mitochondrial γ‐secretase in processing different substrates. We found that a significant proportion of APP is associated with mitochondria in cultured cells and that γ‐secretase cleaves the shedded C‐terminal part of APP identified as C83 associated with the outer membrane of mitochondria (OMM). Moreover, we have established the topology of the C83 in the OMM and found the APP intracellular domain (AICD) to be located inside mitochondria. Our data show for the first time that APP is a substrate for the mitochondrial γ‐secretase and that AICD is produced inside mitochondria. Thus, we provide a mechanistic view of the mitochondria‐associated APP metabolism where AICD, P3 peptide and potentially Aβ are produced locally and may contribute to mitochondrial dysfunction in AD.—Pavlov, P. F., Wiehager, B., Sakai, J., Frykman, S., Behbahani, H., Winblad, B., Ankarcrona, M. Mitochondrial γ‐secretase participates in the metabolism of mitochondria‐associated amyloid precursor protein. FASEB J. 25, 78–88 (2011). www.fasebj.org

Effects of apolipoprotein E (apoE) isoforms, β-amyloid (Aβ) and apoE/Aβ complexes on Protein Kinase C-α (PKC-α) translocation and amyloid precursor protein (APP) processing in human SH-SY5Y neuroblastoma cells and fibroblasts

We investigated the effects of different apolipoprotein E (apoE) isoforms, Abeta (1-42), and apoE/Abeta complexes on PKC-alpha translocation and APP processing in human SH-SY5Y neuroblastoma cells and fibroblasts. Treatment of cells with either 10 nM apoE3 or apoE4, 10 microM Abeta (1-42), or apoE/Abeta complexes induced significant translocation of PKC-alpha in both cell types. Effects were seen using both human recombinant apoE and apoE loaded into beta-very low density lipoprotein (beta-VLDL) particles. Time course (5-24 h) studies of APP processing revealed that some conditions induced transient or moderate increases in the secretion of proteins detected by 22C11. In contrast, the secretion of alpha-secretase cleaved APP was either not modified or transiently decreased, as determined by immunoblotting with the antibody 6E10. These results suggest that apoE, Abeta (1-42) and apoE/Abeta complexes can modulate PKC activity but do not have major consequences for APP processing. These effects could contribute to the reported PKC alterations seen in AD. However, it is unlikely that the contribution of different apoE isoforms to AD pathology occurs via effects on APP processing.

Mitofusin-2 knockdown increases ER-mitochondria contact and decreases amyloid β-peptide production.

Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria‐associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM‐associated proteins and enhanced ER to mitochondria Ca2+ transfer from ER to mitochondria in Alzheimers disease (AD) and amyloid β‐peptide (Aβ)‐related neuronal models. Here, we report that siRNA knockdown of mitofusin‐2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca2+ transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra‐ and extracellular Aβ40 and Aβ42. Analysis of γ‐secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ‐secretase complex function. Amyloid‐β precursor protein (APP), β‐site APP‐cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER–mitochondria contact affects γ‐secretase activity and Aβ generation. Increased ER–mitochondria contact results in lower γ‐secretase activity suggesting a new mechanism by which Aβ generation can be controlled.

Differential role of Presenilin-1 and -2 on mitochondrial membrane potential and oxygen consumption in mouse embryonic fibroblasts.

Increasing evidence indicates that mitochondrial alterations contribute to the neuronal death in Alzheimers disease (AD). Presenilin 1 (PS1) and Presenilin 2 (PS2) mutations have been shown to sensitize cells to apoptosis by mechanisms suggested to involve impaired mitochondrial function. We have previously detected active γ‐secretase complexes in mitochondria. We investigated the impact of PS/γ‐secretase on mitochondrial function using mouse embryonal fibroblasts derived from wild‐type, PS1−/−, PS2−/− and PS double knock‐out (PSKO) embryos. Measurements of mitochondrial membrane potential (ΔΨm) showed a higher percentage of fully functional mitochondria in PS1−/− and PSwt as compared to PS2−/− and PSKO cells. This result was evident both in whole cell preparations and in isolated mitochondria. Interestingly, pre‐treatment of isolated mitochondria with the γ‐secretase inhibitor L‐685,458 resulted in a decreased population of mitochondria with high ΔΨm in PSwt and PS1−/− cells, indicating that PS2/γ‐secretase activity can modify ΔΨm. PS2−/− cells showed a significantly lower basal respiratory rate as compared to other cell lines. However, all cell lines demonstrated competent bioenergetic function. These data point toward a specific role of PS2/γ‐secretase activity for proper mitochondrial function and indicate interplay between PS1 and PS2 in mitochondrial functionality.

β-amyloid peptides enhance binding of the calcium mobilising second messengers, inositol(1,4,5)trisphosphate and inositol-(1,3,4,5)tetrakisphosphate to their receptor sites in rat cortical membranes

We studied the effects of the beta-amyloid (A beta) peptides A beta-(1-40), A beta-(25-35-NH2) and A beta-(25-35-COOH) on binding of the phosphoinositide derived, calcium mobilising, second messengers inositol(1,4,5)-trisphosphate (Ins(1,4,5)P3) and inositol(1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) to their receptor sites in rat cerebral cortical membranes. All three peptides gave statistically significant dose-dependent increases in both [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 binding. A beta-(1-40) and A beta-(25-35-NH2) enhanced [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 binding to a similar extent. In comparison, A beta-(25-35-COOH) gave much greater enhancements of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 binding. However, a component of the latter appeared to be due to the formation of pelletable A beta-(25-35-COOH)/[3H]Ins(1,3,4,5)P4 aggregates, that occurred in the absence of membranes. These results raise the possibility that A beta affects calcium homeostasis by a direct action on [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 receptor sites.