Search

Dimebon, a drug currently being evaluated in multiple Phase III Alzheimer's disease trials, has previously been shown to have effects on isolated mitochondria at muM concentrations. Here the effects of nM concentrations of Dimebon on mitochondrial function were investigated both in primary mouse cortical neurons and human neuroblastoma cells (SH-SY5Y). Under non-stress conditions nM concentrations of Dimebon increased succinate dehydrogenase activity (MTT-assay), mitochondrial membrane potential (DeltaPsim), and cellular ATP levels. Dimebon treatment had no effect on mitochondria DNA content, implying that mitochondrial biogenesis was not induced. Under stress conditions, mitochondria in Dimebon-treated neurons showed increased resistance to elevated intracellular calcium concentrations, thus, maintaining their DeltaPsim throughout the experiment, in contrast to control neurons, which rapidly lost their DeltaPsim. Moreover, we show that serum-starved differentiated SH-SY5Y cells treated with Dimebon had an increased survival rate as compared to untreated cells. In conclusion, these data demonstrate that Dimebon enhances mitochondrial function both in the absence and presence of stress and Dimebon-treated cells are partially protected to maintain cell viability.

gamma-Secretase is a key enzyme involved in the processing of the beta-amyloid precursor protein into amyloid beta-peptides (Abeta). Abeta accumulates and forms plaques in Alzheimer's disease (AD) brains. A progressive neurodegeneration and cognitive decline occurs during the course of the disease, and Abeta is believed to be central for the molecular pathogenesis of AD. Apoptosis has been implicated as one of the mechanisms behind the neuronal cell loss seen in AD. We have studied preservation and activity of the gamma-secretase complex during apoptosis in neuroblastoma cells (SH-SY5Y) exposed to staurosporine (STS). We report that the known components (presenilin, Nicastrin, Aph-1 and Pen-2) interact and form active gamma-secretase complexes in apoptotic cells. In addition, the fragments corresponding to the PS1 N-terminal fragment and the caspase-cleaved PS1 C-terminal fragment (PS1-caspCTF) were found to form active gamma-secretase complexes when co-expressed in presenilin (PS) knockout cells. Interestingly, PS1-caspCTF replaced the normal PS1 C-terminal fragment and was co-immunoprecipitated with the gamma-secretase complex in SH-SY5Y cells exposed to STS. In addition, Abeta was detected in medium from apoptotic HEK APP(swe) cells. Together, the data show that gamma-secretase complexes containing PS1-caspCTF are active, and suggest that this proteolytic activity is also important in dying cells and may affect the progression of AD.

Presenilins are often mutated in familial forms of Alzheimer's disease (AD). Such mutations sensitize cells in culture to different apoptotic stimuli eg. staurosporine, calcium ionophore, growth factor withdrawal. The altered responses to apoptotic stimuli in cells carrying presenilin mutations include increased intracellular calcium concentrations and enhanced production of reactive oxygen species. Presenilin mutations also result in increased production of amyloid beta (Abeta) indicating that presenilins participate in the cleavage of amyloid beta-protein precursor (AbetaPP). In fact, presenilin is part of the gamma-secretase complex which together with beta-secretase cleaves AbetaPP and produce Abeta, later forming the senile plaques typical for AD pathology. Here we review the current knowledge about the mechanisms of cell death in AD with focus on the role of presenilin and presenilin mutations in apoptosis. It appears that presenilin and its different fragments, generated after proteolytic cleavage, have a regulatory role in apoptosis. In addition, different studies show that the cellular levels of presenilin are controlled by proteasomal degradation both under normal and stress conditions.

Markers for caspase activation and apoptosis have been shown in brains of Alzheimer's disease (AD) patients and AD-mouse models. In neurons, caspase activation is associated with elevated amyloid β-peptide (Aβ) production. Caspases cleave numerous substrates including presenilin-1 (PS1). The cleavage takes place in the large cytosolic loop of PS1-C-terminal fragment (PS1CTF), generating a truncated PS1CTF lacking half of the loop domain (caspCTF). The loop has been shown to possess important regulatory functions with regard to Aβ40 and Aβ42 production. Previously, we have demonstrated that γ-secretase complexes are active during apoptosis regardless of caspase cleavage in the PS1CTF-loop. Here, a PS1/PS2-knockout mouse blastocyst-derived cell line was used to establish stable or transient cell lines expressing either caspCTF or full-length CTF (wtCTF). We show that caspCTF restores γ-secretase activity and forms active γ-secretase complexes together with Nicastrin, Pen-2, Aph-1 and PS1-N-terminal fragment. Further, caspCTF containing γ-secretase complexes have a sustained capacity to cleave amyloid precursor protein (APP) and Notch, generating APP and Notch intracellular domain, respectively. However, when compared to wtCTF cells, caspCTF cells exhibit increased intracellular production of Aβ42 accompanied by increased intracellular Aβ42/Aβ40 ratio without changing the Aβ secretion pattern. Similarly, induction of apoptosis in wtCTF cells generate a similar shift in intracellular Aβ pattern with increased Aβ42/Aβ40 ratio. In summary, we show that caspase cleavage of PS1 generates a γ-secretase complex that increases the intracellular Aβ42/Aβ40 ratio. This can have implications for AD pathogenesis and suggests caspase inhibitors as potential therapeutic agents. [ABSTRACT FROM AUTHOR]

γ-Secretase is a key enzyme involved in the processing of the β-amyloid precursor protein into amyloid β-peptides (Aβ). Aβ accumulates and forms plaques in Alzheimer's disease (AD) brains. A progressive neurodegeneration and cognitive decline occurs during the course of the disease, and Aβ is believed to be central for the molecular pathogenesis of AD. Apoptosis has been implicated as one of the mechanisms behind the neuronal cell loss seen in AD. We have studied preservation and activity of the γ-secretase complex during apoptosis in neuroblastoma cells (SH-SY5Y) exposed to staurosporine (STS). We report that the known components (presenilin, Nicastrin, Aph-1 and Pen-2) interact and form active γ-secretase complexes in apoptotic cells. In addition, the fragments corresponding to the PS1 N-terminal fragment and the caspase-cleaved PS1 C-terminal fragment (PS1-caspCTF) were found to form active γ-secretase complexes when co-expressed in presenilin (PS) knockout cells. Interestingly, PS1-caspCTF replaced the normal PS1 C-terminal fragment and was co-immunoprecipitated with the γ-secretase complex in SH-SY5Y cells exposed to STS. In addition, Aβ was detected in medium from apoptotic HEK APPswe cells. Together, the data show that γ-secretase complexes containing PS1-caspCTF are active, and suggest that this proteolytic activity is also important in dying cells and may affect the progression of AD. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) affects millions of people worldwide and the number of AD cases will increase with increased life expectancy. Today there is no cure for this devastating and always lethal disease and therefore it is of great interest for patients, relatives and societies to find new drugs that can hinder the disease process. During the progression of AD a substantial amount of neurons degenerate in the brain. The mechanisms of cell death involved in AD have not been fully elucidated. However, there are several reports showing that neurons die partly by apoptosis in the AD brain. Drugs blocking apoptosis could therefore be potentially useful for early prevention of neuronal cell death. Biomarkers for apoptosis should be important tools in the evaluation of drug effects and in the diagnostics of AD. Here we review the current knowledge in the field and discuss potential biomarkers for apoptosis in AD. Copyright © 2005 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Presenilins are mutated in most cases of autosomal dominant inherited forms of early onset Alzheimer''s disease and such mutations are known to sensitize cells to apoptotic stimuli in vitro. Previous studies show that presenilins are primarily located in the endoplasmatic reticulum and cell membranes. Here we report, based on immunoblot analysis and immunoelectron microscopy studies, that PS1 is also located in mitochondrial membranes. For these studies we used tissue sections and subcellular fractions of rat brain and liver. Immunogold labeling of sections show that PS1 is predominantly located in the inner membrane of mitochondria. The function of PS1 in mitochondrial membranes is presently unknown. PS1 mutations may make cells more vulnerable to apoptotic stimuli due to dysfunction of this protein at the mitochondrial level. [Copyright &y& Elsevier]

Mitochondria are central in the regulation of cell death. Apart from providing the cell with ATP, mitochondria also harbor several death factors that are released upon apoptotic stimuli. Alterations in mltochondrial functions, increased oxidative stress, and neurons dying by apoptosis have been detected in Alzheimer's disease patients. These findings suggest that mitochondria may trigger the abnormal onset of neuronal cell death in Alzhelmer's disease. We previously reported that presenilin 1 (PS1), which is often mutated in familiai forms of Alzheimer's disease, is located in mitochondria and hypothesized that presenilin mutations may sensitize cells to apoptotic stimuli at the mitochondrial level. Presenilin forms an active γ-secretase complex together with Nicastrin (NCT), APH-1, and PEN-2, which among other substrates cleaves the β-amyloid precursor protein (β-APP) generating the amyloid β-peptide and the β-APP intracellular domain. Here we have identified dual targeting sequences (for endoplasmic reticulum and mitochondria) in NCT and showed expression of NCT in mitochondria by immunoelectron microscopy. We also showed that NCT together with APH-1, PEN-2, and PS1 form a high molecular weight complex located in mitochondria. γ-Secretase activity in isolated mitochondria was demonstrated using C83 (α-secretasecleaved C-terminal 83-residue β-APP fragment from BD8 cells lacking presenilin and thus γ-secretase activity) or recombinant C100-Flag (C-terminal 100-residue β-APP fragment) as substrates. Both systems generated an APP intracellular domain, and the activity was inhibited by the γ-secretase inhibitors L-685,458 or Compound E. This novel localization of NCT, PS1, APH-1, and PEN-2 expands the role and importance of γ-secretase activity to mitochondria. [ABSTRACT FROM AUTHOR]