Mitochondrial dysfunction and mitophagy failures in Alzheimer’s disease
Raphael Becquart
Selected article is reference [8]
Alzheimer’s disease (AD) is the world's most prevalent neurological disease. Post-mortem
analyses of AD patients' brains led to the classification of the disease as a "proteinopathy"
characterized by the accumulation of β-amyloid peptides (Aβ) inside neurons and the
extracellular matrix, and by abnormal phosphorylation of the Tau protein (pTau), resulting in
neurofibrillary tangles (NFT) [1].
Aβ is produced following successive cleavages of the amyloid precursor protein (APP)
by β- and γ-secretase enzymes in the amyloidogenic pathway. Aggressive familial forms of AD,
promoted by mutations in the genes encoding APP and presenilins 1 and 2 (PS1/2, components
of the γ-secretase complex), led to the proposal that the amyloidogenic pathway played a major
role in the pathogenesis of AD. Nevertheless, the failure of treatments targeting only Aβ suggests
that other fragments derived from APP cleavage may contribute to AD pathology. The
pathogenic role of the C-terminal fragments of APP (APP-CTF: C99 and C83) has been recently
highlighted in different studies [2].
Mitochondria are the cellular powerhouse whose main function is to produce energy in
the form of ATP. In neurons, ATP is necessary for neurotransmitter release and synaptic
plasticity. The homeostasis of mitochondria requires their recycling by a specific autophagic
process, mitophagy, which takes place in three steps [3]: 1) modification of mitochondrial
proteins to signal dysfunctional mitochondria (phosphorylation or ubiquitination); 2)
mitochondria sequestration in an autophagic vacuole (i.e mitophagosome); and 3) fusion of this
vacuole with lysosomes containing degradative enzymes to digest it.
Altered mitochondrial structure and function have been reported in several studies based
on in vitro and in vivo models of AD [4], and post-mortem analysis of AD patients [5]. These
defects have generally been associated with the presence of Aβ peptide [4]. A recent study
revealed the presence and cleavage of APP in "mitochondria-associated membranes" contact
sites between mitochondria and the endoplasmic reticulum. They also identified APP-CTF in
mitochondria [7]. These observations led the authors to hypothesize a specific role for APP-CTF
in mitochondrial dysfunction in AD [8].
The authors used a human neuroblastoma cell line (SH-SY5) which overexpresses an
APP carrying a double mutation, Lys670Met and Asn671Leu (APPLys670Met, Asn671Leu) also
known as APPswe), which promotes the accumulation of APP-CTF and Aβ [8]. These cells have
larger mitochondria with disorganized cristae and reduced matrix density. To determine how Aβ
and APP-CTF alter mitochondrial structures, APPswe cells were treated with a γ-secretase
inhibitor, thereby blocking Aβ production in favor of APP-CTF. Using subcellular fractionation
and confocal microscopy, they found accumulating APP-CTFs in mitochondria, associated with
a fragmented mitochondrial network and disorganized cristae. This observation was supported by
blocking β-secretase (reducing Aβ and C99 fragment production). The latter indeed restores the
healthy mitochondrial phenotype [8].
In parallel to the alteration of mitochondrial structure, they observed that APPswe cells
with dysfunctional mitochondria had less NDUFB8 subunit (NADH: ubiquinone oxidoreductase
subunit B8) of the mitochondrial respiratory chain complex I, associated with a decrease in its
activity, a decrease in mitochondrial membrane potential, and an increase in the production of
reactive oxygen species in the mitochondria. Furthermore, the authors showed that the
accumulation of APP-CTFs in mitochondria, independent of Aβ, exacerbates the production of
reactive oxygen species. Finally, by analyzing a cell model expressing the C99 fragment of APP
in an inducible manner (SH-SY5Y C99 cells), they confirmed the impact of APP-CTF on
mitochondrial dysfunctions. Indeed, this model shows an accumulation of APP-CTF in the
mitochondria, a defect in the expression and activity of complex I of the respiratory chain, and an
overproduction of reactive oxygen species [8].
The absence of an apoptotic phenotype in SH-SY5Y APPswe and SH-SY5Y C99 cells
treated with the γ-secretase inhibitor led the authors to focus on mitophagy. Using biochemical
approaches combined with subcellular fractionation, they quantified different molecular markers
of mitophagy and analyzed the different steps of the mitophagic process with fluorescent probes.
In SH-SY5Y APPswe cells, the first steps of the mitophagic process were observed: enrichment
of PINK1 (PTEN-induced kinase 1) protein in the mitochondrial fraction, parkin recruitment to
the mitochondria (a ubiquitin ligase), and the conversion of the cytosolic LC3-I form of
MAP1LC3 (microtubule-associated protein 1A/1B-light chain 3) into LC3-II, the form recruited
to the autophagosome membrane. However, they found that mitophagy was inhibited, as
revealed by the absence of degradation of the SQSTM1/p62 protein (sequestosome 1 or p62
ubiquitin-binding protein) and by the accumulation of several mitochondrial proteins (TIMM23,
TOMM20, HSP60, and HSP10). This failure of mitophagy was confirmed as mitochondria could
not fuse to lysosomes. The authors then showed that APP-CTF accumulation, independent of
Aβ, exacerbates mitophagy failure in SH-SY5Y APPswe and SH-SY5Y C99 cells [8].
Postmortem biochemical analysis of brains from patients with advanced non-familial
forms of AD demonstrates accumulation of APP-CTF (C99 and C83) and Aβ in the
mitochondrial fraction, coupled with a defect in mitophagy characterized by decreased amounts
of PINK1 and parkin in the mitochondria, increased LC3-II/LC3-I ratio and SQSTM1/p62
protein. The authors reported a significant correlation between the levels of the C99 fragment
and changes in the mitophagic markers. However, these correlations were weak with levels of
Aβ and pTau. This result validated in human samples the results previously obtained in cellular
and animal study models mimicking genetic forms of AD.
In all, independently of the action of Aβ, the accumulation of APP-CTF fragments leads
to a deterioration of mitochondrial structure and function, and a blockage of the mitochondria
turnover (mitophagy) [8]. The accumulation of defective mitochondria associated with disruption
of mitophagy is common to various neurodegenerative diseases, such as Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and AD. Restoring normal mitophagic
function could therefore be considered a new therapeutic goal in AD and other
neurodegenerative diseases.
This article is relevant to lecture 3 where protein aggregation was discussed. The
cleavage of APP by secretases is not biochemically optimal as Aβ and APP-CTF fragments can
both accidentally result from a naturally occurring reaction that serves homeostatic functions, to
a significant degree where they initiate pathogenesis. A potential explanation for this
suboptimality is the fact that humans have not evolved to live long enough for natural selection
to have developed robust repair systems that work during old age, or optimized metabolic
reactions that do not produce such accumulating molecules.
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