The interaction of amyloidogenic proteins with mitochondrial membranes - role of cardiolipin

Abstract

BACKGROUND Neurodegenerative proteinopathies like Alzheimer's disease (AD) and Parkinson's disease (PD) are increasingly linked to direct damage to cellular membranes by aggregate complexes of amyloidogenic proteins, such as the amyloid-beta (A~) peptide, tau and a-synuclein (as). Although not related to human disease, aggregates ofthe N-terminal domain of the Eschericia coli HypF (HypF-N) protein are also membrane-active and cytotoxic. Mitochondria, abundantly located at neuronal synapses, are critically impaired in both AD and PD, leading to bioenergetics failure.

OBJECTIVES In the present study, the focus of the investigation was amyloid toxicity by tau and as proteins to mitochondrial membranes. In particular, it was especially important to probe a mechanistic role for cardiolipin (CL), a non-bilayer forming acidic phospholipid unique to mitochondrial membranes, in mediating amyloid protein interaction with mitochondria. Another aspect of the present work involved validating a select group of small-molecule compounds as potential drug leads for enhancing the resilience of the mitochondrial membrane barrier against insult by amyloid aggregates.

METHODOLOGY With this aim, an in vitro minimalist model system was used to eschew cellular complexity and provide a powerful means for obtaining mechanistic insights. Thus, mitochondria were freshly isolated from human neuroblastoma SH-SYSY cells and incubated with physiological (low micromolar) concentrations of tau, as, and HypF-N proteins aggregated according to established protocols. Mitochondria were subjected to assays reflecting outer or inner mitochondrial membrane integrity: organelle swelling, Cytochrome (Cyt) c release (CCR), and depolarisation of the inner mitochondrial membrane potential (LltJIm) . Further, direct binding of amyloid protein to mitochondria was assessed by immunoblotting and, in the case IV of tau, using liposomes having CL-rich membranes simulating the lipid environment of mitochondrial contact sites and the inner mitochondrial membrane.

RESULTS Aggregate exposure instigated disruption of mitochondrial structural integrity as evidenced by abundant swelling, robust efflux of Cyt c from the mitochondria, and loss of the Ll'Pm (the latter not in the case of tau). An important result from a mechanistic point of view is that pharmacological inhibitors of the mitochondrial permeability transition pore (mPTP) complex could not rescue these phenotypes. Together with the mitochondrial binding studies, the data describes a scenario in which the amyloid proteins physically associate with mitochondria and possibly directly porate the mitochondrial membranes. Regarding amyloid-membrane affinity, a key finding is that the phospholipid CL appears to function as a leitmotif in targeting of amyloid proteins to mitochondrial membranes. Such a mechanism could involve either direct CL binding or sensing of, and insertion into, lipid packing and curvature defects created by the cone-shaped CL molecules. Finally, the diphenyl- pyrazole anti-aggregator anle138b, the polyphenol rosmarinic acid, and the theaflavin-rich extract from black tea, were the most effective three compounds in protecting mitochondrial organelles from perturbation by toxic amyloid entities. They may thus represent important drug leads to alleviate mitochondrial dysfunction in neurodegenerative diseases.

CONCLUSION The data presented here advances a conceptual model in which CL-rich mitochondrial membranes, such as the outer membrane contact sites and the inner membrane, represent a generic target of aggregate complexes of amyloidogenic proteins. In the cytosol, toxic amyloid entities of proteins such as as and tau, would be free to target mitochondria by interacting with CL or by inserting into lipid defects created by the CL molecules, leading to compromised mitochondrial structural integrity. Hence, efforts on developing drugs that target mitochondrial lipids, and especially CL, might constitute a new strategy for pharmacological therapy of neurodegenerative proteinopathies.