The Broad Impact of TOM40 on Neurodegenerative Diseases in Aging

Abstract

Mitochondrial dysfunction is an important factor in the pathogenesis of age-related diseases, including neurodegenerative diseases like Alzheimer's and Parkinson's spectrum disorders. A polymorphism in Translocase of the Outer Mitochondrial Membrane - 40 kD (TOMM40) is associated with risk and age-of onset of late-onset AD, and is the only nuclear- encoded gene identified in genetic studies to date that presumably contributes to LOAD-related mitochondria dysfunction. In this review, we describe the TOM40-mediated mitochondrial protein import mechanism, and discuss the evidence linking TOM40 with Alzheimer's (AD) and Parkinson's (PD) diseases. All but 36 of the >~1,500 mitochondrial proteins are encoded by the nucleus and are synthesized on cytoplasmic ribosomes, and most of these are imported into mitochondria through the TOM complex, of which TOM40 is the central pore, mediating communication between the cytoplasm and the mitochondrial interior. APP enters and obstructs the TOM40 pore, inhibiting import of OXPHOS-related proteins and disrupting the mitochondrial redox balance. Other pathogenic proteins, such as Aβ and alpha-synuclein, readily pass through the pore and cause toxic effects by directly inhibiting mitochondrial enzymes. Healthy mitochondria normally import and degrade the PD-related protein Pink1, but Pink1 exits mitochondria if the membrane potential collapses and initiates Parkin-mediated mitophagy. Under normal circumstances, this process helps clear dysfunctional mitochondria and contributes to cellular health, but PINK1 mutations associated with PD exit mitochondria with intact membrane potentials, disrupting mitochondrial dynamics, leading to pathology. Thus, TOM40 plays a central role in the mitochondrial dysfunction that underlies age-related neurodegenerative diseases. Learning about the factors that control TOM40 levels and activity, and how TOM40, specifically, and the TOM complex, generally, interacts with potentially pathogenic proteins, will provide deeper insights to AD and PD pathogenesis, and possibly new targets for preventative and/or therapeutic treatments.

Keywords: APOE; Alzheimer’s Disease; Mitochondria; PARK2; PINK1; Parkinson’s Disease; Regulation Of Gene Expression; SNCA; TOM Complex; TOMM40.

Figure 1

a: Schematic representation of normal mitochondrial protein uptake. Proteins with cleavable terminal targeting signals (positively charged, red) are targeted to the TIM23 complex for release into the inner membrane or into the matrix. Some matrix proteins travel from TIM23 to the Mia40/Erv1 complex for redox-mediated folding. Proteins with multiple membrane-spanning domains have internal targeting sequences (uncharged, red); these are targeted to the TIM22 complex, for lateral release into the inner membrane. Outer membrane β-barrel proteins, such as TOM40 itself, contain non-cleavable targeting domains and are transferred from TOM to intermembrane space chaperones via SAM (not shown) to TOB, required for insertion into the outer membrane. Outer membrane proteins with a-helical segments use multiple pathways for insertion into the outer membrane, some involving MIM1, but none involving TOM. b: Model of the possible hijacking of mitochondrial protein uptake by pathogenic proteins. Pink1, α-synuclein and APP possess alternative mitochondrial targeting signals. Pink1, is targeted to the TIM23 complex where it is subject to proteolytic degradation by the Mitochondrial Proteolytic Peptidase and possibly other proteases. If the Δψ collapses, wild-type Pink1, escapes and elicits Park2 binding to mitochondria, leading eventually to autophagy of the damaged mitochondria. PD pathogenic mutant proteins escape proteolysis and exit mitochondria, even in the presence of an intact membrane potential, leading to loss of healthy mitochondria. α-synuclein targets inner membrane proteins, such as cytochrome c oxidase. Aβ peptides enter mitochondria through the TOM40 pore and target matrix and inner membrane proteins. APP enters and blocks the TOM40 channel and interferes with mitochondrial oxidative processes.

Figure 2

Levels of GRP75 and TOM40 (A) and Complex IV activity (B) in control (transfected with an empty vector) and TOM40 over-expressing cells. Cultures at 75 –85% confluence were harvested in RIPA buffer supplemented with protease inhibitors and processed for determination of GRP75 and TOM40 by Western blotting (A) and Complex IV amount/activity using an ELISA/activity kit from MitoSciences according to the manufacturer’s instructions. Both HeLaC3 and TOM Mix cultures were processed simultaneously.

Figure 3

Complex I (A) and ODGH (B) levels in control (transfected with an empty vector) and TOM40 over-expressing cell lines. Cultures were harvested at 75 – 85% confluence in RIPA buffer without protease inhibitors (A) or fortified with protease inhibitors (B). After one freeze-thaw cycle the respective mitochondrial enzyme complexes were measured using activity/ELISA methodology with kits from MitoSciences according to the manufacturer’s instructions. C: ROS levels were determined in control and TOM40 over-expressing cells after a mild hypoglycemic shock. Growth medium was removed from cultures once they had attained 75 – 85% confluence and was replaced with either fresh medium containing 25 mM glucose (standard) or 5 mM glucose (low glucose). After 24 hours the medium was replaced with Earl’s buffered salts solution containing 5 mM malate, 5 mM pyruvate and 10μM 5’,6’-chloromethoxy-2,7-dichlorodihydro fluorescein diacetate to quantify ROS. For all three experiments, all cell lines were processed simultaneously.