Kambiz Alavian, PhD

The research in my laboratory revolves around the central theme of mitochondrial bioenergetic efficiency. The processes of ATP synthesis through oxidative phosphorylation and mitochondrial substrate production for anabolic growth both require an electrochemical (proton) gradient across the mitochondrial inner membrane. The proton gradient is produced as a result of redox reactions by the electron transport chain (ETC). Futile ion leak currents across the inner membrane impede ATP production by the mitochondrial F1Fo ATP synthase as well as the passive and active transport of substrates, including pyruvate, phosphate and glutamate into the mitochondrial matrix. The leak currents, therefore, not only cause inefficiency in energy production, they may also interfere with cellular growth by limiting mitochondrial substrate levels required for downstream anabolic processes. In recent years, we have found that this process is regulated by a number of neuroprotective oncogenes. The goal of research in my laboratory is to continue to understand the basic molecular mechanisms, including serine metabolism, involved in mitochondrial energetic efficiency and the connections between this process to proliferation of adult and pediatric brain tumor cells and neuronal physiology. We will also pursue the results of our recent drug discovery project to develop novel compounds for modulation of mitochondrial ion leak currents for treatment of glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG).

Neuronal metabolic plasticity

The efficient regulation of cellular energy production by mitochondria is dependent upon the integrity of the inner and outer mitochondrial membranes and the maintenance of the proton-motive force that drives the production of adenosine triphosphate (ATP) by the F1Fo ATP synthase complex. The proton gradient produced as a function of the activity of mitochondrial electron transport chain is also required for pumping of cations, including sodium and potassium from matrix, as well as passive and active transport of substrates, required for cellular and synaptic growth, repair and regeneration across the inner membrane. Under physiological conditions mitochondrial membranes contain a number of ion conductance pathways, including proton leak (uncoupling) currents, which comprise a major energy sink (up to 50% in healthy tissue). While baseline leak currents exist in healthy tissue, some conductance pathways seem to be functionally expressed or suppressed only under pathophysiological conditions. Mitochondrial dysfunction through increased activity of the ion leak channels is particularly problematic in neurons, which have exceptional energy requirements. Neurons require the physical translocation of mitochondria along axons and dendrites to synapses, providing numerous additional opportunities for disruption of critical energy supplies. Neuronal mitochondrial stress, as a result of trauma, aging and adverse environmental or genetic factors, would result in increased risk for degeneration and loss of the energetic capacity for growth, repair and regeneration. We have described an ion leak channel associated with the F1Fo ATP synthase that is activated by calcium or ROS and is regulated by the anti-apoptotic member of the Bcl-2 family, Bcl-xL. In my laboratory we have been investigating the molecular interactions that contribute to this mechanism of mitochondrial bioenergetic efficiency.

- The role of Parkinson’s disease gene DJ1 in modulation of neuronal energy metabolism. We have recently identified the early onset Parkinson’s disease gene DJ1 (PARK7) as a modulator of neuronal metabolic efficiency through direct interaction with the mitochondrial F1Fo ATP synthase. We have also found that the DJ1 associated regulatory complex is disrupted by the PD-associated mutations, resulting in reduced mitochondrial efficiency. The adequate levels of DJ1 are required at mitochondria to meet locally increased ATP demand under stress or intense neuronal and synaptic activity. Our unpublished data also demonstrate that the 14-3-3 protein is the main regulator of the subcellular localization and the bioenergetic functions of DJ1. We are currently characterizing the effect of this novel function of DJ1 and 14-3-3 on the bioenergetics of resting, active and degenerating neurons through measurements of cellular ATP levels, oxygen consumption and metabolomics studies.

- The role of Bcl-x_L in long-term synaptic potentiation. Synaptic potentiation produces larger and more frequently firing synapses that have higher metabolic demands compared to resting synapses. Our previous work has demonstrated that even in the absence of high frequency stimulation, over-expression of the anti-apoptotic molecule, Bcl-x_L, increases the number and size of synapses, localizes mitochondria to presynaptic sites, increases mitochondrial biomass, and enhances ATP production. Our unpublished data have shown that Bcl-x_L is crucial for events that ready the synapse for long-term potentiation (LTP), as Bcl-x_L overexpression increases the number of resting GluR1 AMPA receptors. Bcl-x_L overexpression increases the amplitude of mEPSCs, consistent with a role for Bcl-x_L in increasing responses to spontaneous transmitter release through effects on postsynaptic AMPA receptor insertion. The aim of this project is to test the novel hypothesis that Bcl-x_L interacts with the mitochondrial ATP synthase to close the inner membrane proton leak during high intensity synaptic stimulation. This will enhance mitochondrial metabolic efficiency in a long-lasting fashion. In aging and in neurodegenerative conditions, a decline in mitochondrial function has been correlated with decreased neuronal and synaptic function followed by a progressive neuronal loss.

- The connection between the degeneration of midbrain dopaminergic neurons and mitochondrial energy production. The selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) is the pathological hallmark of Parkinson’s disease. The dopaminergic neurons in the neighbouring ventral tegmental area (VTA) are relatively resistant to cell death. Several groups have characterized the gene/protein expression profile of the SNpc vs. VTA neurons. A number of differentially expressed proteins localize to mitochondria. There is also evidence that the mitochondria of the two types of dopaminergic neurons are different in their membrane potential and function. This project aims to identify the mitochondrial processes that contribute to vulnerability of SNpc neurons by comparing them to their VTA counterparts and to determine whether instability in the mitochondrial membrane potential predisposes neurons to degeneration.

- Mitochondrial control of protein translation in Fragile X. We have found that fragile X mental retardation protein (FMRP) localizes to the mitochondria and that enhanced mitochondrial efficiency reverses the defect in protein translation in mouse and human models of FX. We have also found that Bcl-x_L represses protein translation, similarly to FMRP, in a similar manner to or in collaboration with FMRP to increase oxidative metabolism acutely during stimulus dependent synaptogenesis. We have employed ATP measurements, mitochondrial ion channel recordings, and single synapse oxygen consumption studies to characterise the role of mitochondrial efficiency in protein translation. The failure to increase the use of oxidative metabolism by synapses during development or plasticity seems to contribute to abnormal synaptic phenotypes and abnormal synaptic function as illustrated by Fmr1^-/y mutant animals. We predict that rescuing these mitochondrial metabolic defects by pharmacological treatment or genetic ATP synthase channel modulation will normalize protein translation levels, synaptic phenotypes and behavior in the Fmr1^-/y mutant mouse. In particular, we are planning to test the effect of the candidate drugs that modulate the metabolic efficiency of the mitochondria (see Theme 3) in Fmr1^-/y mutant mice.

Mitochondrial efficiency and proliferation of adult and pediatric brain tumors

Resistance to cell death and reprogramming of energy metabolism are two prominent features of cancer cells. Mitochondria are known to be intimately linked to both these features and are believed to play a key role in a diversity of processes contributing to the metabolic plasticity and growth of cancer cells. While most of the energy of normal cells is provided by mitochondrial oxidative phosphorylation (OXPHOS), under normoxic or hypoxic conditions many cancer cell types obtain a major portion of their cellular ATP through glycolysis, a less efficient means of energy production. In such cells typically 40 to 60% of ATP generation may occur via glycolysis and mitochondria generate the remainder of the energy requirements. Mitochondrial substrates are also utilized in tumour cells for anabolic growth. The process of ATP production through OXPHOS and substrates production for anabolic growth both require generation of an electrochemical gradient across the mitochondrial inner membrane. Ion leak currents across the inner membrane, therefore, impede ATP production and slow down exchange of substrates required for proliferation and growth (Figure-1). The complete loss of membrane potential through the opening of the mitochondrial permeability transition pore may ultimately result in cell death.

- The role of mitochondrial metabolic efficiency in proliferation of brain tumor cells. A metabolic shift from mitochondrial OXPHOS toward aerobic glycolysis is believed to be a prominent feature of many cancer cell types. This feature of cancer cells, however, is not universal and the degree to which the tumour cells rely on different means of energy production is highly variable. We recently compared the metabolic profile of low-grade (astrocytoma) to the high-grade glioma cells (including GBM and DIPG) by performing metabolomics, gene expression analysis and biochemical studies. We found that while glycolysis is highly active in both grade 3 and grade 4 cell types, the glioma (grade 4) cells also rely heavily on their mitochondria for ATP production and for anabolic processes. We also demonstrated that this process involves enhanced mitochondrial coupling and efficiency in ATP synthesis through closure of the inner membrane leak channels. We are currently examining the source of the leak currents.

- The connection between serine metabolism and the TCA cycle Anaplerosis. In addition to modulation of ATP production efficiency, the mitochondrial ion leak channel regulate the exchange of ions and substrates including pyruvate and glutamate across the inner membrane. These substrates are utilized for not only ATP production but for maintaining the TCA cycle anaplerotic flux required for biosynthesis of lipids, amino acids and nucleotides. Through a metabolomics study we have demonstrated an inverse correlation between the rate of serine metabolism and the mitochondrial pyruvate and glutamate uptake. Our data indicates that the grade 3 astrocytoma cells, with a low rate of proliferation, have a higher rate of serine and glycine metabolism in comparison to their grade 4 counterparts. The decrease in serine metabolism is mainly due to downregulation of a key enzyme phosphohydroxythreonine aminotransferase in the grade 4 glioma cells. The downregulation of serine metabolism in turn leads to upregulation of pyruvate production. The excess pyruvate is utilized by mitochondria for ATP production and for the biosynthetic processes required for proliferation. Since the mitochondrial pyruvate uptake depends on the mitochondrial membrane potential, modulation of the ion leak channels would change the growth rate of the glioblastoma cells. We are currently investigating this concept for development of novel therapeutics for treatment of glioblastoma multiforme tumors.