Sarika Srivastava

PGC-1α/β induced expression partially compensates for respiratory chain defects in cells from patients with mitochondrial disorders

Members of the peroxisome proliferator-activated receptor gamma coactivator (PGC) family are potent inducers of mitochondrial biogenesis. We have tested the potential effect of increased mitochondrial biogenesis in cells derived from patients harboring oxidative phosphorylation defects due to either nuclear or mitochondrial DNA mutations. We found that the PGC-1alpha and/or PGC-1beta expression improved mitochondrial respiration in cells harboring a complex III or IV deficiency as well as in transmitochondrial cybrids harboring mitochondrial encephalomyopathy lactic acidosis and stroke A3243G tRNA((Leu)UUR) gene mutation. The respiratory function improvement was found to be associated with increased levels of mitochondrial components per cell, although this increase was not homogeneous. These results reinforce the concept that increased mitochondrial biogenesis is a promising venue for the treatment of mitochondrial diseases.

Central Presynaptic Terminals Are Enriched in ATP but the Majority Lack Mitochondria

Synaptic neurotransmission is known to be an energy demanding process. At the presynapse, ATP is required for loading neurotransmitters into synaptic vesicles, for priming synaptic vesicles before release, and as a substrate for various kinases and ATPases. Although it is assumed that presynaptic sites usually harbor local mitochondria, which may serve as energy powerhouse to generate ATP as well as a presynaptic calcium depot, a clear role of presynaptic mitochondria in biochemical functioning of the presynapse is not well-defined. Besides a few synaptic subtypes like the mossy fibers and the Calyx of Held, most central presynaptic sites are either en passant or tiny axonal terminals that have little space to accommodate a large mitochondrion. Here, we have used imaging studies to demonstrate that mitochondrial antigens poorly co-localize with the synaptic vesicle clusters and active zone marker in the cerebral cortex, hippocampus and the cerebellum. Confocal imaging analysis on neuronal cultures revealed that most neuronal mitochondria are either somatic or distributed in the proximal part of major dendrites. A large number of synapses in culture are devoid of any mitochondria. Electron micrographs from neuronal cultures further confirm our finding that the majority of presynapses may not harbor resident mitochondria. We corroborated our ultrastructural findings using serial block face scanning electron microscopy (SBFSEM) and found that more than 60% of the presynaptic terminals lacked discernible mitochondria in the wild-type mice hippocampus. Biochemical fractionation of crude synaptosomes into mitochondria and pure synaptosomes also revealed a sparse presence of mitochondrial antigen at the presynaptic boutons. Despite a low abundance of mitochondria, the synaptosomal membranes were found to be highly enriched in ATP suggesting that the presynapse may possess alternative mechanism/s for concentrating ATP for its function. The potential mechanisms including local glycolysis and the possible roles of ATP-binding synaptic proteins such as synapsins, are discussed.

Emerging therapeutic roles for NAD+ metabolism in mitochondrial and age-related disorders

Nicotinamide adenine dinucleotide (NAD+) is a central metabolic cofactor in eukaryotic cells that plays a critical role in regulating cellular metabolism and energy homeostasis. NAD+ in its reduced form (i.e. NADH) serves as the primary electron donor in mitochondrial respiratory chain, which involves adenosine triphosphate production by oxidative phosphorylation. The NAD+/NADH ratio also regulates the activity of various metabolic pathway enzymes such as those involved in glycolysis, Kreb’s cycle, and fatty acid oxidation. Intracellular NAD+ is synthesized de novo from l-tryptophan, although its main source of synthesis is through salvage pathways from dietary niacin as precursors. NAD+ is utilized by various proteins including sirtuins, poly ADP-ribose polymerases (PARPs) and cyclic ADP-ribose synthases. The NAD+ pool is thus set by a critical balance between NAD+ biosynthetic and NAD+ consuming pathways. Raising cellular NAD+ content by inducing its biosynthesis or inhibiting the activity of PARP and cADP-ribose synthases via genetic or pharmacological means lead to sirtuins activation. Sirtuins modulate distinct metabolic, energetic and stress response pathways, and through their activation, NAD+ directly links the cellular redox state with signaling and transcriptional events. NAD+ levels decline with mitochondrial dysfunction and reduced NAD+/NADH ratio is implicated in mitochondrial disorders, various age-related pathologies as well as during aging. Here, I will provide an overview of the current knowledge on NAD+ metabolism including its biosynthesis, utilization, compartmentalization and role in the regulation of metabolic homoeostasis. I will further discuss how augmenting intracellular NAD+ content increases oxidative metabolism to prevent bioenergetic and functional decline in multiple models of mitochondrial diseases and age-related disorders, and how this knowledge could be translated to the clinic for human relevance.

The Mitochondrial Basis of Aging and Age-Related Disorders

Aging is a natural phenomenon characterized by progressive decline in tissue and organ function leading to increased risk of disease and mortality. Among diverse factors that contribute to human aging, the mitochondrial dysfunction has emerged as one of the key hallmarks of aging process and is linked to the development of numerous age-related pathologies including metabolic syndrome, neurodegenerative disorders, cardiovascular diseases and cancer. Mitochondria are central in the regulation of energy and metabolic homeostasis, and harbor a complex quality control system that limits mitochondrial damage to ensure mitochondrial integrity and function. The intricate regulatory network that balances the generation of new and removal of damaged mitochondria forms the basis of aging and longevity. Here, I will review our current understanding on how mitochondrial functional decline contributes to aging, including the role of somatic mitochondrial DNA (mtDNA) mutations, reactive oxygen species (ROS), mitochondrial dynamics and quality control pathways. I will further discuss the emerging evidence on how dysregulated mitochondrial dynamics, mitochondrial biogenesis and turnover mechanisms contribute to the pathogenesis of age-related disorders. Strategies aimed to enhance mitochondrial function by targeting mitochondrial dynamics, quality control, and mitohormesis pathways might promote healthy aging, protect against age-related diseases, and mediate longevity.

Prediction of the maximal stability temperature of monomeric globular proteins solely from amino acid sequence

Globular protein thermostability is characterized the cold denaturation, maximal stability (T ms) and heat denaturation temperatures. For mesophilic globular proteins, T ms typically ranges from −25°C to +35°C. We show that the indirect estimate of T ms from calorimetry and the direct estimate from chemical denaturation performed in a range of temperatures are in close agreement. The heat capacity change of unfolding per mol residue (ΔC p) alone is shown to accurately predict T ms. ΔC p and hence T ms can be predicted solely from the protein sequence. The average difference in free energy of unfolding at the observed and predicted values of T ms is 1.0 kcal mol−1, which is small compared to typical values of the total free energy of unfolding.

Mitochondrial DNA structure and function

Publisher Summary This chapter discusses mitochondrial DNA (mtDNA) structures and its functions. The human mtDNA is representative of mammalian mitochondrial genomes. Comparison of the nucleotide sequences of mammal mtDNA revealed some degree of conservation in the promoter regions as well as in three other regions (termed “Conserved Sequence Blocks,” or CSB I, CSB II, and CSB III). The mammalian mitochondrial genome has two modes of replication. The first one involves the asymmetric replication of the leading and lagging strands. The second one, based on the observation of replication intermediates in two-dimensional (2D) gels suggested that replication, in a certain number of mtDNA molecules, involves coupled leading- and lagging-strand synthesis. Many factors involved in mammalian mtDNA replication have been characterized. Because of its probable prokaryote origin, in many aspects, the mitochondrion behaves as an independent entity living inside an eukaryotic cell. All basic processes associated with life (DNA maintenance, transcription, and translation) occur inside the organelle. However, the vast majority of the factors involved in promoting and controlling these processes are borrowed from the cytoplasm, where nuclear-coded proteins are synthesized.

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimers and Parkinsons diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of <200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

X-linked intellectual disability gene CASK regulates postnatal brain growth in a non-cell autonomous manner

The phenotypic spectrum among girls with heterozygous mutations in the X-linked intellectual disability (XLID) gene CASK (calcium/calmodulin-dependent serine protein kinase) includes postnatal microcephaly, ponto-cerebellar hypoplasia, seizures, optic nerve hypoplasia, growth retardation and hypotonia. Although CASK knockout mice were previously reported to exhibit perinatal lethality and a 3-fold increased apoptotic rate in the brain, CASK deletion was not found to affect neuronal physiology and their electrical properties. The pathogenesis of CASK associated disorders and the potential function of CASK therefore remains unknown. Here, using Cre-LoxP mediated gene excision experiments; we demonstrate that deleting CASK specifically from mouse cerebellar neurons does not alter the cerebellar architecture or function. We demonstrate that the neuron-specific deletion of CASK in mice does not cause perinatal lethality but induces severe recurrent epileptic seizures and growth retardation before the onset of adulthood. Furthermore, we demonstrate that although neuron-specific haploinsufficiency of CASK is inconsequential, the CASK mutation associated human phenotypes are replicated with high fidelity in CASK heterozygous knockout female mice (CASK(+/-)). These data suggest that CASK-related phenotypes are not purely neuronal in origin. Surprisingly, the observed microcephaly in CASK(+/-) animals is not associated with a specific loss of CASK null brain cells indicating that CASK regulates postnatal brain growth in a non-cell autonomous manner. Using biochemical assay, we also demonstrate that CASK can interact with metabolic proteins. CASK knockdown in human cell lines cause reduced cellular respiration and CASK(+/-) mice display abnormalities in muscle and brain oxidative metabolism, suggesting a novel function of CASK in metabolism. Our data implies that some phenotypic components of CASK heterozygous deletion mutation associated disorders represent systemic manifestation of metabolic stress and therefore amenable to therapeutic intervention.

Two microcephaly-associated novel missense mutations in CASK specifically disrupt the CASK–neurexin interaction

Deletion and truncation mutations in the X-linked gene CASK are associated with severe intellectual disability (ID), microcephaly and pontine and cerebellar hypoplasia in girls (MICPCH). The molecular origin of CASK-linked MICPCH is presumed to be due to disruption of the CASK–Tbr-1 interaction. This hypothesis, however, has not been directly tested. Missense variants in CASK are typically asymptomatic in girls. We report three severely affected girls with heterozygous CASK missense mutations (M519T (2), G659D (1)) who exhibit ID, microcephaly, and hindbrain hypoplasia. The mutation M519T results in the replacement of an evolutionarily invariant methionine located in the PDZ signaling domain known to be critical for the CASK–neurexin interaction. CASKM519T is incapable of binding to neurexin, suggesting a critically important role for the CASK–neurexin interaction. The mutation G659D is in the SH3 (Src homology 3) domain of CASK, replacing a semi-conserved glycine with aspartate. We demonstrate that the CASKG659D mutation affects the CASK protein in two independent ways: (1) it increases the protein’s propensity to aggregate; and (2) it disrupts the interface between CASK’s PDZ (PSD95, Dlg, ZO-1) and SH3 domains, inhibiting the CASK–neurexin interaction despite residing outside of the domain deemed critical for neurexin interaction. Since heterozygosity of other aggregation-inducing mutations (e.g., CASKW919R) does not produce MICPCH, we suggest that the G659D mutation produces microcephaly by disrupting the CASK–neurexin interaction. Our results suggest that disruption of the CASK–neurexin interaction, not the CASK–Tbr-1 interaction, produces microcephaly and cerebellar hypoplasia. These findings underscore the importance of functional validation for variant classification.

Cellular Adaptations to Oxidative Phosphorylation Defects in Cancer

Mitochondrial DNA (mtDNA) somatic mutations or mutations in nuclear genes encoding mitochondrial proteins important for the assembly, activity, or maintenance of the individual oxidative phosphorylation (OXPHOS) complexes have been observed in tumors. Although the functional consequence of such mutations is unclear at the moment, retrograde signaling in response to OXPHOS defects can activate various nuclear genes and signaling pathways that alter mitochondrial function, tumor invasion, metastasis, redox-sensitive pathways, programmed cell death pathways, calcium signaling pathways, and cellular pathways leading to global changes in cellular morphology and architecture. In addition, we have found that some cancer cell lines harboring deleterious mtDNA mutations upregulate the expression of members of the peroxisome-proliferator activated γ coactivator 1 family of coactivators, probably to sustain the necessary ATP production for cell proliferation. In this chapter, we describe such cellular adaptations and changes in response to OXPHOS defects that are associated with a variety of cancer cell types.