Narayan G. Avadhani

Mitochondrial Signaling: The Retrograde Response

Mitochondrial retrograde signaling is a pathway of communication from mitochondria to the nucleus that influences many cellular and organismal activities under both normal and pathophysiological conditions. In yeast it is used as a sensor of mitochondrial dysfunction that initiates readjustments of carbohydrate and nitrogen metabolism. In both yeast and animal cells, retrograde signaling is linked to TOR signaling, but the precise connections are unclear. In mammalian cells, mitochondrial dysfunction sets off signaling cascades through altered Ca(2+) dynamics, which activate factors such as NFkappaB, NFAT, and ATF. Retrograde signaling also induces invasive behavior in otherwise nontumorigenic cells implying a role in tumor progression.

Function of Mitochondrial Stat3 in Cellular Respiration

Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3–/– cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the proteins function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.

Mitochondrial Import and Accumulation of α-Synuclein Impair Complex I in Human Dopaminergic Neuronal Cultures and Parkinson Disease Brain

α-Synuclein, a protein implicated in the pathogenesis of Parkinson disease (PD), is thought to affect mitochondrial functions, although the mechanisms of its action remain unclear. In this study we show that the N-terminal 32 amino acids of human α-synuclein contain cryptic mitochondrial targeting signal, which is important for mitochondrial targeting of α-synuclein. Mitochondrial imported α-synuclein is predominantly associated with the inner membrane. Accumulation of wild-type α-synuclein in the mitochondria of human dopaminergic neurons caused reduced mitochondrial complex I activity and increased production of reactive oxygen species. However, these defects occurred at an early time point in dopaminergic neurons expressing familial α-synuclein with A53T mutation as compared with wild-type α-synuclein. Importantly, α-synuclein that lacks mitochondrial targeting signal failed to target to the mitochondria and showed no detectable effect on complex I function. The PD relevance of these results was investigated using mitochondria of substantia nigra, striatum, and cerebellum of postmortem late-onset PD and normal human brains. Results showed the constitutive presence of ∼14-kDa α-synuclein in the mitochondria of all three brain regions of normal subjects. Mitochondria of PD-vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of α-synuclein and decreased complex I activity. Analysis of mitochondria from PD brain and α-synuclein expressing dopaminergic neuronal cultures using blue native gel electrophoresis and immunocapture technique showed the association of α-synuclein with complex I. These results provide evidence that mitochondrial accumulated α-synuclein may interact with complex I and interfere with its functions.

Accumulation of Amyloid Precursor Protein in the Mitochondrial Import Channels of Human Alzheimer’s Disease Brain Is Associated with Mitochondrial Dysfunction

Mitochondrial dysfunction is one of the major intracellular lesions of Alzheimer’s disease (AD). However, the causative factors involved in the mitochondrial dysfunction in human AD are not well understood. Here we report that nonglycosylated full-length and C-terminal truncated amyloid precursor protein (APP) accumulates exclusively in the protein import channels of mitochondria of human AD brains but not in age-matched controls. Furthermore, in AD brains, mitochondrially associated APP formed stable ∼480 kDa complexes with the translocase of the outer mitochondrial membrane 40 (TOM40) import channel and a super complex of ∼620 kDa with both mitochondrial TOM40 and the translocase of the inner mitochondrial membrane 23 (TIM23) import channel TIM23 in an “Nin mitochondria–Cout cytoplasm” orientation. Accumulation of APP across mitochondrial import channels, which varied with the severity of AD, inhibited the entry of nuclear-encoded cytochrome c oxidase subunits IV and Vb proteins, which was associated with decreased cytochrome c oxidase activity and increased levels of H2O2. Regional distribution of mitochondrial APP showed higher levels in AD-vulnerable brain regions, such as the frontal cortex, hippocampus, and amygdala. Mitochondrial accumulation of APP was also observed in the cholinergic, dopaminergic, GABAergic, and glutamatergic neuronal types in the category III AD brains. The levels of translocationally arrested mitochondrial APP directly correlated with mitochondrial dysfunction. Moreover, apolipoprotein genotype analysis revealed that AD subjects with the E3/E4 alleles had the highest content of mitochondrial APP. Collectively, these results suggest that abnormal accumulation of APP across mitochondrial import channels, causing mitochondrial dysfunction, is a hallmark of human AD pathology.

Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells

Alzheimers amyloid precursor protein 695 (APP) is a plasma membrane protein, which is known to be the source of the toxic amyloid β (Aβ) peptide associated with the pathogenesis of Alzheimers disease (AD). Here we demonstrate that by virtue of its chimeric NH2-terminal signal, APP is also targeted to mitochondria of cortical neuronal cells and select regions of the brain of a transgenic mouse model for AD. The positively charged residues at 40, 44, and 51 of APP are critical components of the mitochondrial-targeting signal. Chemical cross-linking together with immunoelectron microscopy show that the mitochondrial APP exists in NH2-terminal inside transmembrane orientation and in contact with mitochondrial translocase proteins. Mutational studies show that the acidic domain, which spans sequence 220–290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side. Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism. These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions.

Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter‐organelle crosstalk

We have investigated the mechanism of mitochondrial–nuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m‐chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Δψm) and electron transport‐coupled ATP synthesis, which was also accompanied by an elevated steady‐state cytosolic Ca2+ level ([Ca2+]i). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor‐1 (RyR‐1), hence potentiating the Ca2+ release in response to its modulator, caffeine; (ii) enhanced levels of Ca2+‐responsive factors calineurin, calcineurin‐dependent NFATc (cytosolic counterpart of activated T‐cell‐specific nuclear factor) and c‐Jun N‐terminal kinase (JNK)‐dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF‐κB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady‐state [Ca2+]i were normalized in genetically reverted cells which contain near‐normal mtDNA levels. We propose that the mitochondria‐to‐nucleus stress signaling occurs through cytosolic [Ca2+]i changes, which are likely to be due to reduced ATP and Ca2+ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

Mitochondria-to-nucleus stress signaling induces phenotypic changes, tumor progression and cell invasion.

Recently we showed that partial depletion of mitochondrial DNA (genetic stress) or treatment with mitochondrial‐specific inhibitors (metabolic stress) induced a stress signaling that was associated with increased cytoplasmic‐free Ca2+ [Ca2+]c. In the present study we show that the mitochondria‐to‐nucleus stress signaling induces invasive phenotypes in otherwise non‐invasive C2C12 myoblasts and human pulmonary carcinoma A549 cells. Tumor‐specific markers cathepsin L and transforming growth factor β (TGFβ) are overexpressed in cells subjected to mitochondrial genetic as well as metabolic stress. C2C12 myoblasts subjected to stress showed 4‐ to 6‐fold higher invasion through reconstituted Matrigel membrane as well as rat tracheal xenotransplants in Scid mice. Activation of Ca2+‐dependent protein kinase C (PKC) under both genetic and metabolic stress conditions was associated with increased cathepsin L gene expression, which contributes to increased invasive property of cells. Reverted cells with ∼70% of control cell mtDNA exhibited marker mRNA contents, cell morphology and invasive property closer to control cells. These results provide insights into a new pathway by which mitochondrial DNA and membrane damage can contribute to tumor progression and metastasis.

Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells

We have investigated mechanisms of mitochondrial stress-induced phenotypic changes and cell invasion in tumorigenic but poorly invasive human pulmonary carcinoma A549 cells that were partly depleted of mitochondrial DNA (mtDNA). Depletion of mtDNA (genetic stress) caused a markedly lower electron transport-coupled ATP synthesis, loss of mitochondrial membrane potential, elevation of steady state [Ca2+]c, and notably induction of both glycolysis and gluconeogenic pathway enzymes. Markers of tumor invasion, cathepsin L and TGFβ1, were overexpressed; calcium-dependent MAP kinases (ERK1 and ERK2) and calcineurin were activated. The levels of anti-apoptotic proteins Bcl2 and Bcl-XL were increased, and the cellular levels of pro-apoptotic proteins Bid and Bax were reduced. Both mtDNA-depleted cells (genetic stress) and control cells treated with carbonyl cyanide m-chlorophenylhydrazone (metabolic stress) exhibited higher invasive behavior than control cells in a Matrigel basement membrane matrix assay system. MtDNA-depleted cells stably expressing anti-sense cathepsin L RNA, TGFβ1 RNA, or treated with specific inhibitors showed reduced invasion. Reverted cells with 80% of control cell mtDNA exhibited marker protein levels, cell morphology and invasive property closer to control cells. Our results suggest that the mitochondria-to-nucleus signaling pathway operating through increased [Ca2+]c plays an important role in cancer progression and metastasis.

A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis.

Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.

Mitochondria to nucleus stress signaling a distinctive mechanism of NFκB/Rel activation through calcineurin-mediated inactivation of IκBβ

Mitochondrial genetic and metabolic stress causes activation of calcineurin (Cn), NFAT, ATF2, and NFκB/Rel factors, which collectively alter the expression of an array of nuclear genes. We demonstrate here that mitochondrial stress–induced activation of NFκB/Rel factors involves inactivation of IκBβ through Cn-mediated dephosphorylation. Phosphorylated IκBβ is a substrate for Cn phosphatase, which was inhibited by FK506 and RII peptide. Chemical cross-linking and coimmunoprecipitation show that NFκB/Rel factor–bound IκBβ forms a ternary complex with Cn under in vitro and in vivo conditions that was sensitive to FK506. Results show that phosphorylation at S313 and S315 from the COOH-terminal PEST domain of IκBβ is critical for binding to Cn. Mutations at S313/S315 of IκBβ abolished Cn binding, inhibited Cn-mediated increase of Rel proteins in the nucleus, and had a dominant-negative effect on the mitochondrial stress–induced expression of RyR1 and cathepsin L genes. Our results show the distinctive nature of mitochondrial stress–induced NFκB/Rel activation, which is independent of IKKα and IKKβ kinases and affects gene target(s) that are different from cytokine and TNFα-induced stress signaling. The results provide new insights into the role of Cn as a critical link between Ca2+ signaling and NFκB/Rel activation.