iPSC-Derived Neurons from Patients with POLG Mutations Exhibit Decreased Mitochondrial Content and Dendrite Simplification.

Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase gamma, result in clinical syndromes characterized by mitochondrial DNA (mtDNA) depletion in affected tissues with variable organ involvement. The brain is one of the most affected organs, and symptoms include intractable seizures, developmental delay, dementia, and ataxia. Patient-derived induced pluripotent stem cells (iPSCs) provide opportunities to explore mechanisms in affected cell types and potential therapeutic strategies. Fibroblasts from two patients were reprogrammed to create new iPSC models of POLG-related mitochondrial diseases. Compared with iPSC-derived control neurons, mtDNA depletion was observed upon differentiation of the POLG-mutated lines to cortical neurons. POLG-mutated neurons exhibited neurite simplification with decreased mitochondrial content, abnormal mitochondrial structure and function, and increased cell death. Expression of the mitochondrial kinase PTEN-induced kinase 1 (PINK1) mRNA was decreased in patient neurons. Overexpression of PINK1 increased mitochondrial content and ATP:ADP ratios in neurites, decreasing cell death and rescuing neuritic complexity. These data indicate an intersection of polymerase gamma and PINK1 pathways that may offer a novel therapeutic option for patients affected by this spectrum of disorders.

Chronic treatment with the complex I inhibitor MPP+ depletes endogenous PTEN-induced kinase 1 (PINK1) via up-regulation of Bcl-2-associated athanogene 6 (BAG6).

Mitochondrial dysfunction is implicated in sporadic and familial Parkinson's disease (PD). However, the mechanisms that impair homeostatic responses to mitochondrial dysfunction remain unclear. Previously, we found that chronic, low-dose administration of the mitochondrial complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+) dysregulates mitochondrial fission-fusion, mitophagy, and mitochondrial biogenesis. Given that PTEN-induced kinase 1 (PINK1) regulates mitochondrial function, dynamics, and turnover, we hypothesized that alterations in endogenous PINK1 levels contribute to depletion of mitochondria during chronic complex I injury. Here we found that chronic MPP+ treatment of differentiated SH-SY5Y neuronal cells significantly decreases PINK1 expression prior to reductions in other mitochondrial components. Furthermore, Bcl2-associated athanogene 6 (BAG6, BAT3, or Scythe), a protein involved in protein quality control and degradation, was highly up-regulated during the chronic MPP+ treatment. BAG6 interacted with PINK1, and BAG6 overexpression decreased the half-life of PINK1. Conversely, siRNA-mediated BAG6 knockdown prevented chronic MPP+ stress-induced loss of PINK1, reversed MPP+-provoked mitochondrial changes, increased cell viability, and prevented MPP+-induced dendrite shrinkage in primary neurons. These results indicate that BAG6 up-regulation during chronic complex I inhibition contributes to mitochondrial pathology by decreasing the levels of endogenous PINK1. Given that recessive mutations in PINK1 cause familial PD, the finding of accelerated PINK1 degradation in the chronic MPP+ model suggests that PINK1 loss of function represents a point of convergence between the neurotoxic and genetic causes of PD.

PINK1 Interacts with VCP/p97 and Activates PKA to Promote NSFL1C/p47 Phosphorylation and Dendritic Arborization in Neurons.

While PTEN-induced kinase 1 (PINK1) is well characterized for its role in mitochondrial homeostasis, much less is known concerning its ability to prevent synaptodendritic degeneration. Using unbiased proteomic methods, we identified valosin-containing protein (VCP) as a major PINK1-interacting protein. RNAi studies demonstrate that both VCP and its cofactor NSFL1C/p47 are necessary for the ability of PINK1 to increase dendritic complexity. Moreover, PINK1 regulates phosphorylation of p47, but not the VCP co-factor UFD1. Although neither VCP nor p47 interact directly with PKA, we found that PINK1 binds and phosphorylates the catalytic subunit of PKA at T197 [PKAcat(pT197)], a site known to activate the PKA holoenzyme. PKA in turn phosphorylates p47 at a novel site (S176) to regulate dendritic complexity. Given that PINK1 physically interacts with both the PKA holoenzyme and the VCP-p47 complex to promote dendritic arborization, we propose that PINK1 scaffolds a novel PINK1-VCP-PKA-p47 signaling pathway to orchestrate dendritogenesis in neurons. These findings highlight an important mechanism by which proteins genetically implicated in Parkinson's disease (PD; PINK1) and frontotemporal dementia (FTD; VCP) interact to support the health and maintenance of neuronal arbors.

ERK-mediated phosphorylation of TFAM downregulates mitochondrial transcription: implications for Parkinson's disease.

Mitochondrial transcription factor A (TFAM) regulates mitochondrial biogenesis, which is downregulated by extracellular signal-regulated protein kinases (ERK1/2) in cells treated chronically with the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+). We utilized mass spectrometry to identify ERK1/2-dependent TFAM phosphorylation sites. Mutation of TFAM at serine 177 to mimic phosphorylation recapitulated the effects of MPP+ in decreasing the binding of TFAM to the light strand promoter, suppressing mitochondrial transcription. Mutant TFAM was unable to affect respiratory function or rescue the effects of MPP+ on respiratory complexes. These data disclose a novel mechanism by which ERK1/2 regulates mitochondrial function through direct phosphorylation of TFAM.

Beyond the mitochondrion: cytosolic PINK1 remodels dendrites through protein kinase A.

The subcellular compartmentalization of kinase activity allows for regulation of distinct cellular processes involved in cell differentiation or survival. The PTEN-induced kinase 1 (PINK1), which is linked to Parkinson's disease, is a neuroprotective kinase localized to cytosolic and mitochondrial compartments. While mitochondrial targeting of PINK1 is important for its activities regulating mitochondrial homeostasis, the physiological role of the cytosolic pool of PINK1 remains unknown. Here, we demonstrate a novel role for cytosolic PINK1 in neuronal differentiation/neurite maintenance. Over-expression of wild-type PINK1, but not a catalytically inactive form of PINK1(K219M), promoted neurite outgrowth in SH-SY5Y cells and increased dendritic lengths in primary cortical and midbrain dopaminergic neurons. To identify the subcellular pools of PINK1 involved in promoting neurite outgrowth, we transiently transfected cells with PINK1 constructs designed to target PINK1 to the outer mitochondrial membrane (OMM-PINK1) or restrict PINK1 to the cytosol (ΔN111-PINK1). Both constructs blocked cell death associated with loss of endogenous PINK1. However, transient expression of ΔN111-PINK1, but not of OMM-PINK1 or ΔN111-PINK1(K219M), promoted dendrite outgrowth in primary neurons, and rescued the decreased dendritic arborization of PINK1-deficient neurons. Mechanistically, the cytosolic pool of PINK1 regulated neurite morphology through enhanced anterograde transport of dendritic mitochondria and amplification of protein kinase A-related signaling pathways. Our data support a novel role for PINK1 in regulating dendritic morphogenesis.

Distinct mechanisms for induction and tolerance regulate the immediate early genes encoding interleukin 1ß and tumor necrosis factor α.

Interleukin-1ß and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPß and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators.

After the banquet: mitochondrial biogenesis, mitophagy, and cell survival.

Mitochondria are highly dynamic organelles of crucial importance to the proper functioning of neuronal, cardiac and other cell types dependent upon aerobic efficiency. Mitochondrial dysfunction has been implicated in numerous human conditions, to include cancer, metabolic diseases, neurodegeneration, diabetes, and aging. In recent years, mitochondrial turnover by macroautophagy (mitophagy) has captured the limelight, due in part to discoveries that genes linked to Parkinson disease regulate this quality control process. A rapidly growing literature is clarifying effector mechanisms that underlie the process of mitophagy; however, factors that regulate positive or negative cellular outcomes have been less studied. Here, we review the literature on two major pathways that together may determine cellular adaptation vs. cell death in response to mitochondrial dysfunction. Mitochondrial biogenesis and mitophagy represent two opposing, but coordinated processes that determine mitochondrial content, structure, and function. Recent data indicate that the capacity to undergo mitochondrial biogenesis, which is dysregulated in disease states, may play a key role in determining cell survival following mitophagy-inducing injuries. The current literature on major pathways that regulate mitophagy and mitochondrial biogenesis is summarized, and mechanisms by which the interplay of these two processes may determine cell fate are discussed. We conclude that in primary neurons and other mitochondrially dependent cells, disruptions in any phase of the mitochondrial recycling process can contribute to cellular dysfunction and disease. Given the emerging importance of crosstalk among regulators of mitochondrial function, autophagy, and biogenesis, signaling pathways that coordinate these processes may contribute to therapeutic strategies that target or regulate mitochondrial turnover and regeneration.

TRAF6 protein couples Toll-like receptor 4 signaling to Src family kinase activation and opening of paracellular pathway in human lung microvascular endothelia.

Gram-negative bacteria release lipopolysaccharide (LPS) into the bloodstream. Here, it engages Toll-like receptor (TLR) 4 expressed in human lung microvascular endothelia (HMVEC-Ls) to open the paracellular pathway through Src family kinase (SFK) activation. The signaling molecules that couple TLR4 to the SFK-driven barrier disruption are unknown. In HMVEC-Ls, siRNA-induced silencing of TIRAP/Mal and overexpression of dominant-negative TIRAP/Mal each blocked LPS-induced SFK activation and increases in transendothelial [(14)C]albumin flux, implicating the MyD88-dependent pathway. LPS increased TRAF6 autoubiquitination and binding to IRAK1. Silencing of TRAF6, TRAF6-dominant-negative overexpression, or preincubation of HMVEC-Ls with a cell-permeable TRAF6 decoy peptide decreased both LPS-induced SFK activation and barrier disruption. LPS increased binding of both c-Src and Fyn to GST-TRAF6 but not to a GST-TRAF6 mutant in which the three prolines in the putative Src homology 3 domain-binding motif (amino acids 461-469) were substituted with alanines. A cell-permeable decoy peptide corresponding to the same proline-rich motif reduced SFK binding to WT GST-TRAF6 compared with the Pro → Ala-substituted peptide. Finally, LPS increased binding of activated Tyr(P)(416)-SFK to GST-TRAF6, and preincubation of HMVEC-Ls with SFK-selective tyrosine kinase inhibitors, PP2 and SU6656, diminished TRAF6 binding to c-Src and Fyn. During the TRAF6-SFK association, TRAF6 catalyzed Lys(63)-linked ubiquitination of c-Src and Fyn, whereas SFK activation increased tyrosine phosphorylation of TRAF6. The TRAF6 decoy peptide blocked both LPS-induced SFK ubiquitination and TRAF6 phosphorylation. Together, these data indicate that the proline-rich Src homology 3 domain-binding motif in TRAF6 interacts directly with activated SFKs to couple LPS engagement of TLR4 to SFK activation and loss of barrier integrity in HMVEC-Ls.

TRAF6 is autoinhibited by an intramolecular interaction which is counteracted by trans-ubiquitination.

The tumor necrosis factor (TNF) receptor associated factor (TRAF) class of intracellular signal transducers is responsible for mediating many of the activation events initiated by TNF receptor (TNFR) and Toll-like/Interleukin-1, -17, and -18 receptor (TIR) families. Investigation of the mechanism by which TRAF6 is activated has demonstrated that two critical domains of the molecule required for activation and downstream signaling are involved in an interaction which renders the molecule inactive and structurally closed, as well as incapable of auto-ubiquitination. Contrary to its assumed role as a direct mediator of protein-protein interaction, TRAF auto-ubiquitination is a means of sustaining an open conformation active in downstream signaling. Furthermore, the inferred cis-function of TRAF auto-ubiquitination is now demonstrated to act in trans and requires both the RING-Zinc (RZ) fingers region and coiled-coil domain. We also observed that both the RZ fingers region and the MATH domain are targets for ubiquitination. Although TRAF6 ubiquitination has emerged as a hallmark of activation, trans-ubiquitination induced by two TRAF6 muteins is insufficient for NF-kappaB activation.

Phosphorylation of IRF8 in a pre-associated complex with Spi-1/PU.1 and non-phosphorylated Stat1 is critical for LPS induction of the IL1B gene.

Rapid induction of transcription is known to be mediated by factors which bind DNA following post-translational modification. We report here that non-tyrosine phosphorylated (NTP)-Stat1 is involved in a cooperative interaction with Spi-1/PU.1 and IRF8 to form a pre-associated, poised complex for IL1B gene induction. A double point mutation at a putative STAT binding site, which overlaps this composite Spi-1 x IRF8 site located in the LPS and IL-1 response element (LILRE), inhibited human IL1B LPS-dependent reporter activity to about 10 percent of the control wild type vector. Chromatin immunoprecipitation revealed stimulation-independent constitutive binding of IRF8, Spi-1 and NTP-Stat1 at the LILRE, while binding of C/EBP beta was activated at an adjacent C/EBP beta site after LPS stimulation. In contrast to Stat1, IRF8 was tyrosine phosphorylated following LPS treatment. Supporting the involvement of NTP-Stat1, LPS-induced IL1B reporter activity in monocytes was enhanced by ectopic expression of NTP-Stat1 Y701F. In contrast, co-expression of a Y211F IRF8 mutein functioned as a dominant-negative inhibitor of LPS-induced IL1B reporter activity. In vitro DNA binding using extracts from LPS-treated monocytes confirmed that the LILRE enhancer constitutively binds a trimolecular complex containing IRF8, Spi-1 and NTP-Stat1. Binding studies using in vitro-expressed proteins revealed that NTP-Stat1 enhanced the binding of Spi-1 and IRF8 to the LILRE. Co-expression of TRAF6, an LPS surrogate, with Spi-1 and IRF8 enhanced IL1B reporter activity in HEK293R cells, which was dramatically reduced when Y211F IRF8 was co-expressed. These results suggest that the rapid transcriptional induction of an important inflammatory gene is dependent upon constitutive cooperative binding of a Spi-1 x IRF8 x NTP-Stat1 complex to the LILRE, which primes the gene for immediate induction following IRF8 phosphorylation. Phosphorylation of chromatin pre-associated factors like IRF8 may be an important strategy for the rapid transcriptional activation of genes involved in innate immunity.

TRAF6 activation of PI 3-kinase-dependent cytoskeletal changes is cooperative with Ras and is mediated by an interaction with cytoplasmic Src.

Interleukin 1 (IL-1) has been implicated in the reorganization of the actin cytoskeleton. An expression vector encoding a PKB/Akt pleckstrin-homology domain fused to a fluorescent protein was used to detect phosphoinositide 3-kinase (PI 3-kinase) products. It was observed that PI 3-kinase was activated either by treatment with IL-1 or by expression of either TRAF6, Src, MyD88 or dominant-positive PI 3-kinase, and resulted in the formation of long filopodia-like cellular protrusions that appeared to branch at membrane sites consisting of clusters of phosphoinositide. This depended upon a TRAF6 polyproline motif and Src catalytic activity, and was blocked by inhibitors of PI 3-kinase, Src and Ras. Using both conventional and split fluorescent protein probes fused to expressed TRAF6 and Src in living cells, the polyproline sequence of TRAF6 and the Src-homology 3 (SH3) domain of Src were shown to be required for interaction between these two proteins. Interaction occurred within the cytoplasm, and not at either the cell membrane or cytoplasmic sequestosomes. In addition, co-transfection of vectors expressing fluorescent-protein-fused TRAF6 and non-fluorescent MyD88, IRAK1 and IRAK2 revealed an inverse correlation between increased sequestosome formation and activation of both PI 3-kinase and NF-kappaB. Although a key factor in TRAF6-dependent activation of PI 3-kinase, ectopic expression of Src was insufficient for NF-kappaB activation and, in contrast to NF-kappaB, was not inhibited by IRAK2.

Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos.

Osteoclasts are specialized macrophages that resorb bone. Mice lacking the AP-1 component c-Fos are osteopetrotic because of a lack of osteoclast differentiation and show an increased number of macrophages. The nature of the critical function of c-Fos in osteoclast differentiation is not known. Microarray analysis revealed that Nfatc1, another key regulator of osteoclastogenesis, was down-regulated in Fos(-/-) osteoclast precursors. Chromatin immunoprecipitation assay showed that c-Fos bound to the Nfatc1 and Acp5 promoters in osteoclasts. In vitro promoter analyses identified nuclear factor of activated T-cells (NFAT)/AP-1 sites in the osteoclast-specific Acp5 and Calcr promoters. Moreover, in Fos(-/-) precursors gene transfer of an active form of NFAT restored transcription of osteoclast-specific genes in the presence of receptor activator of the NF-kappaB ligand (RANKL), rescuing bone resorption. In the absence of RANKL, however, Fos(-/-) precursors were insensitive to NFAT-induced osteoclastogenesis unlike wild-type precursors. These data indicate that lack of Nfatc1 expression is the cause of the differentiation block in Fos(-/-) osteoclast precursors and that transcriptional induction of Nfatc1 is a major function of c-Fos in osteoclast differentiation.

sFlt-1 gene therapy of follicular thyroid carcinoma.

Tumor progression largely depends on blood supply and neovessel formation, and angiogenesis is emerging as a promising target for cancer therapy. Vascular endothelial growth factor (VEGF), a major proangiogenic molecule, stimulates angiogenesis via promoting endothelial proliferation, survival and migration. VEGF has been found to be up-regulated in various types of tumors and to be associated with tumor progression and poor prognosis. Inhibition of VEGF or its signaling pathway has been shown to suppress tumor angiogenesis and tumor growth. In the present study, we tested the antiangiogenic and antitumor effects of soluble VEGF receptor-1 [soluble Flt (sFlt)-1] on the growth of follicular thyroid carcinoma (FTC). We constructed a 293 embryonic kidney cell line (293-Flt1-3d) that expresses sFlt-1, which is composed of the first three extracellular domains of Flt-1. The 293-Flt1-3d cells inhibited the in vitro growth of human umbilical vein endothelial cells in a paracrine manner. The in vivo antitumor and antiangiogenic activities of the 293-Flt1-3d cells were tested. When 293-Flt1-3d cells were inoculated at a site remote to the FTC-133 tumor transplant, the growth of FTC-133 tumors were inhibited by 70.37%, as compared with the control treatment with 293 cells expressing control gene LacZ. Immunohistochemical analysis of microvessel densities in treated tumors demonstrated that 293-Flt1-3d cells robustly suppressed intratumoral angiogenesis. Our data suggest that a mammalian cell-mediated approach could effectively deliver sFlt-1 gene therapy and inhibit tumor angiogenesis and tumor growth.