Clueless regulates aPKC activity and promotes self-renewal cell fate in Drosophila lgl mutant larval brains

Asymmetric cell division of Drosophila neural stem cells or neuroblasts is an important process which gives rise to two different daughter cells, one of which is the stem cell itself and the other, a committed or differentiated daughter cell. During neuroblast asymmetric division, atypical Protein Kinase C (aPKC) activity is tightly regulated; aberrant levels of activity could result in tumorigenesis in third instar larval brain. We identified clueless (clu), a genetic interactor of parkin (park), as a novel regulator of aPKC activity. It preferentially binds to the aPKC/Bazooka/Partition Defective 6 complex and stabilizes aPKC levels. In clu mutants, Miranda (Mira) and Numb are mislocalized in small percentages of dividing neuroblasts. Adult mutants are short-lived with severe locomotion defects. Clu promotes tumorigenesis caused by loss of function of lethal(2) giant larvae (lgl) in the larval brain. Removal of clu in lgl mutants rescues Mira and Numb mislocalization and restores the enlarged brain size. Western blot analyses indicate that the rescue is due to the down-regulation of aPKC levels in the lgl clu double mutant. Interestingly, the phenotype of the park mutant, which causes Parkinson's Disease-like symptoms in adult flies, is reminiscent of that of clu in neuroblast asymmetric division. Our study provides the first clue for the potential missing pathological link between temporally separated neurogenesis and neurodegeneration events; the minor defects during early neurogenesis could be a susceptible factor contributing to neurodegenerative diseases at later stages of life.     

Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal

Asymmetric cell division is a mechanism for generating cell diversity as well as maintaining stem cell homeostasis in both Drosophila and mammals. In Drosophila, larval neuroblasts are stem cell-like progenitors that divide asymmetrically to generate neurons of the adult brain. Mitotic neuroblasts localize atypical protein kinase C (aPKC) to their apical cortex. Cortical aPKC excludes cortical localization of Miranda and its cargo proteins Prospero and Brain tumor, resulting in their partitioning into the differentiating, smaller ganglion mother cell (GMC) where they are required for neuronal differentiation. In addition to aPKC, the kinases Aurora-A and Polo also regulate neuroblast self-renewal, but the phosphatases involved in neuroblast self-renewal have not been identified. Here we report that aPKC is in a protein complex in vivo with Twins, a Drosophila B-type protein phosphatase 2A (PP2A) subunit, and that Twins and the catalytic subunit of PP2A, called Microtubule star (Mts), are detected in larval neuroblasts. Both Twins and Mts are required to exclude aPKC from the basal neuroblast cortex: twins mutant brains, twins mutant single neuroblast mutant clones, or mts dominant negative single neuroblast clones all show ectopic basal cortical localization of aPKC. Consistent with ectopic basal aPKC is the appearance of supernumerary neuroblasts in twins mutant brains or twins mutant clones. We conclude that Twins/PP2A is required to maintain aPKC at the apical cortex of mitotic neuroblasts, keeping it out of the differentiating GMC, and thereby maintaining neuroblast homeostasis.     

Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids

Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψ_m) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψ_m in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis.     

The Proapoptotic F-box Protein Fbxl7 Regulates Mitochondrial Function by Mediating the Ubiquitylation and Proteasomal Degradation of Survivin

Fbxl7, a component of the Skp1·Cul1·F-box protein type ubiquitin E3 ligase, regulates mitotic cell cycle progression. Here we demonstrate that overexpression of Fbxl7 in lung epithelia decreases the protein abundance of survivin, a member of the inhibitor of apoptosis family. Fbxl7 mediates polyubiquitylation and proteasomal degradation of survivin by interacting with Glu-126 within its carboxyl-terminal α helix. Furthermore, both Lys-90 and Lys-91 within survivin serve as ubiquitin acceptor sites. Ectopically expressed Fbxl7 impairs mitochondrial function, whereas depletion of Fbxl7 protects mitochondria from actions of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of oxidative phosphorylation. Compared with wild-type survivin, cellular expression of a survivin mutant protein deficient in its ability to interact with Fbxl7 (E126A) and a ubiquitylation-resistant double point mutant (KK90RR/KK91RR) rescued mitochondria to a larger extent from damage induced by overexpression of Fbxl7. Therefore, these data suggest that the Skp1·Cul1·F-box protein complex subunit Fbxl7 modulates mitochondrial function by controlling the cellular abundance of survivin. The results raise opportunities for F-box protein targeting to preserve mitochondrial function.     

Drosophila APC2 and APC1 have overlapping roles in the larval brain despite their distinct intracellular localizations

The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and mammals have two APC family members. We further explored the functions of the Drosophila APCs using the larval brain as a model. We found that both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions. Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. Our experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins.     

Long-Term Live Cell Imaging and Automated 4D Analysis of Drosophila Neuroblast Lineages

The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.     

Proteasomes Associated with the Blm10 Activator Protein Antagonize Mitochondrial Fission through Degradation of the Fission Protein Dnm1

The conserved Blm10/PA200 activators bind to the proteasome core particle gate and facilitate turnover of peptides and unfolded proteins in vitro. We report here that Blm10 is required for the maintenance of functional mitochondria. BLM10 expression is induced 25-fold upon a switch from fermentation to oxidative metabolism. In the absence of BLM10, Saccharomyces cerevisiae cells exhibit a temperature-sensitive growth defect under oxidative growth conditions and produce colonies with dysfunctional mitochondria at high frequency. Loss of BLM10 leads to reduced respiratory capacity, increased mitochondrial oxidative damage, and reduced viability in the presence of oxidative stress or death stimuli. In the absence of BLM10, increased fragmentation of the mitochondrial network under oxidative stress is observed indicative of elevated activity of the mitochondrial fission machinery. The degradation of Dnm1, the main factor mediating mitochondrial fission, is impaired in the absence of BLM10 in vitro and in vivo. These data suggest that the mitochondrial functional and morphological changes observed are related to elevated Dnm1 levels. This hypothesis is supported by the finding that cells that constitutively overexpress DNM1 display the same mitochondrial defects as blm10Δ cells. The data are consistent with a model in which Blm10 proteasome-mediated turnover of Dnm1 is required for the maintenance of mitochondrial function and provides cytoprotection under conditions that induce increased mitochondrial damage and programmed cell death.     

Mutation of the central nervous system neuroblast proliferation repressor ana leads to defects in larval olfactory behavior

In the developing nervous system, interactions between glia and immature neurons or neuroblasts regulate axon pathfinding, migration, and cell division, and therefore affect structure and function. Glial control of neuroblast cell division has been documented by studies of the anachronism (ana) gene of Drosophila melanogaster. ana encodes a glycoprotein which, in the developing larval central nervous system, is secreted by glia that neighbor regulated neuroblasts. Mutations in ana lead to premature neuroblast proliferation in the larval brain. Examination of lacZ expression from an ana enhancer trap line as well as detection of the ana protein show that ana is also expressed in the larval antennal-maxillary complex (AMC) at all larval stages. As previously reported for the central nervous system, ana expression in the AMC appears to be confined to glial cells. Larval olfactory system function in ana mutants was assayed in a behavioral paradigm. When tested with the three different chemoattractants, third instar ana⁹ mutant larvae showed diminished olfactory response compared to controls. Examination of a second ana allele revealed aberrant olfactory response to ethyl acetate, demonstrating that more than one mutation in ana can give rise to abnormal larval olfactory behavior. Assays of early first instar ana⁹ mutant larvae revealed defective olfactory behavior, implying that the olfactory phenotype stems from early larval AMC and/or embryonic origins. This is consistent with proliferation analysis in the early larval AMC region which uncovered a significantly higher number of S-phase cells in ana⁹ mutants. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 199–211, 1997     

Cortical aPKC kinase activity distinguishes neural stem cells from progenitor cells by ensuring asymmetric segregation of Numb

During asymmetric stem cell division, polarization of the cell cortex targets fate determinants unequally into the sibling daughters, leading to regeneration of a stem cell and production of a progenitor cell with restricted developmental potential. In mitotic neural stem cells (neuroblasts) in fly larval brains, the antagonistic interaction between the polarity proteins Lethal (2) giant larvae (Lgl) and atypical Protein Kinase C (aPKC) ensures self-renewal of a daughter neuroblast and generation of a progenitor cell by regulating asymmetric segregation of fate determinants. In the absence of lgl function, elevated cortical aPKC kinase activity perturbs unequal partitioning of the fate determinants including Numb and induces supernumerary neuroblasts in larval brains. However, whether increased aPKC function triggers formation of excess neuroblasts by inactivating Numb remains controversial. To investigate how increased cortical aPKC function induces formation of excess neuroblasts, we analyzed the fate of cells in neuroblast lineage clones in lgl mutant brains. Surprisingly, our analyses revealed that neuroblasts in lgl mutant brains undergo asymmetric division to produce progenitor cells, which then revert back into neuroblasts. In lgl mutant brains, Numb remained localized in the cortex of mitotic neuroblasts and failed to segregate exclusively into the progenitor cell following completion of asymmetric division. These results led us to propose that elevated aPKC function in the cortex of mitotic neuroblasts reduces the function of Numb in the future progenitor cells. We identified that the acyl-CoA binding domain containing 3 protein (ACBD3) binding region is essential for asymmetric segregation of Numb in mitotic neuroblasts and suppression of the supernumerary neuroblast phenotype induced by increased aPKC function. The ACBD3 binding region of Numb harbors two aPKC phosphorylation sites, serines 48 and 52. Surprisingly, while the phosphorylation status at these two sites directly impinged on asymmetric segregation of Numb in mitotic neuroblasts, both the phosphomimetic and non-phosphorylatable forms of Numb suppressed formation of excess neuroblasts triggered by increased cortical aPKC function. Thus, we propose that precise regulation of cortical aPKC kinase activity distinguishes the sibling cell identity in part by ensuring asymmetric partitioning of Numb into the future progenitor cell where Numb maintains restricted potential independently of regulation by aPKC.     

Mitochondrial targeting of quinones: Therapeutic implications

Mitochondrial oxidative damage contributes to a range of degenerative diseases. Ubiquinones have been shown to protect mitochondria from oxidative damage, but only a small proportion of externally administered ubiquinone is taken up by mitochondria. Conjugation of the lipophilic triphenylphosphonium cation to a ubiquinone moiety has produced a compound, MitoQ, which accumulates selectively into mitochondria. MitoQ passes easily through all biological membranes and, because of its positive charge, is accumulated several hundred-fold within mitochondria driven by the mitochondrial membrane potential. MitoQ protects mitochondria against oxidative damage in vitro and following oral delivery, and may therefore form the basis for mitochondria-protective therapies.     

Mitochondrial Fragmentation Due to Inhibition of Fusion Increases Cyclin B through Mitochondrial Superoxide Radicals

During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G₁-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G₂- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the ‘mitotic mitochondrial fragmentation’ checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R⁺ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. We provide evidence for the importance of mitochondrial superoxide in this process. Our results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.     

The SCFSlimb E3 ligase complex regulates asymmetric division to inhibit neuroblast overgrowth

Drosophila larval brain neuroblasts divide asymmetrically to balance between self‐renewal and differentiation. Here, we demonstrate that the SCF^Slimb E3 ubiquitin ligase complex, which is composed of Cul1, SkpA, Roc1a and the F‐box protein Supernumerary limbs (Slimb), inhibits ectopic neuroblast formation and regulates asymmetric division of neuroblasts. Hyperactivation of Akt leads to similar neuroblast overgrowth and defects in asymmetric division. Slimb associates with Akt in a protein complex, and SCF^Slimb acts through SAK and Akt to inhibit neuroblast overgrowth. Moreover, Beta‐transducin repeat containing, the human ortholog of Slimb, is frequently deleted in highly aggressive gliomas, suggesting a conserved tumor suppressor‐like function.     

Mitochondrial division prevents neurodegeneration

Mitochondrial division is mediated by the conserved dynamin-related GTPase DNM1L/DRP1. DNM1L assembles onto the surface of mitochondria and constricts this tubular organelle. Alterations in mitochondrial division are linked to many neurodegenerative diseases. However, the in vivo function of mitochondrial division is poorly understood. In our recent paper, we studied the physiological role of mitochondrial division in postmitotic neurons using the cre-loxP system. We found that the loss of DNM1L resulted in increased oxidative damage in mitochondria, impaired respiration and neurodegeneration in postmitotic neurons. Suggesting a decrease in mitochondrial turnover, mitophagy-related proteins such as LC3, SQSTM1/p62 and ubiqutin accumulated in division-defective mitochondria. These findings suggest that mitochondrial division functions as an important quality control mechanism that suppresses oxidative damage and neurodegeneration in vivo

     

Mitochondrial Targeting of Cytochrome P450 (CYP) 1B1 and Its Role in Polycyclic Aromatic Hydrocarbon-induced Mitochondrial Dysfunction

We report that polycyclic aromatic hydrocarbon (PAH)-inducible CYP1B1 is targeted to mitochondria by sequence-specific cleavage at the N terminus by a cytosolic Ser protease (polyserase 1) to activate the cryptic internal signal. Site-directed mutagenesis, COS-7 cell transfection, and in vitro import studies in isolated mitochondria showed that a positively charged domain at residues 41–48 of human CYP1B1 is part of the mitochondrial (mt) import signal. Ala scanning mutations showed that the Ser protease cleavage site resides between residues 37 and 41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that was attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene. In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (adrenodoxin) and ferredoxin reductase showed high aryl hydrocarbon hydroxylase activity. Administration of benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzodioxin induced similar mitochondrial functional abnormalities and oxidative stress in the lungs of wild-type mice and Cyp1a1/1a2-null mice, but the effects were markedly blunted in Cyp1b1-null mice. These results confirm a role for CYP1B1 in inducing PAH-mediated mitochondrial dysfunction. The role of mitochondrial CYP1B1 was assessed using A549 lung epithelial cells stably expressing shRNA against NADPH-cytochrome P450 oxidoreductase or mitochondrial adrenodoxin. Our results not only show conservation of the endoprotease cleavage mechanism for mitochondrial import of family 1 CYPs but also reveal a direct role for mitochondrial CYP1B1 in PAH-mediated oxidative and chemical damage to mitochondria.     

Reperfusion Promotes Mitochondrial Dysfunction following Focal Cerebral Ischemia in Rats

Background and Purpose

Mitochondrial dysfunction has been implicated in the cell death observed after cerebral ischemia, and several mechanisms for this dysfunction have been proposed. Reperfusion after transient cerebral ischemia may cause continued and even more severe damage to the brain. Many lines of evidence have shown that mitochondria suffer severe damage in response to ischemic injury. The purpose of this study was to observe the features of mitochondrial dysfunction in isolated mitochondria during the reperfusion period following focal cerebral ischemia.

Methods

Male Wistar rats were subjected to focal cerebral ischemia. Mitochondria were isolated using Percoll density gradient centrifugation. The isolated mitochondria were fixed for electron microscopic examination; calcium-induced mitochondrial swelling was quantified using spectrophotometry. Cyclophilin D was detected by Western blotting. Fluorescent probes were used to selectively stain mitochondria to measure their membrane potential and to measure reactive oxidative species production using flow cytometric analysis.

Results

Signs of damage were observed in the mitochondrial morphology after exposure to reperfusion. The mitochondrial swelling induced by Ca²⁺ increased gradually with the increasing calcium concentration, and this tendency was exacerbated as the reperfusion time was extended. Cyclophilin D protein expression peaked after 24 hours of reperfusion. The mitochondrial membrane potential was decreased significantly during the reperfusion period, with the greatest decrease observed after 24 hours of reperfusion. The surge in mitochondrial reactive oxidative species occurred after 2 hours of reperfusion and was maintained at a high level during the reperfusion period.

Conclusions

Reperfusion following focal cerebral ischemia induced significant mitochondrial morphological damage and Ca²⁺-induced mitochondrial swelling. The mechanism of this swelling may be mediated by the upregulation of the Cyclophilin D protein, the destruction of the mitochondrial membrane potential and the generation of excessive reactive oxidative species.     

Adaptations of Energy Metabolism Associated with Increased Levels of Mitochondrial Cholesterol in Niemann-Pick Type C1-deficient Cells

Niemann-Pick type C1 (NPC1) is a late endosomal transmembrane protein, which, together with NPC2 in the endosome lumen, mediates the transport of endosomal cholesterol to the plasma membrane and endoplasmic reticulum. Loss of function of NPC1 or NPC2 leads to cholesterol accumulation in late endosomes and causes neuronal dysfunction and neurodegeneration. Recent studies indicate that cholesterol also accumulates in mitochondria of NPC1-deficient cells and brain tissue and that NPC1 deficiency leads to alterations in mitochondrial function and energy metabolism. Here, we have investigated the effects of increased mitochondrial cholesterol levels on energy metabolism, using RNA interference to deplete Chinese hamster ovary cells of NPC1 alone or in combination with MLN64, which mediates endosomal cholesterol transport to mitochondria. Mitochondrial cholesterol levels were also altered by depletion of NPC2 in combination with the expression of NPC2 mutants. We found that the depletion of NPC1 increased lactate secretion, decreased glutamine-dependent mitochondrial respiration, and decreased ATP transport across mitochondrial membranes. These metabolic alterations did not occur when transport of endosomal cholesterol to mitochondria was blocked. In addition, the elevated mitochondrial cholesterol levels in NPC1-depleted cells and in NPC2-depleted cells expressing mutant NPC2 that allows endosomal cholesterol trafficking to mitochondria were associated with increased expression of the antioxidant response factor Nrf2. Antioxidant treatment not only prevented the increase in Nrf2 mRNA levels but also prevented the increased lactate secretion in NPC1-depleted cells. These results suggest that mitochondrial cholesterol accumulation can increase oxidative stress and in turn cause increased glycolysis to lactate and other metabolic alterations.