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1.  Mitochondrial function in neuronal cells depends on p97/VCP/Cdc48-mediated quality control 
Maintaining mitochondrial function is essential for neuronal survival and offers protection against neurodegeneration. Ubiquitin-mediated, proteasome-dependent protein degradation in the form of outer mitochondrial membrane associated degradation (OMMAD) was shown to play roles in maintenance of mitochondria on the level of proteostasis, but also mitophagy and cell death. Recently, the AAA-ATPase p97/VCP/Cdc48 was recognized as part of OMMAD acting as retrotranslocase of ubiquitinated mitochondrial proteins for proteasomal degradation. Thus, p97 likely plays a major role in mitochondrial maintenance. Support for this notion comes from mitochondrial dysfunction associated with amyotrophic lateral sclerosis and hereditary inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) caused by p97 mutation. Using SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ treated with mitochondrial toxins rotenone, 6-OHDA, or Aβ-peptide as model for neuronal cells suffering from mitochondrial dysfunction, we found mitochondrial fragmentation under normal and stress conditions was significantly increased upon inactivation of p97. Furthermore, inactivation of p97 resulted in loss of mitochondrial membrane potential and increased production of reactive oxygen species (ROS). Under additional stress conditions, loss of mitochondrial membrane potential and increased ROS production was even more pronounced. Loss of mitochondrial fidelity upon inactivation of p97 was likely due to disturbed maintenance of mitochondrial proteostasis as the employed treatments neither induced mitophagy nor cell death. This was supported by the accumulation of oxidatively-damaged proteins on mitochondria in response to p97 inactivation. Dysfunction of p97 under normal and stress conditions in neuron-like cells severely impacts mitochondrial function, thus supporting for the first time a role for p97 as a major component of mitochondrial proteostasis.
PMCID: PMC4313695
mitochondria; p97/VCP; neuronal cells; quality control
2.  Role of Membrane Association and Atg14-Dependent Phosphorylation in Beclin-1-Mediated Autophagy 
Molecular and Cellular Biology  2013;33(18):3675-3688.
During autophagy, a double membrane envelops cellular material for trafficking to the lysosome. Human beclin-1 and its yeast homologue, Atg6/Vps30, are scaffold proteins bound in a lipid kinase complex with multiple cellular functions, including autophagy. Several different Atg6 complexes exist, with an autophagy-specific form containing Atg14. However, the roles of Atg14 and beclin-1 in the activation of this complex remain unclear. We here addressed the mechanism of beclin-1 complex activation and reveal two critical steps in this pathway. First, we identified a unique domain in beclin-1, conserved in the yeast homologue Atg6, which is involved in membrane association and, unexpectedly, controls autophagosome size and number in yeast. Second, we demonstrated that human Atg14 is critical in controlling an autophagy-dependent phosphorylation of beclin-1. We map these novel phosphorylation sites to serines 90 and 93 and demonstrate that phosphorylation at these sites is necessary for maximal autophagy. These results help clarify the mechanism of beclin-1 and Atg14 during autophagy.
PMCID: PMC3753860  PMID: 23878393
3.  Anti-inflammatory response following uptake of apoptotic bodies by meningothelial cells 
Meningothelial cells (MECs) are the cellular components of the meninges. As such, they provide important barrier function for the central nervous system (CNS) building the interface between neuronal tissue and the cerebrospinal fluid (CSF), and are also part of the immune response of the CNS.
Human, immortalized MECs were analyzed by flow cytometry and confocal microscopy to study the uptake of apoptotic cells. Furthermore, cytokine and chemokine production by MECs was analyzed by cytokine array and ELISA.
We found that MECs are highly active phagocytes able of ingesting and digesting large amounts of apoptotic cells. Furthermore, the uptake of apoptotic cells by MECs was immune suppressive via inhibiting the secretion of pro-inflammatory and chemoattractant cytokines and chemokines IL-6, IL-8, IL-16, MIF, and CXCL1, while increasing the secretion of anti-inflammatory IL-1 receptor antagonist by MECs.
MECs respond with the secretion of anti-inflammatory cytokines and chemokines following the uptake of apoptotic cells potentially connecting these cells to processes important for the shut-down of immune responses in the brain.
PMCID: PMC3941933  PMID: 24565420
Meningothelial cells; Apoptotic cells; Cytokines; Cerebrospinal fluid; Central nervous system; Optic nerve
4.  MARCH5 inactivation supports mitochondrial function during neurodegenerative stress 
Neuronal cell death is accompanied by mitochondrial dysfunction with mitochondrial maintenance critical to neuronal survival. The mitochondrial ubiquitin ligase MARCH5 has dual roles in the upkeep of mitochondrial function. MARCH5 is involved in targeted degradation of proteins harmful to mitochondria and impacts mitochondrial morphology upstream of the fission protein Drp1. In a neuronal cell model, dominant-negative MARCH5 prevents mitochondrial fragmentation during neurodegenerative stress induced by the neuron-specific reactive oxygen generator 6-hydroxydopamine, the complex I inhibitor rotenone or Alzheimer’s-related amyloid beta peptide. In addition, preservation of mitochondrial function in terms of membrane potential and lower reactive oxygen generation was observed following inactivation of MARCH5. Our findings connect MARCH5 to neuronal stress responses and further emphasize the link between mitochondrial dynamics and function.
PMCID: PMC3794199  PMID: 24133412
MARCH5; mitochondria; Aβ; neurodegeneration; mitochondrial quality control
5.  Inactivation of MARCH5 Prevents Mitochondrial Fragmentation and Interferes with Cell Death in a Neuronal Cell Model 
PLoS ONE  2012;7(12):e52637.
To study the impact of the mitochondrial ubiquitin ligase MARCH5 on mitochondrial morphology and induction of apoptosis using an in vitro model of neuronal precursor cells exposed to glaucoma-relevant stress conditions.
RGC5 cells transfected with expression constructs for MARCH5, MARCH5H43W, Dpr1K38A or vector control were exposed to either elevated pressure of 30 mmHg, oxidative stress caused by mitochondrial electron transport chain (ETC) inhibition, or hypoxia-reoxygenation conditions. Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay. Induction of apoptotic cell death in these cells was determined by analyzing the release of cytochrome c from mitochondria into the cytosol and flow cytometry.
Exposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5. In cells expressing inactive MARCH5H43W or inactive DrpK38A, mitochondrial fragmentation was significantly blocked and mitochondrial morphology was comparable to that of control cells under normal conditions. Exposure of RGC5 cells to elevated pressure or oxidative stress conditions induced apoptotic cell death as assessed by cytochrome c release and DNA staining, while expression of dominant-negative MARCH5H43W or Drp1K38A did significantly delay cell death.
Preventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.
PMCID: PMC3526576  PMID: 23285122
6.  Bcl-xL retrotranslocates Bax from the mitochondria into the cytosol 
Cell  2011;145(1):104-116.
The Bcl-2 family member Bax translocates from the cytosol to mitochondria where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by pro-survival Bcl-2 proteins, but how they inhibit Bax remains controversial, because they neither co-localize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-xL in detergents and cell free MOMP activity, but unexpectedly induce Bax accumulation on mitochondria. Fluorescence Loss in Photobleaching (FLIP) reveals constant retrotranslocation of wt Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on pro-survival Bcl-2 family proteins and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-xL inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax.
PMCID: PMC3070914  PMID: 21458670
7.  PGC-1α Determines Light Damage Susceptibility of the Murine Retina 
PLoS ONE  2012;7(2):e31272.
The peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) proteins are key regulators of cellular bioenergetics and are accordingly expressed in tissues with a high energetic demand. For example, PGC-1α and PGC-1β control organ function of brown adipose tissue, heart, brain, liver and skeletal muscle. Surprisingly, despite their prominent role in the control of mitochondrial biogenesis and oxidative metabolism, expression and function of the PGC-1 coactivators in the retina, an organ with one of the highest energy demands per tissue weight, are completely unknown. Moreover, the molecular mechanisms that coordinate energy production with repair processes in the damaged retina remain enigmatic. In the present study, we thus investigated the expression and function of the PGC-1 coactivators in the healthy and the damaged retina. We show that PGC-1α and PGC-1β are found at high levels in different structures of the mouse retina, most prominently in the photoreceptors. Furthermore, PGC-1α knockout mice suffer from a striking deterioration in retinal morphology and function upon detrimental light exposure. Gene expression studies revealed dysregulation of all major pathways involved in retinal damage and apoptosis, repair and renewal in the PGC-1α knockouts. The light-induced increase in apoptosis in vivo in the absence of PGC-1α was substantiated in vitro, where overexpression of PGC-1α evoked strong anti-apoptotic effects. Finally, we found that retinal levels of PGC-1 expression are reduced in different mouse models for retinitis pigmentosa. We demonstrate that PGC-1α is a central coordinator of energy production and, importantly, all of the major processes involved in retinal damage and subsequent repair. Together with the observed dysregulation of PGC-1α and PGC-1β in retinitis pigmentosa mouse models, these findings thus imply that PGC-1α might be an attractive target for therapeutic approaches aimed at retinal degeneration diseases.
PMCID: PMC3278422  PMID: 22348062
8.  Meningothelial Cells React to Elevated Pressure and Oxidative Stress 
PLoS ONE  2011;6(5):e20142.
Meningothelial cells (MECs) are the cellular components of the meninges enveloping the brain. Although MECs are not fully understood, several functions of these cells have been described. The presence of desmosomes and tight junctions between MECs hints towards a barrier function protecting the brain. In addition, MECs perform endocytosis and, by the secretion of cytokines, are involved in immunological processes in the brain. However, little is known about the influence of pathological conditions on MEC function; e.g., during diseases associated with elevated intracranial pressure, hypoxia or increased oxidative stress.
We studied the effect of elevated pressure, hypoxia, and oxidative stress on immortalized human as well as primary porcine MECs. We used MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) bioreduction assays to assess the proliferation of MECs in response to treatment and compared to untreated control cells. To assess endocytotic activity, the uptake of fluorescently labeled latex beads was analyzed by fluorescence microscopy.
We found that exposure of MECs to elevated pressure caused significant cellular proliferation and a dramatic decrease in endocytotic activity. In addition, mild oxidative stress severely inhibited endocytosis.
Elevated pressure and oxidative stress impact MEC physiology and might therefore influence the microenvironment of the subarachnoid space and thus the cerebrospinal fluid within this compartment with potential negative impact on neuronal function.
PMCID: PMC3096659  PMID: 21611150
9.  The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division 
The Journal of Cell Biology  2007;178(1):71-84.
We identify a mitochondrial E3 ubiquitin ligase, MARCH5, as a critical regulator of mitochondrial fission. MARCH5 RING mutants and MARCH5 RNA interference induce an abnormal elongation and interconnection of mitochondria indicative of an inhibition of mitochondrial division. The aberrant mitochondrial phenotypes in MARCH5 RING mutant–expressing cells are reversed by ectopic expression of Drp1, but not another mitochondrial fission protein Fis1. Moreover, as indicated by abnormal clustering and mitochondrial accumulation of Drp1, as well as decreased cellular mobility of YFP-Drp1 in cells expressing MARCH5 RING mutants, MARCH5 activity regulates the subcellular trafficking of Drp1, likely by impacting the correct assembly at scission sites or the disassembly step of fission complexes. Loss of this activity may account for the observed mitochondrial division defects. Finally, MARCH5 RING mutants and endogenous Drp1, but not wild-type MARCH5 or Fis1, co-assemble into abnormally enlarged clusters in a Drp1 GTPase-dependent manner, suggesting molecular interactions among these proteins. Collectively, our data suggest a model in which mitochondrial division is regulated by a MARCH5 ubiquitin-dependent switch.
PMCID: PMC2064424  PMID: 17606867

Results 1-9 (9)