Mitochondrial network adaptations of microglia reveal sex-specific stress response after injury and UCP2 knockout.

Mitochondrial networks remodel their connectivity, content, and subcellular localization to support optimized energy production in conditions of increased environmental or cellular stress. Microglia rely on mitochondria to respond to these stressors, however our knowledge about mitochondrial networks and their adaptations in microglia in vivo is limited. Here, we generate a mouse model that selectively labels mitochondria in microglia. We identify that mitochondrial networks are more fragmented with increased content and perinuclear localization in vitro vs. in vivo. Mitochondrial networks adapt similarly in microglia closest to the injury site after optic nerve crush. Preventing microglial UCP2 increase after injury by selective knockout induces cellular stress. This results in mitochondrial hyperfusion in male microglia, a phenotype absent in females due to circulating estrogens. Our results establish the foundation for mitochondrial network analysis of microglia in vivo, emphasizing the importance of mitochondrial-based sex effects of microglia in other pathologies.

BAX activation in mouse retinal ganglion cells occurs in two temporally and mechanistically distinct steps.

BACKGROUND: Pro-apoptotic BAX is a central mediator of retinal ganglion cell (RGC) death after optic nerve damage. BAX activation occurs in two stages including translocation of latent BAX to the mitochondrial outer membrane (MOM) and then permeabilization of the MOM to facilitate the release of apoptotic signaling molecules. As a critical component of RGC death, BAX is an attractive target for neuroprotective therapies and an understanding of the kinetics of BAX activation and the mechanisms controlling the two stages of this process in RGCs is potentially valuable in informing the development of a neuroprotective strategy. METHODS: The kinetics of BAX translocation were assessed by both static and live-cell imaging of a GFP-BAX fusion protein introduced into RGCs using AAV2-mediated gene transfer in mice. Activation of BAX was achieved using an acute optic nerve crush (ONC) protocol. Live-cell imaging of GFP-BAX was achieved using explants of mouse retina harvested 7 days after ONC. Kinetics of translocation in RGCs were compared to GFP-BAX translocation in 661W tissue culture cells. Permeabilization of GFP-BAX was assessed by staining with the 6A7 monoclonal antibody, which recognizes a conformational change in this protein after MOM insertion. Assessment of individual kinases associated with both stages of activation was made using small molecule inhibitors injected into the vitreous either independently or in concert with ONC surgery. The contribution of the Dual Leucine Zipper-JUN-N-Terminal Kinase cascade was evaluated using mice with a double conditional knock-out of both Mkk4 and Mkk7. RESULTS: ONC induces the translocation of GFP-BAX in RGCs at a slower rate and with less intracellular synchronicity than 661W cells, but exhibits less variability among mitochondrial foci within a single cell. GFP-BAX was also found to translocate in all compartments of an RGC including the dendritic arbor and axon. Approximately 6% of translocating RGCs exhibited retrotranslocation of BAX immediately following translocation. Unlike tissue culture cells, which exhibit simultaneous translocation and permeabilization, RGCs exhibited a significant delay between these two stages, similar to detached cells undergoing anoikis. Translocation, with minimal permeabilization could be induced in a subset of RGCs using an inhibitor of Focal Adhesion Kinase (PF573228). Permeabilization after ONC, in a majority of RGCs, could be inhibited with a broad spectrum kinase inhibitor (sunitinib) or a selective inhibitor for p38/MAPK14 (SB203580). Intervention of DLK-JNK axis signaling abrogated GFP-BAX translocation after ONC. CONCLUSIONS: A comparison between BAX activation kinetics in tissue culture cells and in cells of a complex tissue environment shows distinct differences indicating that caution should be used when translating findings from one condition to the other. RGCs exhibit both a delay between translocation and permeabilization and the ability for translocated BAX to be retrotranslocated, suggesting several stages at which intervention of the activation process could be exploited in the design of a therapeutic strategy.

BAX activation in mouse retinal ganglion cells occurs in two temporally and mechanistically distinct steps.

Background Pro-apoptotic BAX is a central mediator of retinal ganglion cell (RGC) death after optic nerve damage. BAX activation occurs in two stages including translocation of latent BAX to the mitochondrial outer membrane (MOM) and then permeabilization of the MOM to facilitate the release of apoptotic signaling molecules. As a critical component of RGC death, BAX is an attractive target for neuroprotective therapies and an understanding of the kinetics of BAX activation and the mechanisms controlling the two stages of this process in RGCs is potentially valuable in informing the development of a neuroprotective strategy. Methods The kinetics of BAX translocation were assessed by both static and live-cell imaging of a GFP-BAX fusion protein introduced into RGCs using AAV2-mediated gene transfer in mice. Activation of BAX was achieved using an acute optic nerve crush (ONC) protocol. Live-cell imaging of GFP-BAX was achieved using explants of mouse retina harvested 7 days after ONC. Kinetics of translocation in RGCs were compared to GFP-BAX translocation in 661W tissue culture cells. Permeabilization of GFP-BAX was assessed by staining with the 6A7 monoclonal antibody, which recognizes a conformational change in this protein after MOM insertion. Assessment of individual kinases associated with both stages of activation was made using small molecule inhibitors injected into the vitreous either independently or in concert with ONC surgery. The contribution of the Dual Leucine Zipper-JUN-N-Terminal Kinase cascade was evaluated using mice with a double conditional knock-out of both Mkk4 and Mkk7 . Results ONC induces the translocation of GFP-BAX in RGCs at a slower rate and with less intracellular synchronicity than 661W cells, but exhibits less variability among mitochondrial foci within a single cell. GFP-BAX was also found to translocate in all compartments of an RGC including the dendritic arbor and axon. Approximately 6% of translocating RGCs exhibited retrotranslocation of BAX immediately following translocation. Unlike tissue culture cells, which exhibit simultaneous translocation and permeabilization, RGCs exhibited a significant delay between these two stages, similar to detached cells undergoing anoikis. Translocation, with minimal permeabilization could be induced in a subset of RGCs using an inhibitor of Focal Adhesion Kinase (PF573228). Permeabilization after ONC, in a majority of RGCs, could be inhibited with a broad spectrum kinase inhibitor (sunitinib) or a selective inhibitor for p38/MAPK14 (SB203580). Intervention of DLK-JNK axis signaling abrogated GFP-BAX translocation after ONC. Conclusions A comparison between BAX activation kinetics in tissue culture cells and in cells of a complex tissue environment shows distinct differences indicating that caution should be used when translating findings from one condition to the other. RGCs exhibit both a delay between translocation and permeabilization and the ability for translocated BAX to be retrotranslocated, suggesting several stages at which intervention of the activation process could be exploited in the design of a therapeutic strategy.

Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment.

Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells, allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy, likely due to the cells' heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding by introducing four point mutations (K531E, R576Q, K493S, and K459S), resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette for use in combination with the Cx3cr1 CreERT2 mouse line. Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency.

Increased Susceptibility and Intrinsic Apoptotic Signaling in Neurons by Induced HDAC3 Expression.

Purpose: Inhibition or targeted deletion of histone deacetylase 3 (HDAC3) is neuroprotective in a variety neurodegenerative conditions, including retinal ganglion cells (RGCs) after acute optic nerve damage. Consistent with this, induced HDAC3 expression in cultured cells shows selective toxicity to neurons. Despite an established role for HDAC3 in neuronal pathology, little is known regarding the mechanism of this pathology. Methods: Induced expression of an HDAC3-mCherry fusion protein in mouse RGCs was accomplished by transduction with AAV2/2-Pgk-HDAC3-mCherry. Increased susceptibility to optic nerve damage in HDAC3-mCherry expressing RGCs was evaluated in transduced mice that received acute optic nerve crush surgery. Expression of HDAC3-FLAG or HDAC3-mCherry was induced by nucleofection or transfection of plasmids into differentiated or undifferentiated 661W tissue culture cells. Immunostaining for cleaved caspase 3, localization of a GFP-BAX fusion protein, and quantitative RT-PCR was used to evaluate HDAC3-induced damage. Results: Induced expression of exogenous HDAC3 in RGCs by viral-mediated gene transfer resulted in modest levels of cell death but significantly increased the sensitivity of these neurons to axonal damage. Undifferentiated 661W retinal precursor cells were resilient to induced HDAC3 expression, but after differentiation, HDAC3 induced GFP-BAX recruitment to the mitochondria and BAX/BAK dependent activation of caspase 3. This was accompanied by an increase in accumulation of transcripts for the JNK2/3 kinases and the p53-regulated BH3-only gene Bbc3/Puma. Cell cycle arrest of undifferentiated 661W cells did not increase their sensitivity to HDAC3 expression. Conclusions: Collectively, these results indicate that HDAC3-induced toxicity to neurons is mediated by the intrinsic apoptotic pathway.

Microglia enable mature perineuronal nets disassembly upon anesthetic ketamine exposure or 60-Hz light entrainment in the healthy brain.

Perineuronal nets (PNNs), components of the extracellular matrix, preferentially coat parvalbumin-positive interneurons and constrain critical-period plasticity in the adult cerebral cortex. Current strategies to remove PNN are long-lasting, invasive, and trigger neuropsychiatric symptoms. Here, we apply repeated anesthetic ketamine as a method with minimal behavioral effect. We find that this paradigm strongly reduces PNN coating in the healthy adult brain and promotes juvenile-like plasticity. Microglia are critically involved in PNN loss because they engage with parvalbumin-positive neurons in their defined cortical layer. We identify external 60-Hz light-flickering entrainment to recapitulate microglia-mediated PNN removal. Importantly, 40-Hz frequency, which is known to remove amyloid plaques, does not induce PNN loss, suggesting microglia might functionally tune to distinct brain frequencies. Thus, our 60-Hz light-entrainment strategy provides an alternative form of PNN intervention in the healthy adult brain.

The Influence of Mitochondrial Dynamics and Function on Retinal Ganglion Cell Susceptibility in Optic Nerve Disease.

The important roles of mitochondrial function and dysfunction in the process of neurodegeneration are widely acknowledged. Retinal ganglion cells (RGCs) appear to be a highly vulnerable neuronal cell type in the central nervous system with respect to mitochondrial dysfunction but the actual reasons for this are still incompletely understood. These cells have a unique circumstance where unmyelinated axons must bend nearly 90° to exit the eye and then cross a translaminar pressure gradient before becoming myelinated in the optic nerve. This region, the optic nerve head, contains some of the highest density of mitochondria present in these cells. Glaucoma represents a perfect storm of events occurring at this location, with a combination of changes in the translaminar pressure gradient and reassignment of the metabolic support functions of supporting glia, which appears to apply increased metabolic stress to the RGC axons leading to a failure of axonal transport mechanisms. However, RGCs themselves are also extremely sensitive to genetic mutations, particularly in genes affecting mitochondrial dynamics and mitochondrial clearance. These mutations, which systemically affect the mitochondria in every cell, often lead to an optic neuropathy as the sole pathologic defect in affected patients. This review summarizes knowledge of mitochondrial structure and function, the known energy demands of neurons in general, and places these in the context of normal and pathological characteristics of mitochondria attributed to RGCs.

Characteristics of intracellular propagation of mitochondrial BAX recruitment during apoptosis.

Recent advancements in live cell imaging technologies have identified the phenomenon of intracellular propagation of late apoptotic events, such as cytochrome c release and caspase activation. The mechanism, prevalence, and speed of apoptosis propagation remain unclear. Additionally, no studies have demonstrated propagation of the pro-apoptotic protein, BAX. To evaluate the role of BAX in intracellular apoptotic propagation, we used high speed live-cell imaging to visualize fluorescently tagged-BAX recruitment to mitochondria in four immortalized cell lines. We show that propagation of mitochondrial BAX recruitment occurs in parallel to cytochrome c and SMAC/Diablo release and is affected by cellular morphology, such that cells with processes are more likely to exhibit propagation. The initiation of propagation events is most prevalent in the distal tips of processes, while the rate of propagation is influenced by the 2-dimensional width of the process. Propagation was rarely observed in the cell soma, which exhibited near synchronous recruitment of BAX. Propagation velocity is not affected by mitochondrial volume in segments of processes, but is negatively affected by mitochondrial density. There was no evidence of a propagating wave of increased levels of intracellular calcium ions. Alternatively, we did observe a uniform increase in superoxide build-up in cellular mitochondria, which was released as a propagating wave simultaneously with the propagating recruitment of BAX to the mitochondrial outer membrane.

BAX-Depleted Retinal Ganglion Cells Survive and Become Quiescent Following Optic Nerve Damage.

Removal of the Bax gene from mice completely protects the somas of retinal ganglion cells (RGCs) from apoptosis following optic nerve injury. This makes BAX a promising therapeutic target to prevent neurodegeneration. In this study, Bax+/- mice were used to test the hypothesis that lowering the quantity of BAX in RGCs would delay apoptosis following optic nerve injury. RGCs were damaged by performing optic nerve crush (ONC) and then immunostaining for phospho-cJUN, and quantitative PCR were used to monitor the status of the BAX activation mechanism in the months following injury. The apoptotic susceptibility of injured cells was directly tested by virally introducing GFP-BAX into Bax-/- RGCs after injury. The competency of quiescent RGCs to reactivate their BAX activation mechanism was tested by intravitreal injection of the JNK pathway agonist, anisomycin. Twenty-four weeks after ONC, Bax+/- mice had significantly less cell loss in their RGC layer than Bax+/+ mice 3 weeks after ONC. Bax+/- and Bax+/+ RGCs exhibited similar patterns of nuclear phospho-cJUN accumulation immediately after ONC, which persisted in Bax+/- RGCs for up to 7 weeks before abating. The transcriptional activation of BAX-activating genes was similar in Bax+/- and Bax+/+ RGCs following ONC. Intriguingly, cells deactivated their BAX activation mechanism between 7 and 12 weeks after crush. Introduction of GFP-BAX into Bax-/- cells at 4 weeks after ONC showed that these cells had a nearly normal capacity to activate this protein, but this capacity was lost 8 weeks after crush. Collectively, these data suggest that 8-12 weeks after crush, damaged cells no longer displayed increased susceptibility to BAX activation relative to their naïve counterparts. In this same timeframe, retinal glial activation and the signaling of the pro-apoptotic JNK pathway also abated. Quiescent RGCs did not show a timely reactivation of their JNK pathway following intravitreal injection with anisomycin. These findings demonstrate that lowering the quantity of BAX in RGCs is neuroprotective after acute injury. Damaged RGCs enter a quiescent state months after injury and are no longer responsive to an apoptotic stimulus. Quiescent RGCs will require rejuvenation to reacquire functionality.

Targeting microglia with lentivirus and AAV: Recent advances and remaining challenges.

Microglia have emerged as a critical component of neurodegenerative diseases. Genetic manipulation of microglia can elucidate their functional impact in disease. In neuroscience, recombinant viruses such as lentiviruses and adeno-associated viruses (AAVs) have been successfully used to target various cell types in the brain, although effective transduction of microglia is rare. In this review, we provide a short background of lentiviruses and AAVs, and strategies for designing recombinant viral vectors. Then, we will summarize recent literature on successful microglial transductions in vitro and in vivo, and discuss the current challenges. Finally, we provide guidelines for reporting the efficiency and specificity of viral targeting in microglia, which will enable the microglial research community to assess and improve methodologies for future studies.

AAV2-Mediated Transduction of the Mouse Retina After Optic Nerve Injury.

Purpose: Gene therapy of retinal ganglion cells (RGCs) has promise as a powerful therapeutic for the rescue and regeneration of these cells after optic nerve damage. However, early after damage, RGCs undergo atrophic changes, including gene silencing. It is not known if these changes will deleteriously affect transduction and transgene expression, or if the therapeutic protein can influence reactivation of the endogenous genome. Methods: Double-transgenic mice carrying a Rosa26-(LoxP)-tdTomato reporter, and a mutant allele for the proapoptotic Bax gene were reared. The Bax mutant blocks apoptosis, but RGCs still exhibit nuclear atrophy and gene silencing. At times ranging from 1 hour to 4 weeks after optic nerve crush (ONC), eyes received an intravitreal injection of AAV2 virus carrying the Cre recombinase. Successful transduction was monitored by expression of the tdTomato reporter. Immunostaining was used to localize tdTomato expression in select cell types. Results: Successful transduction of RGCs was achieved at all time points after ONC using AAV2 expressing Cre from the phosphoglycerate kinase (Pgk) promoter, but not the CMV promoter. ONC promoted an increase in the transduction of cell types in the inner nuclear layer, including Müller cells and rod bipolar neurons. There was minimal evidence of transduction of amacrine cells and astrocytes in the inner retina or optic nerve. Conclusions: Damaged RGCs can be transduced and at least some endogenous genes can be subsequently activated. Optic nerve damage may change retinal architecture to allow greater penetration of an AAV2 virus to transduce several additional cell types in the inner nuclear layer.

Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis.

The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies.

BAX to basics: How the BCL2 gene family controls the death of retinal ganglion cells.

Retinal ganglion cell (RGC) death is the principal consequence of injury to the optic nerve. For several decades, we have understood that the RGC death process was executed by apoptosis, suggesting that there may be ways to therapeutically intervene in this cell death program and provide a more direct treatment to the cells and tissues affected in diseases like glaucoma. A major part of this endeavor has been to elucidate the molecular biological pathways active in RGCs from the point of axonal injury to the point of irreversible cell death. A major component of this process is the complex interaction of members of the BCL2 gene family. Three distinct family members of proteins orchestrate the most critical junction in the apoptotic program of RGCs, culminating in the activation of pro-apoptotic BAX. Once active, BAX causes irreparable damage to mitochondria, while precipitating downstream events that finish off a dying ganglion cell. This review is divided into two major parts. First, we summarize the extent of knowledge of how BCL2 gene family proteins interact to facilitate the activation and function of BAX. This area of investigation has rapidly changed over the last few years and has yielded a dramatically different mechanistic understanding of how the intrinsic apoptotic program is run in mammalian cells. Second, we provided a comprehensive analysis of nearly two decades of investigation of the role of BAX in the process of RGC death, much of which has provided many important insights into the overall pathophysiology of diseases like glaucoma.

Spink2 modulates apoptotic susceptibility and is a candidate gene in the Rgcs1 QTL that affects retinal ganglion cell death after optic nerve damage.

The Rgcs1 quantitative trait locus, on mouse chromosome 5, influences susceptibility of retinal ganglion cells to acute damage of the optic nerve. Normally resistant mice (DBA/2J) congenic for the susceptible allele from BALB/cByJ mice exhibit susceptibility to ganglion cells, not only in acute optic nerve crush, but also to chronic inherited glaucoma that is characteristic of the DBA/2J strain as they age. SNP mapping of this QTL has narrowed the region of interest to 1 Mb. In this region, a single gene (Spink2) is the most likely candidate for this effect. Spink2 is expressed in retinal ganglion cells and is increased after optic nerve damage. This gene is also polymorphic between resistant and susceptible strains, containing a single conserved amino acid change (threonine to serine) and a 220 bp deletion in intron 1 that may quantitatively alter endogenous expression levels between strains. Overexpression of the different variants of Spink2 in D407 tissue culture cells also increases their susceptibility to the apoptosis-inducing agent staurosporine in a manner consistent with the differential susceptibility between the DBA/2J and BALB/cByJ strains.

c-Jun N-terminal kinase regulates mGluR-dependent expression of post-synaptic FMRP target proteins.

Fragile X syndrome (FXS) is caused by the loss of functional fragile X mental retardation protein (FMRP). Loss of FMRP results in an elevated basal protein expression profile of FMRP targeted mRNAs, a loss of local metabotropic glutamate receptor (mGluR)-regulated protein synthesis, exaggerated long-term depression and corresponding learning and behavioral deficits. Evidence shows that blocking mGluR signaling in FXS models ameliorates these deficits. Therefore, understanding the signaling mechanisms downstream of mGluR stimulation may provide additional therapeutic targets for FXS. Kinase cascades are an integral mechanism regulating mGluR-dependent protein translation. The c-Jun N-terminal kinase (JNK) pathway has been shown to regulate mGluR-dependent nuclear transcription; however, the involvement of JNK in local, synaptic signaling has not been explored. Here, we show that JNK is both necessary and sufficient for mGluR-dependent expression of a subset of FMRP target proteins. In addition, JNK activity is basally elevated in fmr1 knockout mouse synapses, and blocking JNK activity reduces the over-expression of post-synaptic proteins in these mice. Together, these data suggest that JNK may be an important signaling mechanism downstream of mGluR stimulation, regulating FMRP-dependent protein synthesis. Furthermore, local, post-synaptic dysregulation of JNK activity may provide a viable target to ameliorate the deficits involved in FXS. Expression of many FMRP target proteins is enhanced in FXS. Here, we evaluated the role of JNKs in FXS. We found that JNK signaling is activated upon mGluR stimulation in wild-type neurons. Conversely, JNK activity is basally elevated in fmr1 knockout. Inhibiting JNK reduced the expression of FMRP target proteins and driving JNK activity increased the expression of these proteins.

Sh3rf2/POSHER protein promotes cell survival by ring-mediated proteasomal degradation of the c-Jun N-terminal kinase scaffold POSH (Plenty of SH3s) protein.

We report that Sh3rf2, a homologue of the pro-apoptotic scaffold POSH (Plenty of SH3s), acts as an anti-apoptotic regulator for the c-Jun N-terminal kinase (JNK) pathway. siRNA-mediated knockdown of Sh3rf2 promotes apoptosis of neuronal PC12 cells, cultured cortical neurons, and C6 glioma cells. This death appears to result from activation of JNK signaling. Loss of Sh3rf2 triggers activation of JNK and its target c-Jun. Also, apoptosis promoted by Sh3rf2 knockdown is inhibited by dominant-negative c-Jun as well as by a JNK inhibitor. Investigation of the mechanism by which Sh3rf2 regulates cell survival implicates POSH, a scaffold required for activation of pro-apoptotic JNK/c-Jun signaling. In cells lacking POSH, Sh3rf2 knockdown is unable to activate JNK. We further find that Sh3rf2 binds POSH to reduce its levels by a mechanism that requires the RING domains of both proteins and that appears to involve proteasomal POSH degradation. Conversely, knockdown of Sh3rf2 promotes the stabilization of POSH protein and activation of JNK signaling. Finally, we show that endogenous Sh3rf2 protein rapidly decreases following several different apoptotic stimuli and that knockdown of Sh3rf2 activates the pro-apoptotic JNK pathway in neuronal cells. These findings support a model in which Sh3rf2 promotes proteasomal degradation of pro-apoptotic POSH in healthy cells and in which apoptotic stimuli lead to rapid loss of Sh3rf2 expression, and consequently to stabilization of POSH and JNK activation and cell death. On the basis of these observations, we propose the alternative name POSHER (POSH-eliminating RING protein) for the Sh3rf2 protein.