Apoptosis-induced translocation of nesprin-2 from the nuclear envelope to mitochondria is associated with mitochondrial dysfunction.

Accumulating evidence suggests that the nuclear envelope (NE) is not just a target, but also a mediator of apoptosis. We showed recently that the NE protein nesprin-2 has pro-apoptotic activity, which involves its subcellular redistribution and Bcl-2 proteins. Here we further characterize the pro-apoptotic activity of nesprin-2 focusing on its redistribution. We assessed the redistribution kinetics of endogenous nesprin-2 tagged with GFP relative to apoptosis-associated mitochondrial dysfunction. The results show apoptosis-induced GFP-nesprin-2G redistribution occurred by two different modes - complete and partial, both lead to appearance of nesprin-2G near the mitochondria. Moreover, GFP-nesprin-2 redistribution is associated with reduction in mitochondrial membrane potential and mitochondrial outer membrane permeabilization and precedes the appearance of morphological features of apoptosis. Our results show that nesprin-2G redistribution and translocation near mitochondria is an early apoptotic effect associated with mitochondrial dysfunction, which may be responsible for the pro-apoptotic function of nesprin-2.

LINC complex protein nesprin-2 has pro-apoptotic activity via Bcl-2 family proteins.

The apoptotic intrinsic pathway is initiated by perforation of the mitochondrial outer membrane by the effector pro-apoptotic proteins of the Bcl-2 family, Bax and Bak. Bax and Bak need to be activated, a process facilitated by the action of BH3-only pro-apoptotic members of the Bcl-2 family. The latter either directly activates the effector proteins or antagonizes the action of pro-survival Bcl-2 family members such as Bcl-xL. The nuclear envelope is a known target of the apoptotic machinery; however, it may also act as mediator of apoptosis. We showed previously that the nuclear envelope protein nesprin-2, a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex, can bind to Bax in close proximity to the mitochondria and that the binding increases in apoptotic cells. We now show that depleting nesprin-2 inhibits the apoptotic mitochondrial pathway as measured by Bax and Bak activation and cytochrome c release. This survival effect was Bcl-xL-dependent. Nesprin-2 depletion also inhibited spontaneous exposure of the N-terminus of Bak in cells lacking Bcl-xL and increased the presence of Bcl-xL and Bax in the mitochondria. These results indicate that nesprin-2 promotes Bak activation and regulates mitochondrial translocation/retrotranslocation of Bcl-2 family proteins. Our findings demonstrate a new apoptotic pathway whereby the nuclear envelope, via nesprin-2, regulates apoptosis.

Distinct spatiotemporal features of microglia and monocyte-derived macrophages in glioma.

Gliomas are the most frequent primary tumors of the brain. Glioma progression is regulated by the tumor microenvironment, which is mainly composed of tumor-associated microglia (TA-MG) and monocyte-derived macrophages (MDM). Recent studies have highlighted the distinct properties of these cells in glioma progression. However, their spatiotemporal alteration during tumor progression has not been fully explored. Using a genetic lineage tracing approach, we show that TA-MG and MDMs differ in their spatiotemporal distribution and interaction with other components of the glioma microenvironment. MDM were present only inside the tumor, whereas TA-MG accumulated both outside and inside the tumor. However, TA-MG was eliminated from the tumor mass as the tumor progressed. Depletion of MDM led to enhanced occupancy of TA-MG in the tumor core, indicating that TA-MG elimination was regulated by MDM. TA-MG and MDM are heterogeneous cell populations whose compositions and properties can change during tumor progression. Finally, MG, TA-MG and MDM were enriched in the perivascular area (PVA) compared to more distal blood vessel-associated areas. However, inside the tumor, the MDM enrichment in PVA was higher than that in TA-MG. Collectively, we established that TA-MG and MDM exhibit different spatiotemporal features in glioma, suggesting distinctive roles during tumor progression.

Regulatory feedback cycle of the insulin-degrading enzyme and the amyloid precursor protein intracellular domain: Implications for Alzheimer's disease.

One of the major pathological hallmarks of Alzheimer´s disease (AD) is an accumulation of amyloid-ß (Aß) in brain tissue leading to formation of toxic oligomers and senile plaques. Under physiological conditions, a tightly balanced equilibrium between Aß-production and -degradation is necessary to prevent pathological Aß-accumulation. Here, we investigate the molecular mechanism how insulin-degrading enzyme (IDE), one of the major Aß-degrading enzymes, is regulated and how amyloid precursor protein (APP) processing and Aß-degradation is linked in a regulatory cycle to achieve this balance. In absence of Aß-production caused by APP or Presenilin deficiency, IDE-mediated Aß-degradation was decreased, accompanied by a decreased IDE activity, protein level, and expression. Similar results were obtained in cells only expressing a truncated APP, lacking the APP intracellular domain (AICD) suggesting that AICD promotes IDE expression. In return, APP overexpression mediated an increased IDE expression, comparable results were obtained with cells overexpressing C50, a truncated APP representing AICD. Beside these genetic approaches, also AICD peptide incubation and pharmacological inhibition of the γ-secretase preventing AICD production regulated IDE expression and promoter activity. By utilizing CRISPR/Cas9 APP and Presenilin knockout SH-SY5Y cells results were confirmed in a second cell line in addition to mouse embryonic fibroblasts. In vivo, IDE expression was decreased in mouse brains devoid of APP or AICD, which was in line with a significant correlation of APP expression level and IDE expression in human postmortem AD brains. Our results show a tight link between Aß-production and Aß-degradation forming a regulatory cycle in which AICD promotes Aß-degradation via IDE and IDE itself limits its own production by degrading AICD.

Apoptotic stress induces Bax-dependent, caspase-independent redistribution of LINC complex nesprins.

The canonical function of Bcl-2 family proteins is to regulate mitochondrial membrane integrity. In response to apoptotic signals the multi-domain pro-apoptotic proteins Bax and Bak are activated and perforate the mitochondrial outer membrane by a mechanism which is inhibited by their interaction with pro-survival members of the family. However, other studies have shown that Bax and Bak may have additional, non-canonical functions, which include stress-induced nuclear envelope rupture and discharge of nuclear proteins into the cytosol. We show here that the apoptotic stimuli cisplatin and staurosporine induce a Bax/Bak-dependent degradation and subcellular redistribution of nesprin-1 and nesprin-2 but not nesprin-3, of the linker of nucleoskeleton and cytoskeleton (LINC) complex. The degradation and redistribution were caspase-independent and did not occur in Bax/Bak double knockout (DKO) mouse embryo fibroblasts (MEFs). Re-expression of Bax in Bax/Bak DKO MEFs restored stress-induced redistribution of nesprin-2 by a mechanism which requires Bax membrane localization and integrity of the α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. We found that nesprin-2 interacts with Bax in close proximity to perinuclear mitochondria in mouse and human cells. This interaction requires the mitochondrial targeting and N-terminal region but not the BH3 domain of Bax. Our results identify nesprin-2 as a Bax binding partner and also a new function of Bax in impairing the integrity of the LINC complex.

CD38 in cancer-associated fibroblasts promotes pro-tumoral activity.

Primary and metastatic melanoma progression are supported by a local microenvironment comprising, inter alia, of cancer-associated fibroblasts (CAFs). We previously reported in orthotropic/syngeneic mouse models that the stromal ectoenzyme CD38 participates in melanoma growth and metastasis. The results presented here suggest that CD38 is a novel regulator of CAFs' pro-tumorigenic functions. Orthotopic co-implantation of CD38 deficient fibroblasts and B16F10 melanoma cells limited tumor size, compared with CD38-expressing fibroblasts. Intrinsically, CAF-CD38 promoted migration of primary fibroblasts toward melanoma cells. Further, in vitro paracrine effects of CAF-CD38 fostered tumor cell migration and invasion as well as endothelial cell tube formation. Mechanistically, we report that CAF-CD38 drives the protein expression of an angiogenic/pro-metastatic signature, which includes VEGF-A, FGF-2, CXCL-12, MMP-9, and HGF. Data suggest that CAF-CD38 fosters tumorigenesis by enabling the production of pro-tumoral factors that promote cell invasion, migration, and angiogenesis.

The nuclear envelope: target and mediator of the apoptotic process.

Apoptosis is characterized by the destruction of essential cell organelles, including the cell nucleus. The nuclear envelope (NE) separates the nuclear interior from the cytosol. During apoptosis, the apoptotic machinery, in particular caspases, increases NE permeability by cleaving its proteins, such as those of the nuclear pore complex (NPC) and the nuclear lamina. This in turns leads to passive diffusion of cytosolic apoptogenic proteins, such as caspases and nucleases, through NPCs into the nucleus and the subsequent breakdown of the NE and destruction of the nucleus. However, NE leakiness at early stages of the apoptotic process can also occur in a caspase-independent manner, where Bax, by a non-canonical action, promotes transient and repetitive localized generation and subsequent rupture of nuclear protein-filled nuclear bubbles. This NE rupture leads to discharge of apoptogenic nuclear proteins from the nucleus to the cytosol, a process that can contribute to the death process. Therefore, the NE may play a role as mediator of cell death at early stages of apoptosis. The NE can also serve as a platform for assembly of complexes that regulate the death process. Thus, the NE should be viewed as both a mediator of the cell death process and a target.

Stromal CD38 regulates outgrowth of primary melanoma and generation of spontaneous metastasis.

The outgrowth of primary melanoma, the deadliest skin cancer, and generation of metastasis is supported by the tumor microenvironment (TME) which includes non-cancerous cells. Since the TME plays an important role in melanoma pathogenesis, its targeting is a promising therapeutic approach. Thus, it is important to identify proteins in the melanoma TME that may serve as therapeutic targets. Here we show that the nicotinamide adenine dinucleotide glycohydrolase CD38 is a suitable target for this purpose. Loss of CD38 in the TME as well as inhibition of its enzymatic activity restrained outgrowth of primary melanoma generated by two transplantable models of melanoma, B16F10 and Ret-mCherry-sorted (RMS) melanoma cells. Pathological analysis indicated that loss of CD38 increased cell death and reduced the amount of cancer-associated fibroblasts (CAFs) and blood vessels. Importantly, in addition to inhibiting outgrowth of primary melanoma tumors, loss of CD38 also inhibited spontaneous occurrence of RMS pulmonary and brain metastasis. The underlying mechanism may involve, at least in the brain, inhibition of metastasis expansion, since loss of CD38 inhibited the outgrowth of B16F10 and RMS brain tumors that were generated by direct intracranial implantation. Collectively, our results suggest that targeting CD38 in the melanoma TME provides a new therapeutic approach for melanoma treatment.

Increased Degree of Unsaturation in the Lipid of Antifungal Cationic Amphiphiles Facilitates Selective Fungal Cell Disruption.

Antimicrobial cationic amphiphiles derived from aminoglycosides act through cell membrane permeabilization but have limited selectivity for microbial cell membranes. Herein, we report that an increased degree of unsaturation in the fatty acid segment of antifungal cationic amphiphiles derived from the aminoglycoside tobramycin significantly reduced toxicity to mammalian cells. A collection of tobramycin-derived cationic amphiphiles substituted with C18 lipid chains varying in degree of unsaturation and double bond configuration were synthesized. All had potent activity against a panel of important fungal pathogens including strains with resistance to a variety of antifungal drugs. The tobramycin-derived cationic amphiphile substituted with linolenic acid with three cis double bonds (compound 6) was up to an order of magnitude less toxic to mammalian cells than cationic amphiphiles composed of lipids with a lower degree of unsaturation and than the fungal membrane disrupting drug amphotericin B. Compound 6 was 12-fold more selective (red blood cell hemolysis relative to antifungal activity) than compound 1, the derivative with a fully saturated lipid chain. Notably, compound 6 disrupted the membranes of fungal cells without affecting the viability of cocultured mammalian cells. This study demonstrates that the degree of unsaturation and the configuration of the double bond in lipids of cationic amphiphiles are important parameters that, if optimized, result in compounds with broad spectrum and potent antifungal activity as well as reduced toxicity toward mammalian cells.

Incipient Melanoma Brain Metastases Instigate Astrogliosis and Neuroinflammation.

Malignant melanoma is the deadliest of skin cancers. Melanoma frequently metastasizes to the brain, resulting in dismal survival. Nevertheless, mechanisms that govern early metastatic growth and the interactions of disseminated metastatic cells with the brain microenvironment are largely unknown. To study the hallmarks of brain metastatic niche formation, we established a transplantable model of spontaneous melanoma brain metastasis in immunocompetent mice and developed molecular tools for quantitative detection of brain micrometastases. Here we demonstrate that micrometastases are associated with instigation of astrogliosis, neuroinflammation, and hyperpermeability of the blood-brain barrier. Furthermore, we show a functional role for astrocytes in facilitating initial growth of melanoma cells. Our findings suggest that astrogliosis, physiologically instigated as a brain tissue damage response, is hijacked by tumor cells to support metastatic growth. Studying spontaneous melanoma brain metastasis in a clinically relevant setting is the key to developing therapeutic approaches that may prevent brain metastatic relapse. Cancer Res; 76(15); 4359-71. ©2016 AACR.

Intercellular transfer of small RNAs from astrocytes to lung tumor cells induces resistance to chemotherapy.

Brain metastases are resistant to chemotherapy and carry a poor prognosis. Studies have shown that tumor cells are surrounded by activated astrocytes, whose cytoprotective properties they exploit for protection from chemotherapy-induced apoptosis. The mechanism of such astrocytic protection is poorly understood. A non-mutational mechanism of resistance to chemotherapy that is receiving increased attention is the regulation of gene translation mediated by small noncoding RNAs (sRNAs), and particularly microRNAs (miRNAs). With the aim of examining the role of astrocytic sRNAs in promoting resistance of human lung tumor PC14 cells to chemotherapy-induced apoptosis, here we used a miRNA microarray to compare sRNA profiles of human lung tumor cells cultured with and without astrocytes. We found that sRNAs are transferred from astrocytes to PC14 cells in a contact-dependent manner. Transfer was rapid, reaching a plateau after only 6 hours in culture. The sRNA transfer was inhibited by the broad-spectrum gap-junction antagonist carbenoxolone, indicating that transfer occurs via gap junctions. Among the transferred sRNAs were several that are implicated in survival pathways. Enforced expression of these sRNAs in PC14 cells increased their resistance to the chemotherapeutic agent paclitaxel. These novel findings might be of clinical relevance for the treatment of patients with brain metastases.

Regulation of alternative splicing at the single-cell level.

Alternative splicing is a key cellular mechanism for generating distinct isoforms, whose relative abundances regulate critical cellular processes. It is therefore essential that inclusion levels of alternative exons be tightly regulated. However, how the precision of inclusion levels among individual cells is governed is poorly understood. Using single-cell gene expression, we show that the precision of inclusion levels of alternative exons is determined by the degree of evolutionary conservation at their flanking intronic regions. Moreover, the inclusion levels of alternative exons, as well as the expression levels of the transcripts harboring them, also contribute to this precision. We further show that alternative exons whose inclusion levels are considerably changed during stem cell differentiation are also subject to this regulation. Our results imply that alternative splicing is coordinately regulated to achieve accuracy in relative isoform abundances and that such accuracy may be important in determining cell fate.

Exploring the Effects of Glycosylation and Etherification of the Side Chains of the Anticancer Drug Mitoxantrone.

Herein we report the synthesis and biological evaluation of symmetric and asymmetric analogues of the DNA intercalating drug mitoxantrone (MTX) in which the side chains of the parent drug were modified through glycosylation or methyl etherification. Several analogues with glycosylated side chains exhibited higher DNA affinity than the parent MTX. The most potent in vitro cytotoxicity was observed for MTX analogue 8 (1,4-dimethoxy-5,8-bis[2-(2-methoxyethylamino)ethylamino]anthracene-9,10-dione) with methoxy ether containing side chains. Treatment of melanoma-bearing mice with MTX or analogue 8 decreased the intraperitoneal tumor burden relative to untreated mice; the effect of 8 was less pronounced than that of MTX. In vitro metabolism assays of MTX with rabbit liver S9 fraction gave rise to several metabolites; almost no metabolites were detected for MTX analogue 8. The results presented indicate that derivatization of the MTX side chain primary hydroxy groups may result in a significant improvement in DNA affinity and lower susceptibility to the formation of potentially toxic metabolites.

Alzheimer's disease pathology is attenuated in a CD38-deficient mouse model.

OBJECTIVE: Alzheimer's disease (AD)-associated dementia is due to tissue damage caused by amyloid ß (Aß) deposition within the brain and by accompanying neuroinflammation. The nicotinamide adenine dinucleotide (NAD) glycohydrolase CD38, which is expressed by neurons, astrocytes, and microglial cells, regulates inflammatory and repair processes in the brain and other tissues by degrading NAD and repressing the activity of other NAD-consuming enzymes and by producing NAD-derived metabolites that regulate calcium signaling and migration of inflammatory cells. Given the role of CD38 in neuroinflammation and repair, we examined the effect of CD38 deletion on AD pathology. METHODS: We crossed APPswePS1ΔE9 (APP.PS) mice with Cd38(-) (/) (-) mice to generate AD-prone CD38-deficient animals (APP.PS.Cd38(-) (/) (-) ) and examined AD-related phenotypes in both groups. RESULTS: APP.PS.Cd38(-) (/) (-) mice exhibited significant reductions in Aß plaque load and soluble Aß levels compared to APP.PS mice, and this correlated with improved spatial learning. Although CD38 deficiency resulted in decreased microglia/macrophage (MM) accumulation, the transcription profile of the Cd38(-) (/) (-) and Cd38(+/) (+) MM was similar, suggesting that the decreased Aß burden in APP.PS.Cd38(-) (/) (-) mice was not due to alterations in MM activation/function. Instead, APP.PS.Cd38(-) (/) (-) neuronal cultures secreted less Aß and this reduction was mimicked when APP.PS neuronal cultures were treated with inhibitors that blocked CD38 enzyme activity or the signaling pathways controlled by CD38-derived metabolites. Furthermore, ß- and γ-secretase activity was decreased in APP.PS.Cd38(-) (/) (-) mice, which correlated with decreased Aß production. INTERPRETATION: CD38 regulates AD pathology in the APP.PS model of AD, suggesting that CD38 may be a novel target for AD treatment.

Inhibition of glioma progression by a newly discovered CD38 inhibitor.

Glioma, the most common cancer of the central nervous system, has very poor prognosis and no effective treatment. It has been shown that activated microglia/macrophages in the glioma tumor microenvironment support progression. Hence, inhibition of the supporting effect of these cells may constitute a useful therapeutic approach. Recently, using a syngeneic mouse glioma progression model, we showed that the ectoenzyme CD38 regulated microglia activation and, in addition, that the loss of CD38 from the tumor microenvironment attenuated glioma progression and prolonged the life span of the tumor-bearing mice. These studies, which employed wild-type (WT) and Cd38(-/-) C57BL/6J mice, suggest that inhibition of CD38 in glioma microenvironment may be used as a new therapeutic approach to treat glioma. Our study tested this hypothesis. Initially, we found that the natural anthranoid, 4,5-dihydroxyanthraquinone-2-carboxylic acid (rhein), and its highly water-soluble tri-potassium salt form (K-rhein) are inhibitors of CD38 enzymatic (nicotinamide adenine dinucleotide glycohydrolase) activity (IC50 = 1.24 and 0.84 µM, respectively, for recombinant mouse CD38). Treatment of WT, but not Cd38(-/-) microglia with rhein and K-rhein inhibited microglia activation features known to be regulated by CD38 (lipopolysaccharide/IFN-γ-induced activation, induced cell death and NO production). Furthermore, nasal administration of K-rhein into WT, but not Cd38(-/-) C57BL/6J, mice intracranially injected with GL261 cells substantially and significantly inhibited glioma progression. Hence, these results serve as a proof of concept, demonstrating that targeting CD38 at the tumor microenvironment by small-molecule inhibitors of CD38, for example, K-rhein, may serve as a useful therapeutic approach to treat glioma.

Cellular stress induces Bax-regulated nuclear bubble budding and rupture followed by nuclear protein release.

Cellular stress triggers many pathways including nuclear protein redistribution. We previously discovered that this process is regulated by Bax but the underlying mechanism has not yet been studied. Here we define this mechanism by showing that apoptotic stimuli cause Bax-regulated disturbances in lamin A/C and nuclear envelope (NE)-associated proteins which results in the generation and subsequent rupture of nuclear protein-containing bubbles. The bubbles do not contain DNA and are encapsulated by impaired nuclear pore-depleted NE. Stress-induced generation and rupture of nuclear bubbles ultimately leads to the discharge of nuclear proteins into the cytoplasm. This process precedes morphological changes of apoptosis and occurs independently of caspases. Rescue experiments revealed that this Bax effect is non-canonical, i.e. it requires the BH3 domain and α-helices 5 and 6 but it is not inhibited by Bcl(-)xL. Targeting Bax to the NE by the Klarsicht/ANC-1/Syne-1 homology (KASH) domain effectively triggers the generation and rupture of nuclear bubbles. Overall, our findings provide evidence for a novel stress-response, which is regulated by a non-canonical action of Bax on the NE.

Upregulation of PGC-1α expression by Alzheimer's disease-associated pathway: presenilin 1/amyloid precursor protein (APP)/intracellular domain of APP.

Cleavage of amyloid precursor protein (APP) by ß- and γ-secretase generates amyloid-ß (Aß) and APP intracellular domain (AICD) peptides. Presenilin (PS) 1 or 2 is the catalytic component of the γ-secretase complex. Mitochondrial dysfunction is an established phenomenon in Alzheimer's disease (AD), but the causes and role of PS1, APP, and APP's cleavage products in this process are largely unknown. We studied the effect of these AD-associated molecules on mitochondrial features. Using cells deficient in PSs expression, expressing human wild-type PS1, or PS1 familial AD (FAD) mutants, we found that PS1 affects mitochondrial energy metabolism (ATP levels and oxygen consumption) and expression of mitochondrial proteins. These effects were associated with enhanced expression of the mitochondrial master transcriptional coactivator PGC-1α and its target genes. Importantly, PS1-FAD mutations decreased PS1's ability to enhance PGC-1α mRNA levels. Analyzing the effect of APP and its γ-secretase-derived cleavage products Aß and AICD on PGC-1α expression showed that APP and AICD increase PGC-1α expression. Accordingly, PGC-1α mRNA levels in cells deficient in APP/APLP2 or expressing APP lacking its last 15 amino acids were lower than in control cells, and treatment with AICD, but not with Aß, enhanced PGC-1α mRNA levels in these and PSs-deficient cells. In addition, knockdown of the AICD-binding partner Fe65 reduced PGC-1α mRNA levels. Importantly, APP/AICD increases PGC-1α expression also in the mice brain. Our results therefore suggest that APP processing regulates mitochondrial function and that impairments in the newly discovered PS1/APP/AICD/PGC-1α pathway may lead to mitochondrial dysfunction and neurodegeneration.

Non-canonical function of Bax in stress-induced nuclear protein redistribution.

Bax and Bak (Bax/Bak) are essential pro-apoptotic proteins of the Bcl-2 family that trigger mitochondrial outer membrane permeabilization (MOMP) in a Bcl-2/Bcl-xL-inhibitable manner. We recently discovered a new stress-related function for Bax/Bak-regulation of nuclear protein redistribution (NPR) from the nucleus to cytoplasm. This effect was independent of Bax/Bak N-terminus exposure and not inhibited by Bcl-xL over-expression. Here, we studied the molecular mechanism governing this novel non-canonical response. Wild-type (WT) and mutant versions of Bax were re-expressed in Bax/Bak double-knockout mouse embryonic fibroblasts and their ability to promote NPR, apoptotic events, and changes in lamin A mobility was examined. Our results show that, in this system, Bax expression was sufficient to restore NPR such as in WT cells undergoing apoptosis. This activity of Bax was uncoupled from cytochrome c release from the mitochondria (indicative of MOMP) and required its membrane localization, α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. Moreover, enrichment of Bax in the nuclear envelope by the so-called Klarsicht/ANC-1/Syne-1 homology domain effectively triggered NPR as in WT Bax, but without inducing MOMP or cell death. Bax-induced NPR was associated with impairment in lamin A mobility, implying a connection between these two nuclear envelope-associated events. Overall, the results indicate a new MOMP-independent, stress-induced Bax function on the nuclear envelope.

CD38 deficiency in the tumor microenvironment attenuates glioma progression and modulates features of tumor-associated microglia/macrophages.

Gliomas are the most frequent primary tumors of the brain, and for highly malignant gliomas there is no successful treatment. The tumor microenvironment contains large numbers of infiltrating microglia and macrophages (MM). There is increasing evidence that the tumor-associated MM support glioma expansion. CD38 is a multifunctional ectoenzyme that uses nicotinamide adenine dinucleotide as a substrate to generate second messengers. Previously we showed that CD38 deficiency modulates microglial "activation" and impaired recovery from head trauma by a microglia-associated mechanism. In view of the supportive role of MM in glioma progression and the role of CD38 in microglia activation, we hypothesize that deficiency of CD38 in the tumor microenvironment would inhibit glioma progression. Using the syngeneic GL261 model of glioma progression in wild-type and CD38 null mice, we show here that CD38 deficiency significantly attenuates glioma expansion and prolongs the life span of the glioma-bearing mice. The CD38 deficiency effect was associated with increased cell death and decreased metalloproteinase-12 expression in the tumor mass, as well as modulation of the tumor-induced MM properties, as indicated by a reduction in the expression of the MM marker F4/80 and matrix metalloproteinases. Our results thus suggest that CD38 participates in the tumor-supporting action of MM and that targeting CD38 might be a potential therapeutic approach for glioma treatment.

γ-Secretase component presenilin is important for microglia ß-amyloid clearance.

OBJECTIVE: The cleavage of amyloid precursor protein by γ-secretase is an important aspect of the pathogenesis of Alzheimer's disease. γ-Secretase also cleaves other membrane proteins (eg, Notch), which control cell development and homeostasis. Presenilin 1 and 2 are considered important determinants of the γ-secretase catalytic site. Our aim was to investigate whether γ-secretase can be important for microglial phagocytosis of Alzheimer's disease ß-amyloid. METHODS: We investigated the role of γ-secretase in microglia activity toward ß-amyloid phagocytosis in cell culture using γ-secretase inhibitors and small hairpin RNA and presenilin-deficient mice. RESULTS: We found that γ-secretase inhibitors impair microglial activity as measured in gene expression, protein levels, and migration ability, which resulted in a reduction of soluble ß-amyloid phagocytosis. Moreover, microglia deficient in presenilin 1 and 2 showed impairment in phagocytosis of soluble ß-amyloid. Dysfunction in the γ-secretase catalytic site led to an impairment in clearing insoluble ß-amyloid from brain sections taken from an Alzheimer's disease mouse model when compared to microglia from wild-type mice. INTERPRETATION: We suggest for the first time, a dual role for γ-secretase in Alzheimer's disease. One role is the cleavage of the amyloid precursor protein for pathologic ß-amyloid production and the other is to regulate microglia activity that is important for clearing neurotoxic ß-amyloid deposits. Further studies of γ-secretase-mediated cellular pathways in microglia may provide useful insights into the development of Alzheimer's disease and other neurodegenerative diseases, providing future avenues for therapeutic intervention.