Immunohistochemical Characterization of Phosphorylated Ubiquitin in the Mouse Hippocampus

ABSTRACTMitochondrial autophagy (mitophagy) is an essential and evolutionarily conserved process that maintains mitochondrial integrity via the removal of damaged or superfluous mitochondria in eukaryotic cells. Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and Parkin promote mitophagy and function in a common signaling pathway. PINK1-mediated ubiquitin phosphorylation at Serine 65 (Ser65-pUb) is a key event in the efficient execution of PINK1/Parkin-dependent mitophagy. However, few studies have used immunohistochemistry to analyze Ser65-pUb in the mouse. Here, we examined the immunohistochemical characteristics of Ser65-pUb in the mouse hippocampus. Some hippocampal cells were Ser65-pUb positive, whereas the remaining cells expressed no or low levels of Ser65-pUb. PINK1 deficiency resulted in a decrease in the density of Ser65-pUb-positive cells, consistent with a previous hypothesis based on in vitro research. Interestingly, Ser65-pUb-positive cells were detected in hippocampi lacking PINK1 expression. The CA3 pyramidal cell layer and the dentate gyrus (DG) granule cell layer exhibited significant reductions in the density of Ser65-pUb-positive cells in PINK1-deficient mice. Moreover, Ser65-pUb immunoreactivity colocalized predominantly with neuronal markers. These findings suggest that Ser65-pUb may serve as a biomarker of in situ PINK1 signaling in the mouse hippocampus; however, the results should be interpreted with caution, as PINK1 deficiency downregulated Ser65-pUb only partially.

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Identification and Functional Analysis of Apoptotic Protease Activating Factor-1 (Apaf-1) from Spodoptera litura

Insects ◽

10.3390/insects12010064 ◽

2021 ◽

Vol 12 (1) ◽

pp. 64

Author(s):

Haihao Ma ◽

Xiumei Yan ◽

Lin Yan ◽

Jingyan Zhao ◽

Jiping Song ◽

...

Keyword(s):

Spodoptera Litura ◽

Actinomycin D ◽

Titration Calorimetry ◽

Apoptosis Pathway ◽

Downstream Effector ◽

Lepidopteran Insects ◽

Evolutionarily Conserved ◽

Effector Caspases ◽

And Function

Apoptotic protease activating factor-1 (Apaf-1) is an adaptor molecule, essential for activating initiator caspase and downstream effector caspases, which directly cause apoptosis. In fruit flies, nematodes, and mammals, Apaf-1 has been extensively studied. However, the structure and function of Apaf-1 in Lepidoptera remain unclear. This study identified a novel Apaf-1 from Spodoptera litura, named Sl-Apaf-1. Sl-Apaf-1 contains three domains: a CARD domain, as well as NOD and WD motifs, and is very similar to mammalian Apaf-1. Interference of Sl-apaf-1 expression in SL-1 cells blocked apoptosis induced by actinomycin D. Overexpression of Sl-apaf-1 significantly enhances apoptosis induced by actinomycin D in Sf9/SL-1/U2OS cells, suggesting that the function of Sl-Apaf-1 is evolutionarily conserved. Furthermore, Sl-Apaf-1 could interact with Sl-caspase-5 (a homologue of mammalian caspase-9) and yielded a binding affinity of 1.37 × 106 M–1 according isothermal titration calorimetry assay. Initiator caspase (procaspase-5) of S. litura could be activated by Sl-Apaf-1 (without WD motif) in vitro, and the activated Sl-caspase-5 could cleave Sl-procaspase-1 (a homologue of caspase-3 in mammals), which directly caused apoptosis. This study demonstrates the key role of Sl-Apaf-1 in the apoptosis pathway, suggesting that the apoptosis pathway in Lepidopteran insects and mammals is conserved.

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BMI1 promotes cardiac fibrosis in ischemia-induced heart failure via the PTEN-PI3K/Akt-mTOR signaling pathway

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00487.2018 ◽

2019 ◽

Vol 316 (1) ◽

pp. H61-H69 ◽

Cited By ~ 11

Author(s):

Wenbo Yang ◽

Zhijun Wu ◽

Ke Yang ◽

Yanxin Han ◽

Yanjia Chen ◽

...

Keyword(s):

Cardiac Fibrosis ◽

Mammalian Target Of Rapamycin ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatase And Tensin Homolog ◽

B Lymphoma ◽

Tensin Homolog ◽

And Migration ◽

Proliferation And Migration

Cardiac fibrosis has been known to play an important role in the etiology of heart failure after myocardial infarction (MI). B lymphoma Mo-MLV insertion region 1 homolog (BMI1), a transcriptional repressor, is important for fibrogenesis in the kidneys. However, the effect of BMI1 on ischemia-induced cardiac fibrosis remains unclear. BMI1 was strongly expressed in the infarct region 1 wk post-MI in mice and was detected by Western blot and histological analyses. Lentivirus-mediated overexpression of BMI1 significantly promoted cardiac fibrosis, worsened cardiac function 4 wk after the intervention in vivo, and enhanced the proliferation and migration capabilities of fibroblasts in vitro , whereas downregulation of BMI1 decreased cardiac fibrosis and prevented cardiac dysfunction in mice 4 wk post-MI in vivo. Furthermore, upregulated BMI1 inhibited phosphatase and tensin homolog (PTEN) expression, enhanced phosphatidylinositol 3-kinase (PI3K) expression, and increased the phosphorylation level of Akt and mammalian target of rapamycin (mTOR) in mice 4 wk after lentiviral infection, which was in accordance with the changes seen in their infarcted myocardial tissues. At the same time, the effects of BMI1 on cardiac fibroblasts were reversed in vitro when these cells were exposed to NVP-BEZ235, a dual-kinase (PI3K/mTOR) inhibitor. In conclusion, BMI1 is associated with cardiac fibrosis and dysfunction after MI by regulating cardiac fibroblast proliferation and migration, and these effects could be partially explained by the regulation of the PTEN-PI3K/Akt-mTOR pathway. NEW & NOTEWORTHY Ischemia-induced B lymphoma Mo-MLV insertion region 1 homolog (BMI1) significantly promoted cardiac fibrosis and worsened cardiac function in vivo, whereas downregulation of BMI1 decreased cardiac fibrosis and prevented cardiac dysfunction in myocardial infarcted mice. BMI1 also enhanced proliferation and migration capabilities of fibroblasts in vitro; these effects were reversed by NVP-BEZ235. Effects of BMI1 on cardiac fibrosis could be partially explained by regulation of the phosphatase and tensin homolog-phosphatidylinositol 3-kinase/Akt-mammalian target of rapamycin pathway.

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Targeting TORC2 in multiple myeloma with a new mTOR kinase inhibitor

Blood ◽

10.1182/blood-2010-05-285726 ◽

2010 ◽

Vol 116 (22) ◽

pp. 4560-4568 ◽

Cited By ~ 86

Author(s):

Bao Hoang ◽

Patrick Frost ◽

Yijiang Shi ◽

Eileen Belanger ◽

Angelica Benavides ◽

...

Keyword(s):

Multiple Myeloma ◽

Kinase Inhibitor ◽

Mammalian Target Of Rapamycin ◽

Target Of Rapamycin ◽

Phosphatase And Tensin Homolog ◽

Akt Phosphorylation ◽

Tensin Homolog ◽

Complex Protein ◽

Preclinical Work

Although preclinical work with rapalogs suggests potential in treatment of multiple myeloma (MM), they have been less successful clinically. These drugs allostearically inhibit the mammalian target of rapamycin kinase primarily curtailing activity of the target of rapamycin complex (TORC)1. To assess if the mammalian target of rapamycin within the TORC2 complex could be a better target in MM, we tested a new agent, pp242, which prevents activation of TORC2 as well as TORC1. Although comparable to rapamycin against phosphorylation of the TORC1 substrates p70S6kinase and 4E-BP-1, pp242 could also inhibit phosphorylation of AKT on serine 473, a TORC2 substrate, while rapamycin was ineffective. pp242 was also more effective than rapamycin in achieving cytoreduction and apoptosis in MM cells. In addition, pp242 was an effective agent against primary MM cells in vitro and growth of 8226 cells in mice. Knockdown of the TORC2 complex protein, rictor, was deleterious to MM cells further supporting TORC2 as the critical target for pp242. TORC2 activation was frequently identified in primary specimens by immunostaining for AKT phosphorylation on serine 473. Potential mechanisms of up-regulated TORC2 activity in MM were stimulation with interleukin-6 or insulin-like growth factor 1, and phosphatase and tensin homolog or RAS alterations. Combining pp242 with bortezomib led to synergistic anti-MM effects. These results support TORC2 as a therapeutic target in MM.

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Podocyte-specific knockin of PTEN protects kidney from hyperglycemia

AJP Renal Physiology ◽

10.1152/ajprenal.00575.2017 ◽

2018 ◽

Vol 314 (6) ◽

pp. F1096-F1107 ◽

Cited By ~ 10

Author(s):

Huizhen Wang ◽

Ziwei Feng ◽

Jianteng Xie ◽

Feng Wen ◽

Menglei Jv ◽

...

Keyword(s):

Diabetic Kidney Disease ◽

Therapeutic Strategy ◽

Renal Protection ◽

Phosphatase And Tensin Homolog ◽

Tensin Homolog ◽

Matrix Expansion ◽

Interfering Rna

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has proven to be downregulated in podocytes challenged with high glucose (HG), and knockout of PTEN in podocytes aggravated the progression of diabetic kidney disease (DKD). However, whether podocyte-specific knockin of PTEN protects the kidney against hyperglycemia in vivo remains unknown. The inducible podocyte-specific PTEN knockin (PPKI) mice were generated by crossing newly created transgenic loxP-stop- loxP-PTEN mice with podocin-iCreERT2 mice. Diabetes mellitus was induced in mice by intraperitoneal injection of streptozotocin at a dose of 150 mg/kg. In vitro, small interfering RNA and adenovirus interference were used to observe the role of PTEN in HG-treated podocytes. Our data demonstrated that PTEN was markedly reduced in the podocytes of patients with DKD and focal segmental glomerulosclerosis, as well as in those of db/db mice. Interestingly, podocyte-specific knockin of PTEN significantly alleviated albuminuria, mesangial matrix expansion, effacement of podocyte foot processes, and incrassation of glomerular basement membrane in diabetic PPKI mice compared with wild-type diabetic mice, whereas no alteration was observed in the level of blood glucose. The potential renal protection of overexpressed PTEN in podocytes was partly attributed with an improvement in autophagy and motility and the inhibition of apoptosis. Our results showed that podocyte-specific knockin of PTEN protected the kidney against hyperglycemia in vivo , suggesting that targeting PTEN might be a novel and promising therapeutic strategy against DKD.

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Dynamics and function of CXCR4 in formation of the granule cell layer during hippocampal development

Scientific Reports ◽

10.1038/s41598-017-05738-7 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 3

Author(s):

Yuka Mimura-Yamamoto ◽

Hiroshi Shinohara ◽

Taichi Kashiwagi ◽

Toru Sato ◽

Seiji Shioda ◽

...

Keyword(s):

Granule Cell ◽

Cell Layer ◽

Granule Cell Layer ◽

Hippocampal Development ◽

Dynamics And Function ◽

And Function

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Ovarian Fragmentation and AKT Stimulation for Expansion of Fertile Lifespan

Frontiers in Reproductive Health ◽

10.3389/frph.2021.636771 ◽

2021 ◽

Vol 3 ◽

Author(s):

Kim Cat Tuyen Vo ◽

Kazuhiro Kawamura

Keyword(s):

Ovarian Reserve ◽

Enzyme Inhibitor ◽

Premature Ovarian Insufficiency ◽

Hippo Signaling ◽

Follicle Growth ◽

Ovarian Insufficiency ◽

Phosphatase And Tensin Homolog ◽

Primordial Follicles ◽

Tensin Homolog

Since the first baby was born after in vitro fertilization, the female infertility treatment has been well-developed, yielding successful outcomes. However, successful pregnancies for patients with premature ovarian insufficiency and diminished ovarian reserve are still difficult and diverse therapies have been suggested to improve the chances to have their genetically linked offspring. Recent studies demonstrated that the activation Akt pathway by using a phosphatase and tensin homolog enzyme inhibitor and a phosphatidylinositol-3 kinase stimulator can activate dormant primordial follicles in both mice and human ovaries. Subsequent researches suggested that the disruption of Hippo signaling pathway by ovarian fragmentation increased the expression of downstream growth factors and secondary follicle growth. Based on the combination of ovarian fragmentation and Akt stimulation, the in vitro activation (IVA) approach has resulted in successful follicle growth and live births in premature ovarian insufficiency patients. The approach with disruption of Hippo signaling only was also shown to be effective for treating poor ovarian responders with diminishing ovarian reserve, including advanced age women and cancer patients undergoing sterilizing treatments. This review aims to summarize the effectiveness of ovarian fragmentation and Akt stimulation on follicle growth and the potential of IVA in extending female fertile lifespan.

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miR-375 Inhibits Differentiation of Neurites by Lowering HuD Levels

Molecular and Cellular Biology ◽

10.1128/mcb.00316-10 ◽

2010 ◽

Vol 30 (17) ◽

pp. 4197-4210 ◽

Cited By ~ 83

Author(s):

Kotb Abdelmohsen ◽

Emmette R. Hutchison ◽

Eun Kyung Lee ◽

Yuki Kuwano ◽

Mihee M. Kim ◽

...

Keyword(s):

Mrna Stability ◽

Untranslated Region ◽

Neuronal Development ◽

Rna Binding ◽

Developmentally Regulated ◽

Cytoskeleton Organization ◽

Evolutionarily Conserved ◽

Mouse Hippocampus ◽

Regulated Gene Expression ◽

And Function

ABSTRACT Neuronal development and plasticity are maintained by tightly regulated gene expression programs. Here, we report that the developmentally regulated microRNA miR-375 affects dendrite formation and maintenance. miR-375 overexpression in mouse hippocampus potently reduced dendrite density. We identified the predominantly neuronal RNA-binding protein HuD as a key effector of miR-375 influence on dendrite maintenance. Heterologous reporter analysis verified that miR-375 repressed HuD expression through a specific, evolutionarily conserved site on the HuD 3′ untranslated region. miR-375 overexpression lowered both HuD mRNA stability and translation and recapitulated the effects of HuD silencing, which reduced the levels of target proteins with key functions in neuronal signaling and cytoskeleton organization (N-cadherin, PSD-95, RhoA, NCAM1, and integrin α1). Moreover, the increase in neurite outgrowth after brain-derived neurotrophic factor (BDNF) treatment was diminished by miR-375 overexpression; this effect was rescued by reexpression of miR-375-refractory HuD. Our findings indicate that miR-375 modulates neuronal HuD expression and function, in turn affecting dendrite abundance.

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Loss of phosphatase and tensin homolog (PTEN) in myeloid cells controls inflammatory bone destruction by regulating the osteoclastogenic potential of myeloid cells

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2013-203486 ◽

2013 ◽

Vol 74 (1) ◽

pp. 227-233 ◽

Cited By ~ 26

Author(s):

Stephan Blüml ◽

Martin Friedrich ◽

Tobias Lohmeyer ◽

Emine Sahin ◽

Victoria Saferding ◽

...

Keyword(s):

Myeloid Cells ◽

Bone Destruction ◽

Precursor Cells ◽

Nuclear Factor ◽

Phosphatase And Tensin Homolog ◽

Tensin Homolog ◽

Inflammatory Conditions ◽

Local Bone

ObjectiveLocal bone destruction in rheumatic diseases, which often leads to disability and severely reduced quality of life, is almost exclusively mediated by osteoclasts. Therefore, it is important to understand pathways regulating the generation of osteoclasts. Here, we analysed the impact of the Phosphoinositide-3-Kinase (PI3K)/Phosphatase and tensin homolog (PTEN) axis on osteoclast generation and bone biology under basal and inflammatory conditions.MethodsWe analysed osteoclastogenesis of wildtype (wt) and PTEN−/− cells in vitro and in vivo, pit resorption and qPCR of osteoclasts in vitro. Mice with a myeloid cell-specific deletion of PTEN and wt littermate mice were investigated by bone histomorphometry and clinical and histological assessment in the human tumour necrosis factor (TNF)-transgenic (hTNFtg) arthritis model.ResultsWe show that myeloid-specific PTEN−/− mice display increased osteoclastogenesis in vitro and in vivo compared to wt mice. Loss of PTEN did not affect the generation or survival of osteoclast precursor cells. However, PTEN deficiency greatly enhanced receptor activator of nuclear factor κ-B ligand (RANKL)-induced expression of the master transcription factor of osteoclastogenesis, nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), resulting in markedly increased terminal differentiation of osteoclasts in vitro. We also observed increased osteoclastogenesis under inflammatory conditions in the hTNFtg mouse model of arthritis, where hTNFtg/myeloid-specific PTEN−/− mice displayed enhanced local bone destruction as well as osteoclast formation in the inflamed joints. The extent of synovial inflammation, however, as well as recruitment of osteoclast precursor cells was not different between wt and myeloid-specific PTEN−/− mice.ConclusionsThese data demonstrate that loss of PTEN and, therefore, sustained PI3-Kinase signalling in myeloid cells especially, elevates the osteoclastogenic potential of myeloid cells, leading to enhanced inflammatory local bone destruction. Therefore, although our study allows no direct translational conclusion since we used a conditional knockout approach, the therapeutic targeting of the PI3-Kinase pathway may be of benefit in preventing structural joint damage.

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Ovarian Follicles Rescued 3 Days after Cyclophosphamide Treatment in Adolescent Mice: An Experimental Study Aiming at Maximizing Methods for Fertility Preservation through In Vitro Follicle Culture

International Journal of Molecular Sciences ◽

10.3390/ijms20246190 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6190

Author(s):

Amandine Anastácio ◽

Max Waterstone ◽

Xia Hao ◽

Catherine Poirot ◽

Kenny A. Rodriguez-Wallberg

Keyword(s):

Fertility Preservation ◽

Survival Rates ◽

Terminal Deoxynucleotidyl Transferase ◽

Controlled Study ◽

Phosphatase And Tensin Homolog ◽

Follicle Culture ◽

Tensin Homolog ◽

Cyclophosphamide Treatment ◽

In Vitro Follicle Culture

There is currently a lack of knowledge about the feasibility of performing procedures for fertility preservation after chemotherapy treatment has been initiated. In this experimental controlled study using adolescent mice, we aimed to investigate if the chance of rescuing and growing in vitro secondary follicles (SeF) would be affected three days after a single injection of cyclophosphamide (CPA). The main outcomes included were: (1) The number of SeF with good morphologic quality obtained per ovary 3 days after CPA injection, (2) SeF development in culture, (3) small follicle density (SFD) on histology, and (4) apoptosis markers, including terminal deoxynucleotidyl transferase dUTP nick end-labelling (TUNEL), mRNA expression, and distribution of p 53 upregulated modulator of apoptosis (Puma) and phosphatase and tensin homolog (Pten). We found a 60% reduction of SeF obtained per ovary in all CPA-treated groups vs. controls. However, in vitro survival rates at culture day 12 and antrum formation were similar among all groups. On histology, SFD was only significantly reduced in the high CPA dose group. Apoptotic cells were mainly found in large growing follicles of CPA groups. Our study indicates the feasibility of SeF isolation and in vitro follicle culture 3 days following CPA treatment and a still preserved SFD, particularly following a low-dose CPA treatment.

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PTEN Function at the Interface between Cancer and Tumor Microenvironment: Implications for Response to Immunotherapy

International Journal of Molecular Sciences ◽

10.3390/ijms21155337 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5337 ◽

Cited By ~ 1

Author(s):

Fabiana Conciatori ◽

Chiara Bazzichetto ◽

Italia Falcone ◽

Ludovica Ciuffreda ◽

Gianluigi Ferretti ◽

...

Keyword(s):

Tumor Microenvironment ◽

Current Knowledge ◽

Predictive Biomarkers ◽

Point Of View ◽

Solid Cancer ◽

Host Immune System ◽

Phosphatase And Tensin Homolog ◽

Tensin Homolog ◽

And Function ◽

Promising Development

Mounting preclinical and clinical evidence indicates that rewiring the host immune system in favor of an antitumor microenvironment achieves remarkable clinical efficacy in the treatment of many hematological and solid cancer patients. Nevertheless, despite the promising development of many new and interesting therapeutic strategies, many of these still fail from a clinical point of view, probably due to the lack of prognostic and predictive biomarkers. In that respect, several data shed new light on the role of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN) in affecting the composition and function of the tumor microenvironment (TME) as well as resistance/sensitivity to immunotherapy. In this review, we summarize current knowledge on PTEN functions in different TME compartments (immune and stromal cells) and how they can modulate sensitivity/resistance to different immunological manipulations and ultimately influence clinical response to cancer immunotherapy.

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