Lionel M.L. Chow

Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells

It was previously reported that the ciliary epithelium (CE) of the mammalian eye contains a rare population of cells that could produce clonogenic self-renewing pigmented spheres in culture. Based on their ability to up-regulate genes found in retinal neurons, it was concluded that these sphere-forming cells were retinal stem cells. This conclusion raised the possibility that CE-derived retinal stem cells could help to restore vision in the millions of people worldwide who suffer from blindness associated with retinal degeneration. We report here that human and mouse CE-derived spheres are made up of proliferating pigmented ciliary epithelial cells rather than retinal stem cells. All of the cells in the CE-derived spheres, including the proliferating cells, had molecular, cellular, and morphological features of differentiated pigmented CE cells. These differentiated cells ectopically expressed nestin when exposed to growth factors and low levels of pan-neuronal markers such as beta-III-tubulin. Although the cells aberrantly expressed neuronal markers, they retained their pigmented CE cell morphology and failed to differentiate into retinal neurons in vitro or in vivo. Our results provide an example of a differentiated cell type that can form clonogenic spheres in culture, self-renew, express progenitor cell markers, and initiate neuronal differentiation that is not a stem or progenitor cell. More importantly, our findings highlight the importance of shifting the focus away from studies on CE-derived spheres for cell-based therapies to restore vision in the degenerating retina and improving techniques for using ES cells or retinal precursor cells.

Cooperativity within and among Pten, p53, and Rb Pathways Induces High-Grade Astrocytoma in Adult Brain

Mutations in the PTEN, TP53, and RB1 pathways are obligate events in the pathogenesis of human glioblastomas. We induced various combinations of deletions in these tumor suppressors in astrocytes and neural precursors in mature mice, resulting in astrocytomas ranging from grade III to grade IV (glioblastoma). There was selection for mutation of multiple genes within a pathway, shown by somatic amplifications of genes in the PI3K or Rb pathway in tumors in which Pten or Rb deletion was an initiating event. Despite multiple mutations within PI3K and Rb pathways, elevated Mapk activation was not consistent. Gene expression profiling revealed striking similarities to subclasses of human diffuse astrocytoma. Astrocytomas were found within and outside of proliferative niches in the adult brain.

Prox1 is required for granule cell maturation and intermediate progenitor maintenance during brain neurogenesis.

The transcription factor Prox1 plays a crucial role in intermediate progenitor maintenance and hippocampal neuron differentiation during adult neurogenesis in the dentate gyrus region of the hippocampus.

Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK

Significance Cancer cells reprogram their metabolism for optimal growth and survival. AMPK-activated protein kinase (AMPK) is a key energy sensor that controls many metabolic pathways including metabolic reprogramming. However, its role in cancer is poorly understood. Some studies claim that it has a tumor suppressor role while others show its protumor role. Two AMPK-activating compounds (including metformin, now in many clinical trials) are widely used to suppress cancer cell proliferation. We found that AMPK is abundantly expressed in high-grade gliomas and, in contrast to popular belief, these two AMPK activators suppressed glioma cell proliferation through unique AMPK-independent mechanisms. The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis—two crucial arms of cancer growth—AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40’s association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.

MELK-Dependent FOXM1 Phosphorylation is Essential for Proliferation of Glioma Stem Cells

Glioblastoma multiforme (GBM) is a life‐threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem‐like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase‐dependent manner. This MELK‐dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK‐driven FOXM1 activation is regulated by the binding and subsequent trans‐phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA‐mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK‐mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first‐line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC‐derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1‐dependent manner and thus, this protein complex is a potential therapeutic target for GBM. STEM Cells 2013;31:1051–1063

Rapid cell-cycle reentry and cell death after acute inactivation of the retinoblastoma gene product in postnatal cochlear hair cells

Unlike lower vertebrates, mammals are unable to replace damaged mechanosensory hair cells (HCs) in the cochlea. Recently, ablation of the retinoblastoma protein (Rb) in undifferentiated mouse HC precursors was shown to cause cochlear HC proliferation and the generation of new HCs, raising the hope that inactivation of Rb in postmitotic HCs could trigger cell division and regenerate functional HCs postnatally. Here, we acutely inactivated Rb in nearly all cochlear HCs of newborn mice, using a newly developed HC-specific inducible Cre mouse line. Beginning 48 h after Rb deletion, ≈40% of HCs were in the S and M phases of the cell cycle, demonstrating an overriding role for Rb in maintaining the quiescent state of postnatal HCs. Unlike Rb-null HC precursors, such HCs failed to undergo cell division and died rapidly. HC clusters were restricted to the less differentiated cochlear regions, consistent with differentiation-dependent roles of Rb. Moreover, outer HCs expressed the maturation marker prestin, suggesting an embryonic time window for Rb-dependent HC specification. We conclude that Rb plays essential and age-dependent roles during HC proliferation and differentiation, and, in contrast to previous hypotheses, cell death after forced cell-cycle reentry presents a major challenge for mammalian HC regeneration from residual postnatal HCs.

Requirement of the SH3 and SH2 domains for the inhibitory function of tyrosine protein kinase p50csk in T lymphocytes.

Previous studies from our laboratory have shown that the cytosolic tyrosine protein kinase p50csk is involved in the negative regulation of T-cell activation (L.M. L. Chow, M. Fournel, D. Davidson, and A. Veillette, Nature [London] 365:156-160, 1993). This function most probably reflects the ability of Csk to phosphorylate the inhibitory carboxy-terminal tyrosine of p56lck and p59fynT, two Src-related enzymes abundantly expressed in T lymphocytes. Herein, we have attempted to better understand the mechanisms by which Csk participates in the inhibitory phase of T-cell receptor signalling. Our results demonstrated that the Src homology 3 (SH3) and SH2 domains of p50csk are crucial for its negative impact on T-cell receptor-mediated signals. As these two sequences were not essential for phosphorylation of the carboxy-terminal tyrosine of a Src-like product in yeast cells, we postulated that they mediate protein-protein interactions allowing the recruitment of p50csk in the vicinity of activated Lck and/or FynT in T cells. In complementary studies, it was observed that linkage of a constitutive membrane targeting signal to the amino terminus of Csk rescued the deleterious impact of a point mutation in the SH2 domain of p50csk. This observation suggested that the SH2 sequence is in part necessary to translocate p50csk from the cytoplasm to the plasma membrane, where Src-related enzymes are located. Nevertheless, constitutive membrane localization was unable to correct the effect of complete deletion of the SH3 or SH2 sequence, implying that these domains provide additional functions necessary for the biological activity of p50csk.

Digital sorting of complex tissues for cell type-specific gene expression profiles

BackgroundCellular heterogeneity is present in almost all gene expression profiles. However, transcriptome analysis of tissue specimens often ignores the cellular heterogeneity present in these samples. Standard deconvolution algorithms require prior knowledge of the cell type frequencies within a tissue or their in vitro expression profiles. Furthermore, these algorithms tend to report biased estimations.ResultsHere, we describe a Digital Sorting Algorithm (DSA) for extracting cell-type specific gene expression profiles from mixed tissue samples that is unbiased and does not require prior knowledge of cell type frequencies.ConclusionsThe results suggest that DSA is a specific and sensitivity algorithm in gene expression profile deconvolution and will be useful in studying individual cell types of complex tissues.

Inducible Cre recombinase activity in mouse cerebellar granule cell precursors and inner ear hair cells.

A transgenic mouse line expressing the CreERTM fusion protein under the control of the Math1 enhancer was generated. Expression of the transgene in the postnatal mouse was restricted to hair cells of the inner ear and granule neurons in the external granule layer of the cerebellum in a temporally regulated manner. Cre activity was virtually nonexistent in uninduced mice; however, treatment of newborn pups with tamoxifen, leading to nuclear translocation of the fusion protein, resulted in efficient recombination at LoxP sites in the appropriate cell types. Up to two thirds of cerebellar granule neurons and 80–90% of cochlear hair cells underwent Cre‐specific recombination. This mouse line provides a powerful tool to dissect gene function at early and late stages in development of the cerebellum and inner ear. Developmental Dynamics 235:2991–2998, 2006.

Inducible Cre recombinase activity in mouse mature astrocytes and adult neural precursor cells.

Two transgenic mouse lines expressing an inducible form of the Cre recombinase (CreERTM) under the control of the human GFAP promoter have been generated and characterized. In adult mice, expression of the fusion protein is largely confined to astrocytes in all regions of the central nervous system. Minimal spontaneous Cre activity was detected and recombination was efficiently induced by intraperitoneal administration of tamoxifen in adult mice. The pattern of recombination closely mirrored that of transgene expression. The percentage of astrocytes undergoing recombination varied from region to region ranging from 35% to 70% while a much smaller portion (<1%) of oligodendrocytes and neural precursor cells showed evidence of Cre activity. These mouse lines will provide important tools to dissect gene function in glial cells and in gliomagenesis.