Faculty Bibliography

BACKGROUND:Tamoxifen is an oestrogen receptor modulator that is widely used for the treatment of early stage breast cancer and reduction of recurrences. Tamoxifen is also used as a powerful research tool for controlling gene expression in the context of the Cre/loxP site-specific recombination system in conditional mutant mice. METHODS:To determine whether the administration of tamoxifen affects Plasmodium growth and/or disease outcome in malaria, in vitro studies assessing the effect of tamoxifen and its active metabolite 4-hydroxytamoxifen on Plasmodium falciparum blood stages were performed. Tamoxifen effects were also evaluated in vivo treating C57/B6 mice infected with Plasmodium berghei (ANKA strain), which is the standard animal model for the study of cerebral malaria. RESULTS:Tamoxifen and its active metabolite, 4-hydroxytamoxifen, show activity in vitro against P. falciparum (16.7 to 5.8 µM IC50, respectively). This activity was also confirmed in tamoxifen-treated mice infected with P. berghei, which show lower levels of parasitaemia and do not develop signs of cerebral malaria, compared to control mice. Mice treated with tamoxifen for 1 week and left untreated for an additional week before infection showed similar parasitaemia levels and signs of cerebral malaria as control untreated mice. CONCLUSIONS:Tamoxifen and its active metabolite, 4-hydroxytamoxifen, have significant activity against the human parasite P. falciparum in vitro and the rodent parasite P. berghei in vivo. This activity may be useful for prevention of malaria in patients taking this drug chronically, but also represents a major problem for scientists using the conditional mutagenic Cre/LoxP system in the setting of rodent malaria. Allowing mice to clear tamoxifen before starting a Plasmodium infection allows the use the Cre/LoxP conditional mutagenic system to investigate gene function in specific tissues.

The eukaryotic translation initiation factor 2a (eIF2a) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2a kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2a phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low-input, low sequencing depth method, EpiMethylTag, that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.

Nervous system development is instructed by genetic programs and refined by distinct mechanisms that couple neural activity to gene expression. How these processes are integrated remains poorly understood. Here, we report that the regulated release of insulin-like peptides (ILPs) during development of the C. elegans nervous system accomplishes such an integration. We find that the p38 MAP kinase PMK-3, which is required for the differentiation of chemosensory BAG neurons, limits an ILP signal that represses expression of a BAG neuron fate. ILPs are released from BAGs themselves in an activity-dependent manner during development, indicating that ILPs constitute an autocrine signal that regulates the differentiation of BAG neurons. Expression of a specialized neuronal fate is, therefore, coordinately regulated by a genetic program that sets levels of ILP expression during development and by neural activity, which regulates ILP release. Autocrine signals of this kind might have general and conserved functions as integrators of deterministic genetic programs with activity-dependent mechanisms during neurodevelopment.

For neural systems to function effectively, the numbers of each cell type must be proportioned properly during development. We found that conditional knockout of the mouse homeobox genes En1 and En2 in the excitatory cerebellar nuclei neurons (eCN) leads to reduced postnatal growth of the cerebellar cortex. A subset of medial and intermediate eCN are lost in the mutants, with an associated cell non-autonomous loss of their presynaptic partner Purkinje cells by birth leading to proportional scaling down of neuron production in the postnatal cerebellar cortex. Genetic killing of embryonic eCN throughout the cerebellum also leads to loss of Purkinje cells and reduced postnatal growth but throughout the cerebellar cortex. Thus, the eCN play a key role in scaling the size of the cerebellum by influencing the survival of their Purkinje cell partners, which in turn regulate production of granule cells and interneurons via the amount of sonic hedgehog secreted.

Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g. stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membrane-less metastable (shape-changing) condensates in the cytoplasm. In our studies we used the same cell lines and methods as used by previous investigators but concluded that wt MxA formed variably-sized spherical or irregular bodies, filaments and even a reticulum distinct from ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of GFP-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24±0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase respectively in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-α-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature, and provide evidence for membrane-less MxA biomolecular condensates in the uninfected cell cytoplasm.IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation, and further report that MxA forms membrane-less metastable (shape-changing) condensates in the cytoplasm consisting of variably-sized spherical or irregular bodies, filaments and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1-3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.

Brain insulin resistance is a key pathological feature contributing to obesity, diabetes and neurodegenerative disorders, including Alzheimer's Disease (AD). Besides to classic transcriptional mechanism mediated by hormones, posttranscriptional regulation has recently been shown to regulate a number of signaling pathways that could lead to metabolic diseases. Here, we show that miR-7, an abundant miRNA in the brain, targets Insulin Receptor (INSR), Insulin Receptor Substrate-2 (IRS-2) and Insulin Degrading Enzyme (IDE), key regulators of insulin homeostatic functions in the Central Nervous System (CNS) and the pathology of AD. In this study, we found that insulin and Liver X receptor (LXR) activators promote the expression of the intronic miR-7-1 in vitro and in vivo, along with its host gene HNRNPK, an RNA binding protein (RBP) that is involved in insulin action at the posttranscriptional level. Our data show that miR-7 expression is altered in the brains of diet-induced obese mice. Moreover, we found that miR-7 levels are also elevated in brain of AD patients, which inversely correlates with the expression of its target genes IRS-2 and IDE. Furthermore, overexpression of miR-7 increased the levels of extracellular Aβ in neuronal cells and impaired the clearance of extracellular Aβ by microglial cells. Taken together, these results represent a novel branch of insulin action through the HNRNPK-miR-7 axis and highlight the possible implication of these posttranscriptional regulators in a range of diseases underlying metabolic dysregulation in the brain, from diabetes to Alzheimer's disease.

The cerebellum undergoes major rapid growth during the third trimester and early neonatal stage in humans, making it vulnerable to injuries in pre-term babies. Experiments in mice have revealed a remarkable ability of the neonatal cerebellum to recover from injuries around birth. In particular, recovery following irradiation-induced ablation of granule cell precursors (GCPs) involves adaptive reprogramming of Nestin-expressing glial progenitors (NEPs). Sonic hedgehog signaling is required for the initial step in NEP reprogramming; however, the full spectrum of developmental signaling pathways that promote NEP-driven regeneration is not known. Since the growth regulatory Hippo pathway has been implicated in the repair of several tissue types, we tested whether Hippo signaling is involved in regeneration of the cerebellum. Using mouse models, we found that the Hippo pathway transcriptional co-activator YAP1 (Yes-associated protein 1) but not TAZ (transcriptional coactivator with PDZ binding motif, or WWTR1) is required in NEPs for full recovery of cerebellar growth following irradiation one day after birth. Although Yap1 plays only a minor role during normal development in differentiation of NEPs or GCPs, the size of the cerebellum, and in particular the internal granule cell layer produced by GCPs, is significantly reduced in Yap1 mutants after irradiation, and the organization of Purkinje cells and Bergmann glial fibers is disrupted. The initial proliferative response of Yap1 mutant NEPs to irradiation is normal and the cells migrate to the GCP niche, but subsequently there is increased cell death of GCPs and altered migration of granule cells, possibly due to defects in Bergmann glia. Moreover, loss of Taz along with Yap1 in NEPs does not abrogate regeneration or alter development of the cerebellum. Our study provides new insights into the molecular signaling underlying postnatal cerebellar development and regeneration.

Previous work has reported the important links between cellular bioenergetics and the development of chronic kidney disease, highlighting the potential for targeting metabolic functions to regulate disease progression. More recently, it has been shown that alterations in fatty acid oxidation (FAO) can have an important impact on the progression of kidney disease. In this work, we demonstrate that loss of miR-33, an important regulator of lipid metabolism, can prevent the repression of FAO in fibrotic kidneys and reduce lipid accumulation. These changes were associated with a dramatic reduction in the extent of fibrosis induced in two different mouse models of kidney disease. These effects were not related to changes in circulating leukocytes, as bone marrow transplant from miR-33 deficient animals did not have a similar impact on disease progression. Most importantly, targeted delivery of miR-33 peptide nucleic acid (PNA) inhibitors to the kidney and other acidic microenvironments was accomplished using pH low insertion peptides (pHLIP) as a carrier. This was effective at both increasing the expression of factors involved in FAO and reducing the development of fibrosis. Together, these findings suggest that miR-33 may be an attractive therapeutic target for the treatment of chronic kidney disease.

Mammalian focal adhesion proteins Pinch1 and Pinch2 regulate integrin activation and cell-extracellular matrix adhesion and migration. Here, we show that deleting Pinch1 in osteocytes and mature osteoblasts using the 10-kb mouse Dmp1-Cre and Pinch2 globally (double KO; dKO) results in severe osteopenia throughout life, while ablating either gene does not cause bone loss, suggesting a functional redundancy of both factors in bone. Pinch deletion in osteocytes and mature osteoblasts generates signals that inhibit osteoblast and bone formation. Pinch-deficient osteocytes and conditioned media from dKO bone slice cultures contain abundant sclerostin protein and potently suppress osteoblast differentiation in primary BM stromal cells (BMSC) and calvarial cultures. Pinch deletion increases adiposity in the BM cavity. Primary dKO BMSC cultures display decreased osteoblastic but enhanced adipogenic, differentiation capacity. Pinch loss decreases expression of integrin β3, integrin-linked kinase (ILK), and α-parvin and increases that of active caspase-3 and -8 in osteocytes. Pinch loss increases osteocyte apoptosis in vitro and in bone. Pinch loss upregulates expression of both Rankl and Opg in the cortical bone and does not increase osteoclast formation and bone resorption. Finally, Pinch ablation exacerbates hindlimb unloading-induced bone loss and impairs active ulna loading-stimulated bone formation. Thus, we establish a critical role of Pinch in control of bone homeostasis.