[Show abstract][Hide abstract] ABSTRACT: Alterations in Hedgehog (Hh) signaling lead to birth defects and cancers including medulloblastoma, the most common pediatric brain tumor. Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling, acquired drug resistance and tumor relapse call for additional therapeutic targets. Here we show that phosphodiesterase 4D (PDE4D) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth. PDE4D interacts directly with Neuropilins, positive regulators of Hh pathway. The Neuropilin ligand Semaphorin3 enhances this interaction, promoting PDE4D translocation to the plasma membrane and cAMP degradation. The consequent inhibition of protein kinase A (PKA) enhances Hh transduction. In the developing cerebellum, genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors. In mouse medulloblastoma allografts, PDE4D inhibitors suppress Hh transduction and inhibit tumor growth. Our findings reveal a new regulatory mechanism of Hh transduction, and highlight PDE4D as a promising target to treat Hh-related tumors.
[Show abstract][Hide abstract] ABSTRACT: The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP-activated protein kinase (AMPK) is an important sensor of energy stores and controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.
[Show abstract][Hide abstract] ABSTRACT: Accumulation of the signaling protein Smoothened (Smo) in the membrane of primary cilia is an essential step in Hedgehog (Hh) signal transduction, yet the molecular mechanisms of Smo movement and localization are poorly understood. Using ultrasensitive single-molecule tracking with high spatial/temporal precision (30 nm/10 ms), we discovered that binding events disrupt the primarily diffusive movement of Smo in cilia at an array of sites near the base. The affinity of Smo for these binding sites was modulated by the Hh pathway activation state. Activation, by either a ligand or genetic loss of the negatively acting Hh receptor Patched-1 (Ptch), reduced the affinity and frequency of Smo binding at the base. Our findings quantify activation-dependent changes in Smo dynamics in cilia and highlight a previously unknown step in Hh pathway activation.
No preview · Article · Jun 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method's utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.
[Show abstract][Hide abstract] ABSTRACT: Hedgehog (Hh) signal transduction is necessary for the development of most mammalian tissues and can go awry and cause birth defects or cancer. Hh signaling was initially described in Drosophila, and much of what we know today about mammalian Hh signaling was directly guided by discoveries in the fly. Indeed, Hh signaling is a wonderful example of the use of non-vertebrate model organisms to make basic discoveries that lead to new disease treatment. The first pharmaceutical to treat hyperactive Hh signaling in Basal Cell Carcinoma was released in 2012, approximately 30 years after the isolation of Hh mutants in Drosophila. The study of Hh signaling has been greatly facilitated by the imaginal wing disc, a tissue with terrific experimental advantages. Studies using the wing disc have led to an understanding of Hh ligand processing, packaging into particles for transmission, secretion, reception, signal transduction, target gene activation, and tissue patterning. Here we describe the imaginal wing disc, how Hh patterns this tissue, and provide methods to use wing discs to study Hh signaling in Drosophila. The tools and approaches we highlight form the cornerstone of research efforts in many laboratories that use Drosophila to study Hh signaling, and are essential for ongoing discoveries.
[Show abstract][Hide abstract] ABSTRACT: Insulin-producing cells (IPCs) in the Drosophila brain produce and release insulin-like peptides (ILPs) to hemolymph. ILPs are crucial for growth and regulation of metabolic activity in flies, functions analogous to those of mammalian insulin and insulin-like growth factors (IGFs). To identify components functioning in IPCs to control ILP production, we employed genomic and candidate gene approaches. We used laser microdissection and mRNA sequencing to characterize the transcriptome of larval IPCs. IPCs highly express many genes homologous to genes active in insulin-producing beta cells of the mammalian pancreas. The genes in common encode insulin-like peptides and proteins that control insulin metabolism, storage, secretion, and beta cell proliferation, and some not previously linked to insulin production or beta cell function. Among these novelties is unc-104, a Kinesin 3 family gene, which is more highly expressed in IPCs compared to most other neurons. Knockdown of unc-104 in IPCs impaired ILP secretion and reduced peripheral insulin signaling. Unc-104 appears to transport ILPs along axons. As a complementary approach, we tested dominant-negative Rab genes to find Rab proteins required in IPCs for ILP production or secretion. Rab1 was identified as crucial for ILP trafficking in IPCs. Inhibition of Rab1 in IPCs increased circulating sugar levels, delayed development, and lowered weight and body size. Immunofluorescence labeling of Rab1 showed its tight association with ILP2 in the Golgi of IPCs. Unc-104 and Rab1 join other proteins required for ILP transport in IPCs.
[Show abstract][Hide abstract] ABSTRACT: The Hedgehog (Hh) signaling pathway has been implicated in the most common childhood brain tumor, medulloblastoma (MB). Given the toxicity of post-surgical treatments for MB, continued need exists for new, targeted therapies. Based upon our finding that Neuropilin (Nrp) transmembrane proteins are required for Hh signal transduction, we investigated the role of Nrp in MB cells. Cultured cells derived from a mouse Ptch
;LacZ MB (Med1-MB), effectively modeled the Hh pathway-related subcategory of human MBs in vitro. Med1-MB cells maintained constitutively active Hh target gene transcription, and consistently formed tumors within one month after injection into mouse cerebella. The proliferation rate of Med1-MBs in culture was dependent upon Nrp2, while reducing Nrp1 function had little effect. Knockdown of Nrp2 prior to cell implantation significantly increased mouse survival, compared to transfection with a non-targeting siRNA. Knocking down Nrp2 specifically in MB cells avoided any direct effect on tumor vascularization. Nrp2 should be further investigated as a potential target for adjuvant therapy in patients with MB.
No preview · Article · Sep 2013 · Journal of Neuro-Oncology
[Show abstract][Hide abstract] ABSTRACT: Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to αvβ3, αvβ5, and α5β1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α5β1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F-Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F-Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5-Fc as targeted molecular probes for brain tumor imaging.
Preview · Article · Aug 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Understanding neurodegenerative disease progression and its treatment requires the systematic characterization and manipulation of relevant cell types and molecular pathways. The neurodegenerative lysosomal storage disorder Niemann-Pick disease type C (NPC) is highly amenable to genetic approaches that allow exploration of the disease biology at the organismal, cellular and molecular level. Although NPC is a rare disease, genetic analysis of the associated neuropathology promises to provide insight into the logic of disease neural circuitry, selective neuron vulnerability and neural-glial interactions. The ability to control the disorder cell-autonomously and in naturally occurring spontaneous animal models that recapitulate many aspects of the human disease allows for an unparalleled dissection of the disease neurobiology in vivo. Here, we review progress in mouse-model-based studies of NPC disease, specifically focusing on the subtype that is caused by a deficiency in NPC1, a sterol-binding late endosomal membrane protein involved in lipid trafficking. We also discuss recent findings and future directions in NPC disease research that are pertinent to understanding the cellular and molecular mechanisms underlying neurodegeneration in general.
No preview · Article · Aug 2013 · Disease Models and Mechanisms
[Show abstract][Hide abstract] ABSTRACT: Nucleostemin 3 (NS3) is an evolutionarily conserved protein with profound roles in cell growth and viability. Here we analyze cell autonomous and non-cell autonomous growth control roles of NS3 in Drosophila, and demonstrate its GTPase activity using genetic and biochemical assays. Two null alleles of ns3, and RNAi, demonstrate the necessity of NS3 for cell autonomous growth. A hypomorphic allele highlights the hypersensitivity of neurons to lowered NS3 function. We propose that NS3 is the functional ortholog of yeast and human Lsg1, which promotes release of the nuclear export adapter from the large ribosomal subunit. Release of the adapter and its recycling to the nucleus are essential for sustained production of ribosomes. The ribosome biogenesis role of NS3 is essential for proper rates of translation in all tissues and is necessary for functions of growth-promoting neurons.
[Show abstract][Hide abstract] ABSTRACT: During preimplantation development, the embryo must establish totipotency and enact the earliest differentiation choices, processes that involve extensive chromatin modification. To identify novel developmental regulators, we screened for genes that are preferentially transcribed in the pluripotent inner cell mass (ICM) of the mouse blastocyst. Genes that encode chromatin remodeling factors were prominently represented in the ICM, including Chd1l, a member of the Snf2 gene family. Chd1l is developmentally regulated and expressed in embryonic stem (ES) cells, but its role in development has not been investigated. Here we show that inhibiting Chd1l protein production by microinjection of antisense morpholinos causes arrest prior to the blastocyst stage. Despite this important function in vivo, Chd1l is non-essential for cultured ES cell survival, pluripotency, or differentiation, suggesting that Chd1l is vital for events in embryos that are distinct from events in ES cells. Our data reveal a novel role for the chromatin remodeling factor Chd1l in the earliest cell divisions of mammalian development.
[Show abstract][Hide abstract] ABSTRACT: Medulloblastoma (MB) cells arise from granule neuron precursors (GNPs) that have lost growth control. During normal development, GNPs divide in response to Sonic hedgehog (SHH), a ligand that binds to the Patched (PTCH) receptor on GNPs. If one copy of the Ptch gene is lost, as in human Gorlin's syndrome and in Ptch(+/-) mice, MBs may form. Proper transduction of the SHH signal critically depends on primary cilia. Loss of primary cilia results in improper signal reception and failure to properly activate SHH target genes. KIF3a, part of a kinesin motor, is required for formation of primary cilia. Here we use tamoxifen-induced ablation of Kif3a in GNPs of postnatal Ptch(+/-) mouse cerebella to show that KIF3a is necessary for MB formation. To investigate the importance of primary cilia in established tumors, we deleted Kif3a from cultured cells and from tumor cell grafts. The loss of Kif3a from established tumors led to their growth arrest and regression. MBs behave as if they are addicted to the presence of primary cilia. These results underscore the potential utility of agents that disrupt cilia for treatment of Hh pathway-related MBs.
[Show abstract][Hide abstract] ABSTRACT: Background:
Airway cilia must be physically oriented along the longitudinal tissue axis for concerted, directional motility that is essential for proper mucociliary clearance.
We show that planar cell polarity (PCP) signaling specifies directionality and orients respiratory cilia. Within all airway epithelial cells, a conserved set of PCP proteins shows interdependent, asymmetric junctional localization; nonautonomous signaling coordinates polarization between cells; and a polarized microtubule (MT) network is likely required for asymmetric PCP protein localization. We find that basal bodies dock after polarity of PCP proteins is established and are polarized nearly simultaneously, and that refinement of basal body/cilium orientation continues during airway epithelial development. Unique to mature multiciliated cells, we identify PCP-regulated, planar polarized MTs that originate from basal bodies and interact, via their plus ends, with membrane domains associated with the PCP proteins Frizzled and Dishevelled. Disruption of MTs leads to misoriented cilia.
A conserved PCP pathway orients airway cilia by communicating polarity information from asymmetric membrane domains at the apical junctions, through MTs, to orient the MT and actin-based network of ciliary basal bodies below the apical surface.
Preview · Article · Oct 2012 · Current biology: CB
[Show abstract][Hide abstract] ABSTRACT: Background
The immune system has been implicated in neurodegeneration during development and disease. In various studies, the absence of complement (that is, C1q deficiency) impeded the elimination of apoptotic neurons, allowing survival. In the genetic lysosomal storage disease Niemann-Pick C (NPC), caused by loss of NPC1 function, the expression of complement system components, C1q especially, is elevated in degenerating brain regions of Npc1-/- mice. Here we test whether complement is mediating neurodegeneration in NPC disease.
In normal mature mice, C1q mRNA was found in neurons, particularly cerebellar Purkinje neurons (PNs). In Npc1-/- mice, C1q mRNA was additionally found in activated microglia, which accumulate during disease progression and PN loss. Interestingly, C1q was not enriched on or near degenerating neurons. Instead, C1q was concentrated in other brain regions, where it partially co-localized with a potential C1q inhibitor, chondroitin sulfate proteoglycan (CSPG). Genetic deletion of C1q, or of the downstream complement pathway component C3, did not significantly alter patterned neuron loss or disease progression. Deletion of other immune response factors, a Toll-like receptor, a matrix metalloprotease, or the apoptosis facilitator BIM, also failed to alter neuron loss.
We conclude that complement is not involved in the death and clearance of neurons in NPC disease. This study supports a view of neuroinflammation as a secondary response with non-causal relationship to neuron injury in the disease. This disease model may prove useful for understanding the conditions in which complement and immunity do contribute to neurodegeneration in other disorders.
Full-text · Article · Sep 2012 · Journal of Neuroinflammation
[Show abstract][Hide abstract] ABSTRACT: The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.
[Show abstract][Hide abstract] ABSTRACT: Chronic systemic inflammation is thought to be a major contributor to metabolic and neurodegenerative diseases. Since inflammatory components are shared among different disorders, targeting inflammation is an attractive option for mitigating disease. To test the significance of inflammation in the lipid storage disorder (LSD) Niemann-Pick C (NPC), we deleted the macrophage inflammatory gene Mip1a/Ccl3 from NPC diseased mice. Deletion of Ccl3 had been reported to delay neuronal loss in Sandhoff LSD mice by inhibiting macrophage infiltration. For NPC mice, in contrast, deleting Ccl3 did not retard neurodegeneration and worsened the clinical outcome. Depletion of visceral tissue macrophages also did not alter central nervous system (CNS) pathology and instead increased liver injury, suggesting a limited macrophage infiltration response into the CNS and a beneficial role of macrophage activity in visceral tissue. Prevention of neuron loss or liver injury, even at late stages in the disease, was achieved through specific rescue of NPC disease in neurons or in liver epithelial cells, respectively. Local epithelial cell correction was also sufficient to reduce the macrophage-associated pathology in lung tissue. These results demonstrate that elevated inflammation and macrophage activity does not necessarily contribute to neurodegeneration and tissue injury, and LSD defects in immune cells may not preclude an appropriate inflammatory response. We conclude that inflammation remains secondary to neuronal and epithelial cell dysfunction and does not irreversibly contribute to the pathogenic cascade in NPC disease. Without further exploration of possible beneficial roles of inflammatory mediators, targeting inflammation may not be therapeutically effective at ameliorating disease severity.
Full-text · Article · Apr 2012 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system.
Full-text · Article · Nov 2011 · Genes & development