Dopamine pathway: ETS make a neuron

The ETS family of transcription factors bind to a conserved cis-regulatory element in dopamine responsive genes to promote neuronal differentiation.

Although dopaminergic neurons derive from a variety of precursor cells, they all express the five genes that are required for dopamine synthesis and transport. However, the mechanism that activates the transcription of these dopamine pathway genes has not yet been defined. In Nature, Flames and Hobert now report that a shared cis-regulatory element called the 'dopamine motif' controls expression of dopamine pathway genes in all dopaminergic cell types in Caenorhabditis elegans.

The authors fused a gfp (green fluorescent protein) reporter to the promoters for two genes expressed only in dopamine neurons: dopamine transporter (dat-1) and tyrosine hydroxylase (cat-2). Mutational analysis showed that a small conserved cis-regulatory module (CRM) in each promoter region was necessary and sufficient for GFP expression in all dopamine neurons.

Bioinformatics analysis predicted that six types of transcription factors could bind to this CRM. But mutagenesis revealed that only ETS family transcription factors bound to the motif in dopaminergic neurons. After studying each of the ten ETS family members, the authors determined that AST-1 alone was required for dopamine neuron differentiation. Loss of ast-1 blocked dopaminergic differentiation, whereas ectopic re-expression of ast-1 rescued dopamine neuron specification and induced dopamine motif activation in unrelated 5HT neurons.

Vertebrate dopamine pathway genes possess conserved motifs, suggesting that this mechanism is broadly conserved between organisms. In mice, ectopic expression of the ETS family member ETV1 was sufficient to induce terminal differentiation in primary cultures of olfactory bulb dopaminergic neurons, and could also rescue ast-1 deficiency in C. elegans. Conversely, loss of Etv1 reduced the number of dopaminergic neurons in the olfactory bulb. Thus dopamine neuron differentiation in the olfactory bulb may result from the selectivity of the CRM for Etv1.

These data show that neuronal differentiation in vertebrates and invertebrates is regulated by specific ETS family transcription factors binding to a common cis-regulatory element in the promoters of all dopamine-pathway genes. The expression of all five genes was severely reduced or destroyed in ast-1 mutants. The transcription factors AST-1 and Etv1 are necessary for the terminal differentiation of dopaminergic neurons, but cannot be considered sufficient, as both are also expressed in non-dopamine neurons. Could cell-type-specific regulation be achieved in vivo by distinct members of the ETS family? Further research will be necessary to determine whether the differences seen in terminal differentiation between various cell types are the result of specific co-factors.

Rachel Davis
Scitable

Reference:
Flames, N. and Hobert, O.
Gene regulatory logic of dopamine neuron differentiation
Nature 458, 885-889 (2009)
Full text | PDF | Subscribe to Nature

Neural development: Proteoglycans go forth to multiply

Sonic hedgehog requires interactions with proteoglycans to achieve cell proliferation, but not tissue patterning.

The hedgehog pathway is critical for control of tissue patterning and cell proliferation during development. Proteoglycans are known to influence hedgehog signaling but the mechanisms are little understood. Now, in Nature Neuroscience, Chan et al. reveal that proteoglycan interactions with the mammalian morphogen Sonic hedgehog (Shh) selectively affect proliferation but not patterning responses during neural development.

Many proteins bind to proteoglycans, which makes the defects observed after the loss of a specific proteoglycan difficult to attribute to a particular signaling pathway. Shh contains a proteoglycan-binding domain, and this can be mutated to prevent high-affinity proteoglycan-Shh interactions, without altering the affinity of Shh for its receptor. This mutation allows the Shh-proteoglycan interaction to be specifically isolated. The authors created a mouse containing this mutant Shh (Shh^Ala) and checked that Shh expression levels were unaffected. Shh^Ala mice were smaller overall than wild-type littermates, with particular reductions in the size of their brain, spinal cord and eyes. Unlike mice lacking Shh, however, Shh^Ala animals have no patterning defects, indicating that the Shh-proteoglycan interactions regulate tissue growth. Neural precursor cell proliferation was reduced in the brain and spinal cord, in Shh^Ala embryos and adult mice.

Precursor cell proliferation is often localized in proliferative zones termed mitogenic niches. Granule cell precursors proliferate extensively in the external granule cell layer (EGL). Using organotypic slice cultures, Chan et al. found that the wild type EGL, but not the Shh^Ala EGL, provides a mitogenic niche that promotes proliferation of either wild-type or Shh^Ala granule cell precursors. Immunostaining of Shh in the EGL of Shh^Ala mice was also reduced. These results indicate the importance of Shh-proteoglycan interactions for localizing Shh to proliferative zones. Shh-proteoglycan interactions also alter intracellular signaling cascades, as demonstrated by the much greater proliferative increase induced in granule cell precursor cultures by Shh than Shh^Ala.

The expression of Shh-responsive genes was analyzed, with three clusters of genes apparent: one was induced similarly by Shh^Ala and wild-type, implicating a proteoglycan-independent Shh signaling pathway; a second cluster was induced to a greater extent by Shh^Ala than wild-type; induction of the third by Shh^Ala was reduced compared with wild-type. Many of the target genes altered by Shh^Ala stimulation have been implicated in proliferation and oncogenesis. In a standard assay for Shh activity, there was no effect on the dose-response curve of Shh^Ala compared with wild-type; however, the ability to bind proteoglycan was shown to be important for the kinetics of signaling, with Shh^Ala levels in cultured cells declining more rapidly than wild-type Shh after stimulation.

This study shows that Shh-proteoglycan interactions are critical for mitogenic responses to Shh. It ensures the correct localization of Shh, the induction of specific intracellular signaling pathways and altered kinetics of the signaling protein. Proliferation in the absence of differentiation is a hallmark of tumor biology, and the hedgehog pathway may promote oncogenesis when mitosis is stimulated without the required patterning.

Emma Leah
Functional Glycomics Gateway

Reference:
Chan, J. A. et al.
Proteoglycan interactions with Sonic Hedgehog specify mitogenic responses
Nature Neuroscience 12, 409-417 (2009)
Full text | PDF | Subscribe to Nature Neuroscience

Protein kinase D1: A (sling)shot against directed migration

PKD1 inhibits the activation of cofilin at the cell front via slingshot phosphatases, thus blocking the formation of free F-actin barbed ends and directed cell migration.

Rapid actin remodeling at the leading edge drives the formation of cell protrusions. The F-actin depolymerization and severing factor ADF/cofilin (cofilin) plays a crucial role as it increases the number of free barbed ends that can be nucleated by the Arp2/3 complex. The slingshot (SSH) phosphatases activate cofilin, but how they are regulated upstream remains elusive. In Nature Cell Biology, Peter Storz and colleagues now report that Protein Kinase D1 (PKD1) affects cofilin activity by regulating the slingshot phosphatase SSH1L.

The authors used both immunochemistry and FRET to show that PKD1 binds directly to F-actin at the edge of lamellipodia in vivo and in membrane ruffles in migration-competent tumor cells. Furthermore, PKD1 was found to co-localize with SSH1L at F-actin structures in the periphery of membrane protrusions and in the cytoplasm.

Using an antibody that recognizes PKD substrates, the authors identified SSHL1 as a substrate for PKD1. PKD1 specifically phosphorylated SSHL1 at Ser 978 both in vitro and in vivo. Interestingly, Ser 978 phosphorylation occurred downstream of the small GTPase RhoA, a known regulator of directed cell migration. Loss of PKD1 function blocked RhoA-mediated phosphorylation of SSHL1, showing that PKD1 regulates SSHL1 downstream of RhoA.

The authors next addressed the functional significance of SSHL1 phosphorylation. Following expression of active PKD1, SSHL1 predominantly localized in the cytoplasm, whereas the localization of a non-phosphorylatable SSHL1 mutant remained unchanged. Kinase-dead PKD1 also failed to induce SSHL1 redistribution. Ser 978 lies within an SSH1L motif that is capable of binding both F-actin and 14-3-3 proteins. PKD1-dependent phosphorylation of Ser 978 promoted its binding to 14-3-3 proteins and abolished binding to F-actin. Thus, PKD1 controls F-actin localization of SSHL1 at membrane ruffles and lamellipodia by regulating 14-3-3 binding.

The authors found that increased PKD1 activity enhanced cofilin phosphorylation at the SSHL1 target site Ser 3. This effect is dependent on SSH1L phosphorylation at Ser 978, suggesting that PKD1 inhibits SSHL1-mediated cofilin activation. In accordance with these results, PKD1 activation blocked the formation of free actin barbed ends and impaired directed cell migration in invasive tumor cells by decreasing both velocity and directionality.

This work shows that PKD1 regulates the SSHL1-cofilin pathway that controls F-actin remodeling at the cell front during directed migration. Chemotaxis also requires cofilin inactivation in areas other than the lamellipodium; however, whether PKD1 functions during other stages of the cofilin cycle remains to be determined.

Kim Baumann
Cell Migration Gateway

Reference:
Eiseler, T., Döppler, H., Yan, I. K., Kitatani, K., Mizuno, K. & Storz, P.
Protein kinase D1 regulates cofilin mediated F-actin reorganization and cell motility via slingshot
Nature Cell Biology advance online publication, 29 March 2009 (DOI 10.1038/ncb1861)
Full text | PDF | Subscribe to Nature Cell Biology

Energy homeostasis: AMPing up SIRT1

AMPK responds to energy deprivation by increasing cellular NAD⁺ levels, which activates SIRT1 and stimulates transcription of genes important for mitochondrial biogenesis and fatty-acid oxidation.

Fluctuations in the intracellular ratio of ATP/AMP cause AMP-activated protein kinase (AMPK) to shut off ATP-consuming pathways and stimulate ATP-producing catabolic pathways. AMPK is important for mitochondrial biogenesis, as energy deprivation stimulates mitochondrial gene expression in an AMPK-dependent manner. However, the signaling pathway linking AMPK activation to gene expression has not yet been fully elucidated. In Nature, Johan Auwerx and colleagues now describe an intersection between the ATP/AMP and NAD⁺ metabolic sensors, and show that AMPK and sirtuin 1 (SIRT1) are both necessary to stimulate the transcription of genes important for the response to energy deprivation.

In response to elevated AMP/ATP levels, AMPK was previously shown to phosphorylate and activate the transcription factor proliferator-activated receptor-γ coactivator 1α (PGC-1α), which promotes transcription of genes important for cellular metabolism. PGC-1α is also activated by SIRT1-mediated deacetylation. In this study, pharmacological or exercise-induced activation of AMPK decreased PGC-1α acetylation in glycolytic, but not oxidative muscle, which correlated with increased PGC-1α target gene expression. However, stimulation of AMPK did not reduce PGC-1α acetylation in SIRT1-deficient C2C12 myotubes and failed to elicit PGC-1α-dependent transcription of genes important for mitochondrial function and lipid metabolism. SIRT1 deficiency also attenuated an AMPK-driven increase in O₂ consumption. Thus, the AMPK-mediated response to energy deprivation requires SIRT1.

AMPK did not associate with or phosphorylate SIRT1 in vitro, indicating an indirect mechanism of regulation. SIRT1 deacetylase activity is known to be stimulated by NAD⁺ levels, and the authors found that AMPK activation raised the NAD⁺/NADH ratio via mitochondrial fatty-acid oxidation. The elevated NAD⁺/NADH ratio also correlated with increased PGC-1α deacetylation. Intriguingly, a PGC-1α mutant lacking the two AMPK phosphorylation sites was not efficiently deacetylated and could not stimulate mitochondrial gene expression, suggesting that AMPK phosphorylation primes PGC-1α for subsequent SIRT1-mediated deacetylation.

Similar to PGC-1α, the transcription factors FOXO1 and FOXO3a are also targets for AMPK-mediated phosphorylation and SIRT1-stimulated deacetylation. Interestingly, FOXO1 and FOXO3a were deacetylated following AMPK activation, suggesting that this pathway might be a conserved mechanism that provides SIRT1 substrate specificity and prevents unintended deacetylation. These results document the intersection of two key pathways involved in energy homeostasis. As emerging evidence also points to a role for SIRT1 agonists in the indirect activation of AMPK, it will also be interesting to determine whether this pathway can be regulated through positive feedback.

Emily J. Chenette
Signaling Gateway

Reference:
Cantó, C. et al.
AMPK regulates energy expenditure by modulating NAD⁺ metabolism and SIRT1 activity
Nature advance online publication, 4 March 2009 (DOI 10.1038/nature07813)
Full text | PDF | Subscribe to Nature