Nicolas Fritz

Schwann Cell Precursors from Nerve Innervation Are a Cellular Origin of Melanocytes in Skin

Current opinion holds that pigment cells, melanocytes, are derived from neural crest cells produced at the dorsal neural tube and that migrate under the epidermis to populate all parts of the skin. Here, we identify growing nerves projecting throughout the body as a stem/progenitor niche containing Schwann cell precursors (SCPs) from which large numbers of skin melanocytes originate. SCPs arise as a result of lack of neuronal specification by Hmx1 homeobox gene function in the neural crest ventral migratory pathway. Schwann cell and melanocyte development share signaling molecules with both the glial and melanocyte cell fates intimately linked to nerve contact and regulated in an opposing manner by Neuregulin and soluble signals including insulin-like growth factor and platelet-derived growth factor. These results reveal SCPs as a cellular origin of melanocytes, and have broad implications on the molecular mechanisms regulating skin pigmentation during development, in health and pigmentation disorders.

Biochemistry of calcium oscillations.

Cytosolic calcium (Ca2+) oscillations are vastly flexible cell signals that convey information regulating numerous cellular processes. The frequency and amplitude of the oscillating signal can be varied infinitely by concerted actions of Ca2+ transporters and Ca2+-binding proteins to encode specific messages that trigger downstream molecular events. High frequency cytosolic Ca2+ oscillations regulate fast responses, such as synaptic transmission and secretion, whereas low frequency oscillations regulate slow processes, such as fertilization and gene transcription. Thus, the cell exploits Ca2+ oscillations as a signalling carrier to transduce vital information that controls its behaviour. Here, we review the underlying biochemical mechanisms responsible for generating and discriminating cytosolic Ca2+ oscillations.

CXXC5 Is a Novel BMP4-regulated Modulator of Wnt Signaling in Neural Stem Cells

Bone morphogenetic proteins such as BMP4 are essential for proper development of telencephalic forebrain structures and induce differentiation of telencephalic neural stem cells into a variety of cellular fates, including astrocytic, neuronal, and mesenchymal cells. Little is yet understood regarding the mechanisms that underlie the spatiotemporal differences in progenitor response to BMP4. In a screen designed to identify novel targets of BMP4 signaling in telencephalic neural stem cells, we found the mRNA levels of the previously uncharacterized factor CXXC5 reproducibly up-regulated upon BMP4 stimulation. In vivo, CXXC5 expression overlapped with BMP4 adjacent to Wnt3a expression in the dorsal regions of the telencephalon, including the developing choroid plexus. CXXC5 showed partial homology with Idax, a related protein previously shown to interact with the Wnt-signaling intermediate Dishevelled (Dvl). Indeed CXXC5 and Dvl co-localized in the cytoplasm and interacted in co-immunoprecipitation experiments. Moreover, fluorescence resonance energy transfer (FRET) experiments verified that CXXC5 and Dvl2 were located in close spatial proximity in neural stem cells. Studies of the functional role of CXXC5 revealed that overexpression of CXXC5 or exposure to BMP4 repressed the levels of the canonical Wnt signaling target Axin2, and CXXC5 attenuated Wnt3a-mediated increase in TOPflash reporter activity. Accordingly, RNA interference of CXXC5 attenuated the BMP4-mediated decrease in Axin2 levels and facilitated the response to Wnt3a in neural stem cells. We propose that CXXC5 is acting as a BMP4–induced inhibitor of Wnt signaling in neural stem cells.

α-synuclein assemblies sequester neuronal α3-Na+/K+-ATPase and impair Na+ gradient

Extracellular α‐synuclein (α‐syn) assemblies can be up‐taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that α‐syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic‐based approach, we identify the α3‐subunit of Na+/K+‐ATPase (NKA) as a cell surface partner of α‐syn assemblies. The interaction strength depended on the state of α‐syn, fibrils being the strongest, oligomers weak, and monomers none. Mutations within the neuron‐specific α3‐subunit are linked to rapid‐onset dystonia Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). We show that freely diffusing α3‐NKA are trapped within α‐syn clusters resulting in α3‐NKA redistribution and formation of larger nanoclusters. This creates regions within the plasma membrane with reduced local densities of α3‐NKA, thereby decreasing the efficiency of Na+ extrusion following stimulus. Thus, interactions of α3‐NKA with extracellular α‐syn assemblies reduce its pumping activity as its mutations in RDP/AHC.

Crucial Role of Type 2 Inositol 1,4,5-Trisphosphate Receptors for Acetylcholine-Induced Ca2+ Oscillations in Vascular Myocytes

Objective—The aim of this study was to correlate the expression of InsP3R subtypes in native vascular and visceral myocytes with specific Ca2+-signaling patterns. Methods and Results—By Western blot and immunostaining, we showed that rat portal vein expressed InsP3R1 and InsP3R2 but not InsP3R3, whereas rat ureter expressed InsP3R1 and InsP3R3 but not InsP3R2. Acetylcholine induced single Ca2+ responses in all ureteric myocytes but only in 50% of vascular myocytes. In the remaining vascular myocytes, the first transient peak was followed by Ca2+ oscillations. By correlating Ca2+ signals and immunostaining, we revealed that oscillating vascular cells expressed both InsP3R1 and InsP3R2 whereas nonoscillating vascular cells expressed only InsP3R1. Acetylcholine-induced oscillations were not affected by inhibitors of ryanodine receptors, Ca2+-ATPases, Ca2+ influx, and mitochondrial Ca2+ uniporter but were inhibited by intracellular infusion of heparin. Using specific antibodies against InsP3R subtypes, we showed that acetylcholine-induced Ca2+ oscillations were specifically blocked by the anti-InsP3R antibody. These data were supported by antisense oligonucleotides targeting InsP3R2, which selectively inhibited Ca2+ oscillations. Conclusions—Our results suggest that in native smooth muscle cells, a differential expression of InsP3R subtypes encodes specific InsP3-mediated Ca2+ responses and that the presence of the InsP3R2 subtype is required for acetylcholine-induced Ca2+ oscillations in vascular myocytes.

Ryanodine receptor subtype 2 encodes Ca2+ oscillations activated by acetylcholine via the M2 muscarinic receptor/cADP-ribose signalling pathway in duodenum myocytes

In this study, we characterized the signalling pathway activated by acetylcholine that encodes Ca2+ oscillations in rat duodenum myocytes. These oscillations were observed in intact myocytes after removal of external Ca2+, in permeabilized cells after abolition of the membrane potential and in the presence of heparin (an inhibitor of inositol 1,4,5-trisphosphate receptors) but were inhibited by ryanodine, indicating that they are dependent on Ca2+ release from intracellular stores through ryanodine receptors. Ca2+ oscillations were selectively inhibited by methoctramine (a M2 muscarinic receptor antagonist). The M2 muscarinic receptor-activated Ca2+ oscillations were inhibited by 8-bromo cyclic adenosine diphosphoribose and inhibitors of adenosine diphosphoribosyl cyclase (ZnCl2 and anti-CD38 antibody). Stimulation of ADP-ribosyl cyclase activity by acetylcholine was evaluated in permeabilized cells by measuring the production of cyclic guanosine diphosphoribose (a fluorescent compound), which resulted from the cyclization of nicotinamide guanine dinucleotide. As duodenum myocytes expressed the three subtypes of ryanodine receptors, an antisense strategy revealed that the ryanodine receptor subtype 2 alone was required to initiate the Ca2+ oscillations induced by acetylcholine and also by cyclic adenosine diphosphoribose and rapamycin (a compound that induced uncoupling between 12/12.6 kDa FK506-binding proteins and ryanodine receptors). Inhibition of cyclic adenosine diphosphoribose-induced Ca2+ oscillations, after rapamycin treatment, confirmed that both compounds interacted with the ryanodine receptor subtype 2. Our findings show for the first time that the M2 muscarinic receptor activation triggered Ca2+ oscillations in duodenum myocytes by activation of the cyclic adenosine diphosphoribose/FK506-binding protein/ryanodine receptor subtype 2 signalling pathway.

Inositol 1,4,5-Trisphosphate Receptor Subtype-Specific Regulation of Calcium Oscillations

Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca2+) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are intracellular Ca2+ release channels that mediate Ca2+ release from endoplasmic reticulum (ER) Ca2+ stores. The three IP3R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP3R by the endogenous modulators IP3, Ca2+, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP3R subtype in shaping cytosolic Ca2+ oscillations.

MYC proteins promote neuronal differentiation by controlling the mode of progenitor cell division

The role of MYC proteins in somatic stem and progenitor cells during development is poorly understood. We have taken advantage of a chick in vivo model to examine their role in progenitor cells of the developing neural tube. Our results show that depletion of endogenous MYC in radial glial precursors (RGPs) is incompatible with differentiation and conversely, that overexpression of MYC induces neurogenesis independently of premature or upregulated expression of proneural gene programs. Unexpectedly, the neurogenic function of MYC depends on the integrity of the polarized neural tissue, in contrast to the situation in dissociated RGPs where MYC is mitogenic. Within the polarized RGPs of the neural tube, MYC drives differentiation by inhibiting Notch signaling and by increasing neurogenic cell division, eventually resulting in a depletion of progenitor cells. These results reveal an unexpected role of MYC in the control of stemness versus differentiation of neural stem cells in vivo.

RyR1-specific requirement for depolarization-induced Ca2+ sparks in urinary bladder smooth muscle

Ryanodine receptor subtype 1 (RyR1) has been primarily characterized in skeletal muscle but several studies have revealed its expression in smooth muscle. Here, we used Ryr1-null mice to investigate the role of this isoform in Ca2+ signaling in urinary bladder smooth muscle. We show that RyR1 is required for depolarization-induced Ca2+ sparks, whereas RyR2 and RyR3 are sufficient for spontaneous or caffeine-induced Ca2+ sparks. Immunostaining revealed specific subcellular localization of RyR1 in the superficial sarcoplasmic reticulum; by contrast, RyR2 and RyR3 are mainly expressed in the deep sarcoplasmic reticulum. Paradoxically, lack of depolarization-induced Ca2+ sparks in Ryr1–/– myocytes was accompanied by an increased number of cells displaying spontaneous or depolarization-induced Ca2+ waves. Investigation of protein expression showed that FK506-binding protein (FKBP) 12 and FKBP12.6 (both of which are RyR-associated proteins) are downregulated in Ryr1–/– myocytes, whereas expression of RyR2 and RyR3 are unchanged. Moreover, treatment with rapamycin, which uncouples FKBPs from RyR, led to an increase of RyR-dependent Ca2+ signaling in wild-type urinary bladder myocytes but not in Ryr1–/– myocytes. In conclusion, although decreased amounts of FKBP increase Ca2+ signals in Ryr1–/– urinary bladder myocytes the depolarization-induced Ca2+ sparks are specifically lost, demonstrating that RyR1 is required for depolarization-induced Ca2+ sparks and suggesting that the intracellular localization of RyR1 fine-tunes Ca2+ signals in smooth muscle.

Calcium Signaling in Neocortical Development

The calcium ion (Ca2+) is an essential second messenger that plays a pivotal role in neurogenesis. In the ventricular zone (VZ) of the neocortex, neural stem cells linger to produce progenitor cells and subsequently neurons and glial cells, which together build up the entire adult brain. The radial glial cells, with their characteristic radial fibers that stretch from the inner ventricular wall to the outer cortex, are known to be the neural stem cells of the neocortex. Migrating neurons use these radial fibers to climb from the proliferative VZ in the inner part of the brain to the outer layers of the cortex, where differentiation processes continue. To establish the complex structures that constitute the adult cerebral cortex, proliferation, migration, and differentiation must be tightly controlled by various signaling events, including cytosolic Ca2+ signaling. During development, cells regularly exhibit spontaneous Ca2+ activity that stimulates downstream effectors, which can elicit these fundamental cell processes. Spontaneous Ca2+ activity during early neocortical development depends heavily on gap junctions and voltage dependent Ca2+ channels, whereas later in development neurotransmitters and synapses exert an influence. Here, we provide an overview of the literature on Ca2+ signaling and its impact on cell proliferation, migration, and differentiation in the neocortex. We point out important historical studies and review recent progress in determining the role of Ca2+ signaling in neocortical development.