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Lyso‐phosphatidyl β‐D‐glucoside (lysoPtdGlc) is during embryonic development a neurite growth cone guidance cue associated with G‐protein coupled receptor 55, and has been found to be involved in a variety of physiological and pathological events, including inflammatory pain, and cancer. Hence, the ability to prepare large quantities of lysoPtdGlc is an important objective. Herein, we report the convergent chemical synthesis of lysoPtdGlc and its glyceryl epimer (S‐lysoPtdGlc). High stereocontrol during β‐glycosyl H‐phosphonate construction was achieved using a microfluidic approach, and common but undesirable regioisomerization due to acyl migration on the glyceryl subunit was successfully suppressed during deprotection. Purification of the final product was optimized to obtain sufficiently large amounts of lysoPtdGlc and S‐lysoPtdGlc for biological assaying purposes. [ABSTRACT FROM AUTHOR]

Skin development is tightly temporally coordinated with its sensory innervation, which consists of the peripheral branches of the dorsal root ganglion (DRG) axons. Various studies suggest that the skin produces a long‐range attractant for the sensory axons. However, the exact identity of the guidance cue(s) remains unclear. To reveal the detailed molecular mechanism that controls DRG axon guidance and targeting, manipulation of specific skin layers at specific time points are required. To test a variety of attractants that can be expressed in specific skin layers at specific timepoints, we combined in utero electroporation with the Tol2 transposon system to induce long‐term transgene expression in the developing mouse skin, including in the highly proliferative epidermal stem cells (basal layer) and their descendants (spinous and granular layer cells). The plasmid solution was injected as close to the hindpaw plantar surface as possible. Immediately, electric pulses were passed through the embryo to transduce the plasmid DNA into hindpaw skin cells. Balancing outcome measurements including: embryo survival, transfection efficiency, and the efficiency of transgene integration into host cells, we found that IUE was best performed on E13.5, and using an electroporation voltage of 34V. After immunostaining embryonic and early postnatal skin tissue sections for keratinocyte and sensory axon markers, we observe the growth of axons into skin epidermal layers including areas expressing EGFP. Therefore, this method is useful for studying the interaction between axon growth and epidermal cell division/differentiation. [ABSTRACT FROM AUTHOR]

Lysophosphatidyl glucoside (LPGlc) is a structurally unique glycolipid that acts as a guidance cue for extending axons during central nervous system development by activating the class A G protein coupled receptor (GPR) 55 of spinal cord sensory axons. GPR55 not only plays an important role during development, but is also implicated in many disease states, rendering molecules that target GPR55 of widespread interest. In this study, we developed synthetic access to a novel class of LPGlc analogues featuring a squaryl diamide group as surrogate for the phosphodiester. We report the facile synthesis of a series of LPGlc analogues, their GPR dependent biological activity and a systematic analysis of the structure–activity relationship in regards to GPR55 modulation. The lead compound featuring identical configuration at all stereocenters compared to natural LPGlc exhibits an activity to repel axons of dorsal root ganglion (DGR) nociceptive neurons. [ABSTRACT FROM AUTHOR]

Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/ threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WTor the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus,LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5-LMTK1-Rab11 is a novel signaling pathway involved in axonal outgrowth. [ABSTRACT FROM AUTHOR]

Graded distributions of extracellular cues guide developing axons toward their targets. A network of second messengers - Ca(2+) and cyclic nucleotides - shapes cue-derived information into either attractive or repulsive signals that steer growth cones bidirectionally. Emerging evidence suggests that such guidance signals create a localized imbalance between exocytosis and endocytosis, which in turn redirects membrane, adhesion and cytoskeletal components asymmetrically across the growth cone to bias the direction of axon extension. These recent advances allow us to propose a unifying model of how the growth cone translates shallow gradients of environmental information into polarized activity of the steering machinery for axon guidance. [ABSTRACT FROM AUTHOR]

Asymmetric Ca2+ signals across the growth cone mediate attractive or repulsive axon guidance depending on the occurrence of Ca2+- induced Ca2+ release (CICR) through ryanodine receptors (RyRs). Although the neuronal isoform of nitric oxide (NO) synthase (nNOS) is highly expressed in developing dorsal root ganglion (DRG) neurons, the role of NO in axon guidance remains essentially unknown. Here we report that the NO-cGMP pathway negatively regulates CICR to control the directional polarity of DRG axon guidance. Intracellular levels of NO and cGMP depend on extracellular substrates: laminin activates the NO-cGMP pathway, whereas the adhesion molecule L1 does not. The activity of NO and cGMP determines the turning direction of growth cones with respect to asymmetric Ca2+ signals that are produced by photolysing caged Ca2+. The Ca2+ signals cause growth cone repulsion on a laminin substrate, which is converted to attraction by pharmacological blockade of the NO-cGMP pathway or genetic deletion of nNOS. Conversely, Ca2+-induced growth cone attraction on an L1 substrate is converted to repulsion by increasing NO levels. Such NO-mediated switching of turning direction involves the regulation of CICR through RyRs. Furthermore, growth cone repulsion induced by an extracellular gradient of a physiological cue, neurotrophin-4, is dependent on Ca2+ signals and converted to attraction by inhibiting the NO-cGMP pathway. These results suggest that, on contact with different adhesive environments, growth cones can change their turning responses to axon guidance cues by modulating CICR via endogenous NO and cGMP. [ABSTRACT FROM AUTHOR]

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam-/-) died within 24 h after birth. Whole-body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Furthermore, a medulla-spinal cord preparation of Dscam-/- mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory neurons located in the rostral ventrolateral medulla and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam-/- mice. Dscam-/- mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam-/- mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosagedependent manner. [ABSTRACT FROM AUTHOR]