Role of neurotrophins in pregnancy, delivery and postpartum.

Neurotrophins (NTs) are a family of polypeptides whose functions have been extensively studied in the past two decades. In particular, Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF) play a major role in the development, nutrition and growth of the central and peripheral nervous system and in the pathogenesis of neurodegenerative, cardiometabolic and (auto)immune diseases. However, NGF and BDNF have subtle functions for follicular development, implantation, and placentation. This short narrative review summarizes the existing evidence, published between 2000 and 2019, about the role of NTs in many different conditions that might affect women during and after pregnancy such as preeclampsia, gestational diabetes, obesity, depression, anxiety, smoking and alcohol abuse. Literature suggests that the dysregulation of synthesis and release of NTs may lead to decisive effects on both maternal and fetal health. Some piece of evidences was found about a possible association between NGF/BDNF and breastfeeding. Additional studies on human models are necessary to further characterize the role of NTs in life-changing experiences like labor and delivery.

Ral GTPases in Schwann cells promote radial axonal sorting in the peripheral nervous system.

Small GTPases of the Rho and Ras families are important regulators of Schwann cell biology. The Ras-like GTPases RalA and RalB act downstream of Ras in malignant peripheral nerve sheath tumors. However, the physiological role of Ral proteins in Schwann cell development is unknown. Using transgenic mice with ablation of one or both Ral genes, we report that Ral GTPases are crucial for axonal radial sorting. While lack of only one Ral GTPase was dispensable for early peripheral nerve development, ablation of both RalA and RalB resulted in persistent radial sorting defects, associated with hallmarks of deficits in Schwann cell process formation and maintenance. In agreement, ex vivo-cultured Ral-deficient Schwann cells were impaired in process extension and the formation of lamellipodia. Our data indicate further that RalA contributes to Schwann cell process extensions through the exocyst complex, a known effector of Ral GTPases, consistent with an exocyst-mediated function of Ral GTPases in Schwann cells.

Essential Role of Linx/Islr2 in the Development of the Forebrain Anterior Commissure.

Linx is a member of the leucine-rich repeat and immunoglobulin family of membrane proteins which has critical roles in the development of the peripheral nervous system and forebrain connectivity. A previous study showed that Linx is expressed in projection neurons in the cortex and in cells that comprise the passage to the prethalamus that form the internal capsule, indicating the involvement of Linx in axon guidance and cell-cell communication. In this study, we found that Linx-deficient mice develop severe hydrocephalus and die perinatally by unknown mechanisms. Importantly, mice heterozygous for the linx gene exhibited defects in the development of the anterior commissure in addition to hydrocephalus, indicating haploinsufficiency of the linx gene in forebrain development. In N1E-115 neuroblastoma cells and primary cultured hippocampal neurons, Linx depletion led to impaired neurite extension and an increase in cell body size. Consistent with this, but of unknown significance, we found that Linx interacts with and upregulates the activity of Rho-kinase, a modulator of many cellular processes including cytoskeletal organization. These data suggest a role for Linx in the regulation of complex forebrain connectivity, and future identification of its extracellular ligand(s) will help clarify this function.

TNFa/TNFR2 signaling is required for glial ensheathment at the dorsal root entry zone.

Somatosensory information from the periphery is routed to the spinal cord through centrally-projecting sensory axons that cross into the central nervous system (CNS) via the dorsal root entry zone (DREZ). The glial cells that ensheath these axons ensure rapid propagation of this information. Despite the importance of this glial-axon arrangement, how this afferent nerve is assembled during development is unknown. Using in vivo, time-lapse imaging we show that as centrally-projecting pioneer axons from dorsal root ganglia (DRG) enter the spinal cord, they initiate expression of the cytokine TNFalpha. This induction coincides with ensheathment of these axons by associated glia via a TNF receptor 2 (TNFR2)-mediated process. This work identifies a signaling cascade that mediates peripheral glial-axon interactions and it functions to ensure that DRG afferent projections are ensheathed after pioneer axons complete their navigation, which promotes efficient somatosensory neural function.

Immunolocalization of glutaryl-CoA dehydrogenase (GCDH) in adult and embryonic rat brain and peripheral tissues.

Glutaryl-CoA dehydrogenase (GCDH) is a mitochondrial enzyme that is involved in the degradation of tryptophan, lysine and hydroxylysine. Deficient enzyme activity leads to glutaric aciduria type-I (GA-I). This neurometabolic disease usually manifests with acute encephalopathic crises and striatal neuronal death in early childhood leading to an irreversible dystonic-dyskinetic movement disorder. Fronto-temporal atrophy and white matter changes are already present in the pre-symptomatic period. No detailed information on GCDH expression during embryonic development and in adulthood was available so far. Using immunofluorescence microscopy and cell-type-specific markers to localize GCDH in different tissues, we describe the differential cellular localization of GCDH in adult rat brain and peripheral organs as well as its spatiotemporal expression pattern. During embryonic development GCDH was predominantly expressed in neurons of the central and peripheral nervous system. Significant expression levels were found in epithelial cells (skin, intestinal and nasal mucosa) of rat embryos at different developmental stages. Besides the expected strong expression in liver, GCDH was found to be significantly expressed in neurons of different brain regions, renal proximal tubules, intestinal mucosa and peripheral nerves of adult rats. GCDH was found widely expressed in embryonic and adult rat tissues. In rat embryos GCDH is predominantly expressed in brain implying an important role for brain development. Interestingly, GCDH was found to be significantly expressed in different other organs (e.g. kidney, gut) in adult rats probably explaining the evolving phenotype in GA-I patients.

Involvement of IKAP in peripheral target innervation and in specific JNK and NGF signaling in developing PNS neurons.

A splicing mutation in the ikbkap gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we attempted to elucidate the role of IKAP in PNS development in the chick embryo and found that IKAP is required for proper axonal outgrowth, branching, and peripheral target innervation. Moreover, we demonstrate that IKAP colocalizes with activated JNK (pJNK), dynein, and ß-tubulin at the axon terminals of dorsal root ganglia (DRG) neurons, and may be involved in transport of specific target derived signals required for transcription of JNK and NGF responsive genes in the nucleus. These results suggest the novel role of IKAP in neuronal transport and specific signaling mediated transcription, and provide, for the first time, the basis for a molecular mechanism behind the FD phenotype.

The insulin receptor is required for the development of the Drosophila peripheral nervous system.

The Insulin Receptor (InR) in Drosophila presents features conserved in its mammalian counterparts. InR is required for growth; it is expressed in the central and embryonic nervous system and modulates the time of differentiation of the eye photoreceptor without altering cell fate. We show that the InR is required for the formation of the peripheral nervous system during larval development and more particularly for the formation of sensory organ precursors (SOPs) on the fly notum and scutellum. SOPs arise in the proneural cluster that expresses high levels of the proneural proteins Achaete (Ac) and Scute (Sc). The other cells will become epidermis due to lateral inhibition induced by the Notch (N) receptor signal that prevents its neighbors from adopting a neural fate. In addition, misexpression of the InR or of other components of the pathway (PTEN, Akt, FOXO) induces the development of an abnormal number of macrochaetes that are Drosophila mechanoreceptors. Our data suggest that InR regulates the neural genes ac, sc and sens. The FOXO transcription factor which is localized in the cytoplasm upon insulin uptake, displays strong genetic interaction with the InR and is involved in Ac regulation. The genetic interactions between the epidermal growth factor receptor (EGFR), Ras and InR/FOXO suggest that these proteins cooperate to induce neural gene expression. Moreover, InR/FOXO is probably involved in the lateral inhibition process, since genetic interactions with N are highly significant. These results show that the InR can alter cell fate, independently of its function in cell growth and proliferation.

Calcium-activated potassium channel SK1 is widely expressed in the peripheral nervous system and sensory organs of adult zebrafish.

Sensory cells contain ion channels involved in the organ-specific transduction mechanisms that convert different types of stimuli into electric energy. Here we focus on small-conductance calcium-activated potassium channel 1 (SK1) which plays an important role in all excitable cells acting as feedback regulators in after-hyperpolarization. This study was undertaken to analyze the pattern of expression of SK1 in the zebrafish peripheral nervous system and sensory organs using RT-PRC, Westernblot and immunohistochemistry. Expression of SK1 mRNA was observed at all developmental stages analyzed (from 10 to 100 days post fertilization, dpf), and the antibody used identified a protein with a molecular weight of 70kDa, at 100dpf (regarded to be adult). Cell expressing SK1 in adult animals were neurons of dorsal root and cranial nerve sensory ganglia, sympathetic neurons, sensory cells in neuromasts of the lateral line system and taste buds, crypt olfactory neurons and photoreceptors. Present results report for the first time the expression and the distribution of SK1 in the peripheral nervous system and sensory organs of adult zebrafish, and may contribute to set zebrafish as an interesting experimental model for calcium-activated potassium channels research. Moreover these findings are of potential interest because the potential role of SK as targets for the treatment of neurological diseases and sensory disorders.

WNK1/HSN2 mutation in human peripheral neuropathy deregulates KCC2 expression and posterior lateral line development in zebrafish (Danio rerio).

Hereditary sensory and autonomic neuropathy type 2 (HSNAII) is a rare pathology characterized by an early onset of severe sensory loss (all modalities) in the distal limbs. It is due to autosomal recessive mutations confined to exon "HSN2" of the WNK1 (with-no-lysine protein kinase 1) serine-threonine kinase. While this kinase is well studied in the kidneys, little is known about its role in the nervous system. We hypothesized that the truncating mutations present in the neural-specific HSN2 exon lead to a loss-of-function of the WNK1 kinase, impairing development of the peripheral sensory system. To investigate the mechanisms by which the loss of WNK1/HSN2 isoform function causes HSANII, we used the embryonic zebrafish model and observed strong expression of WNK1/HSN2 in neuromasts of the peripheral lateral line (PLL) system by immunohistochemistry. Knocking down wnk1/hsn2 in embryos using antisense morpholino oligonucleotides led to improper PLL development. We then investigated the reported interaction between the WNK1 kinase and neuronal potassium chloride cotransporter KCC2, as this transporter is a target of WNK1 phosphorylation. In situ hybridization revealed kcc2 expression in mature neuromasts of the PLL and semi-quantitative RT-PCR of wnk1/hsn2 knockdown embryos showed an increased expression of kcc2 mRNA. Furthermore, overexpression of human KCC2 mRNA in embryos replicated the wnk1/hsn2 knockdown phenotype. We validated these results by obtaining double knockdown embryos, both for wnk1/hsn2 and kcc2, which alleviated the PLL defects. Interestingly, overexpression of inactive mutant KCC2-C568A, which does not extrude ions, allowed a phenocopy of the PLL defects. These results suggest a pathway in which WNK1/HSN2 interacts with KCC2, producing a novel regulation of its transcription independent of KCC2's activation, where a loss-of-function mutation in WNK1 induces an overexpression of KCC2 and hinders proper peripheral sensory nerve development, a hallmark of HSANII.

The role of "anti-inflammatory" cytokines in axon regeneration.

The injured central and peripheral nervous system (CNS and PNS) are difficult to regenerate due to the presence of growth inhibitory molecules which are upregulated around the lesion site. In addition, a strong inflammatory response triggering the production of so-called "pro"- and "anti-inflammatory" cytokines, adds to this dilemma. Both pro- and anti-inflammatory cytokines are involved in the regulation of diverse signaling pathways. One of the main aims to induce regeneration is to promote axonal outgrowth and stimulate the formation of new connections. Anti-inflammatory cytokines as modulators of neurite plasticity and outgrowth are of pivotal importance in neuroregeneration with different effects reported. Here we summarize the most relevant information about IL-4, IL-10, IL-13, LIF and TGF-ß focusing on their direct and indirect role in axonal outgrowth.

The lysosomal sialic acid transporter sialin is required for normal CNS myelination.

Salla disease and infantile sialic acid storage disease are autosomal recessive lysosomal storage disorders caused by mutations in the gene encoding sialin, a membrane protein that transports free sialic acid out of the lysosome after it is cleaved from sialoglycoconjugates undergoing degradation. Accumulation of sialic acid in lysosomes defines these disorders, and the clinical phenotype is characterized by neurodevelopmental defects, including severe CNS hypomyelination. In this study, we used a sialin-deficient mouse to address how loss of sialin leads to the defect in myelination. Behavioral analysis of the sialin(-/-) mouse demonstrates poor coordination, seizures, and premature death. Analysis by histology, electron microscopy, and Western blotting reveals a decrease in myelination of the CNS but normal neuronal cytoarchitecture and normal myelination of the PNS. To investigate potential mechanisms underlying CNS hypomyelination, we studied myelination and oligodendrocyte development in optic nerves. We found reduced numbers of myelinated axons in optic nerves from sialin(-/-) mice, but the myelin that was present appeared grossly normal. Migration and density of oligodendrocyte precursor cells were normal; however, a marked decrease in the number of postmitotic oligodendrocytes and an associated increase in the number of apoptotic cells during the later stages of myelinogenesis were observed. These findings suggest that a defect in maturation of cells in the oligodendrocyte lineage leads to increased apoptosis and underlies the myelination defect associated with sialin loss.

PICK1 expression in the Drosophila central nervous system primarily occurs in the neuroendocrine system.

The protein interacting with C kinase 1 (PICK1) protein was first identified as a novel binding partner for protein kinase C. PICK1 contains a membrane-binding BAR domain and a PDZ domain interacting with many synaptic proteins, including the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR2 and the dopamine transporter. PICK1 is strongly implicated in GluR2 trafficking and synaptic plasticity. In mammals, PICK1 has been characterized extensively in cell culture studies. To study PICK1 in an intact system, we characterized PICK1 expression immunohistochemically in the adult and larval Drosophila central nervous system. PICK1 was found in cell bodies in the subesophageal ganglion, the antennal lobe, the protocerebrum, and the neuroendocrine center pars intercerebralis. The cell types that express PICK1 were identified using GAL4 enhancer trap lines. The PICK1-expressing cells form a subpopulation of neurons. PICK1 immunoreactivity was neither detected in glutamatergic nor in dopaminergic neurons. Also, we observed PICK1 expression in only a few GABAergic neurons, located in the antennal lobe. In contrast, we detected robust PICK1 immunolabeling of peptidergic neurons in the neuroendocrine system, which express the transcription factor DIMM and the amidating enzyme peptidylglycine-alpha-hydroxylating monooxygenase (PHM). The PICK1-positive cells include neurosecretory cells that produce the insulin-like peptide dILP2. PICK1 expression in insulin-producing cells also occurs in mammals, as it was also observed in a rat insulinoma cell line derived from pancreatic beta-cells. At the subcellular level, PICK1 was found in the perinuclear zone but surprisingly not in synaptic domains. We conclude that PICK1 may serve an important role in the neuroendocrine system both in insects and vertebrates.

LIG family receptor tyrosine kinase-associated proteins modulate growth factor signals during neural development.

Genome-wide screens were performed to identify transmembrane proteins that mediate axonal growth, guidance and target field innervation of somatosensory neurons. One gene, Linx (alias Islr2), encoding a leucine-rich repeat and immunoglobulin (LIG) family protein, is expressed in a subset of developing sensory and motor neurons. Domain and genomic structures of Linx and other LIG family members suggest that they are evolutionarily related to Trk receptor tyrosine kinases (RTKs). Several LIGs, including Linx, are expressed in subsets of somatosensory and motor neurons, and select members interact with TrkA and Ret RTKs. Moreover, axonal projection defects in mice harboring a null mutation in Linx resemble those in mice lacking Ngf, TrkA, and Ret. In addition, Linx modulates NGF-TrkA- and GDNF-GFRalpha1/Ret-mediated axonal extension in cultured sensory and motor neurons, respectively. These findings show that LIGs physically interact with RTKs and modulate their activities to control axonal extension, guidance and branching.

Origin and distribution of the femoral nerve in fetuses of zebu-crossed bovines

In this study the femoral nerve origin and distribution was assessed through the dissection of 30 fetuses of zebu-crossed bovines, 20 males and 10 females. These animals samples fixation in 10% formaldehyde aqueous medium occurred either by subcutaneous, intra-muscle, and intra-cavity injections in different sites, or by immersion of the mentioned pieces in vessels containing the same medium. The femoral nerve originated from the forth (L4), fifth (L5), and sixth (L6) spinal lumbar ventral branches in 14 animals (46.7%), from L4 and L5 in 13 samples (43.3%), and L5 and L6 in three cases (10%). In the course of its way, on both antimeres the mentioned nerve was branched to the greater psoas (100%), iliac (100%), pectinal (56.7%), femoral quadriceps (100%), muscles, and gave off saphena nerve, which gave branches for the pectinal (43.3%) and sartorius (100%) muscles and continued distally along the saphena artery to spread on the medial face of the knee and leg medial articulation skin. Statistically, there was no significant difference between the muscle branch frequencies given by the femoral nerve to the right and left antimeres. The obtained results related to the femoral nerve origin and distribution in fetuses of zebu-crossed bovines generally presented common characteristics with the ruminant corresponding data found in the literature, and this information is important as the basis for clinical or surgical approaches involving the studied structures.

Neuregulin-1/ErbB signaling serves distinct functions in myelination of the peripheral and central nervous system.

Understanding the control of myelin formation by oligodendrocytes is essential for treating demyelinating diseases. Neuregulin-1 (NRG1) type III, an EGF-like growth factor, is essential for myelination in the PNS. It is thus thought that NRG1/ErbB signaling also regulates CNS myelination, a view suggested by in vitro studies and the overexpression of dominant-negative ErbB receptors. To directly test this hypothesis, we generated a series of conditional null mutants that completely lack NRG1 beginning at different stages of neural development. Unexpectedly, these mice assemble normal amounts of myelin. In addition, double mutants lacking oligodendroglial ErbB3 and ErbB4 become myelinated in the absence of any stimulation by neuregulins. In contrast, a significant hypermyelination is achieved by transgenic overexpression of NRG1 type I or NRG1 type III. Thus, NRG1/ErbB signaling is markedly different between Schwann cells and oligodendrocytes that have evolved an NRG/ErbB-independent mechanism of myelination control.

Promotion of peripheral nerve growth by collagen scaffolds loaded with collagen-targeting human nerve growth factor-beta.

Nerve growth factor (NGF) plays a critical role in neuronal development and regeneration. However, the lack of efficient NGF delivery system limits its clinical application. We reported that a peptide deduced from collagenase, TKKTLRT, fused with NGF-beta could develop a collagen based NGF targeting delivery system. Our results showed that this peptide could allow fused NGF-beta bind to collagen specifically. In addition, we found that the polypeptide could result in a 2.3-fold increase in the expression level and a significant improvement of bioactivity of fused NGF-beta. In the in vivo function study, collagen membranes loaded with the collagen binding NGF enhanced the nerve growth. Thus, the targeting wound repair system could be important for the repair of peripheral nerve injury.

Expression patterns of MLC1 protein in the central and peripheral nervous systems.

Mutations in MLC1 cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), a disorder characterized clinically by macrocephaly, deterioration of motor functions, epilepsy and mental decline. Recent studies have detected MLC1 mRNA and protein in astroglial processes. In addition, our group previously reported MLC1 expression in some neurons in the adult mouse brain. Here we performed an exhaustive study of the expression pattern of MLC1 in the developing mouse brain by means of optic and electron microscopy. In the central nervous system, MLC1 was detected mainly in axonal tracts early in development. In addition, MLC1 was also observed in the peripheral nervous system and in several sensory epithelia, as retina or saccula maculae. Post-embedding immunogold experiments indicated that MLC1 is localized in astrocyte-astrocyte junctions, but not in the perivascular membrane, indicating that MLC1 is not a component of the dystrophin-glycoprotein complex. In neurons, MLC1 is located at the plasma membrane and vesicular structures. Our data provide a mouse MLC1 expression map that could be useful to understand the phenotype of MLC patients, and suggested that MLC disease is caused by an astrocytic and a neuronal dysfunction.

IGF-1 stimulates de novo fatty acid biosynthesis by Schwann cells during myelination.

Schwann cell (SC) differentiation to the myelinating phenotype is characterized by the elaboration of a lipid-rich membrane and the expression of myelin-specific proteins. Insulin-like growth factor-1 (IGF-1) has been identified as a growth factor that stimulates the early events of myelination in SCs that signals via the PI3K/Akt pathway. Given the role of IGF-1 in promoting myelination, we performed studies to determine if the fatty acid biosynthetic pathway was a target of IGF-1 signaling in the formation of myelin membrane in dorsal root ganglion neuron/Schwann cell (DRG/SC) cocultures. We report that the fatty acid profile of lipid extracts of cocultures treated with IGF-1 match that reported for native myelin membrane by electrospray mass spectroscopy analysis. We also demonstrate de novo fatty acid biosynthesis in response to IGF-1 treatment in DRG/SC cocultures metabolically labeled with (13)C-acetate as a carbon source for fatty acid synthesis. Consistent with this finding, Western blot analysis of lysates from both cocultures and purified SCs reveal that IGF-1 stimulates two key fatty acid synthesizing enzymes. Additionally, we show that stimulation of fatty acid synthesizing enzymes is mediated by the PI3K/Akt signaling pathway. We also show that the fatty acid synthesizing enzymes and associated signaling pathways are elevated during the period of myelin membrane formation in sciatic nerve. Collectively, these findings demonstrate that IGF-1 plays an important regulatory function during myelin membrane formation.