Neuronally differentiated macula densa cells regulate tissue remodeling and regeneration in the kidney.

Tissue regeneration is limited in several organs, including the kidney, contributing to the high prevalence of kidney disease globally. However, evolutionary and physiological adaptive responses and the presence of renal progenitor cells suggest an existing remodeling capacity. This study uncovered endogenous tissue remodeling mechanisms in the kidney that were activated by the loss of body fluid and salt and regulated by a unique niche of a minority renal cell type called the macula densa (MD). Here, we identified neuronal differentiation features of MD cells that sense the local and systemic environment and secrete angiogenic, growth, and extracellular matrix remodeling factors, cytokines and chemokines, and control resident progenitor cells. Serial intravital imaging, MD nerve growth factor receptor and Wnt mouse models, and transcriptome analysis revealed cellular and molecular mechanisms of these MD functions. Human and therapeutic translation studies illustrated the clinical potential of MD factors, including CCN1, as a urinary biomarker and therapeutic target in chronic kidney disease. The concept that a neuronally differentiated key sensory and regulatory cell type responding to organ-specific physiological inputs controls local progenitors to remodel or repair tissues may be applicable to other organs and diverse tissue-regenerative therapeutic strategies.

Vascular smooth muscle cell-derived nerve growth factor regulates sympathetic collateral branching to innervate blood vessels in embryonic skin.

Blood vessels serve as intermediate conduits for the extension of sympathetic axons towards target tissues, while also acting as crucial targets for their homeostatic processes encompassing the regulation of temperature, blood pressure, and oxygen availability. How sympathetic axons innervate not only blood vessels but also a wide array of target tissues is not clear. Here we show that in embryonic skin, after the establishment of co-branching between sensory nerves and blood vessels, sympathetic axons invade the skin alongside these sensory nerves and extend their branches towards these blood vessels covered by vascular smooth muscle cells (VSMCs). Our mosaic labeling technique for sympathetic axons shows that collateral branching predominantly mediates the innervation of VSMC-covered blood vessels by sympathetic axons. The expression of nerve growth factor (NGF), previously known to induce collateral axon branching in culture, can be detected in the vascular smooth muscle cell (VSMC)-covered blood vessels, as well as sensory nerves. Indeed, VSMC-specific Ngf knockout leads to a significant decrease of collateral branching of sympathetic axons innervating VSMC-covered blood vessels. These data suggest that VSMC-derived NGF serves as an inductive signal for collateral branching of sympathetic axons innervating blood vessels in the embryonic skin.

A functional circuit formed by the autonomic nerves and myofibroblasts controls mammalian alveolar formation for gas exchange.

Alveolar formation increases the surface area for gas exchange. A molecular understanding of alveologenesis remains incomplete. Here, we show that the autonomic nerve and alveolar myofibroblast form a functional unit in mice. Myofibroblasts secrete neurotrophins to promote neurite extension/survival, whereas neurotransmitters released from autonomic terminals are necessary for myofibroblast proliferation and migration, a key step in alveologenesis. This establishes a functional link between autonomic innervation and alveolar formation. We also discover that planar cell polarity (PCP) signaling employs a Wnt-Fz/Ror-Vangl cascade to regulate the cytoskeleton and neurotransmitter trafficking/release from the terminals of autonomic nerves. This represents a new aspect of PCP signaling in conferring cellular properties. Together, these studies offer molecular insight into how autonomic activity controls alveolar formation. Our work also illustrates the fundamental principle of how two tissues (e.g., nerves and lungs) interact to build alveoli at the organismal level.

NKCC1 Deficiency in Forming Hippocampal Circuits Triggers Neurodevelopmental Disorder: Role of BDNF-TrkB Signalling.

The time-sensitive GABA shift from excitatory to inhibitory is critical in early neural circuits development and depends upon developmentally regulated expression of cation-chloride cotransporters NKCC1 and KCC2. NKCC1, encoded by the SLC12A2 gene, regulates neuronal Cl- homeostasis by chloride import working opposite KCC2. The high NKCC1/KCC2 expression ratio decreases in early neural development contributing to GABA shift. Human SLC12A2 loss-of-function mutations were recently associated with a multisystem disorder affecting neural development. However, the multisystem phenotype of rodent Nkcc1 knockout models makes neurodevelopment challenging to study. Brain-Derived Neurotrophic Factor (BDNF)-NTRK2/TrkB signalling controls KCC2 expression during neural development, but its impact on NKCC1 is still controversial. Here, we discuss recent evidence supporting BDNF-TrkB signalling controlling Nkcc1 expression and the GABA shift during hippocampal circuit formation. Namely, specific deletion of Ntrk2/Trkb from immature mouse hippocampal dentate granule cells (DGCs) affects their integration and maturation in the hippocampal circuitry and reduces Nkcc1 expression in their target region, the CA3 principal cells, leading to premature GABA shift, ultimately influencing the establishment of functional hippocampal circuitry and animal behaviour in adulthood. Thus, immature DGCs emerge as a potential therapeutic target as GABAergic transmission is vital for specific neural progenitors generating dentate neurogenesis in early development and the mature brain.

Heart neurons use clock genes to control myocyte proliferation.

Neurons can regulate the development, pathogenesis, and regeneration of target organs. However, the role of neurons during heart development and regeneration remains unclear. We genetically inhibited sympathetic innervation in vivo, which resulted in heart enlargement with an increase in cardiomyocyte number. Transcriptomic and protein analysis showed down-regulation of the two clock gene homologs Period1/Period2 (Per1/Per2) accompanied by up-regulation of cell cycle genes. Per1/Per2 deletion increased heart size and cardiomyocyte proliferation, recapitulating sympathetic neuron­deficient hearts. Conversely, increasing sympathetic activity by norepinephrine treatment induced Per1/Per2 and suppressed cardiomyocyte proliferation. We further found that the two clock genes negatively regulate myocyte mitosis entry through the Wee1 kinase pathway. Our findings demonstrate a previously unknown link between cardiac neurons and clock genes in regulation of cardiomyocyte proliferation and heart size and provide mechanistic insights for developing neuromodulation strategies for cardiac regen5eration.

NGF-TrkA signaling dictates neural ingrowth and aberrant osteochondral differentiation after soft tissue trauma.

Pain is a central feature of soft tissue trauma, which under certain contexts, results in aberrant osteochondral differentiation of tissue-specific stem cells. Here, the role of sensory nerve fibers in this abnormal cell fate decision is investigated using a severe extremity injury model in mice. Soft tissue trauma results in NGF (Nerve growth factor) expression, particularly within perivascular cell types. Consequently, NGF-responsive axonal invasion occurs which precedes osteocartilaginous differentiation. Surgical denervation impedes axonal ingrowth, with significant delays in cartilage and bone formation. Likewise, either deletion of Ngf or two complementary methods to inhibit its receptor TrkA (Tropomyosin receptor kinase A) lead to similar delays in axonal invasion and osteochondral differentiation. Mechanistically, single-cell sequencing suggests a shift from TGFß to FGF signaling activation among pre-chondrogenic cells after denervation. Finally, analysis of human pathologic specimens and databases confirms the relevance of NGF-TrkA signaling in human disease. In sum, NGF-mediated TrkA-expressing axonal ingrowth drives abnormal osteochondral differentiation after soft tissue trauma. NGF-TrkA signaling inhibition may have dual therapeutic use in soft tissue trauma, both as an analgesic and negative regulator of aberrant stem cell differentiation.

Immature Dentate Granule Cells Require Ntrk2/Trkb for the Formation of Functional Hippocampal Circuitry.

Early in brain development, impaired neuronal signaling during time-sensitive windows triggers the onset of neurodevelopmental disorders. GABA, through its depolarizing and excitatory actions, drives early developmental events including neuronal circuit formation and refinement. BDNF/TrkB signaling cooperates with GABA actions. How these developmental processes influence the formation of neural circuits and affect adult brain function is unknown. Here, we show that early deletion of Ntrk2/Trkb from immature mouse hippocampal dentate granule cells (DGCs) affects the integration and maturation of newly formed DGCs in the hippocampal circuitry and drives a premature shift from depolarizing to hyperpolarizing GABAergic actions in the target of DGCs, the CA3 principal cells of the hippocampus, by reducing the expression of the cation-chloride importer Nkcc1. These changes lead to the disruption of early synchronized neuronal activity at the network level and impaired morphological maturation of CA3 pyramidal neurons, ultimately contributing to altered adult hippocampal synaptic plasticity and cognitive processes.

A Neurotrophic Mechanism Directs Sensory Nerve Transit in Cranial Bone.

The flat bones of the skull are densely innervated during development, but little is known regarding their role during repair. We describe a neurotrophic mechanism that directs sensory nerve transit in the mouse calvaria. Patent cranial suture mesenchyme represents an NGF (nerve growth factor)-rich domain, in which sensory nerves transit. Experimental calvarial injury upregulates Ngf in an IL-1ß/TNF-α-rich defect niche, with consequent axonal ingrowth. In calvarial osteoblasts, IL-1ß and TNF-α stimulate Ngf and downstream NF-κB signaling. Locoregional deletion of Ngf delays defect site re-innervation and blunted repair. Genetic disruption of Ngf among LysM-expressing macrophages phenocopies these observations, whereas conditional knockout of Ngf among Pdgfra-expressing cells does not. Finally, inhibition of TrkA catalytic activity similarly delays re-innervation and repair. These results demonstrate an essential role of NGF-TrkA signaling in bone healing and implicate macrophage-derived NGF-induced ingrowth of skeletal sensory nerves as an important mediator of this repair.

Liver-specific deletion of IGF2 mRNA binding protein-2/IMP2 reduces hepatic fatty acid oxidation and increases hepatic triglyceride accumulation.

Insulin-like growth factor 2 mRNA-binding proteins 1-3 (IGF2BP1-3, also known as IMP1-3) contribute to the regulation of RNAs in a transcriptome-specific context. Global deletion of the mRNA-binding protein insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2 or IMP2) in mice causes resistance to obesity and fatty liver induced by a high-fat diet (HFD), whereas liver-specific IMP2 overexpression results in steatosis. To better understand the role of IMP2 in hepatic triglyceride metabolism, here we crossed mice expressing albumin-Cre with mice bearing a floxed Imp2 gene to generate hepatocyte-specific IMP2 knockout (LIMP2 KO) mice. Unexpectedly, the livers of LIMP2 KO mice fed an HFD accumulated more triglyceride. Although hepatocyte-specific IMP2 deletion did not alter lipogenic gene expression, it substantially decreased the levels of the IMP2 client mRNAs encoding carnitine palmitoyltransferase 1A (CPT1A) and peroxisome proliferator-activated receptor α (PPARα). This decrease was associated with their more rapid turnover and accompanied by significantly diminished rates of palmitate oxidation by isolated hepatocytes and liver mitochondria. HFD-fed control and LIMP2 KO mice maintained a similar glucose tolerance and insulin sensitivity up to 6 months; however, by 6 months, blood glucose and serum triglycerides in LIMP2 KO mice were modestly elevated but without evidence of liver damage. In conclusion, hepatocyte-specific IMP2 deficiency promotes modest diet-induced fatty liver by impairing fatty acid oxidation through increased degradation of the IMP2 client mRNAs PPARα and CPT1A This finding indicates that the previously observed marked protection against fatty liver conferred by global IMP2 deficiency in mice is entirely due to their reduced adiposity.

Pancreatic islet chromatin accessibility and conformation reveals distal enhancer networks of type 2 diabetes risk.

Genetic variants affecting pancreatic islet enhancers are central to T2D risk, but the gene targets of islet enhancer activity are largely unknown. We generate a high-resolution map of islet chromatin loops using Hi-C assays in three islet samples and use loops to annotate target genes of islet enhancers defined using ATAC-seq and published ChIP-seq data. We identify candidate target genes for thousands of islet enhancers, and find that enhancer looping is correlated with islet-specific gene expression. We fine-map T2D risk variants affecting islet enhancers, and find that candidate target genes of these variants defined using chromatin looping and eQTL mapping are enriched in protein transport and secretion pathways. At IGF2BP2, a fine-mapped T2D variant reduces islet enhancer activity and IGF2BP2 expression, and conditional inactivation of IGF2BP2 in mouse islets impairs glucose-stimulated insulin secretion. Our findings provide a resource for studying islet enhancer function and identifying genes involved in T2D risk.

IMP2 Increases Mouse Skeletal Muscle Mass and Voluntary Activity by Enhancing Autocrine Insulin-Like Growth Factor 2 Production and Optimizing Muscle Metabolism.

Insulin-like growth factor 2 (IGF2) mRNA binding protein 2 (IMP2) was selectively deleted from adult mouse muscle; two phenotypes were observed: decreased accrual of skeletal muscle mass after weaning and reduced wheel-running activity but normal forced treadmill performance. Reduced wheel running occurs when mice are fed a high-fat diet but is normalized when mice consume standard chow. The two phenotypes are due to altered output from different IMP2 client mRNAs. The reduced fiber size of IMP2-deficient muscle is attributable, in part, to diminished autocrine Igf2 production; basal tyrosine phosphorylation of the insulin and IGF1 receptors is diminished, and Akt1 activation is selectively reduced. Gsk3α is disinhibited, and S536-phosphorylated ε subunit of eukaryotic initiation factor 2B [eIF2Bε(S536)] is hyperphosphorylated. Protein synthesis is reduced despite unaltered mTOR complex 1 activity. The diet-dependent reduction in voluntary exercise is likely due to altered muscle metabolism, as contractile function is normal. IMP2-deficient muscle exhibits reduced fatty acid oxidation, due to a reduced abundance of mRNA of peroxisome proliferator-activated receptor α (PPARα), an IMP2 client, and PPARα protein. IMP2-deficient muscle fibers treated with a mitochondrial uncoupler to increase electron flux, as occurs with exercise, exhibit reduced oxygen consumption from fatty acids, with higher oxygen consumption from glucose. The greater dependence on muscle glucose metabolism during increased oxygen demand may promote central fatigue and thereby diminish voluntary activity.

IGF2 mRNA binding protein-2 is a tumor promoter that drives cancer proliferation through its client mRNAs IGF2 and HMGA1.

The gene encoding the Insulin-like Growth Factor 2 mRNA binding protein 2/IMP2 is amplified and overexpressed in many human cancers, accompanied by a poorer prognosis. Mice lacking IMP2 exhibit a longer lifespan and a reduced tumor burden at old age. Herein we show in a diverse array of human cancer cells that IMP2 overexpression stimulates and IMP2 elimination diminishes proliferation by 50-80%. In addition to its known ability to promote the abundance of Insulin-like Growth Factor 2/IGF2, we find that IMP2 strongly promotes IGF action, by binding and stabilizing the mRNA encoding the DNA binding protein HMGA1, a known oncogene. HMGA1 suppresses the abundance of IGF binding protein 2/IGFBP2 and Grb14, inhibitors of IGF action. IMP2 stabilization of HMGA1 mRNA plus IMP2 stimulated IGF2 production synergistically drive cancer cell proliferation and account for IMP2's tumor promoting action. IMP2's ability to promote proliferation and IGF action requires IMP2 phosphorylation by mTOR.

NGF-TrkA signaling in sensory nerves is required for skeletal adaptation to mechanical loads in mice.

Sensory nerves emanating from the dorsal root extensively innervate the surfaces of mammalian bone, a privileged location for the regulation of biomechanical signaling. Here, we show that NGF-TrkA signaling in skeletal sensory nerves is an early response to mechanical loading of bone and is required to achieve maximal load-induced bone formation. First, the elimination of TrkA signaling in mice harboring mutant TrkAF592A alleles was found to greatly attenuate load-induced bone formation induced by axial forelimb compression. Next, both in vivo mechanical loading and in vitro mechanical stretch were shown to induce the profound up-regulation of NGF in osteoblasts within 1 h of loading. Furthermore, inhibition of TrkA signaling following axial forelimb compression was observed to reduce measures of Wnt/ß-catenin activity in osteocytes in the loaded bone. Finally, the administration of exogenous NGF to wild-type mice was found to significantly increase load-induced bone formation and Wnt/ß-catenin activity in osteocytes. In summary, these findings demonstrate that communication between osteoblasts and sensory nerves through NGF-TrkA signaling is essential for load-induced bone formation in mice.

Neurotrophin Signaling Is Required for Glucose-Induced Insulin Secretion.

Insulin secretion by pancreatic islet ß cells is critical for glucose homeostasis, and a blunted ß cell secretory response is an early deficit in type 2 diabetes. Here, we uncover a regulatory mechanism by which glucose recruits vascular-derived neurotrophins to control insulin secretion. Nerve growth factor (NGF), a classical trophic factor for nerve cells, is expressed in pancreatic vasculature while its TrkA receptor is localized to islet ß cells. High glucose rapidly enhances NGF secretion and increases TrkA phosphorylation in mouse and human islets. Tissue-specific deletion of NGF or TrkA, or acute disruption of TrkA signaling, impairs glucose tolerance and insulin secretion in mice. We show that internalized TrkA receptors promote insulin granule exocytosis via F-actin reorganization. Furthermore, NGF treatment augments glucose-induced insulin secretion in human islets. These findings reveal a non-neuronal role for neurotrophins and identify a new regulatory pathway in insulin secretion that can be targeted to ameliorate ß cell dysfunction.

Retinol Dehydrogenase-10 Regulates Pancreas Organogenesis and Endocrine Cell Differentiation via Paracrine Retinoic Acid Signaling.

Vitamin A-derived retinoic acid (RA) signals are critical for the development of several organs, including the pancreas. However, the tissue-specific control of RA synthesis in organ and cell lineage development has only poorly been addressed in vivo. Here, we show that retinol dehydrogenase-10 (Rdh10), a key enzyme in embryonic RA production, has important functions in pancreas organogenesis and endocrine cell differentiation. Rdh10 was expressed in the developing pancreas epithelium and surrounding mesenchyme. Rdh10 null mutant mouse embryos exhibited dorsal pancreas agenesis and a hypoplastic ventral pancreas with retarded tubulogenesis and branching. Conditional disruption of Rdh10 from the endoderm caused increased mortality, reduced body weight, and lowered blood glucose levels after birth. Endodermal Rdh10 deficiency led to a smaller dorsal pancreas with a reduced density of early glucagon+ and insulin+ cells. During the secondary transition, the reduction of Neurogenin3+ endocrine progenitors in the mutant dorsal pancreas accounted for fewer α- and ß-cells. Changes in the expression of α- and ß-cell-specific transcription factors indicated that Rdh10 might also participate in the terminal differentiation of endocrine cells. Together, our results highlight the importance of both mesenchymal and epithelial Rdh10 for pancreogenesis and the first wave of endocrine cell differentiation. We further propose a model in which the Rdh10-expressing exocrine tissue acts as an essential source of RA signals in the second wave of endocrine cell differentiation.

NGF-TrkA Signaling by Sensory Nerves Coordinates the Vascularization and Ossification of Developing Endochondral Bone.

Developing tissues dictate the amount and type of innervation they require by secreting neurotrophins, which promote neuronal survival by activating distinct tyrosine kinase receptors. Here, we show that nerve growth factor (NGF) signaling through neurotrophic tyrosine kinase receptor type 1 (TrkA) directs innervation of the developing mouse femur to promote vascularization and osteoprogenitor lineage progression. At the start of primary ossification, TrkA-positive axons were observed at perichondrial bone surfaces, coincident with NGF expression in cells adjacent to centers of incipient ossification. Inactivation of TrkA signaling during embryogenesis in TrkA(F592A) mice impaired innervation, delayed vascular invasion of the primary and secondary ossification centers, decreased numbers of Osx-expressing osteoprogenitors, and decreased femoral length and volume. These same phenotypic abnormalities were observed in mice following tamoxifen-induced disruption of NGF in Col2-expressing perichondrial osteochondral progenitors. We conclude that NGF serves as a skeletal neurotrophin to promote sensory innervation of developing long bones, a process critical for normal primary and secondary ossification.

IGF2BP2/IMP2-Deficient mice resist obesity through enhanced translation of Ucp1 mRNA and Other mRNAs encoding mitochondrial proteins.

Although variants in the IGF2BP2/IMP2 gene confer risk for type 2 diabetes, IMP2, an RNA binding protein, is not known to regulate metabolism. Imp2(-/-) mice gain less lean mass after weaning and have increased lifespan. Imp2(-/-) mice are highly resistant to diet-induced obesity and fatty liver and display superior glucose tolerance and insulin sensitivity, increased energy expenditure, and better defense of core temperature on cold exposure. Imp2(-/-) brown fat and Imp2(-/-) brown adipocytes differentiated in vitro contain more UCP1 polypeptide than Imp2(+/+) despite similar levels of Ucp1 mRNA; the Imp2(-/-)adipocytes also exhibit greater uncoupled oxygen consumption. IMP2 binds the mRNAs encoding Ucp1 and other mitochondrial components, and most exhibit increased translational efficiency in the absence of IMP2. In vitro IMP2 inhibits translation of mRNAs bearing the Ucp1 untranslated segments. Thus IMP2 limits longevity and regulates nutrient and energy metabolism in the mouse by controlling the translation of its client mRNAs.

Synaptic mechanisms of adenosine A2A receptor-mediated hyperexcitability in the hippocampus.

Adenosine inhibits excitatory neurons widely in the brain through adenosine A1 receptor, but activation of adenosine A2A receptor (A2A R) has an opposite effect promoting discharge in neuronal networks. In the hippocampus A2A R expression level is low, and the receptor's effect on identified neuronal circuits is unknown. Using optogenetic afferent stimulation and whole-cell recording from identified postsynaptic neurons we show that A2A R facilitates excitatory glutamatergic Schaffer collateral synapses to CA1 pyramidal cells, but not to GABAergic inhibitory interneurons. In addition, A2A R enhances GABAergic inhibitory transmission between CA1 area interneurons leading to disinhibition of pyramidal cells. Adenosine A2A R has no direct modulatory effect on GABAergic synapses to pyramidal cells. As a result adenosine A2A R activation alters the synaptic excitation - inhibition balance in the CA1 area resulting in increased pyramidal cell discharge to glutamatergic Schaffer collateral stimulation. In line with this, we show that A2A R promotes synchronous pyramidal cell firing in hyperexcitable conditions where extracellular potassium is elevated or following high-frequency electrical stimulation. Our results revealed selective synapse- and cell type specific adenosine A2A R effects in hippocampal CA1 area. The uncovered mechanisms help our understanding of A2A R's facilitatory effect on cortical network activity.

Innate immunity. A Spaetzle-like role for nerve growth factor ß in vertebrate immunity to Staphylococcus aureus.

Many key components of innate immunity to infection are shared between Drosophila and humans. However, the fly Toll ligand Spaetzle is not thought to have a vertebrate equivalent. We have found that the structurally related cystine-knot protein, nerve growth factor ß (NGFß), plays an unexpected Spaetzle-like role in immunity to Staphylococcus aureus infection in chordates. Deleterious mutations of either human NGFß or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA) were associated with severe S. aureus infections. NGFß was released by macrophages in response to S. aureus exoproteins through activation of the NOD-like receptors NLRP3 and NLRP4 and enhanced phagocytosis and superoxide-dependent killing, stimulated proinflammatory cytokine production, and promoted calcium-dependent neutrophil recruitment. TrkA knockdown in zebrafish increased susceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFß-TRKA signaling in pathogen-specific host immunity.

Genetic dissection of TrkB activated signalling pathways required for specific aspects of the taste system.

BACKGROUND: Neurotrophin-4 (NT-4) and brain derived neurotrophic factor (BDNF) bind to the same receptor, Ntrk2/TrkB, but play distinct roles in the development of the rodent gustatory system. However, the mechanisms underlying these processes are lacking. RESULTS: Here, we demonstrate, in vivo, that single or combined point mutations in major adaptor protein docking sites on TrkB receptor affect specific aspects of the mouse gustatory development, known to be dependent on BDNF or NT-4. In particular, mice with a mutation in the TrkB-SHC docking site had reduced gustatory neuron survival at both early and later stages of development, when survival is dependent on NT-4 and BDNF, respectively. In addition, lingual innervation and taste bud morphology, both BDNF-dependent functions, were altered in these mutants. In contrast, mutation of the TrkB-PLCγ docking site alone did not affect gustatory neuron survival. Moreover, innervation to the tongue was delayed in these mutants and taste receptor expression was altered. CONCLUSIONS: We have genetically dissected pathways activated downstream of the TrkB receptor that are required for specific aspects of the taste system controlled by the two neurotrophins NT-4 and BDNF. In addition, our results indicate that TrkB also regulate the expression of specific taste receptors by distinct signalling pathways. These results advance our knowledge of the biology of the taste system, one of the fundamental sensory systems crucial for an organism to relate to the environment.