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.

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.

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.

TrkB signalling pathways in LTP and learning.

Understanding the mechanisms that underlie learning is one of the most fascinating and central aims of neurobiological research. Hippocampal long-term potentiation (LTP) is widely regarded as a prime candidate for the cellular mechanism of learning. The receptor tyrosine kinase TrkB (also known as NTRK2), known primarily for its function during PNS and CNS development, has emerged in recent years as a potent regulator of hippocampal LTP. Here I describe efforts to understand the signalling pathways and molecular mechanisms that underlie the involvement of TrkB in LTP and learning.

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.

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.

GABAergic neurons regulate lateral ventricular development via transcription factor Pax5.

Postmortem studies have revealed a downregulation of the transcription factor Pax5 in GABAergic neurons in bipolar disorder, a neurodevelopmental disorder, raising the question whether Pax5 in GABAergic neurons has a role in normal brain development. In a genetic approach to study functions of Pax5 in GABAergic neurons, Pax5 was specifically deleted in GABAergic neurons from Pax5 floxed mice using a novel Gad1-Cre transgenic mouse line expressing Cre recombinase in Gad1-positive, that is, GABAergic neurons. Surprisingly, these mice developed a marked enlargement of the lateral ventricles at approximately 7 weeks of age, which was lethal within 1-2 weeks of its appearance. This hydrocephalus phenotype was observed in mice homozygous or heterozygous for the Pax5 conditional knockout, with a gene dosage-dependent penetrance. By QTL (quantitative trait loci) mapping, a 3.5 Mb segment on mouse chromosome 4 flanked by markers D4Mit237 and D4Mit214 containing approximately 92 genes including Pax5 has previously been linked to differences in lateral ventricular size. Our findings are consistent with Pax5 being a relevant gene underlying this QTL phenotype and demonstrate that Pax5 in GABAergic neurons is essential for normal ventricular development.

Loss of NGF-TrkA signaling from the CNS is not sufficient to induce cognitive impairments in young adult or intermediate-aged mice.

Many molecules expressed in the CNS contribute to cognitive functions either by modulating neuronal activity or by mediating neuronal trophic support and/or connectivity. An ongoing discussion is whether signaling of nerve growth factor (NGF) through its high-affinity receptor TrkA contributes to attention behavior and/or learning and memory, based on its expression in relevant regions of the CNS such as the hippocampus, cerebral cortex, amygdala and basal forebrain. Previous animal models carrying either a null allele or transgenic manipulation of Ngf or Trka have proved difficult in addressing this question. To overcome this problem, we conditionally deleted Ngf or Trka from the CNS. Our findings confirm that NGF-TrkA signaling supports survival of only a small proportion of cholinergic neurons during development; however, this signaling is not required for trophic support or connectivity of the remaining basal forebrain cholinergic neurons. Moreover, comprehensive behavioral analysis of young adult and intermediate-aged mice lacking NGF-TrkA signaling demonstrates that this signaling is dispensable for both attention behavior and various aspects of learning and memory.

TrkB (tropomyosin-related kinase B) controls the assembly and maintenance of GABAergic synapses in the cerebellar cortex.

Inhibitory interneurons play a critical role in coordinating the activity of neural circuits. To explore the mechanisms that direct the organization of inhibitory circuits, we analyzed the involvement of tropomyosin-related kinase B (TrkB) in the assembly and maintenance of GABAergic inhibitory synapses between Golgi and granule cells in the mouse cerebellar cortex. We show that TrkB acts directly within each cell-type to regulate synaptic differentiation. TrkB is required not only for assembly, but also maintenance of these synapses and acts, primarily, by regulating the localization of synaptic constituents. Postsynaptically, TrkB controls the localization of a scaffolding protein, gephyrin, but acts at a step subsequent to the localization of a cell adhesion molecule, Neuroligin-2. Importantly, TrkB is required for the localization of an Ig superfamily cell adhesion molecule, Contactin-1, in Golgi and granule cells and the absence of Contactin-1 also results in deficits in inhibitory synaptic development. Thus, our findings demonstrate that TrkB controls the assembly and maintenance of GABAergic synapses and suggest that TrkB functions, in part, through promoting synaptic adhesion.

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.

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.

Disruption of TrkB-mediated phospholipase Cgamma signaling inhibits limbic epileptogenesis.

The BDNF receptor, TrkB, is critical to limbic epileptogenesis, but the responsible downstream signaling pathways are unknown. We hypothesized that TrkB-dependent activation of phospholipase Cgamma1 (PLCgamma1) signaling is the key pathway and tested this in trkB(PLC/PLC) mice carrying a mutation (Y816F) that uncouples TrkB from PLCgamma1. Biochemical measures revealed activation of both TrkB and PLCgamma1 in hippocampi in the pilocarpine and kindling models in wild-type mice. PLCgamma1 activation was decreased in hippocampi isolated from trkB(PLC/PLC) compared with control mice. Epileptogenesis assessed by development of kindling was inhibited in trkB(PLC/PLC) compared with control mice. Long-term potentiation of the mossy fiber-CA3 pyramid synapse was impaired in slices of trkB(PLC/PLC) mice. We conclude that TrkB-dependent activation of PLCgamma1 signaling is an important molecular mechanism of limbic epileptogenesis. Elucidating signaling pathways activated by a cell membrane receptor in animal models of CNS disorders promises to reveal novel targets for specific and effective therapeutic intervention.

BDNF-TrkB signalling in fear learning: from genetics to neural networks.

Discovering the basic mechanisms in fear encoding and expression is important in many fields, including psychology, sociology, medicine, and neuroscience. Effective treatment for fear-based pathology depends on understanding how fear is learned and regulated. Among the molecular systems required for fear learning and amygdalar synaptic plasticity, brain derived neurtrophic factor (BDNF) and its high affinity receptor Ntrk2/TrkB have been shown to play essential roles. Therefore, we will focus this review on three main aspects; first of all, the impact of Bdnf polymorphism on fear related characteristics in humans and animal models. Secondly, we will discuss BDNF-TrkB activity regulation by epigenetic, transcriptional and post-translational events, and finally we will discuss TrkB-BDNF signalling in fear learning. BDNF-TrkB and the signalling activated in this particular form of plasticity are becoming crucial players in fear learning and memory thus highlighting these molecules as potential therapeutic targets in fear-related pathologies.

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.

TrkB modulates fear learning and amygdalar synaptic plasticity by specific docking sites.

Understanding the modulation of the neural circuitry of fear is clearly one of the most important aims in neurobiology. Protein phosphorylation in response to external stimuli is considered a major mechanism underlying dynamic changes in neural circuitry. TrkB (Ntrk2) neurotrophin receptor tyrosine kinase potently modulates synaptic plasticity and activates signal transduction pathways mainly through two phosphorylation sites [Y515/Shc site; Y816/PLCgamma (phospholipase Cgamma) site]. To identify the molecular pathways required for fear learning and amygdalar synaptic plasticity downstream of TrkB, we used highly defined genetic mouse models carrying single point mutations at one of these two sites (Y515F or Y816F) to examine the physiological relevance of pathways activated through these sites for pavlovian fear conditioning (FC), as well as for synaptic plasticity as measured by field recordings obtained from neurons of different amygdala nuclei. We show that a Y816F point mutation impairs acquisition of FC, amygdalar synaptic plasticity, and CaMKII signaling at synapses. In contrast, a Y515F point mutation affects consolidation but not acquisition of FC to tone, and also alters AKT signaling. Thus, TrkB receptors modulate specific phases of fear learning and amygdalar synaptic plasticity through two main phosphorylation docking sites.

PLCγ-activated signalling is essential for TrkB mediated sensory neuron structural plasticity.

BACKGROUND: The vestibular system provides the primary input of our sense of balance and spatial orientation. Dysfunction of the vestibular system can severely affect a person's quality of life. Therefore, understanding the molecular basis of vestibular neuron survival, maintenance, and innervation of the target sensory epithelia is fundamental. RESULTS: Here we report that a point mutation at the phospholipase Cγ (PLCγ) docking site in the mouse neurotrophin tyrosine kinase receptor TrkB (Ntrk2) specifically impairs fiber guidance inside the vestibular sensory epithelia, but has limited effects on the survival of vestibular sensory neurons and growth of afferent processes toward the sensory epithelia. We also show that expression of the TRPC3 cation calcium channel, whose activity is known to be required for nerve-growth cone guidance induced by brain-derived neurotrophic factor (BDNF), is altered in these animals. In addition, we find that absence of the PLCγ mediated TrkB signalling interferes with the transformation of bouton type afferent terminals of vestibular dendrites into calyces (the largest synaptic contact of dendrites known in the mammalian nervous system) on type I vestibular hair cells; the latter are normally distributed in these mutants as revealed by an unaltered expression pattern of the potassium channel KCNQ4 in these cells. CONCLUSIONS: These results demonstrate a crucial involvement of the TrkB/PLCγ-mediated intracellular signalling in structural aspects of sensory neuron plasticity.

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.

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.