The pathological hallmark of Parkinson's disease (PD) is a selective and progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). In the vast majority of cases the appearance of PD is sporadic, and its etiology remains unknown. Several postmortem studies demonstrate reduced levels of brain-derived neurotrophic factor (BDNF) in the SNc of PD patients. Application of BDNF promotes the survival of DA neurons in PD animal models. Here we show that BDNF signaling via its TrkB receptor tyrosine kinase is important for survival of nigrostriatal DA neurons in aging brains. Immunohistochemistry revealed that the TrkB receptor was expressed in DA neurons located in the SNc and ventral tegmental area (VTA). However, a significant loss of DA neurons occurred at 12-24 months of age only in the SNc but not in the VTA of TrkB hypomorphic mice in which the TrkB receptor was expressed at a quarter to a third of the normal amount. The neuronal loss was accompanied by a decrease in dopaminergic axonal terminals in the striatum and by gliosis in both the SNc and striatum. Furthermore, nigrostriatal DA neurons in the TrkB mutant mice were hypersensitive to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial complex I inhibitor that selectively kills DA neurons. These results suggest that BDNF-to-TrkB signaling plays an important role in the long-term maintenance of the nigrostriatal system and that its deficiency may contribute to the progression of PD.

Caspase-2 is the most evolutionarily conserved member in the caspase family of proteases and is constitutively expressed in most cell types including neurons; however, its physiological function remains largely unknown. Here we report that caspase-2 plays a critical role in synaptic plasticity and cognitive flexibility. We found that caspase-2 deficiency led to deficits in dendritic spine pruning, internalization of AMPA receptors and long-term depression. Our results indicate that caspase-2 degrades Rictor, a key mTOR complex 2 (mTORC2) component, to inhibit Akt activation, which leads to enhancement of the GSK3β activity and thereby long-term depression. Furthermore, we found that mice lacking caspase-2 displayed elevated levels of anxiety, impairment in reversal water maze learning, and little memory loss over time. These results not only uncover a caspase-2-mTORC2-Akt-GSK3β signaling pathway, but also suggest that caspase-2 is important for memory erasing and normal behaviors by regulating synaptic number and transmission.

Huntington's disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion within exon1 of the HTT gene. The gene generates two mRNA variants that carry either a short or long 3' untranslated region (3'UTR) while encoding the same protein. It remains unknown whether the two mRNA variants play distinct roles in HD pathogenesis. We found that the long HTT 3'UTR was capable of guiding mRNA to neuronal dendrites, suggesting that some long-form HTT mRNA is transported to dendrites for local protein synthesis. To assay roles of two HTT mRNA variants in cell bodies, we expressed mRNA harboring HTT exon1 containing 23x or 145x CAGs with the short or long 3'UTR. We found that mutant mRNA containing the short 3'UTR produced more protein aggregates and caused more apoptosis in both cultured neurons and HEK293 cells, compared with mutant mRNA containing the long 3'UTR. Although the two 3'UTRs did not affect mRNA stability, we detected higher levels of protein synthesis from mRNA containing the short 3'UTR than from mRNA containing the long 3'UTR. These results indicate that the long HTT 3'UTR suppresses translation. Thus, short-form mutant HTT mRNA will be more efficient in producing toxic protein than its long-form counterpart.

Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase B (TrkB), are implicit in causing obesity. Mutations that reduce BDNF and TrkB expression are associated with obesity in humans and mice. Recently, it was reported that Bdnf gene deletion in the neurons of the paraventricular hypothalamus (PVH) caused positive energy balance and severe obesity in the form of hyperphagia, impaired adaptive thermogenesis, and decreased energy expenditure. Thus, we hypothesize that activation of these neurons will have the opposite effect and provide an opportunity for long-lasting obesity treatment. To specifically activate BDNF-expressing PVH (PVHBDNF) neurons, we injected Cre-dependent adeno-associated virus (AAV) expressing the excitatory DREADD hM3Dq bilaterally into the PVH of Bdnf2A-Cre/+ knock-in mice and then administered clozapine-N-oxide (CNO). Using this technique, we demonstrated that acute activation of these neurons rapidly decreased normal nocturnal feeding and fasting-induced feeding in male and female mice. At thermoneutral temperatures, acute activation also rapidly increased adaptive thermogenesis, increased core body temperature, increased locomotion, increased energy expenditure, and decreased respiratory exchange ratio (RER) in male and female mice. These observations indicate that acute stimulation of PVHBDNF neurons promotes negative energy balance and weight loss. However, the rapid decrease in RER after activation of PVHBDNF neurons was followed by a delayed and prolonged increase in RER that remained elevated for 3 d in female mice. Thus, although acute activation of PVHBDNF neurons promotes negative energy balance in the short term, long-term effects of activation include sexually dimorphic overcompensatory mechanisms that may promote positive energy balance in female mice.

In the developing hypothalamus, the fat-derived hormone leptin stimulates the growth of axons from the arcuate nucleus of the hypothalamus (ARH) to other regions that control energy balance. These projections are significantly reduced in leptin deficient (Lepob/ob ) mice and this phenotype is largely rescued by neonatal leptin treatments. However, treatment of mature Lepob/ob mice is ineffective, suggesting that the trophic action of leptin is limited to a developmental critical period. To temporally delineate closure of this critical period for leptin-stimulated growth, we treated Lepob/ob mice with exogenous leptin during a variety of discrete time periods, and measured the density of Agouti-Related Peptide (AgRP) containing projections from the ARH to the ventral part of the dorsomedial nucleus of the hypothalamus (DMHv), and to the medial parvocellular part of the paraventricular nucleus (PVHmp). The results indicate that leptin loses its neurotrophic potential at or near postnatal day 28. The duration of leptin exposure appears to be important, with 9- or 11-day treatments found to be more effective than shorter (5-day) treatments. Furthermore, leptin treatment for 9 days or more was sufficient to restore AgRP innervation to both the PVHmp and DMHv in Lepob/ob females, but only to the DMHv in Lepob/ob males. Together, these findings reveal that the trophic actions of leptin are contingent upon timing and duration of leptin exposure, display both target and sex specificity, and that modulation of leptin-dependent circuit formation by each of these factors may carry enduring consequences for feeding behavior, metabolism, and obesity risk.

Mutations in the Bdnf gene, which produces transcripts with either short or long 3' untranslated regions (3' UTRs), cause human obesity; however, the precise role of brain-derived neurotrophic factor (BDNF) in the regulation of energy balance is unknown. Here we show the relationship between Bdnf mRNA with a long 3' UTR (long 3' UTR Bdnf mRNA), leptin, neuronal activation and body weight. We found that long 3' UTR Bdnf mRNA was enriched in the dendrites of hypothalamic neurons and that insulin and leptin could stimulate its translation in dendrites. Furthermore, mice harboring a truncated long Bdnf 3' UTR developed severe hyperphagic obesity, which was completely reversed by viral expression of long 3' UTR Bdnf mRNA in the hypothalamus. In these mice, the ability of leptin to activate hypothalamic neurons and inhibit food intake was compromised despite normal activation of leptin receptors. These results reveal a novel mechanism linking leptin action to BDNF expression during hypothalamic-mediated regulation of body weight, while also implicating dendritic protein synthesis in this process.

Obesity and type 2 diabetes (T2D) lead to various life-threatening diseases such as coronary heart disease, stroke, osteoarthritis, asthma, and neurodegeneration. Therefore, extensive research is ongoing to identify novel pathways that can be targeted in obesity/T2D. Deletion of the inositol pyrophosphate (5-IP7) biosynthetic enzyme, inositol hexakisphosphate kinase-1 (IP6K1), protects mice from high fat diet (HFD) induced obesity (DIO) and insulin resistance. Yet, whether this pathway is a valid pharmacologic target in obesity/T2D is not known. Here, we demonstrate that TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine], a pan-IP6K inhibitor, has strong anti-obesity and anti-diabetic effects in DIO mice.

High prevalence of obesity is attributable in part to consumption of highly palatable, fat-rich foods. However, the mechanism controlling dietary fat intake is largely unknown. In this study we investigated the role of brain-derived neurotrophic factor (BDNF) in the control of dietary fat intake in a mouse model that mimics the common human Val-to-Met (Val66Met) polymorphism that impairs BDNF release via the regulated secretory pathway. BdnfMet/Met mice gained weight much faster than wild-type (WT) mice and developed severe obesity due to marked hyperphagia when they were fed HFD. Hyperphagia in these mice worsened when the fat content in their diet was increased. Conversely, mice lacking leptin exhibited similar hyperphagia on chow and HFD. When 2 diets were provided simultaneously, WT and BdnfMet/Met mice showed a comparable preference for the more palatable diet rich in either fat or sucrose, indicating that increased hyperphagia on fat-rich diets in BdnfMet/Met mice is not due to enhanced hedonic drive. In support of this interpretation, WT and BdnfMet/Met mice increased calorie intake to a similar extent during the first day after chow was switched to HFD; however, WT mice decreased HFD intake faster than BdnfMet/Met mice in subsequent days. Furthermore, we found that refeeding after fasting or nocturnal feeding with HFD activated TrkB more strongly than with chow in the hypothalamus of WT mice, whereas TrkB activation under these 2 conditions was greatly attenuated in BdnfMet/Met mice. These results indicate that satiety factors generated during HFD feeding induce BDNF release to suppress excess dietary fat intake.

Highly specific activity-dependent neuronal responses are necessary for modulating synapses to facilitate learning and memory. We present evidence linking a number of important processes involved in regulating synaptic plasticity, suggesting a mechanistic pathway whereby activity-dependent signaling, likely through protein kinase C (PKC)-mediated phosphorylation of HuD, can relieve basal repression of Bdnf mRNA translation in dendrites, allowing for increased TrkB signaling and synaptic remodeling. We demonstrate that the neuronal ELAV family of RNA binding proteins associates in vivo with several Bdnf mRNA isoforms present in the adult brain in an activity-dependent manner, and that one member, HuD, interacts directly with sequences in the long Bdnf 3' untranslated region (3'UTR) and co-localizes with Bdnf mRNA in dendrites of hippocampal neurons. Activation of PKC leads to increased dendritic translation of mRNAs containing the long Bdnf 3'UTR, a process that is dependent on the presence of HuD and its phosphorylation at threonine residues 149 and/or 165. Thus, we found a direct effect of HuD on regulating translation of dendritic Bdnf mRNAs to mediate local and activity-dependent increases in dendritic BDNF synthesis.

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.

Neurotrophins at axonal terminals signal to cell bodies to regulate neuronal development via signaling endosomes containing activated Trk receptor tyrosine kinases and mitogen-activated protein kinases (MAPKs). Requirements for the formation of signaling endosomes remain, however, poorly characterized. Here we show that a novel Trk-interacting protein, NTRAP (neurotrophic factor receptor-associated protein), plays a crucial role in this signaling process. NTRAP interacts with the Trk intracellular domain through its C(2)H(2) zinc fingers in a kinase-dependent manner. It is associated with vesicles, some of which contain markers for signaling endosomes. Inhibition of NTRAP function suppresses neurotrophin-induced neurite outgrowth in PC12 cells by altering TrkA endocytic traffic, inhibiting the formation of endosomes containing persistently active MAPKs. In compartmentalized sensory neuron cultures, down-regulation of NTRAP abolishes the ability of neurotrophins applied to distal axons to activate the transcription factor adenosine 3',5'-monophosphate response element-binding protein (CREB) and to promote neuronal survival. We propose that NTRAP regulates retrograde neurotrophic signaling by controlling the formation of signaling endosomes.

Neurotrophins (NTs), once believed to be neural-specific trophic factors, are now known to also provide developmental cues to non-neural cells. In the ovary, NTs contribute to both the formation and development of follicles. Here we show that oocyte-specific deletion of the Ntrk2 gene that encodes the NTRK2 receptor (NTRK2) for neurotrophin-4/5 and brain-derived neurotrophic factor (BDNF) results in post-pubertal oocyte death, loss of follicular organization, and early adulthood infertility. Oocytes lacking NTRK2 do not respond to gonadotropins with activation of phosphatidylinositol 3-kinase (PI3K)-AKT-mediated signaling. Before puberty, oocytes only express a truncated NTRK2 form (NTRK2.T1), but at puberty full-length (NTRK2.FL) receptors are rapidly induced by the preovulatory gonadotropin surge. A cell line expressing both NTRK2.T1 and the kisspeptin receptor (KISS1R) responds to BDNF stimulation with activation of Ntrk2 expression only if kisspeptin is present. This suggests that BDNF and kisspeptin that are produced by granulosa cells (GCs) of periovulatory follicles act in concert to mediate the effect of gonadotropins on Ntrk2 expression in oocytes. In keeping with this finding, the oocytes of NTRK2-intact mice fail to respond to gonadotropins with increased Ntrk2 expression in the absence of KISS1R. Our results demonstrate that the preovulatory gonadotropin surge promotes oocyte survival at the onset of reproductive cyclicity by inducing oocyte expression of NTRK2.FL receptors that set in motion an AKT-mediated survival pathway. They also suggest that gonadotropins activate NTRK2.FL expression via a dual communication pathway involving BDNF and kisspeptin produced in GCs and their respective receptors NTRK2.T1 and KISS1R expressed in oocytes.

In the striatum, signaling via G protein-coupled neurotransmitter receptors is essential for motor control. Critical to this process is the effector enzyme adenylyl cyclase type 5 (AC5) that produces second messenger cAMP upon receptor-mediated activation by G protein Golf. However, the molecular organization of the Golf-AC5 signaling axis is not well understood. In this study, we report that in the striatum AC5 exists in a stable pre-coupled complex with subunits of Golf heterotrimer. We use genetic mouse models with disruption in individual components of the complex to reveal hierarchical order of interactions required for AC5-Golf stability. We further identify that the assembly of AC5-Golf complex is mediated by PhLP1 chaperone that plays central role in neurotransmitter receptor coupling to cAMP production motor learning. These findings provide evidence for the existence of stable G protein-effector signaling complexes and identify a new component essential for their assembly.

The ventromedial hypothalamic nucleus (VMN) is known to mediate autonomic responses in feeding and reproductive behaviors. To date, the most definitive molecular marker for the VMN is the orphan nuclear receptor steroidogenic factor-1 (SF-1). However, it is unclear whether SF-1 functions in the VMN as it does in peripheral endocrine organ development where loss of SF-1 results in organ agenesis due to apoptosis. Here, we provide evidence that SF-1 has a distinct role in later stages of VMN development by demonstrating the persistence of VMN precursors, the misexpression of an early marker (NKX2-1) concomitant with the absence of a late marker (BDNF neurotrophin), and the complete loss of projections to the bed nucleus of stria terminalis and the amygdala in sf-1 null mice. Our findings demonstrate that SF-1 is required for terminal differentiation of the VMN and suggest that transcriptional targets of SF-1 mediate normal circuitry between the hypothalamus and limbic structures in the telencephalon.

Thalamocortical input to layer 4 carries the major ascending sensory information to the mammalian sensory cortex and is crucial for the function and plasticity of sensory cortical areas. Here we report identification of a Six3-cre transgene that is selectively expressed in layer 4 of sensory cortical areas but not in the thalamus. In the mature somatosensory cortex Cre recombinase expressed from the transgene is able to mediate gene deletion in the overwhelming majority of layer 4 neurons, including GABAergic interneurons. The gene deletion in layer 4 mainly occurs during the first postnatal week. This cre transgene therefore provides a useful tool for examining the role of proteins expressed in layer 4 neurons.

The high affinity tyrosine kinase receptor, TrkB, is the primary receptor for brain derived neurotrophic factor (BDNF) and plays an important role in development, maintenance and plasticity of the striatal output medium size spiny neuron. The striatal BDNF/TrkB system is thereby implicated in many physiologic and pathophysiologic processes, the latter including mood disorders, addiction, and Huntington's disease. We crossed a mouse harboring a transgene directing cre-recombinase expression primarily to postnatal, dorsal striatal medium spiny neurons, to a mouse containing a floxed TrkB allele (fB) mouse designed for deletion of TrkB to determine its role in the adult striatum.

There are two major isoforms of NTRK2 (neurotrophic receptor tyrosine kinase 2, or TrkB), full-length isoform with tyrosine kinase (TK) domain intact (+) and spliced isoform without tyrosine kinase domain (TK(-)). Within each isoform, there exist subtypes with minor modifications of the protein sequences. In human, the NTRK2 mRNA transcripts encoding TK(+) have same 3'UTRs, while the transcripts encoding subtypes of NTRK2 TK(-) have two completely different 3'UTRs. In mouse, the mRNA transcripts encoding same NTRK2 protein sequence for either TK(+) or TK(-) have long or short 3'UTRs, respectively. The physiological functions of these different 3'UTRs are still unknown. Pilocarpine stimulation increased Ntrk2 mRNA levels in soma, while the increase in synaptosome was smaller. FISH results further showed that mouse Ntrk2 transcripts with different 3'UTRs were distributed differently in cultured cortical neurons. The transcripts with long 3'UTR were distributed more in apical dendrites compared with transcripts with short 3'UTR. Our results provide evidence of non-coding 3'UTR function in regulating mRNA distribution in neurons.

Mutations that inactivate negative translation regulators cause autism spectrum disorders (ASD), which predominantly affect males and exhibit social interaction and communication deficits and repetitive behaviors. However, the cells that cause ASD through elevated protein synthesis resulting from these mutations remain unknown. Here we employ conditional overexpression of translation initiation factor eIF4E to increase protein synthesis in specific brain cells. We show that exaggerated translation in microglia, but not neurons or astrocytes, leads to autism-like behaviors in male mice. Although microglial eIF4E overexpression elevates translation in both sexes, it only increases microglial density and size in males, accompanied by microglial shift from homeostatic to a functional state with enhanced phagocytic capacity but reduced motility and synapse engulfment. Consequently, cortical neurons in the mice have higher synapse density, neuroligins, and excitation-to-inhibition ratio compared to control mice. We propose that functional perturbation of male microglia is an important cause for sex-biased ASD.

At the center of the hippocampal tri-synaptic loop are synapses formed between mossy fiber (MF) terminals from granule cells in the dentate gyrus (DG) and proximal dendrites of CA3 pyramidal neurons. However, the molecular mechanism regulating the development and function of these synapses is poorly understood. In this study, we showed that neurotrophin-3 (NT3) was expressed in nearly all mature granule cells but not CA3 cells. We selectively deleted the NT3-encoding Ntf3 gene in the DG during the first two postnatal weeks to generate a Ntf3 conditional knockout (Ntf3-cKO). Ntf3-cKO mice of both sexes had normal hippocampal cytoarchitecture but displayed impairments in contextual memory, spatial reference memory, and nest building. Furthermore, male Ntf3-cKO mice exhibited anxiety-like behaviors, whereas female Ntf3-cKO showed some mild depressive symptoms. As MF-CA3 synapses are essential for encoding of contextual memory, we examined synaptic transmission at these synapses using ex vivo electrophysiological recordings. We found that Ntf3-cKO mice had impaired basal synaptic transmission due to deficits in excitatory postsynaptic currents mediated by AMPA receptors but normal presynaptic function and intrinsic excitability of CA3 pyramidal neurons. Consistent with this selective postsynaptic deficit, Ntf3-cKO mice had fewer and smaller thorny excrescences on proximal apical dendrites of CA3 neurons and lower GluR1 levels in the stratum lucidum area where MF-CA3 synapses reside but normal MF terminals, compared with control mice. Thus, our study indicates that NT3 expressed in the dentate gyrus is crucial for the postsynaptic structure and function of MF-CA3 synapses and hippocampal-dependent memory.

The brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase receptor B (TrkB) are widely expressed in the central nervous system. It is well documented that neurons express BDNF and full-length TrkB (TrkB.FL) as well as a lower level of truncated TrkB (TrkB.T). However, there are conflicting reports regarding the expression of BDNF and TrkB in glial cells, particularly microglia. In this study, we employed a sensitive and reliable genetic method to characterize the expression of BDNF and TrkB in glial cells in the mouse brain. We utilized three Cre mouse strains in which Cre recombinase is expressed in the same cells as BDNF, TrkB.FL, or all TrkB isoforms, and crossed them to Cre-dependent reporter mice to label BDNF- or TrkB-expressing cells with soma-localized EGFP. We performed immunohistochemistry with glial cell markers to examine the expression of BDNF and TrkB in microglia, astrocytes, and oligodendrocytes. Surprisingly, we found no BDNF- or TrkB-expressing microglia in examined CNS regions, including the somatomotor cortex, hippocampal CA1, and spinal cord. Consistent with previous studies, most astrocytes only express TrkB.T in the hippocampus of adult brains. Moreover, there are a small number of astrocytes and oligodendrocytes that express BDNF in the hippocampus, the function of which is to be determined. We also found that oligodendrocyte precursor cells, but not mature oligodendrocytes, express both TrkB.FL and TrkB.T in the hippocampus of adult mice. These results not only clarify the expression of BDNF and TrkB in glial cells but also open opportunities to investigate previously unidentified roles of BDNF and TrkB in astrocytes and oligodendrocytes.