Leptin stimulates synaptogenesis in hippocampal neurons via KLF4 and SOCS3 inhibition of STAT3 signaling.

Normal development of neuronal connections in the hippocampus requires neurotrophic signals, including the cytokine leptin. During neonatal development, leptin induces formation and maturation of dendritic spines, the main sites of glutamatergic synapses in the hippocampal neurons. However, the molecular mechanisms for leptin-induced synaptogenesis are not entirely understood. In this study, we reveal two novel targets of leptin in developing hippocampal neurons and address their role in synaptogenesis. First target is Kruppel-Like Factor 4 (KLF4), which we identified using a genome-wide target analysis strategy. We show that leptin upregulates KLF4 in hippocampal neurons and that leptin signaling is important for KLF4 expression in vivo. Furthermore, KLF4 is required for leptin-induced synaptogenesis, as shKLF4 blocks and upregulation of KLF4 phenocopies it. We go on to show that KLF4 requires its signal transducer and activator of transcription 3 (STAT3) binding site and thus potentially blocks STAT3 activity to induce synaptogenesis. Second, we show that leptin increases the expression of suppressor of cytokine signaling 3 (SOCS3), another well-known inhibitor of STAT3, in developing hippocampal neurons. SOCS3 is also required for leptin-induced synaptogenesis and sufficient to stimulate it alone. Finally, we show that constitutively active STAT3 blocks the effects of leptin on spine formation, while the targeted knockdown of STAT3 is sufficient to induce it. Overall, our data demonstrate that leptin increases the expression of both KLF4 and SOCS3, inhibiting the activity of STAT3 in the hippocampal neurons and resulting in the enhancement of glutamatergic synaptogenesis during neonatal development.

Leptin-induced spine formation requires TrpC channels and the CaM kinase cascade in the hippocampus.

Leptin is a critical neurotrophic factor for the development of neuronal pathways and synaptogenesis in the hypothalamus. Leptin receptors are also found in other brain regions, including the hippocampus, and a postnatal surge in leptin correlates with a time of rapid growth of dendritic spines and synapses in the hippocampus. Leptin is critical for normal hippocampal dendritic spine formation as db/db mice, which lack normal leptin receptor signaling, have a reduced number of dendritic spines in vivo. Leptin also positively influences hippocampal behaviors, such as cognition, anxiety, and depression, which are critically dependent on dendritic spine number. What is not known are the signaling mechanisms by which leptin initiates spine formation. Here we show leptin induces the formation of dendritic protrusions (thin headless, stubby and mushroom shaped spines), through trafficking and activation of TrpC channels in cultured hippocampal neurons. Leptin-activation of the TrpC current is dose dependent and blocked by targeted knockdown of the leptin receptor. The nonselective TrpC channel inhibitors SKF96365 and 2-APB or targeted knockdown of TrpC1 or 3, but not TrpC5, channels also eliminate the leptin-induced current. Leptin stimulates the phosphorylation of CaMKIγ and ß-Pix within 5 min and their activation is required for leptin-induced trafficking of TrpC1 subunits to the membrane. Furthermore, we show that CaMKIγ, CaMKK, ß-Pix, Rac1, and TrpC1/3 channels are all required for both the leptin-sensitive current and leptin-induced spine formation. These results elucidate a critical pathway underlying leptin's induction of dendritic morphological changes that initiate spine and excitatory synapse formation.

Using FPbase: The Fluorescent Protein Database.

FPbase is a database of fluorescent proteins and their characteristics and a set of online tools that facilitate searching the database and performing experiments with fluorescent probes. This chapter serves as a general reference for using and searching the database and a guide to some of the more commonly used tools including the spectra viewer, custom microscope pages, and FRET calculator. Important caveats when evaluating the data are also discussed.

The Troyer syndrome protein spartin mediates selective autophagy of lipid droplets.

Lipid droplets (LDs) are crucial organelles for energy storage and lipid homeostasis. Autophagy of LDs is an important pathway for their catabolism, but the molecular mechanisms mediating LD degradation by selective autophagy (lipophagy) are unknown. Here we identify spartin as a receptor localizing to LDs and interacting with core autophagy machinery, and we show that spartin is required to deliver LDs to lysosomes for triglyceride mobilization. Mutations in SPART (encoding spartin) lead to Troyer syndrome, a form of complex hereditary spastic paraplegia1. Interfering with spartin function in cultured human neurons or murine brain neurons leads to LD and triglyceride accumulation. Our identification of spartin as a lipophagy receptor, thus, suggests that impaired LD turnover contributes to Troyer syndrome development.

Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo.

Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its expression in mouse brain was monitored by quantitative RT-PCR (RT-qPCR). Pilocarpine-induced seizures led to a robust, rapid, and transient increase in the primary transcript of miR-132 (pri-miR-132) followed by a subsequent rise in mature microRNA (miR-132). Activation of neurons in the hippocampus, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h of stimulation. Expression levels of primary and mature-miR-132 increased significantly between postnatal Days 10 and 24. We conclude that miR-132 is an activity-dependent microRNA in vivo, and may contribute to the long-lasting proteomic changes required for experience-dependent neuronal plasticity.

LDAF1 and Seipin Form a Lipid Droplet Assembly Complex.

Lipid droplets (LDs) originate from the endoplasmic reticulum (ER) to store triacylglycerol (TG) and cholesterol esters. The ER protein seipin was shown to localize to ER-LD contacts soon after LDs form, but what determines the sites of initial LD biogenesis in the ER is unknown. Here, we identify TMEM159, now re-named lipid droplet assembly factor 1 (LDAF1), as an interaction partner of seipin. Together, LDAF1 and seipin form an ∼600 kDa oligomeric complex that copurifies with TG. LDs form at LDAF1-seipin complexes, and re-localization of LDAF1 to the plasma membrane co-recruits seipin and redirects LD formation to these sites. Once LDs form, LDAF1 dissociates from seipin and moves to the LD surface. In the absence of LDAF1, LDs form only at significantly higher cellular TG concentrations. Our data suggest that the LDAF1-seipin complex is the core protein machinery that facilitates LD biogenesis and determines the sites of their formation in the ER.

Age-dependent effects of environmental enrichment on spatial reference memory in male mice.

Although environmental enrichment has been shown to improve various types of memory in young and aging mice, no study has directly compared the degree to which enrichment improves memory at different ages throughout the lifespan in male mice. Therefore, the present study investigated the effects of long-term continuous enrichment in young (3 months), middle-aged (15 months), and aged (21 months) male C57BL/6 mice. Spatial reference memory was tested in the Morris water maze. Results demonstrate that 24h/day environmental enrichment for approximately 6 weeks significantly improved spatial memory in the Morris water maze in aged males, but not in young or middle-aged males. These data also indicate that 24h exposure to complex enriched housing conditions increases the magnitude of enrichment-induced improvements in memory among aged mice relative to those previously reported by this lab and others.

Navigating challenges in the application of superresolution microscopy.

In 2014, the Nobel Prize in Chemistry was awarded to three scientists who have made groundbreaking contributions to the field of superresolution (SR) microscopy (SRM). The first commercial SR microscope came to market a decade earlier, and many other commercial options have followed. As commercialization has lowered the barrier to using SRM and the awarding of the Nobel Prize has drawn attention to these methods, biologists have begun adopting SRM to address a wide range of questions in many types of specimens. There is no shortage of reviews on the fundamental principles of SRM and the remarkable achievements made with these methods. We approach SRM from another direction: we focus on the current practical limitations and compromises that must be made when designing an SRM experiment. We provide information and resources to help biologists navigate through common pitfalls in SRM specimen preparation and optimization of image acquisition as well as errors and artifacts that may compromise the reproducibility of SRM data.

Superresolution microscopy of the ß-carboxysome reveals a homogeneous matrix.

Carbon fixation in cyanobacteria makes a major contribution to the global carbon cycle. The cyanobacterial carboxysome is a proteinaceous microcompartment that protects and concentrates the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a paracrystalline lattice, making it possible for these organisms to fix CO2 from the atmosphere. The protein responsible for the organization of this lattice in beta-type carboxysomes of the freshwater cyanobacterium Synechococcus elongatus, CcmM, occurs in two isoforms thought to localize differentially within the carboxysome matrix. Here we use wide-field time-lapse and three-dimensional structured illumination microscopy (3D-SIM) to study the recruitment and localization of these two isoforms. We demonstrate that this superresolution technique is capable of distinguishing the localizations of the outer protein shell of the carboxysome and its internal cargo. We develop an automated analysis pipeline to analyze and quantify 3D-SIM images and generate a population-level description of the carboxysome shell protein, RuBisCO, and CcmM isoform localization. We find that both CcmM isoforms have similar spatial and temporal localization, prompting a revised model of the internal arrangement of the ß-carboxysome.

Polo-like kinase-dependent phosphorylation of the synaptonemal complex protein SYP-4 regulates double-strand break formation through a negative feedback loop.

The synaptonemal complex (SC) is an ultrastructurally conserved proteinaceous structure that holds homologous chromosomes together and is required for the stabilization of pairing interactions and the completion of crossover (CO) formation between homologs during meiosis I. Here, we identify a novel role for a central region component of the SC, SYP-4, in negatively regulating formation of recombination-initiating double-strand breaks (DSBs) via a feedback loop triggered by crossover designation in C. elegans. We found that SYP-4 is phosphorylated dependent on Polo-like kinases PLK-1/2. SYP-4 phosphorylation depends on DSB formation and crossover designation, is required for stabilizing the SC in pachytene by switching the central region of the SC from a more dynamic to a less dynamic state, and negatively regulates DSB formation. We propose a model in which Polo-like kinases recognize crossover designation and phosphorylate SYP-4 thereby stabilizing the SC and making chromosomes less permissive for further DSB formation.

Multifocus structured illumination microscopy for fast volumetric super-resolution imaging.

We here report for the first time the synergistic implementation of structured illumination microscopy (SIM) and multifocus microscopy (MFM). This imaging modality is designed to alleviate the problem of insufficient volumetric acquisition speed in super-resolution biological imaging. SIM is a wide-field super-resolution technique that allows imaging with visible light beyond the classical diffraction limit. Employing multifocus diffractive optics we obtain simultaneous wide-field 3D imaging capability in the SIM acquisition sequence, improving volumetric acquisition speed by an order of magnitude. Imaging performance is demonstrated on biological specimens.

Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration.

Integrin αß heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding ß-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, µm-scale measurements.

Assessing camera performance for quantitative microscopy.

Charge-coupled device and, increasingly, scientific complementary metal oxide semiconductor cameras are the most common digital detectors used for quantitative microscopy applications. Manufacturers provide technical specification data on the average or expected performance characteristics for each model of camera. However, the performance of individual cameras may vary, and many of the characteristics that are important for quantitation can be easily measured. Though it may seem obvious, it is important to remember that the digitized image you collect is merely a representation of the sample itself--and no camera can capture a perfect representation of an optical image. A clear understanding and characterization of the sources of noise and imprecision in your camera are important for rigorous quantitative analysis of digital images. In this chapter, we review the camera performance characteristics that are most critical for generating accurate and precise quantitative data and provide a step-by-step protocol for measuring these characteristics in your camera.

Leptin induces hippocampal synaptogenesis via CREB-regulated microRNA-132 suppression of p250GAP.

Leptin acts in the hippocampus to enhance cognition and reduce depression and anxiety. Cognitive and emotional disorders are associated with abnormal hippocampal dendritic spine formation and synaptogenesis. Although leptin has been shown to induce synaptogenesis in the hypothalamus, its effects on hippocampal synaptogenesis and the mechanism(s) involved are not well understood. Here we show that leptin receptors (LepRs) are critical for hippocampal dendritic spine formation in vivo because db/db mice lacking the long form of the leptin receptor (LepRb) have reduced spine density on CA1 and CA3 neurons. Leptin promotes the formation of mature spines and functional glutamate synapses on hippocampal pyramidal neurons in both dissociated and slice cultures. These effects are blocked by short hairpin RNAs specifically targeting the LepRb and are absent in cultures from db/db mice. Activation of the LepR leads to cAMP response element-binding protein (CREB) phosphorylation and initiation of CREB-dependent transcription via the MAPK kinase/Erk pathway. Furthermore, both Mek/Erk and CREB activation are required for leptin-induced synaptogenesis. Leptin also increases expression of microRNA-132 (miR132), a well-known CREB target, which is also required for leptin-induced synaptogenesis. Last, leptin suppresses the expression of p250GAP, a miR132 target, and this suppression is obligatory for leptin's effects as is the downstream target of p250GAP, Rac1. LepRs appear to be critical in vivo as db/db mice have lowered hippocampal miR132 levels and elevated p250GAP expression. In conclusion, we identify a novel signaling pathway by which leptin increases synaptogenesis through inducing CREB transcription and increasing microRNA-mediated suppression of p250GAP activity, thus removing a known inhibitor of Rac1-stimulated synaptogenesis.

A genome-wide screen of CREB occupancy identifies the RhoA inhibitors Par6C and Rnd3 as regulators of BDNF-induced synaptogenesis.

Neurotrophin-regulated gene expression is believed to play a key role in long-term changes in synaptic structure and the formation of dendritic spines. Brain-derived neurotrophic factor (BDNF) has been shown to induce increases in dendritic spine formation, and this process is thought to function in part by stimulating CREB-dependent transcriptional changes. To identify CREB-regulated genes linked to BDNF-induced synaptogenesis, we profiled transcriptional occupancy of CREB in hippocampal neurons. Interestingly, de novo motif analysis of hippocampal ChIP-Seq data identified a non-canonical CRE motif (TGGCG) that was enriched at CREB target regions and conferred CREB-responsiveness. Because cytoskeletal remodeling is an essential element of the formation of dendritic spines, within our screens we focused our attention on genes previously identified as inhibitors of RhoA GTPase. Bioinformatic analyses identified dozens of candidate CREB target genes known to regulate synaptic architecture and function. We showed that two of these, the RhoA inhibitors Par6C (Pard6A) and Rnd3 (RhoE), are BDNF-induced CREB-regulated genes. Interestingly, CREB occupied a cluster of non-canonical CRE motifs in the Rnd3 promoter region. Lastly, we show that BDNF-stimulated synaptogenesis requires the expression of Par6C and Rnd3, and that overexpression of either protein is sufficient to increase synaptogenesis. Thus, we propose that BDNF can regulate formation of functional synapses by increasing the expression of the RhoA inhibitors, Par6C and Rnd3. This study shows that genome-wide analyses of CREB target genes can facilitate the discovery of new regulators of synaptogenesis.

MicroRNA132 modulates short-term synaptic plasticity but not basal release probability in hippocampal neurons.

MicroRNAs play important regulatory roles in a broad range of cellular processes including neuronal morphology and long-term synaptic plasticity. MicroRNA-132 (miR132) is a CREB-regulated miRNA that is induced by neuronal activity and neurotrophins, and plays a role in regulating neuronal morphology and cellular excitability. Little is known about the effects of miR132 expression on synaptic function. Here we show that overexpression of miR132 increases the paired-pulse ratio and decreases synaptic depression in cultured mouse hippocampal neurons without affecting the initial probability of neurotransmitter release, the calcium sensitivity of release, the amplitude of excitatory postsynaptic currents or the size of the readily releasable pool of synaptic vesicles. These findings are the first to demonstrate that microRNAs can regulate short-term plasticity in neurons.

Amyloid precursor protein overexpression depresses excitatory transmission through both presynaptic and postsynaptic mechanisms.

Overexpression of the amyloid precursor protein (APP) in hippocampal neurons leads to elevated beta-amyloid peptide (Abeta) production and consequent depression of excitatory transmission. The precise mechanisms underlying APP-induced synaptic depression are poorly understood. Uncovering these mechanisms could provide insight into how neuronal function is compromised before cell death during the early stages of Alzheimer's disease. Here we verify that APP up-regulation leads to depression of transmission in cultured hippocampal autapses; and we perform whole-cell recording, FM imaging, and immunocytochemistry to identify the specific mechanisms accounting for this depression. We find that APP overexpression leads to postsynaptic silencing through a selective reduction of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated currents. This effect is likely mediated by Abeta because expression of mutant APP incapable of producing Abeta did not depress transmission. In addition, although we eliminate presynaptic silencing as a mechanism underlying APP-mediated inhibition of transmission, we did observe an Abeta-induced presynaptic deficit in vesicle recycling with sustained stimulation. These findings demonstrate that APP elevation disrupts both presynaptic and postsynaptic compartments.

Different types of environmental enrichment have discrepant effects on spatial memory and synaptophysin levels in female mice.

Environmental enrichment paradigms that incorporate cognitive stimulation, exercise, and motor learning benefit memory and synaptic plasticity across the rodent lifespan. However, the contribution each individual element of the enriched environment makes to enhancing memory and synaptic plasticity has yet to be delineated. Therefore, the current study tested the effects of three of these elements on memory and synaptic protein levels. Young female C57BL/6 mice were given 3h of daily exposure to either rodent toys (cognitive stimulation) or running wheels (exercise), or daily acrobatic training for 6 weeks prior to and throughout behavioral testing. Controls were group housed, but did not receive enrichment. Spatial working and reference memory were tested in a water-escape motivated radial arm maze. Levels of the presynaptic protein synaptophysin were then measured in frontoparietal cortex, hippocampus, striatum, and cerebellum. Exercise, but not cognitive stimulation or acrobat training, improved spatial working memory relative to controls, despite the fact that both exercise and cognitive stimulation increased synaptophysin levels in the neocortex and hippocampus. These data suggest that exercise alone is sufficient to improve working memory, and that enrichment-induced increases in synaptophysin levels may not be sufficient to improve working memory in young females. Spatial reference memory was unaffected by enrichment. Acrobat training had no effect on memory or synaptophysin levels, suggesting a minimal contribution of motor learning to the mnemonic and neuronal benefits of enrichment. These results provide the first evidence that different elements of the enriched environment have markedly distinct effects on spatial memory and synaptic alterations.