James Cooper Sears

Imaging brain learning and memory circuit kinase signaling is a monumental challenge. Theseparation ofphases-basedactivityreporter ofkinase (SPARK) biosensors allow circuit-localized studies of multiple interactive kinasesin vivo, including protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) signaling. In the precisely-mappedDrosophilabrain learning/memory circuit, we find PKA and ERK signaling differentially enriched in distinct Kenyon cell connectivity nodes. We discover that potentiating normal circuit activity induces circuit-localized PKA and ERK signaling, expanding kinase function within new presynaptic and postsynaptic domains. Activity-induced PKA signaling shows extensive overlap with previously selective ERK signaling nodes, while activity-induced ERK signaling arises in new connectivity nodes. We find targeted synaptic transmission blockade in Kenyon cells elevates circuit-localized ERK induction in Kenyon cells with normally high baseline ERK signaling, suggesting lateral and feedback inhibition. We discover overexpression of the pathway-linking Meng-Po (human SBK1) serine/threonine kinase to improve learning acquisition and memory consolidation results in dramatically heightened PKA and ERK signaling in separable Kenyon cell circuit connectivity nodes, revealing both synchronized and untapped signaling potential. Finally, we find that a mechanically-induced epileptic seizure model (easily shocked“bang-sensitive” mutants) has strongly elevated, circuit-localized PKA and ERK signaling. Both sexes were used in all experiments, except for the hemizygous male-only seizure model. Hyperexcitable, learning-enhanced, and epileptic seizure models have comparably elevated interactive kinase signaling, suggesting a common basis of use-dependent induction. We conclude that PKA and ERK signaling modulation is locally coordinated in use-dependent spatial circuit dynamics underlying seizure susceptibility linked to learning/memory potential.Significance StatementCritical protein kinases act in learning/memory circuits to enable experience-dependent plasticity. This work images kinase signalingin vivoto elucidate circuit-level organization relative to mapped connectivity nodes. We discover different kinases exhibit heightened signaling in different circuit domains, which is dramatically reorganized by use-dependent synaptic transmission and circuit activity levels. We discover the newest learning/memory kinase, Meng-Po/SBK1, co-regulates different kinases within distinct circuit connectivity nodes. Targeted over-expression of Meng-Po/SBK1 improves learning and memory, and elevates kinase signaling, indicating untapped kinase signaling potential may enhance capabilities to learn and remember. Linked heightened seizure susceptibility similarly increases kinase signaling in learning/memory circuitry, indicating a trade-off of benefit for instability. This work mechanistically connects hyperexcitation seizure susceptibility and learning/memory via circuit-level kinase signaling.

Dynamic functional connectivity within brain circuits requires coordination of intercellular signaling and intracellular signal transduction. Critical roles for cAMP-dependent Protein Kinase A (PKA) signaling are well established in the Drosophila Mushroom Body (MB) learning and memory circuitry, but local PKA activity within this well-mapped neuronal network is uncharacterized. Here, we use an in vivo PKA activity sensor (PKA-SPARK) to test spatiotemporal regulatory requirements in the MB axon lobes. We find immature animals have little detectable PKA activity, whereas post-critical period adults show high field-selective activation primarily in just 3/16 defined output regions. In addition to the age-dependent PKA activity in distinct α’/β’ lobe nodes, females show sex-dependent elevation compared to males in these same restricted regions. Loss of neural cell body Fragile X Mental Retardation Protein (FMRP) and Rugose (human Neurobeachin) suppresses localized PKA activity, whereas over-expression of MB lobe PKA-synergist Meng-Po (human SBK1) promotes PKA activity. Elevated Meng-Po subverts the PKA age-dependence, with elevated activity in immature animals, and spatial-restriction, with striking γ lobe activity. Testing circuit signaling requirements with temperature-sensitive shibire (human Dynamin) blockade, we find broadly expanded PKA activity within the MB lobes. Using transgenic tetanus toxin to block MB synaptic output, we find greatly heightened PKA activity in virtually all MB lobe fields, although the age-dependence is maintained. We conclude spatiotemporally restricted PKA activity signaling within this well-mapped learning/memory circuit is age-dependent and sex-dependent, driven by FMRP-Rugose pathway activation, temporally promoted by Meng-Po kinase function, and restricted by output neurotransmission providing network feedback. Significance Statement Learning and memory requires the coordination of cell-cell circuit interactions with the appropriate downstream signal transduction. Plasticity regulation via cAMP-dependent PKA activation is among the best-characterized signaling pathways, but until recently it was very challenging to localize PKA signaling within brain circuits. We employ a new PKA biosensor to visualize local signaling in the Drosophila Mushroom Body; a network model consolidating ∼2000 Kenyon Cells into ∼16 output fields per brain hemisphere. We discover heightened PKA activity in just a few circuit regions, with age-dependent and sex-dependent requirements. Learning/memory mutants linked to intellectual and autism spectrum disorders reduce PKA activity. In contrast, PKA-synergist Meng-Po increases PKA activity. Neurotransmission blockade elevates and expands PKA activity, showing network feedback restricts localized signaling.

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of Fragile X Mental Retardation Protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. We focus here on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain Mushroom Body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized Giant Fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the activity-dependent repression of translation. In the well-characterized Drosophila neuromuscular junction (NMJ) model, developmental synaptogenesis and activity-dependent synaptic remodeling both require extracellular matrix metalloproteinase (MMP) enzymes interacting with the heparan sulfate proteoglycan (HSPG) glypican Dally-like Protein (Dlp) to restrict trans-synaptic Wnt signaling, with FXS synaptogenic defects alleviated by both MMP and HSPG reduction. This new mechanistic axis spanning from activity to FMRP to HSPG-dependent MMP regulation modulates activity-dependent synaptogenesis. We discuss future directions for these mechanisms, and intersecting research priorities for FMRP in glial and signaling interactions.

Introduction: The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast tumors compared to normal breast tissue. Cdc42 regulates key processes that are critical for mammary gland morphogenesis and become disrupted during the development, progression, and metastasis of breast cancer. However, the contribution of Cdc42 to normal and neoplastic mammary gland development in vivo remains poorly understood. We were therefore interested in investigating the effects of Cdc42 overexpression on mammary gland morphogenesis as a first step toward understanding how its overexpression may contribute to mammary tumorigenesis. Methods: We developed a tetracycline-regulatable Cdc42 overexpression mouse model in which Cdc42 can be inducibly overexpressed in the developing mammary gland. The effects of Cdc42 overexpression during postnatal mammary gland development were investigated using in vivo and in vitro approaches, including morphometric analysis of wholemounted mammary glands, quantification of histological markers, and primary mammary epithelial cell (MEC) functional and biochemical assays. Results: Analysis of Cdc42-overexpressing mammary glands revealed abnormal terminal end bud (TEB) morphologies, characterized by hyperbudding and trifurcation, and increased side branching within the ductal tree. Quantification of markers of proliferation and apoptosis suggested that these phenotypes were not due to increased cell proliferation or survival. Rather, Cdc42 overexpressing MECs were more migratory and contractile and formed dysmorphic, invasive acini in three-dimensional cultures. Cdc42 and RhoA activities, phosphorylated myosin light chain, and MAPK signaling, which contribute to migration and invasion, were markedly elevated in Cdc42 overexpressing MECs. Interestingly, Cdc42 overexpressing mammary glands displayed several features associated with altered epithelial-stromal interactions, which are known to regulate branching morphogenesis. These included increased stromal thickness and collagen deposition, and stromal cells isolated from Cdc42 overexpressing mammary glands exhibited elevated mRNA expression of extracellular matrix proteins and remodeling enzymes. Conclusions: These data suggest that Cdc42 overexpression disrupts mammary gland branching morphogenesis by altering Rho GTPase and MAPK signaling, leading to increased MEC contractility and migration in association with stromal alterations. Our studies provide insight into how aberrant Cdc42 expression may contribute to mammary tumorigenesis.

Rho GTPases are overexpressed and hyperactivated in many cancers, including breast cancer. Rho proteins, as well as their regulators and effectors, have been implicated in mitosis, and their altered expression promotes mitotic defects and aneuploidy. Previously, we demonstrated that p190B Rho GTPase activating protein (RhoGAP) deficiency inhibits ErbB2-induced mammary tumor formation in mice. Here we describe a novel role for p190B as a regulator of mitosis. We found that p190B localized to centrosomes during interphase and mitosis, and that it is differentially phosphorylated during mitosis. Knockdown of p190B expression in MCF-7 and Hela cells increased the incidence of aberrant microtubule-kinetochore attachments at metaphase, lagging chromosomes at anaphase, and micronucleation, all of which are indicative of aneuploidy. Cell cycle analysis of p190B deficient MCF-7 cells revealed a significant increase in apoptotic cells with a concomitant decrease in cells in G1 and S phase, suggesting that p190B deficient cells die at the G1 to S transition. Chemical inhibition of the Rac GTPase during mitosis reduced the incidence of lagging chromosomes in p190B knockdown cells to levels detected in control cells, suggesting that aberrant Rac activity in the absence of p190B promotes chromosome segregation defects. Taken together, these data suggest that p190B regulates chromosome segregation and apoptosis in cancer cells. We propose that disruption of mitosis may be one mechanism by which p190B deficiency inhibits tumorigenesis.

Introduction: Rho GTPases are overexpressed and hyperactivated in human breast cancers. Deficiency of p190B RhoGAP, a major inhibitor of the Rho GTPases, inhibits mouse mammary tumor virus long terminal repeat (MMTV)-Neu/ErbB2 mammary tumor formation and progression in part through effects within the stromal environment, suggesting that p190B function is pro-tumorigenic. To further investigate the potential pro-tumorigenic actions of p190B, we examined the effects of exogenous p190B expression within the mammary epithelium on MMTV-Neu tumor formation and progression. Methods: Tetracycline (tet)-regulatable p190B transgenic mice were bred to MMTV-Neu mice, and the effects of exogenous p190B expression on tumor latency, multiplicity, growth rates, angiogenesis, and metastasis were examined. The effects of exogenous p190B expression on cell-matrix adhesion and invasion were tested using non-transformed primary mammary epithelial cells (MECs). Rho GTPase activity, oxidative stress as an indicator of reactive oxygen species (ROS) production, and downstream signaling pathways were analyzed. Results: Altered p190B expression resulted in a two-fold increase in tumor multiplicity and a three-fold increase in metastases compared to control mice indicating that exogenous p190B expression in the mammary epithelium promotes MMTV-Neu mammary tumor formation and progression. Interestingly, non-transformed primary MECs expressing exogenous p190B displayed increased adhesion to laminin and type IV collagen and formed invasive structures in a three-dimensional culture assay. Ras related C3 botulinum toxin 1 (Rac1)-GTP levels were elevated in p190B transgenic tumors whereas Ras homologous A (RhoA) and cell division cycle 42 (Cdc42)-GTP levels were not significantly altered. Rac1 activity affects production of ROS, which regulate transformation, metastasis, and oxidative stress. Protein carbonylation, which is indicative of oxidative stress, was elevated 1.75-fold in p190B transgenic tumors as compared to control tumors suggesting that exogenous p190B expression may affect Rac1-dependent ROS production. Conclusions: These studies indicate that paradoxically, p190B RhoGAP, a major inhibitor of the Rho GTPases in vitro, has pro-tumorigenic functions that enhance MMTV-Neu induced mammary tumor formation and metastasis. Furthermore, exogenous p190B expression enhances cell adhesion and invasion, which may facilitate metastasis. Rac1 activity and oxidative stress are elevated in tumors expressing exogenous p190B suggesting that p190B may promote tumorigenesis through a Rac1/ROS dependent mechanism.