Natural products reveal cancer cell dependence on oxysterol-binding proteins.

Cephalostatin 1, OSW-1, ritterazine B and schweinfurthin A are natural products that potently, and in some cases selectively, inhibit the growth of cultured human cancer cell lines. The cellular targets of these small molecules have yet to be identified. We have discovered that these molecules target oxysterol binding protein (OSBP) and its closest paralog, OSBP-related protein 4L (ORP4L)--proteins not known to be involved in cancer cell survival. OSBP and the ORPs constitute an evolutionarily conserved protein superfamily, members of which have been implicated in signal transduction, lipid transport and lipid metabolism. The functions of OSBP and the ORPs, however, remain largely enigmatic. Based on our findings, we have named the aforementioned natural products ORPphilins. Here we used ORPphilins to reveal new cellular activities of OSBP. The ORPphilins are powerful probes of OSBP and ORP4L that will be useful in uncovering their cellular functions and their roles in human diseases.

Social memory deficit caused by dysregulation of the cerebellar vermis.

Social recognition memory (SRM) is a key determinant of social interactions. While the cerebellum emerges as an important region for social behavior, how cerebellar activity affects social functions remains unclear. We selectively increased the excitability of molecular layer interneurons (MLIs) to suppress Purkinje cell firing in the mouse cerebellar vermis. Chemogenetic perturbation of MLIs impaired SRM without affecting sociability, anxiety levels, motor coordination or object recognition. Optogenetic interference of MLIs during distinct phases of a social recognition test revealed the cerebellar engagement in the retrieval, but not encoding, of social information. c-Fos mapping after the social recognition test showed that cerebellar manipulation decreased brain-wide interregional correlations and altered network structure from medial prefrontal cortex and hippocampus-centered to amygdala-centered modules. Anatomical tracing demonstrated hierarchical projections from the central cerebellum to the social brain network integrating amygdalar connections. Our findings suggest that the cerebellum organizes the neural matrix necessary for SRM.

Graded Control of Climbing-Fiber-Mediated Plasticity and Learning by Inhibition in the Cerebellum.

Purkinje cell dendrites convert excitatory climbing fiber input into signals that instruct plasticity and motor learning. Modulation of instructive signaling may increase the range in which learning is encoded, yet the mechanisms that allow for this are poorly understood. We found that optogenetic activation of molecular layer interneurons (MLIs) that inhibit Purkinje cells suppressed climbing-fiber-evoked dendritic Ca2+ spiking. Inhibitory suppression of Ca2+ spiking depended on the level of MLI activation and influenced the induction of associative synaptic plasticity, converting climbing-fiber-mediated potentiation of parallel fiber-evoked responses into depression. In awake mice, optogenetic activation of floccular climbing fibers in association with head rotation produced an adaptive increase in the vestibulo-ocular reflex (VOR). However, when climbing fibers were co-activated with MLIs, adaptation occurred in the opposite direction, decreasing the VOR. Thus, MLIs can direct a continuous spectrum of plasticity and learning through their influence on Purkinje cell dendritic Ca2+ signaling.

Using c-kit to genetically target cerebellar molecular layer interneurons in adult mice.

The cerebellar system helps modulate and fine-tune motor action. Purkinje cells (PCs) provide the sole output of the cerebellar cortex, therefore, any cerebellar involvement in motor activity must be driven by changes in PC firing rates. Several different cell types influence PC activity including excitatory input from parallel fibers and inhibition from molecular layer interneurons (MLIs). Similar to PCs, MLI activity is driven by parallel fibers, therefore, MLIs provide feed-forward inhibition onto PCs. To aid in the experimental assessment of how molecular layer inhibition contributes to cerebellar function and motor behavior, we characterized a new knock-in mouse line with Cre recombinase expression under control of endogenous c-kit transcriptional machinery. Using these engineered c-Kit mice, we were able to obtain high levels of conditional MLI transduction in adult mice using Cre-dependent viral vectors without any PC or granule cell labeling. We then used the mouse line to target MLIs for activity perturbation in vitro using opto- and chemogenetics.

Scaffolding function of PAK in the PDK1-Akt pathway.

Many extracellular signals stimulate phosphatidylinositol-3-kinase, which in turn activates the Rac1 GTPase, the protein kinase Akt and the Akt Thr 308 upstream kinase PDK1. Active Rac1 stimulates a number of events, including substrate phosphorylation by a subgroup of the PAK family of kinases. The combined effects of Rac1, PDK1 and Akt are crucial for cell migration, growth, survival, metabolism and tumorigenesis. Here we show that Rac1 stimulates a second, kinase-independent function of PAK1. The PAK1 kinase domain serves as a scaffold to facilitate Akt stimulation by PDK1 and to aid recruitment of Akt to the membrane. PAK differentially activates subpopulations of Akt. These findings reveal scaffolding functions of PAK that regulate the efficiency, localization and specificity of the PDK1-Akt pathway.