Three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT).

Optical methods capable of manipulating neural activity with cellular resolution and millisecond precision in three dimensions will accelerate the pace of neuroscience research. Existing approaches for targeting individual neurons, however, fall short of these requirements. Here we present a new multiphoton photo-excitation method, termed three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT), which allows precise, simultaneous photo-activation of arbitrary sets of neurons anywhere within the addressable volume of a microscope. This technique uses point-cloud holography to place multiple copies of a temporally focused disc matching the dimensions of a neurons cell body. Experiments in cultured cells, brain slices, and in living mice demonstrate single-neuron spatial resolution even when optically targeting randomly distributed groups of neurons in 3D. This approach opens new avenues for mapping and manipulating neural circuits, allowing a real-time, cellular resolution interface to the brain

Precise multimodal optical control of neural ensemble activity.

Understanding brain function requires technologies that can control the activity of large populations of neurons with high fidelity in space and time. We developed a multiphoton holographic approach to activate or suppress the activity of ensembles of cortical neurons with cellular resolution and sub-millisecond precision. Since existing opsins were inadequate, we engineered new soma-targeted (ST) optogenetic tools, ST-ChroME and IRES-ST-eGtACR1, optimized for multiphoton activation and suppression. Employing a three-dimensional all-optical read-write interface, we demonstrate the ability to simultaneously photostimulate up to 50 neurons distributed in three dimensions in a 550 × 550 × 100-µm3 volume of brain tissue. This approach allows the synthesis and editing of complex neural activity patterns needed to gain insight into the principles of neural codes

Complementary networks of cortical somatostatin interneurons enforce layer specific control

The neocortex is functionally organized into layers. Layer four receives the densest bottom up sensory inputs, while layers 2/3 and 5 receive top down inputs that may convey predictive information. A subset of cortical somatostatin (SST) neurons, the Martinotti cells, gate top down input by inhibiting the apical dendrites of pyramidal cells in layers 2/3 and 5, but it is unknown whether an analogous inhibitory mechanism controls activity in layer 4. Using high precision circuit mapping, in vivo optogenetic perturbations, and single cell transcriptional profiling, we reveal complementary circuits in the mouse barrel cortex involving genetically distinct SST subtypes that specifically and reciprocally interconnect with excitatory cells in different layers: Martinotti cells connect with layers 2/3 and 5, whereas non-Martinotti cells connect with layer 4. By enforcing layer-specific inhibition, these parallel SST subnetworks could independently regulate the balance between bottom up and top down input

Distinct Roles of Mechanistic Target of Rapamycin Targets S6K1 and S6K2 in Breast Cancer

The purpose of this article is to compare and contrast the expression, structure, regulation and function of these two S6K homologs in breast cancer

Regulation of anti-apoptotic Bcl-2 family protein Mcl-1 by S6 kinase 2.

The anti-apoptotic Bcl-2 family protein myeloid cell leukemia-1 (Mcl-1) plays an important role in breast cancer cell survival and chemoresistance. We have previously shown that knockdown of the 40S ribosomal protein S6 kinase-2 (S6K2), which acts downstream of the mechanistic target of rapamycin complex 1 (mTORC1), enhanced breast cancer cell death by apoptotic stimuli. The increase in cell death by S6K2 depletion was partly due to inactivation of Akt. In the present study, we investigated if S6K2 regulates Mcl-1, which acts downstream of Akt. Silencing of S6K2 but not S6K1 in T47D cells decreased Mcl-1 level, and potentiated apoptosis induced by TRAIL and doxorubicin. Knockdown of S6K2 also decreased the level of anti-apoptotic Bcl-xl. Depletion of the tumor suppressor protein PDCD4 (programmed cell death 4), which regulates translation of several anti-apoptotic proteins, reversed downregulation of Bcl-xl but not Mcl-1 and failed to reverse the effect of S6K2 knockdown on potentiation of doxorubicin-induced apoptosis. Downregulation of Mcl-1 by S6K2 knockdown was partly restored by the proteasome inhibitor MG132. Overexpression of catalytically-active Akt or knockdown of glycogen synthase kinase-3 (GSK3)-β, a substrate for Akt, had little effect on Mcl-1 downregulation caused by S6K2 deficiency. Silencing of S6K2 increased the level of c-Jun N-terminal kinase (JNK) and knockdown of JNK1 increased basal Mcl-1 level and partly reversed the effect of S6K2 knockdown on Mcl-1 downregulation. JNK1 knockdown also had a modest effect in attenuating the increase in doxorubicin-induced apoptosis caused by S6K2 deficiency. These results suggest that S6K2 regulates apoptosis via multiple mechanisms, and involves both Akt and JNK

Regulation of Autophagy by Kinases

Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

Knockdown of JNK1 attenuates increase in doxorubicin-induced apoptosis caused by S6K2 knockdown.

<p>T47D (A) or HCC1428 (B) cells were transfected with indicated siRNAs for approximately 48 h and then treated with or without doxorubicin for approximately 24 h. Western blot analysis was performed with indicated antibodies. The arrow indicates cleaved PARP. Results are representative of 2 (HCC1428) to 3 (T47D) independent experiments. The bands corresponding to cleaved PARP were quantified using ImageJ and the intensities of cleaved PARP normalized with tubulin are shown.</p