SUMMARY The 22 γ-protocadherins (γ-Pcdhs) potentially specify thousands of distinct homophilic adhesive interactions in the brain. Neonatal lethality of mice lacking the Pcdh-γ gene cluster has, however, precluded analysis of many brain regions. Here, we use a conditional Pcdh-γ allele to restrict mutation to the cerebral cortex and find that, in contrast to other central nervous system phenotypes, loss of γ-Pcdhs in cortical neurons does not affect their survival or result in reduced synaptic density. Instead, mutant cortical neurons exhibit severely reduced dendritic arborization. Mutant cortices have aberrantly high levels of protein kinase C (PKC) activity and of phosphorylated (inactive) myristoylated alanine-rich C-kinase substrate, a PKC target that promotes arborization. Dendrite complexity can be rescued in Pcdh-γ mutant neurons by inhibiting PKC, its upstream activator phospholipase C, or the γ-Pcdh binding partner focal adhesion kinase. Our results reveal a distinct role for the γ-Pcdhs in cortical development and identify a signaling pathway through which they play this role.

Adenosine 5′ triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.

Background:The mitochondrial fission enzyme dynamin-related protein 1 (Drp1) is regulated via reversible phosphorylation of Ser-656. Results: The Drp1 LXVP motif mediates dephosphorylation and activation by calcineurin (CaN), which influences mitochondrial morphology and survival post-injury in neurons. Conclusion:The CaN-Drp1 signaling axis can be detrimental to injured neurons. Significance: The CaN-Drp1 complex may be a target for neuroprotective therapeutic intervention.

Adenosine 5' triphosphate (ATP) is a universal intracellular energy source 1 and an evolutionarily ancient 2 extracellular signal 3-5 . Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR 1.0 responded to relevant ATP concentrations (30 µM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. iATPSnFRs represent promising new reagents for imaging ATP dynamics.Given widespread roles in energy homeostasis and cell signaling 3-5 , several methods have been used to detect ATP using small-molecule chemical approaches 6 , firefly luciferase 7,8 , ion channel-expressing "sniffer" cells 9-11 , voltammetry 12 , and different types of microelectrodes 13,14 . However, these methods lack spatial resolution, cannot be easily used in tissue slices or in vivo, respond to off-target ligands, need unwieldy photon-counting cameras, are imprecise and/or unavoidably damage tissue during probe placement. Importantly, none of these methods can be genetically targeted to specific cells, sub-cellular compartments or whole organisms, and they all lack cellular-scale spatial resolution. Although luciferase is genetically encoded 15 , it requires the exogenous substrate luciferin, addition of which complicates use in tissue slices and in vivo. It also saturates at nanomolar ATP, far lower than concentrations expected during ATP signaling (~1 µM to ~1 mM). Additionally, luciferase's bioluminescent output yields low photon fluxes and rules out cellular-resolution imaging. To address these issues, genetically-encoded fluorescent ATP sensors have been developed, including Perceval and PercevalHR 16,17 , ATeam 18 , and QUEEN 19 . These sensors are valuable, but they have limitations. They either respond to ADP/ATP ratio instead of ATP concentration or are susceptible to optical overlap with cellular sources of auto-fluorescence, and all are incompatible with single-wavelength fluorescence imaging, the workhorse of functional fluorescence microscopy 20 .Our goals were to: 1) develop an ATP sensor that could be used in routine fluorescence imaging, e.g. at GFP's 488 nm excitation and ~525 nm emission and 2) deploy such a sensor on the cell surface and within cells to image ATP. QUEEN is an excitation ratio sensor, ATeam is a fluorescence resonance energy transfer (FRET) sensor, and the Perceval sensors are fluorescence lifetime indicators -all of these methods require customized equipment, substantially slow down imaging rates, complicate use with other reagents, and typically have lower signal-to-noise ratio (SNR)...

The choroid plexus (CP) epithelium develops from the ependyma that lines the ventricular system, and plays a critical role in the development and function of the brain. In addition to being the primary site of CSF production, the CP maintains the blood-CSF barrier via apical tight junctions between epithelial cells. Here we show that the 22-member γ-Protocadherin (γ-Pcdh) family of cell adhesion molecules, which we have implicated previously in synaptogenesis and neuronal survival, is highly expressed by both CP epithelial and ependymal cells, in which γ-Pcdh protein localization is, surprisingly, tightly restricted to the apical membrane. Multi-label immunostaining demonstrates that γ-Pcdhs are excluded from tight junctions, basolateral adherens junctions, and apical cilia tufts. RT-PCR analysis indicates that, as a whole, the CP expresses most members of the Pcdh-γ gene family. Immunostaining using novel monoclonal antibodies specific for single γ-Pcdh proteins shows that individual epithelial cells differ in their apically-localized γ-Pcdh repertoire. Restricted mutation of the Pcdh-γ locus in the choroid plexus and ependyma leads to significant reductions in ventricular volume, without obvious disruptions of epithelial apical-basal polarity. Together, these results suggest an unsuspected role for the γ-Pcdhs in CSF production and demonstrate a surprising molecular heterogeneity in the CP epithelium.