Lipidation states orchestrate CLICK-III/CaMKIγ's stepwise association with Golgi and rafts-enriched membranes and specify its functional coupling to STEF-Rac1-dependent neurite extension.

CLICK-III/CaMKIγ is a lipid-anchored neuronal isoform of multifunctional Ca2+/calmodulin-dependent protein kinases, which mediates BDNF-dependent dendritogenesis in cultured cortical neurons. We found that two distinct lipidation states of CaMKIγ, namely, prenylation and palmitoylation, controlled its association with detergent-resistant microdomains in the dendrites and were essential for its dendritogenic activity. However, the impact of each lipid modification on membrane targeting/trafficking and how it specifies functional coupling leading to polarized changes in neuronal morphology are not clear. Here, we show that prenylation induces membrane anchoring of CaMKIγ, permitting access to the Golgi apparatus, and a subsequent palmitoylation facilitates association with cholesterol-enriched lipid microdomains or lipid rafts, in particular at the Golgi. To specifically test the role of palmitoylated CaMKγ in neurite extension, we identified and took advantage of a cell system, PC12, which, unlike neurons, conveniently lacked CaMKIγ and was deficient in the activity-dependent release of a neuritogenic growth factor while possessing the ability to activate polarized rafts signaling for morphogenesis. This system allowed us to rigorously demonstrate that an activity-dependent, lipid rafts-restricted Rac activation leading to neuritogenesis could be functionally rescued by dually lipidated CaMKIγ expression, revealing that not only prenylation but also palmitoylation is essential for CaMKIγ to activate a compartmentalized STEF-Rac1 pathway. These results shed light on the significance of recruiting prenylated and palmitoylated CaMKIγ into the coalescing signalosomes at lipid rafts together with Rac1 and its specific GEF and STEF and forming a compartmentalized Ca2+ signaling pathway that underlies activity-dependent neuritogenesis and morphogenesis during axodendritic polarization critical for brain development and circuitogenesis.

Förster resonance energy transfer-based kinase mutation phenotyping reveals an aberrant facilitation of Ca2+/calmodulin-dependent CaMKIIα activity in de novo mutations related to intellectual disability.

CaMKIIα plays a fundamental role in learning and memory and is a key determinant of synaptic plasticity. Its kinase activity is regulated by the binding of Ca2+/CaM and by autophosphorylation that operates in an activity-dependent manner. Though many mutations in CAMK2A were linked to a variety of neurological disorders, the multiplicity of its functional substrates renders the systematic molecular phenotyping challenging. In this study, we report a new case of CAMK2A P212L, a recurrent mutation, in a patient with an intellectual disability. To quantify the effect of this mutation, we developed a FRET-based kinase phenotyping strategy and measured aberrance in Ca2+/CaM-dependent activation dynamics in vitro and in synaptically connected neurons. CaMKIIα P212L revealed a significantly facilitated Ca2+/CaM-dependent activation in vitro. Consistently, this mutant showed faster activation and more delayed inactivation in neurons. More prolonged kinase activation was also accompanied by a leftward shift in the CaMKIIα input frequency tuning curve. In keeping with this, molecular phenotyping of other reported CAMK2A de novo mutations linked to intellectual disability revealed aberrant facilitation of Ca2+/CaM-dependent activation of CaMKIIα in most cases. Finally, the pharmacological reversal of CAMK2A P212L phenotype in neurons was demonstrated using an FDA-approved NMDA receptor antagonist memantine, providing a basis for targeted therapeutics in CAMK2A-linked intellectual disability. Taken together, FRET-based kinase mutation phenotyping sheds light on the biological impact of CAMK2A mutations and provides a selective, sensitive, quantitative, and scalable strategy for gaining novel insights into the molecular etiology of intellectual disability.

In utero electroporation and cranial window implantation for in vivo wide-field two-photon calcium imaging using G-CaMP9a transgenic mice.

We present a protocol to prepare mouse cranial window implantation for in vivo two-photon wide-field calcium imaging. This protocol uses G-CaMP9a transgenic mice, which express a genetically encoded calcium indicator with high signal-to-noise ratio. We describe in utero electroporation, followed by headplate fixation and cranial window implantation. This protocol can be used for measuring neural activity and is suitable for long-term imaging in large populations. Moreover, this protocol does not require preparation of Flp-expressing transgenic mice. For complete details on the use and execution of this protocol, please refer to Sakamoto et al. (2022).

Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics.

To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

Balloon-occluded retrograde transvenous obliteration of complex gastric varices assisted by temporary balloon occlusion of the splenic artery.

Six cases of gastric varices with multiple afferent veins, in which balloon-occluded venography of the draining vein showed insufficient filling of gastric varices with contrast medium, were treated by balloon-occluded retrograde transvenous obliteration (BRTO) and temporary balloon occlusion of the splenic artery. The gastric varices were completely filled with sclerosant in all but one patient. No procedure-related complications were encountered. Computed tomography (CT) after the procedure showed complete thrombosis of the varices in five patients and partial thrombosis in one patient. Temporary balloon occlusion of the splenic artery is a useful additional technique for complete obliteration of gastric varices in selected cases.

Partial thrombosis of gastric varices after balloon-occluded retrograde transvenous obliteration: CT findings and endoscopic correlation.

OBJECTIVE: The purpose of our study was to investigate the frequency and outcomes of partial thrombosis of gastric varices after balloon-occluded retrograde transvenous obliteration (BRTO). MATERIALS AND METHODS: We reviewed retrospectively 69 consecutive patients with gastric varices who were followed-up for > 6 months after treatment with BRTO. All patients underwent contrast-enhanced CT and gastroscopy before and after BRTO. Imaging findings of gastric varices with particular attention to afferent veins, degree of thrombosis, and variceal changes were investigated. RESULTS: On the basis of pretherapeutic CT images, gastric varices were classified into two types: simple (< 3 afferent veins) and complex (≥ 3 afferent veins). Initial follow-up CT showed complete thrombosis in 58 patients (84%) and partial thrombosis in 11 (16%). Partial thrombosis was observed more frequently in complex-type varices (25% vs 9%). No regrowth or recurrent varices were observed in completely thrombosed varices. Follow-up endoscopy showed regrowth of gastric varices at 6-24 months after BRTO in five patients; all of these were complex-type and partially thrombosed varices. All five recurrent varices were treated successfully with repeated BRTO. CONCLUSION: Partial thrombosis after BRTO can occur in complex-type gastric varices, which have a higher risk of regrowth. Additional techniques that achieve complete thrombosis are required for long-term efficacy for complex-type gastric varices.