Ratiometric measurement of MAM Ca2+ dynamics using a modified CalfluxVTN.

Mitochondria-associated ER membrane (MAM) is a structure where these calcium-regulating organelles form close physical contact sites for efficient Ca2+ crosstalk. Despite the central importance of MAM Ca2+ dynamics in diverse biological processes, directly and specifically measuring Ca2+ concentrations inside MAM is technically challenging. Here, we develop MAM-Calflux, a MAM-specific BRET-based Ca2+ indicator. The successful application of the bimolecular fluorescence complementation (BiFC) concept highlights Ca2+-responsive BRET signals in MAM. The BiFC strategy imparts dual functionality as a Ca2+ indicator and quantitative structural marker specific for MAM. As a ratiometric Ca2+ indicator, MAM-Calflux estimates steady-state MAM Ca2+ levels. Finally, it enables the visualization of uneven intracellular distribution of MAM Ca2+ and the elucidation of abnormally accumulated MAM Ca2+ from the neurons of Parkinson's disease mouse model in both steady-state and stimulated conditions. Therefore, we propose that MAM-Calflux can be a versatile tool for ratiometrically measuring dynamic inter-organellar Ca2+ communication.

Gcap14 is a microtubule plus-end-tracking protein coordinating microtubule-actin crosstalk during neurodevelopment.

Regulation of microtubule dynamics is required to properly control various steps of neurodevelopment. In this study, we identified granule cell antiserum-positive 14 (Gcap14) as a microtubule plus-end-tracking protein and as a regulator of microtubule dynamics during neurodevelopment. Gcap14 knockout mice exhibited impaired cortical lamination. Gcap14 deficiency resulted in defective neuronal migration. Moreover, nuclear distribution element nudE-like 1 (Ndel1), an interacting partner of Gcap14, effectively corrected the downregulation of microtubule dynamics and the defects in neuronal migration caused by Gcap14 deficiency. Finally, we found that the Gcap14-Ndel1 complex participates in the functional link between microtubule and actin filament, thereby regulating their crosstalks in the growth cones of cortical neurons. Taken together, we propose that the Gcap14-Ndel1 complex is fundamental for cytoskeletal remodeling during neurodevelopmental processes such as neuronal processes elongation and neuronal migration.

Schizophrenia-associated Mitotic Arrest Deficient-1 (MAD1) regulates the polarity of migrating neurons in the developing neocortex.

Although large-scale genome-wide association studies (GWAS) have identified an association between MAD1L1 (Mitotic Arrest Deficient-1 Like 1) and the pathology of schizophrenia, the molecular mechanisms underlying this association remain unclear. In the present study, we aimed to address these mechanisms by examining the role of MAD1 (the gene product of MAD1L1) in key neurodevelopmental processes in mice and human organoids. Our findings indicated that MAD1 is highly expressed during active cortical development and that MAD1 deficiency leads to impairments in neuronal migration and neurite outgrowth. We also observed that MAD1 is localized to the Golgi apparatus and regulates vesicular trafficking from the Golgi apparatus to the plasma membrane, which is required for the growth and polarity of migrating neurons. In this process, MAD1 physically interacts and collaborates with the kinesin-like protein KIFC3 (kinesin family member C3) to regulate the morphology of the Golgi apparatus and neuronal polarity, thereby ensuring proper neuronal migration and differentiation. Consequently, our findings indicate that MAD1 is an essential regulator of neuronal development and that alterations in MAD1 may underlie schizophrenia pathobiology.

DYRK3 phosphorylates SNAPIN to regulate axonal retrograde transport and neurotransmitter release.

Among the five members of the dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK) family, the cellular functions of DYRK3 have not been fully elucidated. Some studies have indicated limited physiological roles and substrates of DYRK3, including promotion of glioblastoma, requirement in influenza virus replication, and coupling of stress granule condensation with mammalian target of rapamycin complex 1 signaling. Here, we demonstrate that serum deprivation causes a decrease in intracellular DYRK3 levels via the proteolytic autophagy pathway, as well as the suppression of DYRK3 gene expression. To further demonstrate how DYRK3 affects cell viability, especially in neurons, we used a yeast two-hybrid assay and identified multiple DYRK3-binding proteins, including SNAPIN, a SNARE-associated protein implicated in synaptic transmission. We also found that DYRK3 directly phosphorylates SNAPIN at the threonine (Thr) 14 residue, increasing the interaction of SNAPIN with other proteins such as dynein and synaptotagmin-1. In central nervous system neurons, SNAPIN is associated with and mediate the retrograde axonal transport of diverse cellular products from the distal axon terminal to the soma and the synaptic release of neurotransmitters, respectively. Moreover, phosphorylation of SNAPIN at Thr-14 was found to positively modulate mitochondrial retrograde transport in mouse cortical neurons and the recycling pool size of synaptic vesicles, contributing to neuronal viability. In conclusion, the present study demonstrates that DYRK3 phosphorylates SNAPIN, positively regulating the dynein-mediated retrograde transport of mitochondria and SNARE complex-mediated exocytosis of synaptic vesicles within the neurons. This finding further suggests that DYRK3 affects cell viability and provides a novel neuroprotective mechanism.

Retinoic acid-induced protein 14 controls dendritic spine dynamics associated with depressive-like behaviors.

Dendritic spines are the central postsynaptic machinery that determines synaptic function. The F-actin within dendritic spines regulates their dynamic formation and elimination. Rai14 is an F-actin-regulating protein with a membrane-shaping function. Here, we identified the roles of Rai14 for the regulation of dendritic spine dynamics associated with stress-induced depressive-like behaviors. Rai14-deficient neurons exhibit reduced dendritic spine density in the Rai14+/- mouse brain, resulting in impaired functional synaptic activity. Rai14 was protected from degradation by complex formation with Tara, and accumulated in the dendritic spine neck, thereby enhancing spine maintenance. Concurrently, Rai14 deficiency in mice altered gene expression profile relevant to depressive conditions and increased depressive-like behaviors. Moreover, Rai14 expression was reduced in the prefrontal cortex of the mouse stress model, which was blocked by antidepressant treatment. Thus, we propose that Rai14-dependent regulation of dendritic spines may underlie the plastic changes of neuronal connections relevant to depressive-like behaviors.

Disrupted-in-schizophrenia 1 enhances the quality of circadian rhythm by stabilizing BMAL1.

Disrupted-in-schizophrenia 1 (DISC1) is a scaffold protein that has been implicated in multiple mental disorders. DISC1 is known to regulate neuronal proliferation, signaling, and intracellular calcium homeostasis, as well as neurodevelopment. Although DISC1 was linked to sleep-associated behaviors, whether DISC1 functions in the circadian rhythm has not been determined yet. In this work, we revealed that Disc1 expression exhibits daily oscillating pattern and is regulated by binding of circadian locomotor output cycles kaput (CLOCK) and Brain and muscle Arnt-like protein-1 (BMAL1) heterodimer to E-box sequences in its promoter. Interestingly, Disc1 deficiency increases the ubiquitination of BMAL1 and de-stabilizes it, thereby reducing its protein levels. DISC1 inhibits the activity of GSK3ß, which promotes BMAL1 ubiquitination, suggesting that DISC1 regulates BMAL1 stability by inhibiting its ubiquitination. Moreover, Disc1-deficient cells and mice show reduced expression of other circadian genes. Finally, Disc1-LI (Disc1 knockout) mice exhibit damped circadian physiology and behaviors. Collectively, these findings demonstrate that the oscillation of DISC1 expression is under the control of CLOCK and BMAL1, and that DISC1 contributes to the core circadian system by regulating BMAL1 stability.

Schizophrenia-associated dysbindin modulates axonal mitochondrial movement in cooperation with p150glued.

Mitochondrial movement in neurons is finely regulated to meet the local demand for energy and calcium buffering. Elaborate transport machinery including motor complexes is required to deliver and localize mitochondria to appropriate positions. Defects in mitochondrial transport are associated with various neurological disorders without a detailed mechanistic information. In this study, we present evidence that dystrobrevin-binding protein 1 (dysbindin), a schizophrenia-associated factor, plays a critical role in axonal mitochondrial movement. We observed that mitochondrial movement was impaired in dysbindin knockout mouse neurons. Reduced mitochondrial motility caused by dysbindin deficiency decreased the density of mitochondria in the distal part of axons. Moreover, the transport and distribution of mitochondria were regulated by the association between dysbindin and p150glued. Furthermore, altered mitochondrial distribution in axons led to disrupted calcium dynamics, showing abnormal calcium influx in presynaptic terminals. These data collectively suggest that dysbindin forms a functional complex with p150glued that regulates axonal mitochondrial transport, thereby affecting presynaptic calcium homeostasis.

Sequential phosphorylation of NDEL1 by the DYRK2-GSK3ß complex is critical for neuronal morphogenesis.

Neuronal morphogenesis requires multiple regulatory pathways to appropriately determine axonal and dendritic structures, thereby to enable the functional neural connectivity. Yet, however, the precise mechanisms and components that regulate neuronal morphogenesis are still largely unknown. Here, we newly identified the sequential phosphorylation of NDEL1 critical for neuronal morphogenesis through the human kinome screening and phospho-proteomics analysis of NDEL1 from mouse brain lysate. DYRK2 phosphorylates NDEL1 S336 to prime the phosphorylation of NDEL1 S332 by GSK3ß. TARA, an interaction partner of NDEL1, scaffolds DYRK2 and GSK3ß to form a tripartite complex and enhances NDEL1 S336/S332 phosphorylation. This dual phosphorylation increases the filamentous actin dynamics. Ultimately, the phosphorylation enhances both axonal and dendritic outgrowth and promotes their arborization. Together, our findings suggest the NDEL1 phosphorylation at S336/S332 by the TARA-DYRK2-GSK3ß complex as a novel regulatory mechanism underlying neuronal morphogenesis.

Dopamine D1 Receptor (D1R) Expression Is Controlled by a Transcriptional Repressor Complex Containing DISC1.

Disrupted-in-Schizophrenia 1 (DISC1) is a scaffold protein implicated in various psychiatric diseases. Dysregulation of the dopamine system has been associated with DISC1 deficiency, while the molecular mechanism is unclear. In this study, we propose a novel molecular mechanism underlying the transcriptional regulation of the dopamine D1 receptor (D1R) in the striatum via DISC1. We verified the increase in D1R at the transcriptional level in the striatum of DISC1-deficient mouse models and altered histone acetylation status at the D1r locus. We identified a functional interaction between DISC1 and Krüppel-like factor 16 (KLF16). KLF16 translocates DISC1 into the nucleus and forms a regulatory complex by recruiting SIN3A corepressor complexes to the D1r locus. Moreover, DISC1-deficient mice have altered D1R-mediated signaling in the striatum and exhibit hyperlocomotion in response to cocaine; the blockade of D1R suppresses these effects. Taken together, our results suggest that nuclear DISC1 plays a critical role in the transcriptional regulation of D1R in the striatal neuron, providing a mechanistic link between DISC1 and dopamine-related psychiatric symptoms.

KIN-4/MAST kinase promotes PTEN-mediated longevity of Caenorhabditis elegans via binding through a PDZ domain.

PDZ domain-containing proteins (PDZ proteins) act as scaffolds for protein-protein interactions and are crucial for a variety of signal transduction processes. However, the role of PDZ proteins in organismal lifespan and aging remains poorly understood. Here, we demonstrate that KIN-4, a PDZ domain-containing microtubule-associated serine-threonine (MAST) protein kinase, is a key longevity factor acting through binding PTEN phosphatase in Caenorhabditis elegans. Through a targeted genetic screen for PDZ proteins, we find that kin-4 is required for the long lifespan of daf-2/insulin/IGF-1 receptor mutants. We then show that neurons are crucial tissues for the longevity-promoting role of kin-4. We find that the PDZ domain of KIN-4 binds PTEN, a key factor for the longevity of daf-2 mutants. Moreover, the interaction between KIN-4 and PTEN is essential for the extended lifespan of daf-2 mutants. As many aspects of lifespan regulation in C. elegans are evolutionarily conserved, MAST family kinases may regulate aging and/or age-related diseases in mammals through their interaction with PTEN.

Regulation of the actin cytoskeleton by the Ndel1-Tara complex is critical for cell migration.

Nuclear distribution element-like 1 (Ndel1) plays pivotal roles in diverse biological processes and is implicated in the pathogenesis of multiple neurodevelopmental disorders. Ndel1 function by regulating microtubules and intermediate filaments; however, its functional link with the actin cytoskeleton is largely unknown. Here, we show that Ndel1 interacts with TRIO-associated repeat on actin (Tara), an actin-bundling protein, to regulate cell movement. In vitro wound healing and Boyden chamber assays revealed that Ndel1- or Tara-deficient cells were defective in cell migration. Moreover, Tara overexpression induced the accumulation of Ndel1 at the cell periphery and resulted in prominent co-localization with F-actin. This redistribution of Ndel1 was abolished by deletion of the Ndel1-interacting domain of Tara, suggesting that the altered peripheral localization of Ndel1 requires a physical interaction with Tara. Furthermore, co-expression of Ndel1 and Tara in SH-SY5Y cells caused a synergistic increase in F-actin levels and filopodia formation, suggesting that Tara facilitates cell movement by sequestering Ndel1 at peripheral structures to regulate actin remodeling. Thus, we demonstrated that Ndel1 interacts with Tara to regulate cell movement. These findings reveal a novel role of the Ndel1-Tara complex in actin reorganization during cell movement.

Disrupted-in-schizophrenia 1 (DISC1) and Syntaphilin collaborate to modulate axonal mitochondrial anchoring.

In neuronal axons, the ratio of motile-to-stationary mitochondria is tightly regulated by neuronal activation, thereby meeting the need for local calcium buffering and maintaining the ATP supply. However, the molecular players and detailed regulatory mechanisms behind neuronal mitochondrial movement are not completely understood. Here, we found that neuronal activation-induced mitochondrial anchoring is regulated by Disrupted-in-schizophrenia 1 (DISC1), which is accomplished by functional association with Syntaphilin (SNPH). DISC1 deficiency resulted in reduced axonal mitochondrial movement, which was partially reversed by concomitant SNPH depletion. In addition, a SNPH deletion mutant lacking the sequence for interaction with DISC1 exhibited an enhanced mitochondrial anchoring effect than wild-type SNPH. Moreover, upon neuronal activation, mitochondrial movement was preserved by DISC1 overexpression, not showing immobilized response of mitochondria. Taken together, we propose that DISC1 in association with SNPH is a component of a modulatory complex that determines mitochondrial anchoring in response to neuronal activation.

Disrupted-in-schizophrenia 1 (DISC1) regulates dysbindin function by enhancing its stability.

Dysbindin and DISC1 are schizophrenia susceptibility factors playing roles in neuronal development. Here we show that the physical interaction between dysbindin and DISC1 is critical for the stability of dysbindin and for the process of neurite outgrowth. We found that DISC1 forms a complex with dysbindin and increases its stability in association with a reduction in ubiquitylation. Furthermore, knockdown of DISC1 or expression of a deletion mutant, DISC1 lacking amino acid residues 403-504 of DISC1 (DISC1(Δ403-504)), effectively decreased levels of endogenous dysbindin. Finally, the neurite outgrowth defect induced by knockdown of DISC1 was partially reversed by coexpression of dysbindin. Taken together, these results indicate that dysbindin and DISC1 form a physiologically functional complex that is essential for normal neurite outgrowth.

Antifungal activity and mode of action of silver nano-particles on Candida albicans.

In this study, the antifungal effects of silver nano-particles (nano-Ag) and their mode of action were investigated. Nano-Ag showed antifungal effects on fungi tested with low hemolytic effects against human erythrocytes. To elucidate the antifungal mode of action of nano-Ag, flow cytometry analysis, a glucose-release test, transmission electron microscopy (TEM) and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH), as a plasma membrane probe, were performed with Candida albicans. The results suggest nano-Ag may exert an antifungal activity by disrupting the structure of the cell membrane and inhibiting the normal budding process due to the destruction of the membrane integrity. The present study indicates nano-Ag has considerable antifungal activity, deserving further investigation for clinical applications.

Fungicidal effect of pleurocidin by membrane-active mechanism and design of enantiomeric analogue for proteolytic resistance.

Pleurocidin (Ple) is a 25-residue peptide which is derived from the skin mucous secretion of the winter flounder (Pleuronectes americanus). In this study, we investigated antifungal effects and its mode of action of Ple on human pathogenic fungi. Ple showed potent antifungal activity with low hemolytic activity. To investigate the antifungal mechanisms of Ple, the cellular localization and membrane interaction of Ple were examined. Protoplast regeneration and membrane-disrupting activity by DPH-labeled membrane support the idea, that Ple exerts fungicidal activity against the human pathogenic fungus Candida albicans with the disruption of a plasma membrane. To aim for which was the application of a therapeutic agent, we designed a synthetic enantiomeric peptide composed of all-d-amino acids to enhance proteolytic resistance. The synthetic all-d-Ple also displayed two-fold more potent antifungal activity than that of all-l-Ple, and its antifungal activity showed proteolytic resistance against various proteases. Therefore, these results suggest a therapeutic potential of all-d-Ple with regard to its proteolytic resistance against human fungal infections.