Fully bioresorbable hybrid opto-electronic neural implant system for simultaneous electrophysiological recording and optogenetic stimulation.

Bioresorbable neural implants based on emerging classes of biodegradable materials offer a promising solution to the challenges of secondary surgeries for removal of implanted devices required for existing neural implants. In this study, we introduce a fully bioresorbable flexible hybrid opto-electronic system for simultaneous electrophysiological recording and optogenetic stimulation. The flexible and soft device, composed of biodegradable materials, has a direct optical and electrical interface with the curved cerebral cortex surface while exhibiting excellent biocompatibility. Optimized to minimize light transmission losses and photoelectric artifact interference, the device was chronically implanted in the brain of transgenic mice and performed to photo-stimulate the somatosensory area while recording local field potentials. Thus, the presented hybrid neural implant system, comprising biodegradable materials, promises to provide monitoring and therapy modalities for versatile applications in biomedicine.

A single-component, light-assisted uncaging switch for endoproteolytic release.

Proteases function as pivotal molecular switches, initiating numerous biological events. Notably, potyviral protease, derived from plant viruses, has emerged as a trusted proteolytic switch in synthetic biological circuits. To harness their capabilities, we have developed a single-component photocleavable switch, termed LAUNCHER (Light-Assisted UNcaging switCH for Endoproteolytic Release), by employing a circularly permutated tobacco etch virus protease and a blue-light-gated substrate, which are connected by fine-tuned intermodular linkers. As a single-component system, LAUNCHER exhibits a superior signal-to-noise ratio compared with multi-component systems, enabling precise and user-controllable release of payloads. This characteristic renders LAUNCHER highly suitable for diverse cellular applications, including transgene expression, tailored subcellular translocation and optochemogenetics. Additionally, the plug-and-play integration of LAUNCHER into existing synthetic circuits facilitates the enhancement of circuit performance. The demonstrated efficacy of LAUNCHER in improving existing circuitry underscores its significant potential for expanding its utilization in various applications.

Establishment and Validation of a Model for Fetal Neural Ischemia Using Necrotic Core-Free Human Spinal Cord Organoids.

Fetal spinal cord ischemia is a serious medical condition that can result in significant neurological damage and adverse outcomes for the fetus. However, the lack of an appropriate experimental model has hindered the understanding of the pathology and the development of effective treatments. In our study, we established a system for screening drugs that affect fetal spinal cord ischemia using spinal cord organoids. Importantly, we produced necrotic core-free human spinal cord organoids (nf-hSCOs) by reducing the organoid size to avoid potential complications of spontaneous necrosis in large organoids. Exposing nf-hSCOs to CoCl2 as a hypoxia mimetic and hypoglycemic conditions resulted in significant neuronal damage, as assessed by multiple assay batteries. By utilizing this model, we tested chemicals that have been reported to exhibit beneficial effects in brain organoid-based ischemia models. Surprisingly, these chemicals did not provide sufficient benefit, and we discovered that rapamycin is a mild neuroprotective reagent for both axon degeneration and neuronal survival. We propose that nf-hSCO is suitable for large-scale screening of fetal neural ischemia due to its scalability, ease of ischemic induction, implementation of quantifiable assay batteries, and the absence of spontaneous necrosis.

Mitochondrial NIR imaging probe mitigating oxidative damage by targeting HDAC6.

Despite the apparent copious fluorescent probes targeting mitochondria, the development of low cytotoxic probes is still needed for improving validation of mitochondrial function assessment. Herein, we report a novel cyanine-based NIR fluorescent probe, T2, which selectively targets mitochondria with significantly low toxicity by modulating the intracellular redox status. Additionally, T2 inhibits oxidative stress-induced cell death in cortical neurons. This study provides new insight into developing low-toxic mitochondrial imaging agents by regulating redox homeostasis.

Symmetry Breaking of Human Pluripotent Stem Cells (hPSCs) in Micropattern Generates a Polarized Spinal Cord-Like Organoid (pSCO) with Dorsoventral Organization.

Axis formation and related spatial patterning are initiated by symmetry breaking during development. A geometrically confined culture of human pluripotent stem cells (hPSCs) mimics symmetry breaking and cell patterning. Using this, polarized spinal cord organoids (pSCOs) with a self-organized dorsoventral (DV) organization are generated. The application of caudalization signals promoted regionalized cell differentiation along the radial axis and protrusion morphogenesis in confined hPSC colonies. These detached colonies grew into extended spinal cord-like organoids, which established self-ordered DV patterning along the long axis through the spontaneous expression of polarized DV patterning morphogens. The proportions of dorsal/ventral domains in the pSCOs can be controlled by the changes in the initial size of micropatterns, which altered the ratio of center-edge cells in 2D. In mature pSCOs, highly synchronized neural activity is separately detected in the dorsal and ventral side, indicating functional as well as structural patterning established in the organoids. This study provides a simple and precisely controllable method to generate spatially ordered organoids for the understanding of the biological principles of cell patterning and axis formation during neural development.

Protein interactome and cell-type expression analyses reveal that cytoplasmic FMR1-interacting protein 1 (CYFIP1), but not CYFIP2, associates with astrocytic focal adhesion.

The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2 × Myc and CYFIP2-3 × Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2 × Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.

Cell position within human pluripotent stem cell colonies determines apical specialization via an actin cytoskeleton-based mechanism.

Human pluripotent stem cells (hPSCs) grow as colonies with epithelial-like features including cell polarity and position-dependent features that contribute to symmetry breaking during development. Our study provides evidence that hPSC colonies exhibit position-dependent differences in apical structures and functions. With this apical difference, edge cells were preferentially labeled with amphipathic dyes, which enabled separation of edge and center cells by fluorescence-activated cell sorting. Transcriptome comparison between center and edge cells showed differential expression of genes related to apicobasal polarization, cell migration, and endocytosis. Accordingly, different kinematics and mechanical dynamics were found between center and edge cells, and perturbed actin dynamics disrupted the position-dependent apical polarity. In addition, our dye-labeling approach could be utilized to sort out a certain cell population in differentiated micropatterned colonies. In summary, hPSC colonies have position-dependent differences in apical structures and properties, and actin dynamics appear to play an important role in the establishment of this position-dependent cell polarity.

Interpreting the Entire Connectivity of Individual Neurons in Micropatterned Neural Culture With an Integrated Connectome Analyzer of a Neuronal Network (iCANN).

The function of a neural circuit can be determined by the following: (1) characteristics of individual neurons composing the circuit, (2) their distinct connection structure, and (3) their neural circuit activity. However, prior research on correlations between these three factors revealed many limitations. In particular, profiling and modeling of the connectivity of complex neural circuits at the cellular level are highly challenging. To reduce the burden of the analysis, we suggest a new approach with simplification of the neural connection in an array of honeycomb patterns on 2D, using a microcontact printing technique. Through a series of guided neuronal growths in defined honeycomb patterns, a simplified neuronal circuit was achieved. Our approach allowed us to obtain the whole network connectivity at cellular resolution using a combination of stochastic multicolor labeling via viral transfection. Therefore, we were able to identify several types of hub neurons with distinct connectivity features. We also compared the structural differences between different circuits using three-node motif analysis. This new model system, iCANN, is the first experimental model of neural computation at the cellular level, providing neuronal circuit structures for the study of the relationship between anatomical structure and function of the neuronal network.

Prevention of mitochondrial impairment by inhibition of protein phosphatase 1 activity in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of motor neurons (MNs) and subsequent muscle weakness. These pathological features are associated with numerous cellular changes, including alteration in mitochondrial morphology and function. However, the molecular mechanisms associating mitochondrial structure with ALS pathology are poorly understood. In this study, we found that Dynamin-related protein 1 (Drp1) was dephosphorylated in several ALS models, including those with SOD1 and TDP-43 mutations, and the dephosphorylation was mediated by the pathological induction of protein phosphatase 1 (PP1) activity in these models. Suppression of the PP1-Drp1 cascade effectively prevented ALS-related symptoms, including mitochondrial fragmentation, mitochondrial complex I impairment, axonal degeneration, and cell death, in primary neuronal culture models, iPSC-derived human MNs, and zebrafish models in vivo. These results suggest that modulation of PP1-Drp1 activity may be a therapeutic target for multiple pathological features of ALS.

The Neomycin Resistance Cassette in the Targeted Allele of Shank3B Knock-Out Mice Has Potential Off-Target Effects to Produce an Unusual Shank3 Isoform.

Variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3), which encodes postsynaptic scaffolds, are associated with brain disorders. The targeted alleles in a few Shank3 knock-out (KO) lines contain a neomycin resistance (Neo) cassette, which may perturb the normal expression of neighboring genes; however, this has not been investigated in detail. We previously reported an unexpected increase in the mRNA expression of Shank3 exons 1-12 in the brains of Shank3B KO mice generated by replacing Shank3 exons 13-16 with the Neo cassette. In this study, we confirmed that the increased Shank3 mRNA in Shank3B KO brains produced an unusual ∼60 kDa Shank3 isoform (Shank3-N), which did not properly localize to the synaptic compartment. Functionally, Shank3-N overexpression altered the dendritic spine morphology in cultured neurons. Importantly, Shank3-N expression in Shank3B KO mice was not a compensatory response to a reduction of full-length Shank3 because expression was still detected in the brain after normalizing the level of full-length Shank3. Moreover, in another Shank3 KO line (Shank3 gKO) with a similar Shank3 exonal deletion as that in Shank3B KO mice but without a Neo cassette, the mRNA expression levels of Shank3 exons 1-12 were lower than those of wild-type mice and Shank3-N was not detected in the brain. In addition, the expression levels of genes neighboring Shank3 on chromosome 15 were altered in the striatum of Shank3B KO but not Shank3 gKO mice. These results suggest that the Neo cassette has potential off-target effects in Shank3B KO mice.

Farnesylation-defective Rheb Increases Axonal Length Independently of mTORC1 Activity in Embryonic Primary Neurons.

Rheb (Ras homolog enriched in the brain) is a small GTPase protein that plays an important role in cell signaling for development of the neocortex through modulation of mTORC1 (mammalian-target-of-rapamycin-complex-1) activity. mTORC1 is known to control various biological processes including axonal growth in forming complexes at the lysosomal membrane compartment. As such, anchoring of Rheb on the lysosomal membrane via the farnesylation of Rheb at its cysteine residue (C180) is required for its promotion of mTOR activity. To test the significance of Rheb farnesylation, we overexpressed a farnesylation mutant form of Rheb, Rheb C180S, in primary rat hippocampal neurons and also in mouse embryonic neurons using in utero electroporation. Interestingly, we found that Rheb C180S maintained promotional effect of axonal elongation similar to the wild-type Rheb in both test systems. On the other hand, Rheb C180S failed to exhibit the multiple axon-promoting effect which is found in wild-type Rheb. The levels of phospho-4EBP1, a downstream target of mTORC1, were surprisingly increased in Rheb C180S transfected neurons, despite the levels of phosphorylated mTOR being significantly decreased compared to control vector transfectants. A specific mTORC1 inhibitor, rapamycin, also could not completely abolish axon elongation characteristics of Rheb C180S in transfected cells. Our data suggests that Rheb in a non-membrane compartment can promote the axonal elongation via phosphorylation of 4EBP1 and through an mTORC1-independent pathway.

A monitoring system for axonal growth dynamics using micropatterns of permissive and Semaphorin 3F chemorepulsive signals.

Neurons reach their correct targets by directional outgrowth of axons, which is mediated by attractive or repulsive cues. Growing axons occasionally cross a field of repulsive cues and stop at intermediate targets on the journey to their final destination. However, it is not well-understood how individual growth cones make decisions, and pass through repulsive territory to reach their permissive target regions. We developed a microcontact printing culture system that could trap individual axonal tips in a permissive dot area surrounded by the repulsive signal, semaphorin 3F (Sema3F). Axons of rat hippocampal neurons on the Sema3F/PLL dot array extended in the checkboard pattern with a significantly slow growth rate. The detailed analysis of the behaviors of axonal growth cones revealed the saccadic dynamics in the dot array system. The trapped axonal tips in the permissive area underwent growth cone enlargement with remarkably spiky filopodia, promoting their escape from the Sema3F constraints with straight extension of axons. This structured axonal growth on the dot pattern was disrupted by increased inter-dot distance, or perturbing intracellular signaling machineries. These data indicate that axons grow against repulsive signals by jumping over the repulsive cues, depending on contact signals and intracellular milieu. Our study suggests that our dot array culture system can be used as a screening system to easily and efficiently evaluate ECM or small molecule inhibitors interfering growth cone dynamics leading to controlling axonal growth.

Drp1-Zip1 Interaction Regulates Mitochondrial Quality Surveillance System.

Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.

High-Performance Acellular Tissue Scaffold Combined with Hydrogel Polymers for Regenerative Medicine.

Decellularization of tissues provides extracellular matrix (ECM) scaffolds for regeneration therapy and an experimental model to understand ECM and cellular interactions. However, decellularization often causes microstructure disintegration and reduction of physical strength, which greatly limits the use of this technique in soft organs or in applications that require maintenance of physical strength. Here, we present a new tissue decellularization procedure, namely CASPER (Clinically and Experimentally Applicable Acellular Tissue Scaffold Production for Tissue Engineering and Regenerative Medicine), which includes infusion and hydrogel polymerization steps prior to robust chemical decellularization treatments. Polymerized hydrogels serve to prevent excessive damage to the ECM while maintaining the sophisticated structures and biological activities of ECM components in various organs, including soft tissues such as brains and embryos. CASPERized tissues were successfully recellularized to stimulate a tissue-regeneration-like process after implantation without signs of pathological inflammation or fibrosis in vivo, suggesting that CASPERized tissues can be used for monitoring cell-ECM interactions and for surrogate organ transplantation.

Integrative Brain Transcriptome Analysis Reveals Region-Specific and Broad Molecular Changes in Shank3-Overexpressing Mice.

Variants of the SH3 and multiple ankyrin repeat domain 3 (SHANK3) gene, encoding excitatory postsynaptic core scaffolding proteins, are causally associated with numerous neurodevelopmental and neuropsychiatric disorders, including autism spectrum disorder (ASD), bipolar disorder, intellectual disability, and schizophrenia (SCZ). Although detailed synaptic changes of various Shank3 mutant mice have been well characterized, broader downstream molecular changes, including direct and indirect changes, remain largely unknown. To address this issue, we performed a transcriptome analysis of the medial prefrontal cortex (mPFC) of adult Shank3-overexpressing transgenic (TG) mice, using an RNA-sequencing approach. We also re-analyzed previously reported RNA-sequencing results of the striatum of adult Shank3 TG mice and of the prefrontal cortex of juvenile Shank3+/ΔC mice with a 50-70% reduction of Shank3 proteins. We found that several myelin-related genes were significantly downregulated specifically in the mPFC, but not in the striatum or hippocampus, of adult Shank3 TG mice by comparing the differentially expressed genes (DEGs) of the analyses side by side. Moreover, we also found nine common DEGs between the mPFC and striatum of Shank3 TG mice, among which we further characterized ASD- and SCZ-associated G protein-coupled receptor 85 (Gpr85), encoding an orphan Gpr interacting with PSD-95. Unlike the mPFC-specific decrease of myelin-related genes, we found that the mRNA levels of Gpr85 increased in multiple brain regions of adult Shank3 TG mice, whereas the mRNA levels of its family members, Gpr27 and Gpr173, decreased in the cortex and striatum. Intriguingly, in cultured neurons, the mRNA levels of Gpr27, Gpr85, and Gpr173 were modulated by the neuronal activity. Furthermore, exogenously expressed GPR85 was co-localized with PSD-95 and Shank3 in cultured neurons and negatively regulated the number of excitatory synapses, suggesting its potential role in homeostatic regulation of excitatory synapses in Shank3 TG neurons. Finally, we performed a gene set enrichment analysis of the RNA-sequencing results, which suggested that Shank3 could affect the directional expression pattern of numerous ribosome-related genes in a dosage-dependent manner. To sum up, these results reveal previously unidentified brain region-specific and broad molecular changes in Shank3-overexpressing mice, further elucidating the complexity of the molecular pathophysiology of SHANK3-associated brain disorders.

Characterization of the zinc-induced Shank3 interactome of mouse synaptosome.

Variants of the SHANK3 gene, which encodes a core scaffold protein of the postsynaptic density of excitatory synapses, have been causally associated with numerous brain disorders. Shank3 proteins directly bind zinc ions through their C-terminal sterile α motif domain, which enhances the multimerization and synaptic localization of Shank3, to regulate excitatory synaptic strength. However, no studies have explored whether zinc affects the protein interactions of Shank3, which might contribute to the synaptic changes observed after zinc application. To examine this, we first purified Shank3 protein complexes from mouse brain synaptosomal lysates that were incubated with different concentrations of ZnCl2, and analyzed them with mass spectrometry. We used strict criteria to identify 71 proteins that specifically interacted with Shank3 when extra ZnCl2 was added to the lysate. To characterize the zinc-induced Shank3 interactome, we performed various bioinformatic analyses that revealed significant associations of the interactome with subcellular compartments, including mitochondria, and brain disorders, such as bipolar disorder and schizophrenia. Together, our results showing that zinc affected the Shank3 protein interactions of in vitro mouse synaptosomes provided an additional link between zinc and core synaptic proteins that have been implicated in multiple brain disorders.

Phosphorylation of CYFIP2, a component of the WAVE-regulatory complex, regulates dendritic spine density and neurite outgrowth in cultured hippocampal neurons potentially by affecting the complex assembly.

Actin dynamics is a critical mechanism underlying many cellular processes in neurons. The heteropentameric WAVE-regulatory complex (WRC), consisting of WAVE, CYFIP1/2, Nap, Abi, and HSPC300, is a key regulator of actin dynamics that activates the Arp2/3 complex to initiate actin polymerization and branching. The WRC is basally inactive because of intermolecular interactions among the components, which can be modulated by bindings of phospholipids and Rac1, and phosphorylations of WAVE and Abi. However, the phosphorylation of other components of WRC and their functional significance remain largely unknown. To address this issue, we focused on CYFIP1/2, in which we found two brain-specific phosphorylation sites (S582 of CYFIP2 and T1068/T1067 of CYFIP1/2) from a publicly available phosphoproteome database. To understand their functional effects, we overexpressed wild-type, phospho-blocking, or phospho-mimetic mutants of CYFIP2 in cultured hippocampal neurons, and found that only T1067A CYFIP2 decreased the density of stubby spines. Moreover, overexpression of wild-type CYFIP2 increased neurite length, but T1067A did not exert this effect. To understand the mechanism, we modeled CYFIP2 phosphorylation in the crystal structure of WRC and found that T1067 phosphorylation could weaken the interaction between CYFIP2 and Nap1 by inducing conformational changes of CYFIP2 α-helical bundles. In the co-immunoprecipitation assay, however, wild-type, T1067A, and T1067E CYFIP2 showed similar interaction levels to Nap1, suggesting that T1067 phosphorylation alone is not sufficient to disrupt the interaction. Considering that the activation of WRC requires disassembly of the complex, our results suggest that T1067 phosphorylation, together with other factors, could contribute toward the activation process.

Localization of dynamin-related protein 1 and its potential role in lamellipodia formation.

Dynamin-related protein1 (Drp1) plays an essential role in mitochondrial fission: Cytosolic Drp1 is translocated to the mitochondria upon stimulus, and oligomerized Drp1 constricts mitochondria by aid of actin filaments. Drp1 completes the fission process with GTP hydrolysis by its own GTPase activity. The importance of actin filament and its interaction with Drp1 in the mitochondrial fission process have been demonstrated. In this study, we found that Drp1 is enriched in the actin-rich leading edge of lamellipodia of mouse embryonic fibroblasts (MEFs) wherein mitochondria or peroxisomes are absent. Mff-binding mutant (A395D) of Drp1, which cannot be recruited to mitochondria, was also localized in lamellipodia, indicating that Drp1 in lamellipodia is not related to mitochondria. When lamellipodia formation was induced by platelet-derived growth factor (PDGF) in MEFs, S616 phosphorylated form of Drp1 was accumulated to the lamellipodia. Inhibition of Drp1 with Mdivi-1 or a specific shRNA significantly decreased PDGF-induced lamellipodia formation or initial cell spreading during re-plating of the cells, respectively. Interestingly, defective lamellipodia formation and cell adhesion caused by Drp1 inhibition were not rescued by supplementing L-carnitine, although it restored mitochondrial energy loss caused by Drp1 inhibition. Collectively, these results favor the idea that Drp1 might play a significant role in lamellipodia formation and cell spreading through a different mechanism from that used for regulating mitochondrial dynamics/function.

Neuropilin 2 Signaling Is Involved in Cell Positioning of Adult-born Neurons through Glycogen Synthase Kinase-3ß (GSK3ß).

Proper positioning of neurons is fundamental for brain functions. However, little is known on how adult-born neurons generated in the hilar side of hippocampal dentate gyrus migrate into the granular cell layer. Because class 3 Semaphorins (Sema3) are involved in dendritic growth of these newborn neurons, we examined whether they are essential for cell positioning. We disrupted Sema3 signaling by silencing neuropilin 1 (NRP1) or 2 (NRP2), the main receptors for Sema3A and Sema3F, in neural progenitors of adult mouse dentate gyrus. Silencing of NRP2, but not NRP1, affected cell positioning of adult newborn neurons. Glycogen synthase kinase-3ß (GSK3ß) knockdown phenocopied this NRP2 silencing-mediated cell positioning defect, but did not affect dendritic growth. Furthermore, GSK3ß is activated upon stimulation with Sema3F, and GSK3ß overexpression rescued the cell positioning phenotypes seen in NRP2-deficient neurons. These results point to a new role for NRP2 in the positioning of neurons during adult hippocampal neurogenesis, acting via the GSK3ß signaling pathway.