Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

The mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) comprising the Period1, 2 (Per1, 2), Cryptochrome1, 2 (Cry1, 2), and Brain and Muscle ARNT-Like 1 (Bmal1) genes. The robustness of the TTFL is attributed to genetic redundancy among some essential clock genes, deterring genetic studies on molecular clocks using genome editing targeting single genes. To manipulate multiple clock genes in a streamlined and efficient manner, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for essential clock genes including Pers, Crys, or Bmal1. First, we tested several single guide RNAs (sgRNAs) targeting individual clock genes in silico and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and packaged into AAVs. CSAC efficiency was evident through protein downregulation in vitro and ablated molecular oscillation ex vivo. We also measured the efficiency of CSAC in vivo by assessing circadian rhythms after injecting CSAC into the suprachiasmatic nuclei of Cas9-expressing knock-in mice. Circadian locomotor activity and body temperature rhythms were severely disrupted in these mice, indicating that our CSAC is a simple yet powerful tool for investigating the molecular clock in vivo. © 2021, The Author(s).1

Optimization of Inkjet-Printed Seed Layer Based Flexible, Transparent Metal Electrode for Bio-Signal Sensing

A micro-electrode array (MEA) is essential in the bio-medical field to measure various bio-signals in vitro and in vivo environments. The transparent MEA allows imaging of cell surfaces and organs inside the body. Also, when we perform light-based modulation, such as optogenetics, higher efficiency in response to light can be obtained with a transparent MEA. Here, instead of well-known direct electrode material printing, we print a polymer seed layer that can induce the formation of transparent ultrathin (< 10 nm) metal electrodes with the merits of fabrication simplicity, low processing temperature, and design customizability. We optimized Au deposition thickness and metal film morphology to form conductive and transparent electrodes on selectively printed polymer seed layer regions. These electrodes show improved impedance at low frequencies compared to well-known thick Au-based electrodes. Finally, we successfully recorded brain signals in vivo by placing the flexible electrode array on the surface of the mouse brain.N

Optimization of Inkjet-Printed Seed Layer Based Flexible, Transparent Metal Electrode for Bio-Signal Sensing

A micro-electrode array (MEA) is essential in the bio-medical field to measure various bio-signals in vitro and in vivo environments. The transparent MEA allows imaging of cell surfaces and organs inside the body. Also, when we perform light-based modulation, such as optogenetics, higher efficiency in response to light can be obtained with a transparent MEA. Here, instead of well-known direct electrode material printing, we print a polymer seed layer that can induce the formation of transparent ultrathin (< 10 nm) metal electrodes with the merits of fabrication simplicity, low processing temperature, and design customizability. We optimized Au deposition thickness and metal film morphology to form conductive and transparent electrodes on selectively printed polymer seed layer regions. These electrodes show improved impedance at low frequencies compared to well-known thick Au-based electrodes. Finally, we successfully recorded brain signals in vivo by placing the flexible electrode array on the surface of the mouse brain. © 2023 IEEE

Presenilin 2 N141I Mutation Induces Hyperimmunity by Immune Cell-specific Suppression of REV-ERBα without Altering Central Circadian Rhythm

Circadian rhythm is a 24-hour cycle of behavioral and physiological changes. Disrupted sleep-wake patterns and circadian dysfunction are common in patients of Alzheimer Disease (AD) and are closely related with neuroinflammation. However, it is not well known how circadian rhythm of immune cells is altered during the progress of AD. Previously, we found presenilin 2 (Psen2 ) N141I mutation, one of familial AD (FAD) risk genes, induces hyperimmunity through the epigenetic repression of REV-ERBα expression in microglia and bone marrow-derived macrophage (BMDM) cells. Here, we investigated whether repression of REV-ERBα is associated with dysfunction of immune cell-endogenous or central circadian rhythm by analyses of clock genes expression and cytokine secretion, bioluminescence recording of rhythmic PER2::LUC expression, and monitoring of animal behavioral rhythm. Psen2 N141I mutation down-regulated REV-ERBα and induced selective over-production of IL-6 (a well-known clock-dependent cytokine) following the treatment of toll-like receptor (TLR) ligands in microglia, astrocytes, and BMDM. Psen2 N141I mutation also lowered amplitude of intrinsic daily oscillation in these immune cells representatives of brain and periphery. Of interest, however, the period of daily rhythm remained intact in immune cells. Furthermore, analyses of the central clock and animal behavioral rhythms revealed that central clock remained normal without down-regulation of REV-ERBα. These results suggest that Psen2 N141I mutation induces hyperimmunity mainly through the suppression of REV-ERBα in immune cells, which have lowered amplitude but normal period of rhythmic oscillation. Furthermore, our data reveal that central circadian clock is not affected by Psen2 N141I mutation. © The Korean Society for Brain and Neural SciencesTRU

Machine learning-based high-frequency neuronal spike reconstruction from low-frequency and low-sampling-rate recordings

Recording neuronal activity using multiple electrodes has been widely used to understand the functional mechanisms of the brain. Increasing the number of electrodes allows us to decode more variety of functionalities. However, handling massive amounts of multichannel electrophysiological data is still challenging due to the limited hardware resources and unavoidable thermal tissue damage. Here, we present machine learning (ML)-based reconstruction of high-frequency neuronal spikes from subsampled low-frequency band signals. Inspired by the equivalence between high-frequency restoration and super-resolution in image processing, we applied a transformer ML model to neuronal data recorded from both in vitro cultures and in vivo male mouse brains. Even with the x8 downsampled datasets, our trained model reasonably estimated high-frequency information of spiking activity, including spike timing, waveform, and network connectivity. With our ML-based data reduction applicable to existing multichannel recording hardware while achieving neuronal signals of broad bandwidths, we expect to enable more comprehensive analysis and control of brain functions. Multichannel neural recording enhances understanding of brain function, but handling large data is challenging. Here, the authors develop machine learning-based high frequency spike reconstruction from subsampled low-frequency neuronal signals.Y

Machine learning-based high-frequency neuronal spike reconstruction from low-frequency and low-sampling-rate recordings

&lt;p&gt;Supplementary Data for&lt;strong&gt; "Machine learning-based high-frequency neuronal spike reconstruction from low-frequency and low-sampling-rate recordings", Hong et al.&lt;/strong&gt;&lt;/p&gt

Long-term in-vivo recording performance of flexible penetrating microelectrode arrays

Objective. Neural interfaces are an essential tool to enable the human body to directly communicate with machines such as computers or prosthetic robotic arms. Since invasive electrodes can be located closer to target neurons, they have advantages such as precision in stimulation and high signal-To-noise ratio (SNR) in recording, while they often exhibit unstable performance in long-Term in-vivo implantation because of the tissue damage caused by the electrodes insertion. In the present study, we investigated the electrical functionality of flexible penetrating microelectrode arrays (FPMAs) up to 3 months in in-vivo conditions. Approach. The in-vivo experiment was performed by implanting FPMAs in five rats. The in-vivo impedance as well as the action potential (AP) amplitude and SNR were analyzed over weeks. Additionally, APs were tracked over time to investigate the possibility of single neuron recording. Main results. It was observed that the FPMAs exhibited dramatic increases in impedance for the first 4 weeks after implantation, accompanied by decreases in AP amplitude. However, the increase/decrease in AP amplitude was always accompanied by the increase/decrease in background noise, resulting in quite consistently maintained SNRs. After 4 weeks of implantation, we observed two distinctive issues regarding long-Term implantation, each caused by chronic tissue responses or by the delamination of insulation layer. The results demonstrate that the FPMAs successfully recorded neuronal signals up to 12 weeks, with very stably maintained SNRs, reduced by only 16.1% on average compared to the first recordings, although biological tissue reactions or physical degradation of the FPMA were present. Significance. The fabricated FPMAs successfully recorded intracortical signals for 3 months. The SNR was maintained up to 3 months and the chronic function of FPMA was comparable with other silicon based implantable electrodes. © 2021 Institute of Physics Publishing. All rights reserved.TRU

Presenilin 2 N141I mutation induces hyperactive immune response through the epigenetic repression of REV-ERB alpha

Hyperimmunity is associated with Alzheimer disease. Here the authors show that the Presenilin 2 N141I mutation causes overproduction of clock-controlled cytokines and memory deficits through suppression of REV-ERB alpha gene by hypermethylation. Hyperimmunity drives the development of Alzheimer disease (AD). The immune system is under the circadian control, and circadian abnormalities aggravate AD progress. Here, we investigate how an AD-linked mutation deregulates expression of circadian genes and induces cognitive decline using the knock-in (KI) mice heterozygous for presenilin 2 N141I mutation. This mutation causes selective overproduction of clock gene-controlled cytokines through the DNA hypermethylation-mediated repression of REV-ERB alpha in innate immune cells. The KI/+ mice are vulnerable to otherwise innocuous, mild immune challenges. The antipsychotic chlorpromazine restores the REV-ERB alpha level by normalizing DNA methylation through the inhibition of PI3K/AKT1 pathway, and prevents the overexcitation of innate immune cells and cognitive decline in KI/+ mice. These results highlight a pathogenic link between this AD mutation and immune cell overactivation through the epigenetic suppression of REV-ERB alpha.TRU