Guidelines for Manufacturing and Application of Organoids: Brain.

This study offers a comprehensive overview of brain organoids for researchers. It combines expert opinions with technical summaries on organoid definitions, characteristics, culture methods, and quality control. This approach aims to enhance the utilization of brain organoids in research. Brain organoids, as three-dimensional human cell models mimicking the nervous system, hold immense promise for studying the human brain. They offer advantages over traditional methods, replicating anatomical structures, physiological features, and complex neuronal networks. Additionally, brain organoids can model nervous system development and interactions between cell types and the microenvironment. By providing a foundation for utilizing the most human-relevant tissue models, this work empowers researchers to overcome limitations of two-dimensional cultures and conduct advanced disease modeling research.

Behavioral characterization of early nicotine withdrawal in the mouse: a potential model of acute dependence.

BACKGROUND: Clinical and preclinical research have demonstrated that short-term exposure to nicotine during the initial experimentation stage can lead to early manifestation of withdrawal-like signs, indicating the state of "acute dependence". As drug withdrawal is a major factor driving the progression toward regular drug intake, characterizing and understanding the features of early nicotine withdrawal may be important for the prevention and treatment of drug addiction. In this study, we corroborate the previous studies by showing that withdrawal-like signs can be precipitated after short-term nicotine exposure in mice, providing a potential animal model of acute dependence on nicotine. RESULTS: To model nicotine exposure from light tobacco use during the initial experimentation stage, mice were treated with 0.5 mg/kg (-)-nicotine ditartrate once daily for 3 days. On the following day, the behavioral tests were conducted after implementing spontaneous or mecamylamine-precipitated withdrawal. In the open field test, precipitated nicotine withdrawal reduced locomotor activity and time spent in the center zone. In the elevated plus maze test, the mecamylamine challenge increased the time spent in the closed arm and reduced the number of entries irrespective of nicotine experience. In the examination of the somatic aspect, precipitated nicotine withdrawal enhanced the number of somatic signs. Finally, nicotine withdrawal did not affect cognitive functioning or social behavior in the passive avoidance, spatial object recognition, or social interaction test. CONCLUSIONS: Collectively, our data demonstrate that early nicotine withdrawal-like signs could be precipitated by the nicotinic antagonist mecamylamine in mice, and that early withdrawal from nicotine primarily causes physical symptoms.

Striatal ZBTB16 Is Associated With Cognitive Deficits in Alzheimer Disease Mice.

PURPOSE: In Alzheimer disease (AD), brain regions such as the cortex and the hippocampus show abundant amyloid load which correlates with cognitive function decline. Prior to the significant development of AD pathophysiology, patients report the manifestation of neuropsychiatric symptoms, indicating a functional interplay between basal ganglia structures and hippocampal regions. Zinc finger and BTB domain-containing protein 16 (ZBTB16) is a transcription factor that controls the expression of downstream genes and the involvement of ZBTB16 in the striatum undergoing pathological aging in AD and the resulting behavioral phenotypes has not yet been explored. METHODS: To study molecular alterations in AD pathogenesis, we analyzed the brain from amyloid precursor protein (APP)/ presenilin 1 (PS1) transgenic mice. The molecular changes in the striatal region of the brain were analyzed via the immunoblotting, and the quantitative RNA sequencing. The cognitive impairments of APP/PS1 mice were assessed via 3 behavioral tests: 3-chamber test, Y-maze test, and noble object recognition test. And multielectrode array experiments for the analysis of the neuronal activity of the striatum in APP/PS1 mice was performed. RESULTS: We found that the alteration in ZBTB16 levels that occurred in the early ages of the pathologically aging striatum coalesces with the disruption of transcriptional dysregulation while causing social memory deficits, anxiety-like behavior. The early ZBTB16 knockdown treatment in the striatum of APP/PS1 mice rescued cognition that continued into later age. CONCLUSION: This study demonstrates that perturbation of transcriptional regulation of ZBTB16 during pathological aging may influence cognitive impairments and reveals a potent approach to targeting the transcriptional regulation of the striatum for the treatment of AD.

Quantitative Sequencing Analysis of the Striatal Transcriptome in a Mouse Model of Alzheimer Disease.

PURPOSE: The purpose of this study was to analyze the transcriptomic changes in the striatum of amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice and uncover its association with the methyl-CpG binding protein 2 (MeCP2) mediated-changes in striatal epigenetic signature during Alzheimer disease (AD) pathological progression. METHODS: To observe transcriptomic alterations in the striatum before the onset of cognitive impairment in APP/PS1 mice, quantitative 3'mRNA sequencing was performed with RNA extracted from the striatum of 6-month-old and 12-month-old wildtype and APP/PS1 mice. In addition, chromatin immunoprecipitation sequencing was conducted with the DNA from wildtype and APP/PS1 mice of the same age as aforementioned. For transcriptomic analysis, comparison terms were constructed based on aging and transgene expression-normal-aging (12-month-old wildtype/6-month-old wildtype), early-AD (6-month-old APP/PS1/6-month-old wildtype), and late-AD (12-month-old APP/PS1/6-month-old wildtype). To compare the changes in biological pathways and networks, we analyzed gene lists from each comparison term via bioinformatics tools including DAVID (Database for Annotation, Visualization, and Integrated Discovery), STRING (Search Tool for the Retrieval of Interacting Genes/Proteins), and SynGO (Synaptic Gene Ontologies). Furthermore, to assume the effect MeCP2 in AD pathological conditions may have on the transcriptome regulation, analysis of the common genes from Quant-Seq and MeCP2-ChIP-Seq was performed. RESULTS: Enriched pathways including immune system and inflammatory response were confirmed in normal- aging and lateAD, respectively. In particular, enriched pathways of gene expression regulation, transcriptional regulation, and protein catabolic pathways were found to be significantly altered in early-AD. MeCP2-bound genes that were significantly altered in the transcriptome were suggested to be target genes that have a role in the striatum of the early-stage AD model. CONCLUSION: This study confirmed that the alteration of the striatal transcriptomic profile in APP/PS1 mice was involved with several biological pathways. Additionally, comparative analysis of the transcriptomic changes and the MeCP2 bound regions found that a group of differentially expressed genes may be regulated under the epigenetic control of MeCP2.

SYNCRIP controls miR-137 and striatal learning in animal models of methamphetamine abstinence.

Abstinence from prolonged psychostimulant use prompts stimulant withdrawal syndrome. Molecular adaptations within the dorsal striatum have been considered the main hallmark of stimulant abstinence. Here we explored striatal miRNA-target interaction and its impact on circulating miRNA marker as well as behavioral dysfunctions in methamphetamine (MA) abstinence. We conducted miRNA sequencing and profiling in the nonhuman primate model of MA abstinence, followed by miRNA qPCR, LC-MS/MS proteomics, immunoassays, and behavior tests in mice. In nonhuman primates, MA abstinence triggered a lasting upregulation of miR-137 in the dorsal striatum but a simultaneous downregulation of circulating miR-137. In mice, aberrant increase in striatal miR-137-dependent inhibition of SYNCRIP essentially mediated the MA abstinence-induced reduction of circulating miR-137. Pathway modeling through experimental deduction illustrated that the MA abstinence-mediated downregulation of circulating miR-137 was caused by reduction of SYNCRIP-dependent miRNA sorting into the exosomes in the dorsal striatum. Furthermore, diminished SYNCRIP in the dorsal striatum was necessary for MA abstinence-induced behavioral bias towards egocentric spatial learning. Taken together, our data revealed circulating miR-137 as a potential blood-based marker that could reflect MA abstinence-dependent changes in striatal miR-137/SYNCRIP axis, and striatal SYNCRIP as a potential therapeutic target for striatum-associated cognitive dysfunction by MA withdrawal syndrome.

Adeno-associated virus (AAV) 9-mediated gene delivery of Nurr1 and Foxa2 ameliorates symptoms and pathologies of Alzheimer disease model mice by suppressing neuro-inflammation and glial pathology.

There is a compelling need to develop disease-modifying therapies for Alzheimer's disease (AD), the most common neuro-degenerative disorder. Together with recent progress in vector development for efficiently targeting the central nervous system, gene therapy has been suggested as a potential therapeutic modality to overcome the limited delivery of conventional types of drugs to and within the damaged brain. In addition, given increasing evidence of the strong link between glia and AD pathophysiology, therapeutic targets have been moving toward those addressing glial cell pathology. Nurr1 and Foxa2 are transcription/epigenetic regulators that have been reported to cooperatively regulate inflammatory and neurotrophic response in glial cells. In this study, we tested the therapeutic potential of Nurr1 and Foxa2 gene delivery to treat AD symptoms and pathologies. A series of functional, histologic, and transcriptome analyses revealed that the combined expression of Nurr1 and Foxa2 substantially ameliorated AD-associated amyloid ß and Tau proteinopathy, cell senescence, synaptic loss, and neuro-inflammation in multiple in vitro and in vivo AD models. Intra-cranial delivery of Nurr1 and Foxa2 genes using adeno-associated virus (AAV) serotype 9 improved the memory and cognitive function of AD model mice. The therapeutic benefits of gene delivery were attained mainly by correcting pathologic glial function. These findings collectively indicate that AAV9-mediated Nurr1 and Foxa2 gene transfer could be an effective disease-modifying therapy for AD.

Neuropathological and behavioral features of an APP/PS1/MAPT (6xTg) transgenic model of Alzheimer's disease.

Alzheimer's disease is associated with various brain dysfunctions, including memory impairment, neuronal loss, astrocyte activation, amyloid-ß plaques, and neurofibrillary tangles. Transgenic animal models of Alzheimer's disease have proven to be invaluable for the basic research of Alzheimer's disease. However, Alzheimer's disease mouse models developed so far do not fully recapitulate the pathological and behavioral features reminiscent of Alzheimer's disease in humans. Here, we investigated the neurobehavioral sequelae in the novel 6xTg mouse model of Alzheimer's disease, which was developed by incorporating human tau containing P301L mutation in the widely used 5xFAD mouse model of Alzheimer's disease. At 11-months-old, 6xTg mice displayed the core pathological processes found in Alzheimer's disease, including accumulation of amyloid-ß plaque, extensive neuronal loss, elevated level of astrocyte activation, and abnormal tau phosphorylation in the brain. At 9 to 11-months-old, 6xTg mice exhibited both cognitive and non-cognitive behavioral impairments relevant to Alzheimer's disease, including memory loss, hyperlocomotion, anxiety-like behavior, depression-like behavior, and reduced sensorimotor gating. Our data suggest that the aged 6xTg mouse model of Alzheimer's disease presents pathological and cognitive-behavioral features reminiscent of Alzheimer's disease in humans. Thus, the 6xTg mouse model of Alzheimer's disease may be a valuable model for studying Alzheimer's disease-relevant non-cognitive behaviors.

Dysfunction of striatal MeCP2 is associated with cognitive decline in a mouse model of Alzheimer's disease.

Rationale: Cerebral Methyl-CpG binding Protein 2 (MeCP2) is involved in several psychiatric disorders that are concomitant with cognitive dysfunction. However, the regulatory function of striatal MeCP2 and its association with Alzheimer's disease (AD) has been largely neglected due to the absence of amyloid plaque accumulation in the striatal region until the later stages of AD progression. Considerable evidence indicates that neuropsychiatric symptoms related to cognitive decline are involved with striatal dysfunction. To this respect, we investigated the epigenetic function of striatal MeCP2 paralleling the pathogenesis of AD. Methods: We investigated the brain from amyloid precursor protein (APP)/presenilin1 (PS1) transgenic mice and postmortem brain samples from normal subjects and AD patients. The molecular changes in the brain, particularly in the striatal regions, were analyzed with thioflavin S staining, immunohistochemistry, immunoblotting, and MeCP2 chromatin immunoprecipitation sequencing (ChIP-seq). The cognitive function of APP/PS1 mice was assessed via three behavioral tests: 3-chamber test (3CT), Y-maze test (YMT), and passive avoidance test (PA). A multi-electrode array (MEA) was performed to analyze the neuronal activity of the striatum in APP/PS1 mice. Results: Striatal MeCP2 expression was increased in the younger (6 months) and older (10 months) ages of APP/PS1 mice, and the genome-wide occupancy of MeCP2 in the younger APP/PS1 showed dysregulated binding patterns in the striatum. Additionally, we confirmed that APP/PS1 mice showed behavioral deficits in multiple cognitive behaviors. Notably, defective cognitive phenotypes and abnormal neuronal activity in old APP/PS1 mice were rescued through the knock-down of striatal MeCP2. Conclusion: We found that the MeCP2-mediated dysregulation of the epigenome in the striatum is linked to the defects in cognitive behavior and neuronal activity in the AD animal model, and that this alteration is initiated even in the very early stages of AD pathogenesis. Together, our data indicates that MeCP2 may be a potential target for the diagnosis and treatment of AD at asymptomatic and symptomatic stages.

Circulating microRNA miR-137 as a stable biomarker for methamphetamine abstinence.

OBJECTIVE: Stimulant use instigates abstinence syndrome in humans. miRNAs are a critical component for the pathophysiology of stimulant abstinence. Here we sought to identify a miRNA marker of methamphetamine abstinence in the circulating extracellular vesicles (cEVs). METHODS: miR-137 in the cEVs was quantified by qPCR in thirty-seven patients under methamphetamine abstinence and thirty-five age-matched healthy controls recruited from 2014 to 2016 from the general adult population in a hospital setting, Seoul, South Korea. Diagnostic power was evaluated by area under curve in the receiver-operating characteristics curve and other multiple statistical parameters. RESULTS: Patients under methamphetamine abstinence exhibited a significant reduction in cEV miR-137. Overall, cEV miR-137 had high potential as a blood-based marker of methamphetamine abstinence. cEV miR-137 retained the diagnostic power irrespective of the duration of methamphetamine abstinence or methamphetamine use. Interestingly, cEV miR-137 interacted with age: Control participants displayed an aging-dependent reduction of cEV miR-137, while methamphetamine-abstinent patients showed an aging-dependent increase in cEV miR-137. Accordingly, cEV miR-137 had variable diagnostic power depending on age, in which cEV miR-137 more effectively discriminated methamphetamine abstinence in the younger population. Duration of methamphetamine use or abstinence, cigarette smoking status, depressive disorder, or antidepressant treatment did not interact with the methamphetamine abstinence-induced reduction of cEV miR-137. CONCLUSION: Our data collectively demonstrated that miR-137 in the circulating extracellular vesicles held high potential as a stable and accurate diagnostic marker of methamphetamine abstinence syndrome.

Claustral MeCP2 Regulates Methamphetamine-induced Conditioned Place Preference in Cynomolgus Monkey.

The claustrum, a brain nucleus located between the cortex and the striatum, has recently been highlighted in drug-related reward processing. Methyl CpG-binding protein-2 (MeCP2) is a transcriptional regulator that represses or activates the expression of the target gene and has been known to have an important role in the regulation of drug addiction in the dopaminergic reward system. The claustrum is an important region for regulating reward processing where most neurons receive dopamine input; additionally, in this region, MeCP2 is also abundantly expressed. Therefore, here, we hypothesized that MeCP2 would be involved in drug addiction control in the Claustrum as well and investigated how claustral MeCP2 regulates drug addiction. To better understand the function of human claustral MeCP2, we established a non-human primate model of methamphetamine (METH) - induced conditioned place preference (CPP). After a habituation of two days and conditioning of ten days, the CPP test was conducted for three days. Interestingly, we confirmed that virus-mediated overexpression of MECP2 in the claustrum showed a significant reduction of METH-induced CPP in the three consecutive days during the testing period. Moreover, they showed a decrease in visit scores (frequency for visit) for the METH-paired room compared to the control group although the scores were statistically marginal. Taken together, we suggest that the claustrum is an important brain region associated with drug addiction, in which MeCP2 may function as a mediator in regulating the response to addictive drugs.

Subanesthetic ketamine rapidly alters medial prefrontal miRNAs involved in ubiquitin-mediated proteolysis.

Ketamine is a dissociative anesthetic and a non-competitive NMDAR antagonist. At subanesthetic dose, ketamine can relieve pain and work as a fast-acting antidepressant, but the underlying molecular mechanism remains elusive. This study aimed to investigate the mode of action underlying the effects of acute subanesthetic ketamine treatment by bioinformatics analyses of miRNAs in the medial prefrontal cortex of male C57BL/6J mice. Gene Ontology and KEGG pathway analyses of the genes putatively targeted by ketamine-responsive prefrontal miRNAs revealed that acute subanesthetic ketamine modifies ubiquitin-mediated proteolysis. Validation analysis suggested that miR-148a-3p and miR-128-3p are the main players responsible for the subanesthetic ketamine-mediated alteration of ubiquitin-mediated proteolysis through varied regulation of ubiquitin ligases E2 and E3. Collectively, our data imply that the prefrontal miRNA-dependent modulation of ubiquitin-mediated proteolysis is at least partially involved in the mode of action by acute subanesthetic ketamine treatment.

Disruption of the astrocyte-neuron interaction is responsible for the impairments in learning and memory in 5XFAD mice: an Alzheimer's disease animal model.

The morphological dynamics of astrocytes are altered in the hippocampus during memory induction. Astrocyte-neuron interactions on synapses are called tripartite synapses. These control the synaptic function in the central nervous system. Astrocytes are activated in a reactive state by STAT3 phosphorylation in 5XFAD mice, an Alzheimer's disease (AD) animal model. However, changes in astrocyte-neuron interactions in reactive or resting-state astrocytes during memory induction remain to be defined. Here, we investigated the time-dependent changes in astrocyte morphology and the number of astrocyte-neuron interactions in the hippocampus over the course of long-term memory formation in 5XFAD mice. Hippocampal-dependent long-term memory was induced using a contextual fear conditioning test in 5XFAD mice. The number of astrocytic processes increased in both wild-type and 5XFAD mice during memory formation. To assess astrocyte-neuron interactions in the hippocampal dentate gyrus, we counted the colocalization of glial fibrillary acidic protein and postsynaptic density protein 95 via immunofluorescence. Both groups revealed an increase in astrocyte-neuron interactions after memory induction. At 24 h after memory formation, the number of tripartite synapses returned to baseline levels in both groups. However, the total number of astrocyte-neuron interactions was significantly decreased in 5XFAD mice. Administration of Stattic, a STAT3 phosphorylation inhibitor, rescued the number of astrocyte-neuron interactions in 5XFAD mice. In conclusion, we suggest that a decreased number of astrocyte-neuron interactions may underlie memory impairment in the early stages of AD.

Chronic nicotine impairs sparse motor learning via striatal fast-spiking parvalbumin interneurons.

Nicotine can diversely affect neural activity and motor learning in animals. However, the impact of chronic nicotine on striatal activity in vivo and motor learning at long-term sparse timescale remains unknown. Here, we demonstrate that chronic nicotine persistently suppresses the activity of striatal fast-spiking parvalbumin interneurons, which mediate nicotine-induced deficit in sparse motor learning. Six weeks of longitudinal in vivo single-unit recording revealed that mice show reduced activity of fast-spiking interneurons in the dorsal striatum during chronic nicotine exposure and withdrawal. The reduced firing of fast-spiking interneurons was accompanied by spike broadening, diminished striatal delta oscillation power, and reduced sample entropy in local field potential. In addition, chronic nicotine withdrawal impaired motor learning with a weekly sparse training regimen but did not affect general locomotion and anxiety-like behavior. Lastly, the excitatory DREADD hM3Dq-mediated activation of striatal fast-spiking parvalbumin interneurons reversed the chronic nicotine withdrawal-induced deficit in sparse motor learning. Taken together, we identified that chronic nicotine withdrawal impairs sparse motor learning via disruption of activity in striatal fast-spiking parvalbumin interneurons. These findings suggest that sparse motor learning paradigm can reveal the subtle effect of nicotine withdrawal on motor function and that striatal fast-spiking parvalbumin interneurons are a neural substrate of nicotine's effect on motor learning.

TMEM16A expression in cholinergic neurons of the medial habenula mediates anxiety-related behaviors.

TMEM16A, a Ca2+ -activated Cl- channel, is known to modulate the excitability of various types of cells; however, its function in central neurons is largely unknown. Here, we show the specific expression of TMEM16A in the medial habenula (mHb) via RNAscope in situ hybridization, immunohistochemistry, and electrophysiology. When TMEM16A is ablated in the mHb cholinergic neurons (TMEM16A cKO mice), the slope of after-hyperpolarization of spontaneous action potentials decreases and the firing frequency is reduced. Reduced mHb activity also decreases the activity of the interpeduncular nucleus (IPN). Moreover, TMEM16A cKO mice display anxiogenic behaviors and deficits in social interaction without despair-like phenotypes or cognitive dysfunctions. Finally, chemogenetic inhibition of mHb cholinergic neurons using the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) approach reveals similar behavioral phenotypes to those of TMEM16A cKO mice. We conclude that TMEM16A plays a key role in anxiety-related behaviors regulated by mHb cholinergic neurons and could be a potential therapeutic target against anxiety-related disorders.

Neutrophil-lymphocyte ratio as a valuable prognostic marker in idiopathic sudden sensorineural hearing loss.

Background: Neutrophil-to-lymphocyte ratio (NLR) has emerged as a marker for degree of inflammation. Inflammation has been regarded as one of the causes of idiopathic sudden sensorineural hearing loss.Objectives: This study investigated the potential association between neutrophil-to-lymphocyte ratio (NLR) and prognosis in patients with idiopathic sudden sensorineural hearing loss (ISSNHL).Material and methods: This retrospective, case-control clinical trial analyzed data from 186 subjects diagnosed with ISSNHL and admitted to Kyung-Hee Medical Center between March 2016 and February 2019. Hematologic and audiometric parameters were measured.Results: NLR showed a linear correlation with hearing recovery in patients with ISSNHL, with hearing gain (dB) = 56.698 - 3.718 × NLR (r2 = 0.451, p = .001). Hearing recovery at all frequencies was numerically higher in patients with low (<6.661) than higher (≥6.661) NLR at all frequencies and was significantly higher at 500, 1000, 2000, 3000, and 4000 Hz. Hearing thresholds at 250, 500, 1000, 2000, 3000, and 4000 Hz in the low NLR group were significantly lower after treatment.Conclusions and significance: The ability to recover from ISSNHL decreases as NLR increases. NLR may be a valuable prognostic marker in patients with ISSNHL.

Methyl-CpG Binding Protein 2 in Alzheimer Dementia.

Despite decades of research on Alzheimer disease, understanding the complexity of the genetic and molecular interactions involved in its pathogenesis remains far from our grasp. Methyl-CpG Binding Protein 2 (MeCP2) is an important epigenetic regulator enriched in the brain, and recent findings have implicated MeCP2 as a crucial player in Alzheimer disease. Here, we provide comprehensive insights into the pathophysiological roles of MeCP2 in Alzheimer disease. In particular, we focus on how the alteration of MeCP2 expression can impact Alzheimer disease through risk genes, amyloid-ß and tau pathology, cell death and neurodegeneration, and cellular senescence. We suggest that Alzheimer disease can be adversely affected by upregulated MeCP2-dependent repression of risk genes (MEF2C, ADAM10, and PM20D1), increased tau accumulation, and neurodegeneration through neuronal cell death (excitotoxicity and apoptosis). In addition, we propose that the progression of Alzheimer disease could be caused by reduced MeCP2-mediated enhancement of astrocytic and microglial senescence and consequent glial SASP (senescence-associated secretory phenotype)-dependent neuroinflammation. We surmise that any imbalance in MeCP2 function would accelerate or cause Alzheimer disease pathogenesis, implying that MeCP2 may be a potential drug target for the treatment and prevention of Alzheimer disease.