Preparation of human astrocytes with potent therapeutic functions from human pluripotent stem cells using ventral midbrain patterning.

INTRODUCTION: Astrocytes are glial-type cells that protect neurons from toxic insults and support neuronal functions and metabolism in a healthy brain. Leveraging these physiological functions, transplantation of astrocytes or their derivatives has emerged as a potential therapeutic approach for neurodegenerative disorders. METHODS: To substantiate the clinical application of astrocyte-based therapy, we aimed to prepare human astrocytes with potent therapeutic capacities from human pluripotent stem cells (hPSCs). To that end, we used ventral midbrain patterning during the differentiation of hPSCs into astrocytes, based on the roles of midbrain-specific factors in potentiating glial neurotrophic/anti-inflammatory activity. To assess the therapeutic effects of human midbrain-type astrocytes, we transplanted them into mouse models of Parkinson's disease (PD) and Alzheimer's disease (AD). RESULTS: Through a comprehensive series of in-vitro and in-vivo experiments, we were able to establish that the midbrain-type astrocytes exhibited the abilities to effectively combat oxidative stress, counter excitotoxic glutamate, and manage pathological protein aggregates. Our strategy for preparing midbrain-type astrocytes yielded promising results, demonstrating the strong therapeutic potential of these cells in various neurotoxic contexts. Particularly noteworthy is their efficacy in PD and AD-specific proteopathic conditions, in which the midbrain-type astrocytes outperformed forebrain-type astrocytes derived by the same organoid-based method. CONCLUSION: The enhanced functions of the midbrain-type astrocytes extended to their ability to release signaling molecules that inhibited neuronal deterioration and senescence while steering microglial cells away from a pro-inflammatory state. This success was evident in both in-vitro studies using human cells and in-vivo experiments conducted in mouse models of PD and AD. In the end, our human midbrain-type astrocytes demonstrated remarkable effectiveness in alleviating neurodegeneration, neuroinflammation, and the pathologies associated with the accumulation of α-synuclein and Amyloid ß proteins.

TRPC4 deletion elicits behavioral defects in sociability by dysregulating expression of microRNA-138-2.

To investigate whether the defects in transient receptor potential canonical 4 (TRPC4), which is strongly expressed in the hippocampus, are implicated in ASD, we examined the social behaviors of mice in which Trpc4 was deleted (Trpc4-/-). Trpc4-/- mice displayed the core symptoms of ASD, namely, social disability and repetitive behaviors. In microarray analysis of the hippocampus, microRNA (miR)-138-2, the precursor of miR-138, was upregulated in Trpc4-/- mice. We also found that binding of Matrin3 (MATR3), a selective miR-138-2 binding nuclear protein, to miR-138-2 was prominently enhanced, resulting in the downregulation of miR-138 in Trpc4-/- mice. Some parameters of the social defects and repetitive behaviors in the Trpc4-/- mice were rescued by increased miR-138 levels following miR-138-2 infusion in the hippocampus. Together, these results suggest that Trpc4 regulates some signaling components that oppose the development of social behavioral deficits through miR-138 and provide a potential therapeutic strategy for ASD.

Autistic-like social deficits in hippocampal MeCP2 knockdown rat models are rescued by ketamine.

Autism or autism spectrum disorder (ASD) is a behavioral syndrome characterized by persistent deficits in social interaction, and repetitive patterns of behavior, interests, or activities. The gene encoding Methyl-CpG binding protein 2 (MeCP2) is one of a few exceptional genes of established causal effect in ASD. Although genetically engineered mice studies may shed light on how MeCP2 loss affects synaptic activity patterns across the whole brain, such studies are not considered practical in ASD patients due to the overall level of impairment, and are technically challenging in mice. For the first time, we show that hippocampal MeCP2 knockdown produces behavioral abnormalities associated with autism-like traits in rats, providing a new strategy to investigate the efficacy of therapeutics in ASD. Ketamine, an N-Methyl-D-aspartate (NMDA) blocker, has been proposed as a possible treatment for autism. Using the MeCP2 knockdown rats in conjunction with a rat model of valproic acid (VPA)-induced ASD, we examined gene expression and ASD behaviors upon ketamine treatment. We report that the core symptoms of autism in MeCP2 knockdown rats with social impairment recovered dramatically following a single treatment with ketamine. [BMB Reports 2022; 55(5): 238-243].

Etoposide Induces Mitochondrial Dysfunction and Cellular Senescence in Primary Cultured Rat Astrocytes.

Brain aging is an inevitable process characterized by structural and functional changes and is a major risk factor for neurodegenerative diseases. Most brain aging studies are focused on neurons and less on astrocytes which are the most abundant cells in the brain known to be in charge of various functions including the maintenance of brain physical formation, ion homeostasis, and secretion of various extracellular matrix proteins. Altered mitochondrial dynamics, defective mitophagy or mitochondrial damages are causative factors of mitochondrial dysfunction, which is linked to age-related disorders. Etoposide is an anti-cancer reagent which can induce DNA stress and cellular senescence of cancer cell lines. In this study, we investigated whether etoposide induces senescence and functional alterations in cultured rat astrocytes. Senescence-associated ß-galactosidase (SA-ß-gal) activity was used as a cellular senescence marker. The results indicated that etoposide-treated astrocytes showed cellular senescence phenotypes including increased SA-ß-gal-positive cells number, increased nuclear size and increased senescence-associated secretory phenotypes (SASP) such as IL-6. We also observed a decreased expression of cell cycle markers, including Phospho- Histone H3/Histone H3 and CDK2, and dysregulation of cellular functions based on wound-healing, neuronal protection, and phagocytosis assays. Finally, mitochondrial dysfunction was noted through the determination of mitochondrial membrane potential using tetramethylrhodamine methyl ester (TMRM) and the measurement of mitochondrial oxygen consumption rate (OCR). These data suggest that etoposide can induce cellular senescence and mitochondrial dysfunction in astrocytes which may have implications in brain aging and neurodegenerative conditions.

Social Interaction Test in Home Cage as a Novel and Ethological Measure of Social Behavior in Mice.

Sociability is the disposition to interact with one another. Rodents have a rich repertoire of social behaviors and demonstrate strong sociability. Various methods have been established to measure the sociability of rodents in simple and direct ways, which includes reciprocal social interaction, juvenile social play, and three-chamber social tests. There are possible confounding factors while performing some of these tasks, such as aggression, avoidance of interaction by the stimulus mouse, exposure to a new environment, and lengthy procedures. The present study devised a method to complement these shortcomings and measure sociability as a group in the home cage setting, which prevents group-housed mice from isolation or exposure to a new environment. The home cage social test can allow high-throughput screening of social behaviors in a short amount of time. We developed two types of home cage setup: a home cage social target interaction test that measures sociability by putting the wire cage in the center area of the cage and a home cage two-choice sociability and social preference test that measures both sociability or social preference by putting cage racks at opposite sides of the cage. Interestingly, our results showed that the two types of home cage setup that we used in this study can extract abnormal social behaviors in various animal models, similar to the three-chamber assay. Thus, this study establishes a new and effective method to measure sociability or social preference that could be a complementary assay to evaluate the social behavior of mice in various setup conditions.

Pharmacological modulation of AMPA receptor rescues social impairments in animal models of autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, featuring social communication deficit and repetitive/restricted behaviors as common symptoms. Its prevalence has continuously increased, but, till now, there are no therapeutic approaches to relieve the core symptoms, particularly social deficit. In previous studies, abnormal function of the glutamatergic neural system has been proposed as a critical mediator and therapeutic target of ASD-associated symptoms. Here, we investigated the possible roles of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in autism symptoms using two well-known autistic animal models, Cntnap2 knockout (KO) mice and in utero valproic acid-exposed ICR (VPA) mice. We found that Cntnap2 KO mice displayed decreased glutamate receptor expression and transmission. Contrarily, VPA mice exhibited increased glutamate receptor expression and transmission. Next, we investigated whether AMPAR modulators (positive-allosteric-modulator for Cntnap2 KO mice and antagonist for VPA mice) can improve autistic symptoms by normalizing the aberrant excitatory transmission in the respective animal models. Interestingly, the AMPAR modulation specifically ameliorated social deficits in both animal models. These results indicated that AMPAR-derived excitatory neural transmission changes can affect normal social behavior. To validate this, we injected an AMPAR agonist or antagonist in control ICR mice and, interestingly, these treatments impaired only the social behavior, without affecting the repetitive and hyperactive behaviors. Collectively, these results provide insight into the role of AMPARs in the underlying pathophysiological mechanisms of ASD, and demonstrate that modulation of AMPAR can be a potential target for the treatment of social behavior deficits associated with ASD.

Tenovin-1 Induces Senescence and Decreases Wound-Healing Activity in Cultured Rat Primary Astrocytes.

Brain aging induces neuropsychological changes, such as decreased memory capacity, language ability, and attention; and is also associated with neurodegenerative diseases. However, most of the studies on brain aging are focused on neurons, while senescence in astrocytes has received less attention. Astrocytes constitute the majority of cell types in the brain and perform various functions in the brain such as supporting brain structures, regulating blood-brain barrier permeability, transmitter uptake and regulation, and immunity modulation. Recent studies have shown that SIRT1 and SIRT2 play certain roles in cellular senescence in peripheral systems. Both SIRT1 and SIRT2 inhibitors delay tumor growth in vivo without significant general toxicity. In this study, we investigated the role of tenovin-1, an inhibitor of SIRT1 and SIRT2, on rat primary astrocytes where we observed senescence and other functional changes. Cellular senescence usually is characterized by irreversible cell cycle arrest and induces senescence- associated ß-galactosidase (SA-ß-gal) activity. Tenovin-1-treated astrocytes showed increased SA-ß-gal-positive cell number, senescence-associated secretory phenotypes, including IL-6 and IL-1ß, and cell cycle-related proteins like phospho-histone H3 and CDK2. Along with the molecular changes, tenovin-1 impaired the wound-healing activity of cultured primary astrocytes. These data suggest that tenovin-1 can induce cellular senescence in astrocytes possibly by inhibiting SIRT1 and SIRT2, which may play particular roles in brain aging and neurodegenerative conditions.

Effects of Intraperitoneal N-methyl-D-aspartate (NMDA) Administration on Nociceptive/Repetitive Behaviors in Juvenile Mice.

Dysregulation of excitatory neurotransmission has been implicated in the pathogenesis of neuropsychiatric disorders. Pharmacological inhibition of N-methyl-D-aspartate (NMDA) receptors is widely used to model neurobehavioral pathologies and underlying mechanisms. There is ample evidence that overstimulation of NMDA-dependent neurotransmission may induce neurobehavioral abnormalities, such as repetitive behaviors and hypersensitization to nociception and cognitive disruption, pharmacological modeling using NMDA has been limited due to the induction of neurotoxicity and blood brain barrier breakdown, especially in young animals. In this study, we examined the effects of intraperitoneal NMDA-administration on nociceptive and repetitive behaviors in ICR mice. Intraperitoneal injection of NMDA induced repetitive grooming and tail biting/licking behaviors in a dose- and age-dependent manner. Nociceptive and repetitive behaviors were more prominent in juvenile mice than adult mice. We did not observe extensive blood brain barrier breakdown or neuronal cell death after peritoneal injection of NMDA, indicating limited neurotoxic effects despite a significant increase in NMDA concentration in the cerebrospinal fluid. These findings suggest that the observed behavioral changes were not mediated by general NMDA toxicity. In the hot plate test, we found that the latency of paw licking and jumping decreased in the NMDA-exposed mice especially in the 75 mg/kg group, suggesting increased nociceptive sensitivity in NMDA-treated animals. Repetitive behaviors and increased pain sensitivity are often comorbid in psychiatric disorders (e.g., autism spectrum disorder). Therefore, the behavioral characteristics of intraperitoneal NMDA-administered mice described herein may be valuable for studying the mechanisms underlying relevant disorders and screening candidate therapeutic molecules.

Sex-specific Behavioral Features of Rodent Models of Autism Spectrum Disorder.

Sex is an important factor in understanding the clinical presentation, management, and developmental trajectory of children with neuropsychiatric disorders. While much is known about the clinical and neurobehavioral profiles of males with neuropsychiatric disorders, surprisingly little is known about females in this respect. Animal models may provide detailed mechanistic information about sex differences in autism spectrum disorder (ASD) in terms of manifestation, disease progression, and development of therapeutic options. This review aims to widen our understanding of the role of sex in autism spectrum disorder, by summarizing and comparing behavioral characteristics of animal models. Our current understanding of how differences emerge in boys and girls with neuropsychiatric disorders is limited: Information derived from animal studies will stimulate future research on the role of biological maturation rates, sex hormones, sex-selective protective (or aggravating) factors and psychosocial factors, which are essential to devise sex precision medicine and to improve diagnostic accuracy. Moreover, there is a strong need of novel strategies to elucidate the major mechanisms leading to sex-specific autism features, as well as novel models or methods to examine these sex differences.

Social support rescues acute stress-induced cognitive impairments by modulating ERK1/2 phosphorylation in adolescent mice.

Social support can relieve stress-induced behavioural outcomes, although its underlying molecular mechanisms are not fully understood. Here, we evaluated whether social interactions can prevent the restraint stress (RS)-induced cognitive impairments in male adolescent mice by utilizing molecular, cellular, and behavioural approaches. Acute RS in adolescent ICR mice impaired the working memory in the Y-maze test and memory consolidation and retrieval in the novel-object-recognition test (NORT). In addition, RS increased the extracellular signal-regulated kinases 1/2 phosphorylation (p-ERK1/2) in the prefrontal cortex (PFC) and corticosterone levels in the plasma. Interestingly, these outcomes were normalized by the presence of a conspecific animal (social support) during RS. RS also significantly upregulated the expression levels of known stress-relevant genes such as Egr1, Crh, and Crhr1, which were normalized by social support. Systemic injection of SL327 (an inhibitor of MEK1/2 that also blocks its downstream signal ERK1/2) prior to RS rescued the working memory impairments and the increased p-ERK1/2 while normalizing the expression of Egr1. Our results suggest that social support can alleviate the RS-induced cognitive impairments partly by modulating ERK1/2 phosphorylation and gene transcription in the PFC, and provide novel insights into the molecular mechanisms of the stress-buffering effects of social support.

Modeling crash outcome probabilities at rural intersections: application of hierarchical binomial logistic models.

It is important to examine the nature of the relationships between roadway, environmental, and traffic factors and motor vehicle crashes, with the aim to improve the collective understanding of causal mechanisms involved in crashes and to better predict their occurrence. Statistical models of motor vehicle crashes are one path of inquiry often used to gain these initial insights. Recent efforts have focused on the estimation of negative binomial and Poisson regression models (and related deviants) due to their relatively good fit to crash data. Of course analysts constantly seek methods that offer greater consistency with the data generating mechanism (motor vehicle crashes in this case), provide better statistical fit, and provide insight into data structure that was previously unavailable. One such opportunity exists with some types of crash data, in particular crash-level data that are collected across roadway segments, intersections, etc. It is argued in this paper that some crash data possess hierarchical structure that has not routinely been exploited. This paper describes the application of binomial multilevel models of crash types using 548 motor vehicle crashes collected from 91 two-lane rural intersections in the state of Georgia. Crash prediction models are estimated for angle, rear-end, and sideswipe (both same direction and opposite direction) crashes. The contributions of the paper are the realization of hierarchical data structure and the application of a theoretically appealing and suitable analysis approach for multilevel data, yielding insights into intersection-related crashes by crash type.

The significance of endogeneity problems in crash models: an examination of left-turn lanes in intersection crash models.

Crash prediction models are used for a variety of purposes including forecasting the expected future performance of various transportation system segments with similar traits. The influence of intersection features on safety have been examined extensively because intersections experience a relatively large proportion of motor vehicle conflicts and crashes compared to other segments in the transportation system. The effects of left-turn lanes at intersections in particular have seen mixed results in the literature. Some researchers have found that left-turn lanes are beneficial to safety while others have reported detrimental effects on safety. This inconsistency is not surprising given that the installation of left-turn lanes is often endogenous, that is, influenced by crash counts and/or traffic volumes. Endogeneity creates problems in econometric and statistical models and is likely to account for the inconsistencies reported in the literature. This paper reports on a limited-information maximum likelihood (LIML) estimation approach to compensate for endogeneity between left-turn lane presence and angle crashes. The effects of endogeneity are mitigated using the approach, revealing the unbiased effect of left-turn lanes on crash frequency for a dataset of Georgia intersections. The research shows that without accounting for endogeneity, left-turn lanes 'appear' to contribute to crashes; however, when endogeneity is accounted for in the model, left-turn lanes reduce angle crash frequencies as expected by engineering judgment. Other endogenous variables may lurk in crash models as well, suggesting that the method may be used to correct simultaneity problems with other variables and in other transportation modeling contexts.