N1-methylation of adenosine (m1A) in ND5 mRNA leads to complex I dysfunction in Alzheimer's disease.

One mechanism of particular interest to regulate mRNA fate post-transcriptionally is mRNA modification. Especially the extent of m1A mRNA methylation is highly discussed due to methodological differences. However, one single m1A site in mitochondrial ND5 mRNA was unanimously reported by different groups. ND5 is a subunit of complex I of the respiratory chain. It is considered essential for the coupling of oxidation and proton transport. Here we demonstrate that this m1A site might be involved in the pathophysiology of Alzheimer's disease (AD). One of the pathological hallmarks of this neurodegenerative disease is mitochondrial dysfunction, mainly induced by Amyloid ß (Aß). Aß mainly disturbs functions of complex I and IV of the respiratory chain. However, the molecular mechanism of complex I dysfunction is still not fully understood. We found enhanced m1A methylation of ND5 mRNA in an AD cell model as well as in AD patients. Formation of this m1A methylation is catalyzed by increased TRMT10C protein levels, leading to translation repression of ND5. As a consequence, here demonstrated for the first time, TRMT10C induced m1A methylation of ND5 mRNA leads to mitochondrial dysfunction. Our findings suggest that this newly identified mechanism might be involved in Aß-induced mitochondrial dysfunction.

A bidirectional link between sulfatide and Alzheimer's disease.

Reduced sulfatide level is found in Alzheimer's disease (AD) patients. Here, we demonstrate that amyloid precursor protein (APP) processing regulates sulfatide synthesis and vice versa. Different cell culture models and transgenic mice models devoid of APP processing or in particular the APP intracellular domain (AICD) reveal that AICD decreases Gal3st1/CST expression and subsequently sulfatide synthesis. In return, sulfatide supplementation decreases Aß generation by reducing ß-secretase (BACE1) and γ-secretase processing of APP. Increased BACE1 lysosomal degradation leads to reduced BACE1 protein level in endosomes. Reduced γ-secretase activity is caused by a direct effect on γ-secretase activity and reduced amounts of γ-secretase components in lipid rafts. Similar changes were observed by analyzing cells and mice brain samples deficient of arylsulfatase A responsible for sulfatide degradation or knocked down in Gal3st1/CST. In line with these findings, addition of sulfatides to brain homogenates of AD patients resulted in reduced γ-secretase activity. Human brain APP level shows a significant negative correlation with GAL3ST1/CST expression underlining the in vivo relevance of sulfatide homeostasis in AD.

Comparison of Frailty and Chronological Age as Determinants of the Murine Gut Microbiota in an Alzheimer's Disease Mouse Model.

The ageing of an organism is associated with certain features of functional decline that can be assessed at the cellular level (e.g., reduced telomere length, loss of proteostasis, etc.), but also at the organismic level. Frailty is an independent syndrome that involves increased multidimensional age-related deficits, heightens vulnerability to stressors, and involves physical deficits in mainly the locomotor/muscular capacity, but also in physical appearance and cognition. For sporadic Alzheimer's disease, age per se is one of the most relevant risk factors, but frailty has also been associated with this disease. Therefore, we aimed to answer the two following questions within a cross-sectional study: (1) do Alzheimer's model mice show increased frailty, and (2) what changes of the microbiota occur concerning chronological age or frailty? Indeed, aged 5xFAD mice showed increased frailty compared to wild type littermates. In addition, 5xFAD mice had significantly lower quantities of Bacteroides spp. when only considering frailty, and lower levels of Bacteroidetes in terms of both frailty and chronological age compared to their wild type littermates. Thus, the quality of ageing-as assessed by frailty measures-should be taken into account to unravel potential changes in the gut microbial community in Alzheimer's disease.

Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) originating from the central nervous system (CNS) can enter the blood stream and carry molecules characteristic of disease states. Therefore, circulating CNS-derived EVs have the potential to serve as liquid-biopsy markers for early diagnosis and follow-up of neurodegenerative diseases and brain tumors. Monitoring and profiling of CNS-derived EVs using multiparametric analysis would be a major advance for biomarker as well as basic research. Here, we explored the performance of a multiplex bead-based flow-cytometry assay (EV Neuro) for semi-quantitative detection of CNS-derived EVs in body fluids. METHODS: EVs were separated from culture of glioblastoma cell lines (LN18, LN229, NCH82) and primary human astrocytes and measured at different input amounts in the MACSPlex EV Kit Neuro, human. In addition, EVs were separated from blood samples of small cohorts of glioblastoma (GB), multiple sclerosis (MS) and Alzheimer's disease patients as well as healthy controls (HC) and subjected to the EV Neuro assay. To determine statistically significant differences between relative marker signal intensities, an unpaired samples t-test or Wilcoxon rank sum test were computed. Data were subjected to tSNE, heatmap clustering, and correlation analysis to further explore the relationships between disease state and EV Neuro data. RESULTS: Glioblastoma cell lines and primary human astrocytes showed distinct EV profiles. Signal intensities were increasing with higher EV input. Data normalization improved identification of markers that deviate from a common profile. Overall, patient blood-derived EV marker profiles were constant, but individual EV populations were significantly increased in disease compared to healthy controls, e.g. CD36+EVs in glioblastoma and GALC+EVs in multiple sclerosis. tSNE and heatmap clustering analysis separated GB patients from HC, but not MS patients from HC. Correlation analysis revealed a potential association of CD107a+EVs with neurofilament levels in blood of MS patients and HC. CONCLUSIONS: The semi-quantitative EV Neuro assay demonstrated its utility for EV profiling in complex samples. However, reliable statistical results in biomarker studies require large sample cohorts and high effect sizes. Nonetheless, this exploratory trial confirmed the feasibility of discovering EV-associated biomarkers and monitoring circulating EV profiles in CNS diseases using the EV Neuro assay. Video Abstract.

Extracellular vesicles (EVs) are tiny particles released by cells, carrying unique biomolecules specific to their cell of origin. EVs from the central nervous system (CNS) can reach the blood, where they could serve as liquid-biopsy markers for diagnosing brain diseases like neurodegenerative disorders and tumors. This study evaluated a flow cytometry platform (here termed EV Neuro assay), which can detect multiple EV-associated markers simultaneously, to assess its potential for identifying CNS-derived EVs and disease-specific markers in complex samples including the blood. The study compared different sample materials and methods for isolating EVs. We found distinct EV profiles in EVs derived from glioblastoma and human astrocytes, with signal intensities increasing as more EVs were present. Analyzing serum or plasma from patients with brain diseases and healthy individuals, we observed that EV marker intensities were varying between individuals. Importantly, data normalization improved the identification of disease-specific markers, such as CD36⁺EVs in glioblastoma and GALC⁺EVs in multiple sclerosis, which were significantly higher in disease compared to healthy controls. Advanced clustering analysis techniques effectively distinguished glioblastoma patients from controls. Furthermore, a potential correlation between CD107a⁺EVs and neurofilament levels in multiple sclerosis patients was discovered. Overall, the semi-quantitative EV Neuro assay proved useful for profiling EVs in complex samples. However, for more reliable results in biomarker studies, larger sample cohorts and higher effect sizes are necessary. Nonetheless, this initial trial confirmed the potential of the EV Neuro assay for discovering disease-associated EV markers and monitoring circulating EV profiles in CNS diseases.

Talk to Me-Interplay between Mitochondria and Microbiota in Aging.

The existence of mitochondria in eukaryotic host cells as a remnant of former microbial organisms has been widely accepted, as has their fundamental role in several diseases and physiological aging. In recent years, it has become clear that the health, aging, and life span of multicellular hosts are also highly dependent on the still-residing microbiota, e.g., those within the intestinal system. Due to the common evolutionary origin of mitochondria and these microbial commensals, it is intriguing to investigate if there might be a crosstalk based on preserved common properties. In the light of rising knowledge on the gut-brain axis, such crosstalk might severely affect brain homeostasis in aging, as neuronal tissue has a high energy demand and low tolerance for according functional decline. In this review, we summarize what is known about the impact of both mitochondria and the microbiome on the host's aging process and what is known about the aging of both entities. For a long time, bacteria were assumed to be immortal; however, recent evidence indicates their aging and similar observations have been made for mitochondria. Finally, we present pathways by which mitochondria are affected by microbiota and give information about therapeutic anti-aging approaches that are based on current knowledge.

Commensal bacteria weaken the intestinal barrier by suppressing epithelial neuropilin-1 and Hedgehog signaling.

The gut microbiota influences intestinal barrier integrity through mechanisms that are incompletely understood. Here we show that the commensal microbiota weakens the intestinal barrier by suppressing epithelial neuropilin-1 (NRP1) and Hedgehog (Hh) signaling. Microbial colonization of germ-free mice dampens signaling of the intestinal Hh pathway through epithelial Toll-like receptor (TLR)-2, resulting in decreased epithelial NRP1 protein levels. Following activation via TLR2/TLR6, epithelial NRP1, a positive-feedback regulator of Hh signaling, is lysosomally degraded. Conversely, elevated epithelial NRP1 levels in germ-free mice are associated with a strengthened gut barrier. Functionally, intestinal epithelial cell-specific Nrp1 deficiency (Nrp1ΔIEC) results in decreased Hh pathway activity and a weakened gut barrier. In addition, Nrp1ΔIEC mice have a reduced density of capillary networks in their small intestinal villus structures. Collectively, our results reveal a role for the commensal microbiota and epithelial NRP1 signaling in the regulation of intestinal barrier function through postnatal control of Hh signaling.

The Aging Enteric Nervous System.

The gut and the brain communicate via the nervous system, hormones, microbiota-mediated substances, and the immune system. These intricate interactions have led to the term "gut-brain axis". Unlike the brain-which is somewhat protected-the gut is exposed to a variety of factors throughout life and, consequently, might be either more vulnerable or better adapted to respond to these challenges. Alterations in gut function are common in the elder population and associated with many human pathologies, including neurodegenerative diseases. Different studies suggest that changes in the nervous system of the gut, the enteric nervous system (ENS), during aging may result in gastrointestinal dysfunction and initiate human pathologies of the brain via its interconnection with the gut. This review aims at summarizing the contribution of normal cellular aging to the age-associated physiological changes of the ENS. Morphological alterations and degeneration of the aging ENS are observed in different animal models and humans, albeit with considerable variability. The aging phenotypes and pathophysiological mechanisms of the aging ENS have highlighted the involvement of enteric neurons in age-related diseases of the central nervous system such as Alzheimer's or Parkinson's disease. To further elucidate such mechanisms, the ENS constitutes a promising source of material for diagnosis and therapeutic predictions, as it is more accessible than the brain.

Behavioral Phenotyping of Bbs6 and Bbs8 Knockout Mice Reveals Major Alterations in Communication and Anxiety.

The primary cilium is an organelle with a central role in cellular signal perception. Mutations in genes that encode cilia-associated proteins result in a collection of human syndromes collectively termed ciliopathies. Of these, the Bardet-Biedl syndrome (BBS) is considered one of the archetypical ciliopathies, as patients exhibit virtually all respective clinical phenotypes, such as pathological changes of the retina or the kidney. However, the behavioral phenotype associated with ciliary dysfunction has received little attention thus far. Here, we extensively characterized the behavior of two rodent models of BBS, Bbs6/Mkks, and Bbs8/Ttc8 knockout mice concerning social behavior, anxiety, and cognitive abilities. While learning tasks remained unaffected due to the genotype, we observed diminished social behavior and altered communication. Additionally, Bbs knockout mice displayed reduced anxiety. This was not due to altered adrenal gland function or corticosterone serum levels. However, hypothalamic expression of Lsamp, the limbic system associated protein, and Adam10, a protease acting on Lsamp, were reduced. This was accompanied by changes in characteristics of adult hypothalamic neurosphere cultures. In conclusion, we provide evidence that behavioral changes in Bbs knockout mice are mainly found in social and anxiety traits and might be based on an altered architecture of the hypothalamus.

The Influence of Acitretin on Brain Lipidomics in Adolescent Mice-Implications for Pediatric and Adolescent Dermatological Therapy.

Administration of systemic retinoids such as acitretin has not been approved yet for pediatric patients. An adverse event of retinoid-therapy that occurs with lower prevalence in children than in adults is hyperlipidemia. This might be based on the lack of comorbidities in young patients, but must not be neglected. Especially for the development of the human brain up to young adulthood, dysbalance of lipids might be deleterious. Here, we provide for the first time an in-depth analysis of the influence of subchronic acitretin-administration on lipid composition of brain parenchyma of young wild type mice. For comparison and to evaluate the systemic effect of the treatment, liver lipids were analogously investigated. As expected, triglycerides increased in liver as well as in brain and a non-significant increase in cholesterol was observed. However, specifically brain showed an increase in lyso-phosphatidylcholine and carnitine as well as in sphingomyelin. Group analysis of lipid classes revealed no statistical effects, while single species were tissue-dependently changed: effects in brain were in general more subtly as compared to those in liver regarding the mere number of changed lipid species. Thus, while the overall impact of acitretin seems comparably small regarding brain, the change in individual species and their role in brain development and maturation has to be considered.

The Hidden Role of Non-Canonical Amyloid ß Isoforms in Alzheimer's Disease.

Recent advances have placed the pro-inflammatory activity of amyloid ß (Aß) on microglia cells as the focus of research on Alzheimer's Disease (AD). Researchers are confronted with an astonishing spectrum of over 100 different Aß variants with variable length and chemical modifications. With the exception of Aß1-42 and Aß1-40, the biological significance of most peptides for AD is as yet insufficiently understood. We therefore aim to provide a comprehensive overview of the contributions of these neglected Aß variants to microglia activation. First, the impact of Aß receptors, signaling cascades, scavenger mechanisms, and genetic variations on the physiological responses towards various Aß species is described. Furthermore, we discuss the importance of different types of amyloid precursor protein processing for the generation of these Aß variants in microglia, astrocytes, oligodendrocytes, and neurons, and highlight how alterations in secondary structures and oligomerization affect Aß neurotoxicity. In sum, the data indicate that gene polymorphisms in Aß-driven signaling pathways in combination with the production and activity of different Aß variants might be crucial factors for the initiation and progression of different forms of AD. A deeper assessment of their interplay with glial cells may pave the way towards novel therapeutic strategies for individualized medicine.

Amyloid beta and its naturally occurring N-terminal variants are potent activators of human and mouse formyl peptide receptor 1.

Formyl peptide receptors (FPRs) may contribute to inflammation in Alzheimer's disease through interactions with neuropathological Amyloid beta (Aß) peptides. Previous studies reported activation of FPR2 by Aß1-42, but further investigation of other FPRs and Aß variants is needed. This study provides a comprehensive overview of the interactions of mouse and human FPRs with different physiologically relevant Aß-peptides using transiently transfected cells in combination with calcium imaging. We observed that, in addition to hFPR2, all other hFPRs also responded to Aß1-42, Aß1-40, and the naturally occurring variants Aß11-40 and Aß17-40. Notably, Aß11-40 and Aß17-40 are very potent activators of mouse and human FPR1, acting at nanomolar concentrations. Buffer composition and aggregation state are extremely crucial factors that critically affect the interaction of Aß with different FPR subtypes. To investigate the physiological relevance of these findings, we examined the effects of Aß11-40 and Aß17-40 on the human glial cell line U87. Both peptides induced a strong calcium flux at concentrations that are very similar to those obtained in experiments for hFPR1 in HEK cells. Further immunocytochemistry, qPCR, and pharmacological experiments verified that these responses were primarily mediated through hFPR1. Chemotaxis experiments revealed that Aß11-40 but not Aß17-40 evoked cell migration, which argues for a functional selectivity of different Aß peptides. Together, these findings provide the first evidence that not only hFPR2 but also hFPR1 and hFPR3 may contribute to neuroinflammation in Alzheimer's disease through an interaction with different Aß variants.

Targeting gut microbiota to alleviate neuroinflammation in Alzheimer's disease.

The gut microbiota came into focus within the last years regarding being associated with or even underlying neuropsychiatric diseases. The existence of the gut-brain-axis makes it highly plausible that bacterial metabolites or toxins that escape the intestinal environment or approach the vagal connections towards the brain, exert devastating effects on the central nervous system. In Alzheimer's disease (AD), growing evidence for dysbiotic changes in the gut microbiota is obtained, even though the question for cause or consequence remains open. Nevertheless, using modulation of microbiota to address inflammatory processes seems an attractive therapeutic approach as certain microbial products such as short chain fatty acids have been proven to exert beneficial cognitive effects. In this review, we summarize, contemporary knowledge on neuroinflammation and inflammatory processes within the brain and even more detailed in the gut in AD, try to conclude whom to target regarding human microbial commensals and report on current interventional trials.

Psychobiological Evaluation of Day Clinic Treatment for People Living With Dementia - Feasibility and Pilot Analyses.

Background: Hospitalization is often stressful and burdensome for people living with dementia (PwD) and their informal caregivers (ICs). Day clinic treatment may provide a suitable alternative, but is often precluded by a diagnosis of dementia. Furthermore, it is often caregiver-based ratings that measure treatment success as the validity of self-reports in PwD is critically discussed. We therefore set out to examine the feasibility of psychobiological stress measures in PwD and ICs and to evaluate treatment trajectories considering both the day clinic context and the daily life of the dyads. Method: A total of 40 dyads of PwD (mean age: 78.15 ± 6.80) and their ICs (mean age: 63.85 ± 13.09) completed paper-and-pencil questionnaires (covering stress, depressive symptoms, and caregiver burden among others) in addition to the measurement of hair cortisol concentrations (HCC) at admission, discharge, and follow-up 6 months after day clinic treatment. As part of an ambulatory assessment, for 2 days at the beginning and 2 days at the end of the day clinic treatment, PwD and ICs collected six saliva samples per day for the analysis of salivary cortisol (sCort) and alpha-amylase (sAA). Results: Paper-and-pencil questionnaires and HCC assessments were more feasible than the ambulatory assessment. We found discrepancies between subjective and physiological markers of stress in PwD. Whereas HCC decreased over time, self-reported stress increased. Child-parent dyads reported decreases in neuropsychiatric symptoms, associated burden, and self-reported stress from admission to follow-up. In daily life, both PwD and ICs showed characteristic diurnal profiles of sAA and sCort, however, we found no differences in summary indicators of salivary stress markers over time. Discussion: The psychobiological evaluation was feasible and added informative value, underlining the potential of physiological stress markers to complement self-reports on stress in PwD and to objectively evaluate treatment trajectories. In this sample, HCC was more feasible and acceptable as biological marker of stress compared to saliva samples. Concerning treatment trajectories, differential effects on the dyads were found, with child-parent dyads benefiting more from day clinic treatment compared to spousal dyads.

Resilience and the Gut Microbiome: Insights from Chronically Socially Stressed Wild-Type Mice.

The microbiome is an important player within physiological homeostasis of the body but also in pathophysiological derailments. Chronic social stress is a challenge to the organism, which results in psychological illnesses such as depression in some individuals and can be counterbalanced by others, namely resilient individuals. In this study, we wanted to elucidate the potential contribution of the microbiome to promote resilience. Male mice were subjected to the classical chronic social defeat paradigm. Defeated or undefeated mice were either controls (receiving normal drinking water) or pre-treated with antibiotics or probiotics. Following social defeat, resilient behavior was assessed by means of the social interaction test. Neither depletion nor probiotic-shifted alteration of the microbiome influenced stress-associated behavioral outcomes. Nevertheless, clear changes in microbiota composition due to the defeat stress were observed such as elevated Bacteroides spp. This stress-induced increase in Bacteroides in male mice could be confirmed in a related social stress paradigm (instable social hierarchy) in females. This indicates that while manipulation of the microbiome via the antibiotics- and probiotics-treatment regime used here has no direct impact on modulating individual stress susceptibility in rodents, it clearly affects the microbiome in the second line and in a sex-independent manner regarding Bacteroides.

Psychobiological Monitoring of a Home-Based Dyadic Intervention for People Living with Dementia and Their Caregivers: Added Value to Evaluate Treatment Success and Understand Underlying Mechanisms.

BACKGROUND: Research concerning people living with dementia (PwD) and their informal caregivers (ICs) has recently begun to focus on dyadic aspects of psychosocial interventions. OBJECTIVE: We adapted a dyadic psychosocial intervention and examined its effects on psychobiological stress in daily life. METHODS: Twenty-four PwD-caregiver dyads were visited seven times at home by specialized nursing staff. Momentary subjective stress, salivary cortisol (sCort), and salivary alpha-amylase (sAA) were measured in PwD and ICs before and after each home visit as well as six times per day at two days each at the beginning and end of the intervention as part of an ambulatory assessment. Hair cortisol concentrations (HCC) were measured twice. RESULTS: After each home visit session, ICs reported lower subjective stress. sCort was lower in both ICs and PwD, whereas sAA did not change. In daily life, area under the curve (AUCg) concerning sCort secretion indicated that PwD had lower sCort daily output at the end of the intervention, and AUCg concerning subjective stress indicated that both PwD and ICs reported lower subjective stress than at the beginning of the intervention. AUCg concerning sAA did not change over time in either group. HCC did not vary over time but increased with disease severity. CONCLUSION: The psychosocial intervention reduced psychobiological stress but affected psychobiological stress measures differently in PwD and ICs. In particular, the discrepancy between subjective and physiological markers of stress in PwD emphasizes the added value to evaluate treatment success and understand underlying mechanisms as a complement to self-reports.

Selective targeting of chronic social stress-induced activated neurons identifies neurogenesis-related genes to be associated with resilience in female mice.

Prolonged social stress is a major cause for depression in humans and is associated with a wide range of subsequent pathophysiological changes such as elevated blood pressure. A routinely used model for investigating this kind of stress in mice is the chronic social defeat paradigm where a smaller intruder is exposed to an aggressive inhabitant of a home cage. This model is restricted to males and includes a high proportion of physical stress that might e.g., interfere with immunological aspects of the stress. The prevalence of depression in humans is even higher in women than in men. Therefore, expanding models to female individuals is desirable. We here tested the social instability model as a tool for administering chronic social stress to female C57BL/6J mice and analyzed short-term as well as long-lasting effects. Animals were housed in groups of four and were shuffled two times a week, resulting in a permanent re-structuration of their social hierarchy. While directly after the stress exposure, serum corticosterone was elevated, increased body weight and fat deposits were observed in stressed mice even one year after discontinuation of the stress. At the behavioral level, animals could be stratified into resilient and susceptible animals directly post-stress, but those subgroups were not distinguishable any more in the long-term analysis. To identify molecular contributors to resilience in the here presented social instability induced stress model, Arc-activity dependent trapping of neurons was conducted in Arc-creERT2/sun1sfGFP mice. RNA samples derived from activated nuclei from the ventral hippocampus, a brain region involved in stress-regulation during attacks or explorative behavior of mice, were subjected to a neurogenesis pathway array. While several genes were differentially regulated by stress, in particular, artemin, a neurotrophic factor was upregulated in resilient versus susceptible individuals.

A liquid-culture-based screening approach to study compounds affecting inflammatory processes in Caenorhabditis elegans.

Discovery of biomedical drugs makes use of novel biological sources of limited availability and is often in need of fast, small-scale initial screening approaches. Here, we present a screening, based on the reporter Caenorhabditis elegans strain IG692, for identification of anti- and pro-inflammatory properties. The elaborated workflow is based on cultivation in fluid and by this, allows fast and reproducible seeding in 96 well plates. LPS and dexamethasone served as reliable controls, comparable to application in the human cell line THP-1. This in vivo approach offers a first step for selection of e.g. natural products or for repurposing of compounds from drug libraries and by this can serve as a tool in drug discovery for inflammatory human diseases.

Artemisinin-treatment in pre-symptomatic APP-PS1 mice increases gephyrin phosphorylation at Ser270: a modification regulating postsynaptic GABAAR density.

Artemisinins, a group of plant-derived sesquiterpene lactones, are efficient antimalarial agents. They also share anti-inflammatory and anti-viral activities and were considered for treatment of neurodegenerative disorders like Alzheimer's disease (AD). Additionally, artemisinins bind to gephyrin, the multifunctional scaffold of GABAergic synapses, and modulate inhibitory neurotransmission in vitro. We previously reported an increased expression of gephyrin and GABAA receptors in early pre-symptomatic stages of an AD mouse model (APP-PS1) and in parallel enhanced CDK5-dependent phosphorylation of gephyrin at S270. Here, we studied the effects of artemisinin on gephyrin in the brain of young APP-PS1 mice. We detected an additional increase of gephyrin protein level, elevated gephyrin phosphorylation at Ser270, and an increased amount of GABAAR-γ2 subunits after artemisinin-treatment. Interestingly, the CDK5 activator p35 was also upregulated. Moreover, we demonstrate decreased density of postsynaptic gephyrin and GABAAR-γ2 immunoreactivities in cultured hippocampal neurons expressing gephyrin with alanine mutations at two CDK5 phosphorylation sites. In addition, the activity-dependent modulation of synaptic protein density was abolished in neurons expressing gephyrin lacking one or both of these phosphorylation sites. Thus, our results reveal that artemisinin modulates expression as well as phosphorylation of gephyrin at sites that might have important impact on GABAergic synapses in AD.