Differential Effects of Astrocyte Manipulations on Learned Motor Behavior and Neuronal Ensembles in the Motor Cortex.

Although motor cortex is crucial for learning precise and reliable movements, whether and how astrocytes contribute to its plasticity and function during motor learning is unknown. Here, we report that astrocyte-specific manipulations in primary motor cortex (M1) during a lever push task alter motor learning and execution, as well as the underlying neuronal population coding. Mice that express decreased levels of the astrocyte glutamate transporter 1 (GLT1) show impaired and variable movement trajectories, whereas mice with increased astrocyte Gq signaling show decreased performance rates, delayed response times, and impaired trajectories. In both groups, which include male and female mice, M1 neurons have altered interneuronal correlations and impaired population representations of task parameters, including response time and movement trajectories. RNA sequencing further supports a role for M1 astrocytes in motor learning and shows changes in astrocytic expression of glutamate transporter genes, GABA transporter genes, and extracellular matrix protein genes in mice that have acquired this learned behavior. Thus, astrocytes coordinate M1 neuronal activity during motor learning, and our results suggest that this contributes to learned movement execution and dexterity through mechanisms that include regulation of neurotransmitter transport and calcium signaling.SIGNIFICANCE STATEMENT We demonstrate for the first time that in the M1 of mice, astrocyte function is critical for coordinating neuronal population activity during motor learning. We demonstrate that knockdown of astrocyte glutamate transporter GLT1 affects specific components of learning, such as smooth trajectory formation. Altering astrocyte calcium signaling by activation of Gq-DREADD upregulates GLT1 and affects other components of learning, such as response rates and reaction times as well as trajectory smoothness. In both manipulations, neuronal activity in motor cortex is dysregulated, but in different ways. Thus, astrocytes have a crucial role in motor learning via their influence on motor cortex neurons, and they do so by mechanisms that include regulation of glutamate transport and calcium signals.

Clusterin Neutralizes the Inflammatory and Cytotoxic Properties of Extracellular Histones in Sepsis.

Rationale: Extracellular histones, released into the surrounding environment during extensive cell death, promote inflammation and cell death, and these deleterious roles have been well documented in sepsis. Clusterin (CLU) is a ubiquitous extracellular protein that chaperones misfolded proteins and promotes their removal. Objectives: We investigated whether CLU could protect against the deleterious properties of histones. Methods: We assessed CLU and histone expression in patients with sepsis and evaluated the protective role of CLU against histones in in vitro assays and in vivo models of experimental sepsis. Measurements and Main Results: We show that CLU binds to circulating histones and reduces their inflammatory, thrombotic, and cytotoxic properties. We observed that plasma CLU levels decreased in patients with sepsis and that the decrease was greater and more durable in nonsurvivors than in survivors. Accordingly, CLU deficiency was associated with increased mortality in mouse models of sepsis and endotoxemia. Finally, CLU supplementation improved mouse survival in a sepsis model. Conclusions: This study identifies CLU as a central endogenous histone-neutralizing molecule and suggests that, in pathologies with extensive cell death, CLU supplementation may improve disease tolerance and host survival.

Altered microtubule dynamics and vesicular transport in mouse and human MeCP2-deficient astrocytes.

Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder, characterized by normal post-natal development followed by a sudden deceleration in brain growth with progressive loss of acquired motor and language skills, stereotypic hand movements and severe cognitive impairment. Mutations in the methyl-CpG-binding protein 2 (MECP2) cause more than 95% of classic cases. Recently, it has been shown that the loss of Mecp2 from glia negatively influences neurons in a non-cell-autonomous fashion, and that in Mecp2-null mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern and greatly prolonged lifespan compared with globally null mice. We now report that microtubule (MT)-dependent vesicle transport is altered in Mecp2-deficient astrocytes from newborn Mecp2-deficient mice compared with control wild-type littermates. Similar observation has been made in human MECP2 p.Arg294* iPSC-derived astrocytes. Importantly, administration of Epothilone D, a brain-penetrant MT-stabilizing natural product, was found to restore MT dynamics in Mecp2-deficient astrocytes and in MECP2 p.Arg294* iPSC-derived astrocytes in vitro. Finally, we report that relatively low weekly doses of Epothilone D also partially reversed the impaired exploratory behavior in Mecp2(308/y) male mice. These findings represent a first step toward the validation of an innovative treatment for RTT.

Botensilimab plus balstilimab in relapsed/refractory microsatellite stable metastatic colorectal cancer: a phase 1 trial.

Microsatellite stable metastatic colorectal cancer (MSS mCRC; mismatch repair proficient) has previously responded poorly to immune checkpoint blockade. Botensilimab (BOT) is an Fc-enhanced multifunctional anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody designed to expand therapy to cold/poorly immunogenic solid tumors, such as MSS mCRC. BOT with or without balstilimab (BAL; anti-PD-1 antibody) is being evaluated in an ongoing expanded phase 1 study. The primary endpoint is safety and tolerability, which was evaluated separately in the dose-escalation portion of the study and in patients with MSS mCRC (using combined dose-escalation/dose-expansion data). Secondary endpoints include investigator-assessed RECIST version 1.1-confirmed objective response rate (ORR), disease control rate (DCR), duration of response (DOR) and progression-free survival (PFS). Here we present outcomes in 148 heavily pre-treated patients with MSS mCRC (six from the dose-escalation cohort; 142 from the dose-expansion cohort) treated with BOT and BAL, 101 of whom were considered response evaluable with at least 6 months of follow-up. Treatment-related adverse events (TRAEs) occurred in 89% of patients with MSS mCRC (131/148), most commonly fatigue (35%, 52/148), diarrhea (32%, 47/148) and pyrexia (24%, 36/148), with no grade 5 TRAEs reported and a 12% discontinuation rate due to a TRAE (18/148; data fully mature). In the response-evaluable population (n = 101), ORR was 17% (17/101; 95% confidence interval (CI), 10-26%), and DCR was 61% (62/101; 95% CI, 51-71%). Median DOR was not reached (NR; 95% CI, 5.7 months-NR), and median PFS was 3.5 months (95% CI, 2.7-4.1 months), at a median follow-up of 10.3 months (range, 0.5-42.6 months; data continuing to mature). The combination of BOT plus BAL demonstrated a manageable safety profile with no new immune-mediated safety signals and encouraging clinical activity with durable responses. ClinicalTrials.gov identifier: NCT03860272 .

Altered microtubule dynamics in Mecp2-deficient astrocytes.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by mutations in the gene MECP2 encoding the methyl-CpG binding protein 2. This genetic disease affects predominantly girls and is characterized by a period of normal development that lasts for 8-18 months, followed by neurologic regression affecting both motor and mental abilities. Previous studies performed on brains from RTT subjects and Mecp2-deficient mice showed striking changes in neuronal maturation and dendritic arborization. Recently, we showed that expression of stathmin-like 2 (STMN2) was significantly reduced in fibroblasts from RTT patients, and similar results were obtained in the cerebellum of Mecp2-deficient mice. Because assembly and dynamics of microtubules are known to be modulated by STMN2, we studied microtubule dynamics in brain cells from Mecp2-deficient mice. We observed that Mecp2 deficiency affects microtubule dynamics in astrocytes from Mecp2-deficient mice. Our data reinforce the fact that the loss of Mecp2 in astrocytes may influence the onset and progression of RTT. These results imply that Mecp2 has a stabilizing role in microtubule dynamics and that Mecp2 deficiency, which is associated with STMN2 down-regulation, could lead to impaired microtubule stability, hence explaining the dendritic abnormalities observed in RTT brains.

Molecular Signatures of Response to Mecasermin in Children With Rett Syndrome.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder without effective treatments. Attempts at developing targetted therapies have been relatively unsuccessful, at least in part, because the genotypical and phenotypical variability of the disorder. Therefore, identification of biomarkers of response and patients' stratification are high priorities. Administration of Insulin-like Growth Factor 1 (IGF-1) and related compounds leads to significant reversal of RTT-like symptoms in preclinical mouse models. However, improvements in corresponding clinical trials have not been consistent. A 20-weeks phase I open label trial of mecasermin (recombinant human IGF-1) in children with RTT demonstrated significant improvements in breathing phenotypes. However, a subsequent randomised controlled phase II trial did not show significant improvements in primary outcomes although two secondary clinical endpoints showed positive changes. To identify molecular biomarkers of response and surrogate endpoints, we used RNA sequencing to measure differential gene expression in whole blood samples of participants in the abovementioned phase I mecasermin trial. When all participants (n = 9) were analysed, gene expression was unchanged during the study (baseline vs. end of treatment, T0-T3). However, when participants were subclassified in terms of breathing phenotype improvement, specifically by their plethysmography-based apnoea index, individuals with moderate-severe apnoea and breathing improvement (Responder group) displayed significantly different transcript profiles compared to the other participants in the study (Mecasermin Study Reference group, MSR). Many of the differentially expressed genes are involved in the regulation of cell cycle processes and immune responses, as well as in IGF-1 signalling and breathing regulation. While the Responder group showed limited gene expression changes in response to mecasermin, the MSR group displayed marked differences in the expression of genes associated with inflammatory processes (e.g., neutrophil activation, complement activation) throughout the trial. Our analyses revealed gene expression profiles associated with severe breathing phenotype and its improvement after mecasermin administration in RTT, and suggest that inflammatory/immune pathways and IGF-1 signalling contribute to treatment response. Overall, these data support the notion that transcript profiles have potential as biomarkers of response to IGF-1 and related compounds.

Label-free three-photon imaging of intact human cerebral organoids for tracking early events in brain development and deficits in Rett syndrome.

Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (~2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging.

GSK3ß inhibitor CHIR 99021 modulates cerebral organoid development through dose-dependent regulation of apoptosis, proliferation, differentiation and migration.

Cerebral organoids generated from human pluripotent stem cells (hiPSCs) are unique in their ability to recapitulate human-specific neurodevelopmental events. They are capable of modeling the human brain and its cell composition, including human-specific progenitor cell types; ordered laminar compartments; and both cell-specific transcriptional signatures and the broader telencephalic transcriptional landscape. The serine/threonine kinase, GSK3ß, plays a critical role in neurodevelopment, controlling processes as varied as neurogenesis, morphological changes, polarization, and migration. In the generation of cerebral organoids, inhibition of GSK3ß at low doses has been used to increase organoid size and decrease necrotic core. However, little is known of the effects of GSK3ß inhibition on organoid development. Here, we demonstrate that while low dose of GSK3ß inhibitor CHIR 99021 increases organoid size, higher dose actually reduces organoid size; with the highest dose arresting organoid growth. To examine the mechanisms that may contribute to the phenotypic size differences observed in these treatment groups, we show that low dose of CHIR 99021 increases cell survival, neural progenitor cell proliferation and neuronal migration. A higher dose, however, decreases not only apoptosis but also proliferation, and arrests neural differentiation, enriching the pool of neuroepithelial cells, and decreasing the pools of early neuronal progenitors and neurons. These results reveal new mechanisms of the pleiotropic effects of GSK3ß during organoid development, providing essential information for the improvement of organoid production and ultimately shedding light on the mechanisms of embryonic brain development.

HDAC inhibitor ameliorates behavioral deficits in Mecp2308/y mouse model of Rett syndrome.

Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder. More than 95% of classic RETT syndrome cases result from pathogenic variants in the methyl-CpG binding protein 2 (MECP2) gene. Nevertheless, it has been established that a spectrum of neuropsychiatric phenotypes is associated with MECP2 variants in both females and males. We previously reported that microtubule growth velocity and vesicle transport directionality are altered in Mecp2-deficient astrocytes from newborn Mecp2-deficient mice compared to that of their wild-type littermates suggesting deficit in microtubule dynamics. In this study, we report that administration of tubastatin A, a selective HDAC6 inhibitor, restored microtubule dynamics in Mecp2-deficient astrocytes. We furthermore report that daily doses of tubastatin A reversed early impaired exploratory behavior in male Mecp2308/y mice. These findings are a first step toward the validation of a novel treatment for RTT.

A low-cost 3D printed microfluidic bioreactor and imaging chamber for live-organoid imaging.

Organoids are biological systems grown in vitro and are observed to self-organize into 3D cellular tissues of specific organs. Brain organoids have emerged as valuable models for the study of human brain development in health and disease. Researchers are now in need of improved culturing and imaging tools to capture the in vitro dynamics of development processes in the brain. Here, we describe the design of a microfluidic chip and bioreactor, to enable in situ tracking and imaging of brain organoids on-chip. The low-cost 3D printed microfluidic bioreactor supports organoid growth and provides an optimal imaging chamber for live-organoid imaging, with drug delivery support. This fully isolated design of a live-cell imaging and culturing platform enables long-term live-imaging of the intact live brain organoids as it grows. We can thus analyze their self-organization in a controlled environment with high temporal and spatial resolution.

Differential Expression and Regulation of Brain-Derived Neurotrophic Factor (BDNF) mRNA Isoforms in Brain Cells from Mecp2(308/y) Mouse Model.

Rett syndrome (RTT) is a severe neurodevelopmental disease caused by mutations in methyl-CpG-binding protein 2 (MECP2), which encodes a transcriptional modulator of many genes including BDNF. BDNF comprises nine distinct promoter regions, each triggering the expression of a specific transcript. The role of this diversity of transcripts remains unknown. MeCP2 being highly expressed in neurons, RTT was initially considered as a neuronal disease. However, recent studies have shown that MeCP2 was also expressed in astrocytes. Though several studies explored Bdnf IV expression in Mecp2-deficient mice, the differential expression of Bdnf isoforms in Mecp2-deficient neurons and astrocytes was never studied. By using TaqMan technology and a mouse model expressing a truncated Mecp2 (Mecp2(308/y)), we firstly showed in neurons that Bdnf transcripts containing exon I, IIb, IIc, IV, and VI are prominently expressed, whereas in astrocytes, Bdnf transcript containing exon VI is preferentially expressed, suggesting a specific regulation of Bdnf expression at the cellular level. Secondly, we confirmed the repressive role of Mecp2 only on the expression of Bdnf VI in neurons. Our data suggested that the truncated Mecp2 protein maintains its function on Bdnf expression regulation in neurons and in astrocytes. Interestingly, we observed that Bdnf transcripts (I and IXA), regulated by neural activity induced by bicuculline in Mecp2(308/y) neurons, were not affected by histone deacetylase inhibition. In contrast, Bdnf transcripts (IIb, IIc, and VI), regulated by histone deacetylation, were not affected by bicuculline treatment in wild-type and Mecp2(308/y) neurons. All these results reflect the complexity of regulation of Bdnf gene.

Astrocyte Transcriptome from the Mecp2(308)-Truncated Mouse Model of Rett Syndrome.

Mutations in the gene encoding the transcriptional modulator methyl-CpG binding protein 2 (MeCP2) are responsible for the neurodevelopmental disorder Rett syndrome which is one of the most frequent sources of intellectual disability in women. Recent studies showed that loss of Mecp2 in astrocytes contributes to Rett-like symptoms and restoration of Mecp2 can rescue some of these defects. The goal of this work is to compare gene expression profiles of wild-type and mutant astrocytes from Mecp2(308/y) mice (B6.129S-MeCP2/J) by using Affymetrix mouse 2.0 microarrays. Results were confirmed by quantitative real-time RT-PCR and by Western blot analysis. Gene set enrichment analysis utilizing Ingenuity Pathways was employed to identify pathways disrupted by Mecp2 deficiency. A total of 2152 genes were statistically differentially expressed between wild-type and mutated samples, including 1784 coding transcripts. However, only 257 showed fold changes >1.2. We confirmed our data by replicative studies in independent primary cultures of cortical astrocytes from Mecp2-deficient mice. Interestingly, two genes known to encode secreted proteins, chromogranin B and lipocalin-2, showed significant dysregulation. These proteins secreted from Mecp2-deficient glia may exert negative non-cell autonomous effects on neuronal properties, including dendritic morphology. Moreover, transcriptional profiling revealed altered Nr2f2 expression which may explain down- and upregulation of several target genes in astrocytes such as Ccl2, Lcn2 and Chgb. Unraveling Nr2f2 involvement in Mecp2-deficient astrocytes could pave the way for a better understanding of Rett syndrome pathophysiology and offers new therapeutic perspectives.

Mutations in the C-terminus of CDKL5: proceed with caution.

Mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene have been described in girls with Rett-like features and early-onset epileptic encephalopathy including infantile spasms. Milder phenotypes have been associated with sequence variations in the 3'-end of the CDKL5 gene. Identification of novel CDKL5 transcripts coding isoforms characterized by an altered C-terminal region strongly questions the eventual pathogenicity of sequence variations located in the 3'-end of the gene. We investigated a group of 30 female patients with a clinically heterogeneous phenotype ranging from nonspecific intellectual disability to a severe neonatal encephalopathy and identified two heterozygous CDKL5 missense mutations, the previously reported p.Val999Met and the novel mutation p.Pro944Thr. However, these mutations have also been detected in their healthy father. Considering our results and all data from the literature, we suggest that genetic variations beyond the codon 938 in human CDKL5115 protein may have minor or no significance. It is probable that screening of exons 19-21 of the CDKL5 gene is not useful in practical molecular diagnosis of atypical Rett syndrome.

MeCP2 deficiency is associated with impaired microtubule stability.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Previous studies performed on Mecp2-deficient brain showed striking changes in neuronal maturation. We recently showed that MeCP2 deficiency affects microtubule (MT) dynamics in RTT astrocytes. Here, we analyze MT stability in primary fibroblast cultures from patients with RTT syndrome and identify a significant decrease in stability compared to controls. Furthermore, we found that MT stability was reduced both in cells expressing the mutant or the wild-type allele in RTT fibroblasts, suggesting that mutated cells could damage wild-type ones through a non-cell-autonomous pathway. These results suggest that MeCP2 has a stabilizing role on MT dynamics and that its deficiency could lead to impaired MT stability that may explain in part the dendritic abnormalities observed in RTT brains.