Repressing PTB is incapable to convert reactive astrocytes to dopaminergic neurons in a mouse model of Parkinson's disease

Lineage reprograming of resident glia cells to induced dopaminergic neurons (iDAns) holds attractive prospect for cell-replacement therapy of Parkinson's disease (PD). Recently, whether repressing polypyrimidine tract binding protein (PTB) could truly achieve efficient astrocyte-to-iDAn conversion in substantia nigra and striatum aroused widespread controversy. Although reporter+ iDAns were observed by two groups after delivering adeno-associated virus (AAV) expressing a reporter with shRNA or Crispr-CasRx to repress astroglial PTB, the possibility of AAV leaking into endogenous DAns could not be excluded without using a reliable lineage tracing method. By adopting stringent lineage tracing strategy, two other studies showed that neither knockdown nor genetic deletion of quiescent astroglial PTB fails to obtain iDAns under physiological condition. However, the role of reactive astrocyte might be underestimated since upon brain injury, reactive astrocyte could acquire certain stem cell hallmarks which may facilitate the lineage conversion process. Therefore, whether reactive astrocytes could be genuinely converted to DAns after PTB repression in a PD model needs further validation. In this study, we used Aldh1l1-CreERT2-mediated specific astrocyte-lineage tracing method to investigate whether reactive astrocytes could be converted to DAns in the 6-OHDA PD model. However, we found that no astrocyte-originated DAns was generated after effective knockdown of astroglial PTB either in the substantia nigra or in the striatum, while AAV leakage to nearby neurons was observed. Our results further confirmed that repressing PTB is unable to convert astrocytes to DA neurons no matter in physiological or PD-related pathological conditions.

Chemical inhibition of pathological reactive astrocytes promotes neural protection

Disease, injury, and aging induce reactive astrocyte states with pathological functions. In neurodegenerative diseases, inflammatory reactive astrocytes are abundant and contribute to progressive cell loss. Modulating the state or function of these reactive astrocytes thereby represents an attractive therapeutic goal. Leveraging a cellular phenotypic screening platform, we show that chemical inhibitors of HDAC3 effectively block pathological astrocyte reactivity. Inhibition of HDAC3 reduces molecular and functional features of reactive astrocytes in vitro including inflammatory gene expression, cytokine secretion, and antigen presentation. Transcriptional and chromatin mapping studies show that HDAC3 inhibition mediates a switch between pro-inflammatory and anti-inflammatory states, which disarms the pathological functions of reactive astrocytes. Systemic administration of a blood-brain barrier penetrant chemical inhibitor of HDAC3, RGFP966, blocks reactive astrocyte formation and promotes axonal protection in vivo. Collectively, these results establish a platform for discovering chemical modulators of reactive astrocyte states, inform the mechanisms controlling astrocyte reactivity, and demonstrate the therapeutic potential of modulating astrocyte reactivity for neurodegenerative diseases.

Notch1 and Galectin-3 Modulate Cortical Reactive Astrocyte Response After Brain Injury

After a brain lesion, highly specialized cortical astrocytes react, supporting the closure or replacement of the damaged tissue, but fail to regulate neural plasticity. Growing evidence indicates that repair response leads astrocytes to reprogram, acquiring a partially restricted regenerative phenotype in vivo and neural stem cells (NSC) hallmarks in vitro. However, the molecular factors involved in astrocyte reactivity, the reparative response, and their relation to adult neurogenesis are poorly understood and remain an area of intense investigation in regenerative medicine. In this context, we addressed the role of Notch1 signaling and the effect of Galectin-3 (Gal3) as underlying molecular candidates involved in cortical astrocyte response to injury. Notch signaling is part of a specific neurogenic microenvironment that maintains NSC and neural progenitors, and Gal3 has a preferential spatial distribution across the cortex and has a central role in the proliferative capacity of reactive astrocytes. We report that in vitro scratch-reactivated cortical astrocytes from C57Bl/6J neonatal mice present nuclear Notch1 intracellular domain (NICD1), indicating Notch1 activation. Colocalization analysis revealed a subpopulation of reactive astrocytes at the lesion border with colocalized NICD1/Jagged1 complexes compared with astrocytes located far from the border. Moreover, we found that Gal3 increased intracellularly, in contrast to its extracellular localization in non-reactive astrocytes, and NICD1/Gal3 pattern distribution shifted from diffuse to vesicular upon astrocyte reactivation. In vitro, Gal3–/– reactive astrocytes showed abolished Notch1 signaling at the lesion core. Notch1 receptor, its ligands (Jagged1 and Delta-like1), and Hes5 target gene were upregulated in C57Bl/6J reactive astrocytes, but not in Gal3–/– reactive astrocytes. Finally, we report that Gal3–/– mice submitted to a traumatic brain injury model in the somatosensory cortex presented a disrupted response characterized by the reduced number of GFAP reactive astrocytes, with smaller cell body perimeter and decreased NICD1 presence at the lesion core. These results suggest that Gal3 might be essential to the proper activation of Notch signaling, facilitating the cleavage of Notch1 and nuclear translocation of NICD1 into the nucleus of reactive cortical astrocytes. Additionally, we hypothesize that reactive astrocyte response could be dependent on Notch1/Jagged1-Hes5 signaling activation following brain injury.

Blocking microglial activation of reactive astrocytes is neuroprotective in models of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common cause of age-related dementia. Increasing evidence suggests that neuroinflammation mediated by microglia and astrocytes contributes to disease progression and severity in AD and other neurodegenerative disorders. During AD progression, resident microglia undergo proinflammatory activation, resulting in an increased capacity to convert resting astrocytes to reactive astrocytes. Therefore, microglia are a major therapeutic target for AD and blocking microglia-astrocyte activation could limit neurodegeneration in AD. Here we report that NLY01, an engineered exedin-4, glucagon-like peptide-1 receptor (GLP-1R) agonist, selectively blocks β-amyloid (Aβ)-induced activation of microglia through GLP-1R activation and inhibits the formation of reactive astrocytes as well as preserves neurons in AD models. In two transgenic AD mouse models (5xFAD and 3xTg-AD), repeated subcutaneous administration of NLY01 blocked microglia-mediated reactive astrocyte conversion and preserved neuronal viability, resulting in improved spatial learning and memory. Our study indicates that the GLP-1 pathway plays a critical role in microglia-reactive astrocyte associated neuroinflammation in AD and the effects of NLY01 are primarily mediated through a direct action on Aβ-induced GLP-1R+ microglia, contributing to the inhibition of astrocyte reactivity. These results show that targeting upregulated GLP-1R in microglia is a viable therapy for AD and other neurodegenerative disorders.

TMAO Aggregates Neurological Damage Following Ischemic Stroke by Promoting Reactive Astrocytosis and Glial Scar Formation via the Smurf2/ALK5 Axis

Ischemic stroke has been reported to cause significant changes to memory, thinking, and behavior. Intriguingly, recently reported studies have indicated the association of Trimethylamine N-oxide (TMAO) with the acute phase of ischemic stroke. However, the comprehensive underlying mechanism remained unknown. The objective of the present study was to investigate the association between TMAO and recovery of neurological function after ischemic stroke. For this purpose, a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established and treated with TMAO or/and sh-ALK5, followed by the neurological function evaluation. Behaviors of rats were observed through staircase and cylinder tests. Moreover, the expression of Smurf2 and ALK5 was detected by immunohistochemistry while expression of GFAP, Neurocan, and Phosphacan in brain tissues was determined by immunofluorescence. Thereafter, gain- and loss-of-function assays in astrocytes, the proliferation, viability, and migration were evaluated by the EdU, CCK-8, and Transwell assays. Besides, Smurf2 mRNA expression was determined by the RT-qPCR, whereas, Smurf2, ALK5, GFAP, Neurocan, and Phosphacan expression was evaluated by the Western blotting. Finally, the interaction of Smurf2 with ALK5 and ALK5 ubiquitination was assessed by the co-immunoprecipitation. Notably, our results showed that TMAO promoted the proliferation of reactive astrocyte and formation of glial scar in MCAO/R rats. However, this effect was abolished by the Smurf2 overexpression or ALK5 silencing. We further found that TMAO upregulated the ALK5 expression by inhibiting the ubiquitination role of Smurf2. Overexpression of ALK5 reversed the inhibitory effect of Smurf2 on astrocyte proliferation, migration, and viability. Collectively, our work identifies the evolutionarily TMAO/Smurf2/ALK5 signaling as a major genetic factor in the control of reactive astrocyte proliferation and glial scar formation in ischemic stroke, thus laying a theoretical foundation for the identification of ischemic stroke.

Astrocytic Reactivity Triggered by defective Mitophagy Activates NF-kB Signaling and Causes Neurotoxicity in Frontotemporal Dementia Type 3

Background: Frontotemporal dementia type 3 (FTD3) caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and function as well as endosomal-lysosomal fusion in neurons. However, there is a critical knowledge gap in understanding how mutations in CHMP2B affect astrocytes. Hence, we investigated the disease mechanisms in astrocytes derived from hiPSC with mutations in CHMP2B and their impact on neurons.Methods: To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) from FTD3 patients and their CRISPR/Cas 9 gene edited isogenic controls and produced heterozygous and homozygous CHMP2B-mutant hiPSC via CRISPR/Cas 9 knock-in gene editing. Additionally, we confirmed our findings in CHMP2B mutant mice. The hiPSC were subjected to astrocyte differentiation and the mutation dependent effects were investigated using immunocytochemistry, western blot, cytokine assays, transmission electron microscopy, RNA-sequencing and gas chromatography-mass spectrometry. Finally, neurons were exposed to conditioned media of mutant astrocytes and viability, growth and motility were measured.Results: To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our findings include perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. Furthermore, we identified a shift in astrocyte homeostasis triggering a reactive astrocyte phenotype and increased release of toxic cytokines. This cumulates in NF-kB pathway activation with increased production of CHF, LCN2 and C3, which cause neurodegeneration. The neurotoxic effect was investigated by exposing hiPSC-derived neurons to astrocyte-conditioned media, which severely reduced neurite outgrowth capacities. Rescue experiments targeting ROS could restore ROS levels back to normal levels, indicating that the impaired removal of abnormal mitochondria triggers the pathological cascade in CHMP2B mutant astrocytes culminating in the formation of neurotoxic reactive astrocytes.Conclusion :Our data provide mechanistic insights into how defective mitophagy causes impaired mitochondrial fission, leading to the adoption of reactive astrocyte properties with increased cytokine release, NFkB activation and elevated expression of neurotoxic proteins in FTD3.