magnetic-isolation-microglial-cells-from-neonate-mouse-for-primary

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

A distinctive feature of neocortical development is the highly coordinated production of different progenitor cell subtypes, which are critical for ensuring adequate neurogenic outcome and the development of normal neocortical size. To further understand the mechanisms that underlie neocortical growth, we focused our studies on the microcephaly gene Mcph1, and we report here that Mcph1 (1) exerts its functions in rapidly dividing apical radial glial cells (aRGCs) during mouse neocortical development stages that precede indirect neurogenesis; (2) is expressed at mitochondria; and (3) controls the proper proliferation and survival of RGCs, potentially through crosstalk with cellular metabolic pathways involving the stimulation of mitochondrial activity via VDAC1/GRP75 and AKT/HK2/VDAC1 and glutaminolysis via ATF4/PCK2. We currently report the description of a MCPH-gene implication in the interplay between bioenergetic pathways and neocortical growth, thus pointing to alterations of cellular metabolic pathways, in particular glutaminolysis, as a possible cause of microcephalic pathogenesis.

Cell Metabolic Alterations due to Mcph1 Mutation in Microcephaly.

Cerebellar granule neuron progenitors (CGNPs) give rise to the cerebellar granule neurons in the developing cerebellum. Generation of large number of these neurons is made possible by the high proliferation rate of CGNPs in the external granule layer (EGL) in the dorsal cerebellum. Here, we show that upregulation of β-catenin can maintain murine CGNPs in a state of proliferation. Further, we show that β-catenin mRNA and protein levels can be regulated by the mitogen Sonic hedgehog (Shh). Shh signaling led to an increase in the level of the transcription factor N-myc. N-myc was found to bind the β-catenin promoter, and the increase in β-catenin mRNA and protein levels could be prevented by blocking N-myc upregulation downstream of Shh signaling. Furthermore, blocking Wingless-type MMTV integration site (Wnt) signaling by Wnt signaling pathway inhibitor Dickkopf 1 (Dkk-1) in the presence of Shh did not prevent the upregulation of β-catenin. We propose that in culture, Shh signaling regulates β-catenin expression through N-myc and results in increased CGNP proliferation.

Shh-Mediated Increase in β-Catenin Levels Maintains Cerebellar Granule Neuron Progenitors in Proliferation.

A leading cause of preterm birth is the exposure to systemic inflammation (maternal/fetal infection), which leads to neuroinflammation and white matter injury (WMI). A wide range of cytokines and chemokines are expressed and upregulated in oligodendrocytes (OLs) in response to inflammation and numerous reports show that OLs express several receptors for immune related molecules, which enable them to sense inflammation and to react. However, the role of OL immune response in WMI is unclear. Here, we focus our study on toll-like receptor-3 (TLR3) that is activated by double-strand RNA (dsRNA) and promotes neuroinflammation. Despite its importance, its expression and role in OLs remain unclear. We used an in vivo mouse model, which mimics inflammation-mediated WMI of preterm born infants consisting of intraperitoneal injection of IL-1β from P1 to P5. In the IL-1β-treated animals, we observed the upregulation of Tlr3, IL-1β, IFN-β, Ccl2, and Cxcl10 in both PDGFRα+ and O4+ sorted cells. This upregulation was higher in O4+ immature OLs (immOLs) as compared to PDGFRα+ OL precursor cells (OPCs), suggesting a different sensitivity to neuroinflammation. These observations were confirmed in OL primary cultures: cells treated with TLR3 agonist Poly(I:C) during differentiation showed a stronger upregulation of Ccl2 and Cxcl10 compared to cells treated during proliferation and led to decreased expression of myelin genes. Finally, OLs were able to modulate microglia phenotype and function depending on their maturation state as assessed by qPCR using validated markers for immunomodulatory, proinflammatory, and anti-inflammatory phenotypes and by phagocytosis and morphological analysis. These results show that during inflammation the response of OLs can play an autonomous role in blocking their own differentiation: in addition, the immune activation of OLs may play an important role in shaping the response of microglia during inflammation.

The immune-inflammatory response of oligodendrocytes in a murine model of preterm white matter injury: the role of TLR3 activation.

Groundwater is the main source of drinking water for a significant portion of the human population. In many agricultural areas, diffuse pollution such as high levels of total dissolved salts including nitrate, puts the quality of this resource at risk. However, the effect of exposure to these water contaminants on brain development is currently poorly understood. Here we characterised water from a borewell located in an intensely cultivated area (agricultural) or water from a borewell located in a nearby pristine forest. The agricultural borewell water was rich in nitrates with high total dissolved salts. We then studied the consequence of drinking the agricultural water on mouse brain development. For this, the agricultural borewell water or forest water was given to mice for 6 weeks before and during pregnancy and lactation. The brains of the offspring born to these dams were analysed at postnatal day (P)5 and P21 and compared using immunohistochemistry for changes in glial cells, neurons, myelin, and cell death across many brain regions. Brains from offspring born to dams who had been given agricultural water (versus forest control water) were significantly smaller, and at P21 had a significant degeneration of neurons and increased numbers of microglia in the motor cortex, had fewer white matter astrocytes and an increase in cell death, particularly in the dentate gyrus. This study shows that brain development is sensitive to water composition. It points to the importance of assessing neurodevelopmental delays when considering the effect of water contaminated with agricultural run offs on human health. MAIN FINDING: Pregnant and lactating mice were given borewell water from intensely cultivated land. Offspring brains reveal degeneration of neurons and a loss of astrocytes, increase in microglial cells and cell death, pointing to neurodevelopmental problems.

Agricultural groundwater with high nitrates and dissolved salts given to pregnant mice alters brain development in the offspring.

In the premature newborn, perinatal inflammation mediated by microglia contributes significantly to neurodevelopmental injuries including white matter injury (WMI). Brain inflammation alters development through neuroinflammatory processes mediated by activation of homeostatic microglia toward a pro-inflammatory and neurotoxic phenotype. Investigating immune regulators of microglial activation is crucial to find effective strategies to prevent and treat WMI.

Article	Year
Cell Metabolic Alterations due to Mcph1 Mutation in Microcephaly. Cell reports\| PubMed ID: 32294449	2020
Shh-Mediated Increase in β-Catenin Levels Maintains Cerebellar Granule Neuron Progenitors in Proliferation. Cerebellum (London, England)\| PubMed ID: 32495183	2020
The immune-inflammatory response of oligodendrocytes in a murine model of preterm white matter injury: the role of TLR3 activation. Cell death & disease\| PubMed ID: 33558485	2021
Agricultural groundwater with high nitrates and dissolved salts given to pregnant mice alters brain development in the offspring. Ecotoxicology and environmental safety\| PubMed ID: 34418854	2021
miR-146b Protects the Perinatal Brain against Microglia-Induced Hypomyelination. Annals of neurology\| PubMed ID: 34741343	2022

Shyamala Mani

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