Microglia, the unique and motile immune cells of the central nervous system (CNS), function as a security guard in maintaining CNS homeostasis, primarily through calcium signaling. The calcium dynamics in microglia control important functions such as phagocytosis, cytokine release, and migration. Calcium dysregulation in microglia has been linked to several CNS disorders, like Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), and ischemic stroke (IS). Calcium entering through channels such as voltage-gated calcium channels (VGCCs), store-operated calcium entry (SOCE), and transient receptor potential (TRP) channels is essential for microglial activation and pro-inflammatory responses. Under pathological conditions, like the formation of amyloid-β plaques in AD, aggregation of α-synuclein in PD, and oxidative stress in MS, calcium dysregulation exacerbates neuroinflammation, mitochondrial dysfunction, and neurodegeneration. Therapeutic strategies targeting calcium signaling pathways, using calcium channel blockers and antioxidant interventions, show promise for alleviating microglial activation and slowing down disease progression. This review summarizes the underlying mechanisms of microglial calcium dysregulation and potential therapeutic benefits for restoring microglial calcium balance in CNS disorders.

Microglia, the resident brain immune effectors cells, show dynamic activation level changes for most neuropsychiatric diseases, reflecting their complex regulatory function and potential as a therapeutic target. Emerging single-cell molecular biology studies are used to investigate the genetic modification of individual cells to better understand complex gene regulatory pathways. Although multiple protocols for microglia isolation from adult mice are available, it is always challenging to get sufficient purified microglia from a single brain for simultaneous DNA and RNA extraction for subsequent downstream analysis. Moreover, for data comparison between treated and untreated groups, standardized cell isolation techniques are essential to decrease variability. Here, we present a combined method of microglia isolation from a single adult mouse brain, using a magnetic bead-based column separation technique, and a column-based extraction of purified DNA-RNA from the isolated microglia for downstream application. Our current method provides step-by-step instructions accompanied by visual explanations of important steps for isolating DNA-RNA simultaneously from a highly purified microglia population.

BscF is a type III secretion system (T3SS) needle protein from Bordetella pertussis and has previously been shown to induce a sufficient Th1 and Th17 response in human monocytes and mice as a prerequisite for long-lasting protective immunity against pertussis infection. In our current study, we aim to compare the modulation of inflammatory signaling molecules as a direct measure of the immune response to the B. pertussis antigens BscF and Tdap in the presence or absence of the adrenergic receptor agonists phenylephrine (PE) or isoproterenol (ISO) to observe differences that may contribute to the diminished protective immunity of the current acellular pertussis (aP) vaccine, Tdap. Stimulation of human monocyte THP-1 cells with LPS, BscF, and Tdap induced a robust elevation of CCL20, CXCL10, PGE2, and PGF2α among most chemokine and prostanoid members when compared with the control treatment. Treatment with the adrenergic agonist PE or ISO significantly enhanced the BscF- and Tdap-stimulated modulation of CCL20 and CXCL10 but not PGE2 and PGF2α, suggesting that adrenergic modulation of pertussis antigen responses might be a new therapeutic strategy to improve the longevity of pertussis immunity. Stimulation of THP-1 cells with BscF alone initiated significant expression of CXCL10 and PGF2α but not when Tdap was used, suggesting that BscF might be an important pertussis antigen for next-generation pertussis vaccines or when combined with the current aP vaccine. Our data offer opportunities for designing new therapeutic approaches against pertussis infection.

Introduction: Growing recognition of microglia’s role in neurodegenerative disorders has accentuated the need to characterize microglia profiles and their influence on pathogenesis. To understand changes observed in the microglial profile during the progression of synucleinopathies, microglial gene expression and DNA methylation were examined in the mThy1-α-synuclein mouse model.Methods: Disease progression was determined using behavioral tests evaluating locomotor deficits before DNA and RNA extraction at 7 and 10 months from isolated microglia for enzymatic methyl-sequencing and RNA-sequencing.Results: Pathway analysis of these changes at 7 months indicates a pro-inflammatory profile and changes in terms related to synaptic maintenance. Expression and methylation at both 7 and 10 months included terms regarding mitochondrial and metabolic stress. While behavior symptoms progressed at 10 months, we see many previously activated pathways being inhibited in microglia at a later stage, with only 8 of 53 shared pathways predicted to be directionally concordant. Despite the difference in pathway directionality, 21 of the 22 genes that were differentially expressed and annotated to differentially methylated regions at both 7 and 10 months had conserved directionality changes.Discussion: These results highlight a critical period in disease progression, during which the microglia respond to α-synuclein, suggesting a transition in the role of microglia from the early to late stages of the disease.