Rebecca Morris: Exposure to urban particulate matter (UPM) impairs mitochondrial dynamics in BV2 cells, triggering a mitochondrial biogenesis response

The World Health Organisation (WHO) suggests that 99% of the population are habitually exposed to higher than recommended levels of PM originating from various sources such as transport, domestic fuel burning, industry, and human and natural sources (Karagulian et al., 2015). Emerging data suggesting that even exposure to low levels of PM can be injurious (Pérez Velasco & Jarosińska, 2022; Whaley et al., 2021) prompted the WHO to reappraise and reduce their original guidelines (Supplementary Table 1; WHO, 2021). Inhalation is the main route of PM exposure, where smaller particles (PM2.5) can infiltrate the lungs (Thangavel et al., 2022), associated with an increased risk of cardiovascular and respiratory diseases (Arias-Perez et al., 2020; Kim et al., 2018; Miller, 2020). However, there is growing evidence suggesting that PM can have adverse effects on the CNS (Kim, Kim et al., 2020; You et al., 2022), contributing to long-term detrimental effects on brain health (Costa et al., 2020; Morris et al., 2021). Furthermore, exposure to PM in utero increases neurotoxicity and neuroinflammation in rodent offspring models, and epidemiological studies have found an increased risk of autism spectrum disorder especially in males following maternal exposure to PM (Bilbo et al., 2018; Bolton et al., 2013; Rahman et al., 2022).

Microglia are the resident immune cells of the brain and, in addition to their known role in acute defence against infection, contribute to brain development through synaptic pruning and maintenance of brain homeostasis (Mallard et al., 2019). However, aberrant/persistent microglial activation is a hallmark of a number of neonatal pathologies. In the developing brain, activation of microglia is observed following encephalopathy of prematurity as well as in the sequelae of birth asphyxia and neonatal stroke (Fleiss et al., 2021; Mallard et al., 2019). The subsequent neuroinflammatory response contributes to common outcomes for these infants such as neurological, behavioural and motor impairments, which have lifelong consequences (Fleiss et al., 2021).

Mitochondria reside in all brain cell types and, notably, brain mitochondria are some of the most long-lived in the body (20–30 days, compared with 10 days for the liver) therefore requiring significant quality control (Krishna et al., 2021; Menzies & Gold, 1971; Navarro & Boveris, 2004; Stauch et al., 2023). Energy utilisation by the brain is disproportionately high given its size and there is a significant increase in metabolic demand during early years of brain development (Kuzawa et al., 2014). Mitochondria are known for their ability to generate ATP through oxidative phosphorylation, as well as for calcium buffering, cell death pathways and steroid hormone synthesis. In the brain, healthy mitochondrial function supports neurite outgrowth, neurotransmitter release, neurogenesis, neuronal plasticity and synaptic function, and mediates the inflammatory response (Culmsee et al., 2018; Misgeld & Schwarz, 2017). However, the immature brain is more vulnerable to neurotoxicity, especially due to high levels of extracellular iron and decreased antioxidants (Blomgren & Hagberg, 2006); repeated studies have identified impaired mitochondrial dysfunction in preclinical models of neonatal brain injury (Hagberg et al., 2014; Jones & Thornton, 2022).

Mitochondria in the CNS are increasingly being investigated as targets for air pollution (Chew et al., 2020). PM-bound metals can impair mitochondrial structure, disturb mitochondrial membrane potential and calcium buffering capabilities, and induce intracellular mitochondrial reactive oxygen species (mtROS) production and pro-apoptotic factors into the cytoplasm, which are known to trigger cellular stress pathways (Pardo et al., 2020). In vivo, mitochondrial matrix swelling was observed in hippocampal cells from the offspring of mice exposed to PM2.5 during gestation (Zheng et al., 2018). Similarly, in rodents exposed to PM postnatally, there was an increase in neuroinflammation with a concomitant suppression of mitochondrial gene expression in the hippocampus, and a learning and memory impairment (Li et al., 2018; Li et al., 2020).

We previously showed that exposure to urban (U)PM induces neurotoxicity in microglial-like BV2 cells, with augmented neuroinflammation and oxidative stress (Morris et al., 2022). Pretreatment with a mitochondrially targeted antioxidant rescued UPM-induced cell death and reduced mtROS production to control levels. Moreover, UPM exposure increased apoptosis, suggesting rupture of the cristae and damage to mitochondria. Mitochondrial dysfunction may be a target for the pathological mechanisms underpinning brain impairment following microglial UPM exposure. Here we extend these findings to examine the effect of UPM exposure directly on mitochondria dynamics and associated cellular bioenergetics in microglial-like BV2 cells.

Materials and methods

BV2 cell culture and UPM treatment

The female neonatal mouse microglia BV2 cell line was provided by Professor R. Donato (University of Perugia, Italy), cultured as described previously (Morris et al., 2022) and used between passage (P)2 and P22. For treatments, BV2 cells were transferred to medium containing 5% fetal bovine serum (FBS). Stock solutions were prepared by suspending UPM (Sigma, St Louis, MO, USA, #NIST1648A, certified reference material; Wang et al., 2020) in Dulbecco's modified Eagle's medium (DMEM) growth medium and working solutions prepared by sonication as described previously (Morris et al., 2022).

Detection of mitochondrial membrane potential

Changes in mitochondrial membrane potential were analysed by incubation with tetramethylrhodamine (TMRM, 200 nM, ThermoFisher, Waltham, MA, USA). Following incubation (30 min, 37°C/5% CO2), cells were rinsed with phosphate-buffered saline (PBS) and imaged (EVOS M5000). Corrected total cellular fluorescence (CTCF; Bora et al., 2021) was quantified in ImageJ using the formula:

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