As I secure the delicate, finger-tip size mouse brain into the penetratingly cold cryostat and watch the viscous red blood drip off the side of the warm mouse’s body cavity during a perfusion to collect said brain, I feel my curiosity for the brain grow ever greater.
To obtain these small brains, I performed day-long perfusions with my laboratory supervisors. We euthanized mice with carbon dioxide gas and exsanguinated their still-warm bodies, decapitating them then carefully uncapping their skulls to reveal their delicate brains. I then sliced through these brains on the cryostat to compare the differences between microglial and astrocytic mitochondrial morphologies in males and females.
Such a lab procedure is part of my exciting work with the Bilbo/Kingsbury Laboratory in the Department of Pediatrics at the Charlestown Navy Yard Campus of the Massachusetts General Hospital. Our lab examines how neuro-immune interactions shape sex differences in neurodevelopmental disorders such as Autism Spectrum Disorder (ASD). My project involves characterizing sex differences in non-neuronal (i.e. microglial and astrocytic) cell morphologies in mouse models that lack a cytokine: interleukin-10 (IL-10). IL-10 influences the protection of mitochondrial oxidative phosphorylation against immune challenges in macrophages located in the peripheral nervous system (PNS). I work with the goal of discovering more information on how early-life immune challenges impact development and, in particular, ASD development.
ASD is a neurodevelopmental disability characterized by social, behavioral, and communication challenges (Bock & Goode 2003). The disorder especially hampers the ability for a person to relate with others and express one’s feelings and is characterized by repetitive behaviors. ASD exists on a spectrum ranging from mild (e.g. well-developed language and high academic abilities) to severe (e.g. language disability and sensory dysfunction) (Bock & Goode 2003). According to the Centers for Disease Control and Prevention (CDC), around one in 54 children are diagnosed with ASD globally, and the number of children who are diagnosed with ASD increases each year (CDC 2020). Current treatment includes medical therapy targeting behavior-based strategies, but they often vary in their effectiveness in individuals depending on the degree of severity of ASD. (Levy & Griffiths 2017).
Previous studies have shown that, although there is no known cause for ASD, it is a heterogeneous disorder ─ a phenotype presented from an array of different genetic mechanisms ─ seemingly correlated with many genetic mutations (Bezzi et. al. 2016). Yet because of the heterogeneity of ASD, it is difficult to determine which specific genes are at fault (Bezzi et. al. 2016). A newer way of studying ASD has been to examine the impact of the dysfunction of non-neuronal cells on ASD. Non-neuronal cells that reside in the brain, also known as glial cells, are not directly involved in synaptic input but provide support in a wide range of brain functions, such as immune defense and axonal guidance. Microglia are a specific type of glial cell that are the innate immune cells of the brain. Astrocytes are another type of glial cell in the brain that serve many roles, such as assistance with the blood brain barrier and axon guidance. Recent studies have shown that glial cells in the brains of autistic individuals are overactivated and produce excessive inflammatory factors (Bezzi et. al. 2016).
Past research has also demonstrated that there is a defect in mitochondrial oxidative phosphorylation in the cells of patients with ASD (Levy & Griffiths 2017). This has led to the proposal of a biochemical link between defective mitochondrial function and ASD (Levy & Griffiths 2017). Furthermore, mitochondrial deformities take part in many neurological pathologies that seem to be sex-specific (Garnier et. al 2017), and ASD disproportionately affects three to four times as many boys than girls (CDC 2020). Interestingly, some of these mitochondrial defects take place following maternal immune activation events (Smith et. al 2007; Mashour et. al. 2018). Maternal immune activation events (otherwise known as early life immune challenges) occur when an infectious stimulus triggers the immune system in the mother, resulting in cytokines and other immune factors being passed onto the fetus (Minakova & Warner 2018). This establishes a link between this immune reaction and disruptions in the mechanisms of neural circuitry development in the fetal brain (Smith et. al 2007; Mashour et. al. 2018). However, a stronger link between these immune factors and sex differences is necessary to establish causality.
So far, the Bilbo team at MGH has demonstrated that a mouse model of early life immune challenge results in male-specific deficits in social behavior, which makes sense given the male-biased prevalence of ASD. Additionally, they have shown that this early life immune challenge results in male-specific alterations in mitochondrial function, gene expression, and morphology—specifically within glial cells. The lab’s projects focus on discovering 1) a male protective factor or 2) a female protective factor within glial cells that may explain the male-biased prevalence of ASD. The lab has also demonstrated that IL-10 is much more highly expressed in the brains of females than in males throughout early development.
In my research, I hypothesize that female and male mice would exhibit little differences in their microglial mitochondrial morphologies, because if IL-10 is lacking, the lack in this anti-inflammatory cytokine will not be able to protect mitochondrial oxidative phosphorylation against immune challenges, regardless of sex. If this is true, then IL-10 may be this ‘female protective factor’ that protects female microglia against immune challenges early in life, thereby preventing further alterations in neural circuitry important for social behavior in females, and helping to explain the stark difference in the ratio of male to female diagnoses of ASD. I am slicing 52 brains to look for these morphologies.
My project contributes to the greater understanding of how sex fits into the role immune activation plays in early childhood and ASD development. Ultimately, I hope to help shed light on this ‘female protective factor,’ which may provide more information regarding ASD susceptibility in males and females. This in turn would illuminate how mitochondrial morphologies can impact the brain.
About the Author
Mairead Baker is a sophomore at Harvard College concentrating in Neuroscience.
References
Bock, G., & Goode, J. (2003). Autism : Neural basis and treatment possibilities (Novartis Foundation symposium ; 251). Hoboken, N.J.: Wiley.
Boulanger-Bertolus, Julie, Pancaro, Carlo, & Mashour, George A. (2018). Increasing Role of Maternal Immune Activation in Neurodevelopmental Disorders. Frontiers in Behavioral Neuroscience, 12, 230.
Centers for Disease Control and Prevention. “Autism Spectrum Disorder.” (2020). Retrieved from: https://www.cdc.gov/ncbddd/autism/index.html
Griffiths, K. Levy, R. (2017). Oxidative Medicine and Cellular Longevity. Volume 2017, Article ID 4314025
Lombardo, M V, Moon, H M, Su, J, Palmer, T D, Courchesne, E, & Pramparo, T. (2018). Maternal immune activation dysregulation of the fetal brain transcriptome and relevance to the pathophysiology of autism spectrum disorder. Molecular Psychiatry, 23(4), 1001-1013.
Minakova, Elena, & Warner, Barbara B. (2018). Maternal immune activation, central nervous system development and behavioral phenotypes. Birth Defects Research, 110(20), 1539-1550.
Petrelli, Francesco, Pucci, Luca, & Bezzi, Paola. (2016). Astrocytes and Microglia and Their Potential Link with Autism Spectrum Disorders. Frontiers in Cellular Neuroscience, 10, 21.
Smith, Stephen E. P, Li, Jennifer, Garbett, Krassimira, Mirnics, Karoly, & Patterson, Paul H. (2007). Maternal Immune Activation Alters Fetal Brain Development through Interleukin-6. The Journal of Neuroscience, 27(40), 10695-10702.
Ventura-Clapier, Renee, Moulin, Maryline, Piquereau, Jerome, Lemaire, Christophe, Mericskay, Mathias, Veksler, Vladimir, & Garnier, Anne. (2017). Mitochondria: A central target for sex differences in pathologies. Clinical Science (1979), 131(9), 803-822.
To obtain these small brains, I performed day-long perfusions with my laboratory supervisors. We euthanized mice with carbon dioxide gas and exsanguinated their still-warm bodies, decapitating them then carefully uncapping their skulls to reveal their delicate brains. I then sliced through these brains on the cryostat to compare the differences between microglial and astrocytic mitochondrial morphologies in males and females.
Such a lab procedure is part of my exciting work with the Bilbo/Kingsbury Laboratory in the Department of Pediatrics at the Charlestown Navy Yard Campus of the Massachusetts General Hospital. Our lab examines how neuro-immune interactions shape sex differences in neurodevelopmental disorders such as Autism Spectrum Disorder (ASD). My project involves characterizing sex differences in non-neuronal (i.e. microglial and astrocytic) cell morphologies in mouse models that lack a cytokine: interleukin-10 (IL-10). IL-10 influences the protection of mitochondrial oxidative phosphorylation against immune challenges in macrophages located in the peripheral nervous system (PNS). I work with the goal of discovering more information on how early-life immune challenges impact development and, in particular, ASD development.
ASD is a neurodevelopmental disability characterized by social, behavioral, and communication challenges (Bock & Goode 2003). The disorder especially hampers the ability for a person to relate with others and express one’s feelings and is characterized by repetitive behaviors. ASD exists on a spectrum ranging from mild (e.g. well-developed language and high academic abilities) to severe (e.g. language disability and sensory dysfunction) (Bock & Goode 2003). According to the Centers for Disease Control and Prevention (CDC), around one in 54 children are diagnosed with ASD globally, and the number of children who are diagnosed with ASD increases each year (CDC 2020). Current treatment includes medical therapy targeting behavior-based strategies, but they often vary in their effectiveness in individuals depending on the degree of severity of ASD. (Levy & Griffiths 2017).
Previous studies have shown that, although there is no known cause for ASD, it is a heterogeneous disorder ─ a phenotype presented from an array of different genetic mechanisms ─ seemingly correlated with many genetic mutations (Bezzi et. al. 2016). Yet because of the heterogeneity of ASD, it is difficult to determine which specific genes are at fault (Bezzi et. al. 2016). A newer way of studying ASD has been to examine the impact of the dysfunction of non-neuronal cells on ASD. Non-neuronal cells that reside in the brain, also known as glial cells, are not directly involved in synaptic input but provide support in a wide range of brain functions, such as immune defense and axonal guidance. Microglia are a specific type of glial cell that are the innate immune cells of the brain. Astrocytes are another type of glial cell in the brain that serve many roles, such as assistance with the blood brain barrier and axon guidance. Recent studies have shown that glial cells in the brains of autistic individuals are overactivated and produce excessive inflammatory factors (Bezzi et. al. 2016).
Past research has also demonstrated that there is a defect in mitochondrial oxidative phosphorylation in the cells of patients with ASD (Levy & Griffiths 2017). This has led to the proposal of a biochemical link between defective mitochondrial function and ASD (Levy & Griffiths 2017). Furthermore, mitochondrial deformities take part in many neurological pathologies that seem to be sex-specific (Garnier et. al 2017), and ASD disproportionately affects three to four times as many boys than girls (CDC 2020). Interestingly, some of these mitochondrial defects take place following maternal immune activation events (Smith et. al 2007; Mashour et. al. 2018). Maternal immune activation events (otherwise known as early life immune challenges) occur when an infectious stimulus triggers the immune system in the mother, resulting in cytokines and other immune factors being passed onto the fetus (Minakova & Warner 2018). This establishes a link between this immune reaction and disruptions in the mechanisms of neural circuitry development in the fetal brain (Smith et. al 2007; Mashour et. al. 2018). However, a stronger link between these immune factors and sex differences is necessary to establish causality.
So far, the Bilbo team at MGH has demonstrated that a mouse model of early life immune challenge results in male-specific deficits in social behavior, which makes sense given the male-biased prevalence of ASD. Additionally, they have shown that this early life immune challenge results in male-specific alterations in mitochondrial function, gene expression, and morphology—specifically within glial cells. The lab’s projects focus on discovering 1) a male protective factor or 2) a female protective factor within glial cells that may explain the male-biased prevalence of ASD. The lab has also demonstrated that IL-10 is much more highly expressed in the brains of females than in males throughout early development.
In my research, I hypothesize that female and male mice would exhibit little differences in their microglial mitochondrial morphologies, because if IL-10 is lacking, the lack in this anti-inflammatory cytokine will not be able to protect mitochondrial oxidative phosphorylation against immune challenges, regardless of sex. If this is true, then IL-10 may be this ‘female protective factor’ that protects female microglia against immune challenges early in life, thereby preventing further alterations in neural circuitry important for social behavior in females, and helping to explain the stark difference in the ratio of male to female diagnoses of ASD. I am slicing 52 brains to look for these morphologies.
My project contributes to the greater understanding of how sex fits into the role immune activation plays in early childhood and ASD development. Ultimately, I hope to help shed light on this ‘female protective factor,’ which may provide more information regarding ASD susceptibility in males and females. This in turn would illuminate how mitochondrial morphologies can impact the brain.
About the Author
Mairead Baker is a sophomore at Harvard College concentrating in Neuroscience.
References
Bock, G., & Goode, J. (2003). Autism : Neural basis and treatment possibilities (Novartis Foundation symposium ; 251). Hoboken, N.J.: Wiley.
Boulanger-Bertolus, Julie, Pancaro, Carlo, & Mashour, George A. (2018). Increasing Role of Maternal Immune Activation in Neurodevelopmental Disorders. Frontiers in Behavioral Neuroscience, 12, 230.
Centers for Disease Control and Prevention. “Autism Spectrum Disorder.” (2020). Retrieved from: https://www.cdc.gov/ncbddd/autism/index.html
Griffiths, K. Levy, R. (2017). Oxidative Medicine and Cellular Longevity. Volume 2017, Article ID 4314025
Lombardo, M V, Moon, H M, Su, J, Palmer, T D, Courchesne, E, & Pramparo, T. (2018). Maternal immune activation dysregulation of the fetal brain transcriptome and relevance to the pathophysiology of autism spectrum disorder. Molecular Psychiatry, 23(4), 1001-1013.
Minakova, Elena, & Warner, Barbara B. (2018). Maternal immune activation, central nervous system development and behavioral phenotypes. Birth Defects Research, 110(20), 1539-1550.
Petrelli, Francesco, Pucci, Luca, & Bezzi, Paola. (2016). Astrocytes and Microglia and Their Potential Link with Autism Spectrum Disorders. Frontiers in Cellular Neuroscience, 10, 21.
Smith, Stephen E. P, Li, Jennifer, Garbett, Krassimira, Mirnics, Karoly, & Patterson, Paul H. (2007). Maternal Immune Activation Alters Fetal Brain Development through Interleukin-6. The Journal of Neuroscience, 27(40), 10695-10702.
Ventura-Clapier, Renee, Moulin, Maryline, Piquereau, Jerome, Lemaire, Christophe, Mericskay, Mathias, Veksler, Vladimir, & Garnier, Anne. (2017). Mitochondria: A central target for sex differences in pathologies. Clinical Science (1979), 131(9), 803-822.
