Browsing by Author "Pickrell, Alicia M."

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    Deficiency in Parkinson's Disease risk gene CD38 as it relates to glial function: dysregulation of astrocyte genes and bioenergetics as a result of CD38 deficiency
    Hernandez, Raymundo Daniel (Virginia Tech, 2024-01-12)
    Parkinson's disease (PD) is the second most prevalent age-related neurodegenerative disease and currently affects over 8 million people worldwide. The primary features of PD include cognitive, behavioral, and motor function deficits induced primarily by the progressive loss of dopaminergic neurons within the substantia nigra of the basal ganglia (BG). Motor coordination becomes severely affected over the course of the disease, causing patients to experience tremors at rest, bradykinesia, and body rigidity. The availability of treatment options has increased the quality of life for patients experiencing the early stages of PD; however, there exists no cure and treatment options are limited for those experiencing severe, advanced disease symptoms. Genetic studies in PD patients have led to the identification of causative genes, but revealed that less than 20% of cases can be attributed to monogenic variations. Evidence strongly indicates that the majority of PD cases are idiopathic and likely driven due to gene by environmental interactions. Reflective of this idea, recent research efforts have turned to genome-wide association studies (GWAS) to provide indications of gene variations, that while not causative of PD, incur increased risk within patient populations. GWAS findings play a particularly crucial role in neurodegenerative interventions, as early identification of patient risk may allow for preventative therapeutics to delay disease onset or reduce symptom severity. Amongst the many gene variants identified as incurring increased PD risk, single-nucleotide polymorphisms (SNPs) in the loci for CD38 that cause reduced gene expression are consistently identified as increasing risk. The cluster of differentiation 38 (CD38) protein serves two major roles: one as a receptor for immunological response and a second as an ectoenzyme that modulates bioenergetic functions. The particular functions of CD38 are highly relevant to neurodegenerative contexts, as changes in central nervous system (CNS) inflammatory status and means of cellular energy production typically precede pathological indications. In the brain, CD38 expression is most enriched in astrocytes in BG regions, including substantia nigra, midbrain, and striatum. However, it is not known how CD38 deficiency may contribute to astrocytic dysfunction and neuropathological features of PD. This dissertation describes how CD38 influences astrocytic gene expression and cellular bioenergetics. Astrocyte RNA was sequenced from the BG of one-year old male Cd38+/+, Cd38+/-, and Cd38-/- mice by magnetic-activated cell sorting (MACS) to acquire astrocyte isolates. Numerous differentially expressed genes (DEGs) were identified in Cd38 Cd38+/- and Cd38-/- astrocytes that relate to regulation of cellular health, responses to stress, and bioenergetic functions. GO analysis further suggested mitochondrial dysfunction in both Cd38+/- and Cd38-/- astrocytes. In a subsequent set of experiments evaluating mitochondrial function by Seahorse XF96 platform, Cd38+/- and Cd38-/- astrocytes displayed altered bioenergetic function. The results herein demonstrate that astrocytic Cd38 expression regulates cellular function and implicates transcriptional changes associated with the hallmarks of neurodegeneration. These findings serve to provide future direction for studies evaluating the relationship between CD38 function and astrocytes as it relates to neurodegenerative PD risk.
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    Early influences of microbiota on white matter development in germ-free piglets
    Ahmed, Sadia; Travis, Sierrah; Díaz-Bahamonde, Francisca; Porter, Demisha; Henry, Sara; Ravipati, Aditya; Booker, Aryn; Ding, Hanzhang; Ju, Jing; Ramesh, Ashwin; Pickrell, Alicia M.; Wang, Maosen; LaConte, Stephen M.; Howell, Brittany R.; Yuan, Lijuan; Morton, Paul D. (Frontiers, 2021-12-27)
    Abnormalities in the prefrontal cortex (PFC), as well as the underlying white matter (WM) tracts, lie at the intersection of many neurodevelopmental disorders. The influence of microorganisms on brain development has recently been brought into the clinical and research spotlight as alterations in commensal microbiota are implicated in such disorders, including autism spectrum disorders, schizophrenia, depression, and anxiety via the gut-brain axis. In addition, gut dysbiosis is common in preterm birth patients who often display diffuse WM injury and delayed WM maturation in critical tracts including those within the PFC and corpus callosum. Microbial colonization of the gut aligns with ongoing postnatal processes of oligodendrogenesis and the peak of brain myelination in humans; however, the influence of microbiota on gyral WM development remains elusive. Here, we develop and validate a neonatal germ-free swine model to address these issues, as piglets share key similarities in WM volume, developmental trajectories, and distribution to humans. We find significant region-specific reductions, and sexually dimorphic trends, in WM volume, oligodendrogenesis, and mature oligodendrocyte numbers in germ-free piglets during a key postnatal epoch of myelination. Our findings indicate that microbiota plays a critical role in promoting WM development during early life when the brain is vulnerable to environmental insults that can result in an array of disabilities manifesting later in life.
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    EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity
    Sun, Zhixiong; Xu, Xiguang; He, Jianlin; Murray, Alexander; Sun, Ming-an; Wei, Xiaoran; Wang, Xia; McCoig, Emmarose; Xie, Evan; Jiang, Xi; Li, Liwu; Zhu, Jinsong; Chen, Jianjun; Morozov, Alexei; Pickrell, Alicia M.; Theus, Michelle H.; Xie, Hehuang David (2019-08-29)
    Life experience can leave lasting marks, such as epigenetic changes, in the brain. How life experience is translated into storable epigenetic information remains largely unknown. With unbiased data-driven approaches, we predicted that Egr1, a transcription factor important for memory formation, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated thousands of EGR1 binding sites with methylation patterns established during postnatal brain development. More specifically, these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks and activate downstream genes. The frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. In summary, our study reveals EGR1 programs the brain methylome together with TET1 providing new insight into how life experience may shape the brain methylome.
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    EphA4 Influences Blood Brain Barrier Disruption and Endothelial Cell Response following Traumatic Brain Injury in a Mouse Model
    Cash, Alison M. (Virginia Tech, 2022)
    An astonishing number of deaths and related disabilities are attributed to traumatic brain injury (TBI) in the United States per year. Due to the unforeseeable nature of TBI and its association with the sequelae of other neurological comorbidities, research is centered around the secondary responses of brain mechanisms proceeding the initial mechanical injury. Blood brain barrier disruption is a well described driver of this secondary injury response and predictive marker of prognosis following TBI. Although BBB disruption plays a role in subsequent edema, inflammation, and the overall TBI outcome, the molecular mechanisms responsible for its regulation remain to be investigated. A large family of receptor tyrosine kinases, known as Eph receptors, that are important for axon growth and guidance embryonically and early-postnatally have been implicated in brain insults. Previous findings have shown that Eph expression is upregulated at the mRNA and protein level immediately following TBI. Moreover, ablation of Eph receptors on endothelial cells (ECs) revealed improved blood flow to the lesioned cortex in knockout (KO) mice compared to wild type (WT). Based on these results, we hypothesize that Eph receptors negatively regulate BBB permeability leading to neural dysfunction and motor deficits following TBI. To investigate this hypothesis, we characterized the temporal profile of the BBB, evaluated the EC-specific effects of Eph receptors, and used RNA sequencing to assess the cell-specific contributions following TBI in WT compared to KO mice. Our results show that EC-specific loss of Eph expression ameliorated BBB permeability at 6hr, 1-, 4-, and 7-days post injury (dpi) correlating with improved motor function at 7- and 14-dpi. Furthermore, mechanistic studies revealed increased mRNA expression of Tie2, Ang1, and the tight junction proteins Zona Occludens and Occludin in KO mice compared to WT. As well as, connection with neuronal processes. Based off of these findings, we utilized a soluble Tie2 inhibitor to elucidate the influence of Eph receptors on the Tie2/Ang pathway, and their role in mediating the effects seen. Tie2 inhibition of the KO mice revealed similar BBB disruption and lesion volume as WT 1dpi, attenuating the previous protection KO mice demonstrated. Future studies are necessary to understand other pathways that may be implicated in Eph receptor influence on endothelial cells such as inflammatory mediators and neurovascular crosstalk. This data provides evidence that Eph receptors negatively mediate EC response through downstream signaling of the Tie2/Ang pathway and may be a means of therapeutic target in the future.
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    Evaluation of Computational and Experimental Parameters in RNA Bisulfite Sequencing Analysis and Applications in Brain Development Studies
    Johnson, Zachary Austin (Virginia Tech, 2023-09-13)
    Epitranscriptomics, the study of RNA modifications, has become a hotspot of research over the last decade. Over 170 unique modifications have been discovered with a widespread occurrence in a diverse range of RNAs. 5-methylcytosine, m5C, is an evolutionarily conserved and reversable modification that regulates the stability and export of tRNAs, rRNAs, and mRNAs. m5C has recently been implicated in many biological phenomena including tumorigenesis, embryonic cell expansion and differentiation, brain development, and neuronal functions. While we are just beginning to understand the functions of m5C, a gold standard of m5C detection has yet to be established due to the low signal-to-noise presence of m5C. In this work, we utilize RNA bisulfite sequencing as a transcriptome-wide approach to understand the computational and chemical parameters needed to optimize m5C discovery in the mitochondria and the developing brain. In Chapter 1, we systematically evaluate four preparation conditions of bisulfite sequencing to identify potential presence of m5C-mRNAs localized to the mitochondria in neuronal stem cells. In tandem, we utilize unique molecular identifiers and a consortium of control template transcripts to evaluate sources of false positive m5C sites that may emerge from sequencing errors, PCR amplification, and the inadequate bisulfite conversion of transcripts. While improvements to mitochondrial transcript bisulfite conversion and false positive filtering were observed, no mitochondrial mRNAs were identified to be methylated, indicating no or very few methylated cytosines in mitochondrial mRNAs and the need for improved chemical methods to detect mitochondrial m5C-mRNAs if any. In Chapter 2, we employ the computational approaches established in Chapter 1 to survey the m5C landscape of the developing mammalian brain. We discover a general increase in unique m5C sites in mouse whole brain tissue when compared to neuronal cell cultures. Of these sites, we found the post-natal day 0 and 17 brain time points to undergo significant methylation level changes in comparison to the 6-week-old brain. These differentially methylated sites were significantly enriched for brain development, synaptic development, and transcriptional control gene network pathways. In Chapter 3, we expand on our findings in Chapter 2 to understand the impact of m5C reader FMRP and m5C eraser TET1 loss in the mouse post-natal day 17 brain. Among a set of m5C sites identified in wildtype or knockout samples, few were differentially methylated after protein ablation, suggesting m5C may rely on compensatory enzymes. Using FMRP-RNA pulldown assays to validate FMRP binding positions, we identified Ralbp1 to be hypermethylated and overexpressed in Fmr1-KO brain tissues. RalBP1 is a binding protein responsible for the endocytosis of AMPA receptors, a process critical for neuronal long term depression and brain development.
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    The Imbalance of Astrocytic Mitochondrial Dynamics Following Blast-Induced Traumatic Brain Injury
    Guilhaume-Correa, Fernanda; Pickrell, Alicia M.; VandeVord, Pamela J. (MDPI, 2023-01-24)
    Mild blast-induced traumatic brain injury (bTBI) is a modality of injury that has been of major concern considering a large number of military personnel exposed to explosive blast waves. bTBI results from the propagation of high-pressure static blast forces and their subsequent energy transmission within brain tissue. Exposure to this overpressure energy causes a diffuse injury that leads to acute cell damage and, if chronic, leads to detrimental long-term cognitive deficits. The literature presents a neuro-centric approach to the role of mitochondria dynamics dysfunction in bTBI, and changes in astrocyte-specific mitochondrial dynamics have not been characterized. The balance between fission and fusion events is known as mitochondrial dynamics. As a result of fission and fusion, the mitochondrial structure is constantly altering its shape to respond to physiological stimuli or stress, which in turn affects mitochondrial function. Astrocytic mitochondria are recognized to play an essential role in overall brain metabolism, synaptic transmission, and neuron protection. Mitochondria are vulnerable to injury insults, leading to the increase in mitochondrial fission, a mechanism controlled by the GTPase dynamin-related protein (Drp1) and the phosphorylation of Drp1 at serine 616 (p-Drp1s616). This site is critical to mediate the Drp1 translocation to mitochondria to promote fission events and consequently leads to fragmentation. An increase in mitochondrial fragmentation could have negative consequences, such as promoting an excessive generation of reactive oxygen species or triggering cytochrome c release. The aim of the present study was to characterize the unique pattern of astrocytic mitochondrial dynamics by exploring the role of DRP1 with a combination of in vitro and in vivo bTBI models. Differential remodeling of the astrocytic mitochondrial network was observed, corresponding with increases in p-Drp1S616 four hours and seven days post-injury. Further, results showed a time-dependent reactive astrocyte phenotype transition in the rat hippocampus. This discovery can lead to innovative therapeutics targets to help prevent the secondary injury cascade after blast injury that involves mitochondria dysfunction.
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    Mitochondrial Dynamics Alteration in Astrocytes Following Primary Blast-Induced Traumatic Brain Injury
    Guilhaume Correa, Fernanda (Virginia Tech, 2023-01-11)
    Mild blast-induced traumatic brain injury (bTBI) is a modality of injury that has been of major concern considering a large number of military personnel exposed to the blast wave from explosives. bTBI results from the propagation of high-pressure static blast forces and their subsequent energy transmission within brain tissue. Current literature presents a neuro-centric approach to the role of mitochondria dynamics dysfunction in bTBI; however, changes in astrocyte-specific mitochondrial dynamics have not been characterized. As a result of fission and fusion, the mitochondrial structure is constantly altering shape to respond to physiological stimuli or stress insults by adapting structure and function, which are intimately connected. Dysregulation of the protein regulator of mitochondrial fission, DRP1, and upregulation in the phosphorylation of DRP1 at the serine 616 site is reported to play a crucial role in astrocytic mitochondrial dysfunction, favoring fission over fusion post-TBI. Astrocytic mitochondria are starting to be recognized to play an essential role in overall brain metabolism, synaptic transmission, and neuron protection. Mitochondria are vulnerable to injury insults leading to the worsening of mitochondrial fission and increased mitochondrial fragmentation. In this study, a combination of in vitro and in vivo bTBI models were used to examine the effect of blast on astrocytic mitochondrial dynamics. Acute differential remodeling of the astrocytic mitochondrial network was observed, accompanied by an acute (4hr) and sub-acute (7 days) activation of the GTP-protein DRP1. Further, results showed a time-dependent reactive astrocyte phenotype transition in the rat hippocampus. This discovery can lead to innovative therapeutics targets to help prevent secondary injury cascades that involve mitochondria dysfunction.
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    Mitochondrial Quality Control Adaptations Support Malignant Progression of Serous Ovarian Cancer Cells and Spheroids
    Grieco, Joseph Patrick (Virginia Tech, 2022-04-26)
    Serous ovarian cancer is the 5th leading cause of cancer-related deaths in women, with a 30% survival rate when spread into the highly hypoxic and visceral peritoneal cavity. Despite efforts to treat this highly metastatic disease, traditional chemotherapeutic and cytoreductive therapies are unable to diminish or induce cell death of circulating metastases from colonizing secondary sites due to their genetic and histologic heterogeneity and development of drug resistance. The dissemination route for primary metastasis, however, is most often conserved to the peritoneal cavity, which is low in nutrients and hypoxic (1-2% O2). Cells exfoliated from the primary tumor will aggregate during migration, which elicits a survival signal to maintain viability in this environment. The underlying cellular and molecular changes involved with aggregation have yet to be determined. We have previously found that aggregation of murine ovarian surface epithelial (MOSE) cells present a more suppressed metabolic phenotype upon aggregation. My research sought to identify how the mitochondria were internally regulated to support malignant transformation, migration, and invasion through modulation of quality control, mitochondrial dynamics, mitophagy, and mitobiogenesis. We have shown that aggregation of cancer cells supports increased mitochondrial fragmentation localized to the hypoxic core of our spheroid models. Further, aggregation supports enhanced viability through an upregulation of cancer genetic pathways associated with cell death, proliferation, stemness, and epithelial mesenchymal transition (EMT). Nutrient deprivation during migration further enhanced mitochondrial fragmentation and induction of mitophagy to prevent activation of apoptosis. Additionally, we have identified a phenotypic switch from enhanced mitophagy during peritoneal dissemination that supports survival of ovarian cancer cell aggregates to mitochondrial biogenesis during secondary tissue colonization that enables proliferation upon invasion. We have associated these changes with an increased bioenergetic proliferative niche through inhibition of proliferation, migration, and mitochondrial translation. This research has contributed to the understanding for the role of mitophagy as a survival rather than apoptotic signal in cancer cells as adaptation to nutrient-deprived environments, while also identifying how these processes can be reversed upon adhesion to support invasion and metastatic capacity during secondary colonization. This research is significant because it will identify molecular adaptations associated with the viability of disseminating cancer metastases as well as promote novel preventative therapeutics that can be used to limit the mortality of highly aggressive ovarian cancer in women.
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    Modeling Neural Stem Cell Dynamics in Congenital Heart Disease
    Porter, Demisha Donei Lasha (Virginia Tech, 2023-06-28)
    Neural stem/progenitor cells (NSPCs) play a crucial part in the evolutionary development of the human neocortex. During early postnatal development, NSPCs give rise to immature neurons called neuroblasts within the subventricular zone (SVZ) that utilize unique migratory streams to integrate widely in the cerebral cortex. However, the cellular mechanisms enabling these unique migratory routes through the compacted cellular landscape remain unknown. Special emphasis has been placed on understanding the susceptibility of these brain regions to severe conditions such as congenital heart disease (CHD), resulting in poor neurological outcomes. Owing to its reminiscent complexity to humans, the neonatal piglet (Sus scrofa domesticus), which possesses a highly evolved gyrencephalic neocortex and an expansive outer SVZ, provides a powerful translational model system for the study of how heart dysfunction impacts cortical development from both a modern and evolutionary perspective. The present study provides a detailed characterization of neuroblast migration along their associate substrates in the piglet cortex under normal physiological conditions and how reduced oxygenation (i.e., hypoxia) can impact their vulnerability and/or resistance to injury during a critical period of postnatal development. In this thesis, I investigated the spatiotemporal distribution and developmental origin of SVZ-derived neuroblasts. Following BrdU tracing, multiplex labeling, and confocal microscopy, I show that the porcine brain contains populations of newly generated (BrdU+/DCX+) neurons in the prefrontal cortex that are produced postnatally. Regional analyses using immunohistochemical staining for doublecortin (DCX), a marker expressed by immature neurons, revealed that DCX+ clusters co-express markers of neuronal cell migration (PSA-NCAM), GABAergic interneuron marker (GABA+), and specific transcription factors (SCGN+SP8+) associated with the caudal- and lateral ganglionic eminence progenitor domains in the ventral forebrain. Moreover, I found that DCX+ neuroblasts are encased by astrocytic processes and tightly associated with blood vessels in the SVZ. Additionally, this thesis describes the use of chronic hypoxia as a model to profile neuroblast migration along associated substrates in pathological conditions related to CHD. Together, this work serves as a framework for the functional utilization of the neonatal piglet to understand the impact of substrate-dependent neuronal migration on brain maturation and neurodevelopmental diseases.
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    Monocyte proinflammatory phenotypic control by ephrin type A receptor 4 mediates neural tissue damage
    Kowalski, Elizabeth A.; Soliman, Eman; Kelly, Colin; Basso, Erwin Kristobal Gudenschwager; Leonard, John; Pridham, Kevin J.; Ju, Jing; Cash, Alison; Hazy, Amanda; de Jager, Caroline; Kaloss, Alexandra M.; Ding, Hanzhang; Hernandez, Raymundo D.; Coleman, Gabe; Wang, Xia; Olsen, Michelle L.; Pickrell, Alicia M.; Theus, Michelle H. (American Society for Clinical Investigation, 2022-08-08)
    Circulating monocytes have emerged as key regulators of the neuroinflammatory milieu in a number of neuropathological disorders. Ephrin type A receptor 4 (Epha4) receptor tyrosine kinase, a prominent axon guidance molecule, has recently been implicated in the regulation of neuroinflammation. Using a mouse model of brain injury and a GFP BM chimeric approach, we found neuroprotection and a lack of significant motor deficits marked by reduced monocyte/macrophage cortical infiltration and an increased number of arginase-1(+) cells in the absence of BM-derived Epha4. This was accompanied by a shift in monocyte gene profile from pro- to antiinflammatory that included increased Tek (Tie2 receptor) expression. Inhibition of Tie2 attenuated enhanced expression of M2-like genes in cultured Epha4-null monocytes/macrophages. In Epha4-BM-deficient mice, cortical-isolated GFP(+) monocytes/macrophages displayed a phenotypic shift from a classical to an intermediate subtype, which displayed reduced Ly6c(hi) concomitant with increased Ly6c(lo)- and Tie2-expressing populations. Furthermore, clodronate liposome-mediated monocyte depletion mimicked these effects in WT mice but resulted in attenuation of phenotype in Epha4-BM-deficient mice. This demonstrates that monocyte polarization not overall recruitment dictates neural tissue damage. Thus, coordination of monocyte proinflammatory phenotypic state by Epha4 is a key regulatory step mediating brain injury.
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    NAK-associated protein 1/NAP1 activates TBK1 to ensure accurate mitosis and cytokinesis
    Paul, Swagatika; Sarraf, Shireen; Nam, K. H.; Zavar, Leila; DeFoor, Nicole; Biswas, Sahitya; Fritsch, Lauren; Yaron, Tomer; Johnson, Jared; Huntsman, Emily; Cantley, Lewis; Ordureau, Alban; Pickrell, Alicia M. (Rockefeller University Press, 2023-12-07)
    Subcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes errors in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK-associated protein 1 (NAP1) is essential for mitosis, binding to and activating TBK1, which both localize to centrosomes. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but are also associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate phosphorylating NAP1 at S318 to promote its degradation by the ubiquitin proteasomal system. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division.
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    The Non-canonical Function and Regulation of TBK1 in the Cell Cycle
    Paul, Swagatika (Virginia Tech, 2023-10-11)
    Protein kinases play essential roles in orchestrating almost every step during mitosis. Aberrant kinase activity often leads to errors in the cell cycle progression which consequently becomes the underlying cause for developmental defects or abnormal cell proliferation leading to cancer. Tank Binding Kinase 1 (TBK1) is overexpressed in certain cancer types and is activated on the centrosomes during mitosis. Loss of TBK1 impairs cell division resulting in growth defects and the accumulation of multinucleated cells. Therefore, proper activation and localization of TBK1 are essential for mitotic progression. Yet, the upstream regulation of TBK1 and the function of activated TBK1 on the centrosomes is unknown. Also, the cause and consequences of overexpression of TBK1 in cancers remain to be explored. Activation of TBK1 depends on its binding to an adaptor protein which induces a conformational change leading to trans autophoshorylation on serine 172 of its kinase domain. We identified that an established innate immune response protein, NAK Associated Protein1 (NAP1/AZI2), is the adaptor required for binding and activating TBK1 during mitosis. Loss of either NAP1 or TBK1 results in the accumulation of binucleated and multinucleated cells, possibly due to several mitotic and cytokinetic defects seen in these knockout (KO) cells. We establish NAP1 as a cell cycle regulated protein which colocalizes with activated TBK1 on the centrosomes during mitosis. Furthermore, by performing an unbiased quantitative phosphoproteomics analysis during mitosis, the substrates discovered reveal that TBK1 also regulates other known cell cycle regulating kinases such as Aurora A and Aurora B. TBK1 is also an established autophagy protein and since the autophagy machinery is often impaired or remodeled to facilitate rapid cell division, we evaluated the underlying connection between TBK1 activation and autophagy. The data shows that cells lacking the essential autophagy proteins FIP200 or ATG9A exhibit overactivation and mislocalization of TBK1. By using both genetic and pharmacological inhibition of autophagy processes, we found that impaired autophagy leads to a significantly higher number of micronuclei – a hallmark for tumorigenesis that correlates with defects in mitosis and cytokinesis. Taken together our work has uncovered a novel function for the NAP1-TBK1 complex during mitosis and establishes that overactivation and mislocalization of TBK1 is a direct consequence of impaired autophagy which causes micronuclei formation.
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    Peripheral loss of EphA4 ameliorates TBI-induced neuroinflammation and tissue damage
    Kowalski, Elizabeth A.; Chen, Jiang; Hazy, Amanda; Fritsch, Lauren E.; Gudenschwager-Basso, Erwin K.; Chen, Michael; Wang, Xia; Qian, Yun; Zhou, Mingjun; Byerly, Matthew; Pickrell, Alicia M.; Matson, John B.; Allen, Irving C.; Theus, Michelle H. (2019-11-11)
    Background The continuum of pro- and anti-inflammatory response elicited by traumatic brain injury (TBI) is suggested to play a key role in the outcome of TBI; however, the underlying mechanisms remain ill -defined. Methods Here, we demonstrate that using bone marrow chimeric mice and systemic inhibition of EphA4 receptor shifts the pro-inflammatory milieu to pro-resolving following acute TBI. Results EphA4 expression is increased in the injured cortex as early as 2 h post-TBI and on CX3CR1gfp-positive cells in the peri-lesion. Systemic inhibition or genetic deletion of EphA4 significantly reduced cortical lesion volume and shifted the inflammatory profile of peripheral-derived immune cells to pro-resolving in the damaged cortex. These findings were consistent with in vitro studies showing EphA4 inhibition or deletion altered the inflammatory state of LPS-stimulated monocyte/macrophages towards anti-inflammatory. Phosphoarray analysis revealed that EphA4 may regulate pro-inflammatory gene expression by suppressing the mTOR, Akt, and NF-κB pathways. Our human metadata analysis further demonstrates increased EPHA4 and pro-inflammatory gene expression, which correlates with reduced AKT concurrent with increased brain injury severity in patients. Conclusions Overall, these findings implicate EphA4 as a novel mediator of cortical tissue damage and neuroinflammation following TBI.
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    PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis
    Sarraf, Shireen A.; Sideris, Dionisia P.; Giagtzoglou, Nikolaos; Artavanis-Tsakonas, Spyros; Youle, Richard J.; Pickrell, Alicia M. (Cell Press, 2019-10-01)
    PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy. PINK1 and Parkin have been proposed to act as tumor suppressors, as loss-of-function mutations are correlated with enhanced tumorigenesis. However, it is unclear how PINK1 and Parkin act in coordination during mitophagy to influence the cell cycle. Here we show that PINK1 and Parkin genetically interact with proteins involved in cell cycle regulation, and loss of PINK1 and Parkin accelerates cell growth. PINK1- and Parkin-mediated activation of TBK1 at the mitochondria during mitophagy leads to a block in mitosis due to the sequestration of TBK1 from its physiological role at centrosomes during mitosis. Our study supports a diverse role for the far-reaching, regulatory effects of mitochondrial quality control in cellular homeostasis and demonstrates that the PINK1/Parkin pathway genetically interacts with the cell cycle, providing a framework for understanding the molecular basis linking PINK1 and Parkin to mitosis.
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    Remdesivir increases mtDNA copy number causing mild alterations to oxidative phosphorylation
    DeFoor, Nicole; Paul, Swagatika; Li, Shuang; Basso, Erwin K. Gudenschwager; Stevenson, Valentina; Browning, Jack L.; Prater, Anna K.; Brindley, Samantha; Tao, Ge; Pickrell, Alicia M. (Springer, 2023-12-01)
    SARS-CoV-2 causes the severe respiratory disease COVID-19. Remdesivir (RDV) was the first fast-tracked FDA approved treatment drug for COVID-19. RDV acts as an antiviral ribonucleoside (adenosine) analogue that becomes active once it accumulates intracellularly. It then diffuses into the host cell and terminates viral RNA transcription. Previous studies have shown that certain nucleoside analogues unintentionally inhibit mitochondrial RNA or DNA polymerases or cause mutational changes to mitochondrial DNA (mtDNA). These past findings on the mitochondrial toxicity of ribonucleoside analogues motivated us to investigate what effects RDV may have on mitochondrial function. Using in vitro and in vivo rodent models treated with RDV, we observed increases in mtDNA copy number in Mv1Lu cells (35.26% increase ± 11.33%) and liver (100.27% increase ± 32.73%) upon treatment. However, these increases only resulted in mild changes to mitochondrial function. Surprisingly, skeletal muscle and heart were extremely resistant to RDV treatment, tissues that have preferentially been affected by other nucleoside analogues. Although our data suggest that RDV does not greatly impact mitochondrial function, these data are insightful for the treatment of RDV for individuals with mitochondrial disease.
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    The Role of STING Signaling in Central Nervous System Infection and Neuroinflammatory Disease
    Fritsch, Lauren; Kelly, Colin; Pickrell, Alicia M. (Wiley, 2023-01-12)
    The cyclic guanosine monophosphate–adenosine monophosphate (GMP-AMP) synthase-Stimulator of Interferon Genes (cGAS-STING) pathway is a critical innate immune mechanism for detecting the presence of double-stranded DNA (dsDNA) and prompting a robust immune response. Canonical cGAS-STING activation occurs when cGAS, a predominantly cytosolic pattern recognition receptor, binds microbial DNA to promote STING activation. Upon STING activation, transcription factors enter the nucleus to cause the production of Type I interferons, inflammatory cytokines whose primary function is to prime the host for viral infection by producing a number of antiviral interferon-stimulated genes. While the pathway was originally described in viral infection, more recent studies have implicated cGAS-STING signaling in a number of different contexts, including autoimmune disease, cancer, injury, and neuroinflammatory disease. This review focuses on how our understanding of the cGAS-STING pathway has evolved over time with an emphasis on the role of STING-mediated neuroinflammation and infection in the nervous system. We discuss recent findings on how STING signaling contributes to the pathology of pain, traumatic brain injury, and stroke, as well as how mitochondrial DNA may promote STING activation in common neurodegenerative diseases. We conclude by commenting on the current knowledge gaps that should be filled before STING can be an effective therapeutic target in neuroinflammatory disease. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology Infectious Diseases > Molecular and Cellular Physiology Immune System Diseases > Molecular and Cellular Physiology.
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    The Role of STING-Mediated Neuroinflammation in Traumatic Brain Injury
    Fritsch, Lauren Elizabeth (Virginia Tech, 2022-09-23)
    Despite its prevalence, there are currently zero treatments available for traumatic brain injuries (TBI). Neuroinflammation is a key aspect of the secondary injury process, but remains poorly understood. Recent work has shown that Type I Interferons, inflammatory cytokines typically produced in response to viral infection, are present in the post-mortem brains of human TBI patients. The cyclic GMP-AMP Synthase- Stimulator of Interferon Genes (cGAS-STING) pathway is one of the primary methods of producing Type I IFNs; therefore, this work sought to evaluate the role of the cGAS-STING pathway in a murine controlled cortical impact (CCI) model of TBI. Using cGAS knockout (KO) or STING KO mice, we show that global loss of either protein results in substantial neuroprotection. One day after injury, animals have reduced lesion size, cell death, and inflammatory cytokine production, as well as reduced motor deficits several days after injury. We also determined that mitochondrial DNA (mtDNA) is present in the cytosol of injured cortical cells, indicating it is available to bind cGAS, a cytosolic pattern recognition receptor. To determine whether brain-resident or peripheral immune cells are responsible for detrimental cGAS-STING signaling after TBI, we utilized bone marrow chimeric animals lacking STING in either the brain or hematopoietic cells and animals lacking STING specifically in microglia. We found that both microglia and peripheral immune cells contribute to STING signaling after neurotrauma, and that loss of STING in either cell population is beneficial. Taken together, this work demonstrates that canonical, cGAS-dependent STING signaling occurs primarily in microglia and peripheral immune cells, resulting in detrimental neuroinflammatory events after TBI.
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    Type I Interferon Response Is Mediated by NLRX1-cGAS-STING Signaling in Brain Injury
    Fritsch, Lauren E.; Ju, Jing; Basso, Erwin Kristobal Gudenschwager; Soliman, Eman; Paul, Swagatika; Chen, Jiang; Kaloss, Alexandra M.; Kowalski, Elizabeth A.; Tuhy, Taylor C.; Somaiya, Rachana Deven; Wang, Xia; Allen, Irving C.; Theus, Michelle H.; Pickrell, Alicia M. (Frontiers, 2022-02-25)
    Background: Inflammation is a significant contributor to neuronal death and dysfunction following traumatic brain injury (TBI). Recent evidence suggests that interferons may be a key regulator of this response. Our studies evaluated the role of the Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) signaling pathway in a murine model of TBI. Methods: Male, 8-week old wildtype, STING knockout (−/−), cGAS−/−, and NLRX1−/− mice were subjected to controlled cortical impact (CCI) or sham injury. Histopathological evaluation of tissue damage was assessed using non-biased stereology, which was complemented by analysis at the mRNA and protein level using qPCR and western blot analysis, respectively. Results: We found that STING and Type I interferon-stimulated genes were upregulated after CCI injury in a bi-phasic manner and that loss of cGAS or STING conferred neuroprotection concomitant with a blunted inflammatory response at 24 h post-injury. cGAS−/− animals showed reduced motor deficits 4 days after injury (dpi), and amelioration of tissue damage was seen in both groups of mice up to 14 dpi. Given that cGAS requires a cytosolic damage- or pathogen-associated molecular pattern (DAMP/PAMP) to prompt downstream STING signaling, we further demonstrate that mitochondrial DNA is present in the cytosol after TBI as one possible trigger for this pathway. Recent reports suggest that the immune modulator NLR containing X1 (NLRX1) may sequester STING during viral infection. Our findings show that NLRX1 may be an additional regulator that functions upstream to regulate the cGAS-STING pathway in the brain. Conclusions: These findings suggest that the canonical cGAS-STING-mediated Type I interferon signaling axis is a critical component of neural tissue damage following TBI and that mtDNA may be a possible trigger in this response.