Browsing by Author "Reed, Makenna M."

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    Channels and Transporters in Astrocyte Volume Regulation in Health and Disease
    (Cell Physiol Biochem Press, 2022) Reed, Makenna M.; Blazer-Yost, Bonnie; Biology, School of Science
    Astrocytes are the second most abundant cell type in the central nervous system and serve various functions, many of which maintain homeostasis of the intracellular milieu in the face of constant change. In order to accomplish these important functions, astrocytes must regulate their cell volume. In astrocytes, cell volume regulation involves multiple channels and transporters, including AQP4, TRPV4, TRPM4, VRAC, Na+/K+ ATPase, NKCC1 and Kir4.1. AQP4 is a bidirectional water channel directly involved in astrocyte cell volume regulation. AQP4 also forms heteromultimeric complexes with other channels and transporters involved in cell volume regulation. TRPV4, a mechanosensitive channel in involved in osmotic regulation in various cell types, forms a complex with AQP4 to decrease cell volume in response to cell swelling. TRPM4 also forms a complex with AQP4 and SUR1 in response to injury resulting in cell swelling. Another complex forms between Na+/K+ ATPase, AQP4, and mGluR5 to regulate the perisynaptic space. NKCC1 is a co-transporter involved in cell volume increases either independently through cotransport of water or a functional interaction with AQPs. VRAC is implicated in regulatory volume decreases and may also functionally interact with AQP4. Although Kir4.1 colocalizes with AQP4, its role in cell volume regulation is debated. In diseases where fluid/electrolyte homeostasis is disturbed such as stroke, ischemic injury, inflammation, traumatic brain injury and hydrocephalus, cell volume regulation is challenged, sometimes past the point of recovery. Thus, a greater understanding of signaling pathways which regulate transport proteins as well as the functional and physical interactions that exist between transporters will provide a basis for the development of pharmaceutical targets to treat these prevalent and often devastating diseases.
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    Characterizing Changes in the Brain During Hydrocephalic Development and Exploring Potential Treatment Strategies
    (2024-05) Reed, Makenna M.; Blazer-Yost, Bonnie; Belecky-Adams, Teri; Cummins, Theodore; Baucum, A. J.; Jantzie, Lauren
    A neurological disorder, hydrocephalus, has an estimated global pediatric prevalence of 380,000 new cases each year [1]. It is a family of diseases that can occur at any age when cerebrospinal fluid builds up within the ventricles of the brain. Thus, the only available treatments are surgical, invasive, and prone to complications. There is a global need for successful treatment strategies without brain surgery. Choroid plexus epithelial cells (CPEC) are responsible for production of cerebrospinal fluid (CSF). Ependymal cells line the ventricles and play roles in CSF maintenance and waste clearance. Astrocytes perform various functions, one being blood-brain barrier (BBB) maintenance. Collectively these cells contribute to brain fluid/electrolyte regulation and barrier integrity. Increased glial fibrillary acidic protein (GFAP) fluorescence, a marker of activated astrocytes, appeared in hydrocephalic (Tmem67-/-) animals by immunohistochemistry as early as postnatal day (P)10. The tight junction proteins expressed in choroid plexus (CP); claudin-1 (Cl-1) and zona occludin 1 (ZO-1) fluorescent intensity increased in P15 hydrocephalic animals compared to wildtype (Tmem67+/+). These cells also contain aquaporins (AQP), aquaporin-1 (AQP1) and aquaporin-4 (AQP4), important in regulating CSF and interstitial fluid (ISF). Increased fluorescent intensity of AQP4 in the subventricular zone and increased AQP1 apical localization and protein amount in the CP was observed in hydrocephalic animals at postnatal day (P)15. Many of these may be targeted for the treatment of hydrocephalus. However, there is no consensus in pathological findings between models of hydrocephalus and these finding may not translate to common pharmacological targets. A transient receptor potential cation channel, subfamily vanilloid, member 4 (TRPV4) antagonist (RN1734) ameliorates hydrocephalus in a rat model of congenital hydrocephalus (Tmem67 model). It was hypothesized that targeting this mechanosensitive ion channel may slow production of CSF by targeting the CP. However, hydrocephalus pathology can have various effects on the brain. Astrocytes were visualized using fluorescent immunohistochemistry of glial fibrillary acidic protein (GFAP) and RN1734 did not seem to change immunoreactivity to wildtype untreated levels. Increased immunoreactivity of TRPV4 and AQP1 was observed in CP of untreated and RN1734 treated Tmem67-/- rats. AQP4 and TRPV4 immunoreactivity increased in the subventricular zone and periventricular white matter (WM) of hydrocephalic rats. With RN1734, TRPV4 immunoreactivity, but not AQP4, had similar immunoreactivity to wildtype untreated. Increased GFAP and AQP immunoreactivity may indicate residual inflammation in the Tmem67-/- rats. More experiments must be done to further elucidate TRPV4’s role in hydrocephalus pathology. Serum and glucocorticoid-regulated kinase 1 (SGK1) is a kinase implicated in cell volume regulation and CSF production. SI113, an SGK1 inhibitor, ameliorates hydrocephalus in the Tmem67 rodent model. The goal of this study was to determine if SI113 could be used with a new solvent other than dimethyl sulfoxide (DMSO), which can have possible toxic effects. 1-methyl-2-pyrrolidinone (NMP) has high solubility and ability to cross the BBB. These studies showed that NMP as a solvent did not have adverse effects on body weight, however thus far, it has not ameliorated hydrocephalus significantly at the concentration used in this study. There is a possibility that the concentration in NMP that we used was not efficacious enough. CSF and blood plasma samples from animals treated with SI113 24 hours and 30 minutes before euthanasia will be used to investigate the concentration of SI113 that remains in the circulation and the amount that crosses the BBB and blood-cerebrospinal fluid (BCSFB) barriers. We hope that the results will inform dosage for our future studies. Future studies may also examine SI113 mechanism of action in hydrocephalus. This thesis addresses hydrocephalus cell and molecular pathology in the Tmem67 model and examines potential treatment strategies. Future directions include comparing models of hydrocephalus to find common treatment strategies in the hope to find pharmaceutical strategies to better manage human hydrocephalus.
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    TRPV4 antagonists ameliorate ventriculomegaly in a rat model of hydrocephalus
    (American Society for Clinical Investigation, 2020-09-17) Hochstetler, Alexandra E.; Smith, Hillary M.; Preston, Daniel C.; Reed, Makenna M.; Territo, Paul R.; Shim, Joon W.; Fulkerson, Daniel; Blazer-Yost, Bonnie L.; Biology, School of Science
    Hydrocephalus is a serious condition that impacts patients of all ages. The standards of care are surgical options to divert, or inhibit production of, cerebrospinal fluid; to date, there are no effective pharmaceutical treatments, to our knowledge. The causes vary widely, but one commonality of this condition is aberrations in salt and fluid balance. We have used a genetic model of hydrocephalus to show that ventriculomegaly can be alleviated by inhibition of the transient receptor potential vanilloid 4, a channel that is activated by changes in osmotic balance, temperature, pressure and inflammatory mediators. The TRPV4 antagonists do not appear to have adverse effects on the overall health of the WT or hydrocephalic animals.