Chapter 2



Characterization of Seizure Threshold in System xc- Astrocyte-Conditional Null Mice

© (Myles Morgan, May 14th 2020)

Abstract

System xc- (Sxc-) is a cellular cystine/glutamate antiporter that is constitutively expressed by astrocytes in the central nervous system. It functions in the maintenance of extracellular glutamate levels and serves an important role in controlling synaptic strength and neuronal excitability. Previously, we determined that mice globally null for Sxc- (SLC7a11sut/sut mice) had lower convulsive seizure thresholds ─ that is, had a greater percentage of convulsive seizures ─ than their wild type (SLC7a11+/+) littermates following acute challenge with the chemoconvulsant pentylenetetrazole (PTZ). Herein, we explore whether this excitability phenotype occurs in Sxc- astrocyte-conditional null mice. Mice for study were generated using standard Cre-Lox technology. Slc7a11fl/fl mice were crossed with mGFAP-Cre transgenic mice (JAX Stock #012887) to obtain heterozygous breeders. Subsequent progeny ─ SLC7a11+/+ (n=22; 12M, 10F), SLC7a11fl/fl (n=23; 9M, 14F), mGFAP-Cre-SLC7a11+/+ (n=12; 8M, 4F), and mGFAP-Cre SLC7a11fl/fl mice (n=12; 19M, 13F) ─ were administered a single systemic dose of PTZ (either 43 mg/kg or 50 mg/kg) at 12-14 weeks of age and the maximal seizure score recorded as follows: 0 = no behavioral change, 1 = hypomobility, 2 = myoclonus, 3 = generalized convulsion with righting reflex, 4 = generalized convulsion without righting reflex. Additionally, the convulsive seizure index was determined by dividing the number of mice with a maximum seizure score ≥ 3 by the total number of mice injected. Latency to both first seizure as well as convulsive seizure were also recorded. A one-way ANOVA revealed no statistical differences between genotypes for seizure score, latency to seizure, or latency to convulsion in either female or male mice receiving either 43 or 50 mg/kg PTZ (i.p.). However, chi-square analysis revealed a significant difference in the convulsive index between SLC7a11+/+ and all other variants (SLC7a11fl/fl, mGFAP-Cre-SLC7a11+/+, and mGFAP-Cre-SLC7a11fl/fl) in female mice at 50 mg/kg but not 43 mg/kg. Particularly, only 20% of female SLC7a11+/+ mice developed convulsive seizures following PTZ, whereas the convulsive indices for SLC7a11fl/fl and mGFAP-Cre-SLC7a11+/+ female mice were 100%, and 70% for mGFAP-Cre SLC7a11fl/fl mice. No differences between genotypes were found in the convulsive index for male mice at any dose. However, both male and female mGFAP-Cre-SLC7a11fl/fl mice took longer to experience their first non-convulsive and convulsive seizure compared to their mGFAP-Cre-SLC7a11+/+ littermates at 50 mg/kg PTZ. Additionally, at 43 mg/kg PTZ male and female mGFAP-Cre-SLC7a11+/+ took longer than mGFAP-Cre-SLC7a11fl/fl mice to experience their first convulsive seizure. Taken together, these data suggest that the change in excitability observed in SLC7a11sut/sut mice may not be fully recapitulated in mGFAP-Cre SLC7a11fl/fl mice of either sex. Final conclusions await completion of a full dose response curve (supported by NINDS R01NS105767 to SJH and JAH).

Executive Summary

Epilepsy is a highly prevalent neurological disease that impacts more than 70 million people globally (Thijs, Surges, O’Brien, & Sander, 2019). The disease is characterized by the presence of seizures, which are brief episodic events of abnormal synchronous electrical activity in brain resulting from neuronal circuit dysfunction (Arabadzisz, Antal, Parpan, Emri, & Fritschy, 2005). The brain naturally strives to maintain a balance between excitatory and inhibitory activity ─ between glutamate and gamma aminobutyric acid (GABA) signaling, respectively ─ which is essential for maintaining the stability of neuronal networks, thus preventing seizures. This balance is achieved, in part, by the maintenance of homeostatic levels of the main excitatory amino acid neurotransmitter glutamate, the process of which is largely carried out via astrocyte transporters.

Particularly, the extracellular glutamate concentration must be tightly regulated as excess signaling can lead to abnormal synchronous activity (seizures) or cell death (excitotoxic neuronal injury), whereas sufficient signaling is necessary for normal transmission of excitatory signals (Marcaggi & Attwell, 2004). One astrocyte transporter of significance is System xc- (Sxc-), which is important for maintaining the basal extracellular levels of glutamate via facilitating its release (Baker, Xi, Shen, Swanson, & Kalivas, 202; De Bundel et al., 2011).

In brain, loss of Sxc- function is associated with both a reduction in extracellular glutamate levels and a compensatory increase in the abundance of postsynaptic glutamate receptors (Augustin, Grosjean, Chen, Sheng, & Featherstone, 2007). This latter result could explain the hyperexcitability phenotype, manifest by a decrease in seizure threshold that we uncovered in mice globally null for Sxc- (Chintala et al., 2005) when they were systemically administered a chemical, pentylenetetrazole (PTZ), that elicits seizures in mice (Sears, unpublished observations). In brain, Sxc- is highly enriched in a cell type known as astrocytes [pic](Ottestad-Hansen et al., 2018; Zhang et al., 2014). Thus, the aim of this study was to determine whether mice with no Sxc- expression in their astrocyte cell population would also be hyperexcitable in response to the PTZ.

In this study we used both male and female mice of various genetic makeup at ages 12-14 weeks. Animals designated as SLC7a11+/+; SLC7a11fl/fl and mGFAP-Cre-SLC7a11+/+ represent mice that express functional Sxc- whereas mGFAP-Cre SLC7a11fl/fl mice are Sxc- astrocyte conditional null mice. The most appropriate wild-type control mice for the astrocyte conditional null is mGFAP-Cre-SLC7a11+/+. Mice were observed for 30 min following PTZ administration. Latency to non-convulsive and convulsive seizure were recorded as was the maximal seizure score attained, which was assigned using the standard 5-point Racine scoring system (Racine, 1972). Assignment of a Stage 1 means the mouse demonstrated behavioral arrest and staring. These seizures can evolve into brief myoclonic jerks or twitches (Stage 2) that can further progress into full-bodied clonic or tonic-clonic seizures without (Stage 3) or with (Stage 4) loss of righting reflex (Racine, 1972). Furthermore, a convulsive index was calculated by dividing the number of mice in a cohort that experienced a convulsive seizure (score ≥ 3) by the total number of mice dosed.

Overall, no difference was found between the seizure severity behavioral score, latency to first seizure or latency to convulsion, or convulsive seizure indices between male mGFAP-Cre-SLC7a11+/+ (functional astrocytic Sxc-) and mGFAP-Cre SLC7a11fl/fl (astrocyte-conditional null for Sxc-) mice at 50 mg/kg PTZ. However, while not statistically different, male mGFAP-Cre-SLC7a11fl/fl mice took 46.1% longer than male mGFAP-Cre-SLC7a11+/+ mice to experience their first seizure, which suggest that the seizure threshold of these mice are enhanced. In females, we find the opposite. While seizure severity — behavioral score, latency to convulsant seizure, and the convulsive seizure index — were not different between mGFAP-Cre-SLC7a11+/+ and mGFAP-Cre-SLC7a11fl/fl mice, the latency to first seizure of female mGFAP-Cre SLC7a11fl/fl mice was 106% faster than the time associated with mGFAP-Cre-SLC7a11+/+, suggesting that the seizure threshold of astrocyte conditional null mice is lowered. Regarding the lower dose of 43 mg/kg PTZ, the seizure score, latency to first seizure, latency to convulsion, and convulsive index were not found to statistically differ between male and female mGFAP-Cre-SLC7a11+/+ and mGFAP-Cre-SLC7a11fl/fl mice. However, mGFAP-Cre-SLC7a11+/+ took longer to seize as compared to mGFAP-Cre-SLC7a11fl/fl mice in both males and females. This difference in latency to seizure between mGFAP-Cre-SLC7a11+/+ and mGFAP-Cre-SLC7a11fl/fl mice was also found regarding convulsion, but only in male mice.

Thus, overall the results presented herein did suggest that male Sxc- astrocyte conditional nulls did not fully recapitulate the hyperexcitability phenotype seen in both sexes of the SLC7a11sut/sut mice. This lack of full replication is due to the fact that female mGFAP-Cre-SLC7a11fl/fl mice, seem to experience this hyperexcitability phenotype given their more rapid response to 50 mg/kg of PTZ in comparison to female mGFAP-Cre-SLC7a11+/+ mice. On the other hand, male mGFAP-Cre-SLC7a11fl/fl experienced their first seizure quicker than male mGFAP-Cre-SLC7a11+/+ mice also opposing the hyperexcitability phenotype. Additionally, the full interpretation of these data are complicated by the fact that, at least in female mice, manipulation of the SLC7a11 gene, whether by addition of lox P sites flanking exon 2 of the SLC7a11 gene or via introduction of Cre-recombinase under the control of the mouse GFAP promoter, was shown to reduce seizure threshold as evidenced by the increase in convulsive seizure indices of all mice save for SLC7a11+/+ mice.

Apart from seizure threshold alterations between genotypes in either sex, differences between sexes also occurred at the lower but not higher dose of PTZ regarding convulsion index, leading us to conclude that overall female mice a higher seizure threshold than male mice but this occurs over a narrow and specific dose range. Further supporting the conclusion that loss of system xc- only within astrocytes do not fully recapitulate the hyperexcitability found in global nulls.

While uncontrollable circumstances prevented the completion of my study, the data presented represent a preliminary understanding of the role that astrocytic system xc- plays in the seizure threshold. However, it is necessary for these experiments to be redone with a larger sample size. Present data also raise a cautionary note for all biological researchers using the Cre-lox system to manipulate gene expression, as this alone may affect the seizure threshold making interpretation of the data difficult.

Table of Contents

Abstract……………………………………….……………….…………... iii

Executive Summary………………………….……………….…………… v

Acknowledgements…………..……………………………………………. x

Chapter 1: Introduction ……………………………………………… 1

Chapter 2: Materials and Methods ……………………………………… 4

Chapter 3: Figure Legends ……………………………………………… 7

Chapter 4: Results ………………………………………………………11

Chapter 5: Discussion ……………………………………………… 16

Works Cited.……………………………………………………………… 20

Acknowledgements (Optional)

I would first like to thank my mentor and principal investigator of the lab I have spent many hours working in, Dr. Sandra Hewett for the years of mentorship and guidance it took for me to get to this point within my research career. Additionally, I would like to thank my lab mates who have been there for me throughout my time at Syracuse, answering and asking thought provoking questions while also always up for a good laugh. I would also like to thank my friends who have supported me throughout my college journey, I have learned so much from you all and you continue to inspire and drive me to do my best. I would like to thank my family who have always supported me throughout my life, including my academic endeavors. I would like to thank the SOURCE for helping fund this project. Lastly, I would like to thank the Biology Distinction program and the Renée Crown Honors program for pushing me to achieve more with my education.

Chapter 1

Introduction

Epilepsy is a highly prevalent neurological disease that impacts more than 70 million people globally (Thijs, Surges, O’Brien, & Sander, 2019). The disease is characterized by the presence of seizures, which are brief episodic events of abnormal synchronous electrical activity in brain resulting from neuronal circuit dysfunction (Arabadzisz, Antal, Parpan, Emri, & Fritschy, 2005). One is diagnosed with epilepsy if they fulfil any of the following criteria: 1) having had two or more unprovoked seizures, that is one that is not associated with trauma or infection, occurring within a 24 hr or greater time span; 2) having had an unprovoked seizure with the likelihood of additional seizures will occur, for instance because of the presence of a tumor (recurrence risk between 60-90%); or 3) having what is termed an epilepsy syndrome, for instance Benign Epilepsy with Centro-Temporal Spikes (Fisher, 2015). The brain naturally strives to maintain a balance between excitatory and inhibitory activity ─ between glutamate and gamma aminobutyric acid (GABA) signaling, respectively ─ which is essential for maintaining the stability of neuronal networks, thus preventing seizures. This balance is achieved, in part, by the maintenance of homeostatic levels of the main excitatory amino acid neurotransmitter glutamate, the process of which is largely carried out via astrocyte transporters.

Astrocytes are a category of glial cell that aid in neuronal health and maintenance in a variety of different ways. Astrocytes are the most abundant cell type within the brain; functioning in the formation and maintenance of the blood brain barrier, in the production and release of antioxidants, and in the release and uptake of the major excitatory neurotransmitter, glutamate (Haydon & Carmignoto, 2006; Vainchtein & Molofsky, 2020). The extracellular glutamate concentration must be tightly regulated as excess signaling can lead to abnormal synchronous activity (seizures) or cell death (excitotoxic neuronal injury), whereas sufficient signaling is necessary for normal transmission of excitatory signals (Marcaggi & Attwell, 2004). Importantly, astrocytes maintain optimal excitatory/inhibitory balance via uptake and release of glutamate. Uptake of glutamate into astrocytes is facilitated by the excitatory amino acid transporters (EAATs) 1 and 2, with the latter contributing to the vast majority of uptake (Haugeto et al., 1996). Consistent with its importance in maintaining optimal glutamate levels, loss of EAAT2 in mice results in seizures, followed by death, due excess glutamate in the extracellular space (Tanaka et al., 1997). Interestingly, EAATs are also reduced in the hippocampi of individuals with Temporal Lobe Epilepsy, suggesting that excess extracellular glutamate levels contribute to these recurring seizures (Rakhade & Loeb, 2008; SARAC et al., 2009). Less studied is the role of another astrocyte transporter known as System xc- (Sxc-), which is important for maintaining the basal extracellular levels of glutamate via facilitating its release (Baker, Xi, Shen, Swanson, & Kalivas, 2002; De Bundel et al., 2011).

In brain, Sxc- is an astrocyte-expressed heterodimeric antiporter, which imports cystine and exports glutamate in a 1:1 ratio (Sato, Tamba, Ishii, & Bannai, 1999). Sxc- consists of the substrate specific light-chain subunit xCT, and the heavy-chain subunit 4F2hc (Sato et al., 1999). This export of glutamate contributes to the ambient levels found in the synaptic cleft (Augustin et al., 2007; De Bundel et al., 2011; Baker et al., 2002). In brain, loss of Sxc- function is associated with both a reduction in extracellular glutamate levels and a compensatory increase in the abundance of postsynaptic glutamate receptors (Augustin, Grosjean, Chen, Sheng, & Featherstone, 2007). This latter result could explain the hyperexcitability phenotype, manifest by a decrease in seizure threshold, of SLC7a11sut/sut mice, which was uncovered when seizures were provoked by administration of a chemoconvulsant (Sears, unpublished observations). SLC7a11sut/sut mice have a natural mutation in the SLC7a11 gene which encodes for xCT, the functional subunit of Sxc- (Chintala et al., 2005), meaning that they lack expression of this antiporter in all of their tissues. However, since Sxc- expression in the central nervous system occurs primarily in the astrocyte, the goal of this project was to determine if the seizure threshold in mice lacking Sxc- only in the astrocyte cell population is also reduced.

Chapter 2

Materials and Methods

Materials and Methods: These studies were all conducted in accordance with the National Institute of Health guidelines for the use and care of experimental animals as approved by the Institutional Animal Care and Use Committee of Syracuse University.

Animals Housing: All mice were housed three to five per cage on a 12 hr light/dark schedule (7 am/7pm), bred and maintained in the Laboratory Animal Care facility of Syracuse University, which is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Standard mouse chow and water were provided ad libitum.

Breeding: Purchased female hemizygous mGFAP-Cre line 77.6 mice (The Jackson laboratory 024098) were breed with a male mouse carrying an SLC7a11 gene in which exon 2 was flanked by loxP sites (SLC7a11fl/fl). The resulting male SLC7a11+/fl mice were bred with female mGFAPCre- SLC7a11 +/fl . This process created all relevant genotypes including WT (SLC7a11+/+; SLC7a11fl/fl and mGFAP-Cre-SLC7a11+/+) and AcKOs (mGFAP-Cre SLC7a11fl/fl).

Genotyping: Mice were weaned at 21 days and ear punched for identification. Genotyping of mice was performed by PCR analysis of genomic DNA isolated from tail biopsies. Tails were first dissolved in a tissue lysis buffer containing 1M Tris, 5M NaCl, 0.2% SDS, 0.5M EDTA, and 100(g/mL proteinase K (Invitrogen, Carlsbad, CA) for 48-72 hours at 55◦C, DNA was precipitated by centrifugation in isopropanol for six minutes at 10,000xg. The DNA was then purified by centrifugation in 70% ethanol for six minutes at 10,000xg. The resulting pellet was resuspended in DNAse-free H20 and the concentration of DNA measured using a Nanodrop microvolume spectrophotometer. Primers pairs for target DNA are as follows: WT primers (1100 bp for WT allelles or 1255 bp for floxed allelle) 5’- AAC AGC TCT AGG CAG ACG TG -3’ (forward), 5’- TCA GCT ACC CTG CCT CAA AC -3’; Cre primers (400 bp) 5’-TCC ATA AAG GCC CTG ACA TC -3’ ,5’- TGC GAA CCT CAT CAC TCG T-3’ ; Cre internal control primers (200 bp) 5’- CAA ATG TTG CTT GTC TGG TG -3’, 5’-GTC AGT CGA GTG CAC AGT TT -3’.

Acute PTZ: Male and female SLC7a11+/+; SLC7a11fl/fl; mGFAP-Cre-SLC7a11+/+; mGFAP-Cre SLC7a11fl/fl mice (12-14 weeks) were administered an acute dose of pentylenetetrazole

Pentylenetetrazole (PTZ) (Sigma Aldrich Chemicals, St. Louis, MO) was made fresh on the day of the experiment by dissolving in saline at 100 mg/ml. Solutions were filter sterilized. Each day for five days prior to injection, mice were acclimated to handling by rubbing their lower abdomen. On the day of experimentation, mice were moved into the procedure room, weighed, and then allowed to acclimatize to the new environment for at least one hour. A single dose of PTZ was administered intraperitoneally (i.p.) in a volume of 10 ml/kg body weight. Both male and female mice (12-14 weeks old) were used. Investigator was blind to mouse’s genotype at time of experimentation and during the 30 min behavioral analysis (detailed below) Mice genotypes were re-confirmed at the end of each study.

Behavioral Analysis: Mice are observed for 30 min following PTZ administration. Time to non-convulsive and convulsive seizure were recorded and a maximal seizure score assigned using the standard 5-point Racine scoring system [pic](Claycomb et al., 2011). Within seconds of a systemic administration, PTZ generates seizures consisting of behavioral arrest and immobility, unresponsiveness and staring (Stage 1). Depending upon the dose, these seizures will evolve into brief myoclonic jerks or twitches (Stage 2) that can further progress into full-bodied clonic seizures and tonic-clonic seizures without (Stage 3) or with (Stage 4) loss of righting reflex [pic]( Claycomb et al., 2011). To ensure unbiased scoring, all seizures were scored by an observer blinded to genotype.

Descriptive PTZ-induced Seizure Scoring System

|Seizure |Abbreviated |Detailed |

|Score |Description |Description |

|0 |Normal behavior |Frequent ambulation/exploration, sniffing, rearing, peer interactions, grooming, eating, |

| | |digging, climbing are common. |

|1 | |Mice will socially isolate self and disregard peers; body will be positioned close to |

| | |bottom of cage. The overall behavioral picture will be dominated by bouts (>10 sec in |

| |Hypomobility |duration) of staring and motionlessness that may be interrupted by brief sniffing or |

| |and hypoactivity |ambulation. |

|2 | |Animal exhibit at least two isolated myoclonic seizures typically involving axial |

| |Repeated myoclonus |muscles, commonly seen as neck flexion. Straub tail (dorsiflexion) is also common, as in |

| | |an increase in locomotor activity (compared to score 1). |

|3 | |Clonic seizures involve forelimbs and neck while the animal assumes an upright posture |

| |Convulsive seizure with intact |using hind limbs to support body weight. |

| |righting reflex | |

|4 | |Clonic seizures involve both forelimbs and hind limbs preventing the maintenance of |

| |Convulsive seizure with loss of|upright posture. Infrequently, these seizures can be associated with violent running and |

| |righting reflex |jumping episodes and tonic hind limb extension. |

Table adapted from descriptions in [pic](Ferraro et al., 1999; Racine, 1972; Pitkanen, 2006).

Statistical Analysis: All statistical analyses were run using GraphPad Prism Version 8.4.2. If sample size permitted, one-way analysis of variance (ANOVA) was used to compare seizure scores, latency to seizure, and latency to convulsion between genotypes. The convulsive index between genotypes were assessed using Chi-Square analysis when sample size permitted. Significance was set at p < 0.05.

Chapter 3

Figure Legends

Figure 1. Impact that Cre-Lox variations have on seizure severity and latency to seizure of mice treated with 50 mg/kg PTZ.

A, B) Seizure Severity: Male (A) and female (B) +/+ and fl/fl mice with (+ Cre) and without Cre (-Cre) were administered 50 mg/kg PTZ, i.p. Seizure severity over a 30 min period was scored based on the 5-point Racine scoring system as described in Methods. Each symbol is representative of the maximal seizure score for a single mouse, while the horizontal line represents the median seizure score for that particular genotype. For males, one-way ANOVA revealed no significant differences between genotypes. For females, low sample size prevented statistical assessment.

C, D) Time to Seizure: Each symbol represents the time (in seconds) it took for the male (C) mice in (A) or female (D) mice in (B) to experience their first seizure after administration of 50 mg/kg of PTZ, whereas the horizontal line represents the mean value for that genotype. For male mice, one-way ANOVA revealed no significant differences between genotypes. For female mice, low sample size prevented statistical assessment.

Figure 2. Impact that Cre-Lox variations have on the convulsive seizure activity and latency of mice treated with 50mg/kg PTZ.

A, B) Convulsion Incidence: Male (A) and female (B) +/+ and fl/fl mice with (+ Cre) and without Cre (-Cre) were administered 50 mg/kg PTZ, i.p. and behavioral seizures scored using the 5-point Racine scoring system as described in Methods. The bars represent the number of male and female mice that experienced a convulsive seizure (score ≥ 3) as a percentage of the total mice dosed (fraction within each bar). * represents values that are significant different from +/+ (-Cre) mice as determined by Chi-square analysis. Significance was set at p ................
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