The Effects of a Novel Inhibitor of Tumor Necrosis Factor (TNF) Alpha on Prepulse Inhibition and Microglial Activation in Two Distinct Rodent Models of Schizophrenia.

Increased neuroinflammation has been shown in individuals diagnosed with schizophrenia (SCHZ). This study evaluated a novel immune modulator (PD2024) that targets the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) to alleviate sensorimotor gating deficits and microglial activation employing two different rodent models of SCHZ. In Experiment 1, rats were neonatally treated with saline or the dopamine D2-like agonist quinpirole (NQ; 1 mg/kg) from postnatal day (P) 1-21 which produces increases of dopamine D2 receptor sensitivity throughout the animal's lifetime. In Experiment 2, rats were neonatally treated with saline or the immune system stimulant polyinosinic:polycytidylic acid (Poly I:C) from P5-7. Neonatal Poly I:C treatment mimics immune system activation associated with SCHZ. In both experiments, rats were raised to P30 and administered a control diet or a novel TNFα inhibitor PD2024 (10 mg/kg) in the diet from P30 until P67. At P45-46 and from P60-67, animals were behaviorally tested on auditory sensorimotor gating as measured through prepulse inhibition (PPI). NQ or Poly I:C treatment resulted in PPI deficits, and PD2024 treatment alleviated PPI deficits in both models. Results also revealed that increased hippocampal and prefrontal cortex microglial activation produced by neonatal Poly I:C was significantly reduced to control levels by PD2024. In addition, a separate group of animals neonatally treated with saline or Poly I:C from P5-7 demonstrated increased TNFα protein levels in the hippocampus but not prefrontal cortex, verifying increased TNFα in the brain produced by Poly I:C. Results from this study suggests that that brain TNFα is a viable pharmacological target to treat the neuroinflammation known to be associated with SCHZ.


Introduction
Individuals diagnosed with schizophrenia (SCHZ) have been shown to have increased in ammation in the brain CNS, particularly within the amygdala, hippocampus, nucleus accumbens, and prefrontal cortex (1)(2)(3). In ammation is the rst reaction of the body's immune system, which is a complex response of the host to tissue injury such as infection or physical insult (4). Prenatal maternal infections with pathogens such as herpes simplex, Toxoplasma gondii, or cytomegalovirus are associated with SCHZ in adult offspring (5-7). Further, autoimmune diseases in children or early childhood infections have been associated with higher incidence of SCHZ (8,9). The ensuing in ammation damages key components in the brain involved in neuronal plasticity, and ultimately contribute to cytokine and neurotransmitter abnormalities. It is hypothesized that changes in the immune system are a driving factor behind both psychotic relapses and the macroscopic brain changes that occur in SCHZ, including the characteristic enlarged ventricle size and reductions in gray matter volume, whole-brain volume, as well as abnormalities in white matter (10).
Pro-in ammatory cytokines are recruited from the periphery in elevated levels in response to infection or insult that result in in ammation. In ammation may interrupt normal processes in the blood brain barrier (BBB), which may contribute to the unusual peripheral recruitment pattern (11). Therefore, it becomes essential to further understand and investigate this in ammatory response in individuals diagnosed with SCHZ to potentially serve as a new drug target. In the abnormal in ammatory response for patients diagnosed with SCHZ, increases of tumor necrosis factor-alpha (TNFα) levels, a pro-in ammatory cytokine, have been shown to be increased in the periphery and in the CNS (12,13). TNFα is found in leukocytes and is involved in the acute phase reaction of in ammation. It is responsible for the regulation of a variety of immune cells that respond to in ammatory signals. Elevated levels of TNFα directly in uence the state of microglia cells. Microglia are the primary immune cells of the CNS and constantly survey their local environment for signs of cellular stress (14). Under normal conditions, microglia exist as anti-in ammatory, neuroprotective agents, characterized as the "M2 state." However, upon activation by TNFα secretion, microglia switch to the "M1 state," which is pro-in ammatory and neurotoxic. Microglia in the M1 activation state lead to overexpression of pro-in ammatory cytokines and reactive oxygen species (ROS), resulting in synaptic loss and neuronal death (3).
Although targeting neuroin ammation and the development of new anti-in ammatories has become a novel therapeutic approach to treat SCHZ, an increase of dopamine D2 receptor sensitivity still remains as a central hallmark of the disease. Our laboratory has established a rodent model of SCHZ based on increases in dopamine D2 receptor sensitivity through neonatal treatment of the dopamine D2-like agonist quinpirole to rats (15). The neonatal quinpirole rat model shows behavioral, neurobiological, and genetic consistencies that mimic features of individuals diagnosed with SCHZ (16). However, it is not yet known whether the neonatal quinpirole rat model displays neuroin ammatory features observed in human SCHZ.
In the current study, we will evaluate the effects of a novel, orally bioavailable TNFα modulator, on auditory sensorimotor gating as measured through prepulse inhibition (PPI) in two rodent models of SCHZ: The neonatal quinpirole model and the polyinsinic:polycytidylic acid (Poly I:C) model. The rationale for employing two models that mimic different aspects of the disease is to perform a more rigorous and thorough evaluation of the TNFα modulator. The neonatal quinpirole model mimics the increases of dopamine D2 receptor sensitivity present in SCHZ, whereas the Poly I:C model mimics neuroin ammation and the increase in immune response during early brain development that has been shown to increase the incidence of SCHZ. PPI is a behavioral task that involves the presentation of an auditory prepulse (73, 76, or 82dB) presented shortly (100 ms) before a startling auditory stimulus (120 dB). Therefore, the prepulse predicts the startling auditory stimulus, and over presentations of these stimuli the animal acquires the ability to inhibit the startle response. De cits in PPI are a behavioral hallmark of SCHZ. In addition, we will analyze the effects of a novel TNFα inhibitor on microglial activation in brain areas known to be critical in the circuitry that underlies PPI. Past work has shown that neonatal poly I:C treatment to rats increases proin ammatory cytokines and increases microglial activation in the hippocampus and prefrontal cortex (17,18)

Subjects.
A total of 78 male Sprague-Dawley rats that were the offspring of 15 female Sprague-Dawley dams were subjects across both experiments. Females were not included in this study because of past work that has reported equivocal results relative to sex differences on the auditory sensorimotor gating task in different models of SCHZ and/or neuroin ammation (19,20). One rat from each litter were assigned to each drug/diet condition to control for within litter variance. The day of birth was counted as postnatal day (P)0. Regardless of neonatal drug treatment, all animals were housed with the female dam from P1-21 and socially housed from P22-30. Beginning on P30, the control or experimental diet was presented, and all animals were singly housed from P30 until the P67, when brain tissue was harvested. Animals were housed in a climate-controlled vivarium with a 12 h on/off light/dark cycle throughout the experiment. Procedures used in this study were approved by the University Committee on Animal Care at East Tennessee State University and animal facilities are accredited by the American Association of Laboratory Animal Care (AAALAC).

PD2024.
PD2024 is part of an isoindoline-based drug technology that P2D Bioscience, Inc (Cincinnati, OH) licensed from the NIH and is being developed to treat dementia. Past work from Gabbita and colleagues reported that oral PD2024 engaged brain TNFα, improved cognitive performance, and reduced the core phosphotau tangle immune-histopathology in a mouse model of Alzheimer's Disease (21). Experiment 1: Drug/Diet Treatment.
In Experiment 1, rats were neonatally treated with saline or the dopamine D2-like agonist quinpirole. A total of 24 rats were neonatally i.p. administered saline or quinpirole (1 mg/kg) from postnatal day (P)1-21, identical to past work with this model (see 16  In Experiment 2, rats were neonatally i.p. administered either saline or Polyinosinic:Polycytidylic acid (Poly I:C) from P5-7, which has been shown to result in de cits in auditory sensorimotor gating as measured by PPI as well as increased microglial activation (18). In both experiments, beginning at P30, rats were singly housed with enrichment and given either regular chow or 10 mg/kg PD2024 (P2D Bioscience, Inc., Cincinnati, OH) mixed in their diet (Dyets, Inc., Bethlehem, PA). These diets were presented until the end of the experiment when brain tissue was harvested at P67. The rationale for the use of this dose of PD2024 is that Gabbita and colleagues have shown that this dose, given through the diet, was effective to reduce in ammation as measured by microglial activation in a mouse model of Alzheimer's Disease (3xTg; 21).

Experiment 2b.
Finally, in Experiment 2b, a separate group of 17 male Sprague-Dawley rats were administered saline (N=8) or Poly I:C (N=9) from P5-7, and raised to P30. These animals were not given any diet manipulation.
For this group of animals, brain tissue was harvested at P30-33 and the hippocampus and prefrontal cortex were dissected away and ash frozen on dry ice. The brain tissue from these animals was analyzed for TNFα levels using an ELISA kit (Biomatik, Wilmington, DE) to verify that TNFα was increased by neonatal Poly I:C treatment when the diet manipulation was initiated.
Group codes.
With each neonatal drug treatment manipulation, there were four different conditions. In Experiment 1 the treatment groups were noted as NS PD2024, NQ PD2024, NS Control, NQ Control, with the rst acronym representing neonatal drug treatment (NS-Neonatal Saline; NQ-Neonatal Quinpirole) and the second term representing which diet the group received beginning on P30. Likewise, in animals treated with Poly I:C, there were also four different conditions that were noted as NS PD2024, Poly IC PD2024, NS Control, and Poly IC Control with the rst acronym representing neonatal drug treatment (NS-Neonatal Saline; Poly IC) and the second term representing which diet the group received beginning on P30.

Experiments 1 & 2: PPI Methods.
Animals were behaviorally tested once daily on PPI and at two different time points: once during development adolescent days P45 and 46, and again in adulthood (P60-65). We opted to use two different developmental time points to analyze the effects of PD2024 over a developmental time course. The equipment used for PPI behavioral testing was from Kinder Scienti c, Inc., (Poway, CA, USA). Rats were administered three different, randomly assigned trial types, which included pulse trials, prepulse trials, and no stimulus trials. A pulse trial is a 120 dB startle pulse administered alone. A prepulse trial is an auditory stimulus (click) that is 3, 6, or 12 dB above the 70 dB background noise (i.e. 73, 76, and 82dB). A no stimulus trial is a trial in which no stimulus is given. All animals underwent 25 randomized trials which include 5 pulse, 5 no stimulus, and 15 prepulse trials (5 trials of each 73, 76, and 82db). Animals were placed into a stainless-steel dome (height = 8 cm) that was attached to a platform (11 cm wide x 15 cm long) mounted on a stainless steel ellipse in a sound attenuating chamber (28 cm tall x 30 cm wide x 36 cm in depth). The behavioral response was recorded during a 250 ms window immediately after the stimulus was presented, and was measured in Newtons, the SI (International System of Units) unit for force. On each daily session, the animals were placed into the dome and given a 5 min habituation period with only the background noise (70 dB white noise) presented. Experiment 2a: Immunohistochemistry (IHC) for microglial activation in the hippocampus and prefrontal cortex.
We performed IHC only on animals neonatally treated with Poly I:C (Experiment 2), because it is well established that Poly I:C administered during either gestation (23) or neonatal development results in increases of microglial activation in the brain (Ribeiro et al 2013). On P67-69, Poly I:C animals were deeply anesthetized and intracardially perfused with 4% paraformaldehyde. Brain tissue was removed and stored in 20% sucrose for approximately 48 h, and then transferred to a clean vial and stored at -80 o C. Tissues were coronally sectioned on a Leica cryostat at 50 μm thickness and stored at -20ºC until immunolabeling was performed. We chose to analyze the hippocampus and prefrontal cortex because their established roles in sensorimotor gating (24).
Free oating sections were mounted on glass slides and coverslipped using mounting media. Iba1 (Wako Chemicals USA, Richmond, VA) was the primary antibody used for IHC labeling of microglial cells and AlexaFluor488 conjugated Anti-Rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was the secondary antibody tagged with GFP to emit uorescence. Slides were examined under a Leica TCS SP8 inverted confocal microscope at a magni cation of 40x. A total of four images were captured per brain region (4 PFC, 2 CA1 & 2 CA3 of the Hip). NIH ImageJ software was used to quantify images of the prefrontal cortex and CA1 and CA3 regions of the dorsal hippocampus. For each brain section, average sampled cell body uorescence was measured. The freehand tool in ImageJ was used to draw around a given microglia cell body. Integrated Density from ImageJ was used to determine average cell body uorescence as well as overall eld uorescence. At random, ve GFP-stained microglia cells were selected per given eld for the measurement of average cell body uorescence.
Animals neonatally treated with saline or Poly IC (2 mg/kg) brain tissue was taken between P30-33. This time point was chosen because this is when the dietary manipulation was begun. Once tissue was harvested, the dorsal hippocampus, ventral hippocampus, and medial frontal cortex were dissected away. Tissue samples were further diluted 1:50 before being assayed. The standards and samples were incubated at room temperature for 1 hour. The monoclonal antibody was then added to each well plate, incubated at room temperature (1 h), which was followed by incubation (1 h) with the conjugate.
Visualization was achieved by adding tetramethybenzidine (TMB) one solution to each well followed by an incubation period of 20 minutes at room temperature, and this reaction was stopped by adding the stop solution to each well and plates were read within 5 minutes of stopping the reaction. Optical density was measured using a Bio-Tek ELx 800 microplate reader (Winooski, VT).
Statistical Analysis.
For weight gain analysis in each experiment, a three-way ANOVA was used with neonatal drug treatment, diet (control or PD2024), and day of development (P30, P40, P50, P60) was used as the repeated measure. For analysis of PPI, the two experiments were separately analyzed. In each experiment, a three-way ANOVA was used with neonatal drug treatment, diet (control or PD2024), and prepulse (73, 76, or 82 dB) as between subjects factors. A two-way ANOVA (neonatal drug treatment, diet) was used to analyze IHC immuno uorescence in each brain area. The Newman-Keuls post hoc test (p=0.05) was used to analyze any signi cant interactions in both experiments.

Results
Weight Gain. The weight of animals across neonatal drug treatments and diet from P30-60 are presented in Table 1 for Experiment 1 and 2. A three-way ANOVA revealed no significant main effects or interactions. Therefore, regardless of neonatal drug treatment and diet condition, there was no significant main effects of interactions of the PD2024 diet on normal weight gain in each neonatal drug treatment condition. produced a deficit in control animals at these same prepulse intensities. This is not necessarily unexpected because PD2024 is designed to modulate TNFα in an abnormal system which, in this case, resulted in deficits in PPI. Regardless of the brain area, rats administered poly I:C during the neonatal period demonstrated a significant increase in fluorescence intensity in microglia which was reduced to saline control levels by PD2024. These data indicate that PD2024 was effective to reduce the increase inflammatory response within microglia across three brain areas that are important in both working memory and auditory sensorimotor gating.
TNFα results for adolescent rats neonatally treated with saline or poly I:C are presented in Figure 4. An independent t-test was performed to analyze each brain area, and Page 10/17 revealed a significant main effect of group in the dorsal hippocampus t(12)=2.21, p<.05 as well as the ventral hippocampus t(12)=3.16, p<.01. Although poly I:C increased absolute TNFα levels in the frontal cortex, it was not statistically significant due to high variability in the poly I:C-treated group (N=5 for this brain area). These results are consistent with recent work (25) that recently demonstrated maternal poly I:C significantly increased TNFα in the brain, but did not affect other pro-inflammatory cytokines.

Discussion
The present study demonstrated that a modulator of the pro-in ammatory cytokine TNFα, PD2024, presented in the diet during adolescence and early adulthood successfully alleviated de cits in auditory sensorimotor gating as measured by PPI in rats neonatally treated with the dopamine D2-like agonist quinpirole or the immune stimulant poly I:C. These results demonstrate the utility of PD2024 to alleviate PPI de cits in two well-validated but distinct rodent models of SCHZ that are related to either increase in dopamine D2 receptor functioning or early immune system activation. These ndings are consistent with several past studies that have shown that the anti-in ammatories are effective to treat SCHZ. Likely, the most impactful of these studies utilized minocycline, which also decreases TNFα expression (26).
Minocycline was successful in alleviating PPI de cits in the poly I:C rodent model of SCHZ (23,27). Finally, it was established that neonatal Poly I:C results in a signi cant increase in TNFα in the hippocampus, consistent with a recent study by Clark et al (25) that demonstrated increases in TNFα protein, but no changes in other pro-in ammatory cytokines, in the brain after maternal (GD15) acute infusion of Poly I:C (5 mg/kg). Since the known target of PD2024 is to modulate TNFα, this would at least suggest that its action on this pro-in ammatory cytokine is involved in mediating the effects observed in the present study.
PD2024 resulted in PPI de cits in the control group in adolescent rats in the present study, however, this de cit dissipated in adulthood. The mechanism for this effect is unknown, but interestingly, it was consistent across both models, indicating that there may be an interaction of PD2024 with neurobiological changes occurring during the adolescent period. However, this is really not a major concern, because PD2024 is being developed as a treatment only for individuals diagnosed with SCHZ who possess an abnormal Results here are consistent with past work, including work that has administered poly I:C neonatally (18), as we did here, as well as maternal exposure to poly I:C. Interestingly, maternal (37,38) exposure to poly I:C has demonstrated dopamine hyperfunction, and structural abnormalities including reduced hippocampal and prefrontal cortical volumes in poly I:C offspring. All of these characteristics are hallmarks of SCHZ (3).
An advantage of both of these models is that these alterations emerge in the offspring during adolescence, which is consistent with clinical observations. A critical observation in any model is that treatments that typically alleviate some of the behavioral abnormalities of SCHZ are also observed in the model. Importantly, clozapine (18) as well as risperidone (39) have been shown to alleviate behavioral de cits observed in rats developmentally treated with poly I:C.
We analyzed changes in microglia only in animals neonatally treated with poly I:C. The rationale for this analysis was that it was known that poly I:C results in microglial activation (40,41). A primary focus of this study was to demonstrate that PD2024 would reduce this microglial activation. Poly I:C did in fact increase microglial activation which was reduced to control levels by PD2024. The increase in microglial activation produced by neonatal Poly I:C treatment is consistent with past work and was observed in both hippocampus and prefrontal cortex, whereas past work had observed this effect in hippocampus, prefrontal cortex, and striatum. Presumably, PD2024 reduced this activation through modulating TNFα, however, it is also known that PD2024 increases cholinergic activity in the brain (Gabbita, unpublished observations). Increased nicotinic acetylcholinergic receptor activity has been associated with antiin ammatory properties (42). Interestingly, it has been discovered that acute postnatal Poly I:C (5 mg/kg) administered on P5 reduced binding at muscarinic receptors, suggesting a decrease in cholinergic activity due to neonatal Poly I:C treatment. Cholinergic agonists, especially at the nicotinic alpha7 receptor, have been shown to reduce PPI de cits and cognitive impairments in individuals diagnosed with SCHZ (43,44). Therefore, PD2024 could be working through multiple mechanisms to alleviate PPI de cits observed in the present study.
Recently, the involvement of the immune system has increasingly been implicated in the behavioral pathological processes of SCHZ (45)(46)(47)(48). It has been hypothesized that changes in the immune system is a driving factor behind both psychotic relapses and the macroscopic brain changes that occur in SCHZ, including the characteristic enlarged ventricle size and reductions in gray matter volume, whole-brain volume, as well as the known abnormalities in white matter (10). McEvoy et al. recently published an analysis of in ammatory markers analysis of blood samples from SCHZ patients enrolled in the Clinical Antipsychotic Trials in Intervention Effectiveness (CATIE) project, which were suggestive that in ammation is associated with SCHZ-related psychopathology and cognition and has differential effects of substance use in SCHZ (49). Although the results at this point are not conclusive, there are supporting data to indicate that the anti-in ammatory minocycline improved PANSS scores relative to placebo (For review see, 50,51) and more recently meta-analyses published in 2017 from both Solmi et al 52) and Xiang et al (53) reported minocycline's superiority over placebo, especially on total and negative symptom scores.
Increases in dopamine D2 receptor sensitivity has long been established as a neurobiological abnormality in SCHZ (3). Support for this notion comes from the fact that all antipsychotic medications antagonize the dopamine D2 receptor with some a nity, and all of antipsychotic drugs have been shown to alleviate, at the very least, the positive symptoms of SCHZ (54). Over the past 15 years, our laboratory has investigated behavioral and neurobiological mechanisms in the neonatal quinpirole model, many that are consistent with SCHZ (16). However, this is the rst demonstration that a drug designed to modulate a proin ammatory cytokine was effective at alleviating behavioral de cits in this model. On the other hand, we have published several studies analyzing nicotine in this model because of the high incidence of cigarette smoking in the population diagnosed with SCHZ (55). As mentioned above, the known anti-in ammatory properties of nicotine may be playing a role in this model as well, although we have yet to verify there is neuroin ammation in the neonatal quinpirole model. Nicotine is known to alleviate behavioral impairments in SCHZ, including cognitive de cits (56). Therefore, this may represent a mechanism by which nicotine is working in SCHZ, since it has recently been established neuroin ammation occurs in SCHZ.

Conclusions
This study revealed that PD2024, a novel compound, directed at modulating the pro-in ammatory cytokine TNFα was effective at alleviating de cits in auditory sensorimotor gating, a behavioral hallmark of SCHZ. In addition, this compound also reduced microglial activation in the hippocampus, a brain area that plays a critical role in the symptoms expressed in individuals diagnosed with SCHZ. Future work is designed to evaluate the underlying molecular mechanism by which PD2024 lowers TNFα and advance the compound's use as a possible adjunctive therapy to treat SCHZ.