IDO-1 inhibition improves outcome after fluid percussion injury in adult male rats.

The enzyme indoleamine 2,3 dioxygenase 1 (IDO1) catalyzes the rate-limiting step in the kynurenine pathway (KP) which produces both neuroprotective and neurotoxic metabolites. Neuroinflammatory signals produced as a result of pathological conditions can increase production of IDO1 and boost its enzymatic capacity. IDO1 and the KP have been implicated in behavioral recovery after human traumatic brain injury (TBI), but their roles in experimental models of TBI are for the most part unknown. We hypothesized there is an increase in KP activity in the fluid percussion injury (FPI) model of TBI, and that administration of an IDO1 inhibitor will improve neurological recovery. In this study, adult male Sprague Dawley rats were subjected to FPI or sham injury and received twice-daily oral administration of the IDO1 inhibitor PF-06840003 (100 mg/kg) or vehicle control. FPI resulted in a significant increase in KP activity, as demonstrated by an increased ratio of kynurenine: tryptophan, in the perilesional neocortex and ipsilateral hippocampus 3 days postinjury (DPI), which normalized by 7 DPI. The increase in KP activity was prevented by PF-06840003. IDO1 inhibition also improved memory performance as assessed in the Barnes maze and anxiety behaviors as assessed in open field testing in the first 28 DPI. These results suggest increased KP activity after FPI may mediate neurological dysfunction, and IDO1 inhibition should be further investigated as a potential therapeutic target to improve recovery.

Development and Optimization of a Target Engagement Model of Brain IDO Inhibition for Alzheimer's Disease.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO1) inhibition is a promising target as an Alzheimer's disease (AD) Disease-modifying therapy capable of downregulating immunopathic neuroinflammatory processes. METHODS: To aid in the development of IDO inhibitors as potential AD therapeutics, we optimized a lipopolysaccharide (LPS) based mouse model of brain IDO1 inhibition by examining the dosedependent and time-course of the brain kynurenine:tryptophan (K:T) ratio to LPS via intraperitoneal dosing. RESULTS: We determined the optimal LPS dose to increase IDO1 activity in the brain, and the ideal time point to quantify the brain K:T ratio after LPS administration. We then used a brain penetrant tool compound, EOS200271, to validate the model, determine the optimal dosing profile and found that a complete rescue of the K:T ratio was possible with the tool compound. CONCLUSION: This LPS-based model of IDO1 target engagement is a useful tool that can be used in the development of brain penetrant IDO1 inhibitors for AD. A limitation of the present study is the lack of quantification of potential clinically relevant biomarkers in this model, which could be addressed in future studies.

Design, synthesis, and biological evaluation of furosemide analogs as therapeutics for the proteopathy and immunopathy of Alzheimer's disease.

ß-Amyloid (Aß) triggered proteopathic and immunopathic processes are a postulated cause of Alzheimer's disease (AD). Monomeric Aß is derived from amyloid precursor protein, whereupon it aggregates into various assemblies, including oligomers and fibrils, which disrupt neuronal membrane integrity and induce cellular damage. Aß is directly neurotoxic/synaptotoxic, but may also induce neuroinflammation through the concomitant activation of microglia. Previously, we have shown that furosemide is a known anthranilate-based drug with the capacity to downregulate the proinflammatory microglial M1 phenotype and upregulate the anti-inflammatory M2 phenotype. To further explore the pharmacologic effects of furosemide, this study reports a series of furosemide analogs that target both Aß aggregation and neuroinflammation, thereby addressing the combined proteopathic-immunopathic pathogenesis of AD. Forty compounds were synthesized and evaluated. Compounds 3c, 3g, and 20 inhibited Aß oligomerization; 33 and 34 inhibited Aß fibrillization. 3g and 34 inhibited the production of TNF-α, IL-6, and nitric oxide, downregulated the expression of COX-2 and iNOS, and promoted microglial phagocytotic activity, suggesting dual activity against Aß aggregation and neuroinflammation. Our data demonstrate the potential therapeutic utility of the furosemide-like anthranilate platform in the development of drug-like molecules targeting both the proteopathy and immunopathy of AD.

A Series of 2-((1-Phenyl-1H-imidazol-5-yl)methyl)-1H-indoles as Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a promising therapeutic target in cancer immunotherapy and neurological disease. Thus, searching for highly active inhibitors for use in human cancers is now a focus of widespread research and development efforts. In this study, we report the structure-based design of 2-(5-imidazolyl)indole derivatives, a series of novel IDO1 inhibitors which have been designed and synthesized based on our previous study using N1-substituted 5-indoleimidazoles. Among these, we have identified one with a strong IDO1 inhibitory activity (IC50 =0.16 µM, EC50 =0.3 µM). Structural-activity relationship (SAR) and computational docking simulations suggest that a hydroxyl group favorably interacts with a proximal Ser167 residue in Pocket A, improving IDO1 inhibitory potency. The brain penetrance of potent compounds was estimated by calculation of the Blood Brain Barrier (BBB) Score and Brain Exposure Efficiency (BEE) Score. Many compounds had favorable scores and the two most promising compounds were advanced to a pharmacokinetic study which demonstrated that both compounds were brain penetrant. We have thus discovered a flexible scaffold for brain penetrant IDO1 inhibitors, exemplified by several potent, brain penetrant, agents. With this promising scaffold, we provide herein a basis for further development of brain penetrant IDO1 inhibitors.

Age-related deterioration of motor function in male and female 5xFAD mice from 3 to 16 months of age.

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to age-related cognitive and sensori-motor dysfunction. There is an increased understanding that motor dysfunction contributes to overall AD severity, and a need to ameliorate these impairments. The 5xFAD mouse develops the neuropathology, cognitive and motor impairments observed in AD, and thus may be a valuable animal model to study motor deficits in AD. Therefore, we assessed age-related changes in motor ability of male and female 5xFAD mice from 3 to 16 months of age, using a battery of behavioral tests. At 9-10 months, 5xFAD mice showed reduced body weight, reduced rearing in the open-field and impaired performance on the rotarod compared to wild-type controls. At 12-13 months, 5xFAD mice showed reduced locomotor activity on the open-field, and impaired balance on the balance beam. At 15-16 months, impairments were also seen in grip strength. Although sex differences were observed at specific ages, the development of motor dysfunction was similar in male and female mice. Given the 5xFAD mouse is commonly on a C57BL/6 × SJL hybrid background, a subset of mice may be homozygous recessive for the Dysf im mutant allele, which leads to muscular weakness in SJL mice and may exacerbate motor dysfunction. We found small effects of Dysf im on motor function, suggesting that Dysf im contributes little to motor dysfunction in 5xFAD mice. We conclude that the 5xFAD mouse may be a useful model to study mechanisms that produce motor dysfunction in AD, and to assess the efficacy of therapeutics on ameliorating motor impairment.

Effects of the Novel IDO Inhibitor DWG-1036 on the Behavior of Male and Female 3xTg-AD Mice.

The kynurenine pathway metabolizes tryptophan into nicotinamide adenine dinucleotide, producing a number of intermediary metabolites, including 3-hydroxy kynurenine and quinolinic acid, which are involved in the neurodegenerative mechanisms that underlie Alzheimer's disease (AD). Indolamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of this pathway, is increased in AD, and it has been hypothesized that blocking this enzyme may slow the progression of AD. In this study, we treated male and female 3xTg-AD and wild-type mice with the novel IDO inhibitor DWG-1036 (80 mg/kg) or vehicle (distilled water) from 2 to 6 months of age and then tested them in a battery of behavioral tests that measured spatial learning and memory (Barnes maze), working memory (trace fear conditioning), motor coordination and learning (rotarod), anxiety (elevated plus maze), and depression (tail suspension test). The 3xTg-AD mice treated with DWG-1036 showed better memory in the trace fear conditioning task and significant improvements in learning but poorer spatial memory in the Barnes maze. DWG-1036 treatment also ameliorated the behaviors associated with increased anxiety in the elevated plus maze and depression-like behaviors in the tail suspension test in 3xTg-AD mice. However, the effects of DWG-1036 treatment on the behavioral tasks were variable, and sex differences were apparent. In addition, high doses of DWG-1036 resulted in reduced body weight, particularly in females. Taken together, our results suggest that the kynurenine pathway is a promising target for treating AD, but more work is needed to determine the effective compounds, examine sex differences, and understand the side effects of the compounds.

Impaired Spatial Learning and Memory in Middle-Aged Mice with Kindling-Induced Spontaneous Recurrent Seizures.

Temporal lobe epilepsy is the most common and often drug-resistant type of epilepsy in the adult and aging populations and has great diversity in etiology, electro-clinical manifestations, and comorbidities. Kindling through repeated brief stimulation of limbic structures is a commonly used model of temporal lobe epilepsy. Particularly, extended kindling can induce spontaneous recurrent seizures in several animal species. However, kindling studies in middle-aged, aging, or aged animals remain scarce, and currently, little is known about kindling-induced behavioral changes in middle-aged/aging animals. We therefore attempted to provide more information in this area using a mouse model of extended hippocampal kindling. We conducted experiments in middle-aged mice (C57BL/6, male, 12-14 months of age) to model new-onset epilepsy in adult/aging populations. Mice experienced twice daily hippocampal stimulations or handling manipulations for 60-70 days and then underwent continuous electroencephalogram (EEG)-video monitoring to detect spontaneous recurrent seizures. Extended kindled mice consistently exhibited spontaneous recurrent seizures with mean incidences of 6-7 events per day, and these seizures featured EEG discharges and corresponding convulsions. The handling control mice showed neither seizure nor aberrant EEG activity. The two groups of mice underwent the Morris water maze test of spatial learning and memory 1-2 weeks after termination of the kindling stimulation or handling manipulation. During visible platform trials, the kindled mice took a longer distance and required more time than the control mice to find the platform. During hidden platform trials, the kindled mice showed no improvement over 5-day trials in finding the platform whereas the control mice improved significantly. During probe tests in which the hidden platform was removed, the kindled mice spent less time than the controls searching in the correct platform location. There were no significant differences between the kindled and control mice with respect to swim speed or total locomotor activity in an open-field test. Together, these observations indicate that the extended kindled mice with spontaneous recurrent seizures are impaired in spatial learning and memory as assessed by the Morris water maze test. We postulate that the extended hippocampal kindling in middle-aged mice may help explore epileptogenic mechanisms and comorbidities potentially relevant to new-onset temporal lobe epilepsy in adult and aging patients. Limitations and confounds of our present experiments are discussed to improve future examinations of epileptic comorbidities in extended kindled mice.

Identification of Potent Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors Based on a Phenylimidazole Scaffold.

Inhibition of indoleamine 2,3-dioxygenase (IDO1) is an attractive immunotherapeutic approach for the treatment of a variety of cancers. Dysregulation of this enzyme has also been implicated in other disorders including Alzheimer's disease and arthritis. Herein, we report the structure-based design of two related series of molecules: N1-substituted 5-indoleimidazoles and N1-substituted 5-phenylimidazoles. The latter (and more potent) series was accessed through an unexpected rearrangement of an imine intermediate during a Van Leusen imidazole synthesis reaction. Evidence for the binding modes for both inhibitor series is supported by computational and structure-activity relationship studies.

Susceptibility to hippocampal kindling seizures is increased in aging C57 black mice.

The incidence of seizures increases with old age. Stroke, dementia and brain tumors are recognized risk factors for new-onset seizures in the aging populations and the incidence of these conditions also increased with age. Whether aging is associated with higher seizure susceptibility in the absence of the above pathologies remains unclear. We used classic kindling to explore this issue as the kindling model is highly reproducible and allows close monitoring of electrographic and motor seizure activities in individual animals. We kindled male young and aging mice (C57BL/6 strain, 2-3 and 18-22 months of age) via daily hippocampal CA3 stimulation and monitored seizure activity via video and electroencephalographic recordings. The aging mice needed fewer stimuli to evoke stage-5 motor seizures and exhibited longer hippocampal afterdischarges and more frequent hippocampal spikes relative to the young mice, but afterdischarge thresholds and cumulative afterdischarge durations to stage 5 motor seizures were not different between the two age groups. While hippocampal injury and structural alterations at cellular and micro-circuitry levels remain to be examined in the kindled mice, our present observations suggest that susceptibility to hippocampal CA3 kindling seizures is increased with aging in male C57 black mice.

Adaptive Processes in Thalamus and Cortex Revealed by Silencing of Primary Visual Cortex during Contrast Adaptation.

Visual adaptation illusions indicate that our perception is influenced not only by the current stimulus but also by what we have seen in the recent past. Adaptation to stimulus contrast (the relative luminance created by edges or contours in a scene) induces the perception of the stimulus fading away and increases the contrast detection threshold in psychophysical tests [1, 2]. Neural correlates of contrast adaptation have been described throughout the visual system including the retina [3], dorsal lateral geniculate nucleus (dLGN) [4, 5], primary visual cortex (V1) [6], and parietal cortex [7]. The apparent ubiquity of adaptation at all stages raises the question of how this process cascades across brain regions [8]. Focusing on V1, adaptation could be inherited from pre-cortical stages, arise from synaptic depression at the thalamo-cortical synapse [9], or develop locally, but what is the weighting of these contributions? Because contrast adaptation in mouse V1 is similar to classical animal models [10, 11], we took advantage of the optogenetic tools available in mice to disentangle the processes contributing to adaptation in V1. We disrupted cortical adaptation by optogenetically silencing V1 and found that adaptation measured in V1 now resembled that observed in dLGN. Thus, the majority of adaptation seen in V1 neurons arises through local activity-dependent processes, with smaller contributions from dLGN inheritance and synaptic depression at the thalamo-cortical synapse. Furthermore, modeling indicates that divisive scaling of the weakly adapted dLGN input can predict some of the emerging features of V1 adaptation.

Early detection of cognitive deficits in the 3xTg-AD mouse model of Alzheimer's disease.

Which behavioral test is the most sensitive for detecting cognitive deficits in the 3xTg-AD at 6.5 months of age? The 3xTg-AD mouse model of Alzheimer's disease (AD) has three transgenes (APPswe, PS1M146V, and Tau P301L) which cause the development of amyloid beta plaques, neurofibrillary tangles, and cognitive deficits with age. In order to determine which task is the most sensitive in the early detection of cognitive deficits, we compared male and female 3xTg-AD and B6129SF2 wildtype mice at 6.5 months of age on a test battery including spontaneous alternation in the Y-Maze, novel object recognition, spatial memory in the Barnes maze, and cued and contextual fear conditioning. The 3xTg-AD mice had impaired learning and memory in the Barnes maze but performed better than B6129SF2 wildtype mice in the Y-Maze and in contextual fear conditioning. Neither genotype demonstrated a preference in the novel object recognition task nor was there a genotype difference in cued fear conditioning but females performed better than males. From our results we conclude that the 3xTg-AD mice have mild cognitive deficits in spatial learning and memory and that the Barnes maze was the most sensitive test for detecting these cognitive deficits in 6.5-month-old mice.

Analysis of motor function in 6-month-old male and female 3xTg-AD mice.

The 3xTg-AD mouse has high validity as a model of Alzheimer's disease (AD) because it develops both amyloid beta plaques and neurofibrillary tangles. Human patients with AD typically develop motor deficits, which worsen as the disease progresses, but 3xTg-AD mice have been reported to show enhanced motor abilities. We investigated the motor behaviour phenotype of male and female 3xTg-AD and B6129SF2 wildtype mice on a battery of motor behaviours at 6 months of age. Compared to wildtype mice, the 3xTg-AD mice had enhanced motor performance on the Rotarod, but worse performance on the grid suspension task. In gait analysis 3xTg-AD mice had a longer stride length and made more foot slips on the balance beam than wildtype mice. There was no overall difference in voluntary wheel-running activity between genotypes, but there was a disruption in circadian activity rhythm in 3xTg-AD mice. In some motor tasks, such as the Rotarod and balance beam, females appeared to perform better than males, but this sex differences was accounted for by differences in body weight. Our results indicate that while the 3xTg-AD mice show enhanced performance on the Rotarod, they have poorer performance on other motor behaviour tasks, indicating that their motor behaviour phenotype is more complex than previously reported. The presence of the P301L transgene may explain the enhancement of Rotarod performance but the poorer performance on other motor behaviour tasks may be due to other transgenes.

Spatiotemporal specificity of contrast adaptation in mouse primary visual cortex.

Prolonged viewing of high contrast gratings alters perceived stimulus contrast, and produces characteristic changes in the contrast response functions of neurons in the primary visual cortex (V1). This is referred to as contrast adaptation. Although contrast adaptation has been well-studied, its underlying neural mechanisms are not well-understood. Therefore, we investigated contrast adaptation in mouse V1 with the goal of establishing a quantitative description of this phenomenon in a genetically manipulable animal model. One interesting aspect of contrast adaptation that has been observed both perceptually and in single unit studies is its specificity for the spatial and temporal characteristics of the stimulus. Therefore, in the present work we determined if the magnitude of contrast adaptation in mouse V1 neurons was dependent on the spatial frequency and temporal frequency of the adapting grating. We used protocols that were readily comparable with previous studies in cats and primates, and also a novel contrast ramp stimulus that characterized the spatial and temporal specificity of contrast adaptation simultaneously. Similar to previous work in higher mammals, we found that contrast adaptation was strongest when the spatial frequency and temporal frequency of the adapting grating matched the test stimulus. This suggests similar mechanisms underlying contrast adaptation across animal models and indicates that the rapidly advancing genetic tools available in mice could be used to provide insights into this phenomenon.

Maternal genotype influences behavioral development of 3×Tg-AD mouse pups.

Transgenic mice are a valuable tool in the investigation of neurodegenerative disorders such as Alzheimer's disease. The triple transgenic mouse (3×Tg-AD) is a model of Alzheimer's disease that possesses age-related amyloid beta plaques, neurofibrillary tangles and cell death as well as cognitive decline. Because maternal effects may interact with pup genotype in determining behavior phenotypes, we used a cross-fostering paradigm to investigate the effects of maternal genotype on behavioral development of the 3×Tg-AD mouse model and its wildtype control (B6129S1F2) from 2 to 24 days of age. Developmental patterns of behavior were influenced by both pup and maternal genotype. The 3×Tg-AD mice were delayed in sensory reflexes, showed less activity and poorer habituation to a novel object, but showed advanced development of motor reflexes compared to wildtype pups. While there were no differences in levels of maternal care between transgenic and control mothers, maternal genotype affected the development of several pup reflexes (body weight, hindlimb grasp reflex, loss of crossed extensor reflex, vibrissae response, righting reflex) and the number of horizontal and vertical beam breaks in an open field. This study is the first to examine neurobehavioral development and maternal behavior in a mouse model of Alzheimer's disease, and highlights the importance of investigating the consequences of early environmental experience as well as genetic manipulation on behavioral development.

Age-related changes in visual acuity, learning and memory in the APPswe/PS1dE9 mouse model of Alzheimer's disease.

Mouse models of Alzheimer's disease (AD) are often tested for learning and memory deficits using visuo-spatial tasks such as the Morris water maze. Performance on these tasks is dependent on vision and the APPswe/PS1dE9 mouse model has amyloid beta plaques in their retinas which might influence their performance in these tasks. In a visual learning task, old (20-26 months) transgenic mice and their wildtype littermates of both sexes had poorer visual ability than young (5-8 months) mice and old transgenic mice had poorer visual acuity than old wildtype mice. Old transgenic mice also had deficits in visuo-spatial learning and memory on the Morris water maze. The transgenic mice had no deficits in the conditioned odour preference or conditioned taste aversion memory tests at any age. These results indicate that the old APPswe/PS1dE9 mice and their wildtype littermates both have a deficit in their visual ability and that visually dependent measures alone should not be used to assess learning and memory in this strain.