Differential transcriptomic alterations in nasal versus lung tissue of acrolein-exposed rats.

Introduction: Acrolein is a significant component of anthropogenic and wildfire emissions, as well as cigarette smoke. Although acrolein primarily deposits in the upper respiratory tract upon inhalation, patterns of site-specific injury in nasal versus pulmonary tissues are not well characterized. This assessment is critical in the design of in vitro and in vivo studies performed for assessing health risk of irritant air pollutants. Methods: In this study, male and female Wistar-Kyoto rats were exposed nose-only to air or acrolein. Rats in the acrolein exposure group were exposed to incremental concentrations of acrolein (0, 0.1, 0.316, 1 ppm) for the first 30 min, followed by a 3.5 h exposure at 3.16 ppm. In the first cohort of male and female rats, nasal and bronchoalveolar lavage fluids were analyzed for markers of inflammation, and in a second cohort of males, nasal airway and left lung tissues were used for mRNA sequencing. Results: Protein leakage in nasal airways of acrolein-exposed rats was similar in both sexes; however, inflammatory cells and cytokine increases were more pronounced in males when compared to females. No consistent changes were noted in bronchoalveolar lavage fluid of males or females except for increases in total cells and IL-6. Acrolein-exposed male rats had 452 differentially expressed genes (DEGs) in nasal tissue versus only 95 in the lung. Pathway analysis of DEGs in the nose indicated acute phase response signaling, Nrf2-mediated oxidative stress, unfolded protein response, and other inflammatory pathways, whereas in the lung, xenobiotic metabolism pathways were changed. Genes associated with glucocorticoid and GPCR signaling were also changed in the nose but not in the lung. Discussion: These data provide insights into inhaled acrolein-mediated sex-specific injury/inflammation in the nasal and pulmonary airways. The transcriptional response in the nose reflects acrolein-induced acute oxidative and cytokine signaling changes, which might have implications for upper airway inflammatory disease susceptibility.

Serum metabolome and liver transcriptome reveal acrolein inhalation-induced sex-specific homeostatic dysfunction.

Acrolein, a respiratory irritant, induces systemic neuroendocrine stress. However, peripheral metabolic effects have not been examined. Male and female WKY rats were exposed to air (0 ppm) or acrolein (3.16 ppm) for 4 h, followed by immediate serum and liver tissue collection. Serum metabolomics in both sexes and liver transcriptomics in males were evaluated to characterize the systemic metabolic response. Of 887 identified metabolites, > 400 differed between sexes at baseline. An acrolein biomarker, 3-hydroxypropyl mercapturic acid, increased 18-fold in males and 33-fold in females, indicating greater metabolic detoxification in females than males. Acrolein exposure changed 174 metabolites in males but only 50 in females. Metabolic process assessment identified higher circulating free-fatty acids, glycerols, and other lipids in male but not female rats exposed to acrolein. In males, acrolein also increased branched-chain amino acids, which was linked with metabolites of nitrogen imbalance within the gut microbiome. The contribution of neuroendocrine stress was evident by increased corticosterone in males but not females. Male liver transcriptomics revealed acrolein-induced over-representation of lipid and protein metabolic processes, and pathway alterations including Sirtuin, insulin-receptor, acute-phase, and glucocorticoid signaling. In sum, acute acrolein inhalation resulted in sex-specific serum metabolomic and liver transcriptomic derangement, which may have connections to chronic metabolic-related diseases.

Reducing brain kynurenic acid synthesis precludes kynurenine-induced sleep disturbances.

Patients with neurocognitive disorders often battle sleep disturbances. Kynurenic acid is a tryptophan metabolite of the kynurenine pathway implicated in the pathology of these illnesses. Modest increases in kynurenic acid, an antagonist at glutamatergic and cholinergic receptors, result in cognitive impairments and sleep dysfunction. We explored the hypothesis that inhibition of the kynurenic acid synthesising enzyme, kynurenine aminotransferase II, may alleviate sleep disturbances. At the start of the light phase, adult male and female Wistar rats received systemic injections of either: (i) vehicle; (ii) kynurenine (100 mg kg-1 ; i.p.); (iii) the kynurenine aminotransferase II inhibitor, PF-04859989 (30 mg kg-1 ; s.c.); or (iv) PF-04859989 and kynurenine in combination. Kynurenine and kynurenic acid levels were evaluated in the plasma and brain. Separate animals were implanted with electroencephalogram and electromyogram telemetry devices to record polysomnography, and evaluate the vigilance states wake, rapid eye movement sleep and non-rapid eye movement sleep following each treatment. Kynurenine challenge increased brain kynurenic acid and resulted in reduced rapid eye movement sleep duration, non-rapid eye movement sleep delta power and sleep spindles. PF-04859989 reduced brain kynurenic acid formation when given prior to kynurenine, prevented disturbances in rapid eye movement sleep and sleep spindles, and enhanced non-rapid eye movement sleep. Our findings suggest that reducing kynurenic acid in conditions where the kynurenine pathway is activated may serve as a potential strategy for improving sleep dynamics.

Time of Day-Dependent Alterations in Hippocampal Kynurenic Acid, Glutamate, and GABA in Adult Rats Exposed to Elevated Kynurenic Acid During Neurodevelopment.

Hypofunction of glutamatergic signaling is causally linked to neurodevelopmental disorders, including psychotic disorders like schizophrenia and bipolar disorder. Kynurenic acid (KYNA) has been found to be elevated in postmortem brain tissue and cerebrospinal fluid of patients with psychotic illnesses and may be involved in the hypoglutamatergia and cognitive dysfunction experienced by these patients. As insults during the prenatal period are hypothesized to be linked to the pathophysiology of psychotic disorders, we presently utilized the embryonic kynurenine (EKyn) paradigm to induce a prenatal hit. Pregnant Wistar dams were fed chow laced with kynurenine to stimulate fetal brain KYNA elevation from embryonic day 15 to embryonic day 22. Control dams (ECon) were fed unlaced chow. Plasma and hippocampal tissue from young adult (postnatal day 56) ECon and EKyn male and female offspring were collected at the beginning of the light (Zeitgeber time, ZT 0) and dark (ZT 12) phases to assess kynurenine pathway metabolites. Hippocampal tissue was also collected at ZT 6 and ZT 18. In separate animals, in vivo microdialysis was conducted in the dorsal hippocampus to assess extracellular KYNA, glutamate, and γ-aminobutyric acid (GABA). Biochemical analyses revealed no changes in peripheral metabolites, yet hippocampal tissue KYNA levels were significantly impacted by EKyn treatment, and increased in male EKyn offspring at ZT 6. Interestingly, extracellular hippocampal KYNA levels were only elevated in male EKyn offspring during the light phase. Decreases in extracellular glutamate levels were found in the dorsal hippocampus of EKyn male and female offspring, while decreased GABA levels were present only in males during the dark phase. The current findings suggest that the EKyn paradigm may be a useful tool for investigation of sex- and time-dependent changes in hippocampal neuromodulation elicited by prenatal KYNA elevation, which may influence behavioral phenotypes and have translational relevance to psychotic disorders.

Prenatal Kynurenine Elevation Elicits Sex-Dependent Changes in Sleep and Arousal During Adulthood: Implications for Psychotic Disorders.

Dysregulation of the kynurenine pathway (KP) of tryptophan catabolism has been implicated in psychotic disorders, including schizophrenia and bipolar disorder. Kynurenic acid (KYNA) is a KP metabolite synthesized by kynurenine aminotransferases (KATs) from its biological precursor kynurenine and acts as an endogenous antagonist of N-methyl-D-aspartate and α7-nicotinic acetylcholine receptors. Elevated KYNA levels found in postmortem brain tissue and cerebrospinal fluid of patients are hypothesized to play a key role in the etiology of cognitive symptoms observed in psychotic disorders. Sleep plays an important role in memory consolidation, and sleep disturbances are common among patients. Yet, little is known about the effect of altered KP metabolism on sleep-wake behavior. We presently utilized a well-established experimental paradigm of embryonic kynurenine (EKyn) exposure wherein pregnant dams are fed a diet laced with kynurenine the last week of gestation and hypothesized disrupted sleep-wake behavior in adult offspring. We examined sleep behavior in adult male and female offspring using electroencephalogram and electromyogram telemetry and determined sex differences in sleep and arousal in EKyn offspring. EKyn males displayed reduced rapid eye movement sleep, while female EKyn offspring were hyperaroused compared to controls. We determined that EKyn males maintain elevated brain KYNA levels, while KYNA levels were unchanged in EKyn females, yet the activity levels of KAT I and KAT II were reduced. Our findings indicate that elevated prenatal kynurenine exposure elicits sex-specific changes in sleep-wake behavior, arousal, and KP metabolism.

Application of Machine Learning to Sleep Stage Classification.

Sleep studies are imperative to recapitulate phenotypes associated with sleep loss and uncover mechanisms contributing to psychopathology. Most often, investigators manually classify the polysomnography into vigilance states, which is time-consuming, requires extensive training, and is prone to inter-scorer variability. While many works have successfully developed automated vigilance state classifiers based on multiple EEG channels, we aim to produce an automated and openaccess classifier that can reliably predict vigilance state based on a single cortical electroencephalogram (EEG) from rodents to minimize the disadvantages that accompany tethering small animals via wires to computer programs. Approximately 427 hours of continuously monitored EEG, electromyogram (EMG), and activity were labeled by a domain expert out of 571 hours of total data. Here we evaluate the performance of various machine learning techniques on classifying 10-second epochs into one of three discrete classes: paradoxical, slow-wave, or wake. Our investigations include Decision Trees, Random Forests, Naive Bayes Classifiers, Logistic Regression Classifiers, and Artificial Neural Networks. These methodologies have achieved accuracies ranging from approximately 74% to approximately 96%. Most notably, the Random Forest and the ANN achieved remarkable accuracies of 95.78% and 93.31%, respectively. Here we have shown the potential of various machine learning classifiers to automatically, accurately, and reliably classify vigilance states based on a single EEG reading and a single EMG reading.

The Role of Lysosomes in a Broad Disease-Modifying Approach Evaluated across Transgenic Mouse Models of Alzheimer's Disease and Parkinson's Disease and Models of Mild Cognitive Impairment.

Many neurodegenerative disorders have lysosomal impediments, and the list of proposed treatments targeting lysosomes is growing. We investigated the role of lysosomes in Alzheimer's disease (AD) and other age-related disorders, as well as in a strategy to compensate for lysosomal disturbances. Comprehensive immunostaining was used to analyze brains from wild-type mice vs. amyloid precursor protein/presenilin-1 (APP/PS1) mice that express mutant proteins linked to familial AD. Also, lysosomal modulation was evaluated for inducing synaptic and behavioral improvements in transgenic models of AD and Parkinson's disease, and in models of mild cognitive impairment (MCI). Amyloid plaques were surrounded by swollen organelles positive for the lysosome-associated membrane protein 1 (LAMP1) in the APP/PS1 cortex and hippocampus, regions with robust synaptic deterioration. Within neurons, lysosomes contain the amyloid ß 42 (Aß42) degradation product Aß38, and this indicator of Aß42 detoxification was augmented by Z-Phe-Ala-diazomethylketone (PADK; also known as ZFAD) as it enhanced the lysosomal hydrolase cathepsin B (CatB). PADK promoted Aß42 colocalization with CatB in lysosomes that formed clusters in neurons, while reducing Aß deposits as well. PADK also reduced amyloidogenic peptides and α-synuclein in correspondence with restored synaptic markers, and both synaptic and cognitive measures were improved in the APP/PS1 and MCI models. These findings indicate that lysosomal perturbation contributes to synaptic and cognitive decay, whereas safely enhancing protein clearance through modulated CatB ameliorates the compromised synapses and cognition, thus supporting early CatB upregulation as a disease-modifying therapy that may also slow the MCI to dementia continuum.

Potential Alzheimer's Disease Therapeutics Among Weak Cysteine Protease Inhibitors Exhibit Mechanistic Differences Regarding Extent of Cathepsin B Up-Regulation and Ability to Block Calpain.

Cysteine protease inhibitors have long been part of drug discovery programs for Alzheimer's disease (AD), traumatic brain injury (TBI), and other disorders. Select inhibitors reduce accumulating proteins and AD pathology in mouse models. One such compound, Z-Phe-Aladiazomethylketone (PADK), exhibits a very weak IC50 (9-11 µM) towards cathepsin B (CatB), but curiously PADK causes marked up-regulation of the Aß-degrading CatB and improves spatial memory. Potential therapeutic and weak inhibitor E64d (14 µM IC50) also up-regulates CatB. PADK and E64d were compared regarding the blockage of calcium-induced cytoskeletal deterioration in brain samples, monitoring the 150-kDa spectrin breakdown product (SBDP) known to be produced by calpain. PADK had little to no effect on SBDP production at 10-100 µM. In contrast, E64d caused a dose-dependent decline in SBDP levels with an IC50 of 3-6 µM, closely matching its reported potency for inhibiting µ-calpain. Calpain also cleaves the cytoskeletal organizing protein gephyrin, producing 49-kDa (GnBDP49) and 18-kDa (GnBDP18) breakdown products. PADK had no apparent effect on calcium-induced gephyrin fragments whereas E64d blocked their production. E64d also protected the parent gephyrin in correspondence with reduced BDP levels. The findings of this study indicate that PADK's positive and selective effects on CatB are consistent with human studies showing exercise elevates CatB and such elevation correlates with improved memory. On the other hand, E64d exhibits both marginal CatB enhancement and potent calpain inhibition. This dual effect may be beneficial for treating AD. Alternatively, the potent action on calpain-related pathology may explain E64d's protection in AD and TBI models.