Mitochondrial dysfunction precedes hippocampal IL-1ß transcription and cognitive impairments after low-dose lipopolysaccharide injection in aged mice.

Acute cognitive impairments termed delirium often occur after inflammatory insults in elderly patients. While previous preclinical studies suggest mitochondria as a target for reducing neuroinflammation and cognitive impairments after LPS injection, fewer studies have evaluated the effects of a low-grade systemic inflammation in the aged brain. Thus, to identify the significance of mitochondrial dysfunction after a clinically relevant systemic inflammatory stimulus, we injected old-aged mice (18-20 months) with low-dose lipopolysaccharide (LPS, 0.04 mg/kg). LPS injection reduced mitochondrial respiration in the hippocampus 24 h after injection (respiratory control ratio [RCR], state3u/state4o; control = 2.82 ± 0.19, LPS = 2.57 ± 0.08). However, gene expression of the pro-inflammatory cytokine IL-1ß was increased (RT-PCR, control = 1.00 ± 0.30; LPS = 2.01 ± 0.67) at a more delayed time point, 48 h after LPS injection. Such changes were associated with cognitive impairments in the Barnes maze and fear chamber tests. Notably, young mice were unaffected by low-dose LPS, suggesting that mitochondrial dysfunction precedes neuroinflammation and cognitive decline in elderly patients following a low-grade systemic insult. Our findings highlight mitochondria as a potential therapeutic target for reducing delirium in elderly patients.

Application of recombinase polymerase amplification with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick assay for Bactrocera correcta.

BACKGROUND: Bactrocera correcta is a quarantine pest that negatively impacts the fruit and vegetable industry. Differentiating B. correcta from similar species, especially in non-adult stages, remains challenging. Rapid molecular identification techniques, such as recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick (MIRA-LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces two methods for the rapid visual identification of B. correcta. RESULTS: Bactrocera correcta specific RPA primers, CRISPR RNA (crRNA), and the LFD probe were designed based on the cox1 genes. The RPA reaction conditions were optimized (at 37 °C for 8 min) for effective template DNA amplification. Two nucleic acid detection methods were established to visualize RPA. In the RPA-CRISPR/Cas12a system, the optimal LbCas12a/crRNA concentration ratio was 200:400 nmol L-1. Successful amplification was determined by the presence or absence of green fluorescence following 15 min incubation at 37 °C. The MIRA-LFD system achieved precise identification of the target species within 4 min at 37 °C. Both methods exhibited high specificity and sensitivity, allowing for detection from 1.0 × 10-1 ng µL-1 of DNA. Combined with rapid DNA extraction, rapid identification of individual B. correcta at different developmental stages was achieved, enhancing the practicality and convenience of the established methods. CONCLUSION: Our research findings demonstrate that both the RPA-CRISPR/Cas12a and MIRA-LFD methods for B. correcta detection was accurate and rapid (within 30 min and 10 min, respectively), at 37 °C. Our methods do not rely on expensive equipment, thus possess high practical value, providing improved identification solutions for port quarantine pests and field applications. © 2024 Society of Chemical Industry.

Repeated ketamine anesthesia during neurodevelopment upregulates hippocampal activity and enhances drug reward in male mice.

Early exposures to anesthetics can cause long-lasting changes in excitatory/inhibitory synaptic transmission (E/I imbalance), an important mechanism for neurodevelopmental disorders. Since E/I imbalance is also involved with addiction, we further investigated possible changes in addiction-related behaviors after multiple ketamine anesthesia in late postnatal mice. Postnatal day (PND) 16 mice received multiple ketamine anesthesia (35 mg kg-1, 5 days), and behavioral changes were evaluated at PND28 and PND56. Although mice exposed to early anesthesia displayed normal behavioral sensitization, we found significant increases in conditioned place preference to both low-dose ketamine (20 mg kg-1) and nicotine (0.5 mg kg-1). By performing transcriptome analysis and whole-cell recordings in the hippocampus, a brain region involved with CPP, we also discovered enhanced neuronal excitability and E/I imbalance in CA1 pyramidal neurons. Interestingly, these changes were not found in female mice. Our results suggest that repeated ketamine anesthesia during neurodevelopment may influence drug reward behavior later in life.

Reactive microglia and mitochondrial unfolded protein response following ventriculomegaly and behavior defects in kaolin-induced hydrocephalus.

Ventriculomegaly induced by the abnormal accumulation of cerebrospinal fluid (CSF) leads to hydrocephalus, which is accompanied by neuroinflammation and mitochondrial oxidative stress. The mitochondrial stress activates mitochondrial unfolded protein response (UPRmt), which is essential for mitochondrial protein homeostasis. However, the association of inflammatory response and UPRmt in the pathogenesis of hydrocephalus is still unclear. To assess their relevance in the pathogenesis of hydrocephalus, we established a kaolin-induced hydrocephalus model in 8-week-old male C57BL/6J mice and evaluated it over time. We found that kaolin-injected mice showed prominent ventricular dilation, motor behavior defects at the 3-day, followed by the activation of microglia and UPRmt in the motor cortex at the 5-day. In addition, PARP-1/NF-κB signaling and apoptotic cell death appeared at the 5-day. Taken together, our findings demonstrate that activation of microglia and UPRmt occurs after hydrocephalic ventricular expansion and behavioral abnormalities which could be lead to apoptotic neuronal cell death, providing a new perspective on the pathogenic mechanism of hydrocephalus. [BMB Reports 2022; 55(4): 181-186].

General Anesthesia During Neurodevelopment Reduces Autistic Behavior in Adult BTBR Mice, a Murine Model of Autism.

Preclinical studies suggest that repeated exposure to anesthetics during a critical period of neurodevelopment induces long-term changes in synaptic transmission, plasticity, and behavior. Such changes are of great concern, as similar changes have also been identified in animal models of neurodevelopmental disorders (NDDs) such as autism. Because of overlapping synaptic changes, it is also possible that anesthetic exposures have a more significant effect in individuals diagnosed with NDDs. Thus, we evaluated the effects of early, multiple anesthetic exposures in BTBR mice, an inbred strain that displays autistic behavior. We discovered that three cycles of sevoflurane anesthesia (2.5%, 1 h) with 2-h intervals between each exposure in late postnatal BTBR mice did not aggravate, but instead improved pathophysiological mechanisms involved with autistic behavior. Sevoflurane exposures restored E/I balance (by increasing inhibitory synaptic transmission), and increased mitochondrial respiration and BDNF signaling in BTBR mice. Most importantly, such changes were associated with reduced autistic behavior in BTBR mice, as sociability was increased in the three-chamber test and repetitive behavior was reduced in the self-grooming test. Our results suggest that anesthetic exposures during neurodevelopment may affect individuals diagnosed with NDDs differently.

Auraptene Enhances Junction Assembly in Cerebrovascular Endothelial Cells by Promoting Resilience to Mitochondrial Stress through Activation of Antioxidant Enzymes and mtUPR.

Junctional proteins in cerebrovascular endothelial cells are essential for maintaining the barrier function of the blood-brain barrier (BBB), thus protecting the brain from the infiltration of pathogens. The present study showed that the potential therapeutic natural compound auraptene (AUR) enhances junction assembly in cerebrovascular endothelial cells by inducing antioxidant enzymes and the mitochondrial unfolded protein response (mtUPR). Treatment of mouse cerebrovascular endothelial cells with AUR enhanced the expression of junctional proteins, such as occludin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin), by increasing the levels of mRNA encoding antioxidant enzymes. AUR treatment also resulted in the depolarization of mitochondrial membrane potential and activation of mtUPR. The ability of AUR to protect against ischemic conditions was further assessed using cells deprived of oxygen and glucose. Pretreatment of these cells with AUR protected against damage to junctional proteins, including occludin, claudin-5, ZO-1 and VE-cadherin, accompanied by a stress resilience response regulated by levels of ATF5, LONP1 and HSP60 mRNAs. Collectively, these results indicate that AUR promotes resilience against oxidative stress and improves junction assembly, suggesting that AUR may help maintain intact barriers in cerebrovascular endothelial cells.

Interval-dependent neurotoxicity after multiple ketamine injections in late postnatal mice.

PURPOSE: Measuring the neurotoxic effects of multiple anesthetic exposures during neurodevelopment is complex due to the numerous factors that can affect the outcome. While we recently discovered that the interval between multiple sevoflurane exposures can affect the level of neurotoxicity, the significance of interval for other anesthetic agents is unknown. Thus, we evaluated the significance of dosing interval in the neurotoxic effects of multiple ketamine injections in postnatal day (PND) 17 mice. METHODS: PND17 mice of both sexes were intraperitoneally injected with ketamine (35 mg/kg) three times at short (2 h) or long (24 h) intervals. Changes in synaptic transmission were measured in hippocampal pyramidal neurons 5 days after the last injection, and behavioral changes were assessed at the age of 8 weeks. Values are presented as mean ± SD. RESULTS: Whereas short-interval ketamine injections enhanced excitatory synaptic transmission, as evidenced by an increased frequency of miniature excitatory postsynaptic currents (mEPSCs; ketamine, 0.09 ± 0.07 Hz; control, 0.06 ± 0.03 Hz), long-interval ketamine injections did not; instead, they decreased the amplitude of miniature inhibitory postsynaptic currents (mIPSCs; ketamine, 47.72 ± 6.90 pA; control, 51.21 ± 7.65 pA,). However, only long-interval ketamine injections induced long-term changes in anxiety behavioral in the open-field test (decrease in center duration; ketamine, 400.1 ± 162.8 s; control, 613.3 ± 312.7 s). CONCLUSIONS: Multiple ketamine injections induce interval-dependent, long-lasting synaptic changes and behavioral impairments. Future studies should carefully consider the dosing interval as a significant factor when studying the neurotoxic effects of multiple anesthetic exposures.

General anesthesia activates the mitochondrial unfolded protein response and induces age-dependent, long-lasting changes in mitochondrial function in the developing brain.

General anesthesia induces changes in dendritic spine number and synaptic transmission in developing mice. These changes are rather disturbing, as similar changes are seen in animal models of neurodevelopmental disorders. We previously suggested that mTor-dependent upregulation of mitochondrial function may be involved in such changes. To further understand the significance of mitochondrial changes after general anesthesia during neurodevelopment, we exposed young mice to 2.5 % sevoflurane for 2 h followed by injection of rotenone, a mitochondrial complex I inhibitor. In postnatal day 17 (PND17) mice, intraperitoneal injection of rotenone not only blocked sevoflurane-induced increases in mitochondrial function, it also prevented sevoflurane-induced changes in excitatory synaptic transmission. Interestingly, similar changes were not observed in younger, neonatal mice (PND7). We next assessed whether the mitochondrial unfolded protein response (UPRmt) acted as a link between anesthetic exposure and mitochondrial function. Expression of UPRmt proteins, which help maintain protein-folding homeostasis and increase mitochondrial function, was increased 6 h after sevoflurane exposure. Our results show that a single, brief sevoflurane exposure induces age-dependent changes in mitochondrial function that constitute an important mechanism for the increase in excitatory synaptic transmission in late postnatal mice, and also suggest mitochondria and UPRmt as potential targets for preventing anesthesia toxicity.

Increasing the interval between repeated anesthetic exposures reduces long-lasting synaptic changes in late post-natal mice.

While recent studies strongly suggest that a single, short anesthetic exposure does not affect neurodevelopment, the effects of multiple exposures remain unclear. Unfortunately, studying "multiple exposures" is challenging as it is an extremely heterogeneous descriptor comprising diverse factors. One potentially important, but unrecognized factor is the interval between anesthetic exposures. In order to evaluate the significance of interval, we exposed post-natal day 16, 17 mice to three sevoflurane exposures (2.5%, 1 hr) with short (2 hr) or long (24 hr) intervals. Changes in synaptic transmission, plasticity, protein expression, and behavior were assessed in male and female mice. We discovered that short-interval exposures induced a female-dependent decrease in miniature inhibitory post-synaptic current (mIPSC) frequency 5 days after the last exposure (control: 18.44 ± 2.86 Hz, sevoflurane:14.65 ± 4.54 Hz). Short-interval sevoflurane exposed mice also displayed long-term behavioral deficits at adult age (hypoactivity, anxiety). These behavioral changes were consistent with the sex-dependent changes in inhibitory transmission, as they were more robust in female mice. Although there was no change in learning and memory, short-interval sevoflurane exposures also impaired LTP in a non-sex-dependent manner (control: 171.10 ± 26.90%, sevoflurane: 149.80 ± 26.48 %). Most importantly, we were unable to find long-lasting consequences in mice that received long-interval sevoflurane exposures. Our study provides novel insights regarding the significance of the interval between multiple exposures, and also suggests that the neurotoxic effects of multiple anesthetic exposures may be reduced by simply increasing the interval between each exposure.

Schisandra Extract and Ascorbic Acid Synergistically Enhance Cognition in Mice Through Modulation of Mitochondrial Respiration.

Cognitive decline is observed in aging and neurodegenerative diseases, including Alzheimer's disease (AD) and dementia. Intracellular energy produced via mitochondrial respiration is used in the regulation of synaptic plasticity and structure, including dendritic spine length and density, as well as for the release of neurotrophic factors involved in learning and memory. To date, a few synthetic agents for improving mitochondrial function have been developed for overcoming cognitive impairment. However, no natural compounds that modulate synaptic plasticity by directly targeting mitochondria have been developed. Here, we demonstrate that a mixture of Schisandra chinensis extract (SCE) and ascorbic acid (AA) improved cognitive function and induced synaptic plasticity-regulating proteins by enhancing mitochondrial respiration. Treatment of embryonic mouse hippocampal mHippoE-14 cells with a 4:1 mixture of SCE and AA increased basal oxygen consumption rate. We found that mice injected with the SCE-AA mixture showed enhanced learning and memory and recognition ability. We further observed that injection of the SCE-AA mixture in mice significantly increased expression of postsynaptic density protein 95 (PSD95), an increase that was correlated with enhanced brain-derived neurotrophic factor (BDNF) expression. These results demonstrate that a mixture of SCE and AA improves mitochondrial function and memory, suggesting that this natural compound mixture could be used to alleviate AD and aging-associated memory decline.

The mTOR Inhibitor Rapamycin Prevents General Anesthesia-Induced Changes in Synaptic Transmission and Mitochondrial Respiration in Late Postnatal Mice.

Preclinical animal studies have continuously reported the possibility of long-lasting neurotoxic effects after general anesthesia in young animals. Such studies also show that the neurological changes induced by anesthesia in young animals differ by their neurodevelopmental stage. Exposure to anesthetic agents increase dendritic spines and induce sex-dependent changes of excitatory/inhibitory synaptic transmission in late postnatal mice, a critical synaptogenic period. However, the mechanisms underlying these changes remain unclear. Abnormal activation of the mammalian target of rapamycin (mTOR) signaling pathway, an important regulator of neurodevelopment, has also been shown to induce similar changes during neurodevelopment. Interestingly, previous studies show that exposure to general anesthetics during neurodevelopment can activate the mTOR signaling pathway. This study, therefore, evaluated the role of mTOR signaling after exposing postnatal day (PND) 16/17 mice to sevoflurane, a widely used inhalation agent in pediatric patients. We first confirmed that a 2-h exposure of 2.5% sevoflurane could induce widespread mTOR phosphorylation in both male and female mice. Pretreatment with the mTOR inhibitor rapamycin not only prevented anesthesia-induced mTOR phosphorylation, but also the increase in mitochondrial respiration and male-dependent enhancement of excitatory synaptic transmission. However, the changes in inhibitory synaptic transmission that appear after anesthesia in female mice were not affected by rapamycin pretreatment. Our results suggest that mTOR inhibitors may act as potential therapeutic agents for anesthesia-induced changes in the developing brain.

Endothelial-specific Crif1 deletion induces BBB maturation and disruption via the alteration of actin dynamics by impaired mitochondrial respiration.

Cerebral endothelial cells (ECs) require junctional proteins to maintain blood-brain barrier (BBB) integrity, restricting toxic substances and controlling peripheral immune cells with a higher concentration of mitochondria than ECs of peripheral capillaries. The mechanism underlying BBB disruption by defective mitochondrial oxidative phosphorylation (OxPhos) is unclear in a mitochondria-related gene-targeted animal model. To assess the role of EC mitochondrial OxPhos function in the maintenance of the BBB, we developed an EC-specific CR6-interactin factor1 (Crif1) deletion mouse. We clearly observed defects in motor behavior, uncompacted myelin and leukocyte infiltration caused by BBB maturation and disruption in this mice. Furthermore, we investigated the alteration in the actin cytoskeleton, which interacts with junctional proteins to support BBB integrity. Loss of Crif1 led to reorganization of the actin cytoskeleton and a decrease in tight junction-associated protein expression through an ATP production defect in vitro and in vivo. Based on these results, we suggest that mitochondrial OxPhos is important for the maturation and maintenance of BBB integrity by supplying ATP to cerebral ECs.

PTEN/AKT signaling mediates chemoresistance in refractory acute myeloid leukemia through enhanced glycolysis.

Primary refractory acute myeloid leukemia (AML) and early recurrence of leukemic cells are among the most difficult hurdles to overcome in the treatment of AML. Moreover, uncertainties surrounding the molecular mechanism underlying refractory AML pose a challenge when it comes to developing novel therapeutic drugs. However, accumulating evidence suggests a contribution of phosphatase and tensin homolog (PTEN)/protein kinase B (AKT) signaling to the development of refractory AML. To assess PTEN/AKT signaling in AML, two types of AML cell lines were evaluated, namely control HL60 cells and KG1α cells, a refractory AML cell line that is resistant to idarubicin and cytarabine (AraC) treatment. Changes in the expression level of glycolysis­ and mitochondrial oxidative phosphorylation­related genes and proteins were evaluated by reverse transcription­quantitative polymerase chain reaction and western blot analyses, respectively. The mitochondrial oxygen consumption and extracellular acidification rates were measured using an XF24 analyzer. CCK8 assay and Annexin V/PI staining were used to analyze cell viability and cellular apoptosis, respectively. The PTEN protein was found to be depleted, whereas AKT phosphorylation levels were elevated in KG1α cells compared with HL60 cells. These changes were associated with increased expression of glucose transporter 1 and hexokinase 2, and increased lactate production. AKT inhibition decreased the proliferation of KG1α cells and decreased extracellular acidification without affecting HL60 cells. Notably, AKT inhibition increased the susceptibility of KG1α cells to chemotherapy with idarubicin and AraC. Taken together, the findings of the present study indicate that activation of AKT by PTEN deficiency sustains the refractory AML status through enhancement of glycolysis and mitochondrial respiration, effects that may be rescued by inhibiting AKT activity.

Chloramphenicol Mitigates Oxidative Stress by Inhibiting Translation of Mitochondrial Complex I in Dopaminergic Neurons of Toxin-Induced Parkinson's Disease Model.

Paraquat (PQ), an herbicide considered an environmental contributor to the development of Parkinson's disease (PD), induces dopaminergic neuronal loss through reactive oxygen species (ROS) production and oxidative stress by mitochondrial complex I. Most patients with PQ-induced PD are affected by chronic exposure and require a preventive strategy for modulation of disease progression. To identify drugs that are effective in preventing PD, we screened more than 1000 drugs that are currently used in clinics and in studies employing PQ-treated cells. Of these, chloramphenicol (CP) showed the most powerful inhibitory effect. Pretreatment with CP increased the viability of PQ-treated SN4741 dopaminergic neuronal cells and rat primary cultured dopaminergic neurons compared with control cells treated with PQ only. CP pretreatment also reduced PQ-induced ROS production, implying that mitochondrial complex I is a target of CP. This effect of CP reflected downregulation of the mitochondrial complex I subunit ND1 and diminished PQ recycling, a major mechanism of ROS production, and resulted in the prevention of cell loss. Notably, these effects of CP were not observed in rotenone-pretreated SN4741 cells and Rho-negative cells, in which mitochondrial function is defective. Consistent with these results, CP pretreatment of MPTP-treated PD model mice also ameliorated dopaminergic neuronal cell loss. Our findings indicate that the inhibition of mitochondrial complex I with CP protects dopaminergic neurons and may provide a strategy for preventing neurotoxin-induced PD.

Auraptene Mitigates Parkinson's Disease-Like Behavior by Protecting Inhibition of Mitochondrial Respiration and Scavenging Reactive Oxygen Species.

Current therapeutics for Parkinson's disease (PD) are only effective in providing relief of symptoms such as rigidity, tremors and bradykinesia, and do not exert disease-modifying effects by directly modulating mitochondrial function. Here, we investigated auraptene (AUR) as a potent therapeutic reagent that specifically protects neurotoxin-induced reduction of mitochondrial respiration and inhibits reactive oxygen species (ROS) generation. Further, we explored the mechanism and potency of AUR in protecting dopaminergic neurons. Treatment with AUR significantly increased the viability of substantia nigra (SN)-derived SN4741 embryonic dopaminergic neuronal cells and reduced rotenone-induced mitochondrial ROS production. By inducing antioxidant enzymes AUR treatment also increased oxygen consumption rate. These results indicate that AUR exerts a protective effect against rotenone-induced mitochondrial oxidative damage. We further assessed AUR effects in vivo, investigating tyrosine hydroxylase (TH) expression in the striatum and substantia nigra of MPTP-induced PD model mice and behavioral changes after injection of AUR. AUR treatment improved movement, consistent with the observed increase in the number of dopaminergic neurons in the substantia nigra. These results demonstrate that AUR targets dual pathogenic mechanisms, enhancing mitochondrial respiration and attenuating ROS production, suggesting that the preventative potential of this natural compound could lead to improvement in PD-related neurobiological changes.