ABSTRACT
BACKGROUND: Chronic Gulf War Illness (GWI) is characterized by cognitive and mood impairments, as well as persistent neuroinflammation and oxidative stress. This study aimed to investigate the efficacy of Epidiolex®, a Food and Drug Administration (FDA)-approved cannabidiol (CBD), in improving brain function in a rat model of chronic GWI. METHODS: Six months after exposure to low doses of GWI-related chemicals [pyridostigmine bromide, N,N-diethyl-meta-toluamide (DEET), and permethrin (PER)] along with moderate stress, rats with chronic GWI were administered either vehicle (VEH) or CBD (20 mg/kg, oral) for 16 weeks. Neurobehavioral tests were conducted on 11 weeks after treatment initiation to evaluate the performance of rats in tasks related to associative recognition memory, object location memory, pattern separation, and sucrose preference. The effect of CBD on hyperalgesia was also examined. The brain tissues were processed for immunohistochemical and molecular studies following behavioral tests. RESULTS: GWI rats treated with VEH exhibited impairments in all cognitive tasks and anhedonia, whereas CBD-treated GWI rats showed improvements in all cognitive tasks and no anhedonia. Additionally, CBD treatment alleviated hyperalgesia in GWI rats. Analysis of hippocampal tissues from VEH-treated rats revealed astrocyte hypertrophy and increased percentages of activated microglia presenting NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) complexes as well as elevated levels of proteins involved in NLRP3 inflammasome activation and Janus kinase/signal transducers and activators of the transcription (JAK/STAT) signaling. Furthermore, there were increased concentrations of proinflammatory and oxidative stress markers along with decreased neurogenesis. In contrast, the hippocampus from CBD-treated GWI rats displayed reduced levels of proteins mediating the activation of NLRP3 inflammasomes and JAK/STAT signaling, normalized concentrations of proinflammatory cytokines and oxidative stress markers, and improved neurogenesis. Notably, CBD treatment did not alter the concentration of endogenous cannabinoid anandamide in the hippocampus. CONCLUSIONS: The use of an FDA-approved CBD (Epidiolex®) has been shown to effectively alleviate cognitive and mood impairments as well as hyperalgesia associated with chronic GWI. Importantly, the improvements observed in rats with chronic GWI in this study were attributed to the ability of CBD to significantly suppress signaling pathways that perpetuate chronic neuroinflammation.
Subject(s)
Cannabidiol , Cognitive Dysfunction , Hyperalgesia , Neurogenesis , Neuroinflammatory Diseases , Persian Gulf Syndrome , Animals , Cannabidiol/pharmacology , Cannabidiol/therapeutic use , Rats , Persian Gulf Syndrome/drug therapy , Persian Gulf Syndrome/complications , Male , Hyperalgesia/drug therapy , Neuroinflammatory Diseases/drug therapy , Cognitive Dysfunction/drug therapy , Cognitive Dysfunction/etiology , Neurogenesis/drug effects , Disease Models, Animal , Rats, Sprague-Dawley , Signal Transduction/drug effects , Mood Disorders/drug therapy , Oxidative Stress/drug effects , Hippocampus/drug effects , Pyridostigmine Bromide/pharmacology , Pyridostigmine Bromide/therapeutic useABSTRACT
Background: One of the hallmarks of Alzheimer's disease (AD) is the buildup of amyloid beta-42 (Aß-42) in the brain, which leads to various adverse effects. Therefore, therapeutic interventions proficient in reducing Aß-42-induced toxicity in AD are of great interest. One promising approach is to use extracellular vesicles from human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC-EVs) because they carry multiple therapeutic miRNAs and proteins capable of protecting neurons against Aß-42-induced pathological changes. Therefore, this in vitro study investigated the proficiency of hiPSC-NSC-EVs to protect human neurons derived from two distinct hiPSC lines from Aß-42o-induced neurodegeneration. Methods: We isolated hiPSC-NSC-EVs using chromatographic methods and characterized their size, ultrastructure, expression of EV-specific markers and proficiency in getting incorporated into mature human neurons. Next, mature human neurons differentiated from two different hiPSC lines were exposed to 1 µM Aß-42 oligomers (Aß-42o) alone or with varying concentrations of hiPSC-NSC-EVs. The protective effects of hiPSC-NSC-EVs against Aß-42o-induced neurodegeneration, increased oxidative stress, mitochondrial dysfunction, impaired autophagy, and tau phosphorylation were ascertained using multiple measures and one-way ANOVA with Newman-Keuls multiple comparisons post hoc tests. Results: Significant neurodegeneration was observed when human neurons were exposed to Aß-42o alone. Notably, neurodegeneration was associated with elevated levels of oxidative stress markers malondialdehyde (MDA) and protein carbonyls (PCs), increased expression of proapoptotic Bax and Bad genes and proteins, reduced expression of the antiapoptotic gene and protein Bcl-2, increased expression of genes encoding mitochondrial complex proteins, decreased expression of autophagy-related proteins Beclin-1 and microtubule-associated protein 1 light chain 3B, and increased phosphorylation of tau. However, the addition of an optimal dose of hiPSC-NSC-EVs (6 x 10 9 EVs) to human neuronal cultures exposed to Aß-42o significantly reduced the extent of neurodegeneration, along with diminished levels of MDA and PCs, normalized expressions of Bax, Bad, and Bcl-2, and genes linked to mitochondrial complex proteins, and reduced tau phosphorylation. Conclusions: The findings demonstrate that an optimal dose of hiPSC-NSC-EVs could significantly decrease the degeneration of human neurons induced by Aß-42o. The results also support further research into the effectiveness of hiPSC-NSC-EVs in AD, particularly their proficiency in preserving neurons and slowing disease progression.
ABSTRACT
Chronic neuroinflammation represents a prominent hallmark of Alzheimer's disease (AD). While moderately activated microglia are pivotal in clearing amyloid beta (Aß), hyperactivated microglia perpetuate neuroinflammation. Prior investigations have indicated that the elimination of â¼80% of microglia through a month-long inhibition of the colony-stimulating factor 1 receptor (CSF1R) during the advanced stage of neuroinflammation in 5xFamilial AD (5xFAD) mice mitigates synapse loss and neurodegeneration without impacting Aß levels. Furthermore, prolonged CSF1R inhibition diminished the development of parenchymal plaques. Nonetheless, the immediate effects of short-term CSF1R inhibition during the early stages of neuroinflammation on residual microglial phenotype or metabolic fitness are unknown. Therefore, we investigated the effects of 10-day CSF1R inhibition in three-month-old female 5xFAD mice, a stage characterized by the onset of neuroinflammation and minimal Aß plaques. We observed â¼65% microglia depletion in the hippocampus and cerebral cortex. The leftover microglia demonstrated a noninflammatory phenotype, with highly branched and ramified processes and reduced NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome complexes. Moreover, plaque-associated microglia were reduced in number with diminished Clec7a (dectin-1) expression. Additionally, both microglia and neurons displayed reduced mechanistic target of rapamycin (mTOR) signaling and autophagy. Biochemical assays validated the inhibition of NLRP3 inflammasome activation, decreased mTOR signaling, and enhanced autophagy. However, short-term CSF1R inhibition did not influence Aß plaques, soluble Aß-42 levels, or hippocampal neurogenesis. Thus, short-term CSF1R inhibition during the early stages of neuroinflammation in 5xFAD mice promotes the retention of homeostatic microglia with diminished inflammasome activation and mTOR signaling, alongside increased autophagy.
ABSTRACT
Antiinflammatory extracellular vesicles (EVs) derived from human induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) hold promise as a disease-modifying biologic for Alzheimer's disease (AD). This study directly addressed this issue by examining the effects of intranasal administrations of hiPSC-NSC-EVs to 3-month-old 5xFAD mice. The EVs were internalized by all microglia, which led to reduced expression of multiple genes associated with disease-associated microglia, inflammasome, and interferon-1 signaling. Furthermore, the effects of hiPSC-NSC-EVs persisted for two months post-treatment in the hippocampus, evident from reduced microglial clusters, inflammasome complexes, and expression of proteins and/or genes linked to the activation of inflammasomes, p38/mitogen-activated protein kinase, and interferon-1 signaling. The amyloid-beta (Aß) plaques, Aß-42, and phosphorylated-tau concentrations were also diminished, leading to better cognitive and mood function in 5xFAD mice. Thus, early intervention with hiPSC-NSC-EVs in AD may help maintain better brain function by restraining the progression of adverse neuroinflammatory signaling cascades.
ABSTRACT
Extracellular vesicles (EVs) released by human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSCs) are enriched with miRNAs and proteins capable of mediating robust antiinflammatory activity. The lack of tumorigenic and immunogenic properties and ability to permeate the entire brain to incorporate into microglia following intranasal (IN) administrations makes them an attractive biologic for curtailing chronic neuroinflammation in neurodegenerative disorders. We tested the hypothesis that IN administrations of hiPSC-NSC-EVs can alleviate chronic neuroinflammation and cognitive impairments induced by the peripheral lipopolysaccharide (LPS) challenge. Adult male, C57BL/6J mice received intraperitoneal injections of LPS (0.75 mg/kg) for seven consecutive days. Then, the mice received either vehicle (VEH) or hiPSC-NSC-EVs (~ 10 × 109 EVs/administration, thrice over 6 days). A month later, mice in all groups were investigated for cognitive function with behavioral tests and euthanized for histological and biochemical studies. Mice receiving VEH after LPS displayed deficits in associative recognition memory, temporal pattern processing, and pattern separation. Such impairments were associated with an increased incidence of activated microglia presenting NOD-, LRR-, and pyrin domain containing 3 (NLRP3) inflammasomes, elevated levels of NLRP3 inflammasome mediators and end products, and decreased neurogenesis in the hippocampus. In contrast, the various cognitive measures in mice receiving hiPSC-NSC-EVs after LPS were closer to naive mice. Significantly, these mice displayed diminished microglial activation, NLRP3 inflammasomes, proinflammatory cytokines, and a level of neurogenesis matching age-matched naïve controls. Thus, IN administrations of hiPSC-NSC-EVs are an efficacious approach to reducing chronic neuroinflammation-induced cognitive impairments.
Subject(s)
Cognitive Dysfunction , Extracellular Vesicles , Induced Pluripotent Stem Cells , Neural Stem Cells , Humans , Mice , Male , Animals , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Induced Pluripotent Stem Cells/metabolism , Neuroinflammatory Diseases , Lipopolysaccharides/pharmacology , Mice, Inbred NOD , Mice, Inbred C57BL , Inflammation/metabolism , Extracellular Vesicles/metabolism , Microglia/metabolism , Cognitive Dysfunction/etiology , Cognitive Dysfunction/prevention & control , Cognitive Dysfunction/metabolism , Neural Stem Cells/metabolism , Hippocampus/metabolism , NeurogenesisABSTRACT
Introduction: Extracellular vesicles (EVs) released by human-induced pluripotent stem cell (hiPSC)-derived neural stem cells (NSCs) have robust antiinflammatory and neurogenic properties due to therapeutic miRNAs and proteins in their cargo. Hence, hiPSC-NSC-EVs are potentially an excellent biologic for treating neurodegenerative disorders, including Alzheimer's disease (AD). Methods: This study investigated whether intranasally (IN) administered hiPSC-NSC-EVs would quickly target various neural cell types in the forebrain, midbrain, and hindbrain regions of 3-month-old 5xFAD mice, a model of ß-amyloidosis and familial AD. We administered a single dose of 25 × 109 hiPSC-NSC-EVs labeled with PKH26, and different cohorts of naïve and 5xFAD mice receiving EVs were euthanized at 45 min or 6 h post-administration. Results: At 45 min post-administration, EVs were found in virtually all subregions of the forebrain, midbrain, and hindbrain of naïve and 5xFAD mice, with predominant targeting and internalization into neurons, interneurons, and microglia, including plaque-associated microglia in 5xFAD mice. EVs also came in contact with the plasma membranes of astrocytic processes and the soma of oligodendrocytes in white matter regions. Evaluation of CD63/CD81 expression with the neuronal marker confirmed that PKH26 + particles found within neurons were IN administered hiPSC-NSC-EVs. At 6 h post-administration, EVs persisted in all cell types in both groups, with the distribution mostly matching what was observed at 45 min post-administration. Area fraction (AF) analysis revealed that, in both naïve and 5xFAD mice, higher fractions of EVs incorporate into forebrain regions at both time points. However, at 45 min post-IN administration, AFs of EVs within cell layers in forebrain regions and within microglia in midbrain and hindbrain regions were lower in 5xFAD mice than naïve mice, implying that amyloidosis reduces EV penetrance. Discussion: Collectively, the results provide novel evidence that IN administration of therapeutic hiPSC-NSC-EVs is an efficient avenue for directing such EVs into neurons and glia in all brain regions in the early stage of amyloidosis. As pathological changes in AD are observed in multiple brain areas, the ability to deliver therapeutic EVs into various neural cells in virtually every brain region in the early stage of amyloidosis is attractive for promoting neuroprotective and antiinflammatory effects.
ABSTRACT
An optimal intranasal (IN) dose of human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs), 90 min post-traumatic brain injury (TBI), has been reported to prevent the evolution of acute neuroinflammation into chronic neuroinflammation resulting in the alleviation of long-term cognitive and mood impairments. Since hippocampal neurogenesis decline and synapse loss contribute to TBI-induced long-term cognitive and mood dysfunction, this study investigated whether hMSC-EV treatment after TBI can prevent hippocampal neurogenesis decline and synapse loss in the chronic phase of TBI. C57BL6 mice undergoing unilateral controlled cortical impact injury (CCI) received a single IN administration of different doses of EVs or the vehicle at 90 min post-TBI. Quantifying neurogenesis in the subgranular zone-granule cell layer (SGZ-GCL) through 5'-bromodeoxyuridine and neuron-specific nuclear antigen double labeling at ~2 months post-TBI revealed decreased neurogenesis in TBI mice receiving vehicle. However, in TBI mice receiving EVs (12.8 and 25.6 × 109 EVs), the extent of neurogenesis was matched to naive control levels. A similar trend of decreased neurogenesis was seen when doublecortin-positive newly generated neurons were quantified in the SGZ-GCL at ~3 months post-TBI. The above doses of EVs treatment after TBI also reduced the loss of pre-and post-synaptic marker proteins in the hippocampus and the somatosensory cortex. Moreover, at 48 h post-treatment, brain-derived neurotrophic factor (BDNF), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), and phosphorylated cyclic AMP response-element binding protein (p-CREB) levels were downregulated in TBI mice receiving the vehicle but were closer to naïve control levels in TBI mice receiving above doses of hMSC-EVs. Notably, improved BDNF concentration observed in TBI mice receiving hMSC-EVs in the acute phase was sustained in the chronic phase of TBI. Thus, a single IN dose of hMSC-EVs at 90 min post-TBI can ease TBI-induced declines in the BDNF-ERK-CREB signaling, hippocampal neurogenesis, and synapses.
ABSTRACT
Traumatic brain injury (TBI) leads to lasting brain dysfunction with chronic neuroinflammation typified by nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3) inflammasome activation in microglia. This study probed whether a single intranasal (IN) administration of human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) naturally enriched with activated microglia-modulating miRNAs can avert chronic adverse outcomes of TBI. Small RNA sequencing confirmed the enrichment of miRNAs capable of modulating activated microglia in hMSC-EV cargo. IN administration of hMSC-EVs into adult mice ninety minutes after the induction of a unilateral controlled cortical impact injury resulted in their incorporation into neurons and microglia in both injured and contralateral hemispheres. A single higher dose hMSC-EV treatment also inhibited NLRP3 inflammasome activation after TBI, evidenced by reduced NLRP3, apoptosis-associated speck-like protein containing a CARD, activated caspase-1, interleukin-1 beta, and IL-18 levels in the injured brain. Such inhibition in the acute phase of TBI endured in the chronic phase, which could also be gleaned from diminished NLRP3 inflammasome activation in microglia of TBI mice receiving hMSC-EVs. Proteomic analysis and validation revealed that higher dose hMSC-EV treatment thwarted the chronic activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway by IL-18, which decreased the release of proinflammatory cytokines. Inhibition of the chronic activation of NLRP3-p38/MAPK signaling after TBI also prevented long-term cognitive and mood impairments. Notably, the animals receiving higher doses of hMSC-EVs after TBI displayed better cognitive and mood function in all behavioral tests than animals receiving the vehicle after TBI. A lower dose of hMSC-EV treatment also partially improved cognitive and mood function. Thus, an optimal IN dose of hMSC-EVs naturally enriched with activated microglia-modulating miRNAs can inhibit the chronic activation of NLRP3-p38/MAPK signaling after TBI and prevent lasting brain dysfunction.
Subject(s)
Brain Injuries, Traumatic , Extracellular Vesicles , MicroRNAs , Mitogen-Activated Protein Kinase 14 , Animals , Humans , Mice , Brain/metabolism , Brain Injuries, Traumatic/metabolism , Extracellular Vesicles/metabolism , Inflammasomes/metabolism , Interleukin-18/metabolism , MicroRNAs/metabolism , Mitogen-Activated Protein Kinase 14/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Proteomics , Signal Transduction , Mesenchymal Stem CellsABSTRACT
Extracellular vesicles (EVs) shed by human-induced pluripotent stem cell (hiPSC)-derived neural stem cells (hNSC-EVs) have shown potent antiinflammatory properties in a mouse macrophage assay and a mouse model of acute neuroinflammation. They can also quickly permeate the entire brain after intranasal administration, making them attractive as an autologous or allogeneic off-the-shelf product for treating neurodegenerative diseases. However, their ability to modulate activated human microglia and specific proteins and miRNAs mediating antiinflammatory effects of hNSC-EVs are unknown. We investigated the proficiency of hNSC-EVs to modulate activated human microglia and probed the role of the protein pentraxin 3 (PTX3) and the miRNA miR-21-5p within hNSC-EVs in mediating the antiinflammatory effects. Mature microglia generated from hiPSCs (iMicroglia) expressed multiple microglia-specific markers. They responded to lipopolysaccharide (LPS) or interferon-gamma challenge by upregulating tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) mRNA expression and protein release. iMicroglia also exhibited proficiency to phagocytose amyloid-beta (Aß). The addition of hNSC-EVs decreased TNF-α and IL-1ß mRNA expression and the release of TNF-α and IL-1ß by LPS-stimulated iMicroglia (proinflammatory human Microglia). However, the antiinflammatory activity of hNSC-EVs on LPS-stimulated microglia was considerably diminished when the PTX3 or miR-21-5p concentration was reduced in EVs. The results demonstrate that hNSC-EVs are proficient for modulating the proinflammatory human microglia into non-inflammatory phenotypes, implying their utility to treat neuroinflammation in neurodegenerative diseases. Furthermore, the role of PTX3 and miR-21-5p in the antiinflammatory activity of hNSC-EVs provides a new avenue for improving the antiinflammatory effects of hNSC-EVs through PTX3 and/or miR-21-5p overexpression.
ABSTRACT
Persistent cognitive impairment is a primary central nervous system-related symptom in veterans afflicted with chronic Gulf War Illness (GWI). Previous studies in a rat model have revealed that cognitive dysfunction in chronic GWI is associated with neuroinflammation, typified by astrocyte hypertrophy, activated microglia, and enhanced proinflammatory cytokine levels. Studies in a mouse model of GWI have also shown upregulation of several phospholipids that serve as reservoirs of arachidonic acid, a precursor of leukotrienes (LTs). However, it is unknown whether altered LT signaling is a component of chronic neuroinflammatory conditions in GWI. Therefore, this study investigated changes in LT signaling in the brain of rats displaying significant cognitive impairments six months after exposure to GWI-related chemicals and moderate stress. The concentration of cysteinyl LTs (CysLTs), LTB4, and 5-Lipoxygenase (5-LOX), the synthesizing enzyme of LTs, were evaluated. CysLT and LTB4 concentrations were elevated in the hippocampus and the cerebral cortex, along with enhanced 5-LOX expression in neurons and microglia. Such changes were also associated with increased proinflammatory cytokine levels in the hippocampus and the cerebral cortex. Enhanced CysLT and LTB4 levels in the brain could also be gleaned from their concentrations in brain-derived extracellular vesicles in the circulating blood. The circulating blood in GWI rats displayed elevated proinflammatory cytokines with no alterations in CysLT and LTB4 concentrations. The results provide new evidence that a brain-specific increase in LT signaling is another adverse alteration that potentially contributes to the maintenance of chronic neuroinflammation in GWI. Therefore, drugs capable of modulating LT signaling may reduce neuroinflammation and improve cognitive function in GWI. Additional findings demonstrate that altered LT levels in the brain could be tracked efficiently by analyzing brain-derived EVs in the circulating blood.
Subject(s)
Cognitive Dysfunction , Persian Gulf Syndrome , Animals , Brain/metabolism , Cognitive Dysfunction/metabolism , Cytokines/metabolism , Leukotriene B4/metabolism , Leukotrienes/metabolism , Mice , Neuroinflammatory Diseases , Persian Gulf Syndrome/metabolism , Persian Gulf Syndrome/psychology , RatsABSTRACT
Strategies proficient for relieving cognitive impairments in aging and Alzheimer's disease (AD) have an enormous impact. Regular physical exercise (PE) can prevent age-related dementia and slow down AD progression. However, such a lifestyle change is likely not achievable for individuals displaying age-related frailty. Hence, drugs or biologics that could simulate the benefits of PE have received much attention. Previous studies suggested that the fibronectin-domain III containing 5 (FNDC5) underlies the PE-mediated improved cognitive function. A recent study reports that PE-related cognitive benefits in aging and AD are mediated by irisin, the cleaved form of FNDC5 released into the blood after PE. Such a conclusion was apparent from the deletion of irisin through a global knockout of FNDC5, leading to the loss of PE-induced cognitive benefits or inducing memory impairments in adult or aged models. Furthermore, in AD models, peripherally administered irisin mimicked the cognitive benefits of PE by modulating neuroinflammation. This short review discusses the promise of irisin to simulate the cognitive benefits of PE in age- and AD-related dementia. In addition, critical issues such as how blood-borne irisin acts on neural cells, the role of the brain-derived neurotrophic factor in irisin-mediated cognitive benefits, and irisin's ability to inhibit neuroinflammatory cascades in aging and AD are discussed.
Subject(s)
Alzheimer Disease , Cognitive Dysfunction , Aged , Aging , Alzheimer Disease/therapy , Cognition , Cognitive Dysfunction/therapy , Fibronectins , Humans , Memory DisordersABSTRACT
Unrelenting cognitive and mood impairments concomitant with incessant oxidative stress and neuroinflammation are among the significant symptoms of chronic Gulf War Illness (GWI). Curcumin (CUR), an antiinflammatory compound, has shown promise to alleviate brain dysfunction in a model of GWI following intraperitoneal administrations at a high dose. However, low bioavailability after oral treatment has hampered its clinical translation. Therefore, this study investigated the efficacy of low-dose, intermittent, oral polymer nanoparticle encapsulated CUR (nCUR) for improving brain function in a rat model of chronic GWI. Intermittent administration of 10 or 20 mg/Kg nCUR for 8 weeks in the early phase of GWI improved brain function and reduced oxidative stress (OS) and neuroinflammation. We next examined the efficacy of 12-weeks of intermittent nCUR at 10 mg/Kg in GWI animals, with treatment commencing 8 months after exposure to GWI-related chemicals and stress, mimicking treatment for the persistent cognitive and mood dysfunction displayed by veterans with GWI. GWI rats receiving nCUR exhibited better cognitive and mood function associated with improved mitochondrial function and diminished neuroinflammation in the hippocampus. Improved mitochondrial function was evident from normalized expression of OS markers, antioxidants, and mitochondrial electron transport genes, and complex proteins. Lessened neuroinflammation was noticeable from reductions in astrocyte hypertrophy, NF-kB, activated microglia with NLRP3 inflammasomes, and multiple proinflammatory cytokines. Moreover, nCUR treated animals displayed enhanced neurogenesis with a normalized expression of synaptophysin puncta, and multiple genes linked to cognitive dysfunction. Thus, low-dose, intermittent, oral nCUR therapy has promise for improving brain function in veterans with GWI.
ABSTRACT
The expanded lifespan of people, while a positive advance, has also amplified the prevalence of age-related disorders, which include mild cognitive impairment, dementia, and Alzheimer's disease. Therefore, competent therapies that could improve the healthspan of people have great significance. Some of the dietary and pharmacological approaches that augment the lifespan could also preserve improved cognitive function in old age. Metformin, a drug widely used for treating diabetes, is one such candidate that could alleviate age-related cognitive dysfunction. However, the possible use of metformin to alleviate age-related cognitive dysfunction has met with conflicting results in human and animal studies. While most clinical studies have suggested the promise of metformin to maintain better cognitive function and reduce the risk for developing dementia and Alzheimer's disease in aged diabetic people, its efficacy in the nondiabetic population is still unclear. Moreover, a previous animal model study implied that metformin could adversely affect cognitive function in the aged. However, a recent animal study using multiple behavioral tests has reported that metformin treatment in late middle age improved cognitive function in old age. The study also revealed that cognition-enhancing effects of metformin in aged animals were associated with the activation of the energy regulator adenosine monophosphate-activated protein kinase, diminished neuroinflammation, inhibition of the mammalian target of rapamycin signaling, and augmented autophagy in the hippocampus. The proficiency of metformin to facilitate these favorable modifications in the aged hippocampus likely underlies its positive effect on cognitive function. Nonetheless, additional studies probing the outcomes of different doses and durations of metformin treatment at specific windows in the middle and old age across sex in nondiabetic and non-obese prototypes are required to substantiate the promise of metformin to maintain better cognitive function in old age.
ABSTRACT
Persistent cognitive and mood dysfunction is the primary CNS symptom in veterans afflicted with Gulf War Illness (GWI). This study investigated the efficacy of melatonin (MEL) for improving cognitive and mood function with antioxidant, antiinflammatory, and pro-cognitive effects in a rat model of chronic GWI. Six months after exposure to GWI-related chemicals and stress, rats were treated with vehicle or MEL (5, 10, 20, 40, and 80 mg/kg) for eight weeks. Behavioral tests revealed cognitive and mood dysfunction in GWI rats receiving vehicle, which were associated with elevated oxidative stress, reduced NRF2, catalase and mitochondrial complex proteins, astrocyte hypertrophy, activated microglia with NLRP3 inflammasomes, elevated proinflammatory cytokines, waned neurogenesis, and synapse loss in the hippocampus. MEL at 10 mg/kg alleviated simple and associative recognition memory dysfunction and anhedonia, along with reduced oxidative stress, enhanced glutathione and complex III, and reduced NLRP3 inflammasomes, IL-18, TNF-α, and IFN-γ. MEL at 20 mg/kg also normalized NRF2 and catalase and increased microglial ramification. MEL at 40 mg/kg, in addition, reduced astrocyte hypertrophy, activated microglia, NF-kB-NLRP3-caspase-1 signaling, IL-1ß, MCP-1, and MIP-1α. Moreover, MEL at 80 mg/kg activated the BDNF-ERK-CREB signaling pathway, enhanced neurogenesis and diminished synapse loss in the hippocampus, and improved a more complex hippocampus-dependent cognitive function. Thus, MEL therapy is efficacious for improving cognitive and mood function in a rat model of chronic GWI, and MEL's effect was dose-dependent. The study provides the first evidence of MEL's promise for alleviating neuroinflammation and cognitive and mood impairments in veterans with chronic GWI.
Subject(s)
Melatonin , Persian Gulf Syndrome , Animals , Brain-Derived Neurotrophic Factor , Hippocampus , Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Oxidative Stress , RatsABSTRACT
Metformin, a drug widely used for treating diabetes, can prolong the lifespan in several species. Metformin also has the promise to slow down age-related cognitive impairment. However, metformin's therapeutic use as an anti-aging drug is yet to be accepted because of conflicting animal and human studies results. We examined the effects of metformin treatment in late middle age on cognitive function in old age. Eighteen-month-old male C57BL6/J mice received metformin or no treatment for 10 weeks. A series of behavioral tests revealed improved cognitive function in animals that received metformin. Such findings were evident from a better ability for pattern separation, object location, and recognition memory function. Quantification of microglia revealed that metformin treatment reduced the incidence of pathological microglial clusters with alternative activation of microglia into an M2 phenotype, displaying highly ramified processes in the hippocampus. Metformin treatment also seemed to reduce astrocyte hypertrophy. Additional analysis demonstrated that metformin treatment in late middle age increased adenosine monophosphate-activated protein kinase activation, reduced proinflammatory cytokine levels, and the mammalian target of rapamycin signaling, and enhanced autophagy in the hippocampus. However, metformin treatment did not alter neurogenesis or synapses in the hippocampus, implying that improved cognitive function with metformin did not involve enhanced neurogenesis or neosynaptogenesis. The results provide new evidence that metformin treatment commencing in late middle age has promise for improving cognitive function in old age. Modulation of microglia, proinflammatory cytokines, and autophagy appear to be the mechanisms by which metformin facilitated functional benefits in the aged brain.
Subject(s)
Aging/drug effects , Autophagy/drug effects , Cognition/drug effects , Hippocampus/drug effects , Metformin/pharmacology , Microglia/drug effects , Animals , Hippocampus/metabolism , Male , Mice , Mice, Inbred C57BL , Microglia/metabolismABSTRACT
Gulf War Illness (GWI), a chronic multisymptom health problem, afflicts ~30% of veterans served in the first GW. Impaired brain function is among the most significant symptoms of GWI, which is typified by persistent cognitive and mood impairments, concentration problems, headaches, chronic fatigue, and musculoskeletal pain. This review aims to discuss findings from animal prototypes and veterans with GWI on mechanisms underlying its pathophysiology and emerging therapeutic strategies for alleviating brain dysfunction in GWI. Animal model studies have linked brain impairments to incessantly elevated oxidative stress, chronic inflammation, inhibitory interneuron loss, altered lipid metabolism and peroxisomes, mitochondrial dysfunction, modified expression of genes relevant to cognitive function, and waned hippocampal neurogenesis. Furthermore, the involvement of systemic alterations such as the increased intensity of reactive oxygen species and proinflammatory cytokines in the blood, transformed gut microbiome, and activation of the adaptive immune response have received consideration. Investigations in veterans have suggested that brain dysfunction in GWI is linked to chronic activation of the executive control network, impaired functional connectivity, altered blood flow, persistent inflammation, and changes in miRNA levels. Lack of protective alleles from Class II HLA genes, the altered concentration of phospholipid species and proinflammatory factors in the circulating blood have also been suggested as other aiding factors. While some drugs or combination therapies have shown promise for alleviating symptoms in clinical trials, larger double-blind, placebo-controlled trials are needed to validate such findings. Based on improvements seen in animal models of GWI, several antioxidants and anti-inflammatory compounds are currently being tested in clinical trials. However, reliable blood biomarkers that facilitate an appropriate screening of veterans for brain pathology need to be discovered. A liquid biopsy approach involving analysis of brain-derived extracellular vesicles in the blood appears efficient for discerning the extent of neuropathology both before and during clinical trials.
Subject(s)
Brain Diseases , Persian Gulf Syndrome , Animals , Brain Diseases/drug therapy , Brain Diseases/physiopathology , Gulf War , Humans , Inflammation , Neurogenesis , Persian Gulf Syndrome/drug therapy , Randomized Controlled Trials as TopicABSTRACT
Grafting of neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise for brain repair after injury or disease, but safety issues have hindered their clinical application. Employing nano-sized extracellular vesicles (EVs) derived from hiPSC-NSCs appears to be a safer alternative because they likely have similar neuroreparative properties as NSCs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. However, reliable methods for isolation, characterization and testing the biological properties of EVs are critically needed for translation. We investigated signatures of miRNAs and proteins and the biological activity of EVs, isolated from hiPSC-NSCs through a combination of anion-exchange chromatography (AEC) and size-exclusion chromatography (SEC). AEC and SEC facilitated the isolation of EVs with intact ultrastructure and expressing CD9, CD63, CD81, ALIX and TSG 101. Small RNA sequencing, proteomic analysis, pathway analysis and validation of select miRNAs and proteins revealed that EVs were enriched with miRNAs and proteins involved in neuroprotective, anti-apoptotic, antioxidant, anti-inflammatory, blood-brain barrier repairing, neurogenic and Aß reducing activities. Besides, EVs comprised miRNAs and/or proteins capable of promoting synaptogenesis, synaptic plasticity and better cognitive function. Investigations using an in vitro macrophage assay and a mouse model of status epilepticus confirmed the anti-inflammatory activity of EVs. Furthermore, the intranasal administration of EVs resulted in the incorporation of EVs by neurons, microglia and astrocytes in virtually all adult rat and mouse brain regions, and enhancement of hippocampal neurogenesis. Thus, biologically active EVs containing miRNAs and proteins relevant to brain repair could be isolated from hiPSC-NSC cultures, making them a suitable biologic for treating neurodegenerative disorders.
ABSTRACT
Enduring brain dysfunction is amid the highly manifested symptoms in veterans with Gulf War Illness (GWI). Animal studies have established that lasting brain dysfunction in GWI is concomitant with augmented oxidative stress, inflammation, and declined neurogenesis in the brain, and systemic inflammation. We hypothesize that drugs capable of restoring redox homeostasis in GWI will improve cognitive and mood function with modulation of neuroinflammation and neurogenesis. We examined the efficacy of monosodium luminol-GVT (MSL), a drug that promotes redox homeostasis, for improving cognitive and mood function in GWI rats. Young rats were exposed to GWI-related chemicals and moderate restraint stress for four weeks. Four months later, GWI rats received different doses of MSL or vehicle for eight weeks. Behavioral analyses in the last three weeks of treatment revealed that GWI rats receiving higher doses of MSL displayed better cognitive and mood function associated with reinstatement of redox homeostasis. Such restoration was evident from the normalized expression of multiple genes encoding proteins involved in combating oxidative stress in the brain and the return of several oxidative stress markers to control levels in the brain and the circulating blood. Sustained redox homeostasis by MSL also resulted in antiinflammatory and pro-neurogenic effects, which were apparent from reduced densities of hypertrophied astrocytes and activated microglia, and increased neurogenesis with augmented neural stem cell proliferation. Moreover, MSL treatment normalized the concentration of multiple proinflammatory markers in the circulating blood. Thus, MSL treatment reinstated redox homeostasis in an animal model of GWI, which resulted in alleviation of both brain and systemic inflammation, improved neurogenesis, and better cognitive and mood function.
Subject(s)
Neurogenesis/drug effects , Persian Gulf Syndrome/drug therapy , Persian Gulf Syndrome/psychology , Pyridazines/administration & dosage , Affect/drug effects , Animals , Cell Proliferation/drug effects , Cognition/drug effects , Disease Models, Animal , Gulf War , Homeostasis/drug effects , Male , Oxidative Stress , Persian Gulf Syndrome/metabolism , Pyridazines/pharmacology , RatsABSTRACT
An initial precipitating injury in the brain, such as after status epilepticus (SE), evolves into chronic temporal lobe epilepsy (TLE). We investigated changes in the miRNA composition of extracellular vesicles (EVs) in the forebrain after the establishment of SE-induced chronic TLE. We induced SE in young Fischer 344 rats through graded intraperitoneal injections of kainic acid, which resulted in consistent spontaneous recurrent seizures at ~ 3 months post-SE. We isolated EVs from the entire forebrain of chronically epileptic rats and age-matched naïve control animals through an ultracentrifugation method and performed miRNA-sequencing studies to discern changes in the miRNA composition of forebrain-derived EVs in chronic epilepsy. EVs from both naïve and epileptic forebrains displayed spherical or cup-shaped morphology, a comparable size range, and CD63 expression but lacked the expression of a deep cellular marker GM130. However, miRNA-sequencing studies suggested downregulation of 3 miRNAs (miR-187-5p, miR-346, and miR-331-3p) and upregulation of 4 miRNAs (miR-490-5p, miR-376b-3p, miR-493-5p, and miR-124-5p) in EVs from epileptic forebrains with fold changes ranging from 1.5 to 2.4 (p < 0.0006; FDR < 0.05). By using geNorm and Normfinder software, we identified miR-487 and miR-221 as the best combination of reference genes for measurement of altered miRNAs found in the epileptic forebrain through qRT-PCR studies. The validation revealed that only miR-346 and miR-331-3p were significantly downregulated in EVs from the epileptic forebrain. The enrichment pathway analysis of these miRNAs showed an overrepresentation of signaling pathways that are linked to molecular mechanisms underlying chronic epilepsy, including GABA-ergic (miR-346 targets) and mTOR (miR-331-3p targets) systems. Thus, the packaging of two miRNAs into EVs in neural cells is considerably altered in chronic epilepsy. Functional studies on these two miRNAs may uncover their role in the pathophysiology and treatment of TLE.
Subject(s)
Epilepsy, Temporal Lobe/genetics , Extracellular Vesicles/metabolism , MicroRNAs/genetics , Status Epilepticus/genetics , Animals , Disease Models, Animal , Epilepsy, Temporal Lobe/metabolism , Extracellular Vesicles/genetics , Male , Neurons/metabolism , Rats, Inbred F344 , Status Epilepticus/metabolismABSTRACT
Cognitive dysfunction and neuroinflammation are conspicuously observed in Gulf War Illness (GWI). We investigated whether brain inflammation in GWI is associated with activation of high mobility group box-1 (HMGB1) and complement-related proteins in neurons and astrocytes, and brain inflammation can be tracked through neuron-derived extracellular vesicles (NDEVs) and astrocyte-derived EVs (ADEVs) found in the circulating blood. We exposed animals to GWI-related chemicals pyridostigmine bromide, DEET and permethrin, and moderate stress for 28â¯days. We performed behavioral tests 10â¯months post-exposure and quantified activated microglia and reactive astrocytes in the cerebral cortex. Then, we measured the concentration of HMGB1, proinflammatory cytokines, and complement activation-related proteins in the cerebral cortex, and NDEVs and ADEVs in the circulating blood. Cognitive impairments persisted in GWI rats at 10â¯months post-exposure, which were associated with increased density of activated microglia and reactive astrocytes in the cerebral cortex. Moreover, the level of HMGB1 was elevated in the cerebral cortex with altered expression in the cytoplasm of neuronal soma and dendrites as well as the extracellular space. Also, higher levels of proinflammatory cytokines (TNFa, IL-1b, and IL-6), and complement activation-related proteins (C3 and TccC5b-9) were seen in the cerebral cortex. Remarkably, increased levels of HMGB1 and proinflammatory cytokines observed in the cerebral cortex of GWI rats could also be found in NDEVs isolated from the blood. Similarly, elevated levels of complement proteins seen in the cerebral cortex could be found in ADEVs. The results provide new evidence that persistent cognitive dysfunction and chronic neuroinflammation in a model of GWI are linked with elevated HMGB1 concentration and complement activation. Furthermore, the results demonstrated that multiple biomarkers of neuroinflammation could be tracked reliably via analyses of NDEVs and ADEVs in the circulating blood. Execution of such a liquid biopsy approach is especially useful in clinical trials for monitoring the remission, persistence or progression of brain inflammation in GWI patients with drug treatment.