Extracellular vesicles from hiPSC-NSCs can prevent peripheral inflammation-induced cognitive dysfunction with inflammasome inhibition and improved neurogenesis in the hippocampus.

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.

Intranasally administered human MSC-derived extracellular vesicles inhibit NLRP3-p38/MAPK signaling after TBI and prevent chronic brain dysfunction.

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.

Human induced pluripotent stem cell-derived MGE cell grafting after status epilepticus attenuates chronic epilepsy and comorbidities via synaptic integration.

Medial ganglionic eminence (MGE)-like interneuron precursors derived from human induced pluripotent stem cells (hiPSCs) are ideal for developing patient-specific cell therapy in temporal lobe epilepsy (TLE). However, their efficacy for alleviating spontaneous recurrent seizures (SRS) or cognitive, memory, and mood impairments has never been tested in models of TLE. Through comprehensive video- electroencephalographic recordings and a battery of behavioral tests in a rat model, we demonstrate that grafting of hiPSC-derived MGE-like interneuron precursors into the hippocampus after status epilepticus (SE) greatly restrained SRS and alleviated cognitive, memory, and mood dysfunction in the chronic phase of TLE. Graft-derived cells survived well, extensively migrated into different subfields of the hippocampus, and differentiated into distinct subclasses of inhibitory interneurons expressing various calcium-binding proteins and neuropeptides. Moreover, grafting of hiPSC-MGE cells after SE mediated several neuroprotective and antiepileptogenic effects in the host hippocampus, as evidenced by reductions in host interneuron loss, abnormal neurogenesis, and aberrant mossy fiber sprouting in the dentate gyrus (DG). Furthermore, axons from graft-derived interneurons made synapses on the dendrites of host excitatory neurons in the DG and the CA1 subfield of the hippocampus, implying an excellent graft-host synaptic integration. Remarkably, seizure-suppressing effects of grafts were significantly reduced when the activity of graft-derived interneurons was silenced by a designer drug while using donor hiPSC-MGE cells expressing designer receptors exclusively activated by designer drugs (DREADDs). These results implied the direct involvement of graft-derived interneurons in seizure control likely through enhanced inhibitory synaptic transmission. Collectively, the results support a patient-specific MGE cell grafting approach for treating TLE.

Moderate, intermittent voluntary exercise in a model of Gulf War Illness improves cognitive and mood function with alleviation of activated microglia and astrocytes, and enhanced neurogenesis in the hippocampus.

Persistent cognitive and mood impairments in Gulf War Illness (GWI) are associated with chronic neuroinflammation, typified by hypertrophied astrocytes, activated microglia, and increased proinflammatory mediators in the brain. Using a rat model, we investigated whether a simple lifestyle change such as moderate voluntary physical exercise would improve cognitive and mood function in GWI. Because veterans with GWI exhibit fatigue and post-exertional malaise, we employed an intermittent voluntary running exercise (RE) regimen, which prevented exercise-induced stress. The GWI rats were provided access to running wheels three days per week for 13 weeks, commencing ten weeks after the exposure to GWI-related chemicals and stress (GWI-RE group). Groups of age-matched sedentary GWI rats (GWI-SED group) and naïve rats were maintained parallelly. Interrogation of rats with behavioral tests after the 13-week RE regimen revealed improved hippocampus-dependent object location memory and pattern separation function and reduced anxiety-like behavior in the GWI-RE group compared to the GWI-SED group. Moreover, 13 weeks of RE in GWI rats significantly reversed activated microglia with short and less ramified processes into non-inflammatory/antiinflammatory microglia with highly ramified processes and reduced the hypertrophy of astrocytes. Moreover, the production of new neurons in the hippocampus was enhanced when examined eight weeks after the commencement of RE. Notably, increased neurogenesis continued even after the cessation of RE. Collectively, the results suggest that even a moderate, intermittent physical exercise has the promise to improve brain function in veterans with GWI in association with suppression of neuroinflammation and enhancement of hippocampal neurogenesis.

Neuroimmune mechanisms of cognitive impairment in a mouse model of Gulf War illness.

Gulf War Illness (GWI) is a chronic, multi-symptom disorder affecting approximately 30 percent of the nearly 700,000 Veterans of the 1991 Persian Gulf War. GWI-related chemical (GWIC) exposure promotes immune activation that correlates with cognitive impairment and other symptoms of GWI. However, the molecular mechanisms and signaling pathways linking GWIC to inflammation and neurological symptoms remain unclear. Here we show that acute exposure of murine macrophages to GWIC potentiates innate immune signaling and inflammatory cytokine production. Using an established mouse model of GWI, we report that neurobehavioral changes and neuroinflammation are attenuated in mice lacking the cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) and NOD-, LRR- or pyrin domain-containing protein 3 (NLRP3) innate immune pathways. In addition, we report sex differences in response to GWIC, with female mice showing more pronounced cognitive impairment and hippocampal astrocyte hypertrophy. In contrast, male mice display a GWIC-dependent upregulation of proinflammatory cytokines in the plasma that is not present in female mice. Our results indicate that STING and NLRP3 are key mediators of the cognitive impairment and inflammation observed in GWI and provide important new information on sex differences in this model.

Wwox deletion leads to reduced GABA-ergic inhibitory interneuron numbers and activation of microglia and astrocytes in mouse hippocampus.

The association of WW domain-containing oxidoreductase WWOX gene loss of function with central nervous system (CNS) related pathologies is well documented. These include spinocerebellar ataxia, epilepsy and mental retardation (SCAR12, OMIM: 614322) and early infantile epileptic encephalopathy (EIEE28, OMIM: 616211) syndromes. However, there is complete lack of understanding of the pathophysiological mechanisms at play. In this study, using a Wwox knockout (Wwox KO) mouse model (2 weeks old, both sexes) and stereological studies we observe that Wwox deletion leads to a significant reduction in the number of hippocampal GABA-ergic (γ-aminobutyric acid) interneurons. Wwox KO mice displayed significantly reduced numbers of calcium-binding protein parvalbumin (PV) and neuropeptide Y (NPY) expressing interneurons in different subfields of the hippocampus in comparison to Wwox wild-type (WT) mice. We also detected decreased levels of Glutamic Acid Decarboxylase protein isoforms GAD65/67 expression in Wwox null hippocampi suggesting lower levels of GABA synthesis. In addition, Wwox deficiency was associated with signs of neuroinflammation such as evidence of activated microglia, astrogliosis, and overexpression of inflammatory cytokines Tnf-a and Il6. We also performed comparative transcriptome-wide expression analyses of neural stem cells grown as neurospheres from hippocampi of Wwox KO and WT mice thus identifying 283 genes significantly dysregulated in their expression. Functional annotation of transcriptome profiling differences identified 'neurological disease' and 'CNS development related functions' to be significantly enriched. Several epilepsy-related genes were found differentially expressed in Wwox KO neurospheres. This study provides the first genotype-phenotype observations as well as potential mechanistic clues associated with Wwox loss of function in the brain.

Neuroinflammation in Gulf War Illness is linked with HMGB1 and complement activation, which can be discerned from brain-derived extracellular vesicles in the blood.

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 28 days. We performed behavioral tests 10 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 10 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.

Intranasally Administered Human MSC-Derived Extracellular Vesicles Pervasively Incorporate into Neurons and Microglia in both Intact and Status Epilepticus Injured Forebrain.

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stem cells (hMSCs) have great promise as biologics to treat neurological and neurodegenerative conditions due to their robust antiinflammatory and neuroprotective properties. Besides, intranasal (IN) administration of EVs has caught much attention because the procedure is noninvasive, amenable for repetitive dispensation, and leads to a quick penetration of EVs into multiple regions of the forebrain. Nonetheless, it is unknown whether brain injury-induced signals are essential for the entry of IN-administered EVs into different brain regions. Therefore, in this study, we investigated the distribution of IN-administered hMSC-derived EVs into neurons and microglia in the intact and status epilepticus (SE) injured rat forebrain. Ten billion EVs labeled with PKH26 were dispensed unilaterally into the left nostril of naïve rats, and rats that experienced two hours of kainate-induced SE. Six hours later, PKH26 + EVs were quantified from multiple forebrain regions using serial brain sections processed for different neural cell markers and confocal microscopy. Remarkably, EVs were seen bilaterally in virtually all regions of intact and SE-injured forebrain. The percentage of neurons incorporating EVs were comparable for most forebrain regions. However, in animals that underwent SE, a higher percentage of neurons incorporated EVs in the hippocampal CA1 subfield and the entorhinal cortex, the regions that typically display neurodegeneration after SE. In contrast, the incorporation of EVs by microglia was highly comparable in every region of the forebrain measured. Thus, unilateral IN administration of EVs is efficient for delivering EVs bilaterally into neurons and microglia in multiple regions in the intact or injured forebrain. Furthermore, incorporation of EVs by neurons is higher in areas of brain injury, implying that injury-related signals likely play a role in targeting of EVs into neurons, which may be beneficial for EV therapy in various neurodegenerative conditions including traumatic brain injury, stroke, multiple sclerosis, and Alzheimer's disease.

Voluntary Running Exercise-Mediated Enhanced Neurogenesis Does Not Obliterate Retrograde Spatial Memory.

UNLABELLED: Running exercise (RE) improves cognition, formation of anterograde memories, and mood, alongside enhancing hippocampal neurogenesis. A previous investigation in a mouse model showed that RE-induced increased neurogenesis erases retrograde memory (Akers et al., 2014). However, it is unknown whether RE-induced forgetting is common to all species. We ascertained whether voluntary RE-induced enhanced neurogenesis interferes with the recall of spatial memory in rats. Young rats assigned to either sedentary (SED) or running exercise (RE) groups were first subjected to eight learning sessions in a water maze. A probe test (PT) conducted 24 h after the final training session confirmed that animals in either group had a similar ability for the recall of short-term memory. Following this, rats in the RE group were housed in larger cages fitted with running wheels, whereas rats in the SED group remained in standard cages. Animals in the RE group ran an average of 78 km in 4 weeks. A second PT performed 4 weeks after the first PT revealed comparable ability for memory recall between animals in the RE and SED groups, which was evidenced through multiple measures of memory retrieval function. The RE group displayed a 1.5- to 2.1-fold higher hippocampal neurogenesis than SED rats. Additionally, both moderate and brisk RE did not interfere with the recall of memory, although increasing amounts of RE proportionally enhanced neurogenesis. In conclusion, RE does not impair memory recall ability in a rat model despite substantially increasing neurogenesis. SIGNIFICANCE STATEMENT: Running exercise (RE) improves new memory formation along with an increased neurogenesis in the hippocampus. In view of a recent study showing that RE-mediated increased hippocampal neurogenesis promotes forgetfulness in a mouse model, we ascertained whether a similar adverse phenomenon exists in a rat model. Memory recall ability examined 4 weeks after learning confirmed that animals that had run a mean of 78 km and displayed a 1.5- to 2.1-fold increase in hippocampal neurogenesis demonstrated similar proficiency for memory recall as animals that had remained sedentary. Furthermore, both moderate and brisk RE did not interfere with memory recall, although increasing amounts of RE proportionally enhanced neurogenesis, implying that RE has no adverse effects on memory recall.

Intranasally Administered EVs from hiPSC-derived NSCs Alter the Transcriptomic Profile of Activated Microglia and Conserve Brain Function in an Alzheimer's Model.

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.

Residual Microglia Following Short-term PLX5622 Treatment in 5xFAD Mice Exhibit Diminished NLRP3 Inflammasome and mTOR Signaling, and Enhanced Autophagy.

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 ∼80% 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 ∼65% 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.

Pathophysiological basis and promise of experimental therapies for Gulf War Illness, a chronic neuropsychiatric syndrome in veterans.

This article describes the pathophysiology and potential treatments for Gulf War Illness (GWI), which is a chronic neuropsychiatric illness linked to a combination of chemical exposures experienced by service personnel during the first Gulf War in 1991. However, there is currently no effective treatment for veterans with GWI. The article focuses on the current status and efficacy of existing therapeutic interventions in preclinical models of GWI, as well as potential perspectives of promising therapies. GWI stems from changes in brain and peripheral systems in veterans, leading to neurocognitive deficits, as well as physiological and psychological effects resulting from multifaceted changes such as neuroinflammation, oxidative stress, and neuronal damage. Aging not only renders veterans more susceptible to GWI symptoms, but also attenuates their immune capabilities and response to therapies. A variety of experimental models are being used to investigate the pathophysiology and develop therapies that have the ability to alleviate devastating symptoms. Over two dozen therapeutic interventions targeting neuroinflammation, mitochondrial dysfunction, neuronal injury, and neurogenesis are being tested, including agents such as curcumin, curcumin nanoparticles, monosodium luminol, melatonin, resveratrol, fluoxetine, rolipram, oleoylethanolamide, ketamine, levetiracetam, nicotinamide riboside, minocycline, pyridazine derivatives, and neurosteroids. Preclinical outcomes show that some agents have promise, including curcumin, resveratrol, and ketamine, which are being tested in clinical trials in GWI veterans. Neuroprotectants and other compounds such as monosodium luminol, melatonin, levetiracetam, oleoylethanolamide, and nicotinamide riboside appear promising for future clinical trials. Neurosteroids have been shown to have neuroprotective and disease-modifying properties, which makes them a promising medicine for GWI. Therefore, accelerated clinical studies are urgently needed to evaluate and launch an effective therapy for veterans displaying GWI.

A single intranasal dose of human mesenchymal stem cell-derived extracellular vesicles after traumatic brain injury eases neurogenesis decline, synapse loss, and BDNF-ERK-CREB signaling.

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.

Intranasally administered extracellular vesicles from human induced pluripotent stem cell-derived neural stem cells quickly incorporate into neurons and microglia in 5xFAD mice.

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.

Promise of metformin for preventing age-related cognitive dysfunction.

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.

Brain-Specific Increase in Leukotriene Signaling Accompanies Chronic Neuroinflammation and Cognitive Impairment in a Model of Gulf War Illness.

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.

Oral Nano-Curcumin in a Model of Chronic Gulf War Illness Alleviates Brain Dysfunction with Modulation of Oxidative Stress, Mitochondrial Function, Neuroinflammation, Neurogenesis, and Gene Expression.

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.

Metformin treatment in late middle age improves cognitive function with alleviation of microglial activation and enhancement of autophagy in the hippocampus.

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.

Melatonin improves brain function in a model of chronic Gulf War Illness with modulation of oxidative stress, NLRP3 inflammasomes, and BDNF-ERK-CREB pathway in the hippocampus.

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.

Extracellular Vesicles in the Forebrain Display Reduced miR-346 and miR-331-3p in a Rat Model of Chronic Temporal Lobe Epilepsy.

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.