Complement C5a receptor 1 blockade reverses cognitive deficits following cranial radiation therapy for brain cancer.

Cranial radiation therapy (RT) for brain cancers leads to an irreversible decline in cognitive function without an available remedy. Radiation-induced cognitive deficits (RICD) are particularly a pressing problem for the survivors of pediatric and low grade glioma (LGG) patients who often live long post-RT. Radiation-induced elevated neuroinflammation and gliosis, triggered by the detrimental CNS complement cascade, lead to excessive synaptic and cognitive loss. Using intact and brain cancer-bearing mouse models, we now show that targeting anaphylatoxin complement C5a receptor (C5aR1) is neuroprotective against RICD. We used a genetic knockout, C5aR1 KO mouse, and a pharmacologic approach, employing the orally active, brain penetrant C5aR1 antagonist PMX205, to reverse RICD. Irradiated C5aR1 KO and WT mice receiving PMX205 showed significant neurocognitive improvements in object recognition memory and memory consolidation tasks. C5aR1 inhibition reduced microglial activation, astrogliosis, and synaptic loss in the irradiated brain. Importantly, C5aR1 inhibition in the syngeneic, orthotopic astrocytoma, and glioblastoma-bearing mice protected against RICD without interfering with the therapeutic efficacy of RT to reduce tumor volume in vivo . PMX205 is currently in clinical trials for amyotrophic lateral sclerosis (ALS). Thus, C5aR1 inhibition is a translationally feasible approach to address RICD, an unmet medical need.

Extracellular vesicles from GABAergic but not glutamatergic neurons protect against neurological dysfunction following cranial irradiation.

Cranial irradiation used to control brain malignancies invariably leads to progressive and debilitating declines in cognition. Clinical efforts implementing hippocampal avoidance and NMDAR antagonism, have sought to minimize dose to radiosensitive neurogenic regions while normalizing excitatory/inhibitory (E/I) tone. Results of these trials have yielded only marginal benefits to cognition, prompting current studies to evaluate the potential of systemic extracellular vesicle (EV) therapy to restore neurocognitive functionality in the irradiated brain. Here we tested the hypothesis that EVs derived from inhibitory but not excitatory neuronal cultures would prove beneficial to cognition and associated pathology. Rats subjected to a clinically relevant, fractionated cranial irradiation paradigm were given multiple injections of either GABAergic- or glutamatergic-derived EV and subjected to behavioral testing. Rats treated with GABAergic but not glutamatergic EVs showed significant improvements on hippocampal- and cortical-dependent behavioral tasks. While each treatment enhanced levels of the neurotrophic factors BDNF and GDNF, only GABAergic EVs preserved granule cell neuron dendritic spine density. Additional studies conducted with GABAergic EVs, confirmed significant benefits on amygdala-dependent behavior and modest changes in synaptic plasticity as measured by long-term potentiation. These data point to a potentially more efficacious approach for resolving radiation-induced neurological deficits, possibly through a mechanism able to restore homeostatic E/I balance.

More May Not be Better: Enhanced Spacecraft Shielding May Exacerbate Cognitive Decrements by Increasing Pion Exposures during Deep Space Exploration.

The pervasiveness of deep space radiation remains a confounding factor for the transit of humans through our solar system. Spacecraft shielding both protects astronauts but also contributes to absorbed dose through galactic cosmic ray interactions that produce secondary particles. The resultant biological effects drop to a minimum for aluminum shielding around 20 g/cm2 but increase with additional shielding. The present work evaluates for the first time, the impact of secondary pions on central nervous system functionality. The fractional pion dose emanating from thicker shielded spacecraft regions could contribute up to 10% of the total absorbed radiation dose. New results from the Paul Scherrer Institute have revealed that low dose exposures to 150 MeV positive and negative pions, akin to a Mars mission, result in significant, long-lasting cognitive impairments. These surprising findings emphasize the need to carefully evaluate shielding configurations to optimize safe exposure limits for astronauts during deep space travel.

FLASH-RT does not affect chromosome translocations and junction structures beyond that of CONV-RT dose-rates.

BACKGROUND AND PURPOSE: The impact of radiotherapy (RT) at ultra high vs conventional dose rate (FLASH vs CONV) on the generation and repair of DNA double strand breaks (DSBs) is an important question that remains to be investigated. Here, we tested the hypothesis as to whether FLASH-RT generates decreased chromosomal translocations compared to CONV-RT. MATERIALS AND METHODS: We used two FLASH validated electron beams and high-throughput rejoin and genome-wide translocation sequencing (HTGTS-JoinT-seq), employing S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs) in HEK239T cells, to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated after various irradiation doses, dose rates and oxygen tensions (normoxic, 21% O2; physiological, 4% O2; hypoxic, 2% and 0.5% O2). Electron irradiation was delivered using a FLASH capable Varian Trilogy and the eRT6/Oriatron at CONV (0.08-0.13 Gy/s) and FLASH (1x102-5x106 Gy/s) dose rates. Related experiments using clonogenic survival and γH2AX foci in the 293T and the U87 glioblastoma lines were also performed to discern FLASH-RT vs CONV-RT DSB effects. RESULTS: Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Furthermore, RT dose rate modality on U87 cells did not change γH2AX foci numbers at 1- and 24-hours post-irradiation nor did this affect 293T clonogenic survival. CONCLUSION: Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.

Uncovering the Protective Neurologic Mechanisms of Hypofractionated FLASH Radiotherapy.

Implementation of ultra-high dose-rate FLASH radiotherapy (FLASH-RT) is rapidly gaining traction as a unique cancer treatment modality able to dramatically minimize normal tissue toxicity while maintaining antitumor efficacy compared with standard-of-care radiotherapy at conventional dose rate (CONV-RT). The resultant improvements in the therapeutic index have sparked intense investigations in pursuit of the underlying mechanisms. As a preamble to clinical translation, we exposed non-tumor-bearing male and female mice to hypofractionated (3 × 10 Gy) whole brain FLASH- and CONV-RT to evaluate differential neurologic responses using a comprehensive panel of functional and molecular outcomes over a 6-month follow-up. In each instance, extensive and rigorous behavioral testing showed FLASH-RT to preserve cognitive indices of learning and memory that corresponded to a similar protection of synaptic plasticity as measured by long-term potentiation (LTP). These beneficial functional outcomes were not found after CONV-RT and were linked to a preservation of synaptic integrity at the molecular (synaptophysin) level and to reductions in neuroinflammation (CD68+ microglia) throughout specific brain regions known to be engaged by our selected cognitive tasks (hippocampus, medial prefrontal cortex). Ultrastructural changes in presynaptic/postsynaptic bouton (Bassoon/Homer-1 puncta) within these same regions of the brain were not found to differ in response to dose rate. With this clinically relevant dosing regimen, we provide a mechanistic blueprint from synapse to cognition detailing how FLASH-RT reduces normal tissue complications in the irradiated brain. Significance: Functional preservation of cognition and LTP after hypofractionated FLASH-RT are linked to a protection of synaptic integrity and a reduction in neuroinflammation over protracted after irradiation times.

The sparing effect of FLASH-RT on synaptic plasticity is maintained in mice with standard fractionation.

Long-term potentiation (LTP) was used to gauge the impact of conventional and FLASH dose rates on synaptic transmission. Data collected from the hippocampus and medial prefrontal cortex confirmed significant inhibition of LTP after 10 fractions of 3 Gy (30 Gy total) conventional radiotherapy. Remarkably, 10x3Gy FLASH radiotherapy and unirradiated controls were identical and exhibited normal LTP.

Galactic cosmic radiation exposure causes multifaceted neurocognitive impairments.

Technological advancements have facilitated the implementation of realistic, terrestrial-based complex 33-beam galactic cosmic radiation simulations (GCR Sim) to now probe central nervous system functionality. This work expands considerably on prior, simplified GCR simulations, yielding new insights into responses of male and female mice exposed to 40-50 cGy acute or chronic radiations relevant to deep space travel. Results of the object in updated location task suggested that exposure to acute or chronic GCR Sim induced persistent impairments in hippocampus-dependent memory formation and reconsolidation in female mice that did not manifest robustly in irradiated male mice. Interestingly, irradiated male mice, but not females, were impaired in novel object recognition and chronically irradiated males exhibited increased aggressive behavior on the tube dominance test. Electrophysiology studies used to evaluate synaptic plasticity in the hippocampal CA1 region revealed significant reductions in long-term potentiation after each irradiation paradigm in both sexes. Interestingly, network-level disruptions did not translate to altered intrinsic electrophysiological properties of CA1 pyramidal cells, whereas acute exposures caused modest drops in excitatory synaptic signaling in males. Ultrastructural analyses of CA1 synapses found smaller postsynaptic densities in larger spines of chronically exposed mice compared to controls and acutely exposed mice. Myelination was also affected by GCR Sim with acutely exposed mice exhibiting an increase in the percent of myelinated axons; however, the myelin sheathes on small calibur (< 0.3 mm) and larger (> 0.5 mm) axons were thinner when compared to controls. Present findings might have been predicted based on previous studies using single and mixed beam exposures and provide further evidence that space-relevant radiation exposures disrupt critical cognitive processes and underlying neuronal network-level plasticity, albeit not to the extent that might have been previously predicted.

BDNF Augmentation Using Riluzole Reverses Doxorubicin-Induced Decline in Cognitive Function and Neurogenesis.

Cancer-related cognitive impairment (CRCI) considerably affects the quality of life of millions of cancer survivors. Brain-derived neurotrophic factor (BDNF) has been shown to promote survival, differentiation, and maintenance of in vivo dentate neurogenesis, and chemotherapy induces a plethora of physiological and cellular alterations, including a decline in neurogenesis and increased neuroinflammation linked with cognitive impairments. In our clinical studies, breast cancer patients treated with doxorubicin (Adriamycin®, ADR) experienced a significant reduction in the blood levels of BDNF that was associated with a higher risk of CRCI. Our past rodent studies in CRCI have also shown a significant reduction in dentate neurogenesis accompanied by cognitive impairment. In this study, using a female mouse model of ADR-induced cognitive decline, we tested the impact of riluzole (RZ), an orally active BDNF-enhancing medication that is FDA-approved for amyotrophic lateral sclerosis. ADR-treated mice receiving RZ in the drinking water for 1 month showed significant improvements in hippocampal-dependent learning and memory function (spatial recognition), fear extinction memory consolidation, and reduced anxiety-like behavior. RZ prevented chemotherapy-induced reductions of BDNF levels in the hippocampus. Importantly, RZ mitigated chemotherapy-induced loss of newly born, immature neurons, dentate neurogenesis, and neuroinflammation. In conclusion, this data provides pre-clinical evidence for a translationally feasible approach to enhance the neuroprotective effects of RZ treatment to prevent CRCI.

Elucidating the neurological mechanism of the FLASH effect in juvenile mice exposed to hypofractionated radiotherapy.

BACKGROUND: Ultrahigh dose-rate radiotherapy (FLASH-RT) affords improvements in the therapeutic index by minimizing normal tissue toxicities without compromising antitumor efficacy compared to conventional dose-rate radiotherapy (CONV-RT). To investigate the translational potential of FLASH-RT to a human pediatric medulloblastoma brain tumor, we used a radiosensitive juvenile mouse model to assess adverse long-term neurological outcomes. METHODS: Cohorts of 3-week-old male and female C57Bl/6 mice exposed to hypofractionated (2 × 10 Gy, FLASH-RT or CONV-RT) whole brain irradiation and unirradiated controls underwent behavioral testing to ascertain cognitive status four months posttreatment. Animals were sacrificed 6 months post-irradiation and tissues were analyzed for neurological and cerebrovascular decrements. RESULTS: The neurological impact of FLASH-RT was analyzed over a 6-month follow-up. FLASH-RT ameliorated neurocognitive decrements induced by CONV-RT and preserved synaptic plasticity and integrity at the electrophysiological (long-term potentiation), molecular (synaptophysin), and structural (Bassoon/Homer-1 bouton) levels in multiple brain regions. The benefits of FLASH-RT were also linked to reduced neuroinflammation (activated microglia) and the preservation of the cerebrovascular structure, by maintaining aquaporin-4 levels and minimizing microglia colocalized to vessels. CONCLUSIONS: Hypofractionated FLASH-RT affords significant and long-term normal tissue protection in the radiosensitive juvenile mouse brain when compared to CONV-RT. The capability of FLASH-RT to preserve critical cognitive outcomes and electrophysiological properties over 6-months is noteworthy and highlights its potential for resolving long-standing complications faced by pediatric brain tumor survivors. While care must be exercised before clinical translation is realized, present findings document the marked benefits of FLASH-RT that extend from synapse to cognition and the microvasculature.

Impact of IL-21-associated peripheral and brain crosstalk on the Alzheimer's disease neuropathology.

Alzheimer's disease (AD) is associated with dysregulated immune and inflammatory responses. Emerging evidence indicates that peripheral immune activation is linked to neuroinflammation and AD pathogenesis. The present study focuses on determining the role of IL-21 in the pathogenesis of AD using human samples and the 5xFAD mice model. We find that the levels of IL-21 are increased in the periphery of both humans and mice in AD. In addition, the proportions of IL-21 target cells, Tfh and B plasma cells as well as activation of monocytes is increased in PBMCs from AD and mild cognitively impaired (MCI) subjects as compared to age-matched controls, indicating immune activation. In contrast, the percentage of B1 cells that control inflammation is decreased. These changes are due to IL-21 as the expression of IL-21 receptor (IL-21R) is higher on all these cells in AD. Furthermore, treatment with recombinant IL-21 in AD mice also leads to similar alterations in Tfh, B, B1, and macrophages. The effect of IL-21 is not confined to the periphery since increased expression of IL-21R is also observed in both humans and mice hippocampus derived from the AD brains. In addition, mice injected with IL-21 display increased deposition of amyloid beta (Aß) plaques in the brain which is reduced following anti-IL-21R antibody that blocks the IL-21 signaling. Moreover, activation of microglia was enhanced in IL-21-injected mice. In keeping with enhanced microglial activation, we also observed increased production of pro-inflammatory cytokines, IL-18 and IL-6 in IL-21-injected mice. The microglial activation and cytokines were both inhibited following IL-21R blockage. Altogether, IL-21 escalates AD pathology by enhancing peripheral and brain immune and inflammatory responses leading to increased Aß plaque deposition. IL-21 impacts AD neuropathology by enhancing peripheral and neuronal immune activation, inflammation, and Aß plaque deposition. Increased levels of IL-21 in the circulation of AD and MCI subjects enhances the proportions of Tfh and B plasma cells indicative of peripheral immune activation. On the other hand, the proportions of B1 cells that help reduce inflammation and clear Aß are reduced. In addition to the periphery, IL-21 also acts on the brain via IL-21 receptor, IL-21R that displays increased expression in the hippocampi of AD and MCI subjects. IL-21 enhances the activation of microglia, induces the secretion of pro-inflammatory cytokines and deposition of Aß plaques in the brain in AD.

Dissecting Differential Complex Behavioral Responses to Simulated Space Radiation Exposures.

This study has established the impact that space radiation exposure has on the capability of rats to successfully negotiate behavioral tasks of increasing complexity. Rats previously exposed to a low dose (10 cGy) of either 4He ions or a cocktail of 6 ions that simulates the galactic cosmic ray spectrum (GCRSim) were screened initially on an attentional set shifting (ATSET) task that provides a measure of executive function. Rats that exhibited superior ATSET performance were then selected for follow up behavioral assessments designed to evaluate how the cohort of "good performers" would fare when presented with a novel behavioral paradigm termed the Associative Recognition Memory and Interference Touchscreen (ARMIT) task. Central to this approach was to discriminate if/how adaptive problem solving would be impacted by changing the options of associative cues presented over several learning sessions to obtain a reward under time constraints using this newly designed touch screen-based task. Data from these studies indicated that when faced with an increased cognitive load, possibly due to interference from prior associative recognition memories, rats exhibited impairments in their capability to negotiate task dynamics and efficiently engage abstract reasoning. Interestingly, while exposure to the GCRSim adversely impacted problem-solving capabilities, single ion exposure did not, pointing to the nuances of space radiation exposure on CNS functionality. Since the selected behavioral paradigms exhibit strong cross-species correlates, data suggest that rodents succumb to increased task rigor as observed in humans, and make similar mistakes when challenged with the interference of overlapping associative memories. Furthermore, data clearly points to the limitations of over-reliance on a single cognitive endpoint that may underestimate global neurocognitive risk due to space radiation exposure.

Acute, Low-Dose Neutron Exposures Adversely Impact Central Nervous System Function.

A recognized risk of long-duration space travel arises from the elevated exposure astronauts face from galactic cosmic radiation (GCR), which is composed of a diverse array of energetic particles. There is now abundant evidence that exposures to many different charged particle GCR components within acute time frames are sufficient to induce central nervous system deficits that span from the molecular to the whole animal behavioral scale. Enhanced spacecraft shielding can lessen exposures to charged particle GCR components, but may conversely elevate neutron radiation levels. We previously observed that space-relevant neutron radiation doses, chronically delivered at dose-rates expected during planned human exploratory missions, can disrupt hippocampal neuronal excitability, perturb network long-term potentiation and negatively impact cognitive behavior. We have now determined that acute exposures to similar low doses (18 cGy) of neutron radiation can also lead to suppressed hippocampal synaptic signaling, as well as decreased learning and memory performance in male mice. Our results demonstrate that similar nervous system hazards arise from neutron irradiation regardless of the exposure time course. While not always in an identical manner, neutron irradiation disrupts many of the same central nervous system elements as acute charged particle GCR exposures. The risks arising from neutron irradiation are therefore important to consider when determining the overall hazards astronauts will face from the space radiation environment.

Chronic Low Dose Neutron Exposure Results in Altered Neurotransmission Properties of the Hippocampus-Prefrontal Cortex Axis in Both Mice and Rats.

The proposed deep space exploration to the moon and later to Mars will result in astronauts receiving significant chronic exposures to space radiation (SR). SR exposure results in multiple neurocognitive impairments. Recently, our cross-species (mouse/rat) studies reported impaired associative memory formation in both species following a chronic 6-month low dose exposure to a mixed field of neutrons (1 mGy/day for a total dose pf 18 cGy). In the present study, we report neutron exposure induced synaptic plasticity in the medial prefrontal cortex, accompanied by microglial activation and significant synaptic loss in the hippocampus. In a parallel study, neutron exposure was also found to alter fluorescence assisted single synaptosome LTP (FASS-LTP) in the hippocampus of rats, that may be related to a reduced ability to insert AMPAR into the post-synaptic membrane, which may arise from increased phosphorylation of the serine 845 residue of the GluA1 subunit. Thus, we demonstrate for the first time, that low dose chronic neutron irradiation impacts homeostatic synaptic plasticity in the hippocampal-cortical circuit in two rodent species, and that the ability to successfully encode associative recognition memory is a dynamic, multicircuit process, possibly involving compensatory changes in AMPAR density on the synaptic surface.

Upregulation of Vitamin C Transporter Functional Expression in 5xFAD Mouse Intestine.

The process of obtaining ascorbic acid (AA) via intestinal absorption and blood circulation is carrier-mediated utilizing the AA transporters SVCT1 and SVCT2, which are expressed in the intestine and brain (SVCT2 in abundance). AA concentration is decreased in Alzheimer's disease (AD), but information regarding the status of intestinal AA uptake in the AD is still lacking. We aimed here to understand how AA homeostasis is modulated in a transgenic mouse model (5xFAD) of AD. AA levels in serum from 5xFAD mice were markedly lower than controls. Expression of oxidative stress response genes (glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1)) were significantly increased in AD mice jejunum, and this increase was mitigated by AA supplementation. Uptake of AA in the jejunum was upregulated. This increased AA transport was caused by a marked increase in SVCT1 and SVCT2 protein, mRNA, and heterogeneous nuclear RNA (hnRNA) expression. A significant increase in the expression of HNF1α and specific protein 1 (Sp1), which drive SLC23A1 and SLC23A2 promoter activity, respectively, was observed. Expression of hSVCT interacting proteins GRHPR and CLSTN3 were also increased. SVCT2 protein and mRNA expression in the hippocampus of 5xFAD mice was not altered. Together, these investigations reveal adaptive up-regulation of intestinal AA uptake in the 5xFAD mouse model.

Human neural stem cell-derived extracellular vesicles mitigate hallmarks of Alzheimer's disease.

BACKGROUND: Regenerative therapies to mitigate Alzheimer's disease (AD) neuropathology have shown very limited success. In the recent era, extracellular vesicles (EVs) derived from multipotent and pluripotent stem cells have shown considerable promise for the treatment of dementia and many neurodegenerative conditions. METHODS: Using the 5xFAD accelerated transgenic mouse model of AD, we now show the regenerative potential of human neural stem cell (hNSC)-derived EVs on the neurocognitive and neuropathologic hallmarks in the AD brain. Two- or 6-month-old 5xFAD mice received single or two intra-venous (retro-orbital vein, RO) injections of hNSC-derived EVs, respectively. RESULTS: RO treatment using hNSC-derived EVs restored fear extinction memory consolidation and reduced anxiety-related behaviors 4-6 weeks post-injection. EV treatment also significantly reduced dense core amyloid-beta plaque accumulation and microglial activation in both age groups. These results correlated with partial restoration of homeostatic levels of circulating pro-inflammatory cytokines in the AD mice. Importantly, EV treatment protected against synaptic loss in the AD brain that paralleled improved cognition. MiRNA analysis of the EV cargo revealed promising candidates targeting neuroinflammation and synaptic function. CONCLUSIONS: Collectively, these data demonstrate the neuroprotective effects of systemic administration of stem cell-derived EVs for remediation of behavioral and molecular AD neuropathologies.

Detrimental impacts of mixed-ion radiation on nervous system function.

Galactic cosmic radiation (GCR), composed of highly energetic and fully ionized atomic nuclei, produces diverse deleterious effects on the body. In researching the neurological risks of GCR exposures, including during human spaceflight, various ground-based single-ion GCR irradiation paradigms induce differential disruptions of cellular activity and overall behavior. However, it remains less clear how irradiation comprising a mix of multiple ions, more accurately recapitulating the space GCR environment, impacts the central nervous system. We therefore examined how mixed-ion GCR irradiation (two similar 5-6 beam combinations of protons, helium, oxygen, silicon and iron ions) influenced neuronal connectivity, functional generation of activity within neural circuits and cognitive behavior in mice. In electrophysiological recordings we find that space-relevant doses of mixed-ion GCR preferentially alter hippocampal inhibitory neurotransmission and produce related disruptions in the local field potentials of hippocampal oscillations. Such underlying perturbation in hippocampal network activity correspond with perturbed learning, memory and anxiety behavior.

Glia-Selective Deletion of Complement C1q Prevents Radiation-Induced Cognitive Deficits and Neuroinflammation.

The adverse neurocognitive sequelae following clinical radiotherapy (RT) for central nervous system (CNS) malignancies are often long-lasting without any clinical recourse. Despite recent progress, the cellular mechanisms mediating RT-induced cognitive deficits (RICD) are poorly understood. The complement system is an immediate sensor of a disturbed inflammatory environment and a potent mediator of gliosis with a range of nonimmune functions in the CNS, including synaptic pruning, which is detrimental if dysregulated. We hypothesize that complement-mediated changes in glial cell function significantly contribute to RICD. The underlying alterations in CNS complement cascade proteins (C1q, C3), TLR4, and colabeling with glia (IBA1, GFAP) were examined using gene expression, immunofluorescence, and in silico modeling approaches in the adult mouse brain following 9 Gy cranial RT. Three-dimensional volumetric quantification showed elevated molecular signatures of gliosis at short- and long-term post-RT times. We found significant elevations in complement C1q, C3, and TLR4 post-RT accompanied by increased colabeling of astrocytes and microglia. To address the mechanism of RT-induced complement cascade activation, neuroinflammation, and cognitive dysfunction, we used a genetic approach-conditional, microglia-selective C1q (Flox) knockdown mice-to determine whether a glia-specific, upstream complement cascade contributes to RICD. C1q-Flox mice exposed to cranial RT showed no cognitive deficits compared with irradiated WT mice. Further, irradiated C1q-Flox mice were protected from RT-induced microglial activation and synaptic loss, elevation of anaphylatoxin C5a receptor, astrocytic-C3, and microglial-TLR4 expression in the brain. Our findings demonstrate for the first time a microglia-specific mechanism of RICD involving an upstream complement cascade component, C1q. SIGNIFICANCE: Clinically-relevant radiotherapy induces aberrant complement activation, leading to brain injury. Microglia-selective genetic deletion of CNS complement C1q ameliorates radiation-induced cognitive impairments, synaptic loss, and neuroinflammation, highlighting the potential for C1q as a novel therapeutic target.See related commentary by Korimerla and Wahl, p. 1635.

Extracellular Vesicle-Derived miR-124 Resolves Radiation-Induced Brain Injury.

Radiation-induced cognitive dysfunction (RICD) is a progressive and debilitating health issue facing patients following cranial radiotherapy to control central nervous system cancers. There has been some success treating RICD in rodents using human neural stem cell (hNSC) transplantation, but the procedure is invasive, requires immunosuppression, and could cause other complications such as teratoma formation. Extracellular vesicles (EV) are nanoscale membrane-bound structures that contain biological contents including mRNA, miRNA, proteins, and lipids that can be readily isolated from conditioned culture media. It has been previously shown that hNSC-derived EV resolves RICD following cranial irradiation using an immunocompromised rodent model. Here, we use immunocompetent wild-type mice to show that hNSC-derived EV treatment administered either intravenously via retro-orbital vein injection or via intracranial transplantation can ameliorate cognitive deficits following 9 Gy head-only irradiation. Cognitive function assessed on the novel place recognition, novel object recognition, and temporal order tasks was not only improved at early (5 weeks) but also at delayed (6 months) postirradiation times with just a single EV treatment. Improved behavioral outcomes were also associated with reduced neuroinflammation as measured by a reduction in activated microglia. To identify the mechanism of action, analysis of EV cargo implicated miRNA (miR-124) as a potential candidate in the mitigation of RICD. Furthermore, viral vector-mediated overexpression of miR-124 in the irradiated brain ameliorated RICD and reduced microglial activation. Our findings demonstrate for the first time that systemic administration of hNSC-derived EV abrogates RICD and neuroinflammation in cranially irradiated wild-type rodents through a mechanism involving miR-124. SIGNIFICANCE: Radiation-induced neurocognitive decrements in immunocompetent mice can be resolved by systemic delivery of hNSC-derived EVs involving a mechanism dependent on expression of miR-124.

Mitigation of helium irradiation-induced brain injury by microglia depletion.

BACKGROUND: Cosmic radiation exposures have been found to elicit cognitive impairments involving a wide-range of underlying neuropathology including elevated oxidative stress, neural stem cell loss, and compromised neuronal architecture. Cognitive impairments have also been associated with sustained microglia activation following low dose exposure to helium ions. Space-relevant charged particles elicit neuroinflammation that persists long-term post-irradiation. Here, we investigated the potential neurocognitive benefits of microglia depletion following low dose whole body exposure to helium ions. METHODS: Adult mice were administered a dietary inhibitor (PLX5622) of colony stimulating factor-1 receptor (CSF1R) to deplete microglia 2 weeks after whole body helium irradiation (4He, 30 cGy, 400 MeV/n). Cohorts of mice maintained on a normal and PLX5622 diet were tested for cognitive function using seven independent behavioral tasks, microglial activation, hippocampal neuronal morphology, spine density, and electrophysiology properties 4-6 weeks later. RESULTS: PLX5622 treatment caused a rapid and near complete elimination of microglia in the brain within 3 days of treatment. Irradiated animals on normal diet exhibited a range of behavioral deficits involving the medial pre-frontal cortex and hippocampus and increased microglial activation. Animals on PLX5622 diet exhibited no radiation-induced cognitive deficits, and expression of resting and activated microglia were almost completely abolished, without any effects on the oligodendrocyte progenitors, throughout the brain. While PLX5622 treatment was found to attenuate radiation-induced increases in post-synaptic density protein 95 (PSD-95) puncta and to preserve mushroom type spine densities, other morphologic features of neurons and electrophysiologic measures of intrinsic excitability were relatively unaffected. CONCLUSIONS: Our data suggest that microglia play a critical role in cosmic radiation-induced cognitive deficits in mice and, that approaches targeting microglial function are poised to provide considerable benefit to the brain exposed to charged particles.