HA-tag CD63 is a novel conditional transgenic approach to track extracellular vesicle interactions with sperm and their transfer at conception.

Extracellular vesicles (EVs) are a unique mode of intercellular communication capable of specificity in transmitting signals and cargo to coordinate local and distant cellular functions. A key example of this is the essential role that EVs secreted by epithelial cells lining the lumen of the male reproductive tract play in post-spermatogenic sperm maturation. We recently showed in a preclinical mouse model that this fundamental process had a causal role in somatic-to-germline transmission of biological information regarding prior stress experience capable of altering the rate of fetal development. However, critical mechanistic questions remain unanswered as to the processes by which signaling occurs between EVs and sperm, and whether EVs or their cargo are delivered at conception and are detectable in the early embryo. Unfortunately, notable methodological limitations shared across EV biology, particularly in the isolation and labeling of EVs, complicate efforts to answer these important questions as well as questions on EV targeting specificity and mechanisms. In our current studies, we developed a novel approach to track EVs using a conditional transgenic construct designed to label EVs via conditional Cre-induced hemagglutinin (HA) tagging of the EV endogenous tetraspanin, CD63. In our exhaustive validation steps, this internal small molecular weight tag did not affect EV secretion or functionality, a common problem found in the previous design of EV tags using larger molecular weight proteins, including fluorescent proteins. Utilizing a stably transfected immortalized epididymal epithelial cell line, we first validated key parameters of the conditional HA-tagged protein packaged into secreted EVs. Importantly, we systematically confirmed that expression of the CD63-HA had no impact on the production, size distribution, or surface charge of secreted EVs, nor did it alter the tetraspanin or miRNA composition of these EVs. We also utilized the CD63-HA EVs to verify physical interactions with sperm. Finally, using in vitro fertilization we produced some of the first images confirming sperm delivered EV cargo at conception and still detectable in the early-stage embryo. As such, this construct serves as a methodological advance and as a valuable tool, with applications in the study of EV function across biomedical research areas.

Longitudinal Assessment of Sensorimotor Function after Controlled Cortical Impact in Mice: Comparison of Beamwalk, Rotarod, and Automated Gait Analysis Tests.

Traumatic brain injury (TBI) patients are reported to experience long-term sensorimotor dysfunction, with gait deficits evident up to 2 years after the initial brain trauma. Experimental TBI including rodent models of penetrating ballistic-like brain injury and severe controlled cortical impact (CCI) can induce impairments in static and dynamic gait parameters. It is reported that the majority of deficits in gait-related parameters occur during the acute phase post-injury, as functional outcomes return toward baseline levels at chronic time points. In the present study, we carried out a longitudinal analysis of static, temporal and dynamic gait patterns following moderate-level CCI in adult male C57Bl/6J mice using the automated gait analysis apparatus, CatWalk. For comparison, we also performed longitudinal assessment of fine-motor coordination and function in CCI mice using more traditional sensorimotor behavioral tasks such as the beamwalk and accelerating rotarod tasks. We determined that longitudinal CatWalk analysis did not detect TBI-induced deficits in static, temporal, or dynamic gait parameters at acute or chronic time points. In contrast, the rotarod and beamwalk tasks showed that CCI mice had significant motor function impairments as demonstrated by deficits in balance and fine-motor coordination through 28 days post-injury. Stereological analysis confirmed that CCI produced a significant lesion in the parietal cortex at 28 days post-injury. Overall, these findings demonstrate that CatWalk analysis of gait parameters is not useful for assessment of long-term sensorimotor dysfunction after CCI, and that more traditional neurobehavioral tests should be used to quantify acute and chronic deficits in sensorimotor function.

Pubertal adversity alters chromatin dynamics and stress circuitry in the pregnant brain.

Women who have experienced adverse childhood events (ACEs) around puberty are at the greatest risk for neuropsychiatric disorders across the lifespan. This population is exceptionally vulnerable to neuropsychiatric disease presentation during the hormonally dynamic state of pregnancy. We previously established that chronic adversity around puberty in female mice significantly altered their HPA axis function specifically during pregnancy, modeling the effects of pubertal ACEs we also reported in women. We hypothesized that the pregnancy hormone, allopregnanolone, was involved in presentation of the blunted stress response phenotype by its interaction with the molecular programming that had occurred during pubertal adversity experience. Here, in adult mice previously stressed during puberty, allopregnanolone administration was sufficient to reproduce the decreased corticosterone response after acute stress. Examination of neuronal activation and the electrophysiological properties of CRF neurons in the paraventricular nucleus of the hypothalamus (PVN) found no significant changes in synaptic function that corresponded with the blunted HPA axis reactivity. However, at the chromatin level, utilization of ATAC-Seq profiling demonstrated a dramatic remodeling of DNA accessibility in the PVN following pubertal adversity. Altogether, these data establish a potential molecular mechanism whereby adversity during puberty can enact lasting transcriptional control that manifests only during a unique period of the lifespan where dynamic hormonal changes occur. These results highlight a biological process that may impart an increased risk for a highly vulnerable population, whereby pubertal programming of the PVN results in aberrant HPA axis responsiveness when exposed to the hormonal changes unique to pregnancy.

Old age increases microglial senescence, exacerbates secondary neuroinflammation, and worsens neurological outcomes after acute traumatic brain injury in mice.

After traumatic brain injury (TBI), individuals aged over 65 years show increased mortality and worse functional outcomes compared with younger persons. As neuroinflammation is a key pathobiological mechanism of secondary injury after TBI, we examined how aging affects post-traumatic microglial responses and functional outcomes. Young (3-month-old) and aged (18-month-old) male C57Bl/6 mice were subjected to moderate-level controlled cortical impact or sham surgery, and neurological function was evaluated. At 72 hours after injury, brain, blood, and spleen leukocyte counts were assessed ex vivo using flow cytometry. Aged mice demonstrated more severe deficits in forelimb grip strength, balance and motor coordination, spontaneous locomotor activity, and anxiety-like behavior. These animals also exhibited more robust microglial proliferation and significantly higher numbers of brain-infiltrating leukocytes. Microglia in aged mice showed impairments in phagocytic activity and higher production of interleukin-1ß (IL-1ß). Infiltrating myeloid cells in aged TBI mice also had deficits in phagocytosis but showed diminished proinflammatory cytokine production and greater reactive oxygen species production. Expression of several senescence markers (Bcl-2, p16ink4a, p21cip1a, lipofuscin, and H2AX [pS139]) was increased with age and/or TBI in both microglia and injured cortex. Although there was no difference in the number of circulating blood neutrophils as a function of age, young mice exhibited more pronounced TBI-induced splenomegaly and splenic myeloid cell expansion. Thus, worse post-traumatic behavioral outcomes in aged animals are associated with exaggerated microglial responses, increased leukocyte invasion, and upregulation of senescence markers.

Inhibition of miR-155 Limits Neuroinflammation and Improves Functional Recovery After Experimental Traumatic Brain Injury in Mice.

Micro-RNAs (miRs) are short, noncoding RNAs that negatively regulate gene expression at the post-transcriptional level and have been implicated in the pathophysiology of secondary damage after traumatic brain injury (TBI). Among miRs linked to inflammation, miR-155 has been implicated as a pro-inflammatory factor in a variety of organ systems. We examined the expression profile of miR-155, following experimental TBI (controlled cortical impact) in adult male C57Bl/6 mice, as well as the effects of acute or delayed administration of a miR-155 antagomir on post-traumatic neuroinflammatory responses and neurological recovery. Trauma robustly increased miR-155 expression in the injured cortex over 7 days. Similar TBI-induced miR-155 expression changes were also found in microglia/macrophages isolated from the injured cortex at 7 days post-injury. A miR-155 hairpin inhibitor (antagomir; 0.5 nmol), administered intracerebroventricularly (ICV) immediately after injury, attenuated neuroinflammatory markers at both 1 day and 7 days post-injury and reduced impairments in spatial working memory. Delayed ICV infusion of the miR-155 antagomir (0.5 nmol/day), beginning 24 h post-injury and continuing for 6 days, attenuated neuroinflammatory markers at 7 days post-injury and improved motor, but not cognitive, function through 28 days. The latter treatment limited NADPH oxidase 2 expression changes in microglia/macrophages in the injured cortex and reduced cortical lesion volume. In summary, TBI causes a robust and persistent neuroinflammatory response that is associated with increased miR-155 expression in microglia/macrophages, and miR-155 inhibition reduces post-traumatic neuroinflammatory responses and improves neurological recovery. Thus, miR-155 may be a therapeutic target for TBI-related neuroinflammation.