Youth-associated platelet-derived chemokine reverses brain aging through neuroimmune mechanisms.

Age-related loss of brain function has been seen as inevitable, yet recent work leveraging the systemic environment challenges this notion. Schroer et al. demonstrate that youth-associated platelet factor 4 (PF4) partially restores brain function in aged mice while reducing peripheral immune dysfunction, supporting periphery-based approaches to treat age-associated brain disorders.

Neuronal TIMP2 regulates hippocampus-dependent plasticity and extracellular matrix complexity.

Functional output of the hippocampus, a brain region subserving memory function, depends on highly orchestrated cellular and molecular processes that regulate synaptic plasticity throughout life. The structural requirements of such plasticity and molecular events involved in this regulation are poorly understood. Specific molecules, including tissue inhibitor of metalloproteinases-2 (TIMP2) have been implicated in plasticity processes in the hippocampus, a role that decreases with brain aging as expression is lost. Here, we report that TIMP2 is highly expressed by neurons within the hippocampus and its loss drives changes in cellular programs related to adult neurogenesis and dendritic spine turnover with corresponding impairments in hippocampus-dependent memory. Consistent with the accumulation of extracellular matrix (ECM) in the hippocampus we observe with aging, we find that TIMP2 acts to reduce accumulation of ECM around synapses in the hippocampus. Moreover, its deletion results in hindrance of newborn neuron migration through a denser ECM network. A novel conditional TIMP2 knockout (KO) model reveals that neuronal TIMP2 regulates adult neurogenesis, accumulation of ECM, and ultimately hippocampus-dependent memory. Our results define a mechanism whereby hippocampus-dependent function is regulated by TIMP2 and its interactions with the ECM to regulate diverse processes associated with synaptic plasticity.

Corrigendum: Long-Term Cognitive Impairments and Pathological Alterations in a Mouse Model of Repetitive Mild Traumatic Brain Injury.

[This corrects the article DOI: 10.3389/fneur.2014.00012.].

APOE4 confers transcriptomic and functional alterations to primary mouse microglia.

Common genetic variants in more than forty loci modulate risk for Alzheimer's disease (AD). AD risk alleles are enriched within enhancers active in myeloid cells, suggesting that microglia, the brain-resident macrophages, may play a key role in the etiology of AD. A major genetic risk factor for AD is Apolipoprotein E (APOE) genotype, with the ε4/ε4 (E4) genotype increasing risk for AD by approximately 15 fold compared to the most common ε3/ε3 (E3) genotype. However, the impact of APOE genotype on microglial function has not been thoroughly investigated. To address this, we cultured primary microglia from mice in which both alleles of the mouse Apoe gene have been humanized to encode either human APOE ε3 or APOE ε4. Relative to E3 microglia, E4 microglia exhibit altered morphology, increased endolysosomal mass, increased cytokine/chemokine production, and increased lipid and lipid droplet accumulation at baseline. These changes were accompanied by decreased translation and increased phosphorylation of eIF2ɑ and eIF2ɑ-kinases that participate in the integrated stress response, suggesting that E4 genotype leads to elevated levels of cellular stress in microglia relative to E3 genotype. Using live-cell imaging and flow cytometry, we also show that E4 microglia exhibited increased phagocytic uptake of myelin and other substrates compared to E3 microglia. While transcriptomic profiling of myelin-challenged microglia revealed a largely overlapping response profile across genotypes, differential enrichment of genes in interferon signaling, extracellular matrix and translation-related pathways was identified in E4 versus E3 microglia both at baseline and following myelin challenge. Together, our results suggest E4 genotype confers several important functional alterations to microglia even prior to myelin challenge, providing insight into the molecular and cellular mechanisms by which APOE4 may increase risk for AD.

Eosinophils regulate adipose tissue inflammation and sustain physical and immunological fitness in old age.

Adipose tissue eosinophils (ATEs) are important in the control of obesity-associated inflammation and metabolic disease. However, the way in which ageing impacts the regulatory role of ATEs remains unknown. Here, we show that ATEs undergo major age-related changes in distribution and function associated with impaired adipose tissue homeostasis and systemic low-grade inflammation in both humans and mice. We find that exposure to a young systemic environment partially restores ATE distribution in aged parabionts and reduces adipose tissue inflammation. Approaches to restore ATE distribution using adoptive transfer of eosinophils from young mice into aged recipients proved sufficient to dampen age-related local and systemic low-grade inflammation. Importantly, restoration of a youthful systemic milieu by means of eosinophil transfers resulted in systemic rejuvenation of the aged host, manifesting in improved physical and immune fitness that was partially mediated by eosinophil-derived IL-4. Together, these findings support a critical function of adipose tissue as a source of pro-ageing factors and uncover a new role of eosinophils in promoting healthy ageing by sustaining adipose tissue homeostasis.

Leaving the Lights on Using Gamma Entrainment to Protect against Neurodegeneration.

The brain generates natural oscillations from coordinated neuronal activity. Recent work exploring gamma oscillation entrainment raised the possibility that the phenomenon can be exploited to preserve neural function. In this issue of Neuron, Adaikkan et al. (2019) now show that chronic gamma entrainment using visual stimuli protects against several neurodegenerative phenotypes.

Blood-Based Therapies to Combat Aging.

Dysfunction associated with the aging process positions aging as a leading culprit for development of devastating diseases and mounting health-care costs. Many age-associated conditions for which aging increases risk are neurological disorders with no effective treatments, including Alzhei-mer's disease. As the proportion of aged individuals continues to rise in the coming decades, aging-related costs are expected to increase dramatically. Diverse approaches have emerged to meet the clinical need to treat aging and its associated conditions, including those aimed at increasing longevity, slowing the aging process itself, and improving healthspan. An emerging approach takes advantage of molecules circulating in the blood to limit or reverse aspects of aging in various organs throughout the body. Efforts are underway to translate these findings into novel therapeutics that harness the activity of youth-associated molecules present within blood. Here, we discuss the current state of blood-based approaches in this arena. Despite the apparent ease with which blood products might conceivably be applied as treatment paradigms, we propose that challenges nonetheless exist, which may be overcome with mechanistic studies that identify common pathways for targeted therapeutics.

Human umbilical cord plasma proteins revitalize hippocampal function in aged mice.

Ageing drives changes in neuronal and cognitive function, the decline of which is a major feature of many neurological disorders. The hippocampus, a brain region subserving roles of spatial and episodic memory and learning, is sensitive to the detrimental effects of ageing at morphological and molecular levels. With advancing age, synapses in various hippocampal subfields exhibit impaired long-term potentiation, an electrophysiological correlate of learning and memory. At the molecular level, immediate early genes are among the synaptic plasticity genes that are both induced by long-term potentiation and downregulated in the aged brain. In addition to revitalizing other aged tissues, exposure to factors in young blood counteracts age-related changes in these central nervous system parameters, although the identities of specific cognition-promoting factors or whether such activity exists in human plasma remains unknown. We hypothesized that plasma of an early developmental stage, namely umbilical cord plasma, provides a reservoir of such plasticity-promoting proteins. Here we show that human cord plasma treatment revitalizes the hippocampus and improves cognitive function in aged mice. Tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor enriched in human cord plasma, young mouse plasma, and young mouse hippocampi, appears in the brain after systemic administration and increases synaptic plasticity and hippocampal-dependent cognition in aged mice. Depletion experiments in aged mice revealed TIMP2 to be necessary for the cognitive benefits conferred by cord plasma. We find that systemic pools of TIMP2 are necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, arguing for previously unknown roles for TIMP2 in normal hippocampal function. Our findings reveal that human cord plasma contains plasticity-enhancing proteins of high translational value for targeting ageing- or disease-associated hippocampal dysfunction.

Microglial complement receptor 3 regulates brain Aß levels through secreted proteolytic activity.

Recent genetic evidence supports a link between microglia and the complement system in Alzheimer's disease (AD). In this study, we uncovered a novel role for the microglial complement receptor 3 (CR3) in the regulation of soluble ß-amyloid (Aß) clearance independent of phagocytosis. Unexpectedly, ablation of CR3 in human amyloid precursor protein-transgenic mice results in decreased, rather than increased, Aß accumulation. In line with these findings, cultured microglia lacking CR3 are more efficient than wild-type cells at degrading extracellular Aß by secreting enzymatic factors, including tissue plasminogen activator. Furthermore, a small molecule modulator of CR3 reduces soluble Aß levels and Aß half-life in brain interstitial fluid (ISF), as measured by in vivo microdialysis. These results suggest that CR3 limits Aß clearance from the ISF, illustrating a novel role for CR3 and microglia in brain Aß metabolism and defining a potential new therapeutic target in AD.

In vivo assessment of behavioral recovery and circulatory exchange in the peritoneal parabiosis model.

The sharing of circulation between two animals using a surgical procedure known as parabiosis has created a wealth of information towards our understanding of physiology, most recently in the neuroscience arena. The systemic milieu is a complex reservoir of tissues, immune cells, and circulating molecules that is surprisingly not well understood in terms of its communication across organ systems. While the model has been used to probe complex physiological questions for many years, critical parameters of recovery and exchange kinetics remain incompletely characterized, limiting the ability to design experiments and interpret results for complex questions. Here we provide evidence that mice joined by parabiosis gradually recover much physiology relevant to the study of brain function. Specifically, we describe the timecourse for a variety of recovery parameters, including those for general health and metabolism, motor coordination, activity, and sleep behavior. Finally, we describe the kinetics of chimerism for several lymphocyte populations as well as the uptake of small molecules into the brains of mice following parabiosis. Our characterization provides an important resource to those attempting to understand the complex interplay between the immune system and the brain as well as other organ systems.

Traumatic Brain Injury Imaging in the Second Near-Infrared Window with a Molecular Fluorophore.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. A bright, renal-excreted, and biocompatible near-infrared II fluorophore for in vivo imaging of TBI is designed. A transient hypoperfusion in the injured cerebral region, followed by fluorophore leakage, is observed. NIR-II fluorophores can provide noninvasive assessment of TBI.

Blood-Borne Revitalization of the Aged Brain.

In the modern medical era, more diverse and effective treatment options have translated to increased life expectancy. With this increased life span comes increased age-associated disease and the dire need to understand underlying causes so that therapies can be designed to mitigate the burden to health and the economy. Aging exacts a seemingly inevitable multisystem deterioration of function that acts as a risk factor for a variety of age-related disorders, including those that devastate organs of limited regenerative potential, such as the brain. Rather than studying the brain and mechanisms that govern its aging in isolation from other organ systems, an emerging approach is to understand the relatively unappreciated communication that exists between the brain and systemic environment. Revisiting classical methods of experimental physiology in animal models has uncovered surprising regenerative activity in young blood with translational implications for the aging liver, muscle, brain, and other organs. Soluble factors present in young or aged blood are sufficient to improve or impair cognitive function, respectively, suggesting an aging continuum of brain-relevant systemic factors. The age-associated plasma chemokine CCL11 has been shown to impair young brain function while GDF11 has been reported to increase the generation of neurons in aged mice. However, the identities of specific factors mediating memory-enhancing effects of young blood and their mechanisms of action are enigmatic. Here we review brain rejuvenation studies in the broader context of systemic rejuvenation research. We discuss putative mechanisms for blood-borne brain rejuvenation and suggest promising avenues for future research and development of therapies.

CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum.

Fluorescent bioorthogonal smart probes across the visible spectrum will enable sensitive visualization of metabolically labeled molecules in biological systems. Here we present a unified design, based on the principle of photoinduced electron transfer, to access a panel of highly fluorogenic azide probes that are activated by conversion to the corresponding triazoles via click chemistry. Termed the CalFluors, these probes possess emission maxima that range from green to far red wavelengths, and enable sensitive biomolecule detection under no-wash conditions. We used the CalFluor probes to image various alkyne-labeled biomolecules (glycans, DNA, RNA, and proteins) in cells, developing zebrafish, and mouse brain tissue slices.

Effects of the absence of apolipoprotein e on lipoproteins, neurocognitive function, and retinal function.

IMPORTANCE: The identification of a patient with a rare form of severe dysbetalipoproteinemia allowed the study of the consequences of total absence of apolipoprotein E (apoE). OBJECTIVES: To discover the molecular basis of this rare disorder and to determine the effects of complete absence of apoE on neurocognitive and visual function and on lipoprotein metabolism. DESIGN, SETTING, AND PARTICIPANTS: Whole-exome sequencing was performed on the patient's DNA. He underwent detailed neurological and visual function testing and lipoprotein analysis. Lipoprotein analysis was also performed in the Cardiovascular Research Institute, University of California, San Francisco, on blood samples from the proband's mother, wife, 2 daughters, and normolipidemic control participants. MAIN OUTCOME MEASURES: Whole-exome sequencing, lipoprotein analysis, and neurocognitive function. RESULTS: The patient was homozygous for an ablative APOE frameshift mutation (c.291del, p.E97fs). No other mutations likely to contribute to the phenotype were discovered, with the possible exception of two, in ABCC2 (p.I670T) and LIPC (p.G137R). Despite complete absence of apoE, he had normal vision, exhibited normal cognitive, neurological, and retinal function, had normal findings on brain magnetic resonance imaging, and had normal cerebrospinal fluid levels of ß-amyloid and tau proteins. He had no significant symptoms of cardiovascular disease except a suggestion of myocardial ischemia on treadmill testing and mild atherosclerosis noted on carotid ultrasonography. He had exceptionally high cholesterol content (760 mg/dL; to convert to millimoles per liter, multiply by 0.0259) and a high cholesterol to triglycerides ratio (1.52) in very low-density lipoproteins with elevated levels of small-diameter high-density lipoproteins, including high levels of prebeta-1 high-density lipoprotein. Intermediate-density lipoproteins, low-density lipoproteins, and very low-density lipoproteins contained elevated apoA-I and apoA-IV levels. The patient's apoC-III and apoC-IV levels were decreased in very low-density lipoproteins. Electron microscopy revealed large lamellar particles having electron-opaque cores attached to electron-lucent zones in intermediate-density and low-density lipoproteins. Low-density lipoprotein particle diameters were distributed bimodally. CONCLUSIONS AND RELEVANCE: Despite a profound effect on lipoprotein metabolism, detailed neurocognitive and retinal studies failed to demonstrate any defects. This suggests that functions of apoE in the brain and eye are not essential or that redundant mechanisms exist whereby its role can be fulfilled. Targeted knockdown of apoE in the central nervous system might be a therapeutic modality in neurodegenerative disorders.

Aging. Aging-induced type I interferon response at the choroid plexus negatively affects brain function.

Aging-associated cognitive decline is affected by factors produced inside and outside the brain. By using multiorgan genome-wide analysis of aged mice, we found that the choroid plexus, an interface between the brain and the circulation, shows a type I interferon (IFN-I)-dependent gene expression profile that was also found in aged human brains. In aged mice, this response was induced by brain-derived signals, present in the cerebrospinal fluid. Blocking IFN-I signaling within the aged brain partially restored cognitive function and hippocampal neurogenesis and reestablished IFN-II-dependent choroid plexus activity, which is lost in aging. Our data identify a chronic aging-induced IFN-I signature, often associated with antiviral response, at the brain's choroid plexus and demonstrate its negative influence on brain function, thereby suggesting a target for ameliorating cognitive decline in aging.

Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice.

As human lifespan increases, a greater fraction of the population is suffering from age-related cognitive impairments, making it important to elucidate a means to combat the effects of aging. Here we report that exposure of an aged animal to young blood can counteract and reverse pre-existing effects of brain aging at the molecular, structural, functional and cognitive level. Genome-wide microarray analysis of heterochronic parabionts--in which circulatory systems of young and aged animals are connected--identified synaptic plasticity-related transcriptional changes in the hippocampus of aged mice. Dendritic spine density of mature neurons increased and synaptic plasticity improved in the hippocampus of aged heterochronic parabionts. At the cognitive level, systemic administration of young blood plasma into aged mice improved age-related cognitive impairments in both contextual fear conditioning and spatial learning and memory. Structural and cognitive enhancements elicited by exposure to young blood are mediated, in part, by activation of the cyclic AMP response element binding protein (Creb) in the aged hippocampus. Our data indicate that exposure of aged mice to young blood late in life is capable of rejuvenating synaptic plasticity and improving cognitive function.

Long-term cognitive impairments and pathological alterations in a mouse model of repetitive mild traumatic brain injury.

Mild traumatic brain injury (mTBI, also referred to as concussion) accounts for the majority of all traumatic brain injuries. The consequences of repetitive mTBI have become of particular concern for individuals engaged in certain sports or in military operations. Many mTBI patients suffer long-lasting neurobehavioral impairments. In order to expedite pre-clinical research and therapy development, there is a need for animal models that reflect the long-term cognitive and pathological features seen in patients. In the present study, we developed and characterized a mouse model of repetitive mTBI, induced onto the closed head over the left frontal hemisphere with an electromagnetic stereotaxic impact device. Using GFAP-luciferase bioluminescence reporter mice that provide a readout of astrocyte activation, we observed an increase in bioluminescence relative to the force delivered by the impactor after single impact and cumulative effects of repetitive mTBI. Using the injury parameters established in the reporter mice, we induced a repetitive mTBI in wild-type C57BL/6J mice and characterized the long-term outcome. Animals received repetitive mTBI showed a significant impairment in spatial learning and memory when tested at 2 and 6 months after injury. A robust astrogliosis and increased p-Tau immunoreactivity were observed upon post-mortem pathological examinations. These findings are consistent with the deficits and pathology associated with mTBI in humans and support the use of this model to evaluate potential therapeutic approaches.

ApoE influences amyloid-ß (Aß) clearance despite minimal apoE/Aß association in physiological conditions.

Apolipoprotein E gene (APOE) alleles may shift the onset of Alzheimer's disease (AD) through apoE protein isoforms changing the probability of amyloid-ß (Aß) accumulation. It has been proposed that differential physical interactions of apoE isoforms with soluble Aß (sAß) in brain fluids influence the metabolism of Aß, providing a mechanism to account for how APOE influences AD risk. In contrast, we provide clear evidence that apoE and sAß interactions occur minimally in solution and in the cerebrospinal fluid of human subjects, producing apoE3 and apoE4 isoforms as assessed by multiple biochemical and analytical techniques. Despite minimal extracellular interactions with sAß in fluid, we find that apoE isoforms regulate the metabolism of sAß by astrocytes and in the interstitial fluid of mice that received apoE infusions during brain Aß microdialysis. We find that a significant portion of apoE and sAß compete for the low-density lipoprotein receptor-related protein 1 (LRP1)-dependent cellular uptake pathway in astrocytes, providing a mechanism to account for apoE's regulation of sAß metabolism despite minimal evidence of direct interactions in extracellular fluids. We propose that apoE influences sAß metabolism not through direct binding to sAß in solution but through its actions with other interacting receptors/transporters and cell surfaces. These results provide an alternative frame work for the mechanistic explanations on how apoE isoforms influence the risk of AD pathogenesis.

In vivo measurement of apolipoprotein E from the brain interstitial fluid using microdialysis.

BACKGROUND: The APOE4 allele variant is the strongest known genetic risk factor for developing late-onset Alzheimer's disease. The link between apolipoprotein E (apoE) and Alzheimer's disease is likely due in large part to the impact of apoE on the metabolism of amyloid ß (Aß) within the brain. Manipulation of apoE levels and lipidation within the brain has been proposed as a therapeutic target for the treatment of Alzheimer's disease. However, we know little about the dynamic regulation of apoE levels and lipidation within the central nervous system. We have developed an assay to measure apoE levels in the brain interstitial fluid of awake and freely moving mice using large molecular weight cut-off microdialysis probes. RESULTS: We were able to recover apoE using microdialysis from human cerebrospinal fluid (CSF) in vitro and mouse brain parenchyma in vivo. Microdialysis probes were inserted into the hippocampus of wild-type mice and interstitial fluid was collected for 36 hours. Levels of apoE within the microdialysis samples were determined by ELISA. The levels of apoE were found to be relatively stable over 36 hours. No apoE was detected in microdialysis samples from apoE KO mice. Administration of the RXR agonist bexarotene increased ISF apoE levels while ISF Aß levels were decreased. Extrapolation to zero-flow analysis allowed us to determine the absolute recoverable concentration of apoE3 in the brain ISF of apoE3 KI mice. Furthermore, analysis of microdialysis samples by non-denaturing gel electrophoresis determined lipidated apoE particles in microdialysis samples were consistent in size with apoE particles from CSF. Finally, we found that the concentration of apoE in the brain ISF was dependent upon apoE isoform in human apoE KI mice, following the pattern apoE2>apoE3>apoE4. CONCLUSIONS: We are able to collect lipidated apoE from the brain of awake and freely moving mice and monitor apoE levels over the course of several hours from a single mouse. Our technique enables assessment of brain apoE dynamics under physiological and pathophysiological conditions and in response to therapeutic interventions designed to affect apoE levels and lipidation within the brain.