Aging Microglia and Their Impact in the Nervous System.

Aging is the greatest risk factor for neurodegenerative diseases. Microglia are the resident immune cells in the central nervous system (CNS), playing key roles in its normal functioning, and as mediators for age-dependent changes of the CNS, condition at which they generate a hostile environment for neurons. Transforming Growth Factor ß1 (TGFß1) is a regulatory cytokine involved in immuneregulation and neuroprotection, affecting glial cell inflammatory activation, neuronal survival, and function. TGFß1 signaling undergoes age-dependent changes affecting the regulation of microglial cells and can contribute to the pathophysiology of neurodegenerative diseases. This chapter focuses on assessing the role of age-related changes on the regulation of microglial cells and their impact on neuroinflammation and neuronal function, for understanding age-dependent changes of the nervous system.

Microglial reactivity in brainstem chemosensory nuclei in response to hypercapnia.

Microglia, the resident immune cells of the CNS, surveil, detect, and respond to various extracellular signals. Depending on the nature of these signals, an integrative microglial response can be triggered, resulting in a phenotypic transformation. Here, we evaluate whether hypercapnia modifies microglia phenotype in brainstem respiratory-related nuclei. Adult C57BL/6 inbred mice were exposed to 10% CO2 enriched air (hypercapnia), or pure air (control), for 10 or 30 min and immediately processed for immunohistochemistry to detect the ubiquitous microglia marker, ionized calcium binding adaptor molecule 1 (Iba1). Hypercapnia for thirty, but not 10 min reduced the Iba1 labeling percent coverage in the ventral respiratory column (VRC), raphe nucleus (RN), and nucleus tractus solitarius (NTS) and the number of primary branches in VRC. The morphological changes persisted, at least, for 60 min breathing air after the hypercapnic challenge. No significant changes were observed in Iba1+ cells in the spinal trigeminal nucleus (Sp5) and the hippocampus. In CF-1 outbred mice, 10% CO2 followed by 60 min of breathing air, resulted in the reduction of Iba1 labeling percent coverage and the number and length of primary branches in VRC, RN, and NTS. No morphological change was observed in Iba1+ cells in Sp5 and hippocampus. Double immunofluorescence revealed that prolonged hypercapnia increased the expression of CD86, an inflammatory marker for reactive state microglia, in Iba1+ cells in VRC, RN, and NTS, but not in Sp5 and hippocampus in CF-1 mice. By contrast, the expression of CD206, a marker of regulatory state microglia, persisted unmodified. In brainstem, but not in hippocampal microglia cultures, hypercapnia increased the level of IL1ß, but not that of TGFß measured by ELISA. Our results show that microglia from respiratory-related chemosensory nuclei, are reactive to prolonged hypercapnia acquiring an inflammatory-like phenotype.

Age-dependent changes on fractalkine forms and their contribution to neurodegenerative diseases.

The chemokine fractalkine (FKN, CX3CL1), a member of the CX3C subfamily, contributes to neuron-glia interaction and the regulation of microglial cell activation. Fractalkine is expressed by neurons as a membrane-bound protein (mCX3CL1) that can be cleaved by extracellular proteases generating several sCX3CL1 forms. sCX3CL1, containing the chemokine domain, and mCX3CL1 have high affinity by their unique receptor (CX3CR1) which, physiologically, is only found in microglia, a resident immune cell of the CNS. The activation of CX3CR1contributes to survival and maturation of the neural network during development, glutamatergic synaptic transmission, synaptic plasticity, cognition, neuropathic pain, and inflammatory regulation in the adult brain. Indeed, the various CX3CL1 forms appear in some cases to serve an anti-inflammatory role of microglia, whereas in others, they have a pro-inflammatory role, aggravating neurological disorders. In the last decade, evidence points to the fact that sCX3CL1 and mCX3CL1 exhibit selective and differential effects on their targets. Thus, the balance in their level and activity will impact on neuron-microglia interaction. This review is focused on the description of factors determining the emergence of distinct fractalkine forms, their age-dependent changes, and how they contribute to neuroinflammation and neurodegenerative diseases. Changes in the balance among various fractalkine forms may be one of the mechanisms on which converge aging, chronic CNS inflammation, and neurodegeneration.

[Negative effects of the patients' rights law and neuro-rights bill in Chile]. / Efectos negativos en la investigación y el quehacer médico en Chile de la Ley 20.584 y la Ley de Neuroderechos en discusión: la urgente necesidad de aprender de nuestros errores.

Recently, the Chilean Senate approved the main ideas of a constitutional reform and a Neuro-rights bill. This bill aims to protect people from the potential abusive use of "neuro-technologies". Unfortunately, a literal interpretation of this law can produce severe negative effects both in the development of neuroscience research and medical practice in Chile, interfering with current treatments in countless patients suffering from neuropsychiatric diseases. This fear stems from the observation of the negative effects that recent Chilean legislations have produced, which share with the Neuro-Rights Law the attempt to protect vulnerable populations from potential abuse from certain medical interventions. In fact, Law 20,584 promulgated in 2012, instead of protecting the most vulnerable patients "incapacitated to consent", produced enormous, and even possibly irreversible, damage to research in Chile in pathologies that require urgent attention, such as many neuropsychiatric diseases. This article details the effects that Law 20.584 had on research in Chile, how it relates to the Neuro-Rights Law, and the potential negative effects that the latter could have on research and medical practice, if it is not formulated correcting its errors.

Efectos negativos en la investigación y el quehacer médico en Chile de la Ley 20.584 y la Ley de Neuroderechos en discusión: la urgente necesidad de aprender de nuestros errores / Negative effects of the patients' rights law and neuro-rights bill in Chile

Recently, the Chilean Senate approved the main ideas of a constitutional reform and a Neuro-rights bill. This bill aims to protect people from the potential abusive use of "neuro-technologies". Unfortunately, a literal interpretation of this law can produce severe negative effects both in the development of neuroscience research and medical practice in Chile, interfering with current treatments in countless patients suffering from neuropsychiatric diseases. This fear stems from the observation of the negative effects that recent Chilean legislations have produced, which share with the Neuro-Rights Law the attempt to protect vulnerable populations from potential abuse from certain medical interventions. In fact, Law 20,584 promulgated in 2012, instead of protecting the most vulnerable patients "incapacitated to consent", produced enormous, and even possibly irreversible, damage to research in Chile in pathologies that require urgent attention, such as many neuropsychiatric diseases. This article details the effects that Law 20.584 had on research in Chile, how it relates to the Neuro-Rights Law, and the potential negative effects that the latter could have on research and medical practice, if it is not formulated correcting its errors.

The effect of aged microglia on synaptic impairment and its relevance in neurodegenerative diseases.

Microglia serve key functions in the central nervous system (CNS), participating in the establishment and regulation of synapses and the neuronal network, and regulating activity-dependent plastic changes. As the neuroimmune system, they respond to endogenous and exogenous signals to protect the CNS. In aging, one of the main changes is the establishment of inflamm-aging, a mild chronic inflammation that reduces microglial response to stressors. Neuroinflammation depends mainly on the increased activation of microglia. Microglia over-activation may result in a reduced capacity for performing normal functions related to migration, clearance, and the adoption of an anti-inflammatory state, contributing to an increased susceptibility for neurodegeneration. Oxidative stress contributes both to aging and to the progression of neurodegenerative diseases. Increased production of reactive oxygen species (ROS) and neuroinflammation associated with age- and disease-dependent mechanisms affect synaptic activity and neurotransmission, leading to cognitive dysfunction. Astrocytes prevent microglial cell cytotoxicity by mechanisms mediated by transforming growth factor ß1 (TGFß1). However, TGFß1-Smad3 pathway is impaired in aging, and the age-related impairment of TGFß signaling can reduce protective activation while facilitating cytotoxic activation of microglia. A critical analysis on the effect of aging microglia on neuronal function is relevant for the understanding of age-related changes on neuronal function. Here, we present evidence in the context of the "microglial dysregulation hypothesis", which leads to the reduction of the protective functions and increased cytotoxicity of microglia, to discuss the mechanisms involved in neurodegenerative changes and Alzheimer's disease.

The Challenge by Multiple Environmental and Biological Factors Induce Inflammation in Aging: Their Role in the Promotion of Chronic Disease.

The aging process is driven by multiple mechanisms that lead to changes in energy production, oxidative stress, homeostatic dysregulation and eventually to loss of functionality and increased disease susceptibility. Most aged individuals develop chronic low-grade inflammation, which is an important risk factor for morbidity, physical and cognitive impairment, frailty, and death. At any age, chronic inflammatory diseases are major causes of morbimortality, affecting up to 5-8% of the population of industrialized countries. Several environmental factors can play an important role for modifying the inflammatory state. Genetics accounts for only a small fraction of chronic-inflammatory diseases, whereas environmental factors appear to participate, either with a causative or a promotional role in 50% to 75% of patients. Several of those changes depend on epigenetic changes that will further modify the individual response to additional stimuli. The interaction between inflammation and the environment offers important insights on aging and health. These conditions, often depending on the individual's sex, appear to lead to decreased longevity and physical and cognitive decline. In addition to biological factors, the environment is also involved in the generation of psychological and social context leading to stress. Poor psychological environments and other sources of stress also result in increased inflammation. However, the mechanisms underlying the role of environmental and psychosocial factors and nutrition on the regulation of inflammation, and how the response elicited for those factors interact among them, are poorly understood. Whereas certain deleterious environmental factors result in the generation of oxidative stress driven by an increased production of reactive oxygen and nitrogen species, endoplasmic reticulum stress, and inflammation, other factors, including nutrition (polyunsaturated fatty acids) and behavioral factors (exercise) confer protection against inflammation, oxidative and endoplasmic reticulum stress, and thus ameliorate their deleterious effect. Here, we discuss processes and mechanisms of inflammation associated with environmental factors and behavior, their links to sex and gender, and their overall impact on aging.

[Ethical guidelines for medical decision-making during COVID-19 pandemic in Chile]. / Orientaciones éticas para la toma de decisiones médicas en el contexto de la pandemia de COVID-19 en Chile.

The catastrophic emergency experienced by many countries with the COVID-19 pandemic emphasized the importance of bioethics for decision-making, both at the public health (equitable and effective policies) and at the clinical level. At the clinical level, the issues are the fulfillment of medical care demand with adequate health care teams, infrastructure, and supplies, and to cover critical care demands that surpass the available resources. Therefore, ethically correct approaches are required for the allocation of life sustaining resources. There are recommendations for the allocating life support during disasters based on multiple considerations, including ethical ones. However, the ethical criteria of existing guidelines are variable. Ethical principles usually considered are saving the greatest number of lives, saving the greatest number of years of life and the principle of the life cycle or the goal to give each individual equal opportunity to live through the various phases of life. However, the centrality of the human being and the search for the common good should be considered. Knowledge of public perspectives and moral benchmarks on these issues is essential. A successful assignment effort will require everyone's trust and cooperation. Decision making should be planned and discussed in advance, since in-depth deliberation will be extremely complex during the disaster. Our goal is to help the health care teams to wisely allocate resources in shortage periods.

Orientaciones éticas para la toma de decisiones médicas en el contexto de la pandemia de COVID-19 en Chile / Ethical guidelines for medical decision-making during COVID-19 pandemic in Chile

The catastrophic emergency experienced by many countries with the COVID-19 pandemic emphasized the importance of bioethics for decision-making, both at the public health (equitable and effective policies) and at the clinical level. At the clinical level, the issues are the fulfillment of medical care demand with adequate health care teams, infrastructure, and supplies, and to cover critical care demands that surpass the available resources. Therefore, ethically correct approaches are required for the allocation of life sustaining resources. There are recommendations for the allocating life support during disasters based on multiple considerations, including ethical ones. However, the ethical criteria of existing guidelines are variable. Ethical principles usually considered are saving the greatest number of lives, saving the greatest number of years of life and the principle of the life cycle or the goal to give each individual equal opportunity to live through the various phases of life. However, the centrality of the human being and the search for the common good should be considered. Knowledge of public perspectives and moral benchmarks on these issues is essential. A successful assignment effort will require everyone's trust and cooperation. Decision making should be planned and discussed in advance, since in-depth deliberation will be extremely complex during the disaster. Our goal is to help the health care teams to wisely allocate resources in shortage periods.

Impact of Aging in Microglia-Mediated D-Serine Balance in the CNS.

A mild chronic inflammatory state, like that observed in aged individuals, affects microglial function, inducing a dysfunctional phenotype that potentiates neuroinflammation and cytotoxicity instead of neuroprotection in response to additional challenges. Given that inflammatory activation of microglia promotes increased release of D-serine, we postulate that age-dependent inflammatory brain environment leads to microglia-mediated changes on the D-serine-regulated glutamatergic transmission. Furthermore, D-serine dysregulation, in addition to affecting synaptogenesis and synaptic plasticity, appears also to potentiate NMDAR-dependent excitotoxicity, promoting neurodegeneration and cognitive impairment. D-serine dysregulation promoted by microglia could have a role in age-related cognitive impairment and in the induction and progression of neurodegenerative processes like Alzheimer's disease.

What Is Neural Plasticity?

"Neural plasticity" refers to the capacity of the nervous system to modify itself, functionally and structurally, in response to experience and injury. As the various chapters in this volume show, plasticity is a key component of neural development and normal functioning of the nervous system, as well as a response to the changing environment, aging, or pathological insult. This chapter discusses how plasticity is necessary not only for neural networks to acquire new functional properties, but also for them to remain robust and stable. The article also reviews the seminal proposals developed over the years that have driven experiments and strongly influenced concepts of neural plasticity.

Glial Cells and Integrity of the Nervous System.

Today, there is enormous progress in understanding the function of glial cells, including astroglia, oligodendroglia, Schwann cells, and microglia. Around 150 years ago, glia were viewed as a glue among neurons. During the course of the twentieth century, microglia were discovered and neuroscientists' views evolved toward considering glia only as auxiliary cells of neurons. However, over the last two to three decades, glial cells' importance has been reconsidered because of the evidence on their involvement in defining central nervous system architecture, brain metabolism, the survival of neurons, development and modulation of synaptic transmission, propagation of nerve impulses, and many other physiological functions. Furthermore, increasing evidence shows that glia are involved in the mechanisms of a broad spectrum of pathologies of the nervous system, including some psychiatric diseases, epilepsy, and neurodegenerative diseases to mention a few. It appears safe to say that no neurological disease can be understood without considering neuron-glia crosstalk. Thus, this book aims to show different roles played by glia in the healthy and diseased nervous system, highlighting some of their properties while considering that the various glial cell types are essential components not only for cell function and integration among neurons, but also for the emergence of important brain homeostasis.

Microglia Function in the Normal Brain.

The activation of microglia has been recognized for over a century by their morphological changes. Long slender microglia acquire a short sturdy ramified shape when activated. During the past 20 years, microglia have been accepted as an essential cellular component for understanding the pathogenic mechanism of many brain diseases, including neurodegenerative diseases. More recently, functional studies and imaging in mouse models indicate that microglia are active in the healthy central nervous system. It has become evident that microglia release several signal molecules that play key roles in the crosstalk among brain cells, i.e., astrocytes and oligodendrocytes with neurons, as well as with regulatory immune cells. Recent studies also reveal the heterogeneous nature of microglia diverse functions depending on development, previous exposure to stimulation events, brain region of residence, or pathological state. Subjects to approach by future research are still the unresolved questions regarding the conditions and mechanisms that render microglia protective, capable of preventing or reducing damage, or deleterious, capable of inducing or facilitating the progression of neuropathological diseases. This novel knowledge will certainly change our view on microglia as therapeutic target, shifting our goal from their general silencing to the generation of treatments able to change their activation pattern.

Age-Dependent Changes in the Activation and Regulation of Microglia.

As we age, a large number of physiological and molecular changes affect the normal functioning of cells, tissues, and the organism as a whole. One of the main changes is the establishment of a state of systemic inflammatory activation, which has been termed "inflamm-aging"; a mild chronic inflammation of the aging organism that reduces the ability to generate an efficient response against stressor stimuli. As any other system, the nervous system undergoes these aging-related changes; the neuroinflammatory state depends mainly on the dysregulated activation of microglia, the innate immune cells of the central nervous system (CNS) and the principal producers of reactive oxygen species. As the brain ages, microglia acquire a phenotype that is increasingly inflammatory and cytotoxic, generating a hostile environment for neurons. There is mounting evidence that this process facilitates development of neurodegenerative diseases, for which the greatest risk factor is age. In this chapter, we will review key aging-associated changes occurring in the central nervous system, focusing primarily on the changes that occur in aging microglia, the inflammatory and oxidative stressful environment they establish, and their impaired regulation. In addition, we will discuss the effects of aged microglia on neuronal function and their participation in the development of neurodegenerative pathologies such as Parkinson's and Alzheimer's diseases.

Reduction of Blood Amyloid-ß Oligomers in Alzheimer's Disease Transgenic Mice by c-Abl Kinase Inhibition.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of amyloid plaques, which are deposits of misfolded and aggregated amyloid-beta peptide (Aß). The role of the c-Abl tyrosine kinase in Aß-mediated neurodegeneration has been previously reported. Here, we investigated the therapeutic potential of inhibiting c-Abl using imatinib. We developed a novel method, based on a technique used to detect prions (PMCA), to measure minute amounts of misfolded-Aß in the blood of AD transgenic mice. We found that imatinib reduces Aß-oligomers in plasma, which correlates with a reduction of AD brain features such as plaques and oligomers accumulation, neuroinflammation, and cognitive deficits. Cells exposed to imatinib and c-Abl KO mice display decreased levels of ß-CTF fragments, suggesting that an altered processing of the amyloid-beta protein precursor is the most probable mechanism behind imatinib effects. Our findings support the role of c-Abl in Aß accumulation and AD, and propose AD-PMCA as a new tool to evaluate AD progression and screening for drug candidates.

Expression Pattern of Scavenger Receptors and Amyloid-ß Phagocytosis of Astrocytes and Microglia in Culture are Modified by Acidosis: Implications for Alzheimer's Disease.

The pathological hallmarks of Alzheimer's disease (AD) are amyloid-ß (Aß) plaques, neurofibrillary tangles, and glia activation. The pathology also includes vascular amyloidosis and cerebrovascular disease. Vascular compromise can result in hypoperfusion, local tissue hypoxia, and acidosis. Activated microglia and astrocytes can phagocytose Aß through membrane receptors that include scavenger receptors. Changes in glial cells induced by extracellular acidosis could play a role in the development of AD. Here, we assess whether extracellular acidosis changes glial cell properties relevant for Aß clearance capacity. Incubation of glial cells on acidified culture medium (pH 6.9 or 6.5) for 24-48 h resulted in decreased cell diameter, with thinner branches in astrocytes, slight reduction in cell body size in microglia, a transient decrease in astrocyte adhesion to substrates, and a persistent decrease in microglia adhesion compared with control media (pH 7.4). Astrocyte Aß phagocytosis decreased at pH 6.9 and 6.5, whereas microglia phagocytosis only transiently decreased in acidified media. Scavenger receptors class B member I (SR-BI) increased and scavenger receptors-macrophage receptors with collagenous structures (SR-MARCO) decreased in astrocytes cultured at pH 6.5. In contrast, in microglia exposed to pH 6.5, expression of SR-BI and SR-MARCO increased and fatty acid translocase (CD-36) decreased. In conclusion, the acidic environment changed the adhesiveness and morphology of both microglia and astrocytes, but only astrocytes showed a persistent decrease in Aß clearance activity. Expression of scavenger receptors was affected differentially in microglia and astrocytes by acidosis. These changes in scavenger receptor patterns can affect the activation of glia and their contribution to neurodegeneration.

Role of TGFß signaling in the pathogenesis of Alzheimer's disease.

Aging is the main risk factor for Alzheimer's disease (AD); being associated with conspicuous changes on microglia activation. Aged microglia exhibit an increased expression of cytokines, exacerbated reactivity to various stimuli, oxidative stress, and reduced phagocytosis of ß-amyloid (Aß). Whereas normal inflammation is protective, it becomes dysregulated in the presence of a persistent stimulus, or in the context of an inflammatory environment, as observed in aging. Thus, neuroinflammation can be a self-perpetuating deleterious response, becoming a source of additional injury to host cells in neurodegenerative diseases. In aged individuals, although transforming growth factor ß (TGFß) is upregulated, its canonical Smad3 signaling is greatly reduced and neuroinflammation persists. This age-related Smad3 impairment reduces protective activation while facilitating cytotoxic activation of microglia through several cellular mechanisms, potentiating microglia-mediated neurodegeneration. Here, we critically discuss the role of TGFß-Smad signaling on the cytotoxic activation of microglia and its relevance in the pathogenesis of AD. Other protective functions, such as phagocytosis, although observed in aged animals, are not further induced by inflammatory stimuli and TGFß1. Analysis in silico revealed that increased expression of receptor scavenger receptor (SR)-A, involved in Aß uptake and cell activation, by microglia exposed to TGFß, through a Smad3-dependent mechanism could be mediated by transcriptional co-factors Smad2/3 over the MSR1 gene. We discuss that changes of TGFß-mediated regulation could at least partially mediate age-associated microglia changes, and, together with other changes on inflammatory response, could result in the reduction of protective activation and the potentiation of cytotoxicity of microglia, resulting in the promotion of neurodegenerative diseases.

Microglial cell dysregulation in brain aging and neurodegeneration.

Aging is the main risk factor for neurodegenerative diseases. In aging, microglia undergoes phenotypic changes compatible with their activation. Glial activation can lead to neuroinflammation, which is increasingly accepted as part of the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). We hypothesize that in aging, aberrant microglia activation leads to a deleterious environment and neurodegeneration. In aged mice, microglia exhibit an increased expression of cytokines and an exacerbated inflammatory response to pathological changes. Whereas LPS increases nitric oxide (NO) secretion in microglia from young mice, induction of reactive oxygen species (ROS) predominates in older mice. Furthermore, there is accumulation of DNA oxidative damage in mitochondria of microglia during aging, and also an increased intracellular ROS production. Increased ROS activates the redox-sensitive nuclear factor kappa B, which promotes more neuroinflammation, and can be translated in functional deficits, such as cognitive impairment. Mitochondria-derived ROS and cathepsin B, are also necessary for the microglial cell production of interleukin-1ß, a key inflammatory cytokine. Interestingly, whereas the regulatory cytokine TGFß1 is also increased in the aged brain, neuroinflammation persists. Assessing this apparent contradiction, we have reported that TGFß1 induction and activation of Smad3 signaling after inflammatory stimulation are reduced in adult mice. Other protective functions, such as phagocytosis, although observed in aged animals, become not inducible by inflammatory stimuli and TGFß1. Here, we discuss data suggesting that mitochondrial and endolysosomal dysfunction could at least partially mediate age-associated microglial cell changes, and, together with the impairment of the TGFß1-Smad3 pathway, could result in the reduction of protective activation and the facilitation of cytotoxic activation of microglia, resulting in the promotion of neurodegenerative diseases.

Prenatal exposure to inflammatory conditions increases Cx43 and Panx1 unopposed channel opening and activation of astrocytes in the offspring effect on neuronal survival.

Several epidemiological studies indicate that children born from mothers exposed to infections during gestation, have an increased risk to develop neurological disorders, including schizophrenia, autism and cerebral palsy. Given that it is unknown if astrocytes and their crosstalk with neurons participate in the above mentioned brain pathologies, the aim of this work was to address if astroglial paracrine signaling mediated by Cx43 and Panx1 unopposed channels could be affected in the offspring of LPS-exposed dams during pregnancy. Ethidium uptake experiments showed that prenatal LPS-exposure increases the activity of astroglial Cx43 and Panx1 unopposed channels in the offspring. Induction of unopposed channel opening by prenatal LPS exposure depended on intracellular Ca2+ levels, cytokine production and activation of p38 MAP kinase/iNOS pathway. Biochemical assays and Fura-2AM/DAF-FM time-lapse fluorescence images revealed that astrocytes from the offspring of LPS-exposed dams displayed increased spontaneous Ca2+ dynamics and NO production, whereas iNOS levels and release of IL-1ß/TNF-α were also increased. Interestingly, we found that prenatal LPS exposure enhanced the release of ATP through astroglial Cx43 and Panx1 unopposed channels in the offspring, resulting in an increased neuronal death mediated by the activation of neuronal P2X7 receptors and Panx1 channels. Altogether, this evidence suggests that astroglial Cx43 and Panx1 unopposed channel opening induced by prenatal LPS exposure depended on the inflammatory activation profile and the activation pattern of astrocytes. The understanding of the mechanism underlying astrocyte-neuron crosstalk could contribute to the development of new strategies to ameliorate the brain abnormalities induced in the offspring by prenatal inflammation. GLIA 2015;63:2058-2072.