Suppression of NK Cell Activation by JAK3 Inhibition: Implication in the Treatment of Autoimmune Diseases.

Natural killer (NK) cell is an essential cytotoxic lymphocyte in our innate immunity. Activation of NK cells is of paramount importance in defending against pathogens, suppressing autoantibody production and regulating other immune cells. Common gamma chain (γc) cytokines, including IL-2, IL-15, and IL-21, are defined as essential regulators for NK cell homeostasis and development. However, it is inconclusive whether γc cytokine-driven NK cell activation plays a protective or pathogenic role in the development of autoimmunity. In this study, we investigate and correlate the differential effects of γc cytokines in NK cell expansion and activation. IL-2 and IL-15 are mainly responsible for NK cell activation, while IL-21 preferentially stimulates NK cell proliferation. Blockade of Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway by either JAK inhibitors or antibodies targeting γc receptor subunits reverses the γc cytokine-induced NK cell activation, leading to suppression of its autoimmunity-like phenotype in vitro. These results underline the mechanisms of how γc cytokines trigger autoimmune phenotype in NK cells as a potential target to autoimmune diseases.

Interleukins 4 and 21 Protect Anti-IgM Induced Cell Death in Ramos B Cells: Implication for Autoimmune Diseases.

Interleukins 4 (IL-4) and 21 (IL-21) belong to the common gamma chain cytokine family which are highly involved in the progression of autoimmune diseases. While IL-4 is well known to be involved in the suppression of apoptosis of autoreactive B cells, the role played by IL-21 remains unclear. In the current study, we activated the human Burkitt's lymphoma Ramos B cells with anti-IgM to mimic B cell hyperactivation observed in patients of autoimmune diseases. Consistent with other reported findings, anti-IgM led to the downregulation of proteins involved in B cell survival and proliferation, as well as the activation of caspase 3 activity and DNA damage, resulting in apoptotic cell death after 48-hour treatment. Although both IL-4 and IL-21 reversed anti-IgM-induced apoptosis and cell cycle arrest, they did so via different mechanisms: while IL-4 could directly suppress anti-IgM-induced caspase 3 activation and marker indicative of DNA damage, IL-21 could induce B cell proliferation in the presence of anti-IgM. Importantly, IL-21 also suppressed activation induced cell death in human primary B cells. Pre-treatment with clinically validated JAK inhibitors completely reversed the effects of IL-4 and IL-21 to rescue anti-IgM induced cell death and DNA damage. The results indicate the underlying mechanisms of how IL-4 and IL-21 differentially promote survival of hyperactivated B cells and provide hints to treat autoimmune diseases.

Microglial-glucocorticoid receptor depletion alters the response of hippocampal microglia and neurons in a chronic unpredictable mild stress paradigm in female mice.

Chronic psychological stress is one of the most important triggers and environmental risk factors for neuropsychiatric disorders. Chronic stress can influence all organs via the secretion of stress hormones, including glucocorticoids by the adrenal glands, which coordinate the stress response across the body. In the brain, glucocorticoid receptors (GR) are expressed by various cell types including microglia, which are its resident immune cells regulating stress-induced inflammatory processes. To study the roles of microglial GR under normal homeostatic conditions and following chronic stress, we generated a mouse model in which the GR gene is depleted in microglia specifically at adulthood to prevent developmental confounds. We first confirmed that microglia were depleted in GR in our model in males and females among the cingulate cortex and the hippocampus, both stress-sensitive brain regions. Then, cohorts of microglial-GR depleted and wild-type (WT) adult female mice were housed for 3 weeks in a standard or stressful condition, using a chronic unpredictable mild stress (CUMS) paradigm. CUMS induced stress-related behavior in both microglial-GR depleted and WT animals as demonstrated by a decrease of both saccharine preference and progressive ratio breakpoint. Nevertheless, the hippocampal microglial and neural mechanisms underlying the adaptation to stress occurred differently between the two genotypes. Upon CUMS exposure, microglial morphology was altered in the WT controls, without any apparent effect in microglial-GR depleted mice. Furthermore, in the standard environment condition, GR depleted-microglia showed increased expression of pro-inflammatory genes, and genes involved in microglial homeostatic functions (such as Trem2, Cx3cr1 and Mertk). On the contrary, in CUMS condition, GR depleted-microglia showed reduced expression levels of pro-inflammatory genes and increased neuroprotective as well as anti-inflammatory genes compared to WT-microglia. Moreover, in microglial-GR depleted mice, but not in WT mice, CUMS led to a significant reduction of CA1 long-term potentiation and paired-pulse ratio. Lastly, differences in adult hippocampal neurogenesis were observed between the genotypes during normal homeostatic conditions, with microglial-GR deficiency increasing the formation of newborn neurons in the dentate gyrus subgranular zone independently from stress exposure. Together, these findings indicate that, although the deletion of microglial GR did not prevent the animal's ability to respond to stress, it contributed to modulating hippocampal functions in both standard and stressful conditions, notably by shaping the microglial response to chronic stress.

Sex Differences of Microglia and Synapses in the Hippocampal Dentate Gyrus of Adult Mouse Offspring Exposed to Maternal Immune Activation.

Schizophrenia is a psychiatric disorder affecting ∼1% of humans worldwide. It is earlier and more frequently diagnosed in men than woman, and men display more pronounced negative symptoms together with greater gray matter reductions. Our previous findings utilizing a maternal immune activation (mIA) mouse model of schizophrenia revealed exacerbated anxiety-like behavior and sensorimotor gating deficits in adult male offspring that were associated with increased microglial reactivity and inflammation in the hippocampal dentate gyrus (DG). However, both male and female adult offspring displayed stereotypy and impairment of sociability. We hypothesized that mIA may lead to sex-specific alterations in microglial pruning activity, resulting in abnormal synaptic connectivity in the DG. Using the same mIA model, we show in the current study sex-specific differences in microglia and synapses within the DG of adult offspring. Specifically, microglial levels of cluster of differentiation (CD)68 and CD11b were increased in mIA-exposed females. Sex-specific differences in excitatory and inhibitory synapse densities were also observed following mIA. Additionally, inhibitory synaptic tone was increased in DG granule cells of both males and females, while changes in excitatory synaptic transmission occurred only in females with mIA. These findings suggest that phagocytic and complement pathways may together contribute to a sexual dimorphism in synaptic pruning and neuronal dysfunction in mIA, and may propose sex-specific therapeutic targets to prevent schizophrenia-like behaviors.

Alterations in Intrinsic and Synaptic Properties of Hippocampal CA1 VIP Interneurons During Aging.

Learning and memory deficits are hallmarks of the aging brain, with cortical neuronal circuits representing the main target in cognitive deterioration. While GABAergic inhibitory and disinhibitory circuits are critical in supporting cognitive processes, their roles in age-related cognitive decline remain largely unknown. Here, we examined the morphological and physiological properties of the hippocampal CA1 vasoactive intestinal peptide/calretinin-expressing (VIP+/CR+) type 3 interneuron-specific (I-S3) cells across mouse lifespan. Our data showed that while the number and morphological features of I-S3 cells remained unchanged, their firing and synaptic properties were significantly altered in old animals. In particular, the action potential duration and the level of steady-state depolarization were significantly increased in old animals in parallel with a significant decrease in the maximal firing frequency. Reducing the fast-delayed rectifier potassium or transient sodium conductances in I-S3 cell computational models could reproduce the age-related changes in I-S3 cell firing properties. However, experimental data revealed no difference in the activation properties of the Kv3.1 and A-type potassium currents, indicating that transient sodium together with other ion conductances may be responsible for the observed phenomena. Furthermore, I-S3 cells in aged mice received a stronger inhibitory drive due to concomitant increase in the amplitude and frequency of spontaneous inhibitory currents. These age-associated changes in the I-S3 cell properties occurred in parallel with an increased inhibition of their target interneurons and were associated with spatial memory deficits and increased anxiety. Taken together, these data indicate that VIP+/CR+ interneurons responsible for local circuit disinhibition survive during aging but exhibit significantly altered physiological properties, which may result in the increased inhibition of hippocampal interneurons and distorted mnemonic functions.

Microglia in the developing prefrontal cortex of rats show dynamic changes following neonatal disconnection of the ventral hippocampus.

Impaired ventral hippocampal (VH)-prefrontal cortex (PFC) connectivity is implicated in many cognitive and behavioral disorders. Excitotoxic neonatal VH (nVH) lesion in rat pups has been shown to induce synaptic pruning in the PFC as well as behavioral changes of relevance to developmental neuropsychiatric disorders. In the current study, we hypothesized that microglia, immune cells required for proper brain development and plasticity, may play a role in the development of abnormal behaviors in the nVH-lesioned animals. Ibotenic acid-induced nVH lesion was induced in postnatal day (P)7 male rats. Developmental changes in microglial density, morphology, ultrastructure and gene expression were analyzed in the PFC at P20 and P60. Our results revealed increased microglial reactivity and phagocytic activity in the lesioned rats at P20. Increased mRNA levels of C3 and C1q, complement molecules involved in synaptic pruning, were concomitantly observed. Diminished, but maintained, microglial reactivity and reduced antioxidative defenses were identified in lesioned rats at P60. Behavioral deficits were significantly reduced in the post-pubertal rats by suppressing microglial reactivity by a one-week minocycline treatment immediately after the lesion, These results suggest that early-life disconnection of the VH has long-lasting consequences for microglial functions in the connected structures. Alterations in microglia may underlie synaptic reorganization and behavioral deficits observed following neonatal VH disconnection.

Microglial Ultrastructure in the Hippocampus of a Lipopolysaccharide-Induced Sickness Mouse Model.

Sickness behavior is a set of behavioral changes induced by infections and mediated by pro-inflammatory cytokines. It is characterized by fatigue, decreased appetite and weight loss, changes in sleep patterns, cognitive functions, and lost interest in social activity. It can expedite recovery by conserving energy to mount an immune response involving innate immunity. To provide insights into microglial implication in sickness behavior with special focus on cognitive and social impairment, we investigated changes in their ultrastructure and interactions with synapses using a toxemia mouse model. Adult mice were injected with 1 mg/kg lipopolysaccharide (LPS) or saline, and assayed for signs of sickness behavior. LPS treated mice displayed reduced activity in open-field tests 24 h post-injection, while social avoidance and weight gain/loss were not significantly different between treatment groups. Microglia were investigated using electron microscopy to describe changes in their structure and function at nanoscale resolution. Microglial cell bodies and processes were investigated in the hippocampus CA1, a region responsible for learning and memory that is often impacted after peripheral LPS administration. Microglia in LPS treated animals displayed larger cell bodies as well as less complex processes at the time point examined. Strikingly, microglial processes in LPS injected animals were also more likely to contact excitatory synapses and contained more phagocytic material compared with saline injected controls. We have identified at the ultrastructural level significant changes in microglia-synapse interactions shortly after LPS administration, which draws attention to studying the roles of microglia in synaptic rewiring after inflammatory stimuli.

Ibuprofen prevents progression of ataxia telangiectasia symptoms in ATM-deficient mice.

BACKGROUND: Inflammation plays a critical role in accelerating the progression of neurodegenerative diseases, such as Alzheimer's disease (AD) and ataxia telangiectasia (A-T). In A-T mouse models, LPS-induced neuroinflammation advances the degenerative changes found in cerebellar Purkinje neurons both in vivo and in vitro. In the current study, we ask whether ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), can have the opposite effect and delay the symptoms of the disease. METHODS: We tested the beneficial effects of ibuprofen in both in vitro and in vivo models. Conditioned medium from LPS stimulated primary microglia (LM) applied to cultures of dissociated cortical neurons leads to numerous degenerative changes. Pretreatment of the neurons with ibuprofen, however, blocked this damage. Systemic injection of LPS into either adult wild-type or adult Atm-/- mice produced an immune challenge that triggered profound behavioral, biochemical, and histological effects. We used a 2-week ibuprofen pretreatment regimen to investigate whether these LPS effects could be blocked. We also treated young presymptomatic Atm-/- mice to determine if ibuprofen could delay the appearance of symptoms. RESULTS: Adding ibuprofen directly to neuronal cultures significantly reduced LM-induced degeneration. Curiously, adding ibuprofen to the microglia cultures before the LPS challenge had little effect, thus implying a direct effect of the NSAID on the neuronal cultures. In vivo administration of ibuprofen to Atm-/- animals before a systemic LPS immune challenge suppressed cytological damage. The ibuprofen effects were widespread as microglial activation, p38 phosphorylation, DNA damage, and neuronal cell cycle reentry were all reduced. Unfortunately, ibuprofen only slightly improved the LPS-induced behavioral deficits. Yet, while the behavioral symptoms could not be reversed once they were established in adult Atm-/- animals, administration of ibuprofen to young mutant pups prevented their symptoms from appearing. CONCLUSION: Inflammatory processes impact the normal progression of A-T implying that modulation of the immune system can have therapeutic benefit for both the behavioral and cellular symptoms of this neurodegenerative disease.

Nonfunctional mutant Wrn protein leads to neurological deficits, neuronal stress, microglial alteration, and immune imbalance in a mouse model of Werner syndrome.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter lifespan. Yet, little is known about the impact of WRN mutations on the central nervous system in both humans and mouse models of WS. In the current study, we have performed a longitudinal behavioral assessment on mice bearing a Wrn helicase deletion. Behavioral tests demonstrated a loss of motor activity and coordination, reduction in perception, increase in repetitive behavior, and deficits in both spatial and social novelty memories in Wrn mutant mice compared to age-matched wild type mice. These neurological deficits were associated with biochemical and histological changes in the brain of aged Wrn mutant mice. Microglia, resident immune cells that regulate neuronal plasticity and function in the brain, were hyper-ramified in multiple regions involved with the behavioral deficits of Wrn mutant mice. Furthermore, western analyses indicated that Wrn mutant mice exhibited an increase of oxidative stress markers in the prefrontal cortex. Supporting these findings, electron microscopy studies revealed increased cellular aging and oxidative stress features, among microglia and neurons respectively, in the prefrontal cortex of aged Wrn mutant mice. In addition, multiplex immunoassay of serum identified significant changes in the expression levels of several pro- and anti-inflammatory cytokines. Taken together, these findings indicate that microglial dysfunction and neuronal oxidative stress, associated with peripheral immune system alterations, might be important driving forces leading to abnormal neurological symptoms in WS thus suggesting potential therapeutic targets for interventions.

ProMoIJ: A new tool for automatic three-dimensional analysis of microglial process motility.

Microglia, the immune cells of the central nervous system, continuously survey the brain to detect alterations and maintain tissue homeostasis. The motility of microglial processes is indicative of their surveying capacity in normal and pathological conditions. The gold standard technique to study motility involves the use of two-photon microscopy to obtain time-lapse images from brain slices or the cortex of living animals. This technique generates four dimensionally-coded images which are analyzed manually using time-consuming, non-standardized protocols. Microglial process motility analysis is frequently performed using Z-stack projections with the consequent loss of three-dimensional (3D) information. To overcome these limitations, we developed ProMoIJ, a pack of ImageJ macros that perform automatic motility analysis of cellular processes in 3D. The main core of ProMoIJ is formed by two macros that assist the selection of processes, automatically reconstruct their 3D skeleton, and analyze their motility (process and tip velocity). Our results show that ProMoIJ presents several key advantages compared with conventional manual analysis: (1) reduces the time required for analysis, (2) is less sensitive to experimenter bias, and (3) is more robust to varying numbers of processes analyzed. In addition, we used ProMoIJ to demonstrate that commonly performed 2D analysis underestimates microglial process motility, to reveal that only cells adjacent to a laser injured area extend their processes toward the lesion site, and to demonstrate that systemic inflammation reduces microglial process motility. ProMoIJ is a novel, open-source, freely-available tool which standardizes and accelerates the time-consuming labor of 3D analysis of microglial process motility.

Microglia under psychosocial stressors along the aging trajectory: Consequences on neuronal circuits, behavior, and brain diseases.

Mounting evidence indicates the importance of microglia for proper brain development and function, as well as in complex stress-related neuropsychiatric disorders and cognitive decline along the aging trajectory. Considering that microglia are resident immune cells of the brain, a homeostatic maintenance of their effector functions that impact neuronal circuitry, such as phagocytosis and secretion of inflammatory factors, is critical to prevent the onset and progression of these pathological conditions. However, the molecular mechanisms by which microglial functions can be properly regulated under healthy and pathological conditions are still largely unknown. We aim to summarize recent progress regarding the effects of psychosocial stress and oxidative stress on microglial phenotypes, leading to neuroinflammation and impaired microglia-synapse interactions, notably through our own studies of inbred mouse strains, and most importantly, to discuss about promising therapeutic strategies that take advantage of microglial functions to tackle such brain disorders in the context of adult psychosocial stress or aging-induced oxidative stress.

Microglia across the lifespan: from origin to function in brain development, plasticity and cognition.

Microglia are the only immune cells that permanently reside in the central nervous system (CNS) alongside neurons and other types of glial cells. The past decade has witnessed a revolution in our understanding of their roles during normal physiological conditions. Cutting-edge techniques revealed that these resident immune cells are critical for proper brain development, actively maintain health in the mature brain, and rapidly adapt their function to physiological or pathophysiological needs. In this review, we highlight recent studies on microglial origin (from the embryonic yolk sac) and the factors regulating their differentiation and homeostasis upon brain invasion. Elegant experiments tracking microglia in the CNS allowed studies of their unique roles compared with other types of resident macrophages. Here we review the emerging roles of microglia in brain development, plasticity and cognition, and discuss the implications of the depletion or dysfunction of microglia for our understanding of disease pathogenesis. Immune activation, inflammation and various other conditions resulting in undesirable microglial activity at different stages of life could severely impair learning, memory and other essential cognitive functions. The diversity of microglial phenotypes across the lifespan, between compartments of the CNS, and sexes, as well as their crosstalk with the body and external environment, is also emphasised. Understanding what defines particular microglial phenotypes is of major importance for future development of innovative therapies controlling their effector functions, with consequences for cognition across chronic stress, ageing, neuropsychiatric and neurological diseases.

Correction: Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

[This corrects the article DOI: 10.1371/journal.pbio.1002466.].

Correction: Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

[This corrects the article DOI: 10.1371/journal.pbio.1002466.].

Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.

Non-Neuronal Cells Are Required to Mediate the Effects of Neuroinflammation: Results from a Neuron-Enriched Culture System.

Chronic inflammation is associated with activated microglia and reactive astrocytes and plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's. Both in vivo and in vitro studies have demonstrated that inflammatory cytokine responses to immune challenges contribute to neuronal death during neurodegeneration. In order to investigate the role of glial cells in this phenomenon, we developed a modified method to remove the non-neuronal cells in primary cultures of E16.5 mouse cortex. We modified previously reported methods as we found that a brief treatment with the thymidine analog, 5-fluorodeoxyuridine (FdU), is sufficient to substantially deplete dividing non-neuronal cells in primary cultures. Cell cycle and glial markers confirm the loss of ~99% of all microglia, astrocytes and oligodendrocyte precursor cells (OPCs). More importantly, under this milder treatment, the neurons suffered neither cell loss nor any morphological defects up to 2.5 weeks later; both pre- and post-synaptic markers were retained. Further, neurons in FdU-treated cultures remained responsive to excitotoxicity induced by glutamate application. The immunobiology of the FdU culture, however, was significantly changed. Compared with mixed culture, the protein levels of NFκB p65 and the gene expression of several cytokine receptors were altered. Individual cytokines or conditioned medium from ß-amyloid-stimulated THP-1 cells that were, potent neurotoxins in normal, mixed cultures, were virtually inactive in the absence of glial cells. The results highlight the importance of our glial-depleted culture system and identifies and offer unexpected insights into the complexity of -brain neuroinflammation.

Individual Cytokines Modulate the Neurological Symptoms of ATM Deficiency in a Region Specific Manner

Ataxia-telangiectasia (A-T) is a multisystemic neurodegenerative disease of childhood caused by the absence of functional ATM (A-T mutated) protein. The cerebellar cortex has the most obvious neuropathology, yet cells in other brain regions are also abnormal. A-T mouse models have been produced that replicate much, though not all, of the complex A-T phenotype. Nongenetic factors, including modulations of the immune status of the animal, have also recently been found to play a role in the disease phenotype. Here we report that these modulations show both cytokine and brain region specificity. The CNS changes induced by broad-spectrum immune challenges, such as lipopolysaccharide (LPS) injections are a complex mixture of neuroprotective (TNFα) and neurodegenerative (IL1ß) cytokine responses that change over time. For example, LPS first induces a protective response in A-T neurons through activation of tissue repair genes through infiltration of monocytes with M2 phenotype, followed over time by a set of more degenerative responses. Additional phenotypic complexity arises because the neuronal response to an immune challenge is regionally variable; cerebellum and cortex differ in important ways in their patterns of cellular and biochemical changes. Tracking these changes reveals an important though not exclusive role for the MAP kinase pathway. Our findings suggest brain responses to cytokine challenges are temporally and regionally specific and that both features are altered by the absence of ATM. This implies that management of the immune status of A-T patients might have significant clinical benefit.

Correction: The Interaction of the Atm Genotype with Inflammation and Oxidative Stress.

[This corrects the article DOI: 10.1371/journal.pone.0085863.].

The interaction of the atm genotype with inflammation and oxidative stress.

In ataxia-telangiectasia (A-T) the death of neurons is associated with the loss of neuronal cell cycle control. In most Atm(-/-) mouse models, however, these cell cycle anomalies are present but the phenotype of neuronal cell loss found in humans is not. Mouse Atm(-/-) neurons re-enter a cell cycle and replicate their DNA, but they do not die--even months after initiating the cycle. In the current study, we explore whether systemic inflammation or hypoxia-induced oxidative stress can serve as second stressors that can promote cell death in ATM-deficient neurons. We find that after either immune or hypoxic challenge, the levels of cell cycle proteins--PCNA, cyclin A and cyclin B--are significantly elevated in cerebellar Purkinje cells. Both the number of cells that express cell cycle proteins as well as the intensity of the expression levels in each cell is increased in the stressed animals. The cell cycle-positive neurons also increasingly express cell death markers such as activated caspase-3, γ-H2AX and TUNEL staining. Interestingly, nuclear HDAC4 localization is also enhanced in Atm(-/-) Purkinje neurons after the immune challenge suggesting that both genetic and epigenetic changes in Atm(-/-) mice respond to environmental challenges. Our findings support the hypothesis that multiple insults are needed to drive even genetically vulnerable neurons to die a cell cycle-related cell death and point to either inflammation or oxidative stressors as potential contributors to the A-T disease process.