Chronic Stress and Glucocorticoids: From Neuronal Plasticity to Neurodegeneration.

Stress and stress hormones, glucocorticoids (GCs), exert widespread actions in central nervous system, ranging from the regulation of gene transcription, cellular signaling, modulation of synaptic structure, and transmission and glial function to behavior. Their actions are mediated by glucocorticoid and mineralocorticoid receptors which are nuclear receptors/transcription factors. While GCs primarily act to maintain homeostasis by inducing physiological and behavioral adaptation, prolonged exposure to stress and elevated GC levels may result in neuro- and psychopathology. There is now ample evidence for cause-effect relationships between prolonged stress, elevated GC levels, and cognitive and mood disorders while the evidence for a link between chronic stress/GC and neurodegenerative disorders such as Alzheimer's (AD) and Parkinson's (PD) diseases is growing. This brief review considers some of the cellular mechanisms through which stress and GC may contribute to the pathogenesis of AD and PD.

Microglial glucocorticoid receptors play a pivotal role in regulating dopaminergic neurodegeneration in parkinsonism.

Among the pathogenic processes contributing to dopaminergic neuron (DN) death in Parkinson disease (PD), evidence points to non-cell-autonomous mechanisms, particularly chronic inflammation mounted by activated microglia. Yet little is known about endogenous regulatory processes that determine microglial actions in pathological states. We examined the role of glucocorticoid receptors (GRs), activated by glucocorticoids released in response to stress and known to regulate inflammation, in DN survival. Overall GR level was decreased in substantia nigra of PD patients and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. GR changes, specifically in the microglia after MPTP treatment, revealed a rapid augmentation in the number of microglia displaying nuclear localization of GR. Mice with selective inactivation of the GR gene in macrophages/microglia (GR(LysMCre)) but not in DNs (GR(DATCre)) showed increased loss of DNs after MPTP intoxication. This DN loss in GR(LysMCre) mice was not prevented by corticosterone treatment, in contrast to the protection observed in control littermates. Moreover, absence of microglial GRs augmented microglial reactivity and led to their persistent activation. Analysis of inflammatory genes revealed an up-regulation of Toll-like receptors (TLRs) by MPTP treatment, particularly TLR9, the level of which was high in postmortem parkinsonian brains. The regulatory control of GR was reflected by higher expression of proinflammatory genes (e.g., TNF-α) with a concomitant decrease in anti-inflammatory genes (e.g., IL-1R2) in GR(LysMCre) mice. Indeed, in GR(LysMCre) mice, alterations in phosphorylated NF-κB levels indicated its protracted activation. Together, our data indicate that GR is important in curtailing microglial reactivity, and its deregulation in PD could lead to sustained inflammation-mediated DN injury.

Adult neurogenic process in the subventricular zone-olfactory bulb system is regulated by Tau protein under prolonged stress.

OBJECTIVES: The area of the subventricular zone (SVZ) in the adult brain exhibits the highest number of proliferative cells, which, together with the olfactory bulb (OB), maintains constant brain plasticity through the generation, migration and integration of newly born neurons. Despite Tau and its malfunction is increasingly related to deficits of adult hippocampal neurogenesis and brain plasticity under pathological conditions [e.g. in Alzheimer's disease (AD)], it remains unknown whether Tau plays a role in the neurogenic process of the SVZ and OB system under conditions of chronic stress, a well-known sculptor of brain and risk factor for AD. MATERIALS AND METHODS: Different types of newly born cells in SVZ and OB were analysed in animals that lack Tau gene (Tau-KO) and their wild-type littermates (WT) under control or chronic stress conditions. RESULTS: We demonstrate that chronic stress reduced the number of proliferating cells and neuroblasts in the SVZ leading to decreased number of newborn neurons in the OB of adult WT, but not Tau-KO, mice. Interestingly, while stress-evoked changes were not detected in OB granular cell layer, Tau-KO exhibited increased number of mature neurons in this layer indicating altered neuronal migration due to Tau loss. CONCLUSIONS: Our findings suggest the critical involvement of Tau in the neurogenesis suppression of SVZ and OB neurogenic niche under stressful conditions highlighting the role of Tau protein as an essential regulator of stress-driven plasticity deficits.

Glucocorticoid receptor in astrocytes regulates midbrain dopamine neurodegeneration through connexin hemichannel activity.

The precise contribution of astrocytes in neuroinflammatory process occurring in Parkinson's disease (PD) is not well characterized. In this study, using GRCx30CreERT2 mice that are conditionally inactivated for glucocorticoid receptor (GR) in astrocytes, we have examined the actions of astrocytic GR during dopamine neuron (DN) degeneration triggered by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results show significantly augmented DN loss in GRCx30CreERT2 mutant mice in substantia nigra (SN) compared to controls. Hypertrophy of microglia but not of astrocytes was greatly enhanced in SN of these astrocytic GR mutants intoxicated with MPTP, indicating heightened microglial reactivity compared to similarly-treated control mice. In the SN of GR astrocyte mutants, specific inflammation-associated transcripts ICAM-1, TNF-α and Il-1ß as well as TNF-α protein levels were significantly elevated after MPTP neurotoxicity compared to controls. Interestingly, this paralleled increased connexin hemichannel activity and elevated intracellular calcium levels in astrocytes examined in acute midbrain slices from control and mutant mice treated with MPP+ . The increased connexin-43 hemichannel activity was found in vivo in MPTP-intoxicated mice. Importantly, treatment of MPTP-injected GRCx30CreERT2 mutant mice with TAT-Gap19 peptide, a specific connexin-43 hemichannel blocker, reverted both DN loss and microglial activation; in wild-type mice there was partial but significant survival effect. In the SN of post-mortem PD patients, a significant decrease in the number of astrocytes expressing nuclear GR was observed, suggesting the participation of astrocytic GR deregulation of inflammatory process in PD. Overall, these data provide mechanistic insights into GR-modulated processes in vivo, specifically in astrocytes, that contribute to a pro-inflammatory state and dopamine neurodegeneration in PD pathology.

Author Correction: TLR9 activation via microglial glucocorticoid receptors contributes to degeneration of midbrain dopamine neurons.

The originally published version of this Article contained an error in the subheading "Microglial GR does not affect DN loss triggered by TLR4 and TLR7," which was incorrectly given as "Microglial GR does affect DN loss triggered by TLR2 and TLR4". This has now been corrected in both the PDF and HTML versions of the Article.

TLR9 activation via microglial glucocorticoid receptors contributes to degeneration of midbrain dopamine neurons.

Inflammation is a characteristic feature of Parkinson's disease (PD). We examined the role of TLR9 and its regulation by glucocorticoid receptors (GRs) in degeneration of substantia nigra dopamine neurons (DNs). TLR9 agonist, CpG-ODN, induced DN degeneration in mice lacking GR in microglia but not in controls. TLR9 deletion reduced DN loss in neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. GR regulates TLR9 activation during MPTP neurotoxicity as TLR9 antagonist suppressed increased DN loss in microglia/macrophage GR mutant mice. GR absence in microglia enhanced TLR9 translocation to endolysosomes and facilitated its cleavage leading to pro-inflammatory gene expression. GR-dependent TLR9 activation also triggered DN loss following intranigral injection of mitochondrial DNA. Finally, microglial GR sensitivity to A53T-alpha-synuclein induced DN degeneration as well as decreased microglial GR expression observed in SN of PD brain samples, all suggest that reduced microglial GR activity in SN can stimulate TLR9 activation and DN loss in PD pathology.

Macrophage-derived tumor necrosis factor alpha, an early developmental signal for motoneuron death.

Mechanisms inducing neuronal death at defined times during embryogenesis remain enigmatic. We show in explants that a developmental switch occurs between embryonic day 12 (E12) and E13 in rats that is 72-48 hr before programmed cell death. Half the motoneurons isolated from peripheral tissues at E12 escape programmed cell death, whereas 90% of motoneurons isolated at E13 enter a death program. The surrounding somite commits E12 motoneurons to death. This effect requires macrophage cells, is mimicked by tumor necrosis factor alpha (TNFalpha), and is inhibited by anti-TNFalpha antibodies. In vivo, TNFalpha is detected within somite macrophages, and TNF receptor 1 (TNFR1) is detected within motoneurons precisely between E12 and E13. Although motoneuron cell death occurs normally in TNFalpha-/- mice, this process is significantly reduced in explants from TNFalpha-/- and TNFR1-/- mice. Thus, embryonic motoneurons acquire the competence to die, before the onset of programmed cell death, from extrinsic signals such as macrophage-derived TNFalpha

Motoneuron-derived neurotrophin-3 is a survival factor for PAX2-expressing spinal interneurons.

Rat spinal cord interneurons undergo programmed cell death shortly after birth. We investigated here whether cell death of interneurons could be regulated by trophic factors produced by motoneurons, one of their main targets. To test this hypothesis, we studied the effect of the selective destruction of motoneurons on the survival of interneurons in organotypic cultures of embryonic rat spinal cords. Motoneurons were eliminated by an anti-p75(NTR)-specific immunotoxin (192 IgG-saporin). We then observed a decrease of 28% in the number of ventral spinal interneurons immunoreactive (IR) for the homeoprotein PAX2. This was correlated with an increase in the number of apoptotic nuclei in the same area. Because neurotrophin-3 (NT-3) is specifically produced by motoneurons and because interneurons express the NT-3 high-affinity receptor trkC, we examined the role of NT-3 in the survival of PAX2-IR interneurons. Addition of NT-3 to 192 IgG-saporin-treated explants rescued ventral PAX2-IR interneurons. Depletion of secreted NT-3 by anti-NT-3 antibodies induced 66% loss of ventral PAX2-IR interneurons. We conclude that motoneuron-derived NT-3 is a trophic factor for ventral PAX2-IR interneurons.

Inflammation in Parkinson's disease: role of glucocorticoids.

Chronic inflammation is a major characteristic feature of Parkinson's disease (PD). Studies in PD patients show evidence of augmented levels of potent pro-inflammatory molecules e.g., TNF-α, iNOS, IL-1ß whereas in experimental Parkinsonism it has been consistently demonstrated that dopaminergic neurons are particularly vulnerable to activated glia releasing these toxic factors. Recent genetic studies point to the role of immune system in the etiology of PD, thus in combination with environmental factors, both peripheral and CNS-mediated immune responses could play important roles in onset and progression of PD. Whereas microglia, astrocytes and infiltrating T cells are known to mediate chronic inflammation, the roles of other immune-competent cells are less well understood. Inflammation is a tightly controlled process. One major effector system of regulation is HPA axis. Glucocorticoids (GCs) released from adrenal glands upon stimulation of HPA axis, in response to either cell injury or presence of pathogen, activate their receptor, GR. GR regulates inflammation both through direct transcriptional action on target genes and by indirectly inhibiting transcriptional activities of transcriptional factors such as NF-κB, AP-1 or interferon regulatory factors. In PD patients, the HPA axis is unbalanced and the cortisol levels are significantly increased, implying a deregulation of GR function in immune cells. In experimental Parkinsonism, the activation of microglial GR has a crucial effect in diminishing microglial cell activation and reducing dopaminergic degeneration. Moreover, GCs are also known to regulate human brain vasculature as well as blood brain barrier (BBB) permeability, any dysfunction in their actions may influence infiltration of cytotoxic molecules resulting in increased vulnerability of dopamine neurons in PD. Overall, deregulation of glucocorticoid receptor actions is likely important in dopamine neuron degeneration through establishment of chronic inflammation.

Contribution of glucocorticoids and glucocorticoid receptors to the regulation of neurodegenerative processes.

Isolation of glucocorticoids (GCs) from adrenal glands followed by synthesis led rapidly to their first clinical application, about 70 years ago, for treatment of rheumatoid arthritis. To this day GCs are used in diseases that have an inflammatory component. However, their use is carefully monitored because of harmful side effects. GCs are also synonymous with stress and adaptation. In CNS, GC binds and activates high affinity mineralocorticoid receptor (MR) and low affinity glucocorticoid receptor (GR). GR, whose expression is ubiquitous, is only activated when GC levels rise as during circadian peak and in response to stress. Numerous recent studies have yielded important and new insights on the mechanisms concerning pulsatile secretory pattern of GCs as well as various processes that tightly control their synthesis via hypothalamic-pituitary-adrenal (HPA) axis involving regulated release of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) from hypothalamus and pituitary, respectively. GR modulates neuronal functions and viability through both genomic and non-genomic actions, and importantly its transcriptional regulatory activity is tightly locked with GC secretory pattern. There is increasing evidence pointing to involvement of GC-GR in neurodegenerative disorders. Patients with Alzheimer's or Parkinson's or Huntington's disease show chronically high cortisol levels suggesting changes occurring in controls of HPA axis. In experimental models of these diseases, chronic stress or GC treatment was found to exacerbate both the clinical symptoms and neurodegenerative processes. However, recent evidence also shows that GC-GR can exert neuroprotective effects. Thus, for any potential therapeutic strategies in these neurodegenerative diseases we need to understand the precise modifications both in HPA axis and in GR activity and find ways to harness their protective actions.

Neuroinflammation in Parkinson's disease.

Both epidemiological and genetic studies support a role of neuroinflammation in the pathophysiology of Parkinson's disease (PD). Furthermore, post mortem studies confirm the involvement of innate as well as adaptive immunity in the affected brain regions in patients with PD. Indeed, activated microglial cells and T lymphocytes have been detected in the substantia nigra of patients concomitantly with an increased expression of pro-inflammatory mediators. Preclinical investigations conducted in various animal models of PD indicate that inflammatory processes are instrumental in neuronal cell death even though they are unlikely to be a primary cause for neuronal loss. Neuroinflammatory processes in PD are rather involved in self-perpetuating deleterious events that lead to protracted neuronal degeneration. In line with this, recent data indicate that glucocorticoid receptors are important in curtailing microglial reactivity, and deregulation in their activity in PD could lead to sustained inflammation-mediated degeneration. Altogether, neuroinflammatory processes might represent a target for neuroprotection in PD.

5-HT1A-iCre, a new transgenic mouse line for genetic analyses of the serotonergic pathway.

The 5-HT1A receptor not only plays an important role in brain physiology but it may be also implicated in the etiology of behavioral disorders such as pathological anxiety. To further define the role of 5-HT1A receptor-expressing neurons, we generated a transgenic mouse line expressing Cre recombinase in these cells. The 5-HT1A receptor open reading frame was substituted for that of Cre recombinase in a BAC containing the 5-HT1A receptor gene. In adult transgenic brain, Cre expression perfectly matched the distribution of 5-HT1A receptor mRNA. Additionally, Cre-mediated DNA recombination was restricted to neuronal populations that express the receptor, e.g., cerebral cortex, septum, hippocampus, dorsal raphe, thalamic, hypothalamic and amygdaloid nuclei, and spinal cord. Recombination occurred as early as E13 in trigeminal nerve, spinal ganglia and spinal cord. This transgenic line will allow the generation of conditional mutant mice that lack specific gene products along the serotonergic pathways and represents a unique tool for studying 5-HT1A-mediated serotonin signaling in the developing and adult brain.

Ontogeny of tyrosine hydroxylase mRNA expression in mid- and forebrain: neuromeric pattern and novel positive regions.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines and, thus, critical in determining the catecholaminergic phenotype. In this study, we have examined the expression of TH mRNA by in situ hybridization in the embryonic mouse forebrain and midbrain and have mapped its localization according to the neuromeric pattern. We find that early in embryonic development, 10 to 12 days post coitum (dpc), TH mRNA is expressed in ample continuous regions of the neuroepithelium, extending across several neuromeres. However, from 12.5 dpc onward, the expression becomes restricted to discrete regions, which correspond to the dopaminergic nuclei (A8 to A15). In addition to these nuclei previously described, TH mRNA is also observed in regions that do not express this enzyme according to immunohistochemical studies. This difference in relation to protein expression pattern is consequent with the known posttranscriptional regulation of TH expression. The most representative example of a novel positive region is the conspicuous mRNA expression in both medial and lateral ganglionic eminences. This result agrees with reports describing the capacity of striatal stem cells (that is, located at the lateral ganglionic eminence) to become dopaminergic in vitro. Other regions include the isthmic mantle layer and the early floor plate of the midbrain-caudal forebrain. On the whole, the expression map we have obtained opens new perspectives for evolutionary/comparative studies, as well as for therapeutic approaches looking for potentially dopaminergic cells. Developmental Dynamics 234:709-717, 2005. (c) 2005 Wiley-Liss, Inc.

Differentiation-dependent sensitivity to apoptogenic factors in PC12 cells.

We have investigated the role of the mitochondrial pathway during cell death following serum and nerve growth factor (NGF)/dibutyryl cyclic AMP (Bt(2)cAMP) withdrawal in undifferentiated or NGF/Bt(2)cAMP-differentiated PC12 cells, respectively. Holocytochrome c, Smac/DIABLO, and Omi/HtrA2 are released rapidly following trophic factor deprivation in PC12 cells. Bcl-2 and Akt inhibited this release. The protection, however, persisted longer in differentiated PC12 cells. In differentiated, but not undifferentiated cells, Bcl-2 and Akt also inhibited apoptosis downstream of holocytochrome c release. Thus, undifferentiated PC12 cells showed marked sensitivity to induction of apoptosis by microinjected cytochrome c even in the presence of NGF, Bcl-2, or Akt. In contrast, in differentiated cells these factors suppressed cell death. Consistent with these observations, in vitro processing of procaspase 9 in response to cytochrome c was observed in extracts from undifferentiated but not differentiated cells expressing Akt or Bcl-2. Endogenous caspase 9 was cleaved during cell death, whereas dominant negative caspase 9 inhibited cell death. The results from determining the role of inhibitors of apoptosis (IAPs) suggest that acquisition of inhibition by IAPs is part of the differentiation program. Ubiquitin-DeltaN-AVPI Smac/DIABLO induced cell death in differentiated cells only. c-IAP-2 is unregulated in differentiated cells, whereas X-linked IAP levels decreased in these cells coincident with cell death. Moreover, expressing X-linked IAP rendered undifferentiated cells resistant to microinjected cytochrome c. Overall, the inhibitory regulation, of cell death at the level of release of mitochondrial apoptogenic factors and at post-mitochondrial activation of caspase 9 observed in differentiated PC12 cells, is reduced or absent in the undifferentiated counterparts.

Involvement of survival motor neuron (SMN) protein in cell death.

Infantile spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron (SMN)1 gene. We investigated the role of human (h) SMN protein on cell death in PC12 and Rat-1 cells. hSMN prolonged cell survival in PC12 cells deprived of trophic support and in Rat-1 cells induced to die by activation of the proto-oncogene c-Myc, to similar magnitude as Bcl-2 or IAP-2. While hSMN was ineffective in inhibiting apoptosis induced by ultraviolet light (UV) or etoposide treatment in proliferating PC12 or Rat-1 cells, a protective effect was observed in terminally NGF/dBcAMP-differentiated PC12 cells. hSMN inhibited the onset of apoptosis in NGF/dBcAMP-deprived or UV-treated co-differentiated PC12 cells by preventing cytochrome c release and caspase-3 activation, indicating that its effects are through suppression of the mitochondrial apoptotic pathway. Expressing hSMN deleted for exon 7 (Delta7) or for exons 6 and 7 (Delta6/7), or with the SMA point mutant Y272C, resulted in loss of survival function. Moreover, these mutants also exhibited pro-apoptotic effects in Rat-1 cells. The localization pattern of full-length hSMN in PC12 and Rat-1 cells was similar to that of endogenous SMN: granular labelling in the cytoplasm and discrete fluorescence spots in the nucleus, some of which co-localized with p80 coilin, the characteristic marker of Cajal bodies. However, cytoplasmic and nuclear aggregates were often seen with hSMNDelta7, whereas the hSMNDelta6/7 mutant showed homogenous nuclear labelling that excluded the nucleolus. Thus, our results show that the C-terminal region is critical in suppression of apoptosis by SMN.

Molecular mechanisms of neuronal cell death: implications for nuclear factors responding to cAMP and phorbol esters.

Chronic treatment with calcium ionophore A23187 in NGF-differentiated cells results in cell death that is time- and concentration-dependent. Additionally, PC12 cells codifferentiated with NGF and dBcAMP become dependent on these factors for survival and undergo apoptosis when both factors are withdrawn. We show that in both cases there is a prolonged induction of c-Fos which correlates with cell death. Its continual activation in PC12 cells overexpressing c-FosER results in caspase-3 cleavage and rapid cell death. Specific phosphorylation of CREB/CREM(tau) transactivators or their binding to CRE of c-fos was observed. Our results indicate that prolonged c-Fos induction activates p53. There is increased nuclear localization of p53, p21 and Bax levels are induced in NGF/dBcAMP-deprived c-FosER cells, and dominant negative p53 inhibits cell death induced either by serum deprivation or by c-Fos. Overall these data implicate AP-1 as a nuclear target of signal transduction pathways which plays a role in the activation of apoptosis.

Increased expression and redistribution of the antiapoptotic molecule Bcl-xL in Parkinson's disease.

In the present study, we tried to clarify the potentially protective role of Bcl-x(L), an anti-apoptotic member of the Bcl-2 family of proteins, in Parkinson's disease (PD). Using in situ hybridization on human postmortem mesencephalon sections, we show that in PD patients Bcl-x(L) mRNA expression per dopaminergic neuron was almost double that of controls. We also show that, ultrastructurally, this effect may be mediated by a redistribution of Bcl-x(L) from the cytosol to the outer mitochondrial membrane.