Raul Delgado-Morales

Human DNA methylomes of neurodegenerative diseases show common epigenomic patterns.

Different neurodegenerative disorders often show similar lesions, such as the presence of amyloid plaques, TAU-neurotangles and synuclein inclusions. The genetically inherited forms are rare, so we wondered whether shared epigenetic aberrations, such as those affecting DNA methylation, might also exist. The studied samples were gray matter samples from the prefrontal cortex of control and neurodegenerative disease-associated cases. We performed the DNA methylation analyses of Alzheimers disease, dementia with Lewy bodies, Parkinsons disease and Alzheimer-like neurodegenerative profile associated with Downs syndrome samples. The DNA methylation landscapes obtained show that neurodegenerative diseases share similar aberrant CpG methylation shifts targeting a defined gene set. Our findings suggest that neurodegenerative disorders might have similar pathogenetic mechanisms that subsequently evolve into different clinical entities. The identified aberrant DNA methylation changes can be used as biomarkers of the disorders and as potential new targets for the development of new therapies.

Acute and chronic stress differentially regulate cyclin-dependent kinase 5 in mouse brain: implications to glucocorticoid actions and major depression

Stress activates the hypothalamic–pituitary–adrenal axis, which in turn increases circulating glucocorticoid concentrations and stimulates the glucocorticoid receptor (GR). Chronically elevated glucocorticoids by repetitive exposure to stress are implicated in major depression and anxiety disorders. Cyclin-dependent kinase 5 (CDK5), a molecule essential for nervous system development, function and pathogenesis of neurodegenerative disorders, can modulate GR activity through phosphorylation. We examined potential contribution of CDK5 to stress response and pathophysiology of major depression. In mice, acute immobilized stress (AS) caused a biphasic effect on CDK5 activity, initially reducing but increasing afterwards in prefrontal cortex (PFC) and hippocampus (HIPPO), whereas chronic unpredictable stress (CS) strongly increased it in these brain areas, indicating that AS and CS differentially regulate this kinase activity in a brain region-specific fashion. GR phosphorylation contemporaneously followed the observed changes of CDK5 activity after AS, thus CDK5 may in part alter GR phosphorylation upon this stress. In the postmortem brains of subjects with major depression, CDK5 activity was elevated in Brodmann’s area 25, but not in entire PFC and HIPPO. Messenger RNA expression of glucocorticoid-regulated/stress-related genes showed distinct expression profiles in several brain areas of these stressed mice or depressive subjects in which CDK5-mediated changes in GR phosphorylation may have some regulatory roles. Taken together, these results indicate that CDK5 is an integral component of stress response and major depression with regulatory means specific to different stressors, brain areas and diseases in part through changing phosphorylation of GR.

Altered machinery of protein synthesis is region- and stage-dependent and is associated with α-synuclein oligomers in Parkinson's disease.

IntroductionParkinson’s disease (PD) is characterized by the accumulation of abnormal α-synuclein in selected regions of the brain following a gradient of severity with disease progression. Whether this is accompanied by globally altered protein synthesis is poorly documented. The present study was carried out in PD stages 1-6 of Braak and middle-aged (MA) individuals without alterations in brain in the substantia nigra, frontal cortex area 8, angular gyrus, precuneus and putamen.ResultsReduced mRNA expression of nucleolar proteins nucleolin (NCL), nucleophosmin (NPM1), nucleoplasmin 3 (NPM3) and upstream binding transcription factor (UBF), decreased NPM1 but not NPM3 nucleolar protein immunostaining in remaining neurons; diminished 18S rRNA, 28S rRNA; reduced expression of several mRNAs encoding ribosomal protein (RP) subunits; and altered protein levels of initiation factor eIF3 and elongation factor eEF2 of protein synthesis was found in the substantia nigra in PD along with disease progression. Although many of these changes can be related to neuron loss in the substantia nigra, selective alteration of certain factors indicates variable degree of vulnerability of mRNAs, rRNAs and proteins in degenerating sustantia nigra. NPM1 mRNA and 18S rRNA was increased in the frontal cortex area 8 at stage 5-6; modifications were less marked and region-dependent in the angular gyrus and precuneus. Several RPs were abnormally regulated in the frontal cortex area 8 and precuneus, but only one RP in the angular gyrus, in PD. Altered levels of eIF3 and eIF1, and decrease eEF1A and eEF2 protein levels were observed in the frontal cortex in PD. No modifications were found in the putamen at any time of the study except transient modifications in 28S rRNA and only one RP mRNA at stages 5-6. These observations further indicate marked region-dependent and stage-dependent alterations in the cerebral cortex in PD. Altered solubility and α-synuclein oligomer formation, assessed in total homogenate fractions blotted with anti-α-synuclein oligomer-specific antibody, was demonstrated in the substantia nigra and frontal cortex, but not in the putamen, in PD. Dramatic increase in α-synuclein oligomers was also seen in fluorescent-activated cell sorter (FACS)-isolated nuclei in the frontal cortex in PD.ConclusionsAltered machinery of protein synthesis is altered in the substantia nigra and cerebral cortex in PD being the frontal cortex area 8 more affected than the angular gyrus and precuneus; in contrast, pathways of protein synthesis are apparently preserved in the putamen. This is associated with the presence of α-synuclein oligomeric species in total homogenates; substantia nigra and frontal cortex are enriched, albeit with different band patterns, in α-synuclein oligomeric species, whereas α-synuclein oligomers are not detected in the putamen.

Adaptation of the hypothalamic-pituitary-adrenal axis and glucose to repeated immobilization or restraint stress is not influenced by associative signals.

Repeated exposure to the same stressor very often results in a reduction of some prototypical stress responses, namely those related to the hypothalamic-pituitary-adrenal (HPA) and sympatho-medullo-adrenal (SMA) axes. This reduced response to repeated exposure to the same (homotypic) stressor (adaptation) is usually considered as a habituation-like process, and therefore, a non-associative type of learning. However, there is some evidence that contextual cues and therefore associative processes could contribute to adaptation. In the present study we demonstrated in two experiments using adult male rats that repeated daily exposure to restraint (REST) or immobilization on boards (IMO) reduced the HPA (plasma levels of ACTH and corticosterone) and glucose responses to the homotypic stressor and such reduced responses remained intact when all putative cues associated to the procedure (experimenter, way of transporting to the stress room, stress boxes, stress room and colour of the restrainer in the case of REST) were modified on the next day. Therefore, the present results do not favour the view that adaptation after repeated exposure to a stressor may involve associative processes related to signals predicting the imminence of the stressors, but more studies are needed on this issue.

Susceptibility to stress in transgenic mice overexpressing TrkC, a model of panic disorder

Stressful life events increase the susceptibility for subsequent onset of psychiatric disorders in humans. Previous research has implicated neurotrophins in the onset of some stress-related diseases, such as major depression disorder, post-traumatic stress disorder or panic disorder. We have tested the hypothesis that the neurotrophin-3 (NT-3)/TrkC system is a genetic interface mediating the deleterious effects of stress on the initiation of panic disorder and other pathologies. To this aim, we have analyzed the functionality of HPA axis and the behavioral consequences of different types of stressful conditions in a mouse model of panic disorder, which overexpresses TrkC, the high affinity-receptor for NT-3 (TgNTRK3). Our results reveal that TgNTRK3 mice exhibit an altered circadian corticosterone rhythm that is reversed by clonidine treatment, but normal expression of genes involved in the control of the hypothalamus-pituitary-adrenal (HPA) axis (CRH, GR) and normal corticosterone response to acute and chronic stressors. In contrast, they exhibit an altered pattern of activation of stress-related brain areas and showed enhanced anxiety-related behavior and more passive strategies than wild types under some chronic stress conditions. We conclude that TgNTRK3 mice present differences in their response to stress characterized by subtle changes in the HPA axis, marked changes in acute stress-induced brain activation and altered coping strategies, suggesting a key role of TrkC receptor in the stress neural circuitry and in the behavioral consequences of chronic stress.

Opening up the DNA methylome of dementia

Dementia is a complex clinical condition characterized by several cognitive impairments that interfere with patient independence in executing everyday tasks. Various neurodegenerative disorders have dementia in common among their clinical manifestations. In addition, these diseases, such as Alzheimers disease, Parkinsons disease, dementia with Lewy bodies and frontotemporal dementia, share molecular alterations at the neuropathological level. In recent years, the field of neuroepigenetics has expanded massively and it is now clear that epigenetic processes, such as DNA methylation, are mechanisms involved in both normal and pathological brain function. Despite the persistent methodological and conceptual caveats, it has been reported that several genes fundamental to the development of neurodegenerative disorders are deregulated by aberrant methylation patterns of their promoters, and even common epigenetic signatures for some dementia-associated pathologies have been identified. Therefore, understanding the epigenetic mechanisms that are altered in dementia, especially those associated with the initial phases, will allow us not only to understand the etiopathology of dementia and its progression but also to design effective therapies to reduce this global public health problem. This review provides an in-depth summary of our current knowledge about DNA methylation in dementia, focusing exclusively on the analyses performed in human brain.

Epigenetic mechanisms during ageing and neurogenesis as novel therapeutic avenues in human brain disorders

Ageing is the main risk factor for human neurological disorders. Among the diverse molecular pathways that govern ageing, epigenetics can guide age-associated decline in part by regulating gene expression and also through the modulation of genomic instability and high-order chromatin architecture. Epigenetic mechanisms are involved in the regulation of neural differentiation as well as in functional processes related to memory consolidation, learning or cognition during healthy lifespan. On the other side of the coin, many neurodegenerative diseases are associated with epigenetic dysregulation. The reversible nature of epigenetic factors and, especially, their role as mediators between the genome and the environment make them exciting candidates as therapeutic targets. Rather than providing a broad description of the pathways epigenetically deregulated in human neurological disorders, in this review, we have focused on the potential use of epigenetic enzymes as druggable targets to ameliorate neural decline during normal ageing and especially in neurological disorders. We will firstly discuss recent progress that supports a key role of epigenetic regulation during healthy ageing with an emphasis on the role of epigenetic regulation in adult neurogenesis. Then, we will focus on epigenetic alterations associated with ageing-related human disorders of the central nervous system. We will discuss examples in the context of psychiatric disorders, including schizophrenia and posttraumatic stress disorders, and also dementia or Alzheimer’s disease as the most frequent neurodegenerative disease. Finally, methodological limitations and future perspectives are discussed.

Adrenocortical and behavioural response to chronic restraint stress in neurokinin-1 receptor knockout mice.

Brain substance P and its receptor (neurokinin-1, NK1) have a widespread brain distribution and are involved in an important number of behavioural and physiological responses to emotional stimuli. However, the role of NK1 receptors in the consequences of exposure to chronic stress has not been explored. The present study focused on the role of these receptors in the hypothalamic-pituitary-adrenal (HPA) response to daily repeated restraint stress (evaluated by plasma corticosterone levels), as well as on the effect of this procedure on anxiety-like behaviour, spatial learning and memory in the Morris water maze (MWM), a hippocampus-dependent task. Adult null mutant NK1-/- mice, with a C57BL/6J background, and the corresponding wild-type mice showed similar resting corticosterone levels and, also, did not differ in corticosterone response to a first restraint. Nevertheless, adaptation to the repeated stressor was faster in NK1-/- mice. Chronic restraint modestly increased anxiety-like behaviour in the light-dark test, irrespective of genotype. Throughout the days of the MWM trials, NK1-/- mice showed a similar learning rate to that of wild-type mice, but had lower levels of thigmotaxis and showed a better retention in the probe trial. Chronic restraint stress did not affect these variables in either genotype. These results indicate that deletion of the NK1 receptor does not alter behavioural susceptibility to chronic repeated stress in mice, but accelerates adaptation of the HPA axis. In addition, deletion may result in lower levels of thigmotaxis and improved short-term spatial memory, perhaps reflecting a better learning strategy in the MWM.

Not all stressors are equal: behavioral and endocrine evidence for development of contextual fear conditioning after a single session of footshocks but not of immobilization

Exposure of animals to footshocks (FS) in absence of any specific cue results in the development of fear to the compartment where shocks were given (contextual fear conditioning), and this is usually evaluated by time spent freezing. However, the extent to which contextual fear conditioning always develops when animals are exposed to other stressors is not known. In the present work we firstly demonstrated, using freezing, that exposure of adult rats to a single session of FS resulted in short-term and long-term contextual fear conditioning (freezing) that was paralleled by increased hypothalamic-pituitary-adrenal (HPA) activation. In contrast, using a similar design, no HPA or behavioral evidence for such conditioning was found after exposure to immobilization on boards (IMO), despite this stressor being of similar severity as FS on the basis of standard physiological measures of stress, including HPA activation. In a final experiment we directly compared the exposure to the two stressors in the same type of context and tested for the development of conditioning to the context and to a specific cue for IMO (the board). We observed the expected high levels of freezing and the conditioned HPA activation after FS, but not after IMO, regardless of the presence of the board during testing. Therefore, it can be concluded that development of fear conditioning to context or particular cues, as evaluated by either behavioral or endocrine measures, appears to be dependent on the nature of the aversive stimuli, likely to be related to biologically preparedness to establish specific associations.

Mutations in JMJD1C are involved in Rett syndrome and intellectual disability

Purpose:Autism spectrum disorders are associated with defects in social response and communication that often occur in the context of intellectual disability. Rett syndrome is one example in which epilepsy, motor impairment, and motor disturbance may co-occur. Mutations in histone demethylases are known to occur in several of these syndromes. Herein, we aimed to identify whether mutations in the candidate histone demethylase JMJD1C (jumonji domain containing 1C) are implicated in these disorders.Methods:We performed the mutational and functional analysis of JMJD1C in 215 cases of autism spectrum disorders, intellectual disability, and Rett syndrome without a known genetic defect.Results:We found seven JMJD1C variants that were not present in any control sample (~ 6,000) and caused an amino acid change involving a different functional group. From these, two de novo JMJD1C germline mutations were identified in a case of Rett syndrome and in a patient with intellectual disability. The functional study of the JMJD1C mutant Rett syndrome patient demonstrated that the altered protein had abnormal subcellular localization, diminished activity to demethylate the DNA damage-response protein MDC1, and reduced binding to MECP2. We confirmed that JMJD1C protein is widely expressed in brain regions and that its depletion compromises dendritic activity.Conclusions:Our findings indicate that mutations in JMJD1C contribute to the development of Rett syndrome and intellectual disability.Genet Med 18 1, 378–385.