Biochemical and molecular determinants of the subclinical inflammatory mechanisms in Rett syndrome.

To date, Rett syndrome (RTT), a genetic disorder mainly caused by mutations in the X-linked MECP2 gene, is increasingly considered a broad-spectrum pathology, instead of just a neurodevelopmental disease, due to the multitude of peripheral co-morbidities and the compromised metabolic pathways, affecting the patients. The altered molecular processes include an impaired mitochondrial function, a perturbed redox homeostasis, a chronic subclinical inflammation and an improper cholesterol metabolism. The persistent subclinical inflammatory condition was first defined ten years ago, as a previously unrecognized feature of RTT, playing a role in the pathology progress and modulation of phenotypical severity. In light of this, the present work aims at reviewing the current knowledge on the chronic inflammatory status and the altered immune/inflammatory functions in RTT, as well as investigating the emerging mechanisms underlying this condition with a special focus on the latest findings about inflammasome system, autoimmunity responses and intestinal micro- and mycobiota. On these bases, although further research is needed, future therapeutic strategies able to re-establish an adequate immune/inflammatory response could represent potential approaches for RTT patients.

Anti-MOG antibody encephalitis mimicking neurological deterioration in a case of Rett syndrome with MECP2 mutation.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder primarily caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, resulting in developmental regression after normal development during infancy. Transient presentation of many autistic features is also commonly seen in RTT. Anti-myelin oligodendrocyte glycoprotein (MOG)-antibody encephalitis is an acquired relapsing demyelinating syndrome characterized by a variety of neuroinflammatory symptoms. Here, we report a case of anti-MOG antibody encephalitis in a patient with genetically confirmed RTT, which mimicked many of the features of RTT. CASE REPORT: A three-year-old girl presented with subacute verbal and motor dysfunction, along with involuntary movements and marked irritability. Magnetic resonance imaging (MRI) revealed extensive white matter lesions, with anti-MOG antibodies detected in the serum and cerebrospinal fluid, resulting in an initial diagnosis of anti-MOG antibody encephalitis. However, additional testing of the MECP2 gene was performed in response to persistent involuntary hand movements in combination with progressive verbal and motor deterioration. Sequencing analysis revealed a known pathogenic mutation in MEPC2, indicating a concurrent diagnosis of RTT. CONCLUSION: Both RTT and anti-MOG antibody encephalitis are rare conditions. Similarities in disease presentation suggest that anti-MOG antibody encephalitis may mimic many of the symptoms of RTT.

Intestinal Candida parapsilosis isolates from Rett syndrome subjects bear potential virulent traits and capacity to persist within the host.

BACKGROUND: Rett syndrome (RTT) is a neurological disorder mainly caused by mutations in MeCP2 gene. It has been shown that MeCP2 impairments can lead to cytokine dysregulation due to MeCP2 regulatory role in T-helper and T-reg mediated responses, thus contributing to the pro-inflammatory status associated with RTT. Furthermore, RTT subjects suffer from an intestinal dysbiosis characterized by an abnormal expansion of the Candida population, a known factor responsible for the hyper-activation of pro-inflammatory immune responses. Therefore, we asked whether the intestinal fungal population of RTT subjects might contribute the sub-inflammatory status triggered by MeCP2 deficiency. METHODS: We evaluated the cultivable gut mycobiota from a cohort of 50 RTT patients and 29 healthy controls characterizing the faecal fungal isolates for their virulence-related traits, antifungal resistance and immune reactivity in order to elucidate the role of fungi in RTT's intestinal dysbiosis and gastrointestinal physiology. RESULTS: Candida parapsilosis, the most abundant yeast species in RTT subjects, showed distinct genotypic profiles if compared to healthy controls' isolates as measured by hierarchical clustering analysis from RAPD genotyping. Their phenotypical analysis revealed that RTT's isolates produced more biofilm and were significantly more resistant to azole antifungals compared to the isolates from the healthy controls. In addition, the high levels of IL-1ß and IL-10 produced by peripheral blood mononuclear cells and the mixed Th1/Th17 cells population induced by RTT C. parapsilosis isolates suggest the capacity of these intestinal fungi to persist within the host, being potentially involved in chronic, pro-inflammatory responses. CONCLUSIONS: Here we demonstrated that intestinal C. parapsilosis isolates from RTT subjects hold phenotypic traits that might favour the previously observed low-grade intestinal inflammatory status associated with RTT. Therefore, the presence of putative virulent, pro-inflammatory C. parapsilosis strains in RTT could represent an additional factor in RTT's gastrointestinal pathophysiology, whose mechanisms are not yet clearly understood.

Persistent Unresolved Inflammation in the Mecp2-308 Female Mutated Mouse Model of Rett Syndrome.

Rett syndrome (RTT) is a rare neurodevelopmental disorder usually caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). Several Mecp2 mutant mouse lines have been developed recapitulating part of the clinical features. In particular, Mecp2-308 female heterozygous mice, bearing a truncating mutation, are a validated model of the disease. While recent data suggest a role for inflammation in RTT, little information on the inflammatory status in murine models of the disease is available. Here, we investigated the inflammatory status by proteomic 2-DE/MALDI-ToF/ToF analyses in symptomatic Mecp2-308 female mice. Ten differentially expressed proteins were evidenced in the Mecp2-308 mutated plasma proteome. In particular, 5 positive acute-phase response (APR) proteins increased (i.e., kininogen-1, alpha-fetoprotein, mannose-binding protein C, alpha-1-antitrypsin, and alpha-2-macroglobulin), and 3 negative APR reactants were decreased (i.e., serotransferrin, albumin, and apolipoprotein A1). CD5 antigen-like and vitamin D-binding protein, two proteins strictly related to inflammation, were also changed. These results indicate for the first time a persistent unresolved inflammation of unknown origin in the Mecp2-308 mouse model.

Inflammatory protein response in CDKL5-Rett syndrome: evidence of a subclinical smouldering inflammation.

BACKGROUND: Mutations in the cyclin-dependent kinase-like 5 gene cause a clinical variant of Rett syndrome (CDKL5-RTT). A role for the acute-phase response (APR) is emerging in typical RTT caused by methyl-CpG-binding protein 2 gene mutations (MECP2-RTT). No information is, to date, available on the inflammatory protein response in CDKL5-RTT. We evaluated, for the first time, the APR protein response in CDKL5-RTT. METHODS: Protein patterns in albumin- and IgG-depleted plasma proteome from CDKL5-RTT patients were evaluated by two-dimensional gel electrophoresis/mass spectrometry. The resulting data were related to circulating cytokines and compared to healthy controls or MECP2-RTT patients. The effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) were evaluated. RESULTS: CDKL5-RTT mutations resulted in a subclinical attenuated inflammation, specifically characterized by an overexpression of the complement component C3 and CD5 antigen-like, both strictly related to the inflammatory response. Cytokine dysregulation featuring a bulk increase of anti-inflammatory cytokines, predominantly IL-10, could explain the unchanged erythrocyte sedimentation rate and atypical features of inflammation in CDKL5-RTT. Omega-3 PUFAs were able to counterbalance the pro-inflammatory status. CONCLUSION: For the first time, we revealed a subclinical smouldering inflammation pattern in CDKL5-RTT consisting in the coexistence of an atypical APR coupled with a dysregulated cytokine response.

Efficient derivation of microglia-like cells from human pluripotent stem cells.

Microglia, the only lifelong resident immune cells of the central nervous system (CNS), are highly specialized macrophages that have been recognized to have a crucial role in neurodegenerative diseases such as Alzheimer's, Parkinson's and adrenoleukodystrophy (ALD). However, in contrast to other cell types of the human CNS, bona fide microglia have not yet been derived from cultured human pluripotent stem cells. Here we establish a robust and efficient protocol for the rapid production of microglia-like cells from human (h) embryonic stem (ES) and induced pluripotent stem (iPS) cells that uses defined serum-free culture conditions. These in vitro pluripotent stem cell-derived microglia-like cells (termed pMGLs) faithfully recapitulate the expected ontogeny and characteristics of their in vivo counterparts, and they resemble primary fetal human and mouse microglia. We generated these cells from multiple disease-specific cell lines and find that pMGLs derived from an hES model of Rett syndrome are smaller than their isogenic controls. We further describe a platform to study the integration and live behavior of pMGLs in organotypic 3D cultures. This modular differentiation system allows for the study of microglia in highly defined conditions as they mature in response to developmentally relevant cues, and it provides a framework in which to study the long-term interactions of microglia residing in a tissue-like environment.

Transcriptome analysis of human brain tissue identifies reduced expression of complement complex C1Q Genes in Rett syndrome.

BACKGROUND: MECP2, the gene mutated in the majority of Rett syndrome cases, is a transcriptional regulator that can activate or repress transcription. Although the transcription regulatory function of MECP2 has been known for over a decade, it remains unclear how transcriptional dysregulation leads to the neurodevelopmental disorder. Notably, little convergence was previously observed between the genes abnormally expressed in the brain of Rett syndrome mouse models and those identified in human studies. METHODS: Here we carried out a comprehensive transcriptome analysis of human brain tissue from Rett syndrome brain using both RNA-seq and microarrays. RESULTS: We identified over two hundred differentially expressed genes, and identified the complement C1Q complex genes (C1QA, C1QB and C1QC) as a point of convergence between gene expression changes in human and mouse Rett syndrome brain. CONCLUSIONS: The results of our study support a role for alterations in the expression level of C1Q complex genes in RTT pathogenesis.

Rett Syndrome Mutant Neural Cells Lacks MeCP2 Immunoreactive Bands.

Dysfunctions of MeCP2 protein lead to various neurological disorders such as Rett syndrome and Autism. The exact functions of MeCP2 protein is still far from clear. At a molecular level, there exist contradictory data. MeCP2 protein is considered a single immunoreactive band around 75 kDa by western-blot analysis but several reports have revealed the existence of multiple MeCP2 immunoreactive bands above and below the level where MeCP2 is expected. MeCP2 immunoreactive bands have been interpreted in different ways. Some researchers suggest that multiple MeCP2 immunoreactive bands are unidentified proteins that cross-react with the MeCP2 antibody or degradation product of MeCP2, while others suggest that MeCP2 post-transcriptional processing generates multiple molecular forms linked to cell signaling, but so far they have not been properly analyzed in relation to Rett syndrome experimental models. The purpose of this study is to advance understanding of multiple MeCP2 immunoreactive bands in control neural cells and p.T158M MeCP2e1 mutant cells. We have generated stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Application of N- and C- terminal MeCP2 antibodies, and also, RFP antibody minimized concerns about nonspecific cross-reactivity, since they react with the same antigen at different epitopes. We report the existence of multiple MeCP2 immunoreactive bands in control cells, stable wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Also, MeCP2 immunoreactive bands differences were found between wild-type and p.T158M MeCP2e1-RFP mutant expressing cells. Slower migration phosphorylated band around 70kDa disappeared in p.T158M MeCP2e1-RFP mutant expressing cells. These data suggest that threonine 158 could represent an important phosphorylation site potentially involved in protein function. Our results clearly indicate that MeCP2 antibodies have no cross-reactivity with similar epitopes on others proteins, supporting the idea that MeCP2 may exist in multiple different molecular forms and that molecular pattern variations derived from altered post-transcriptional processing may underlay Rett syndrome physiophatology.

Rett syndrome: An autoimmune disease?

Rett syndrome (RTT) is a devastating neurodevelopmental disease, previously included into the autistic spectrum disorders, affecting almost exclusively females (frequency 1:10,000). RTT leads to intellective deficit, purposeful hands use loss and late major motor impairment besides featuring breathing disorders, epilepsy and increased risk of sudden death. The condition is caused in up to 95% of the cases by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene. Our group has shown a number of previously unrecognized features, such as systemic redox imbalance, chronic inflammatory status, respiratory bronchiolitis-associated interstitial lung disease-like lung disease, and erythrocyte morphology changes. While evidence on an intimate involvement of MeCP2 in the immune response is cumulating, we have recently shown a cytokine dysregulation in RTT. Increasing evidence on the relationship between MeCP2 and an immune dysfunction is reported, with, apparently, a link between MECP2 gene polymorphisms and autoimmune diseases, including primary Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, and systemic sclerosis. Antineuronal (i.e., brain proteins) antibodies have been shown in RTT. Recently, high levels of anti-N-glucosylation (N-Glc) IgM serum autoantibodies [i.e., anti-CSF114(N-Glc) IgMs] have been detected by our group in a statistically significant number of RTT patients. In the current review, the Authors explore the current evidence, either in favor or against, the presence of an autoimmune component in RTT.

Antibody recognition in multiple sclerosis and Rett syndrome using a collection of linear and cyclic N-glucosylated antigenic probes.

Antibody detection in autoimmune disorders, such as multiple sclerosis (MS) and Rett syndrome (RTT) can be achieved more efficiently using synthetic peptides. The previously developed synthetic antigenic probe CSF114(Glc), a type I' ß-turn N-glucosylated peptide structure, is able to recognize antibodies in MS and RTT patients' sera as a sign of immune system derangement. We report herein the design, synthesis, conformational analysis, and immunological evaluation of a collection of glycopeptide analogs of CSF114(Glc) to characterize the specific role of secondary structures in MS and RTT antibody recognition. Therefore, we synthesized a series of linear and cyclic short glucosylated sequences, mimicking different ß-turn conformations, which were evaluated in inhibition enzyme-linked immunosorbent assays (ELISA). Calculated IC50 ranking analysis allowed the selection of the candidate octapeptide containing two (S)-2-amino-4-pentynoic acid (L-Pra) residues Ac-Pra-RRN(Glc)GHT-Pra-NH2 , with an IC50 in the nanomolar range. This peptide was adequately modified for solid-phase ELISA (SP-ELISA) and surface plasmon resonance (SPR) experiments. Pra-RRN(Glc)GHT-Pra-NH2 peptide was modified with an alkyl chain linked to the N-terminus, favoring immobilization on solid phase in SP-ELISA and differentiating IgG antibody recognition between patients and healthy blood donors with a high specificity. However, this peptide displayed a loss in IgM specificity and sensitivity. Moreover, an analog was obtained after modification of the octapeptide candidate Ac-Pra-RRN(Glc)GHT-Pra-NH2 to favor immobilization on SPR sensor chips. SPR technology allowed us to determine its affinity (KD = 16.4 nM), 2.3 times lower than the affinity of the original glucopeptide CSF114(Glc) (KD = 7.1 nM).

Brains, Blood, and Guts: MeCP2 Regulates Microglia, Monocytes, and Peripheral Macrophages.

Mutations in methyl-CpG-binding protein 2 (MECP2) underlie most cases of Rett Syndrome, a neurodevelopmental disorder with neurological and somatic impairments. In this issue of Immunity, Cronk et al. (2015) find that macrophages in MeCP2-deficient mice are abnormal in number, as well as in glucocorticoid, hypoxia, and inflammatory responses.

Methyl-CpG Binding Protein 2 Regulates Microglia and Macrophage Gene Expression in Response to Inflammatory Stimuli.

Mutations in MECP2, encoding the epigenetic regulator methyl-CpG-binding protein 2, are the predominant cause of Rett syndrome, a disease characterized by both neurological symptoms and systemic abnormalities. Microglial dysfunction is thought to contribute to disease pathogenesis, and here we found microglia become activated and subsequently lost with disease progression in Mecp2-null mice. Mecp2 was found to be expressed in peripheral macrophage and monocyte populations, several of which also became depleted in Mecp2-null mice. RNA-seq revealed increased expression of glucocorticoid- and hypoxia-induced transcripts in Mecp2-deficient microglia and peritoneal macrophages. Furthermore, Mecp2 was found to regulate inflammatory gene transcription in response to TNF stimulation. Postnatal re-expression of Mecp2 using Cx3cr1(creER) increased the lifespan of otherwise Mecp2-null mice. These data suggest that Mecp2 regulates microglia and macrophage responsiveness to environmental stimuli to promote homeostasis. Dysfunction of tissue-resident macrophages might contribute to the systemic pathologies observed in Rett syndrome.

Interactions of innate and adaptive immunity in brain development and function.

It has been known for decades that the immune system has a tremendous impact on behavior. Most work has described the negative role of immune cells on the central nervous system. However, we and others have demonstrated over the last decade that a well-regulated immune system is needed for proper brain function. Here we discuss several neuro-immune interactions, using examples from brain homeostasis and disease states. We will highlight our understanding of the consequences of malfunctioning immunity on neurodevelopment and will discuss the roles of the innate and adaptive immune system in neurodevelopment and how T cells maintain a proper innate immune balance in the brain surroundings and within its parenchyma. Also, we describe how immune imbalance impairs higher order brain functioning, possibly leading to behavioral and cognitive impairment. Lastly, we propose our hypothesis that some behavioral deficits in neurodevelopmental disorders, such as in autism spectrum disorder, are the consequence of malfunctioning immunity. This article is part of a Special Issue entitled SI: Neuroimmunology in Health And Disease.

Immune dysfunction in Rett syndrome patients revealed by high levels of serum anti-N(Glc) IgM antibody fraction.

Rett syndrome (RTT), a neurodevelopmental disorder affecting exclusively (99%) female infants, is associated with loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2) and, more rarely, cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1). In this study, we aimed to evaluate the function of the immune system by measuring serum immunoglobulins (IgG and IgM) in RTT patients (n = 53) and, by comparison, in age-matched children affected by non-RTT pervasive developmental disorders (non-RTT PDD) (n = 82) and healthy age-matched controls (n = 29). To determine immunoglobulins we used both a conventional agglutination assay and a novel ELISA based on antibody recognition by a surrogate antigen probe, CSF114(Glc), a synthetic N-glucosylated peptide. Both assays provided evidence for an increase in IgM titer, but not in IgG, in RTT patients relative to both healthy controls and non-RTT PDD patients. The significant difference in IgM titers between RTT patients and healthy subjects in the CSF114(Glc) assay (P = 0.001) suggests that this procedure specifically detects a fraction of IgM antibodies likely to be relevant for the RTT disease. These findings offer a new insight into the mechanism underlying the Rett disease as they unveil the possible involvement of the immune system in this pathology.

Subclinical inflammatory status in Rett syndrome.

Inflammation has been advocated as a possible common central mechanism for developmental cognitive impairment. Rett syndrome (RTT) is a devastating neurodevelopmental disorder, mainly caused by de novo loss-of-function mutations in the gene encoding MeCP2. Here, we investigated plasma acute phase response (APR) in stage II (i.e., "pseudo-autistic") RTT patients by routine haematology/clinical chemistry and proteomic 2-DE/MALDI-TOF analyses as a function of four major MECP2 gene mutation types (R306C, T158M, R168X, and large deletions). Elevated erythrocyte sedimentation rate values (median 33.0 mm/h versus 8.0 mm/h, P < 0.0001) were detectable in RTT, whereas C-reactive protein levels were unchanged (P = 0.63). The 2-DE analysis identified significant changes for a total of 17 proteins, the majority of which were categorized as APR proteins, either positive (n = 6 spots) or negative (n = 9 spots), and to a lesser extent as proteins involved in the immune system (n = 2 spots), with some proteins having overlapping functions on metabolism (n = 7 spots). The number of protein changes was proportional to the severity of the mutation. Our findings reveal for the first time the presence of a subclinical chronic inflammatory status related to the "pseudo-autistic" phase of RTT, which is related to the severity carried by the MECP2 gene mutation.

The role of microglia in brain maintenance: implications for Rett syndrome.

The role of microglia in central nervous system (CNS) pathology has been studied extensively, and more recently, examination of microglia in the healthy brain has yielded important insights into their many functions. It was long assumed that microglia were essentially quiescent cells, unless provoked into activation, which was considered a hallmark of disease. More recently, however, it has become increasingly clear that they are extraordinarily dynamic cells, constantly sampling their environment and adjusting to exquisitely delicate stimuli. Along these lines, our laboratory has identified a new and unexpected role for microglial phagocytosis - or lack thereof - in the pathophysiology of Rett syndrome, a neurodevelopmental disease caused by mutation of the gene encoding methyl-CpG binding protein (MECP)2. We have shown that specific expression of wild type Mecp2 in myeloid cells of Mecp2-null mice is sufficient to arrest major symptoms associated with this devastating disease. This beneficial effect, however, is abolished if phagocytic activity of microglia is inhibited. Here, we discuss microglial origins, the role of microglia in brain development and maintenance, and the phenomenon of microglial augmentation by myeloid progenitor cells in the adult brain. Finally, we address in some detail the beneficial roles of microglia as clinical targets in Rett syndrome and other neurological disorders.

Rett syndrome and other autism spectrum disorders--brain diseases of immune malfunction?

Neuroimmunology was once referred to in terms of its pathological connotation only and was generally understood as covering the deleterious involvement of the immune system in various diseases and disorders of the central nervous system (CNS). However, our conception of the function of the immune system in the structure, function, and plasticity of the CNS has undergone a sea change after relevant discoveries over the past two decades, and continues to be challenged by more recent studies of neurodevelopment and cognition. This review summarizes the recent advances in understanding of immune-system participation in the development and functioning of the CNS under physiological conditions. Considering as an example Rett syndrome a devastating neurodevelopmental disease, we offer a hypothesis that might help to explain the part played by immune cells in its etiology, and hence suggests that the immune system might be a feasible therapeutic target for alleviation of some of the symptoms of this and other autism spectrum disorders.

Folate receptor autoantibodies and spinal fluid 5-methyltetrahydrofolate deficiency in Rett syndrome.

Rett syndrome was associated with low cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5MTHF) in 42-50% of European patients whereas approximately 93% of the patients from North-America had a normal CSF 5MTHF status. We determined the CSF folate status in Rett patients living in North- and South-Western Europe and measured serum folate receptor (FR) autoantibodies of the blocking type to explain the reduced folate transport across the choroid plexus. Irrespective of their MECP2 genotype and despite normal plasma folate values, 14 of 33 Rett patients (42%) had low CSF folate levels. Blocking FR autoantibodies were found in 8 of the Rett patients (24%), 6 of whom had low CSF folate levels. FR autoimmunity was primarily found within the group of Rett patients with low CSF folate status with a higher incidence in North-Western Europe. In Rett patients from North-America 74 of 76 girls had higher folate values in both serum and CSF than European patients. The food folate fortification in North-America may account for the higher folate levels and may prevent CFD in these Rett patients. FR autoimmunity occurred predominantly in Rett patients from North-Western Europe and may contribute to cerebral folate deficiency (CFD).