A proteomics approach to further highlight the altered inflammatory condition in Rett syndrome
Graphical abstract
Introduction
Rett syndrome (RTT; OMIM 312750), a severe neurodevelopmental disorder, which predominantly affects females (about 1 per 10,000–15,000 live births), is mainly caused by sporadic loss-of-function mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) [[1], [2], [3]]. Affected patients display prominent neurologic hallmarks, such as a rapid deterioration of the acquired psychomotor skills (i.e., stereotyped hand movements, absent or very limited speech, ataxia), seizures, and autistic-like behavior, which appear after a period of 6–18-months normal development [4,5]. In addition, common co-morbidities comprise sleep disturbances, breathing and cardiac problems, numerous gastrointestinal disorders, scoliosis, and osteopenia [[6], [7], [8], [9], [10]]. Rett syndrome affects different tissues and organs. It is linked to an abnormal muscular tone [11] and a vasomotor instability especially in the lower limbs [12], to a neurodevelopmental arrest followed by a regression phase where patients lose acquired language and motor skills and exhibit intellectual disability and hand stereotypies [[13], [14], [15]] and to a subclinical myocardial dysfunction [16]. Moreover, Rett patients frequently have marked decreases in bone mineral density leading to osteoporotic fractures [17]. Despite the well-known genetic background at the basis of Rett syndrome, the complete pathogenic mechanisms linking MeCP2 deficiency to the symptoms of this broad-spectrum pathology remain still unclear [18].
Other factors can contribute to the complexity of the disease, indeed an aberrant immune response, an impaired redox homeostasis [[19], [20], [21]], a perturbed cholesterol metabolism [22,23], an altered mitochondrial bioenergetics [24], and a subclinical inflammatory status [[25], [26], [27]] were shown to play a key role in RTT pathogenesis and progression. The condition defined as “OxInflammation”, consisting in a harmful cycle between the redox imbalance and the inflammatory status, has been observed at cellular and systemic levels in both RTT patients and animal models [19].
NF-κB is a redox-sensitive transcription factor involved in innate and adaptive immune responses, as well as in inflammation. Dysregulated NF-κB signaling cascade has been associated with the pathogenesis of several diseases including RTT [28,29]. Indeed, in a recent study our group demonstrated an increase of NF-κB p65 nuclear translocation coupled with high interleukin-1β (IL-1β) transcript levels, and a constitutive NLRP3 inflammasome activation (a multi-protein complex involved in innate immune responses) in primary dermal fibroblasts isolated from RTT patients [30]. Along with other evidence [28], this finding corroborates the important role of this pleiotropic transcription factor in the disorder, even in a non-immune cell type, whose involvement in pathological conditions characterized by chronic inflammation is now ever-growing [[31], [32], [33], [34], [35]].
In this study, using an ex-vivo model, i.e. primary dermal fibroblasts from RTT patients, and a proteomic data mining approach, we were able to confirm the involvement of inflammatory condition in RTT. Therefore, our results on one hand demonstrated that fibroblasts are a good model to investigate RTT [36]; on the other hand, they could be translated to immune cells, thus helping to justify the chronic inflammatory state and immune impairment observed in this disorder [26,37].
In this context, proteome analysis focusing on differentially expressed proteins between RTT and control cells is an effective supportive tool to better explore proteins related to inflammatory network. In particular, we have applied a proteomic approach in order to further examine the dysregulated activation of immune system, which may be responsible for damage tissues and organs, increasing the vulnerability of RTT patients to unknown endogenous factors or infections.
Section snippets
Subjects population
The study included two female patients with clinical diagnosis of classic RTT and MECP2 mutations (sample Rett 1 mutation: R255X, sample Rett 2 mutation:R168X) and two healthy subjects (age: 24 ± 12, and 34 ± 8, respectively). RTT diagnosis and inclusion/exclusion criteria were based on the revised RTT nomenclature consensus [38]. All the patients were admitted to the Child Neuropsychiatry Unit of the “Azienda Ospedaliera Universitaria Senese” (AOUS, Siena, Italy). Both RTT patients have
Results
The latest research on RTT has clearly highlighted how a low-grade chronic inflammatory condition, fueled by abnormal immune response, can play a part in the multisystem manifestations observed in RTT patients [19,28]. In our recent study, fibroblasts isolated from skin biopsies of RTT patients showed atypical function of NF-κB p65 pathway and altered expression of NLRP3 inflammasome machinery [30], proving to be a suitable cellular model able to recapitulate, in vitro, the phenotypic features
Discussion
In recent years, a chronic low-grade inflammatory state has emerged as a prominent phenotypic hallmark in RTT. An altered profile of circulating proinflammatory mediators associated with irregularities in cellular immune function including aberrant NLRP3 inflammasome activity appears to contribute to the broad spectrum of metabolic abnormalities recognized in RTT [19,28,30,54]. In an attempt to explain the molecular and cellular mechanisms behind the atypical immune function of RTT, we have
Conclusions
Taken together, all these findings reinforce the concept of the intricate molecular sequelae resulting from the mutation of a single gene such as MECP2. In particular, we found that RTT fibroblasts show a well distinct proteomic profile compared to control cells, highlighting once again the complex biological nature of MeCP2 deficient cells. To determine whether inflammation is an important component that also characterizes the RTT cellular proteome, we focused our attention on proteins linked
Limitations of the study
An eventual limitation of our study is the number of samples analyzed although considering that RTT is a rare disease and that the isolation of fibroblasts derives from a skin biopsy, which is a very invasive procedure and not easy to have access. In addition, considering the complex techniques and expensive consumables, it would not feasible to analyze a large set of patients. Another limitation would be the use of cell not fully immune competent to study inflammation, but we have previously
Declaration of competing interest
The authors declare no competing interests.
Acknowledgments
The authors thank Fondazione Umberto Veronesi (FUV) for the fellowship supporting VC.
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