Title
A20 expression in blood cells of patients affected by neurological diseases
Authors
Perga Simona1, Martire Serena1 *, Montarolo Francesca1 *, Navone Nicole Desirèe1, Andrea Calvo3, Giuseppe Fuda3, Marchet Alberto 2, Leotta Daniela 2, Chiò Adriano3, Bertolotto Antonio1.
* The authors contributed equally to this work.
Affiliations
1 Neurobiology Unit, Neurologia 2 – CReSM (Regional Referring Center of Multiple Sclerosis), Neuroscience Institute Cavalieri Ottolenghi (NICO), University of Turin & AOU San Luigi, Orbassano (TO) Italy
2 Division of Neurology, Martini Hospital, Turin, Italy
3 ‘Rita Levi Montalcini’ Department of Neuroscience, ALS (Amyotrophic Lateral Sclerosis) Expert Center (CRESLA), University of Turin, and ‘Città della Scienza e della Salute’, University Hospital, Turin, Italy.
Running title
A20 gene expression in AD, PD, ALS and MS patients’ whole blood
Corresponding author: Perga Simona
Neurobiology Unit, Neurologia 2 – CRESM (Regional Referring Center of Multiple Sclerosis), Neuroscience Institute Cavalieri Ottolenghi (NICO) University of Turin & AOU San Luigi, Regione Gonzole, 10, 10043 Orbassano (TO) Italy
Telephone: 0039 011 670 66 00 Fax: 0039 011 670 66 21
Key words: A20/TNFAIP3, NF-kB, systemic inflammation, Multiple Sclerosis, Alzheimer’s diseases, Parkinson diseases, Amyotrophic Lateral Sclerosis, gene expression.
Abstract
Background: The acute inflammatory response is normally terminated once the triggering insult is eliminated. If this does not occurs properly, the resolution phase may not be appropriately induced and chronic inflammatory state may be ensued. Growing evidences refer to the chronic inflammation as a contributor to the pathogenesis of several neurodegenerative disorders. For this several key regulatory mechanisms are normally involved in the resolution of inflammation. Among these A20, the most potent inhibitor of the pro-inflammatory NF-kB signaling, plays a key role in the regulation of inflammation. Recently, reduced levels of A20 were demonstrated in peripheral blood cells of patients affected by Multiple Sclerosis (MS), a chronic neurological inflammatory disease.
Objective: Investigate whether chronic inflammatory neurodegenerative disorders such as Alzheimer (AD), Parkinson (PD) and Amyotrophic Lateral Sclerosis (ALS) are characterize by deregulated levels of A20. Methods: A20 transcript levels were determined in whole blood of 20 patients affected by AD, 20 by PD, 36 by ALS in comparison to 40 healthy controls and 34 treatment-naive MS patients.
Results: we confirmed that A20 levels were reduced in whole blood from MS patients compared to HC and we demonstrated that diminished A20 expression was also evident in PD patients.
Conclusion: these results suggest that MS and PD could share a common regulatory mechanism of resolution of inflammation, consisting in the anti-inflammatory signaling involving A20. The deregulation of this braking pathway, due to A20 down-regulation, could contribute to the pathogenesis of these disorders, leading, in turn, to the exacerbation of neurodegenerative processes.
INTRODUCTION
Inflammation is a beneficial host response to external challenge or cellular injury. It leads to the
activation of a complex array of inflammatory mediators that establish both an innate and acquired
immune response, finalizing the restoration of tissue structure and function in order to maintain
the homeostasis [Medzhitov, 2010]. Although beneficial in the setting of host defense, prolonged
or non-resolving inflammatory processes can become unchecked, thus evolving into a detrimental
process to the host and contributing to the development and progression of many chronic
inflammatory disorders, including autoimmune and neurodegenerative diseases [Cunningham e
al., 2005, Perry et al 2007; Waisman 2015]. Hence, inflammation is normally a strictly controlled
process, being self-limiting thanks to the production of specific anti-inflammatory factors that
counterbalance the effect of pro-inflammatory molecules. Several key regulatory mechanisms are
involved in the resolution of this process [Serhan and Savill 2005]. At cellular level, the
pro-inflammatory nuclear factor kB (NF-kB) plays a central role in the regulation of inflammation and
immunity since able to induce inflammatory responses through the recruitment and activation of
various immune cells by inducing the transcription of pro-inflammatory mediators and response
genes [Sun et al., 2013]. However, after danger is eliminated, NF-kB signaling needs to be tightly
down-regulated for the maintenance of tissue homeostasis and the prevention of diseases [Ruland
et al., 2011]. Multiple mechanisms usually ensure the proper termination of NF-kB action,
including negative feedback control. Among these, A20, has now emerged as the most potent
negative regulator of NF-kB by restricting the duration and intensity of its action [Shembade and
Harhaj 2010; Catrysse et al., 2014].
A20 belongs to the ubiquitin proteasome system (UPS), the primary proteolytic quality control
system, and negatively regulates NF-kB signaling by the ubiquitination of different NF-kB signaling
molecules [Wertz et al., 2004; Shembade and Harhaj 2010] that are, in turn, addressed to
proteasome for degradation.
Given its key role in the negative feedback loop modulation of NF-kB, A20, codified by TNFAIP3
gene, is considered a central gatekeeper in inflammation and immunity in both mice and humans
[Majumdar et al., 2014; Coornaert 2009]. A20-deficient mice develop cachexia and severe
multi-organ inflammation causing premature lethality [Lee et al., 2000]. In humans, polymorphisms
within the TNFAIP3 locus are associated with several autoimmune diseases including systemic
lupus erythematosus (SLE), rheumatoid arthritis (RA) [Mele et al., 2014] and recently multiple
sclerosis (MS) [IMSGC et al.,2013], the most prevalent autoimmune disease of the central nervous
system (CNS), characterized by chronic inflammation with immune cells infiltration, demyelination
and axonal damage [Waubant 2014]. In a previous work, we observed an A20 down-regulation in
whole blood and peripheral blood mononuclear cells of MS patients [Gill et al., 2010; Gilli et al.,
2011; Navone et al., 2014], suggesting a deregulation of molecular mechanisms in the disease
underlying anti-inflammatory phenotypes, leading to an over-activation of the immune system.
This hypothesis seemed to be confirmed by our subsequent results, showing a negative correlation
between the A20 gene expression level and the disease course [Gilli et al, 2011].
In the last few years several evidences suggested that systemic inflammation and particularly NF-kB pathway may be a pathogenic factor in the onset and progression of MS [for a review see Mc Guire et al., 2013] and of several neurodegenerative diseases [Kaltschmidt 2005; Lanzillotta 2015] as Parkinson (PD) [Ghosh et al., 2007; Baiguera 2012; Tokunaga 2013], Alzheimer’s disease (AD) [Ascolani et al., 2012; Lian 2015], and amyotrophic lateral sclerosis (ALS) [Prell 2014; Frakes et al., 2014]. Based on these considerations, we hypothesized that a deregulated expression of the NF-kB inhibitor, A20, was likely to be involved in these diseases.
To probe this assumption we studied the A20 gene expression in total blood of patients affected by several neurodegenerative diseases, such as AD, PD and ALS, compared to MS patients and healthy donors.
MATERIALS AND METHODS Enrolled subjects
Thanks to a collaboration established with some Neurology Centers of Piedmont, we enrolled in the study: 40 healthy controls (HC), 20 patients affected by AD, 20 patients affected by PD, 36 patients affected by ALS. Patients with AD and PD were clinically monitored at the Division of Neurology of the Martini Hospital, Turin, while patients affected by ALS were followed up at ‘Rita Levi Montalcini’ Department of Neuroscience, ALS Expert Center (CRESLA), ‘Città della Scienza e della Salute’, University Hospital, Turin. Most of these patients received disease-specific drugs at the time of blood sampling. In particular, patients affected by AD were treated either with donepezil, rivastigmine or memantine alone or combined together; PD patients were treated with levodopa or dopamine agonist, while ALS patients were treated with riluzole alone or combined with symptomatic therapies. In addition 34 treatment-naive patients with newly diagnosis of relapsing-remitting MS (RRMS) according to the revised McDonald criteria [Polman et Al. , 2011] were enrolled at the Regional Referral Center for Multiple Sclerosis (CReSM), San Luigi University Hospital, Orbassano, Italy. Each Center provided anonymized medical records of patients. Healthy donors included in the study were screened to exclude any neurological and non-neurological disease. All the subjects enrolled in the study were from Caucasian origin and they gave a written consent at the time of blood drawing. This study was approved by Piedmont and San Luigi University Hospital Ethical Committee.
Demographic and clinical features of patients and HC are summarized in Table 1. RNA extraction and real-time PCR analysis
Whole blood samples were collected into a Tempus™ Blood RNA Tubes (Thermos Fisher Scientific, MA USA), which allow RNA stabilization. Total RNA was extracted using the ABI Prism 6100 Nucleic Acid Prep Station (Life Technology Monza, Italy), following the manufacturer’s instructions and was reverse-transcribed at final concentration of 10 ng/μL using random hexamer primers. Gene expression analysis was performed by real-time PCR using Applied Biosystems’ TaqMan gene expression products (Life Technology). Transcriptional expression was normalized using glyceraldehyde-3-phosphate dehydrogenase (GA3PDH) as reference gene. For primers and probes, Applied Biosystems’ TaqMan® Assay-on-demand-TM gene expression products were used (TNFAIP3, Hs 00234713_m1; GA3PDH, Hs 99999905_m1). Expression levels of target genes were calculated by the normalized comparative cycle threshold (Ct) method (2-ΔΔCt), using the Universal Human Reference RNA (Stratagene, Santa Clara, California) as calibrator.
Statistical analysis
Continuous data are presented as medians and ranges and discrete data are given as counts and percentages. Chi square tests were performed to compare groups of categorical data. Student's t-test or Mann–Whitney U test were used to compare continuous data as appropriate. The correlation between A20 gene expression levels and clinical and demographical data was assessed fitting linear models. In particular, for the HC group we evaluated the correlation with age at sampling. For PD, AD and ALS groups with the age at the disease onset and the presence of pharmacological treatments at the time of sampling (yes/not). For PD patients, with the presence of a family history of the disease and symptoms such as tremor, rigidity and hypokinesia. For ALS patients, with the presence of a family history of the disease and the involvement of upper and/or lower motor neurons. Statistical significance was considered at p-value<0.05. All analyses were carried out using R version 3.02.
RESULTS
Clinical and demographical data for each cohort of subjects are summarized in Table 1. The median age of AD, PD, and ALS groups was higher compared to HC (Student t-test for each comparison, p<0.05). Conversely the median age of MS group was lower compared to HC (Student t-test, p <0.05). This is due to the late onset of sporadic PD, AD and ALS, with an average age of diagnosis around 65, 62 and 63 years respectively. Contrariwise, the average age of onset of MS is approximately 34 years. However, to exclude possible bias due to ageing, a correlation analysis was performed in the HC group, displaying that A20 expression is not related to age (data not shown).
There were no significant differences in gender distribution among the groups (Student t-test, p>0.05). Gene expression analysis of A20 was performed in whole blood obtained from 40 HC, 34 untreated MS patients, 20 PD patients, 20 AD patients, and 36 ALS patients. A significant reduction of A20 transcript level
was observed in MS patients with respect to HC (p=0.0007) (Figure 1), confirming our previous data [ Gilli et al., 2010 and Gilli et al., 2011]. Interestingly, PD patients showed a reduced A20 level compared to HC (p=0.0001) and even to MS with a trend toward significance (p=0.08) (Figure 1).
Conversely, no statistical significant differences regarding A20 expression was detected between HC and both AD and ALS populations (Student t-test, p>0.05) (Figure 1).
Furthermore correlation analyses between A20 gene expression and disease-specific clinical characteristics were performed (see Materials and Methods section), highlighting no significant results (data not shown). The clinical features of each group are summarized in Table 1. Clinical correlations were not performed for the MS group since we have previously demonstrated that A20 levels negatively correlates with the disease clinical course in terms of disability progression quantified by the Expanded Disability Status Scale (EDSS) and the annualized relapse rate (RR) [Gilli et al 2011].
Discussion
Recently, a great deal of evidence suggested that systemic inflammation triggers exacerbation in the central brain’s ongoing damage in several neurodegenerative diseases [Frank-Cannon 2009; Amor 2014]. Accordingly, systemic inflammation would be involved in the pathogenesis of these disorders, not only as a contributor to the progression of disease processes but rather as an etiological agent [vedi introduzione]. The A20 enzyme plays a key role in the regulation of inflammation and immunity thanks its ability to inhibit the NF-kB signaling by a feedback loop mechanism. It has been widely demonstrated that A20 dysfunction results in excessive inflammation and autoimmunity in both human beings and mouse models [Majumdar 2015; Lee 2000].
Close to the well-known role of A20 signaling in chronic inflammatory and autoimmune diseases, emerging findings shown the involvement of NF-kB pathway in neurodegenerative disorders such as PD, AD and ALS [REF vedi sopra * review] in whose pathogenesis systemic- and neuro-inflammation have a crucial role. Based on the consolidated evidences that A20 pathway is involved in MS and its basal expression has been shown significantly reduced in peripheral whole blood and lympho-monocytes of several independent cohorts of naïve MS patients compare to healthy subjects [Gilli 2010, 2011 and Navone 2014], in this study, for the first time, we aimed to investigate a possible deregulation of blood A20 gene expression levels in other neurological diseases such as PD, AD and ALS in which neuroinflammation plays a relevant role. Here, we confirmed our previous data showing that A20 basal level was reduced in untreated MS patients compared to HC. In addition, we found a noteworthy A20 down-regulation in PD patients compared to both HC and MS patients, while no differences in A20 expression in AD and ALS patients respect to HC were highlighted. To exclude the possibility that the A20 reduction observed in PD patients could be ascribed to a physiological ageing process, we performed a correlation analysis which demonstrated that A20 gene expression is not related to age in HC. Moreover, ALS and AD patients, which have an average age
comparable to PD patients, did not show different gene expression level compared to HC. Finally, the A20 deregulation was observed in two cohorts of patients with different median age, such as MS and PD. The decreased expression of the anti-inflammatory enzyme A20 in whole blood from PD patients is an interesting and disease-specific finding, in line with the literature. In fact, the relationship between systemic inflammation and PD has been demonstrated by several authors [Hirsch 2009; Ferrari 2011]. A genetic variant of IL-1b was associated to an increased risk to develop PD [Wahner 2007]. In agreement, PD patients showed elevated serum levels of TNF- and TNF-receptor 1 compare to control subjects and increased peripheral cytokine production has been shown to influence the disease progression [ Reale 2009;, Scalzo 2009]. NF-kB pathway, has been directly implicated in the pathogenesis of PD as regulator of both inflammation and neuronal apoptosis [Pranski 2013; Ghosh 2007; Baiguera et al., 2012; for a review Ferrari and Tarelli 2011]. Furthermore, previous studies showed that alterations in the ubiquitin-proteasome pathway, in which A20 is involved, can contribute to the development and progression of various neurodegenerative disorders such as PD [Wang 2006; for a review Opattova 2015]. Finally, non-steroidal anti-inflammatory drugs were used in PD patients to decrease the effects of inflammatory reactions showing also a neuroprotective effect [For a review Rees 2011].
We have to consider that the notion of systemic inflammation in the worsening of neurodegenerative processes is related to several neurological diseases and is not specific for PD [for a review Waisman 2015]. However for the first time we reported the specific A20 deregulation in PD, hence we could suppose that several inflammatory pathways not involving A20 activation could contribute to neuroinflammation in different neurodegenerative disorders but that A20 defect is specifically involved in PD and MS.
Consisting with these results, it seems that MS and PD share common inflammatory mechanisms. In fact close to the A20 deregulation, the reduction of another NF-kB inhibitor, the anti-inflammatory orphan receptor Nurr1, has been shown in peripheral blood cells of both MS [Gilli et al., 2010, 2011] and PD [Liu 2012] patients. Based on this observation, we could suppose that an overall reduction of braking molecules able to counteract the inflammatory processes, leads, in turn, to the exacerbation of neurodegenerative processes in these pathologies.
Further studies including a larger cohort population preferentially of naïve patients and the analysis of wider set of molecules in the NF-kB pathways or in other anti-inflammatory pathways are necessary to confirm this hypothesis.
These results, if verified, could help to clarify the mechanisms underlying to the pathogenesis of these diseases and, in turn, could help to address the research towards new targets for anti-inflammatory pharmacological treatments.
Acknowledgment
donors information and patient’s clinical data. The study was supported thanks grants from the Fondazione Italiana Sclerosi Multipla (FISM – Grant number 2010/R/1) from the Italian Ministry of Health (Bando Giovani Ricercatori 2010- Grant number GR-2010-2315964) and the European Community's Health Seventh Framework Programme (FP7/2007–2013 under grant agreement 259867)
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Table 1. Clinical and demographical characteristics of MS patients and HC.
HC MS PD AD ALS
N 40 34 20 20 36
Sex, F (%) 25 (55) 26 (76) 9 (37) 12 (52) 22 (47)
Age, median (range) 55 (35-76) 50 (45-63) 71.5 (41-83) 70 (54-82) 65 (35-88)
Disease onset, median (range) aggiungi 62 (35-75) 65 (51-80) 63
(37-84) Patients treated with specific therapies, N
(%) 0 (0) 20 (100) 20 (100) 97 (92)
Abbreviations: N, number of subjects considered; F, female; %, percentage respect to the total population (N); HC, healthy controls; MS, multiple sclerosis patients; PD, Parkinson’s disease patients; AD, Alzheimer’s disease patients; ALS, Amyotrophic Lateral Sclerosis patients.
Figure Legend
Figure 1. A20 whole blood gene expression levels in MS, PD, AD and ALS patients and HC.
Comparison of median gene expression levels between 40 HC and 34 treatment-naïve MS patients, 20 patients affected by PD, 20 patients affected by AD and 36 patients affected by ALS. The Student t test or Mann–Whitney U test disclosed that A20 was significantly down-regulated in MS and PD patients compared to HC (p=0.0007 and p = 0.0001, respectively). No differences in A20 transcript levels were detected between HC and the other two neurodegenerative diseases, AD and ALS.
