Clinical Relevance of miR-142-3p as Potential Biomarker of Synaptopathy in Multiple Sclerosis
试验速览
- 阶段
- 不适用
- 发起方
- 入组人数
- 1,000
- 试验地点
- 2
- 主要终点
- Clinical disability assessment by Progression Index calculation for correlation analysis with CSF-miR-142-3p levels
研究概览
简要总结
Inflammatory synaptopathy is a prominent pathogenic mechanism in multiple sclerosis (MS) and in its mouse model, which can cause excitotoxic damage by long-lasting excessive synaptic excitation and, consequentially, drives disease progression by leading to motor and cognitive deficits. As synaptopathy occurs early during the disease course and is potentially reversible, it represents an appealing therapeutic target in MS.
Although reliable biomarkers of MS synaptopathy are still missing, recent researches highlighted miR-142-3p as a possible candidate. Indeed, miR-142-3p has been described to promote the IL-1beta-dependent synaptopathy by downregulating GLAST/EAAT1, a crucial glial transporter involved in glutamate homeostasis. Furthermore, mir-142-3p has been suggested as a putative negative MS prognostic factor and a target of current MS disease modifying therapies.
The hypothesis of this study is that miR-142-3p represents a good biomarker for excitotoxic synaptopathy to predict MS course, and, possibly, treatment efficacy at individual level, including both pharmacological strategies and non-pharmacological interventions, like therapeutic transcranial magnetic stimulation (TMS) to ameliorate MS spasticity. To this aim, the role of miR-142-3p in MS synaptopathy, its potential impact on the efficacy of disease-modifying treatments currently used in MS therapy as well as the influence of genetic variants (SNPs) of miR-142-3p and GLAST/EAAT1 coding genes on the responsiveness to therapeutic TMS, will be further investigated in the study. By validating miR-142-3p as potential biomarker of synaptopathy, it is expect to improve MS prognosis and personalized therapies.
Patients with MS, who will undergo neurological assessment, conventional brain MRI scan, and CSF and blood withdrawal for diagnostic and clinical reasons at the Neurology Unit of IRCCS INM-Neuromed will be enrolled in the study. Neurophysiological, biochemical and genetic parameters together with lower limb spasticity will be evaluated. Subjects, who will undergo blood sampling and/or lumbar puncture for clinical suspicions, later on not confirmed, will be recruited as control group.
A subgroup of MS patients showing lower limb spasticity will be included in a two-week repetitive TMS stimulation protocol (iTBS) to correlate the patient responsiveness to this non-pharmacological treatment with MS-significant SNPs of both miR-142-3p and GLAST/EAAT1 coding genes.
详细描述
In the last decade, structural and functional synaptic alterations, collectively known as synaptopathy, have come up as a determinant pathological process contributing to the neurodegenerative damage in multiple sclerosis (MS) and its mouse model, the experimental autoimmune encephalomyelitis (EAE). Since synaptic alteration and loss are reversible, unlike loss of neurons, an early detection could permit a precocious clinical intervention with potentially better therapeutic outcomes but reliable biomarkers are not available yet.
MicroRNAs (miRs) circulating in the cerebrospinal fluids (CSF) are good candidates as possible sensitive biomarkers for MS synaptopathy-driven disease progression. They represent a new class of modulators of gene expression with stable presence in the body fluids and with a critical role in many physiological and pathological processes, especially in the central nervous system. Accordingly, it has been recently demonstrated that miR-142-3p is a crucial component in a detrimental regulatory axis of EAE/MS excitotoxic synaptic dysfunctions, by reducing the level of the glial glutamate aspartate transporter/excitatory amino acid transporter 1 (GLAST/EAAT1) protein. Moreover, miR-142-3p levels are increased in both EAE brains and CSFs of patients with relapsing-remitting MS (RRMS) and correlate with disease progression. Preliminary data also reveal that miR-142-3p is direct target of different pharmacological treatments for MS, while the action of non-pharmacological treatments, as therapeutic transcranial magnetic stimulation (TMS) to ameliorate MS spasticity, is still unknown.
Based on these considerations, a prospective and retrospective cohort study of about six years will be performed to assess whether miR-142-3p is a possible biomarker for MS synaptopathy-driven disease progression (AIM1) and for the efficacy of disease-modifying treatments (DMTs) currently used in MS therapy (AIM2a). Moreover, a genetic screening from peripheral blood will be conducted in order to identify single nucleotide polymorphisms (SNPs) in coding and/or regulating regions of miR-142-3p and GLAST/EAAT1 genes, associated with MS synaptopathy (AIM2b). Finally, a repetitive TMS stimulation protocol (iTBS) will be performed in a subgroup of screened MS patients with lower limb spasticity (interventional substudy) to evaluate the patient responsiveness to the treatment linked to the identified SNPs (AIM2c).
Given the heterogeneity and complexity of MS disease, multivariable approach will permit to dissect miR-142-3p contribution to MS course influenced by synaptopathy (AIM1).
Firstly, miR-142-3p levels in MS CSF (the day of recruitment, T0) will be correlated with other possible variables relevant to disease progression, such as:
研究设计
- 研究类型
- Interventional
- 分配方式
- Non Randomized
- 干预模型
- Parallel
- 主要目的
- Treatment
- 盲法
- None
入排标准
- 年龄范围
- 18 Years 至 65 Years（Adult, Older Adult）
- 性别
- All
- 接受健康志愿者
- 否
入选标准
- •Ability to provide written informed consent to the study;
- •Diagnosis of MS definite according to 2010 revised McDonald's criteria (Polman et al., 2011);
- •Age range 18-65 (included);
- •EDSS range between 0 and 6 (included);
- •Ability to participate to the study protocol.
排除标准
- •Inability to provide written informed consent to the study;
- •Altered blood count;
- •Female with positive pregnancy test at baseline or having active pregnancy plans in the following months after the beginning of the protocol;
- •Contraindications to gadolinium (MRI);
- •Contraindications to TMS;
- •Patients with comorbidities for neurological disease other than MS, included other neurodegenerative chronic diseases or chronic infections (i.e tubercolosis, infectious hepatitis, HIV/AIDS);
- •Unstable medical condition or infections;
- •Use of medications with increased risk of seizures (i.e. Fampridine, 4- Aminopyridine);
- •Concomitant use of drugs that may alter synaptic transmission and plasticity (cannabinoids, L-dopa, antiepiletics, nicotine, baclofen, SSRI, botulinum toxin).
结局指标
主要结局
Clinical disability assessment by Progression Index calculation for correlation analysis with CSF-miR-142-3p levels
时间窗: Changes from T0 (enrollment) to T12 (12 months), T24 (24 months), T36 (36 months), T48 (48 months), T60 (60 months) and T72 (72 months) of follow-up
Clinical disability will be certified by a qualified neurologist through the Progression Index (PI) calculated as EDSS combined with disease duration (EDSS/disease duration). Disease duration is estimated as the number of years from onset to the most recent assessment of disability and EDSS scale ranging from 0 to 10 in 0.5 unit increments that represent higher levels of disability.
CSF concentration of miR-142-3p
时间窗: T0 (enrollment); MS patients vs Control subjects
Quantification of CSF levels of miR-142-3p by qPCR analysis. Relative quantification will be performed by 2\^(-ddCt) method.
Clinical disability assessment by MSFC calculation for correlation analysis with CSF-miR-142-3p levels
时间窗: Changes from T0 (enrollment) to T12 (12 months), T24 (24 months), T36 (36 months), T48 (48 months), T60 (60 months) and T72 (72 months) of follow-up
The Multiple Sclerosis Functional Composite (MSFC) is a three-part composite clinical measure. Three variables were recommended as primary measures: Timed 25-Foot walk; 9-Hole Peg Test; and Paced Auditory Serial Addition Test (PASAT- 3"). The results from each of these three tests are transformed into Z-scores and averaged to yield a composite score for each patient at each time point. There are 3 components: 1. the average scores from the four trials on the 9-HPT; 2. the average scores of two 25-Foot Timed Walk trials; 3. the number correct from the PASAT-3. The scores for these three dimensions are combined to create a single score that can be used to detect change over time. This is done by creating Z-scores for each component. MSFC Score = {Zarm, average + Zleg, average + Zcognitive} / 3.0 (Where Zxxx =Z-score) Increased scores represent deterioration in the 9-HPT and the 25-Foot Timed Walk, whereas decreased scores represent deterioration in the PASAT-3.
Neuroradiological assessment for correlation analysis with CSF-miR-142-3p levels
时间窗: Changes from T0 (enrollment) to T12 (12 months), T24 (24 months), T36 (36 months), T48 (48 months), T60 (60 months) and T72 (72 months) of follow-up
By conventional MRI (1.5 Tesla) the following parameters will be evaluated: dual-echo proton density, FLAIR, T1-WI, T2-WI, and contrast-enhanced T1-WI after intravenous gadolinium (Gd) infusion (0.2 ml/kg). A new Gd+ lesion is defined as a typical area of hyperintense signal on postcontrast T1-WI. A new or newly enlarging lesion on T2-WI is defined as a rounded or oval lesion arising from an area previously considered as normal appearing brain tissue and/or showing an identifiable increase in size from a previously stable-appearing lesion. An active scan is defined as showing any new, enlarging or recurrent lesion(s) on postcontrast T1- and T2-WI.
CSF concentration of soluble molecules
时间窗: T0 (enrollment); MS patients vs Control subjects
Quantification of CSF inflammatory molecules (TNF, IL-1β, IL-6, IL-17, IFN-γ, IL1ra, IL-22, IL-2, IL-2ra, IL-10, IL-4, IL-5, IL-13, IL-12p40, IL-8) by Luminex multiplex assays; neurofilaments, beta amyloid, tau proteins and growth factors (like NGF, PDGF and BDNF) by Luminex multiplex assays. Data will be expressed as pg/ml.
Statistical correlation of miR-142-3p levels in MS CSF with disease and neurophysiological parameters
时间窗: T0 (enrollment), T12 (12 months), T24 (24 months), T36 (36 months), T48 (48 months), T60 (60 months) and T72 (72 months).
To investigate miR-142-3p association with synaptopathy-driven disease progression (measured in terms of clinical or radiological changes and TMS variables), multivariable generalized linear models (GLM) will be applied considering miR level in the CSF as an independent variable adjusting for demographical, clinical, neuroradiological, neurophysiological, biochemical factors and treatments. In the case of unsuccessful identification, Principal Component Analysis (PCA) will be performed to evaluate the miR contribution with other molecules in the CSF (as cytokines, chemokines, growth factors, neurofilaments, beta amyloid and tau protein) to synaptopathy-driven disease progression to reduce the number of variable examined and increase the power of multivariate analysis. Statistical correlations will be repeated on the identified PCA components including miR-142-3p as part of the component. The significance level is established at p\<0.05.
Neurophysiological assessments for correlation analysis with CSF-miR-142-3p levels
时间窗: Changes from T0 (enrollment) to T12 (12 months), T24 (24 months), T36 (36 months), T48 (48 months), T60 (60 months) and T72 (72 months) of follow-up
To assess synaptic excitability by SICI, ICF and LICI, motor thresholds will be calculated at rest as the lowest stimulus intensity able to evoke MEPs of about 50uV in 5 out of 10 consecutive trials (cts), and during a slight voluntary contraction of the target muscle (20-30% of the max voluntary contraction) as the lowest intensity able to evoke MEPs \> 100uV in 5 out of 10 cts. The mean peak-to-peak amplitude of the conditioned MEP (cMEP), at each interstimulus interval (ISI), will be expressed as a percentage of the mean peak-to-peak amplitude of the test MEP (tMEP). PAS-induced LTP-like plasticity will be expressed as changes of the average MEPs size at each time point after PAS compared to the average baseline MEPs size. Before PAS, 25 MEPs, evoked by single TMS pulses over the APB motor hot spot set at an intensity to obtain MEPs size of about 1mV peak-to-peak, will be collected. The same stimulus intensity will be used to obtain 25 MEPs 0', 30' and 60' after PAS.
次要结局
- Genotyping of SNPs in SLC1A3 and MIR-142 genes for correlation analysis with disease parameters(Time Frame: T0 (enrollment); Changes from T0 (enrollment) to T12 (12 months), T24 (24 months), T36 (36 months), T48 (48 months), T60 (60 months) and T72 (72 months) of follow-up)
- Statistical correlation of miR-142-3p levels in MS CSF with patient's responsiveness to disease modifying therapies (DMTs).(Time Frame: T0 (enrollment); Changes from T0 (enrollment) to T12 (12 months), T24 (24 months), T36 (36 months), T48 (48 months), T60 (60 months) and T72 (72 months) of follow-up)
- Statistical correlation of response to iTBS treatment with MS-significant SNPs of both SLC1A3 and MIR-142.(T0 (enrollment); Changes from the starting day (W0) to the end of the 2-week iTBS protocol (W2).)
- Lower limb spasticity assessment by H/M amplitude ratio for the therapeutic TMS substudy(Changes from T0 (enrollment) to T12 (12 months), T24 (24 months), T36 (36 months), T48 (48 months), T60 (60 months) and T72 (72 months) of follow-up; Changes from the starting day (W0) to the end of the 2-week iTBS protocol (W2).)
- Lower limb spasticity assessment by MAS score for the therapeutic TMS substudy(Changes from T0 (enrollment) to T12 (12 months), T24 (24 months), T36 (36 months), T48 (48 months), T60 (60 months) and T72 (72 months) of follow-up; Changes from the starting day (W0) to the end of the 2-week iTBS protocol (W2).)
研究者
Diego Centonze
Haed of Neurology Unit
Neuromed IRCCS