MicroRNA as diagnostic tool and for developing advanced therapies for amyotrophic lateral sclerosis (ALS) of medulla oblongata

Abstract

The patent application describes a microRNA for the diagnosis and advanced therapy development of amyotrophic lateral sclerosis (ALS) of medulla oblongata.

Description

TECHNICAL FIELD

[0001] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting cortical, brainstem and spinal motor neurons, leading to skeletal muscle paralysis and cognitive behavioral impairment. Since the study of riluzole as the first and so far only drug to improve the disease, three decades ago (despite its poor clinical significant efficacy), all subsequent studies were designed using the clinical diagnosis of ALS as the eligibility criterion for patient enrollment. BACKGROUND

[0002] This reflects the hypothesis that any disease subtype, whether familial, sporadic, rapidly or slowly progressive, can have the same pathophysiology and potential response to the same drug. This can be one of the main reasons for the failure of all phase 3 studies so far.

[0003] ALS is a clinically heterogeneous syndrome whose most urgent need is the biological stratification of patients by specific features ranging from aggressive to very slow course, which can be explained by multiple hitherto unknown biological mechanisms. In clinical trials, clinical heterogeneity is usually addressed by dichotomizing into bulbar and spinal forms.

[0004] Bulbar onset, which accounts for about 30% of cases, is the most homogeneous ALS phenotype, both in terms of progression and neuropathological features, and guided the therapeutic effect of riluzole, and more recently of guanabenz (Dalla Bella, E. et al., Brain 144, 2635-2647; 2021).

[0005] The bulbar onset phenotype is the most devastating variant of ALS, with rapid decline in swallowing, speech and shorter survival.

[0006] Its clinical course is characterized by early involvement of upper motor neurons and cognitive behavioral aspects.

[0007] Despite this, the study of bulbar ALS is relatively underrepresented and basic research data is scarce, due to limited availability of biological samples and lack of preclinical models summarizing the human phenotype.

[0008] Regardless of the classification method, bulbar onset is considered a unique phenotype.

[0009] The publication by Benigni Michele et al. (“Identification of miRNAs as Potential Biomarkers in Cerebrospinal Fluid from Amyotrophic Lateral Sclerosis Patients,” NEUROMOLECULAR MEDICINE, HUMANA PRESS, US, vol. 18, no. 4, 27 April 2016, pages 551-560) describes the use of miR-150-5p as a diagnostic biomarker for ALS, but not for the specific diagnosis of medullary-onset ALS.

[0010] The publication by Mignzhu Liu et al. (“The IGF2 intronic miR-483 selectively enhances transcription from IGF2 fetal promoters and enhances tumorigenesis”, GENESAND DEVELOPMENT, vol. 27, no. 23, 1 December 2013, pages 2543-8) describes the use of miR-483-5p as a diagnostic biomarker for ALS, but not for the specific diagnosis of medullary-onset ALS.

[0011] The publication by Liguori Maria et al. (“Dysregulation of MicroRNA and Target Genes Networks in Peripheral Blood of Patients With Sporadic Amyotrophic Lateral Sclerosis,” FRONTIERS IN MOLECULAR NEUROSCIENCE, vol. 11, 28 August 2018) describes miR-185-5p to differentiate the medullary onset of ALS. Summary of the Invention

[0012] The inventors of this patent application have made a surprising discovery that certain microRNAs are indicators of medullary-onset ALS forms; therefore, these can be used as advanced diagnostic tools or for developing advanced therapies targeting this specific subtype of disease.

[0013] The purpose of this invention

[0014] In the first objective of this invention, the use of specific microRNAs in the diagnosis of medullary-onset amyotrophic lateral sclerosis (ALS) is described.

[0015] In a second objective of the present invention, a diagnostic method for diagnosing amyotrophic lateral sclerosis (ALS) with medullary onset is described.

[0016] In a third objective of this invention, the use of the same microRNA as a potential target for developing novel treatments for medullary-onset amyotrophic lateral sclerosis (ALS) is described.

[0017] In one respect, the specific microRNAs described may be used to identify compounds for specific therapies for medullary-onset amyotrophic lateral sclerosis (ALS).

[0018] In a fourth objective of the invention, specific microRNAs are described for evaluating the response to treatment of patients with medullary-onset amyotrophic lateral sclerosis (ALS) using guanethidine or drugs that act on endoplasmic reticulum stress and on cellular responses defined as unfolded protein responses. Attached Figure Description

[0019] Figure 1 The values ​​of downregulated miR-150-5p and miR-483-5p in the serum of patients with medullary-onset ALS are shown.

[0020] Figure 2 The receiver operating characteristic (ROC) for evaluating the sensitivity and specificity of miR-150-5p and miR-483-5p as biomarkers distinguishing subgroups of patients with medullary-onset ALS is shown.

[0021] Figure 3 The results are shown from proteomic analyses of serum from patients with medullary and spinal cord-onset ALS and corresponding age- and sex-related healthy controls. Principal component analysis (PCA) and volcano plots show how the expression levels of identified proteins correlate with phenotype specificity in the medulla oblongata, spinal cord, and healthy controls.

[0022] Figure 4 This study showed that serum miR-106b-5p expression levels were downregulated in patients with medullary-onset ALS after treatment with guanethidine 32 mg / 64 mg, compared to patients treated with placebo / 16 mg. Interestingly, the expression levels of this microRNA tended to normalize and had expression values ​​similar to those observed in the serum of healthy controls. Figure 4As shown, this phenomenon was not confirmed in patients with spinal cord-onset ALS, where the expression level of this molecule remained upregulated relative to healthy controls even after treatment. Detailed Implementation

[0023] In the first objective of this invention, the use of specific microRNAs in the diagnosis of medullary-onset amyotrophic lateral sclerosis (ALS) is described.

[0024] Specifically, the microRNA is:

[0025]

[0026] Specifically, downregulation of miR-150-5p and miR-483-5p is phenotype-specific to medullary-onset amyotrophic lateral sclerosis (ALS) and determines the biological classification of this phenotype.

[0027] For the purposes of this invention, the downregulation of the indicative diagnostic method is the downregulation of one or both of the aforementioned microRNAs, i.e., simultaneous downregulation.

[0028] Therefore, for the second purpose, a diagnostic method is described for diagnosing this specific form of disease in patients suspected of having medullary-onset amyotrophic lateral sclerosis (ALS).

[0029] More specifically, this method includes the step of determining the expression levels of specific miR-150-5p and / or miR-483-5p microRNAs in biological samples isolated from patients suspected of having the disease.

[0030] More specifically, the biological sample isolated from the patient suspected of having the disease is represented by a sample isolated from the patient's serum.

[0031] Expression levels can be determined using techniques known in the art; in particular, they can be determined using real-time PCR (rt-PCR).

[0032] The diagnosis was confirmed when the expression of the microRNA was downregulated.

[0033] For example, the following average results were obtained:

[0034]

[0035] In a third objective of this invention, the use of specific microRNAs as potential targets for developing novel treatments for medullary-onset amyotrophic lateral sclerosis (ALS) is described.

[0036] Specifically, the microRNAs are represented by miR-150-5p and miR-483-5p.

[0037] Regulating the expression of specific microRNAs, miR-150-5p and miR-483-5p, to physiological levels allows for the evaluation and development of novel therapies for medullary-onset amyotrophic lateral sclerosis (ALS).

[0038] In fact, such therapies are effective when they downregulate the expression of the two specific miRNAs, either independently or both.

[0039] In a fourth objective of the invention, the medical use of a specific microRNA for treating patients with medullary-onset amyotrophic lateral sclerosis (ALS) is described, the treatment using guanethidine or drugs that act on endoplasmic reticulum stress and on cellular responses defined as unfolded protein responses.

[0040] Specifically, the medical use is to evaluate the response of patients with medullary-onset amyotrophic lateral sclerosis (ALS) to treatment with guanethidine.

[0041] More specifically, treatment with guanethidine involves administering 32 mg or 64 mg (daily dose) to patients who require guanethidine.

[0042] For the purposes of this invention, the specific microRNA is represented by miR-106b-5p:

[0043]

[0044] Specifically, the medical uses of miR-106b-5p microRNA include assessing the expression level of said microRNA in patient serum samples isolated during treatment with guanethidine.

[0045] The expression levels of the miRNAs will provide an indication of the effectiveness of drug treatment.

[0046] In fact, surprisingly, it was found that the expression level of miR-106b-5p in the serum of patients with medullary-onset amyotrophic lateral sclerosis (ALS) was downregulated after treatment with guanethidine at doses of 32 mg or 64 mg (daily dose).

[0047] Following treatment, this expression level showed a trend similar to that observed in the serum of healthy controls.

[0048] Experimental Section

[0049] To uncover the unique molecular signature of medullary onset of ALS, an integrated serum microRNA and proteomics profile was constructed. In the first exploratory phase, a cohort of 26 ALS patients and 28 healthy controls (HCs) were included in the study, and their serum samples were analyzed using a TaqMan human microRNA array containing 754 miRNAs via microfluidic technology. In a second, separate cohort of 28 ALS patients and 22 HCs, the expression of selected circulating microRNAs from the exploratory phase was validated using real-time PCR with specific TaqMan analysis. Proteomics analysis was performed on serum samples from a subgroup of 14 ALS patients and 6 HCs included in the selected miRNA validation study. The integration of molecular data from the analysis of miRNA expression levels and proteomics data revealed a unique profile of patients with medullary onset ALS.

[0050] Demographic and clinical features

[0051] This study included a total of 54 ALS patients and 50 HC patients who met the diagnostic criteria for ALS. They were recruited from the Motor NeuronDisease Center of the IRCCS Fondazione Istituto Neurologico Carlo Besta in Milan, Italy, as part of the TRANS-ALS project (authorization number: 2015-0023, recipient: GL) and INTERSLA (authorization number: 1157625, recipient: GL). There were no significant differences in sex (exploratory phase p=0.67, validation phase p=0.85) and age (exploratory phase p=0.74, validation phase p=0.62) between ALS and HC patients. The diagnosis of medullary onset was based on the presence of dysarthria or dysphagia, tongue atrophy, and fasciculations within the first 6 months of symptom onset, but without lower motor neuron involvement of the spinal cord, accompanied by or subsequently with signs of upper motor neuron involvement. Pseudobulbar onset, defined as a marked medullary feature due to upper motor neuron involvement, was excluded from all patients receiving transcranial cortical-medullobulatory magnetic stimulation.

[0052] Exploratory phase - Identification of dysregulated microRNAs in serum of patients with bulbar onset ALS .

[0053] Using microfluidic technology, expression profiling of 754 miRNAs was performed on serum samples from 26 ALS and 28 HC patients to identify molecules potentially associated with medullary onset phenotypes. Specifically, differentially expressed microRNAs from the following patients were considered: ALS with medullary onset (7 patients), spinal cord onset (19 patients), spinal cord onset with medullary injury due to ALS progression (16 patients), and spinal cord onset without medullary injury (10 patients). During the exploratory phase, four microRNAs (miR-885-5p, miR-150-5p, miR-483-5p, and miR-342-3p) were significantly downregulated in the serum of patients with medullary onset compared to those with spinal cord onset and HC (Table 1).

[0054] Table 1

[0055]

[0056] No differentially expressed microRNAs were found in ALS patients with spinal cord onset accompanied by medullary injury.

[0057] Validation phase - Identification of miR-150-5p and miR-483-5p circulating as biomarkers of bulbar onset ALS .

[0058] To validate the data obtained in the exploratory phase, four microRNAs selected in the exploratory phase were analyzed by real-time PCR in the serum of another 28 ALS patients and 22 HC patients. The results showed that, compared with patients with spinal cord onset and HC, the expression levels of miR-150-5p and miR-483-5p were significantly downregulated in patients with medullary onset. Figure 1 This is consistent with the results observed in the exploratory phase. Receiver operating characteristic (ROC) curve analysis showed that the expression levels of circulating miRNAs (miR-150-5p and miR483-3p) could distinguish between medullary onset and spinal cord phenotypes with high sensitivity and specificity (miR-150-5p AUC=76% and miR-483-5p AUC=81%) and HC (miR-150-5p AUC=76% and miR-483-5p AUC=96%). Figure 2 To test whether the expression levels of miR-150-5p and miR-483-5p were correlated, Spearman correlation analysis was performed. This analysis found no significant correlation between serum miRNA levels (r=0.26), indicating different pathophysiological roles of the two miRNAs at different regulatory levels.

[0059] Proteomic analysis

[0060] In addition to microRNA expression analysis, extracellular vesicles (EVs) obtained from the serum of 7 patients with medullary-onset ALS, 7 patients with spinal cord-onset ALS, and 6 patients with HC were analyzed using a platform based on liquid-nanochromatography and high-resolution tandem mass spectrometry (nLC-hrMS / MS). A total of 1175 proteins with at least one unique peptide were identified, and unlabeled comparisons were made under the three detection conditions based on the peak intensity of the unique peptide precursor ion. The distribution and abundance of proteins among the groups were assessed using Venn diagrams and box plots, respectively, revealing a high percentage of shared proteins (82%) and very similar normalized abundance distributions among the sample groups. Volcano plots and PCA (…) Figure 3 Three protein clusters were identified, revealing distinct proteomic profiles in patients with medullary-onset ALS compared to those with spinal cord-onset ALS and healthy controls.

[0061] To investigate differentially expressed proteins, three pairs of comparisons were performed (patients with medullary onset vs. Hc; patients with spinal cord onset vs. Hc; patients with medullary onset vs. patients with spinal cord onset), taking into account group abundance values. A confidence threshold of 1.5 was applied to the Log2 fold change (FC), and a confidence threshold of 0.05 was applied to the corrected p-value. A total of 295 proteins were identified that exceeded the set thresholds in at least one comparison: 98 upregulated and 96 downregulated proteins were identified in patients with medullary onset and Hc; 103 upregulated and 100 downregulated proteins were identified in patients with spinal cord onset and Hc; and 80 upregulated and 89 downregulated proteins were identified in patients with medullary onset and spinal cord onset. Proteomics analysis revealed a unique protein profile, with extreme FC and p-values ​​in the medullary onset condition, strongly suggesting that the selected molecular factors may influence this phenotype. Based on the STRING annotation terminology of biological processes, a functional network was constructed to display the connections between protein levels and their biological pathways. Possible interactions between proteins were highlighted, and differential protein expression was shown in patients with medullary ALS compared to those with spinal cord onset. These proteins are involved in gene processing, immune responses, endometrial systems, adhesion and musculoskeletal activity, proliferation, protein metabolism, muscle function, stress response, and energy metabolism. The network displays a unique protein expression profile for patients with medullary onset phenotypes.

[0062] Integration of microRNA and proteomic profiling in serum of patients with bulbar onset ALS

[0063] To identify unique molecular signatures, an integrative analysis was performed between two candidate miRNAs (miR-150-5p and miR-483-5p) and differentially selected proteins expressed in patients with medullary-onset ALS and spinal cord-onset ALS, as well as in HC. Spearman correlation analysis was applied to the abundance of each miRNA and the selected proteins to identify relationships between them. Since miRNAs and RNA-binding proteins (RBPs) have an increasing association on the basis of mutual regulation, positive and negative correlations were analyzed using coefficients (r) ≥ 0.5 or ≤ -0.5 with p < 0.05 as measures of direct and inverse correlation. Three positive correlations and ten negative correlations were identified between miR-150-5p and the selected proteins associated only with the medullary-onset phenotype. It should be noted that a positive correlation was found between miR-150-5p and DDP9 protein, while a negative correlation was found between miRNA and the reticular membrane protein complex (EMC3), grainyhead like transcription factor 2 (GRHL2), and dolichyl-diphosphooligosaccharide-protein glycosyltransferase non-catalytic subunit protein (DDSOT), whose genes are predicted targets of miR-150-5p. Furthermore, 31 negative correlations and 25 positive correlations were found between miR-483-5p and selected proteins expressed only in the medullary-onset phenotype. Interestingly, the predicted miRNA target, namely the DENN domain containing 6A (DENND6A), was positively correlated with miR-483-5p. In patients with medullary-onset ALS, the biological processes and functions associated with the selected molecules include immune responses, endometrial systems, protein metabolism, energy metabolism, synapses, ion homeostasis, adhesion-motor function, muscle, stress responses, apoptosis, genetic information processing, and proliferation. Molecular integration data revealed possible mechanisms mediated by microRNAs at different regulatory levels, confirming the independent and unrelated roles of the two selected microRNAs.

[0064] A functional network was constructed to demonstrate the connections between two microRNAs and positively / negatively correlated proteins, as well as the connections between proteins and annotated biological processes based on STRING annotation. These biological processes include immune responses, endomembrane systems, protein metabolism, energy metabolism, synapses, ion homeostasis, adhesion-motor activity, muscle, stress responses, apoptosis, genetic information processing, and proliferation.

[0065] Analysis of miR-106b-5p expression levels in bulbar onset patients treated with guanabenz

[0066] Serum miR-106b-5p expression levels were analyzed in 12 patients with medullary onset (5 receiving placebo / 16 mg and 7 receiving 32 mg / 64 mg guanethidine) and 14 patients with spinal cord onset (10 receiving placebo / 16 mg and 5 receiving 32 mg / 64 mg guanethidine). Figure 4 Molecular analysis showed that, compared with patients treated with placebo / 16 mg guanethidine, serum miR-106b-5p expression levels in patients with medullary-onset ALS treated with 32 mg / 64 mg guanethidine were normalized. In contrast, miR-106b-5p expression levels remained significantly higher in patients with spinal cord onset and in all treatment groups.

[0067] method

[0068] Healthy patients and controls

[0069] This study included 54 ALS patients with in-depth phenotypic analysis who met the diagnostic criteria for ALS and 50 age- and sex-matched HCs. Patients were grouped into medullary-onset and spinal-onset groups according to published valid criteria. Serum samples from 26 ALS patients and 28 HCs were used for miRNA analysis (exploratory phase), while serum samples from another 28 ALS patients and 22 HCs were used for miRNA validation experiments (validation phase). For proteomics analysis, 7 patients with medullary-onset, 7 patients with spinal-onset, and 6 age- and sex-matched HCs were selected, and all subjects underwent prior miRNA profiling. No significant sex-related differences were observed between ALS and HC in either the exploration (p=0.67) or validation (p=0.85) phases. Comparing ALS and HC, no significant age differences were observed in either the exploration (p=t-test=0.74) or validation (p=t-test=0.62) phases. This study was conducted in accordance with the ethical standards of the Declaration of Helsinki. According to a statement from the World Medical Association, the investigation and use of patient data for research purposes has been approved by the Ethics Committee of the Carlo Besta Institute of Neurology Foundation of IRCCS.

[0070] Genetic screening

[0071] All patients underwent next-generation sequencing (NGS) of ALS-related genes, with deep amplicon sequencing performed using the Sure Select QXT kit (Agilent) for SOD1, FUS, TARDBP, VCP, OPTN, SQSTM1, TUBA4A, PFN1, UBQLN2, and Amplidex PCR repeats. Kit PCR / CE C9ORF72 (Asuragen Inc. Austin TX) was used to detect C9orf72 amplification. DNA was extracted from peripheral blood using standard procedures. Variants were screened according to the following criteria: (i) variants in the coding region or within a 20 bp flank; (ii) deletions or rare variants with an allele frequency (AF) <1% in population databases (dbSNP137, ESP6500, the 1000 Genome Project, and ExAC). Given the very low prevalence of ALS, genetic variants with a frequency >1% in dbSNP, ExomeVariant Server, or ExAC were classified as “benign” (Category 1) and excluded from subsequent analyses. The influence of synonymous or intron variants on the linker site was predicted using at least two linker prediction tools: the NNSplice predictor ( / / www.fruitfly.org / seq tools / splice.html); and ASSP (http: / / wangcomputing.com / assp / ). The best candidate variants were validated using the Sanger method. According to the criteria proposed by the American College of Medical Genetics and Genomics (ACMG), the genetic variants that passed the filtering process were classified as "pathogenic" (class 5), "probably pathogenic" (class 4), "variants of unknown significance" (VUS, class 3), and "probably benign" (class 2).

[0072] MicroRNA profiling and data analysis

[0073] Total RNA was extracted from serum obtained from 26 ALS patients and 28 healthy controls using the miRNeasy serum / plasma kit (Qiagen, Venlo, Netherlands). RNA quality was examined using a 2100 Nano Bioanalyzer (Agilent Technologies), and reverse transcription of the RNA was performed using Megaplex RT Human Pool A and B primers and a MultiScribe reverse transcriptase kit. cDNA corresponding to 170 ng of total RNA was mixed with the TaqMan Universal PCR Master Mix and distributed into each port of the TaqMan Human MicroRNA A and B v2.0 array plates according to the manufacturer's instructions. The arrays were run on a Via 7 rapid real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Human array plates A and B contained primers for 754 miRNAs, including 3 positive control miRNAs and 1 negative control. Relative thresholding (Crt) was applied. Only miRNAs with good amplification quality (amplification score > 1 and Cq confidence > 0.8) were included in the analysis. Differential expression was quantified as a relative quantification using the 2-ΔΔct method, with miR-24 serving as a normalized endogenous control42 and spinal cord SLA or healthy control samples used as the reference group. ΔΔCq was calculated as mean ΔCq (target miRNA in the medullary-onset ALS group) - mean ΔCq (target miRNA in the reference group). Fold change in expression was calculated as 2 - (ΔΔCq). For a decrease in expression in the target group relative to the reference group, it was converted to the negative reciprocal of 2 - (ΔΔCq) to obtain the reduction in fold change in expression.

[0074] Verification of miRNAs by real-time PCR

[0075] Total RNA was extracted from the serum of 28 ALS and 22 HC patients using the miRNeasy serum / plasma kit (Qiagen). RNA quality was checked using a 2100Nano bioanalyzer (Agilent Technologies). RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit and specific primers for selected miRNAs (miR-150-5p, miR-483-5p, miR-885-5p, miR-342-3p, and miR-24 as an endogenous control). Forty-three cDNAs (corresponding to 100 ng of total RNA) were amplified in duplicate by real-time PCR using a universal PCR master mix and pre-designed TaqMan MicroRNA assay on a Via 7 rapid real-time PCR system (Thermo Fisher Scientific). All results were normalized relative to miR-24, and relative miRNA expression levels were calculated using the ΔCt method.

[0076] Protein extraction and enzymatic digestion

[0077] Proteomic analysis was performed on EV serum extracts from ALS patients with medullary onset (N=7), ALS patients with spinal cord onset (N=7), and healthy controls (N=6), which had previously been analyzed for miRNA assays. EVs were isolated from 250 μL of pre-purified serum according to the manufacturer's protocol using the ExoQuick ULTRA kit (System Biosciences, Palo Alto, CA, USA). Briefly, serum samples were incubated with ExoQuick 42 reagent at 4°C for min and then centrifuged at 3000×g for 10 min. The precipitate was collected, resuspended in 500 µL of ExoQuick Ultra swabs, and loaded into a pre-cleaned resin column. After mixing, purified EVs were obtained by centrifugation at 1000×g for 2 min and analyzed using the Qubit™ Protein Assay Kit (Life Technologies Corp., Eugene, OR, USA) to determine protein concentration. For each sample, 50 µg of the protein mixture was reduced / alkylated and enzymatically digested using the Easy Pep Mini MS Sample Preparation Kit (Thermo Fisher Scientific). According to the kit protocol, within less than 3 hours, for each detection condition, peptides were generated, washed to prepare detergent-free samples, and resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA) for nLC-hrMS / MS analysis.

[0078] LC-MS / MS analysis

[0079] Peptide mixtures were analyzed in trap-elute mode using the Eksigent nanoLC-Ultra 2D system (Eksigent, part of AB SCIEX, Dublin, CA, USA) combined with the cHiPLC-nanoflex system. Briefly, for each condition, two technical replicates were performed by injecting 0.8 µg of protein onto the cHiPLC trap (200 µm x 500 µm ChromXP C18-CL, 3 µm, 120 Å, Eksigent, part of AB SCIEX, Dublin, CA, USA) and running the loading pump at a flow rate of 3 µL / min for 10 min in isocratic mode with 0.1% formic acid in water. The captured mixture was then eluted onto a nano cHiPLC column (75 µm x 15 cm ChromXP C18-CL, 3 µm, 120 Å, Eksigent, part of AB SCIEX, Dublin, CA, USA) using an automatic switching of the ten-port valve. A 115-min gradient of eluent B (eluent A: 0.1% formic acid in water; eluent B: 0.1% formic acid in acetonitrile) was employed at a flow rate of 300 nL / min. Specifically, the gradient was 5–15% B over 3 min, 15–30% B over 90 min, 30–40% B over 6 min, 40–95% B over 6 min, and held at 95% B for 11 min. The eluted peptides were analyzed directly on an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific) equipped with an EASY-Spray ion source. Easy spray was obtained using an EASY-Spray emitter (Thermo Fisher Scientific) maintained at 1.6 kV (7 µm IDTransfer Line 20 µm x 50 cm nanoflow), while the ion transfer capillary was maintained at 220 °C. Data correlation acquisition (DDA) was performed, acquiring precursor ions in the m / z range of 375–1,250 at a resolution of 60,000 FWHM (full width at half maximum) (at m / z 200). Precursor fragmentation was performed at a resolution of 15,000 FWHM (at m / z 200) using high-energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 30 eV and dynamic repulsion of 20 seconds. MS and MS / MS data were acquired in profile and centroid modes, respectively, using positive polarity and active isotope exclusion. The insulation width was set to 2 m / z, and the first mass was set to 120 m / z.The mass spectrometer scanning function and solvent gradient of the high performance liquid chromatography were controlled by the Xcalibur version 4.4 data system (Thermo Fisher Scientific) and the Eksigent version 4.3 control software (Eksigent, part of AB SCIEX, Dublin, CA, USA).

[0080] Data management

[0081] All generated raw data were retrieved using the Sequest HT search engine included in Proteome Discoverer software version 2.5 (Thermo Fisher Scientific), in March 2021, from the Homo sapiens proteome database (75,550 records) downloaded from Uniprot (www.uniprot.org). Prior to Sequest HT, an MSPepSearch node was inserted into the processing workflow for an initial search in the spectral library (NIST Human Orbitrap HCD library, 1,127,970 spectra, September 2016). The following criteria were used to identify peptide sequences and associated proteins: a mass tolerance of ±10 ppm for precursor ions and ±0.02 Da for fragment ions; trypsin as an enzyme with a maximum of two non-cleavage tolerances; aminomethylation of cysteine ​​as a fixed modification; and methionine oxidation as a variable modification. Filter nodes were used in conjunction with a target-decoy strategy, taking into account the maximum deltaCN for XCorr > 1.2 and 0.05 based on q-values ​​to provide false discovery rates (FDR) of 1% (strict) and 5% (lenient). Label-free quantification (LFQ) of 43 proteins was performed through non-nested studies with biological and technical reproducibility, using recalibrated spectral files and the Minora algorithm, where peak intensities of precursor ions of unique peptides used for abundance calculations were employed when 60% reproducibility was achieved. A pairwise approach was established for comparing normalized protein abundances between groups of tested samples. Protein grouping and strict retention principles were also applied. Statistical analysis of protein quantification results was performed using a background-based t-test, and differentially expressed proteins (DEPs) between compared experimental groups were screened if a log2 fold change threshold ≥ |1.5| was exceeded and a p-value ≤ 0.05. Volcano plots, heatmaps, and PCA were obtained using predefined functions of Proteome Discoverer 2.5. Cluster analysis was performed on the distances and clusters of all proteins using Euclidean averaging.

[0082] Protein network analysis

[0083] Starting with DEP, a protein-protein interaction (PPI) network (268 nodes and 3,532 boundaries) was constructed experimentally from the STRING45 database, taking into account PPIs defined in the database with scores >0.15. The resulting subnetworks were viewed and analyzed using Cytoscape v. 3.9.1 and its plugins 46 and 47. Proteins were grouped into functional modules using default settings with STRING enrichment enabled. Node colors reflect DEP expression levels under each condition based on normalized abundance clustering values ​​(abundance clustering values ​​normalized to 0-100, with the highest abundance value for each protein set to 100).

[0084] Correlation analysis and functional miRNA-protein-biological process network construction

[0085] Based on the expression and protein abundance of miR-150-5p and miR483-5p, Spearman correlation coefficients and p-values ​​were calculated for each miRNA-protein pair. Spearman correlation coefficients ≥0.5 or ≤-0.5 and p-values ​​<0.05 were considered statistically significant. Cytoscape (v3.9.1)48 was used to create and visualize functional networks that link miRNAs to proteins and link related proteins to related functional biological processes.

[0086] Statistical analysis

[0087] t-tests were used to compare demographic and clinical continuous variable groups, while chi-square tests were used to compare categorical data. In both the exploration and validation phases, post-hoc analyses using the Kruskal-Wallis rank-sum test and Dunn test were performed to conduct multiple comparisons of medullary and spinal cord onset miRNA expression levels in the ALS and HC groups. The Benjamini-Hochberg (BH) false discovery rate (FDR) test was used to correct for multiple tests. Differences with an FDR p-value less than 0.05 were considered statistically significant. Spearman correlation coefficients were used to assess the correlation between miRNA expression levels and clinical characteristics of ALS patients. ROC curves were used to evaluate the sensitivity and specificity of miR-150-5p and miR483-5p in human serum samples as biomarkers distinguishing between medullary and spinal ALS. Statistical analyses were performed using the statistical programming language R, version 3.6, and STATA 11 software.

[0088] Analysis of miR-106b-5p expression levels

[0089] Total RNA was extracted from the serum of 12 patients with medullary onset (5 receiving placebo / 16 mg and 7 receiving 32 mg / 64 mg guanethidine) and 14 patients with spinal cord onset (including 10 receiving placebo / 16 mg and 5 receiving 32 mg / 64 mg guanethidine) using the miRNeasy serum / plasma kit (Qiagen). RNA quality was assessed using a 2100Nano bioanalyzer (Agilent Technologies). RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit and specific primers for selected miRNAs (miR-106b-5p and miR-24 as an endogenous control). cDNA (corresponding to 100 ng of total RNA) was amplified in duplicate by real-time PCR using a Via 7 rapid real-time PCR system (Thermo Fisher Scientific) with a universal PCR master mix and pre-designed TaqMan MicroRNA assay. All results were normalized relative to miR-24, and relative miRNA expression levels were calculated using the ΔCt method.

[0090] The advantages provided by the present invention will become immediately clear from the above description.

[0091] First, it provides an important new advanced diagnostic tool for medullary-onset forms of amyotrophic lateral sclerosis (ALS), which have previously received relatively little attention.

[0092] The microRNAs described are circulating molecules in patient serum, and therefore can be identified using an easy-to-implement, non-invasive method.

[0093] With the help of the content provided in this invention, it will be possible to develop new treatment methods for amyotrophic lateral sclerosis (ALS) with medullary onset, which is the most destructive form of this neurodegenerative disease.

[0094] The method provided by this invention has proven to be more advantageous because it offers greater reliability and effectiveness by means of a broader database being analyzed; furthermore, the method of this invention has been validated from both biological and technical perspectives.

[0095] Therefore, in general, this method is very robust and reliable in distinguishing the forms of ALS with medullary onset.

[0096] Furthermore, by using the identified reactive biomarkers, this invention enables more effective and advanced monitoring of guanethidine in patients with medullary-onset amyotrophic lateral sclerosis (ALS).

Claims

1. Use of a specific microRNA in the diagnosis of amyotrophic lateral sclerosis (ALS) with bulbar onset, wherein said specific microRNA is represented by: 。 2. Use of a specific microRNA according to the preceding claim in the diagnosis of amyotrophic lateral sclerosis (ALS) with bulbar onset, wherein the expression of said microRNA is down-regulated.

3. Use of a specific microRNA in the diagnosis of amyotrophic lateral sclerosis (ALS) with bulbar onset, wherein said expression is measured in an isolated serum sample of a patient suspected of suffering from said disease.

4. Diagnostic method for the diagnosis of amyotrophic lateral sclerosis (ALS) with bulbar onset in a patient suspected of suffering from this specific form of disease, comprising the step of determining the expression of a microRNA in an isolated biological sample of a patient suspected of suffering from said disease, said microRNA being represented by: 。 5. Diagnostic method according to the preceding claim, wherein said expression is determined in an isolated serum sample of said patient.

6. microRNA for medical use for the treatment of a patient suffering from amyotrophic lateral sclerosis (ALS) with bulbar onset, said treatment using guanabenz or a drug acting on endoplasmic reticulum stress and on the cellular response defined as unfolded protein response, wherein said microRNA is represented by: 。 7. microRNA for medical use for the treatment of a patient suffering from amyotrophic lateral sclerosis (ALS) with bulbar onset according to the preceding claim, wherein said guanabenz is administered at a dose of 32 mg or 64 mg per day.

8. microRNA for medical use for the treatment of a patient suffering from amyotrophic lateral sclerosis (ALS) with bulbar onset according to the preceding claim 6 or 7, wherein the expression value of said microRNA is determined.

9. microRNA for medical use for the treatment of a patient suffering from amyotrophic lateral sclerosis (ALS) with bulbar onset according to the preceding claim, wherein said expression value is determined in an isolated serum sample of said patient.

10. Use of a microRNA for the development of a therapeutic method for amyotrophic lateral sclerosis (ALS) with bulbar onset, wherein said microRNA is represented by: 。

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