MicroRNA for the development of diagnostic tools and advanced treatments for bulbar-onset amyotrophic lateral sclerosis (ALS)

Abstract

This invention provides microRNAs for the diagnosis and development of advanced therapies for bulbar-onset amyotrophic lateral sclerosis (ALS).

Description

Technical Field

[0001] Description Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons in the cortex, brainstem, and spinal cord are invaded, causing skeletal muscle paralysis and cognitive-behavioral disorders. In the past 30 years since riluzole was first introduced and remains the only drug to improve the disease so far, although the clinical effects have been poor, all subsequent studies have been conducted using the clinical diagnosis of ALS as the eligibility criteria for patient enrollment.

[0002] This reflects the hypothesis that all disease subtypes, regardless of whether they are familial, sporadic, rapidly progressive, or slowly progressive, share the same pathophysiology and may respond to the same drugs. Perhaps this is one of the main reasons why all previous phase III clinical trials have ended in failure.

Background Art

[0003] ALS is a clinically heterogeneous syndrome, and its most urgent need is to biologically stratify patients by specific symptoms ranging from rapid progression to very slow progression, which are probably explained by some yet unknown biological mechanisms. In clinical trials, clinical heterogeneity is generally resolved by dichotomizing into bulbar-onset and spinal-onset types.

[0004] The bulbar-onset type accounts for about 30% of cases and is the most homogeneous ALS phenotype in terms of both progression rate and neuropathological characteristics, guiding the treatment effects of riluzole and more recently guanabenz (Dalla Bella, E., et al. Brain 144, 2635-2647; 2021).

[0005] The bulbar-onset phenotype is the most severe variant of ALS, accompanied by a rapid decline in swallowing and speech functions and a short survival period.

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

[0007] Nevertheless, bulbar palsy-type ALS is relatively understudied, with limited access to biological samples and a lack of preclinical models that summarize human phenotypes, resulting in scarcity of basic research data.

[0008] Regardless of the classification system, bulbar palsy is recognized as a unique phenotype.

[0009] The paper 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 does not use it for the specific diagnosis of bulbar palsy-type ALS.

[0010] The paper by Mignzhu Liu et al. ("The IGF2 intronic miR-483 selectively enhances transcription from IGF2 fetal promoters and enhances tumorigenesis", GENES AND 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 does not use it for the specific diagnosis of bulbar palsy-onset ALS.

[0011] Liguori Maria et al.'s paper ("Dysregulation of MicroRNA and Targetes Networks in Peripheral Blood of Patients With Sporadic Amyotrophic Lateral Sclerosis," FRONTIERS IN MOLECULAR NEUROSCIENCE, vol.11, 28 August 2018) describes miR-185-5p for distinguishing bulbar palsy-onset ALS. [Overview of the Initiative]

[0012] Summary of the Invention The inventors of this invention have surprisingly discovered that specific microRNAs serve as indicators of bulbar palsy-type ALS. Therefore, these can be used as advanced diagnostic tools or in the development of advanced treatments for this particular disease subtype.

[0013] Purpose of the invention In the first object of the present invention, the use of specific microRNAs in the diagnosis of bulbar-onset amyotrophic lateral sclerosis (ALS) is described.

[0014] In a second object of the present invention, a method for diagnosing bulbar-onset amyotrophic lateral sclerosis (ALS) is described.

[0015] In a third object of the present invention, the use of the same microRNA described above is described as a potential target for developing a new therapeutic approach for bulbar amyotrophic lateral sclerosis (ALS).

[0016] In one embodiment, the described specific microRNAs can be used to identify compounds for the specific treatment of bulbar amyotrophic lateral sclerosis (ALS).

[0017] A fourth object of the present invention describes a pharmaceutical application for evaluating the response of patients with bulbar-onset amyotrophic lateral sclerosis (ALS) to treatment with drugs that act on cellular responses defined as guanabenz or endoplasmic reticulum stress and unfolded protein responses. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 shows the decreased expression levels of miR-150-5p and miR-483-5p in the serum of patients with bulbar palsy-type ALS. [Figure 2] Figure 2 shows the "Receiver Operating Characteristic (ROC)" used to evaluate the sensitivity and specificity of miR-150-5p and miR-483-5p as biomarkers for identifying a subgroup of patients with bulbar palsy-type ALS. [Figure 3] Figure 3 shows the results obtained from proteomic analysis of serum from patients with bulbar palsy and spinal cord onset ALS, as well as from age- and sex-matched healthy controls. Principal component analysis (PCA) and volcano plots show how the expression levels of identified proteins are specifically associated with the bulbar palsy, spinal cord onset, and healthy control phenotypes. [Figure 4] Figure 4 shows that the expression level of miR-106b-5p in the serum of bulbar-onset ALS patients after administration of guanabenz 32 mg / 64 mg was lower than that of patients administered placebo / 16 mg. Interestingly, the expression levels of this microRNA showed a tendency towards normalization, and were close to the values ​​observed in the serum of healthy control groups. As shown in Figure 4, this phenomenon was not observed in spinal cord-onset ALS patients, and their levels remained elevated compared to healthy control groups even after treatment. [Modes for carrying out the invention]

[0019] Detailed description of the invention A first object of the present invention is to describe the use of specific microRNAs in the diagnosis of bulbar-onset amyotrophic lateral sclerosis (ALS).

[0020] In particular, said microRNAs are as follows. TIFF2026518629000002.tif11115

[0021] In particular, the downregulation of miR-150-5p and miR-483-5p is specifically associated with the phenotype of bulbar-onset amyotrophic lateral sclerosis (ALS) and determines the biological classification of such phenotypes.

[0022] For the purposes of the present invention, the downregulation serving as a diagnostic indicator is one or both of the above microRNAs, i.e., simultaneous downregulation.

[0023] Accordingly, according to a second object, a method for diagnosing bulbar-onset amyotrophic lateral sclerosis (ALS) is described, the method for diagnosing bulbar-onset amyotrophic lateral sclerosis (ALS) in a patient suspected of suffering from this specific form of disease.

[0024] More specifically, such a method includes the step of measuring the expression levels of specific miR-150-5p and / or miR-483-5p microRNAs in a biological sample isolated from a patient suspected of having said disease.

[0025] More specifically, said biological sample isolated from a patient suspected of having said disease is a sample isolated from the serum of said patient.

[0026] The expression level can be determined by techniques known in the art; in particular, it can be determined by real-time PCR (rt-PCR).

[0027] When the expression of said microRNA is downregulated, the diagnosis is confirmed.

[0028] For example, the following average results were obtained: TIFF2026518629000003.tif59160

[0029] A third object of the present invention describes the use of specific microRNAs as potential targets for developing novel therapeutic approaches for bulbar amyotrophic lateral sclerosis (ALS).

[0030] In particular, the aforementioned microRNAs are represented by miR-150-5p and miR-483-5p.

[0031] By regulating the expression of specific microRNAs, miR-150-5p and miR-483-5p, to physiological levels, it will be possible to evaluate and develop new treatments for bulbar-onset amyotrophic lateral sclerosis (ALS).

[0032] In fact, such therapies are effective when they have the effect of downregulating the expression of two specific miRNAs, either independently or in combination.

[0033] A fourth object of the present invention describes specific microRNAs for pharmaceutical applications in the treatment of patients with bulbar-onset amyotrophic lateral sclerosis (ALS) that act on guanabenz or cellular responses defined as endoplasmic reticulum stress and unfolded protein responses.

[0034] In particular, the aforementioned pharmaceutical application is to evaluate the response to treatment with guanabenz in patients suffering from bulbar-onset amyotrophic lateral sclerosis (ALS).

[0035] More specifically, treatment with guanabenz includes administering 32 mg or 64 mg of guanabenz (daily dose) to patients who require it.

[0036] For the purposes of the present invention, the specific microRNA is represented by miR-106b-5p: TIFF2026518629000004.tif11169

[0037] In particular, the pharmaceutical applications of miR-106b-5p microRNA include evaluating the expression levels of the microRNA in isolated serum samples from patients being treated with guanabenz.

[0038] The miRNA expression levels mentioned above may provide an indicator of the effectiveness of drug therapy.

[0039] In fact, surprisingly, it was found that administering 32 mg or 64 mg (daily dose) of guanabenz downregulated the expression level of miR-106b-5p in the serum of patients with bulbar-onset amyotrophic lateral sclerosis (ALS).

[0040] These post-treatment expression levels show a similar trend to those observed in the serum of healthy control groups. [Examples]

[0041] Example section To discover the characteristic molecular signature of the bulbar palsy-onset type of ALS, we performed integrated profiling of serum microRNAs and proteomics. In the initial exploratory phase, serum samples from a cohort of 26 ALS patients and 28 healthy controls were analyzed using microfluidics with a TaqMan Human MicroRNA array containing 754 miRNAs. The expression of circulating microRNAs selected in the exploratory phase was validated using real-time PCR with a specific Taqman assay in a second, independent cohort of 28 ALS patients and 22 HC patients. Proteomic analysis was performed on serum from a subset of 14 ALS patients and 6 HC patients included in the selected miRNA validation study. By integrating molecular data obtained from the analysis of miRNA expression levels and proteomics data, a clear profile of bulbar palsy-onset ALS patients was revealed.

[0042] Statistical and clinical characteristics This study, conducted as part of the TRANS-ALS project (GL approval number 2015-0023) and the INTERSLA project (GL approval number 1157625), included 54 ALS patients meeting the diagnostic criteria for ALS and 50 healthy control (HC) participants recruited from the Motor Neuron Disease Center at the IRCCS Fondazione Istituto Neurologico Carlo Besta (Carlo Besta Foundation Neurological Institute) in Milan, Italy. There were no significant differences between the ALS patient group and the healthy control group in terms of sex (exploratory stage p=0.67, validation stage p=0.85) and age (exploratory stage p=0.74, validation stage p=0.62). Bulbar palsy was diagnosed when dysarthria or dysphagia without lower motor neuron spinal cord lesions, tongue atrophy, or muscle contraction were observed within 6 months of symptom onset, and signs of upper motor neuron involvement were present or observed later. Pseudobulbar palsy, characterized by prominent bulbar palsy symptoms due to upper motor neuron lesions, was ruled out in all patients by transcranial cortical bulbar spinal magnetic stimulation.

[0043] Exploration phase - Identification of dysregulated microRNAs in the serum of patients with bulbar palsy-type ALS. Using serum samples from 26 ALS patients and 28 HC subjects, the expression profiles of 754 miRNAs were analyzed by microfluidic synthesis to identify molecules potentially involved in the phenotype of bulbar palsy. Specifically, microRNAs with different expression levels were examined in ALS patients with bulbar palsy (7 cases), spinal cord-induced ALS (19 cases), spinal cord-induced and bulbar palsy due to ALS progression (16 cases), and spinal cord-induced ALS patients without bulbar palsy (10 cases). The results revealed four microRNAs (miR-885-5p, miR-150-5p, miR-483-5p, miR-342-3p) that were significantly downexpressed in the serum of patients with bulbar palsy compared to spinal cord-induced ALS patients and HC (Table 1). [Table 1]

[0044] In patients with spinal cord-onset ALS who also have bulbar palsy, no microRNAs with differing expression levels were observed.

[0045] Verification phase - Identifying circulating miR-150-5p and miR-483-5p as biomarkers for bulbar palsy-type ALS. To validate the data obtained during the exploratory phase, four microRNAs selected during the exploratory phase were analyzed using real-time PCR in the serum of 28 other ALS patients and 22 HC patients. As a result, the expression levels of miR-150-5p and miR-483-5p were significantly lower in bulbar palsy patients compared to spinal cord-onset patients and HC subjects (Figure 1). ROC (Receiver Operating Characteristic) curve analysis showed that the expression levels of the circulating miRNAs miR-150-5p and miR-483-3p could distinguish between spinal cord type and bulbar palsy type with high sensitivity and specificity (miR-150-5p AUC=76%, miR-463-5p AUC=81) and HC (miR-150-5p AUC=76%, miR-463-5p AUC=96%) (Figure 2). To investigate whether the expression levels of miR-150-5p and miR-483-5p are correlated, Spearman's correlation analysis was performed. The results showed no significant correlation between serum miRNA levels (r=0.26). This suggests that these two miRNAs play different pathophysiological roles at different regulatory levels.

[0046] Proteome Analysis In parallel with microRNA expression analysis, proteomic analysis of extracellular vesicles (EVs) obtained from the serum of 7 bulbar-onset ALS patients, 7 spinal cord-onset ALS patients, and 6 HC patients was performed using a platform based on the coupling of liquid nanochromatography and high-resolution tandem mass spectrometry (nLC-hrMS / MS). A total of 1,175 proteins containing at least one unique peptide were identified, and unlabeled comparisons were performed under three conditions based on the peak intensity of unique peptide precursor ions. The distribution and abundance of proteins between groups were evaluated using Venn diagrams and box plots, respectively. The proportion of co-proteins was high (82%), and the normalized abundance distribution was very similar among the sample groups. Volcano plots and PCA (Figure 3) identified three protein clusters indicating that the proteomic profiles of bulbar-onset ALS patients differed from those of spinal cord-onset ALS patients and healthy controls.

[0047] To examine proteins with different expression levels, three groups were compared in pairs (bulbar palsy patients and HC controls, spinal cord disease patients and HC controls, and bulbar palsy patients and spinal cord disease patients). Considering the grouped expression levels, a confidence threshold of 1.5 was applied to the Log2 change (FC) and a corrected p-value of 0.05. In at least one comparison, 295 proteins were identified that exceeded the set threshold. Specifically, 98 elevated proteins and 96 degraded proteins were identified in the bulbar palsy patient group and HC controls; 103 elevated proteins and 100 degraded proteins were identified in the spinal cord disease patient group and HC controls; and 80 elevated proteins and 89 degraded proteins were identified in the bulbar palsy patient group and spinal cord disease patient group. Proteomic analysis revealed clear protein profiles under extreme bulbar palsy conditions, strongly suggesting that selected molecular factors influence this phenotype. A functional network based on STRING annotation terminology for biological processes was constructed to display the linkage between protein levels and their biological pathways. The potential for protein-protein interactions was highlighted, suggesting that proteins expressed differently in spinal cord-onset ALS patients compared to bulbar-onset ALS patients are involved in gene processing, immune responses, intramembrane systems, adhesion and motility, proliferation, protein metabolism, muscle, stress responses, and energy metabolism. This network clearly demonstrates the expression of protein profiles in the phenotype of bulbar-onset ALS.

[0048] Integration of microRNA and proteomics profiles in serum from patients with bulbar palsy-type ALS. We performed an integrated analysis of two candidate miRNAs, miR-150-5p and miR-483-5p, and proteins whose expression differs in spinal cord-onset patients with ALS and HC, discovering distinct molecular signatures. Spearman's correlation analysis was used to identify the correlation between the expression levels of each miRNA and the selected proteins. Because the relationship between miRNAs and RNA-binding proteins (RBPs) is increasingly based on mutual regulation, we analyzed positive and negative correlations using a correlation coefficient (r) ≥ 0.5 or ≤ -0.5 and a p-value < 0.05 as indicators 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 bulbar palsy phenotype. A positive correlation was observed between miR-150-5p and the DDP9 protein, while negative correlations were found between miR-150-5p and its predicted target genes: endoplasmic reticulum membrane protein complex (EMC3), Grainyhead-like transcription factor 2 (GRHL2), and the non-catalyzed subunit protein of dritil-diphosphooligosaccharide-protein glycosyltransferase (DDSOT). Furthermore, 31 negative correlations and 25 positive correlations were observed between miR-483-5p and selected proteins expressed only in bulbar-onset ALS patients. Interestingly, the predicted miRNA target, DENN domain-containing 6A (DENND6A), showed a positive correlation with miR-483-5p. Biological processes and functions associated with the selected molecules in bulbar-onset ALS patients include immune responses, the intomemal system, protein metabolism, energy metabolism, synapses, ion homeostasis, adhesion motility, muscles, stress responses, apoptosis, genetic information processing, and proliferation. The integrated molecular data revealed possible mechanisms mediated by microRNAs at different regulatory levels, confirming the distinct and unrelated actions of two selected microRNAs.

[0049] Based on string annotation, we constructed a functional network displaying the positive / negative correlation between two microRNAs and the corresponding proteins, as well as the protein connections to the annotated biological processes. These biological processes are involved in immune responses, the intomenar system, protein metabolism, energy metabolism, synapses, ion homeostasis, adhesion motility, muscle, stress responses, apoptosis, genetic information processing, and proliferation.

[0050] Analysis of miR-106b-5p expression levels in bulbar palsy patients treated with guanabenz. Serum miR-106b-5p expression levels were analyzed in 12 patients with bulbar palsy (5 treated with placebo / 16 mg, 7 treated with guanabenz 32 mg / 64 mg) and 14 patients with spinal cord palsy (10 treated with placebo / 16 mg, 5 treated with guanabenz 32 mg / 64 mg) (Figure 4). Molecular biological analysis revealed that serum miR-106b-5p expression levels in ALS patients treated with guanabenz 32 mg / 64 mg normalized compared to patients treated with placebo / 16 mg. In contrast, miR-106b-5p expression levels remained significantly higher in spinal cord palsy patients and in all treatment groups.

[0051] method Healthy patients and control group This study included 54 ALS patients with deep phenotypic determination who met the diagnostic criteria for ALS, and 50 age- and sex-matched HC patients. Patients were classified into bulbar palsy-onset and spinal cord-onset based on published valid criteria. Serum samples from 26 ALS patients and 28 HC patients were used for miRNA analysis (exploratory phase), and serum samples from an additional 28 ALS patients and 22 HC patients were used for miRNA validation experiments (validation phase). For proteomics analysis, 7 age- and sex-matched bulbar palsy-onset patients, 7 spinal cord-onset patients, and 6 HC patients were selected and all were analyzed by miRNA profiling. No significant sex differences were observed in ALS and HC at either discovery (p-value = 0.67) or validation (p-value = 0.85). No significant age differences were observed when comparing ALS and HC at either discovery (p-value t-test = 0.74) or validation (p-value t-test = 0.62). This study was conducted in accordance with the ethical standards of the Declaration of Helsinki. The collection and use of patient data for research purposes was approved by the Ethics Committee of the Fondazione IRCCS Istituto Neurologico Carlo Besta, in accordance with the Declaration of the World Medical Association.

[0052] Genetic screening All patients underwent next-generation sequencing (NGS) of ALS-related genes using deep amplicon sequencing of SOD1, FUS, TARDBP, VCP, OPTN, SQSTM1, TUBA4A, PFN1, UBQLN2, and Amplidex PCR repeats using the Sure Select QXT kit (Agilent). Kit PCR / CE C9ORF72 (Asuragen Inc. Austin TX) was used to detect C9orf72 extensions. DNA was extracted from peripheral blood using standard procedures. Variants were filtered according to the following criteria: (i) variants in the coding region or adjacent 20 bp, and (ii) deletions or rare variants with an allele frequency (AF) of less than 1% in population databases (dbSNP137, ESP6500, 1000 Genome Project, ExAC). Given the extremely low prevalence of ALS, gene mutations with a frequency exceeding 1% in dbSNP, Exome Variant Server, or ExAC were classified as "benign" (Class-1) and excluded from further analysis. Computer prediction of the impact of synonymous or intronic mutations on splice junction sites was performed using at least two junction prediction tools: NNSplice prediction tool ( / / www.fruitfly.org / seq tools / splice.html); ASSP (http: / / wangcomputing.com / assp / ). Optimal candidate variants were validated using the Sanger method. Gene mutations that passed the filtering were classified as "pathogenic" (Class-5), "probably pathogenic" (Class-4), "variant of unknown significance" (VUS, Class-3), and "probably benign" (Class-2) according to the criteria proposed by ACMG (American College of Medical Genetics and Genomics).

[0053] MicroRNA profiling and data analysis Total RNA was extracted from serum samples collected from 26 ALS patients and 28 healthy controls using the miRNeasy serum / plasma kit (Qiagen, Venlo, Netherlands). RNA quality was verified using a 2100 Nano Bioanalyzer (Agilent Technologies), and RNA was reverse transcribed using Megaplex RT human pool A and B primers and the MultiScribe reverse transcriptase kit. cDNA equivalent to 170 ng of total RNA was mixed with TaqMan Universal PCR Master Mix and distributed to each port of TaqMan Human MicroRNA A and B v2.0 array boards according to the manufacturer's instructions. Arrays were analyzed using a Viia 7 Fast Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Human array boards A and B contained 754 miRNA primers, including three positive control miRNAs and one negative control. Relative threshold assay (Crt) was applied. Only miRNAs with good amplification quality (amplification score > 1 and Cq confidence > 0.8) were included in the analysis. Expression differences were normalized using miR-24 as the endogenous control (42 samples), and quantified using relative quantification by the 2-ΔΔct method with spinal cord type SLA or healthy control samples as the reference group. ΔΔCq was calculated as mean ΔCq (target miRNA in the bulbar palsy-developing ALS group) - mean ΔCq (target miRNA in the reference group). The expression ratio was calculated as 2-(ΔΔCq). The decrease in expression in the control group compared to the reference group was converted to the negative reciprocal of 2-(ΔΔCq) to show the reduction in the expression ratio change.

[0054] Validation of miRNAs by real-time PCR Total RNA was extracted from the serum of 28 ALS patients and 22 HC subjects using the miRNeasy serum / plasma kit (Qiagen). RNA quality was confirmed using a 2100 Nano Bioanalyzer (Agilent Technologies). RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit with 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 (equivalent to 100 ng of total RNA) were duplicated and amplified using a Viia 7 Fast Real-Time PCR System (Thermo Fisher Scientific) with a universal PCR master mix and a pre-designed TaqMan MicroRNA assay. All results were normalized to miR-24, and relative miRNA expression levels were calculated using the ΔCt method.

[0055] Protein extraction and enzymatic digestion Proteomics analysis was performed using EV serum extracts from bulbar-onset ALS patients (N=7), spinal cord-onset ALS patients (N=7), and healthy controls (N=6), which had been previously analyzed for miRNA experiments. To isolate EV from 250 μL of pre-purified serum, the ExoQuick ULTRA kit (System Biosciences, Palo Alto, CA, USA) was used according to the manufacturer's protocol. In short, serum samples were incubated with ExoQuick42 reagent at 4°C for 10 minutes, followed by centrifugation at 3000 × g for 10 minutes. The pellet was collected, resuspended in a 500 μL ExoQuick Ultra swab, and added to a pre-washed resin column. After mixing, purified EV was collected by centrifugation at 1000 × g for 2 minutes, and the protein concentration was measured using the Qubit™ Protein Analysis Kit (Life Technologies Corp., Eugene, OR, USA). For each sample, 50 μg of protein mixture was reduced / alkylated using the Easy Pep Mini MS Sample Preparation Kit (Thermo Fisher Scientific) and then enzymatically digested. Following the kit protocol, peptides were generated within 3 hours under each condition, washed to prepare surfactant-free samples, resuspended in 0.1% formic acid (Sigma-Aldrich Inc., St. Louis, MO, USA), and subjected to nLC-hrMS / MS analysis.

[0056] LC-MS / MS analysis Peptide mixtures were analyzed using a combination of the Eksigent nanoLC-Ultra 2D system (Eksigent, an affiliate of AB SCIEX Dublin, CA, USA) and the cHiPLC-nanoflex system (Eksigent) in trap-elute mode. Specifically, under each condition, 0.8 μg of protein was injected into a cHiPLC trap (200 μm × 500 μm ChromXP C18-CL, 3 μm, 120 Å, Eksigent, AB SCIEX Dublin, CA, USA), and two technical replications were performed by running a loading pump at a flow rate of 3 μL / min for 10 minutes in isotropic mode using a 0.1% formic acid aqueous solution. Subsequently, the trapped mixture was automatically eluted into a nano cHiPLC column (75 μm × 15 cm ChromXP C18-CL, 3 μm, 120 Å, Eksigent, affiliate of AB SCIEX Dublin, CA, USA) using eluent B (eluent A: 0.1% formic acid in water, eluent B: 0.1% formic acid in acetonitrile) at a flow rate of 300 nL / min for 115 minutes. The depth gradient was maintained at 5–15% B at 3 minutes, 15–30% B at 90 minutes, 30–40% B at 6 minutes, 40–95% B at 6 minutes, and 95% B at 11 minutes. The eluted peptides were directly analyzed using an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific) equipped with an EASY-Spray ion source (Thermo Fisher Scientific). Easy spray ions were acquired using an EASY-Spray emitter (Thermo Fisher Scientific) maintained at 1.6kV (nanoflow 7μm ID transfer line 20μm × 50cm), and the iontophoresis capillary was maintained at 220°C. Data-dependent acquisition (DDA) was performed to acquire precursor ions in the m / z range of 375 to 1,250. The resolution (at m / z 200) was 60,000 FWHM (full width at half maximum).Precursor fragmentation was performed using high-energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 30 eV, a dynamic exclusion time of 20 seconds, and a resolution (m / z 200) of 15,000 FWHM. MS data were acquired in profile mode, and MS / MS data in centroid mode, with positive polarity and active isotope exclusion modes, respectively. The isolation width was set to 2 m / z, and the initial mass was set to 120 m / z. The mass spectrometer scanning function and the solvent gradient for high-performance liquid chromatography were controlled by the Xcalibur version 4.4 data system (Thermo Fisher Scientific) and Eksigent version 4.3 control software (Eksigent, an affiliate of AB SCIEX Dublin, CA, USA), respectively.

[0057] Data Management All generated raw data was searched using the Sequest HT search engine included in Proteome Discoverer software, version 2.5 (Thermo Fisher Scientific), against the Homo sapiens proteome database (75,550 entries) downloaded from Uniprot (www.uniprot.org) in March 2021. An MSPepSearch node was inserted into the processing workflow before Sequest HT to perform a primary search of the spectral library (NIST Human Orbitrap HCD Library, 1127970 spectra, September 2016). The following criteria were used for peptide sequence and associated protein identification: a mass tolerance of ±10 ppm for precursor ions and ±0.02 Da for fragment ions; trypsin as the enzyme, with a maximum of two non-cleavage tolerances; cysteine ​​carbamide methylation as a fixed modification; and methionine oxidation as a variable modification. A percolator node using a target-bait strategy was applied, considering XCorr > 1.2 and a maximum deltaCN of 0.05, and the false detection rate (FDR) was set to 1% (strict) and 5% (relaxed) based on the q value. Label-free quantification (LFQ) of 43 proteins was performed using non-nested studies with biological and technical replication, recalibration of spectral files, and abundance calculation using the Minora algorithm with the peak intensity of the unique peptide precursor ion when 60% of the replication features were reached. Pairwise assays were employed to compare normalized protein abundances between the analyzed sample groups. Protein grouping and a strict omission principle were also applied. Statistical analysis of the protein quantification results was performed using background-based t-tests, and differentially expressed proteins (DEPs) between the compared experimental groups were screened if the log2-multiple change threshold ≥ │1.5│ and the p-value ≤ 0.05 were met. Volcano plots, heatmaps, and PCA were created using the default functions of Proteome Discoverer 2.5. Clustering analysis was performed using the Euclidean averaging method for calculating the distance and aggregation of all proteins.

[0058] Protein network analysis Starting with DEP, we considered experimental and database-defined protein-protein interactions (PPIs) (score > 0.15) using only the STRING45 database and constructed a PPI network (268 nodes, 3,532 borders). The resulting subnetworks were visualized and analyzed using Cytoscape V.3.9.1 and plugins 46 and 47. Proteins were grouped into functional modules by supporting STRING enrichment with the default settings. Node color reflected the DEP expression level under each condition based on normalized abundance cluster values ​​(abundance cluster values ​​were normalized to the range of 0-100, with the highest abundance per protein set to 100).

[0059] Correlation analysis and construction of functional miRNA-protein-biological process networks Based on the expression and protein abundance of miR-150-5p and miR483-5p, Spearman's correlation coefficient and p-value were calculated for each miRNA-protein pair. A Spearman's correlation coefficient of ≥0.5 or ≤-0.5 and a p-value of <0.05 were considered statistically significant. Using Cytoscape (V3.9.1)48, functional networks linking miRNAs and proteins, and related functional biological processes associated with those proteins, were created and displayed.

[0060] statistical analysis Comparisons of demographic and clinical continuous variable groups were performed using t-tests, while categorical data were compared using chi-square tests. Multiple comparisons of miRNA expression levels in the ALS and HC groups with bulbar palsy and spinal cord ALS groups were performed at both the discovery and validation stages, following post-hoc analyses using the Kruskal-Wallis rank-sum test and Dunn's test. The Benjamini-Hockberg (BH) false detection rate (FDR) test was applied to correct for multiple testing. A statistically significant difference was considered to exist if the FDR p-value was less than 0.05. Spearman's correlation coefficient was used to assess the correlation between clinical characteristics of ALS patients and miRNA expression levels. ROC curves were used to evaluate the sensitivity and specificity of miR-150-5p and miR-483-5p in human serum samples as biomarkers that can distinguish patients with bulbar palsy and spinal cord ALS. Statistical analyses were performed using the statistical programming language R (version 3.6) and STATA11 software.

[0061] Analysis of miR-106b-5p expression levels Total RNA was extracted from the serum of 12 patients with bulbar palsy (5 of whom received placebo / 16 mg and 7 received 32 mg / 64 mg guanabenz) and 14 patients with spinal cord palsy (10 of whom received placebo / 16 mg and 5 received 32 mg / 64 mg guanabenz) using the miRNeasy serum / plasma kit (Qiagen). RNA quality was confirmed using a 2100 Nano Bioanalyzer (Agilent Technologies). RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit with primers specific to selected miRNAs (miR-106b-5p and miR-24 as endogenous controls). cDNA (equivalent to 100 ng of total RNA) was double-amplified by real-time PCR using a Viia 7 Fast Real-Time PCR System (Thermo Fisher Scientific) with a universal PCR master mix and a pre-designed TaqMan MicroRNA assay. All results were normalized to miR-24, and relative miRNA expression levels were calculated using the ΔCt method.

[0062] From the above explanation, the advantages provided by the present invention are clear.

[0063] Firstly, this provides an important new diagnostic tool for bulbar-onset amyotrophic lateral sclerosis (ALS), a type of disease that has not received much attention until now.

[0064] Because the described microRNAs are molecules circulating in the patient's serum, they can be identified using easy-to-implement and non-invasive methods.

[0065] The invention provides for the development of a new therapeutic approach for treating bulbar-onset amyotrophic lateral sclerosis (ALS), the most devastating form of this neurodegenerative disease.

[0066] The methods provided by this invention have been demonstrated to have significantly improved reliability and validity based on the analysis of a broader database. Furthermore, the methods of this invention have been validated from both biological and technical perspectives.

[0067] Therefore, overall, this method is very robust and reliable for identifying the morphology of bulbar palsy-type ALS.

[0068] Furthermore, the present invention makes it possible to more effectively and advancedly monitor the treatment of guanabenz in patients with bulbar-onset amyotrophic lateral sclerosis (ALS) by utilizing the identified response biomarkers.

Claims

1. The use of specific microRNAs in the diagnosis of bulbar-onset amyotrophic lateral sclerosis (ALS), wherein the specific microRNAs are as follows: As indicated by, use.

2. The use of a specific microRNA in the diagnosis of bulbar-onset amyotrophic lateral sclerosis (ALS) according to a prior claim, wherein the expression of the microRNA is downregulated.

3. The use of a specific microRNA in the diagnosis of bulbar-onset amyotrophic lateral sclerosis (ALS), wherein the expression is measured in an isolated serum sample from a patient suspected of having the disease.

4. The following microRNAs were found in isolated biological samples from patients suspected of having bulbar-onset amyotrophic lateral sclerosis (ALS): A diagnostic method for diagnosing bulbar palsy-type ALS in a patient suspected of having the specific disease form, comprising the step of determining the expression of [a specific substance].

5. The diagnostic method according to the preceding claim, wherein the expression is determined in an isolated serum sample from the patient.

6. A microRNA for pharmaceutical use in the treatment of patients with bulbar-onset amyotrophic lateral sclerosis (ALS) by guanabenz or by drugs acting on cellular responses defined as endoplasmic reticulum stress and unfolded protein responses, wherein the microRNA is as follows: MicroRNA, as shown by [this symbol].

7. A microRNA for pharmaceutical use in the treatment of a patient suffering from bulbar-onset amyotrophic lateral sclerosis (ALS) according to a prior claim, wherein the guanabenz is administered at a dose of 32 mg or 64 mg per day.

8. A microRNA for pharmaceutical use in the treatment of a patient suffering from bulbar-type amyotrophic lateral sclerosis (ALS) according to claim 6 or 7, wherein the expression level of the microRNA is determined.

9. A microRNA for pharmaceutical use in the treatment of a patient suffering from bulbar-onset amyotrophic lateral sclerosis (ALS) according to a prior claim, wherein the expression level is determined in an isolated serum sample from the patient.

10. The use of microRNA to develop a therapeutic approach for bulbar-onset amyotrophic lateral sclerosis (ALS), wherein the microRNA is as follows: As indicated by, use.

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