Sarcopenia associates with SNAP-25 SNPs and a miRNAs profile which is modulated by structured rehabilitation treatment

Background Sarcopenia is a loss of muscle mass and strength causing disability, morbidity, and mortality in older adults, which is characterized by alterations of the neuromuscular junctions (NMJs). SNAP-25 is essential for the maintenance of NMJ integrity, and the expression of this protein was shown to be modulated by the SNAP-25 rs363050 polymorphism and by a number of miRNAs. Methods We analysed these parameters in a cohort of sarcopenic patients undergoing structured rehabilitation. The rs363050 genotype frequency distribution was analyzed in 177 sarcopenic patients and 181 healthy controls (HC). The concentration of seven miRNAs (miR-451a, miR-425-5p, miR155-5p, miR-421-3p, miR-495-3p, miR-744-5p and miR-93-5p), identified by mouse brain miRNome analysis to be differentially expressed in wild type compared to SNAP-25± heterozygous mice, was analyzed as well by droplet digital PCR (ddPCR) in a subgroup of severe sarcopenic patients undergoing rehabilitation. Results The SNAP-25 rs363050 AA genotype was significantly more common in sarcopenic patients compared to HC (pc = 0.01); miR-451a was significantly up-regulated in these patients before rehabilitation. Rehabilitation modified miRNAs expression, as miR-155-5p, miR-421-3p, miR-451a, miR-425-5p, miR-744-5p and miR-93-5p expression was significantly up-regulated (p < 0.01), whereas that of miR-495-3p was significantly down-regulated (p < 0.001) by rehabilitation. Notably, rehabilitation-associated improvement of the muscle-skeletal SPPB score was significantly associated with the reduction of miR-451a expression. Conclusion These results support the hypothesis of a role for SNAP-25 in sarcopenia and suggest SNAP-25-associated miRNAs as circulatory biomarkers of rehabilitative outcome for sarcopenia. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-021-02989-x.

predominance of type I fibers that leads to atrophy of type II fibers [4], and accumulation of adipose tissue around and between muscle fibers [5]. Because the muscle contractile activity is regulated by the central nervous system at the NMJs, and NMJs are fundamental for the maintenance of the integrity of motor nerve and muscle fibers [6], sarcopenia was hypothesized to have a neurological origin [7]. The structural features of NMJ are similar to those of other chemical synapses, and many of the molecules involved in the formation of the NMJ have also an essential role in its maintenance, such as the SNARE complex [8,9]. The core SNARE synaptosomal-associated protein of 25 kDa (SNAP-25), in particular, could be a key factor in sarcopenia, as it accumulates in the plasma membrane of nerve terminals at NMJ, consistently with its role in regulating exocytosis at peripheral and central synapses [10,11]. To note, adult SNAP-25 ± mice expressing low SNAP-25 protein level, were shown to be characterized by an impaired grip strength [12].
SNAP-25 expression is modulated by the rs363050 polymorphism, which is localized in intron 1 of the gene encoding for SNAP-25, thus, GG homozygosis resulted in a reduced gene expression compared to the AA homozygosis [13]. As the majority of genes, SNAP-25 expression is also regulated by microRNAs (miRNAs), short non-coding RNAs involved in mRNA silencing and post-transcriptional modulation of gene expression [14]. Of note, the differential expression of SNAP-25-related miRNAs as well as polymorphisms in the SNAP-25 gene (i.e. rs363050), which may result in the modulation of SNAP-25 expression, were suggested to play a pathogenic role in different brain diseases including Alzheimer's Disease, Parkinson's Disease and Amyotrophic Lateral Sclerosis [15,16], but also Attention Deficit Hyperactivity Disorder (ADHD) [17][18][19][20][21] and early-onset bipolar disorders [22]. Whether these mechanisms can be involved in sarcopenia is not known. Indeed, even if research on sarcopenia prevention and treatment is developing quickly, many questions are still unanswered. Furthermore, physical exercise was shown to influence miRNA expression [23,24], however to our knowledge no evidence has been reported regarding the impact of structured physical rehabilitation programs on miRNA expression in sarcopenic patients. Here we analysed the possible association of rs3603050 SNAP-25 polymorphism with sarcopenia, whether miRNAs related with the expression of this gene are differentially expressed in sarcopenic patients, and, finally, the possible effect of rehabilitative treatment on miRNAs expression.  [2].

Patients and controls
At the time of recruitment, patients underwent a comprehensive geriatric multidimensional evaluation that included lower extremity function evaluation with the Short Physical Performance Battery (SPPB) [25], cognitive function evaluation with the Mini-Mental State Examination (MMSE) [26] and the Clock Drawing test (CDT) (score range 0-5) [27]. The emotional status was evaluated using the Yesavage Geriatric Depression Scale, which consists of 30 questions and is scored between 0 and 30 points [28]. Comorbidity was evaluated with the Charlson Comorbidity Index (CCI) [29]. Finally, the functional status was assessed using the ADL (activity of daily living or KATZ index) (bathing, dressing, toileting, transfer, feeding, continence) and the IADL (Lawton-Brody instrumental activity of daily living) (telephone use, housekeeping, laundry, medication use, transportation, preparing meal, shopping, handling finances) tests. ADL and IADL scales are scored between 0 to 6 and 0 to 8 points, respectively [30,31].
Subjects with MMSE < 24, undergoing steroid therapy, that were clinically instable, had a concomitant diagnosis of neoplastic or neurodegenerative diseases, or were unable to participate safely in the intervention program, were excluded by the study. miRNA expression was evaluated in a sub-group of hospitalized severe sarcopenic patients (SPPB < 3, n = 45) who were undergoing a 30-days structured rehabilitation treatment as follows: twice day session of 40' in the morning and 30' in the afternoon with assisted mobilization, progressive muscle strengthening, associated with progressivity of the load, standing work proprioceptive postural balance, walking training firstly with an assisted way and then without. For each subject, SPPB was calculated before and after the rehabilitative treatment. A frailty index was computed following the criteria described by Searle et al. [32] and taking into account a wide range of age-related signs, symptoms, disabilities, and diseases. HC were age-and-sex-matched individuals without sarcopenia who had undergone surgery treatment for knee replacement or hip/knee fractures (n = 44). The study conformed to the ethical principles of the Declaration of Helsinki; all subjects gave informed and written consent according to a protocol approved by the ethics committee of IRCCS Fondazione Don Carlo Gnocchi ONLUS (n#9_04/04/2018).

Mouse brain miRNome analyses
All experiments followed the guidelines established by the European Directive 2010/63/EU and the Italian Governing Law 26/2014 (PR-09/2013). All efforts were made to minimize the number of subjects used and their suffering. SNAP-25 wild-type and SNAP-25 heterozygous mice were housed in cages with free access to food and water at 22 °C and with a 12-h alternating light/dark cycle. Genotyping was performed by PCR as described in Washbourne et al. [33]. SNAP25 +/+ and SNAP25 ± brains (12 months old mice) have been collected and stored at -80 °C until use.
miRNome analyses were performed on 20 mg brain samples of 8 female mice (4 wild type and 4 SNAP-25 ± heterozygous mice) by qPCR by Qiagen Genomic Services (Qiagen GmbH, Hilden, Germany). Briefly, after RNA extraction and the reverse transcription into cDNA, the miRCURY LNA miRNA PCR mouse and Rat, panel I (372 miRNAs analyzed, Qiagen) was used for the analysis. miRNA expression data were normalized using the global mean method.

SNAP-25 genotyping
Whole blood was collected for all the subjects and genomic DNA was isolated by phenol-chloroform extraction. Taqman SNP Genotyping Assay (Life Technologies, Foster City, CA, US) was used to type SNP rs363050 of SNAP-25 [34].

Serum miRNA isolation and cDNA reverse transcription
For severe sarcopenic patients also serum was obtained from peripheral blood by centrifugation at 1800g for 10 min. The absence of hemolysis in serum was evaluated by visual inspection and by spectrophotometric measurement of absorbance of hemoglobin at 414 nm [35]. miRNA isolation from 200 μl of serum was semi-automatically performed with a column-based kit (MiRNeasy serum/plasma kit, Qiagen GmbH, Hilden, Germany) by Qiacube (Qiagen GmbH, Hilden, Germany), according to manufacturer's protocol. After quantitation by Qubit (Qiagen GmbH, Hilden, Germany), equal concentration of extracted miRNAs was retro-transcribed in cDNA (miRCURY LNA RT kit, Qiagen GmbH, Hilden, Germany) for all samples. To avoid variations due to sample differences and handling, all the variable involved in the procedure were kept consistent throughout the study.

Quantification of selected circulatory miRNA
miRNA quantitation was performed by droplet digital PCR (ddPCR QX200, Bio-Rad, Hercules, CA, US). Briefly, 3 µl of diluted cDNA (1:25) was mixed with LNATMspecific primers (Qiagen GmbH, Hilden, Germany), and ddPCR EvaGreen Supermix (Bio-Rad, Hercules, CA, US), which was then emulsified with droplet generator oil (Bio-Rad, Hercules, CA, US) using a QX200 droplet generator, according to the manufacturer's instruction. The droplets were then transferred to a 96-well reaction plate and heat-sealed with a pierceable sealing foil sheet (PX1, PCR plate sealer, Bio-Rad, Hercules, CA, US). PCR amplification was performed in sealed 96-well plate using a T100 thermal cycler (Bio-Rad, Hercules, CA, US) as follows: 10 min at 95 °C, 40 cycles at 94 °C for 30-s and at 58° for 60 s, followed by 10 min at 98 °C and a hold at 4 °C. The 96-well plate was then transferred to a QX200 droplet reader (Bio-Rad, Hercules, CA, US). Each well was queried for fluorescence to determine the quantity of positive events (droplets), and the results were displayed as dot plots. The miRNA concentration was expressed as copies/ng of extracted RNA.

Statistical analysis
Normally distributed data were expressed as mean ± standard deviation, and comparisons among groups were analyzed by ANOVA test and Student t-test, when appropriate. Not-normally distributed data were expressed as median and interquartile range (IQR: 25th and 75th percentile), and comparisons were analyzed by Kruskal-Wallis and Mann-Whitney U test, as appropriate, and with Wilcoxon signed-rank test for paired data. Correlations were analyzed using Spearman's correlation coefficient and multiple logistic regression analysis.

Clinical parameters
Demographic and clinical characteristics of the study population are summarized in Table 1 As expected, the median of SPPB was significantly lower in sarcopenic patients (median and Interquartile range: 2; 1-6) compared to HC (11; 10-12) (p < 0.0001). Table 2 summarizes the demographic and clinical characteristics of the subgroup of the severely sarcopenic patients who underwent rehabilitation, and of the age-and-sex-matched HC; circulatory miRNA expression was analyzed in all these individuals. As expected, median SPPB score of the sarcopenic patients increased after the rehabilitative treatment (median and Interquartile range: 2; 1-3) compared to the values observed before rehabilitation (0; 0-1) (p = 0.002). The majority of the subjects presents more than one comorbidity (Additional file 1: Table S1), probably due to the age of enrolled subjects [37]. The mean ± standard deviation Frailty Index of these patients is 0.19 ± 0.13.

SNAP-25 rs363050 genotyping
Initial molecular genotyping analyses showed that the SNAP-25 (A/G) rs363050 polymorphism genotype distribution was in Hardy-Weinberg (HW) equilibrium. Further analyses showed that the overall genotype distribution of SNAP-25 rs363050 was significantly different in sarcopenic patients compared to HC (p = 0.001; d.f.: 2; χ 2 = 12.94). In particular, the SNAP-25 rs363050 AA genotype resulted to be more frequent in sarcopenic patients (40%) compared to HC (27%) (p c = 0.01; OR = 1.85, 95% CI 1.19-2.90) ( Table 3). These differences were not confirmed when analyses were limited to the subgroup of severely diseased patients who were undergoing rehabilitation and in whom circulatory miRNAs were analyzed, probably due to the relatively small sample size (Table 3).

Mouse brain miRNome profiling according to SNAP-25 genotyping
MiRNome expression profile of mouse brain identified in all samples 231 miRNAs, with an average of 258 miRNAs detectable per sample. Among these miRNAs, 2 were significantly deregulated in wild type compared to SNAP-25 ± heterozygous mice: miR-451a and miR-425-5p. Five other miRNAs were differentially expressed as well, as their expression ratio was > 2.5-fold or < − 2.5fold: miR155-5p, miR-421-3p; miR-495-3p, miR-744-5p and miR-93-5p (data not shown). For these reasons, and because all these miRNAs are homologous between mouse and human, the expression of these 7 miRNAs was analyzed in sera of sarcopenic patients and HC.

miRNAs expression in association with SNAP-25 rs363050 genotypes
Before initiation of the rehabilitation protocol, miR-451a expression was significantly higher in sarcopenic patients compared to HC (p = 0.005) (Fig. 1); the expression of the other 6 miRNAs was similar in the two groups. Upon dividing miRNAs results based on the SNAP-25 rs363050 genotype, results showed that miR-421-3p expression was increased in SNAP-25 AA genotype homozygous (median and Interquartile range: 150; 0-241 copies/ng) compared to AG heterozygous (0; 0-0 copies/ ng) (p = 0.03), and rs363050 GG homozygous (0; 0-133 copies/ng) sarcopenic patients before rehabilitation. The statistical differences remained even when patients that were homozygote for the major allele rs363050 AA were compared with those carrying the minor allele both as homozygous and heterozygous rs363050 genotypes (AG + GG) (150; 0-241 vs. 0; 0-41.77 copies/ng; p = 0.02) (Fig. 2). Interestingly, these differences disappeared at the end of rehabilitation. No differences were observed for the other 6 miRNAs in association with SNAP-25 rs363050 genotype. No differences emerged when male and female were analyzed separately (data not shown).
Finally, when the difference of the expression before and after rehabilitative treatment (delta) was calculated for miRNAs in relationship with SPPB, a significant  19:315 negative correlation was found between the delta of miR-451a expression and the delta of SPPB (p < 0.05) (Fig. 3a). This suggests that when, after rehabilitation, the SPPB score increases, miR-451a decreases more, approaching the values observed in healthy controls. This association between miR-451a expression change after rehabilitation and rehabilitative outcome was confirmed also after multiple logistic regression analysis adjusted for age and frailty index (p = 0.05). Confirming this hypothesis, miR-451a expression after rehabilitation was significantly   19:315 lower in patients with a positive variation of physical performance (delta SPPB > 0; n = 34) compared to those with no or negative variation (SPPB ≤ 0) (p < 0.05; n = 11) (Fig. 3b). Importantly, the frailty index was not significantly different between the two groups (delta SPPB > 0: 0.14; 0.14-0.29; delta SPPB ≤ 0: 0.29; 0.12-0.32). Results were similar when analyses were performed in patients and controls after splitting the two groups according to gender (data not shown).

Bioinformatics analysis
Bioinformatics analysis showed that the analyzed miR-NAs target human genes pathways involved in fatty acid metabolism/biosynthesis (p < 0.0001), steroid biosynthesis, and biotin metabolism (Fig. 4). To note, four of the human pathways (MAPK, FoxO, TGFβ and mTOR signalling pathways) were indicated by bioinformatic analysis were previously indicated involved in sarcopenia [38]. Additionally, as most miRNAs have been analyzed in the scenario of oncology research, most pathways show weak association related to cancer.

Discussion
Sarcopenia is a multifactorial and complex disease in which several genes and proteins contribute to the decline of muscle mass and functions. In this study we focused our attention on SNAP-25, a gene whose polymorphisms have been shown to be associated with a number of pathologies, including Alzheimer's and Parkinson's diseases, schizophrenia, attention deficit hyperactivity disorders and epilepsy; our results indicate that SNAP-25 SNPs also play a role in sarcopenia.
Thus, our results for the first time show that sarcopenia is associated with SNAP-25 polymorphisms and with a particular profile of miRNAs that regulate SNAP-25 expression. Notably, data herein also indicate that successful muscle-skeletal rehabilitation outcome correlates with modifications in miRNAs expression, suggesting that miRNAs expression could be used as a biomarker for sarcopenia.
The SNAP-25 protein is a component of the multiproteic SNARE complex and is responsible for the fusion of synaptic vesicles with the plasma membrane during exocytosis, playing a significant role in the maintenance of the NMJ integrity and functionality. For these reasons SNAP-25 was hypothesized to participate to the pathogenic mechanisms involved in sarcopenia [9]. Reduction of SNAP-25 protein levels has been described in psychiatric patients [39] and is responsible for brain circuits hyperexcitability [12], as well as for learning and cognitive defects in the SNAP-25 heterozygous mice [40]. Reduction of SNAP-25 protein level can be the result of specific gene variants, such as the SNAP-25 rs363050 polymorphism, which affects SNAP-25 expression either directly [13] or through an epigenetic mechanisms involving specific miRNAs [16]. Here we analyzed the SNAP-25 rs363050 polymorphism and the expression of 7 circulatory miRNAs, selected because they are differentially expressed in SNAP-25 heterozygous mice (miR-155-5p, miR-421-3p, miR-451a, miR-425-5p, miR-744-5p, miR-93-5p and mir-495-3p), in sarcopenic patients. Interestingly, bioinformatics analyses showed that the target genes of these 7 miRNAs are involved in pathways relevant for fatty acid metabolism and biosynthesis, and thus in muscle fat accumulation: a risk factor for the development of sarcopenia in old persons [41].
The SNAP-25 rs363050 (AA) polymorphism was significantly more frequent in sarcopenic patients; interestingly this SNAP-25 polymorphism was shown to associate with other age-dependent pathologies, including Alzheimer's disease [34,42] and Type 2 Diabetes mellitus [43]. Analyses performed on SNAP-25 expression-modulating miRNAs also showed that miR-451a was more expressed in sarcopenic patients, compared to controls. This miRNA is localized in chromosome 17 (17q11.2) and is known to be associated with different human neoplasia, including gastric cancer [44,45] where it is down-regulated and contributes to tumor formation and progression.
MiR-451a is expressed in skeletal muscle [46], and its expression was shown to be higher in human muscle biopsies of individuals that are low-responders to resistance exercise training [47]. It seems important to underline that this miRNA also plays a critical role in erythrocytes maturation [48]. These results allow to speculate that the increased circulatory level of miR-451a observed in sarcopenic patients could reflect an augmented amount of this miRNA in muscles, or could reflect a compensatory mechanism to induce erythroid maturation, resulting in an increased delivery of oxygen to muscles. Interestingly, a reduction of miR-451a expression was recently described in muscles of non-small cell lung carcinoma patients with cachexia [49], a condition characterized by loss of body weight, systemic inflammation, anorexia, and pronounced loss of skeletal muscle mass [50]. Our analyses were performed in plasma; results confirm the hypothesis that this miRNA can have a role in muscle mass decline diseases. Notably, a statistically significant correlation was detected between the reduction of mir-451a expression and the increase of the change of SPPB score (i.e. the measurement of lower extremity function) after rehabilitation. Importantly, this correlation remains significant even with a multiple logistic regression adjusted with age and frailty index, meaning that this association is not affected by comorbidity and age. This result suggests that measuring the concentration of this circulatory miRNA might be a novel, easily accessible and non-invasive biomarker to evaluate and monitor the efficacy of rehabilitation programs in sarcopenic patients.
Considering SNAP-25 rs363050 polymorphisms, the only one miRNA differentially modulated was miR-421-3p, which was observed to be more expressed in AA homozygous sarcopenic patients compared to those with G allele. miR-421-3p is localized in chromosome X (Xq13.2) and was recently suggested to modulate the expression of bone morphogenetic protein-2 (BMP-2), a protein with an important role in the regulation of the differentiation and function of bone and muscle tissue, as well as in muscle regeneration [51]. MiR-451a and miR-421-3p, the two miRNAs that were observed to be differently expressed in sarcopenia, thus regulate two proteins (SNAP-25 and BMP-2) related to muscle function. This result is intriguing and allows to speculate that these two molecules play an important role in muscle trophism and functionality.
Although the expression of circulatory miRNAs was already investigated in sarcopenia [52][53][54], those analyses dealt with muscle-specific miRNAs (myoMir). To our knowledge this is the first study that analyzes the effects of rehabilitative treatment on non-muscle-specific miR-NAs expression and modulation in sarcopenic patients. Intriguingly, the concentration of six of these miRNAs in sarcopenic patients before treatment was similar to that observed in healthy controls, suggesting that their modulation is due to rehabilitation and not to pathology, an unexpected result that will need to be further analyzed. To note, miR-155-5p was found to be up-regulated in muscles of elderly people [55] whereas, in accordance with our results, its circulatory expression was seen to be down-regulated in sarcopenic patients compared to controls [53].
Overall, these results could facilitate the identification of biomarkers that could allow the early diagnosis of disease, and might be useful to verify the outcome of rehabilitative/pharmacological therapy in sarcopenia, as well as in other disorders, such as oncological diseases.
A limitation of our study is that the sarcopenia screening was performed just by physical performance indices without imaging parameters that are considered the gold standard to measure the actual ratio of muscle and fat.

Conclusions
To summarize, these results show the presence of associations between SNAP-25-related genetics and epigenetics and sarcopenia, indicate that non-muscle-related miRNAs are involved in the pathogenesis of this condition, and suggest that the analysis of circulating miRNAs could be used as a predictive biomarker for the outcome of rehabilitation protocols. Further analyses on larger cohorts of patients are needed to validate our results and to better understand the effects of these miRNAs on protein expression and disease development.