
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

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10.1038/s41598-024-72355-6
Article
Resistance exercise in early-stage ALS patients, ALSFRS-R, Sickness Impact Profile ALS-19, and muscle transcriptome: a pilot study
Jawdat Omar 1
Rucker Jason 2
Nakano Tomoki 34
Takeno Kotaro 3
Statland Jeffery 1
Pasnoor Mamatha 1
Dimachkie Mazen M. 1
Sabus Carla 210
Badawi Yomna 511
Hunt Suzanne L. 16
Tomioka Naoko H. 3
Gunewardena Sumedha 7
Bloomer Clark 8
Wilkins Heather M. 1
Herbelin Laura 19
Barohn Richard J. rbarohn@health.missouri.edu

19
Nishimune Hiroshi HiroshiNishimune@gmail.com

345
1 grid.412016.0 0000 0001 2177 6375 Department of Neurology, University of Kansas Medical Center, Kansas City, USA
2 grid.412016.0 0000 0001 2177 6375 Department of Physical Therapy, Rehabilitation Science, and Athletic Training, University of Kansas Medical Center, Kansas City, USA
3 Laboratory of Neurobiology of Aging, Tokyo Metropolitan Institute for Geriatrics and Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo, 173-0015 Japan
4 https://ror.org/00qg0kr10 grid.136594.c 0000 0001 0689 5974 Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu-shi, Japan
5 grid.412016.0 0000 0001 2177 6375 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, USA
6 grid.412016.0 0000 0001 2177 6375 Department of Biostatistics and Data Science, University of Kansas Medical Center, Kansas City, USA
7 grid.412016.0 0000 0001 2177 6375 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, USA
8 grid.412016.0 0000 0001 2177 6375 Genome Sequencing Facility, University of Kansas Medical Center, Kansas City, USA
9 grid.134936.a 0000 0001 2162 3504 Department of Neurology, University of Missouri, School of Medicine, 1 Hospital Dr, Columbia, MO 65201 USA
10 grid.67033.31 0000 0000 8934 4045 Present Address: Department of Rehabilitation Science, Tufts University School of Medicine, Boston, USA
11 https://ror.org/01an3r305 grid.21925.3d 0000 0004 1936 9000 Present Address: Department of Neuroscience, University of Pittsburgh, Pittsburgh, USA
17 9 2024
17 9 2024
2024
14 2172930 3 2024
5 9 2024
© The Author(s) 2024
2024
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Amyotrophic lateral sclerosis (ALS) patients lack effective treatments to maintain motor and neuromuscular function. This study aimed to evaluate the effect of a home-based exercise program on muscle strength, ALS scores, and transcriptome in ALS patients, Clinical Trials.gov #NCT03201991 (28/06/2017). An open-label, non-randomized pilot clinical trial was conducted in seven individuals with early-stage ALS. Participants were given 3 months of home-based resistance exercise focusing on the quadriceps muscles. The strength of exercised muscle was evaluated using bilateral quadriceps strength with manual muscle testing, handheld dynamometers, five times sit-to-stand, and Timed-Up-and-Go before and after the exercise program. In addition, changes in the Sickness Impact Profile ALS-19 (SIP/ALS-19) as the functional outcome measure and the transcriptome of exercised muscles were compared before and after the exercise. The primary outcome of muscle strength did not change significantly by the exercise program. The exercise program maintained the SIP/ALS-19 and the ALS Functional Rating Scale-Revised (ALSFRS-R). Transcriptome analysis revealed that exercise reverted the expression level of genes decreased in ALS, including parvalbumin. Three months of moderately intense strength and conditioning exercise maintained muscle strength of the exercised muscle and ALSFRS-R scores and had a positive effect on patients’ muscle transcriptome.

Keywords

Amyotrophic lateral sclerosis
Clinical trial
Exercise
Subject terms

Amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis
Next-generation sequencing
John Thompson, Jr. ALS Research Fundhttp://dx.doi.org/10.13039/100000065 National Institute of Neurological Disorders and Stroke R01NS078214 Nishimune Hiroshi http://dx.doi.org/10.13039/501100001691 Japan Society for the Promotion of Science 19K24690 Nishimune Hiroshi issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

The multidisciplinary approach of the ALS clinics has proven to be beneficial for the treatment and care of ALS patients compared to those patients without access to such clinics1–3. These ALS clinics provide occupational and physical therapy, which may lead to practicing some level of physical activity and exercise. Therefore, exercise may benefit the patient’s quality of life. However, exercise for ALS patients has been considered harmful4–7. Several studies have concluded that exercise is a risk factor for ALS5,8–10 while others were inconclusive in determining harm or benefit11,12. Meanwhile, many clinical trials have provided evidence that exercise is not a risk factor for ALS or has beneficial effects for ALS patients13–25. Recent randomized clinical trials have provided evidence for the beneficial effect of resistance exercise or resistance exercise combined with aerobic, endurance training for ALS patients15,20,23. In addition, a large cohort study has concluded that physical activity is not a risk factor for ALS and may be protective against the disease26. Consistently, recent Meta-analysis has concluded that exercise can significantly improve the functional ability and pulmonary function of ALS patients27. Therefore, we designed a pilot clinical trial to evaluate the effect of a moderate-level resistance training program for ALS patients to test the hypothesis that such an exercise regimen ameliorates ALS symptoms. The program focused on training quadriceps muscles, and the primary outcome was assessed by quadriceps muscle strength. The secondary outcome variables are changes in the Sickness Impact Profile ALS-19 (SIP/ALS-19) as the functional outcome measure and changes in the muscle transcriptome as the molecular outcome measure. The purpose of transcriptome analysis is because exercise alters gene expression levels of skeletal muscles as the adaptive response following the mechanical loading to the skeletal muscle28. In addition, strength training has been shown to clinically benefit myotonic dystrophy type 1 patients, and the molecular mechanism has been studied recently by analyzing the transcriptome of the trained patients’ muscles29. The aforementioned clinical trials of exercise treatment for ALS patients have not investigated the effect of exercise on transcriptome changes. Therefore, we analyzed the changes in the muscle transcriptome to study the potential underlying mechanism of exercise effect for ALS patients.

Results

Demographics

This pilot study was planned to enroll seven patients based on a power analysis using representative transcriptome data from an ALS model rodent exercise experiment (see methods section for details). This power analysis was based on relevance to the secondary outcome, transcriptome analysis. Nine patients with early-stage ALS were recruited in this study, and the demographics were two females and seven males, who were all Caucasian race and not Hispanic or Latino. However, one patient withdrew consent in the second month due to experiencing fatigue and unsteadiness during exercise, and another patient stopped participation after the screening visit. Seven patients completed the 3-month exercise program. At enrollment, these seven patients had an age range of 36 to 67 years (median 44, Table 1).Table 1 Vital signs, ALSFRS-R, SIP/ALS-19 of participants at baseline and after the exercise program.

Patient ID	3843-001	3843-002	3843-003	3843-004	3843-005	3843-006	3843-007	3843-008	3843-009	
Gender	Female	Male	Male	Female	Male	Male	Male	Male	Male	
Age	67	66	36	66	41	N/A	46	37	44	
Baseline blood pressure	154/85	132/84	135/98	125/79	147/95		130/91	142/78	135/96	
Baseline pulse	80	63	85	74	76		62	110	90	
Baseline weight		194	210	140	224		221	210	173	
Baseline forced vital capacity	84	73	96	59	96	59	93	62	86	
Baseline ALSFRS-R	35	41	43	40	37		43	39	39	
Baseline SIP/ALS-19	0	1	0	0	2		0	0	1	
Pre-exercise biopsy	+	+	+	+	+	N/A	+	+	+	
Post-exercise Blood Pressure	148/80	125/95	155/110		144/98		130/96	141/76	152/98	
Post-exercise pulse	90	89	91		88		65	62	84	
Post-exercise weight	155	192	211		219		215	202	168	
Post-exercise biopsy	+	+	+	N/A	+	N/A	+	+	+	
Post-ALSFRS-R	36	38	42		32		45	31	40	
Post- SIP/ALS-19	1	2	0		6		1	0	5	

ALS assessments

Among the seven patients, two were bulbar onset and five were limb onset. One patient was familial, and others were sporadic type. The days from diagnosis to enrollment in the exercise program ranged from 41 to 350 days (median 158). Vital signs are shown in Table 1. ALSFRS-R scores varied from 35 to 43 (median 39), and SIP/ALS-19 scores were 0 to 2 (median 0) (Fig. 1, Tables 1, 2). The ALSFRS-R is the widely used rating scale for monitoring the disability progression of ALS patients based on 12 categories of evaluations, giving a score range of 48 for healthy people to decreasing scores for patients30. The SIP/ALS-19 is a set of 19 questions independently chosen from a 136-item Sickness Impact Profile by a panel of ALS specialists, giving a score range of 0 for healthy people to increasing scores for patients31,32. The SIP/ALS-19 corresponds closely to strength and function measures and can be used as an effective surrogate for the ALSFRS32, with the difference that the SIP/ALS-19 also includes questions encompassing the psychological and social domains of quality of life33. After the 3-month exercise program, ALSFRS-R scores were 31 to 45 (median 38), and SIP/ALS-19 scores were 0 to 6 (median 1). The ALSFRS-R scores increased for three patients and decreased for four (Table 1, Fig. 1A). The SIP/ALS-19 scores increased for five patients and remained the same for two (Table 1, Fig. 1B, Supplementary Table S1). The ALSFRS-R and SIP/ALS-19 scores were not statistically different between baseline and post-exercise sessions (Table 2).Fig. 1 Baseline and post-exercise scores for ALSFRS-R and SIP/ALS-19. Graphs show changes of (A) ALSFRS-R and (B) SIP/ALS-19 scores by the exercise program. Patient IDs are indicated on the right side of the data points. Data points above the dotted line reflect an improvement after the exercise program.

Table 2 Functional outcome measures.

		Min	Median	Max	Mean	SD	Wilcoxon matched-pairs test	
ALSFRS-R	Baseline	35	39	43	39.6	2.99		
Post-exercise	31	38	45	37.7	5.12		
Change	− 4	− 1	2	− 1.86	3.67	NS	
SIP/ALS-19	Baseline	0	0	2	0.571	0.787		
Post-exercise	0	1	6	2.14	2.41		
Change	0	1	4	1.57	1.72	NS	
ALSFRS-R and SIP/ALS-19 were from seven participants. The third values of the mean and SD columns indicate the mean and SD of the differences.

Human exercise

The study aimed to determine the muscle strength of the quadriceps femoris muscles before and after the 3-month home exercise program among ambulatory subjects early in the disease process. We assessed bilateral quadriceps strength with a handheld dynamometer and manual muscle testing as quantifiable outcome measures collected at baseline and after 12 weeks of home exercise. The handheld dynamometer results showed an increased median for both knees (Median, right baseline 49.6 lbs to post-exercise 54.3 lbs; left baseline 49.2 lbs to post-exercise 56.1 lbs). The manual muscle testing for knee extension showed no change of median for both knees (Median, right and left baseline 5 to post-exercise 5). In addition, we measured timed five times Sit-to-Stand and Timed-Up-and-Go as functional outcome measures. The median slightly increased for both the timed five times Sit-to-Stand and the Timed-Up-and-Go (Median, Sit-to-Stand, baseline 12.2 s to post-exercise 13 s; Timed-Up-and-Go, baseline 9.15 s to post-exercise 10.7 s). Patient 3843-005 did not participate in the quantifiable and functional outcome measures at the post-exercise session; therefore, these measures were analyzed for six patients who completed both baseline and post-exercise sessions. The compliance of the home exercise intervention program and the adverse effects of the exercise are described in Table 3. The means did not show statistically significant differences for all four tests.Table 3 Summary of compliance for each participant.

	3843-001	3843-002	3843-003	3843-004	
Follow-up number	1	2	3	1	2	3	1	2	3	1	2	3	
Has participant been compliant with the home exercise program?	Yes	Yes	Yes	Yes	Yes	Yes		Yes		Yes	Yes		
Number of sessions missed	0	0	0	0	0	0		3		5	7		
Has participant modified exercise program in order to maintain compliance?	No	No	No	No	No	No		No		Yes	Yes		
If Yes, how	N/A	N/A	N/A	N/A	N/A	N/A		N/A		Skipped exercises	Skipped exercises		
Has participant experienced any adverse effects of exercise?	No	No	No	No	No	No		No		Yes	No		
If Yes, what type?	N/A	N/A	N/A	N/A	N/A	N/A		N/A		Toe injury	N/A		
	3843-005	3843-007	3843-008	3843-009	
Follow-up Number	1	2	3	1	2	3	1	2	3	1	2	3	
Has participant been compliant with the home exercise program?	Yes			Yes	Yes	Yes	Yes	Yes		Yes			
Number of sessions missed	3			2	3	1	0	0		4			
Has participant modified exercise program in order to maintain compliance?	Yes			No	No	No	No	No		No			
If Yes, how	Skipped exercises			N/A	N/A	N/A	N/A	N/A		N/A			
Has participant experienced any adverse effects of exercise?	Yes			No	No	No	No	No		No			
If Yes, what type	LE fasciculations and tremor			N/A	N/A	N/A	N/A	N/A		N/A			
		Did not participate in monthly phone calls with the physical therapist

3843-006 did not participate

	

Transcriptome of exercised muscles of ALS patients

Muscle needle biopsies were obtained from the exercised quadriceps femoris muscles from the same leg of seven patients before and after the exercise program (Patient 3843-001~3, 5, 7~9). The transcriptome of these muscles was analyzed by RNA-seq using the Illumina NovaSeq 6000 sequencing system. First, we evaluated the expression level of 44 clinically validated ALS-related genes and five recently identified genes linked to ALS, which are described in the methods section. In the patient’s muscle, four genes were not detected or detected at a very low level of cpm < 1: DAO, KIF5A, NEFH, and PRPH. The other 45 genes showed expression levels of cpm > 1; however, none of them showed a significant change (fold-change > 1.5 or < − 1.5, p < 0.05) between before and after the exercise program. Thus, the moderate-level exercise program did not change the expression level of genes linked to ALS in the patient’s muscle.

Next, we focused on genes upregulated by the exercise program. The selection criteria for the up-regulated genes are described in the methods section. The network pattern of upregulated genes suggested that it has an inhibitory role for the onset of ALS, which is one of the networks registered in IPA as the network22 onset of ALS (Fig. 2A). The transcriptome of upregulated genes showed a relation with the IPA disease and function categories (gene sets), including skeletal and muscular system development and function, skeletal and muscular disorder, nervous system development and function, neurological disease, cell death and survival, connective tissue development and function, and a total of 11 categories related to muscle (Fig. 2B). Pathway analysis revealed the activation of ‘acute phase response signaling’ as the top and ‘wound healing’ signaling pathway within the top 20 relevant canonical pathways (Fig. 2C). These pathways are related to tissue repair, suggesting that the exercise program activates protective pathways for the muscle. The ‘acute phase response signaling’ category included three genes, FOS, SAA, and SERPINA3, which were selected in Table 4 and explained in the next paragraph.Fig. 2 Network and pathway analyses of exercise-activated genes. (A) The RNA-seq data superimposed on the network ‘onset of ALS’ in the Qiagen IPA. Warm colors indicate genes with increased expression levels in the RNA-seq data, including PVALB on the right. Cold colors and blue dotted lines suggest inhibitory influence and role, including the onset of ALS shown as a blue cross at the center. (B) Diseases and function categories of IPA knowledgebase related to the increased genes of RNA-seq data. Orange highlights indicate categories containing disease or function annotations related to muscle. Yellow highlights indicate categories containing disease or function annotations related to disease and survival of neurons or muscle. (C) List of 20 canonical pathways showing significant activation or inhibition based on the increased genes of RNA-seq data. (D) The top canonical pathway in list C, ‘acute phase response signaling,’ is shown. The yellow arrows indicate the FOS, SAA, and SERPINA3 genes, which are also included in Table 4 as the top 20 genes upregulated after exercise intervention in ALS patients.

Table 4 Top 20 genes upregulated after exercise intervention in ALS patients.

Rank	NCBI gene symbol	NCBI gene full names	Protein location	Fold change (Log2)	ALS research publications	Expression increases by exercise in humans or animals	Expression decreases in ALS and increases after exercise	
1	CLDN1	Claudin 1	Plasma membrane	3.72	+			
2	CPA4	Carboxypeptidase A4	Extracellular space	2.74				
3	NGFR	Nerve growth factor receptor	Plasma membrane	2.59	+			
4	PVALB	Parvalbumin	Cytoplasm	2.53	+	+	+	
5	CHI3L1, YKL-40	Chitinase 3 like 1	Extracellular space	2.48	+	+		
6	SAA1	Serum amyloid A1	Extracellular space	2.46	+	+		
7	EPHB3	EPH receptor B3	Plasma membrane	2.27				
8	SAA2	Serum amyloid A2	Extracellular space	2.24		+		
9	FJX1	Four jointed box 1	Extracellular space	2.21				
10	SAA2-SAA4	SAA2-SAA4 readthrough	Extracellular space	2.1				
11	SCD	Stearoyl-CoA desaturase = delta-9 desaturase	Cytoplasm	2.04	+	+	+	
12	DUSP4	Dual specificity phosphatase 4	Nucleus	1.98				
13	MYBPH	Myosin binding protein H	Cytoplasm	1.96	+			
14	TFPI2	Tissue factor pathway inhibitor 2	Extracellular space	1.96	+			
15	CDC42EP5	CDC42 effector protein 5	Cytoplasm	1.93				
16	FOS (AKA: AP-1, C-FOS, p55)	Fos proto-oncogene, AP-1 transcription factor subunit	Nucleus	1.91	+	+	+	
17	LRRC4	Leucine rich repeat containing 4	Plasma membrane	1.9				
18	SERPINA3	Serpin family A member 3	Extracellular space	1.9	+			
19	PDK4	Pyruvate dehydrogenase kinase 4	Cytoplasm	1.89	+	+		
20	RPS6KA1 (AKA RSK1)	Ribosomal protein S6 kinase A1	Cytoplasm	1.89				
+: shows genes described in ALS research publications, genes known to increase expression level by exercise, and genes known to decrease expression by ALS but recover by exercise.

The upregulated genes were ranked by the fold change value, and the top 20 genes are shown in Table 4. Among these genes, 11 have been described in ALS research publications based on a Pubmed search, and seven genes were known to increase their expression level by exercise in human or animal studies. Within these genes, PVALB (parvalbumin), SCD (stearoyl-CoA desaturase), and FOS (C-FOS or AP-1 transcription factor subunit) are known to decrease their expression levels in ALS patients or ALS animal models and increase after exercise programs. The three genes were further evaluated by RT-qPCR using TaqMan assays. All three genes increased expression level by the exercise program in five out of seven patients, which was consistent with the criteria for selecting these genes from the RNA-seq data. PVALB showed a statistically significant increase among the seven patients by the exercise program (Fig. 3).Fig. 3 Expression level quantification of RNA-seq identified genes. The three genes identified in Table 4, (A) PVALB, (B) SCD, and (C) FOS, were validated using RT-qPCR, normalized to the expression level of GAPDH, and expressed as a relative value to the pre-exercise program for each patient. All three genes showed increased expression among five to six out of seven patients tested. PVALB showed a statistically significant increase (paired t-test, p < 0.05, n = 6).

Discussion

We evaluated whether 12 weeks of a home-based exercise program is beneficial for preserving the muscle strength and function of ALS patients with leg involvement early in the disease process. The primary outcome of muscle strength of the exercised quadriceps and the secondary outcome of the SIP/ALS-19 did not show a significant difference between baseline- and post-exercise-means. The exercise program did not significantly change the ALSFRS-R scores and did not cause serious adverse events. These results suggested that moderate-level exercise was not detrimental and may be beneficial for maintaining the muscle strength and ALS-FRS-R score of early-stage ALS patients. However, these clinical assessments should be interpreted cautiously due to the small sample size and a short trial period.

For the other secondary outcome variables, the exercise program had a beneficial effect on the transcriptome of ALS patients’ skeletal muscles by reverting the expression levels of genes downregulated in ALS patients. The upregulation of PVALB by the exercise program is suggested to have an inhibitory role in the onset of ALS based on the IPA network analysis. The IPA analysis also revealed the activation of an IPA canonical pathway called ‘acute phase response signaling,’ which is a pathway to protect against tissue injury and promote tissue repair (https://www.sciencedirect.com/topics/medicine-and-dentistry/acute-phase-response). This canonical pathway included three of the top 20 increased genes, namely Parvalbumin, Stearoyl-CoA desaturase, and Fos protooncogene. Parvalbumin protein is a high-affinity calcium-binding protein that regulates muscle contraction and relaxation. The PVALB expression level in ALS patients’ muscles is not well known. In SOD1G93A mice, the reduced expression level of PVALB causes perturbed intracellular Ca2+ handling in skeletal muscles34. Therefore, the increased expression level of parvalbumin by the exercise program may contribute to the maintained muscle strength of the exercised muscle. Stearoyl-CoA desaturase enzyme increases triglyceride formation and lipogenesis and improves lipid bilayer fluidity35. The expression level of Stearoyl-CoA desaturase is down-regulated in ALS patients’ skeletal muscles36. Exercise increases the expression level of stearoyl-CoA desaturase and triglyceride in muscles35. Fos protooncogene (C-FOS) protein is a leucine zipper transcription factor, which was down-regulated in microglia of SOD1*G93A mice37. Exercise increases the expression level of C-FOS protein in skeletal muscle38,39, and the AP-1 complex is likely to modulate exercise-induced antioxidant mechanisms38. However, the upregulation of these genes could be a general reaction of muscle to resistance exercise. It may not be a specific genetic link to ALS or a particular response of ALS-affected muscle. A lack of healthy control is the limitation of this study. These findings suggest that the increased expression level of these genes by the exercise program is likely to contribute to the maintenance of muscle function of the patients.

The exercise program in the current study did not alter the expression level of 49 ALS-related genes40,41. However, a previous study reported a positive causal relationship between ALS and physical exercise based on Mendelian randomization, transcriptomics, and ALS risk genotypes40. The difference between the two studies might have risen from the difference in the analyzed materials, which were quadriceps femoris muscles in the current study versus peripheral blood mononuclear cells in the previous study.

Based on the data of this study, moderate-level home-based exercise may be a safe regimen to maintain muscle strength, SIP/ALS-19, and ALSFRS-R scores of early-stage ALS patients. Similarly, we have seen a positive effect of resistance exercise protecting against neuromuscular junction denervation in the SOD1G93A rat model of ALS42. However, the exercise program should be monitored and kept at a moderate level to avoid damage to muscle fibers due to vigorous intensity activities43. The molecular mechanism investigation also revealed potentially beneficial effects of the exercise for the patients, including the inhibition of ALS-related gene network and the activation of PVALB. These gene expression measures could be sensitive biomarkers in addition to respiratory or strength measures because they may quantify the effect of resistant exercise and personalize the treatment to each patient. This pilot study was planned to enroll seven patients based on the justification explained in the results and methods sections. However, the small sample size is a limitation of this pilot study, and a larger-scale clinical trial will be necessary to generalize the effect of resistance exercise on ALS patients.

Methods

Overall study design and inclusion/exclusion criteria

The study, ALS study determining various biomarkers and strength comparison after exercise: ADVANCE, was approved by the University of Kansas Medical Center Institutional Review Board and registered under Clinical Trials.gov as #NCT03201991 (28/06/2017, https://clinicaltrials.gov/study/NCT03201991?titles=ALS). All research was performed in accordance with relevant guidelines/regulations. All participants were provided a written informed consent form stating, “the researchers may publish the results and that they will be presented as group results. Your name will not be used in any publication or presentation about this study.” The participants were provided the opportunity to read the consent form and ask questions when the consent form was discussed with the participants before signing. After the consent form was signed, a copy was provided for the participant. All participants had baseline testing of vital signs and a clinical diagnosis of ALS. Eligible participants performed tests of quantifiable outcome measures described below, 12 weeks of home exercise, followed by tests of quantifiable outcome measures after the exercise program.

The inclusion and exclusion criteria were as follows. Inclusion criteria: (a) Diagnosis of possible, probable, or definite ALS based on the revised El-Escorial criteria, (b) Presence in the ipsilateral leg of either weakness in any muscle group or of active denervation by needle EMG, which is a surrogate marker of early weakness, as we will be performing exercises focused on the quadriceps femoris and obtaining biopsies from that muscle, (c) Ipsilateral quadriceps femoris strength: > = 4, (d) Ambulatory with or without assistance, (e) ALS Functional Rating Scale-Revised (ALSFRS-R) > 30, (f) Forced expiratory vital capacity (FVC) > 50% of predicted since subjects with FVC < 50% of predicted have significant restrictive respiratory compromise resulting in exercise limiting dyspnea. This in turn would be a barrier in completing the study exercises.

Exclusion criteria: (a) ALSFRS-R ≤ 30, (b) Quadriceps femoris strength < 4, (c) Inability to ambulate, (d) More than mild atrophy of quadriceps by visual inspection, (e) Bleeding disorder or taking anticoagulants, (f) Unwilling to comply with exercise and needle muscle biopsy, (g) Not a good research candidate according to the medical opinion of the investigator, such as medical or psychiatric co-morbidities, or suicidal ideation. We planned to enroll seven patients based on a power analysis (power of 95%, significance set at p = 0.05, two tailed) using representative transcriptome data from an ALS model rodent exercise experiment that showed twofold significant difference between exercises versus sedentary (VEGF, fold change 2.2, FDR 0.0002, P < 0.00001).

Exercise program and quantifiable outcome measures

A home-based exercise program focusing on quadriceps femoris muscle strengthening and conditioning was developed by a physical therapist and consisted of the following exercises: bilateral isometric quadriceps contractions, short arc quadriceps exercises, straight leg raises, repeated sit-to-stands, partial squats, and timed wall sits (supplementary online material). All exercises were performed at home each day for 12 weeks, with a goal of 3 sets of 10 repetitions for each exercise. The program was developed to promote general muscle conditioning and encourage daily physical activity. Participants were instructed perform as many of the exercises as they felt comfortable doing at what they perceived to be a moderate level of intensity. They were educated on the signs and symptoms of overexertion (e.g. excessive muscle soreness, fatigue, etc.) and encouraged to avoid any exercises that they felt were uncomfortable or unsafe. Resistance-type exercise was selected based on successful outcome in previous randomized controlled trials15,20. As our program was intended to be home-based, we chose body weight resistance activities, targeting the quadriceps muscle. This muscle was chosen for the feasibility of muscle biopsy sampling. The quadriceps is one of the muscles traditionally chosen for muscle biopsy44.

Participants were provided with a written handout as well as access to a YouTube video exercise guide (https://goo.gl/Wu9pnU). Participants were instructed not to start exercising for three days following the biopsy. Exercises were performed without supervision; therefore, compliance was tracked with a calendar fitness application. The study participants were not assessed of outside physical activity during the trial. Adverse events due to exercise were monitored, including soreness, pain, weakness, fatigue, and falls. There were weekly text messages or emails for encouragement and monthly phone calls with the physical therapist to report adverse events, discuss exercises, review the tracking calendar, problem-solve barriers or challenges, and modify difficulty as appropriate.

Quantifiable outcome measures were bilateral quadriceps strength with manual muscle testing (grades 0 to 5) and a handheld dynamometer collected at baseline and after 12 weeks of home exercise. The functional outcome measures were timed five times Sit-to-Stand, Timed-Up-and-Go, and Sickness Impact Profile ALS-19 (SIP/ALS-19). The SIP/ALS-19 is a set of 19 questions independently chosen from a 136-item Sickness Impact Profile by a panel of ALS specialists33, giving a score range of 0 for healthy people to increasing scores for patients31,32. The ALSFRS-R is the widely used rating scale for monitoring the disability progression of ALS patients based on 12 categories of evaluations, giving a score range of 48 for healthy people to decreasing scores for patients30.

Muscle biopsy

Muscle needle biopsies were obtained from quadriceps femoris muscles of seven ALS patients before and after the exercise intervention. The biopsies were immediately frozen between dry ice at the site of muscle biopsy and stored at − 80 °C until total RNA purification for the transcriptome analysis.

RNA-sequencing

Patient muscle biopsies were homogenized in TRIzol reagent (Invitrogen) and transferred to 5 Prime—Phase Lock Gel–heavy 2 ml tubes (Andwin Scientific) to purify total RNA. RNA quality was checked using the Agilent Bioanalyzer 2100. The Stranded mRNA-Seq was performed using the Illumina NovaSeq 6000 Sequencing System (Illumina, San Diego, CA) at the University of Kansas Medical Center—Genomics Core (Kansas City, KS). Total RNA (500 ng) was used to initiate the Stranded mRNA-Seq library preparation protocol. The total RNA fraction underwent oligo dT bead capture of mRNA, fragmentation, sizing, reverse transcription into cDNA, and ligation with the appropriate indexed adaptors using the TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina RS-122-2101/2102). Following Agilent Bioanalyzer QC of the library preparation and library quantification using the Roche Lightcycler96 with FastStart Essential DNA Green Master (Roche—cat#06402712001), the mRNA-Seq libraries were normalized to 2.125 nM and pooled for multiplexed sequencing. Pooled libraries were denatured with 0.2N NaOH (0.04 N final concentration) and neutralized with 400 mM Tris–HCl pH, resulting in a 380 pm concentration of the library pool for final onboard clonal clustering of the patterned flow cell using the NovaSeq 6000 S1 Reagent Kit—200 cycle (Illumina 20012864). A 2 × 101 cycle sequencing profile with dual index reads is completed using the following sequence profile: Read 1–101 cycles × Index Read 1–8 cycles × Index Read 2–8 cycles × Read 2–101 cycles. Following collection, sequence data is converted from .bcl file format to fastq file format using bcl2fastq software and de-multiplexed into individual sequences for data distribution using a secure FTP site or Illumina BaseSpace for further downstream analysis.

RNA-Seq data analysis

Samples, pre and post-exercise, from seven individuals with ALS were analyzed using RNA-sequencing. Sequencing generated between 25.8 and 28.7 million reads per sample. The read quality was assessed using the FastQC software45. On average, the per sequence quality score measured in the Phred quality scale was above 30 for all the samples. The reads were mapped to the human genome (GRCh38.rel92) using the STAR software, version 2.5.2b46. Between 96–98% of the sequenced reads in the 14 samples mapped to the reference genome, resulting in between 24.8 and 27.7 million mapped reads per sample, of which 94% on average were uniquely mapped reads.

Transcript abundance estimates were calculated using the RSEM47 (version 1.3.0) software. Expression normalization and differential gene expression calculations were performed in edgeR48 (release 2.14) to identify statistically significant differentially expressed genes. EdgeR employs a negative binomial generalized linear model (NB-GLM) for statistical calculations. The edgeR package implements advance empirical Bayes methods to estimate gene-specific biological variation under minimal levels of biological replication. A blocked design, with the subject as the blocking factor, was employed as the design model for the NB-GLM. The RNA composition in each sample was normalized in edgeR using the trimmed mean of M-values (TMM) method. The significance p-vales were adjusted for multiple hypotheses testing by the Benjamini and Hochberg method49 establishing a false discovery rate (FDR) for each gene.

Clinically validated ALS-related 44 genes and recently identified five genes linked to ALS were obtained from these references40,41, which include ALS2, ANG, ANXA11, ARHGEF28, ATXN2, C9orf72, CHCHD10, CHMP2B, CYP27A1, DAO, DCTN1, ERBB4, EWSR1, FIG4, FUS, GBA2, GRN, HNRNPA1, HNRNPA2B1, KIF5A, MAPT, MATR3, NEFH, NEK1, OPTN, PFN1, PRPH, SETX, SIGMAR1, SOD1, SPAST, SPG20, SPG11, SQSTM1, SS18L1, TAF15, TARDBP, TBK1, TUBA4A, UBQLN2, VAPB, VCP, VPS54, VRK1, GLT8D1, GLE1, PPARGC1A, TIA1, C21orf2).

For Fig. 2 and Table 4, upregulated genes were selected based on the following criteria: the expression level increased by the exercise program in five out of seven patients, the difference was statistically significant p < 0.05, and the expression level was at a reliable level of cpm > 1 after the exercise program in four out of seven patients. Biological functional and pathway analysis was performed using the online software Ingenuity Pathway Analysis (IPA, Qiagen)50 on the significantly upregulated genes. For Network generation, the data set containing gene identifiers and corresponding data measurement values was uploaded into the IPA application. Each identifier was mapped to its corresponding entity in QIAGEN's Knowledge Base. The molecules were overlaid onto a global molecular network developed from information contained in the QIAGEN Knowledge Base. Networks of Network Eligible Molecules were then algorithmically generated based on their connectivity50. The Diseases and Functions Analysis identified the biological functions and/or diseases that were most significant from the data set. Molecules from the dataset associated with biological functions and/or diseases in the QIAGEN Knowledge Base were considered for the analysis. A right-tailed Fisher’s Exact Test was used to calculate a p-value determining the probability that each biological function and/or disease assigned to that data set is due to chance alone. A z-score was calculated to indicate the likelihood of an increase or decrease in that disease or function. Canonical pathways analysis identified the pathways from the QIAGEN Ingenuity Pathway Analysis library of canonical pathways that were most significant to the data set. Molecules from the data set associated with a canonical pathway in the QIAGEN Knowledge Base were considered for the analysis. The significance of the association between the data set and the canonical pathway was measured in two ways: (1) A ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to the canonical pathway is displayed, and (2) A right-tailed Fisher’s Exact Test was used to calculate a p-value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone. (3) In many cases, a z-score was calculated to indicate the likelihood of activation or inhibition of that pathway.

Quantitative RT-PCR

We analyzed RNA samples from seven ALS patients obtained before and after the exercise intervention. Total RNAs purified for RNA-seq analysis were used to reverse transcribe 100 ng of total RNA to cDNA using SuperScript IV VILO master mix (Thermo Fisher Scientific). Equal volume cDNAs were used to analyze relative gene expression levels by TaqMan real-time PCR using the QuantStudio 3 real-time PCR system (Thermo Fisher Scientific) in 96-well plates and 10 μl reaction volume using TaqMan fast advance master mix for qPCR (Thermo Fisher Scientific). The cycling conditions were as follows: initial denaturation at 50 °C for 2 min and 95 °C for 20 s, followed by 40 cycles of denaturation at 95 °C for 1 s and annealing/extension at 60 °C for 20 s. Each sample was run in duplicate reactions and a control reaction without reverse transcription, using the comparative CT ΔΔCT method. Three genes were detected using TaqMan assays designed by Thermo Fisher Scientific: PVALB (parvalbumin, Hs00161045_m1), SCD (stearoyl-CoA desaturase, Hs01682761_m1), FOS (C-FOS or AP-1 transcription factor subunit, Hs00170630_m1) and normalized with GAPDH (glyceraldehyde-3-phosphate dehydrogenase, Hs99999905_m1) using the manufacture recommended amplification conditions. Relative quantification analysis was performed using the Design and Analysis 2.6 software (Thermo Fisher Scientific), and RQ values were compared before and after the exercise program.

Statistics

The differences of quantifiable and functional outcome measures between baseline and post-exercise program were assessed using the Wilcoxon matched-pairs signed-rank test. The qPCR results were assessed using a paired t-test. All statistics were performed using GraphPad Prism software version 9. The p and n values are reported in the text.

Supplementary Information

Supplementary Table S1.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72355-6.

Acknowledgements

We thank Andrew J. Heim, MS-CR, CCRP, for his administrative support. This work was supported by the NIH under Grant R01NS078214 and the JSPS-KAKENHI under Grant 19K24690 (H.N.), the John Thompson, Jr. ALS Research Fund (O.J., H.N.), the Genomics Core facility receives support from the NIH under the Grant P30AG035982, U54HD090216, P30GM122731-03, S10OD021743, and UL1TR002366.

Author contributions

Conception and design of the study: O.J., J.R., J.S., C.S., H.M.W., L.H, R.J.B., H.N.; Investigation and acquisition of the data: O.J., J.R., T.N., K.T., J.S., M.P., M.M.D., C.S., Y.B., S.G., C.B., H.M.W., R.J.B., H.N.; Analysis and interpretation of data: O.J., J.R., T.N., K.T., Y.B., S.L.H., N.H.T., S.G., H.M.W., R.J.B., H.N.; Funding acquisition: O.J., R.J.B., H.N.; Writing-original draft preparation: H.N.; Writing-Review, editing, final approval: all authors.

Data availability

The data from this study cannot be made publicly available because no patient approval has been obtained for sharing coded data. The output of statistical analyses will be made available upon reasonable request. RNA-seq data generated during this study are available in the Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) through the GEO series accession number (GSE250455). Other datasets generated in this study are available from the corresponding authors upon reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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