- Email: [email protected]
Transforming growth factor-β plasma levels and its role in amyotrophic lateral sclerosis
Journal Pre-proofs Transforming growth factor-β plasma levels and its role in Amyotrophic Lat‐ eral Sclerosis Tatiana Duque, Marta Gromicho, Ana Catarina Pronto-Laborinho, Mamede de Carvalho PII: DOI: Reference:
S0306-9877(20)30026-8 https://doi.org/10.1016/j.mehy.2020.109632 YMEHY 109632
To appear in:
Medical Hypotheses
Received Date: Revised Date: Accepted Date:
5 January 2020 9 February 2020 13 February 2020
Please cite this article as: T. Duque, M. Gromicho, A.C. Pronto-Laborinho, M. de Carvalho, Transforming growth factor-β plasma levels and its role in Amyotrophic Lateral Sclerosis, Medical Hypotheses (2020), doi: https://doi.org/10.1016/j.mehy.2020.109632
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.
Transforming growth factor-β plasma levels and its role in Amyotrophic Lateral Sclerosis
Tatiana Duque1, Marta Gromicho1, Ana Catarina Pronto-Laborinho1, Mamede de Carvalho1,2 1 Instituto de Fisiologia, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa. Lisbon, Portugal. 2 Department of Neurosciences and Mental Health, Centro Hospitalar Universitário de Lisboa-Norte. Lisbon, Portugal.
Running title: ALS and transforming growth factor-β plasma levels
Word count: words
Keywords: amyotrophic lateral sclerosis; biomarker; predictive value; progression; transforming growth factor-β plasma levels
Corresponding author: Professor Mamede de Carvalho Faculdade de Medicina, Universidade de Lisboa. Av. Professor Egas Moniz, 1648-028 Lisbon, Portugal E –mail: [email protected] Phone and Fax: + 351 21 7805219
1
ABSTRACT Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive muscle paralysis. Respiratory complications are the main cause of death in ALS. For this reason, initial respiratory status and its decline over disease progression are strong independent predictors of survival. Riluzole, a glutamatergic neurotransmission inhibitor, is the only drug that has shown to extend survival. Therefore, both novel molecular biomarkers and treatment strategies are needed. Transforming growth factor-β (TGF-β) family cytokines are important regulators of cell fate affecting both neurogenesis and neurodegeneration. Several studies demonstrate that TGF-β signalling protects neurons from glutamate-mediated excitotoxicity, a recognized mechanism underlying the pathogenesis of various neurodegenerative disorders such as ALS. Recent studies report dysregulations of the TGF-β system as a common feature of neurodegenerative disorders. The upregulation of this system has been linked with ALS progression. We have quantified TGF-β1, TGF-β2 and TGF-β3 serum levels in 23 ALS patients and 12 healthy controls, our preliminary results support the hypothesis that TGF-β3 levels can be a marker disease severity ALS. Further results are necessary to confirm this hypothesis.
2
INTRODUCTION Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease or motor neuron disease (MND), is a neurodegenerative disease characterized by progressive muscle paralysis, due to the degeneration of motor neurons in the primary motor cortex, brainstem and spinal cord [1]. ALS is the most frequent motor neuron disease, affecting 1 per 20,000 people worldwide [2]. There are two types of ALS differentiated by family history: positive, familial-ALS (5% to 10% of the patients); and negative, sporadic-ALS. Diagnosis is primarily determined by clinical examination coupled with nerve conduction studies (NCSs), needle electromyography (EMG), laboratory testing and imaging. In general, initial symptoms may be described as “limb-onset” or “bulbar-onset.” With limb onset, patients may experience difficulty with simple actions, such as buttoning a shirt, stumbling more easily, and experiencing changes in their walking. Patients with bulbar onset may experience challenges with chewing, swallowing, and speaking, such as nasal or slurred speech [3]. Some patients experience respiratory or generalized onset. Extramotor symptoms can also occur, such as cognitive deficits, which are present in more than 10% of patients [4]. Atypical presentation includes emotional lability, weight loss, and fasciculations and cramps without muscle weakness [3]. Diagnosis can, thus, be difficult at onset, because ALS is a clinically heterogeneous disorder. Therefore, in the early phase, misdiagnosis of ALS remains a clinical problem. Disease progression is clinically evaluated by the application of the functional scale ALS-Functional Rating Scale-revised (ALSFRS-R), which is a validated rating instrument for monitoring the progression of disability in patients with
3
ALS. Its rate of decline is a strong independent predictor of survival [5]. No cure has yet been found and median survival is 2-3 years after onset [2]. The management of ALS is mainly supportive and palliative. Respiratory complications are the main cause of death in ALS, in particular, respiratory failure, in spite of effective and early intervention with non-invasive ventilation. For this reason, initial respiratory status and its decline over disease progression are strong independent predictors of survival [6]. Respiratory function is easily accessed by forced vital capacity (FVC), which provides a reliable indication of inspiratory and expiratory muscles function [7]. Both ALSFRS-R and FVC are evaluated at each consultation (every 3-4 months) as recommended in the European Guidelines [8]. Immune and inflammatory abnormalities seem to contribute to the pathogenesis and progression of ALS. Neuronal loss is an inflammation mediated process, in which resident and infiltrating immune cells, secreted inflammatory modulators and expressed surface receptors on activated microglial cells take part. Activated microglial cells, astrocytes and proinflammatory cytokines influence proliferation, cell survival and death of neurons, also reducing neurogenesis [4]. T cells have been linked to immune modulation by promoting microglia to generate a neuroprotective environment. Astrocytes are responsible for regulating glutamate by the activity of the excitatory aminoacid-transporter 2. The excitotoxicity of glutamate occurs when synaptic glutamate is not quickly removed, resulting in the excessive firing of motor neurons, leading to mitochondrial and endoplasmic reticulum stress [3]. Riluzole, a glutamatergic neurotransmission inhibitor, is the only drug that has shown to extend survival. Therefore, new treatment strategies are needed [1].
4
Moreover, for the ALS community, the identification of new molecular biomarkers, with predictive value, is considered a critical priority [9].
HYPOTHESIS TGF-β plasma levels correlate with markers of disease progression in Amyotrophic Lateral Sclerosis (ALS)
a- TGF-β family and physiological processes
Transforming growth factor-β (TGF-β) family cytokines are important regulators of cell fate with pleiotropic roles in developmental and physiological processes. TGF-β signaling influences oligodendrogenesis, myelination and enhances synapse formation, having, thus, an important role in neurogenesis. Several studies have also shown that the TGF-β family is also important in the survival of neurons. Levels of ligands and receptors of TGF-β family are regulated following neural injury and repair, for proper function of CNS [10]. TGF-β has also an important role in immunity, by stimulating the maturation of CD4+CD25+FoxP3+ T cells (Tregs). The TGF-β1, -β2, and -β3 isoforms are expressed by neurons and glial cells, and their receptors are expressed throughout the central nervous system.
b- TGF-β signaling and ALS
5
Several studies demonstrate that TGF-β signaling also protects neurons from glutamate-mediated excitotoxicity, a recognized mechanism underlying the pathogenesis of various neurodegenerative disorders such as ALS [11]. TGF-β superfamily of growth-factors is composed by, approximately, 20 cytokines subdivided into different families, such as TGF-βs and Bone Morphogenetic Proteins (BMPs). Each growth-factor binds to two ligand-specific receptors (Type I and Type II), which contain serine/threonine kinases and the signal is, then, transduced to the nucleus by Smad proteins, through a cascade, known as “canonical pathway”. Smads 2 and 3 are the substrates of Type I TGF-β receptor kinases. Once the ligand binds to the receptors, Type II receptor phosphorylates Type I receptor, which then phosphorylates the Smad proteins. The activated Smad proteins bind to Smad4 (Co-Smad) and the complex translocates to the nucleus, thus, modulating gene transcription and microRNA processing. The TGF- β signal can also be transduced by intracellular Smad-independent pathways, including LIM domain kinase 1 (LIMK1)-actin depolymerizing factor (ADF)-cofilin and mitogen activated protein kinase pathways, the “noncanonical” pathways. TDP43 is one of the several genes that have been found to be mutated in ALS patients. The mutated TDP43 protein tends to aggregate and the intracytoplasmic inclusion bodies not only contain TDP43, but have also been reported to have Smurf2, an E3 ubiquitin ligase that promotes ubiquitindependent degradation of Smad 2/3 proteins, and phosphorylated Smad 2/3 proteins, the transducers of the TGF- β signaling. Thus, this gene mutation has
6
been linked with deregulation of TGF-β and BMP signaling pathways in ALS [10]. Recent studies report dysregulations of the TGF-β system as a common feature of neurodegenerative disorders. The upregulation of this system has been linked with ALS progression, leading to diminished stem cell activity, arrested neurogenesis at late stage ALS and increased proinflammatory cytokines, suggesting a shift from a neuroprotective toward a neurotoxic immune response. Thus, persistent elevated TGF-β system activity interacts with three different systems: the immune response; the activity of the adult neurogenic niche, and fibrotic scarring. ALS disease course may be divided in two distinct immunological phases: (1) a presymptomatic or stable phase, with a predominantly pronounced antiinflammatory T2 immune response and (2) a progressive phase with a tendency toward a proinflammatory T1 immunity. As previously mentioned, normal TGFβ levels enhance Treg cells maturation, therefore, diminishing the activity of effector T cells and preventing excessive inflammatory activity. A recent study has demonstrated that as ALS progresses, Treg cell levels decline, meanwhile increasing effector T cell functions (Fig. 1). High TGF-β serum levels might reflect a compensatory mechanism to fight the neurotoxic Th1-mediated immune response, inducing T cells to differentiate into non-regulatory phenotypes such as proinflammatory Th17 effectors. This way, the upregulation of TGF-β family contributes to the switch from neuroprotective to neurotoxic immune response.
7
While in normal brain tissue TGF-β favors neuronal differentiation and survival, in the damaged brain, high TGF-β levels induce cell cycle arrest and stem cell quiescence, thus preventing neurogenesis. Moreover, TGF-β indirectly affects hippocampal neurogenesis via immune interactions. Fibrosis is a process that involves the replacement of functional tissue by connective tissue mainly composed by extracellular matrix (ECM), therefore affecting normal functioning. Several factors are involved in skeletal muscle fibrosis, one of which is the up-regulation of TGF-β. A recent murine in vivo study has shown that enhanced TGF-β signaling directly enhances muscle fibrosis, through direct induction of a profibrotic phenotype on fibroblasts [10]. Other studies correlate with this, suggesting that several fibro/adipogenic progenitors, which are interstitial populations of mesenchymal cells that participate directly in fatty and fibrous tissue deposition, are more expressed in tissues with higher TGF-β levels [12]. Enhanced fibrotic processes within the central nervous system, therefore, indirectly promote the progression of ALS, by replacing areas of neuronal loss with excessive scar tissue [11]. All these findings suggest that the TGF-β system may, therefore, represent a promising target in treatment of ALS patients [4].
EVALUATION OF THE HYPOTHESIS
In order to identify a putative correlation between TGF-β plasma levels and disease progression in ALS patients, it is important to measure TGF-β plasma levels in ALS patients, in patients with mimicking disorders and in a healthy control group. Moreover, TGF-β plasma levels should be evaluated
8
longitudinally. It is essential do correlate its values with clinical markers of disease progression (FVC and ALSFRS-R rate of decay) during disease progression. Its predictive value as a marker of disease progression, in particular functional decay, respiratory function decline and survival, are crucial targets. The confirmation of its predictive value should stimulate novel compounds able to modulate its expression. In order to test our hypothesis, we have quantified TGF-β1, TGF-β2 and TGFβ3 serum levels by Multiplex Assay in a population of 25 ALS patients and 11 healthy controls. Statistical analysis was performed to test normality of data distribution (Shapiro-Wilk test), compare results in ALS patients vs controls (unpaired t-test), and Pearson correlation coefficient was applied to investigate correlation of TGF-β serum levels with age and functional impairment in the ALS population.
RESULTS ALS population was formed by 13 men and 12 women, with a mean age of 61.4 years (SD=11.2), mean disease duration 29.05 months (SD= 37.4), 18 patients had spinal-onset, 6 bulbar-onset, 1 respiratory-onset, mean ALSFRS-R was 30.45 (SD=6.7). Healthy controls group included 12 subjects, 7 women and 5 men, mean age of 50.8 years (SD=9.6). Groups were well matched for gender (p=0.55), but controls were non-significantly younger (p=0.08). TGF-β1, TGF-β2 and TGF-β3 serum levels in ALS patients and controls (Fig. 2) followed a normal distribution (p>0.1). Even though the difference of TGF- β values in the two populations did not prove to be statistically significant (Table 1), TGF-β3 levels seem to be trendily higher in ALS patients (p=0.19).
9
In control, gender and age did not influence results (p>0.3). In the ALS group, TGF-β3 levels were not dependent on gender (p=0-7) or age (p=64) (Fig 3), and levels were similar between spinal and bulbar-onset patients (p=0.5). However, as theoretically predicted, disease severity (evaluated by ALS-FRS functional scale) was inversely correlated with TGF-β levels (Fig 3). In particular regarding TGF-β1 and TGF-β3 levels, with the same results (r = -0.59, p = 0.013) - Fig. 3.
CONCLUSIONS Because TGF-β has a relevant role in motoneuron survival, as such it should be explored as a biomarker candidate, and as a treatment target. Our preliminary results suggest a dysregulation of the TGF-β system in ALS, leading to higher TGF- β serum levels in ALS patients. Moreover, higher TGF-β levels seem to correlate with disease progression, evaluated by ALSFRS. As TGF-β3 was particularly higher in ALS patients, we intend to investigate this marker in a larger population. We believe our hypothesis could not only contribute to the discovery of early disease markers, but also to new treatment strategies: TGF-β is a promising target in ALS.
10
Fig. 1 The effect of enhanced TGF-β levels in Treg cells maturation and in effector T cells activity. While normal TGF-β levels induce Treg cells maturation, diminishing effector T cells activity, the upregulation of TGF-β leads to Treg cell decline, thus, increasing effector T cells activity.
11
DECLARATION OF CONFLICTING INTERESTS The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
FUNDING This study was partially supported by a grant from GAPIC (nº 20140012) – “22º Program of Educação para a Ciência”
12
REFERENCES [1] Wijesekera LC, Leigh PN. Amyotrophic lateral sclerosis. Orphanet J Rare Dis. 2009; 4:3. [2] Volk AE, Weishaupt JH, Andersen PM, Ludolph AC, Kubisch C. Current knowledge and recent insights into the genetic basis of amyotrophic lateral sclerosis. Med Genet. 2018;30(2):252-258. [3] Hulisz D. Amyotrophic lateral sclerosis: disease state overview. Am J Manag Care. 2018;24(15 Suppl): S320-S326. [4] Peters S, Zitzelsperger E, Kuespert S, et al. The TGF-β System As a Potential Pathogenic Player in Disease Modulation of Amyotrophic Lateral Sclerosis. Front Neurol. 2017; 8:669. [5] Magnus T, Beck M, Giess R, Puls I, Naumann M, Toyka KV. Disease progression in amyotrophic lateral sclerosis: predictors of survival. Muscle Nerve. 2002;25(5):709-714. [6] Stambler N, Charatan M, Cedarbaum JM. Prognostic indicators of survival in ALS. ALS CNTF Treatment Study Group. Neurology. 1998;50(1):66-72. [7] Pinto S, de Carvalho M. Correlation between Forced Vital Capacity and Slow Vital Capacity for the assessment of respiratory involvement in Amyotrophic Lateral Sclerosis: a prospective study. Amyotroph Lateral Scler Frontotemporal Degener. 2017;18(1-2):86-91. [8] Andersen PM, Abrahams S, Borasio GD, de Carvalho M, Chio A, Van Damme P, et al. EFNS guidelines on the clinical management of amyotrophic lateral sclerosis (MALS)–revised report of an EFNS task force. Eur J Neurol 2012; 19; 360-375. [9] van den Berg LH, Sorenson E, Gronseth G, Macklin EA, Andrews J, Baloh RH, Benatar M, et al. Revised Airlie House consensus guidelines for design and implementation
of
ALS
clinical
trials.
Neurology
2019
(DOI:
https://doi.org/10.1212/WNL.0000000000007242). [10] Kashima, R., Hata, A. The role of TGF-β superfamily signaling in neurological disorders. Acta Biochim Biophys Sin (Shanghai) 50, 106-120 (2018). [11] Katsuno M, Adachi H, Banno H, Suzuki K, Tanaka F, Sobue G. Transforming growth factor-β signaling in motor neuron diseases. Curr Mol Med. 2011;11(1):48-56. 13
[12] Gonzalez, D. et al. ALS skeletal muscle shows enhanced TGF-β signaling, fibrosis and induction of fibro/adipogenic progenitor markers. PLOS One 12, e0177649 (2017).
14
Table 1 – Results Groups (number of subjects)
TGF-β1 level
TGF-β2 level
TGF-β3 level
(pg/ml)
(pg/ml)
(pg/ml)
(SD)
(SD)
(SD)
ALS (23)
23424.7 (12082.3)
1632.3 (725.0)
942.5 (404.1)
Healthy controls (12)
20895.4 (12254.5)
1930.0 (834.7)
800.2 (437.5)
0.44
0.71
0.19
P value (unpaired t-test)
TGF-β - Transforming growth factor-β; TGF-β1, TGF-β2, TGF-β3 level - Transforming growth factor-β isoforms Pg/ml – pictogram per milliliter; SD – standard deviation
15
Fig. 1 The effect of enhanced TGF-β levels in Treg cells maturation and in effector T cells activity. While normal TGF-β levels induce Treg cells maturation, diminishing effector T cells activity, the upregulation of TGF-β leads to Treg cell decline, thus, increasing effector T cells activity.
Fig. 2 TGF-β1, TGF-β2 and TGF-β3 serum levels in ALS patients and controls, determined by Multiplex Assay.
Fig. 3 Significant negative correlation was disclosed between TGF-β1 and TGF-β3 levels and ALS severity (ALSFRS-R scale), r = -0.59, p = 0.013. This correlation was not found for TGF-β2 levels (), r = -0.1, p = 0.71).
16
S0306-9877(20)30026-8 https://doi.org/10.1016/j.mehy.2020.109632 YMEHY 109632
To appear in:
Medical Hypotheses
Received Date: Revised Date: Accepted Date:
5 January 2020 9 February 2020 13 February 2020
Please cite this article as: T. Duque, M. Gromicho, A.C. Pronto-Laborinho, M. de Carvalho, Transforming growth factor-β plasma levels and its role in Amyotrophic Lateral Sclerosis, Medical Hypotheses (2020), doi: https://doi.org/10.1016/j.mehy.2020.109632
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.
Transforming growth factor-β plasma levels and its role in Amyotrophic Lateral Sclerosis
Tatiana Duque1, Marta Gromicho1, Ana Catarina Pronto-Laborinho1, Mamede de Carvalho1,2 1 Instituto de Fisiologia, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa. Lisbon, Portugal. 2 Department of Neurosciences and Mental Health, Centro Hospitalar Universitário de Lisboa-Norte. Lisbon, Portugal.
Running title: ALS and transforming growth factor-β plasma levels
Word count: words
Keywords: amyotrophic lateral sclerosis; biomarker; predictive value; progression; transforming growth factor-β plasma levels
Corresponding author: Professor Mamede de Carvalho Faculdade de Medicina, Universidade de Lisboa. Av. Professor Egas Moniz, 1648-028 Lisbon, Portugal E –mail: [email protected] Phone and Fax: + 351 21 7805219
1
ABSTRACT Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive muscle paralysis. Respiratory complications are the main cause of death in ALS. For this reason, initial respiratory status and its decline over disease progression are strong independent predictors of survival. Riluzole, a glutamatergic neurotransmission inhibitor, is the only drug that has shown to extend survival. Therefore, both novel molecular biomarkers and treatment strategies are needed. Transforming growth factor-β (TGF-β) family cytokines are important regulators of cell fate affecting both neurogenesis and neurodegeneration. Several studies demonstrate that TGF-β signalling protects neurons from glutamate-mediated excitotoxicity, a recognized mechanism underlying the pathogenesis of various neurodegenerative disorders such as ALS. Recent studies report dysregulations of the TGF-β system as a common feature of neurodegenerative disorders. The upregulation of this system has been linked with ALS progression. We have quantified TGF-β1, TGF-β2 and TGF-β3 serum levels in 23 ALS patients and 12 healthy controls, our preliminary results support the hypothesis that TGF-β3 levels can be a marker disease severity ALS. Further results are necessary to confirm this hypothesis.
2
INTRODUCTION Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease or motor neuron disease (MND), is a neurodegenerative disease characterized by progressive muscle paralysis, due to the degeneration of motor neurons in the primary motor cortex, brainstem and spinal cord [1]. ALS is the most frequent motor neuron disease, affecting 1 per 20,000 people worldwide [2]. There are two types of ALS differentiated by family history: positive, familial-ALS (5% to 10% of the patients); and negative, sporadic-ALS. Diagnosis is primarily determined by clinical examination coupled with nerve conduction studies (NCSs), needle electromyography (EMG), laboratory testing and imaging. In general, initial symptoms may be described as “limb-onset” or “bulbar-onset.” With limb onset, patients may experience difficulty with simple actions, such as buttoning a shirt, stumbling more easily, and experiencing changes in their walking. Patients with bulbar onset may experience challenges with chewing, swallowing, and speaking, such as nasal or slurred speech [3]. Some patients experience respiratory or generalized onset. Extramotor symptoms can also occur, such as cognitive deficits, which are present in more than 10% of patients [4]. Atypical presentation includes emotional lability, weight loss, and fasciculations and cramps without muscle weakness [3]. Diagnosis can, thus, be difficult at onset, because ALS is a clinically heterogeneous disorder. Therefore, in the early phase, misdiagnosis of ALS remains a clinical problem. Disease progression is clinically evaluated by the application of the functional scale ALS-Functional Rating Scale-revised (ALSFRS-R), which is a validated rating instrument for monitoring the progression of disability in patients with
3
ALS. Its rate of decline is a strong independent predictor of survival [5]. No cure has yet been found and median survival is 2-3 years after onset [2]. The management of ALS is mainly supportive and palliative. Respiratory complications are the main cause of death in ALS, in particular, respiratory failure, in spite of effective and early intervention with non-invasive ventilation. For this reason, initial respiratory status and its decline over disease progression are strong independent predictors of survival [6]. Respiratory function is easily accessed by forced vital capacity (FVC), which provides a reliable indication of inspiratory and expiratory muscles function [7]. Both ALSFRS-R and FVC are evaluated at each consultation (every 3-4 months) as recommended in the European Guidelines [8]. Immune and inflammatory abnormalities seem to contribute to the pathogenesis and progression of ALS. Neuronal loss is an inflammation mediated process, in which resident and infiltrating immune cells, secreted inflammatory modulators and expressed surface receptors on activated microglial cells take part. Activated microglial cells, astrocytes and proinflammatory cytokines influence proliferation, cell survival and death of neurons, also reducing neurogenesis [4]. T cells have been linked to immune modulation by promoting microglia to generate a neuroprotective environment. Astrocytes are responsible for regulating glutamate by the activity of the excitatory aminoacid-transporter 2. The excitotoxicity of glutamate occurs when synaptic glutamate is not quickly removed, resulting in the excessive firing of motor neurons, leading to mitochondrial and endoplasmic reticulum stress [3]. Riluzole, a glutamatergic neurotransmission inhibitor, is the only drug that has shown to extend survival. Therefore, new treatment strategies are needed [1].
4
Moreover, for the ALS community, the identification of new molecular biomarkers, with predictive value, is considered a critical priority [9].
HYPOTHESIS TGF-β plasma levels correlate with markers of disease progression in Amyotrophic Lateral Sclerosis (ALS)
a- TGF-β family and physiological processes
Transforming growth factor-β (TGF-β) family cytokines are important regulators of cell fate with pleiotropic roles in developmental and physiological processes. TGF-β signaling influences oligodendrogenesis, myelination and enhances synapse formation, having, thus, an important role in neurogenesis. Several studies have also shown that the TGF-β family is also important in the survival of neurons. Levels of ligands and receptors of TGF-β family are regulated following neural injury and repair, for proper function of CNS [10]. TGF-β has also an important role in immunity, by stimulating the maturation of CD4+CD25+FoxP3+ T cells (Tregs). The TGF-β1, -β2, and -β3 isoforms are expressed by neurons and glial cells, and their receptors are expressed throughout the central nervous system.
b- TGF-β signaling and ALS
5
Several studies demonstrate that TGF-β signaling also protects neurons from glutamate-mediated excitotoxicity, a recognized mechanism underlying the pathogenesis of various neurodegenerative disorders such as ALS [11]. TGF-β superfamily of growth-factors is composed by, approximately, 20 cytokines subdivided into different families, such as TGF-βs and Bone Morphogenetic Proteins (BMPs). Each growth-factor binds to two ligand-specific receptors (Type I and Type II), which contain serine/threonine kinases and the signal is, then, transduced to the nucleus by Smad proteins, through a cascade, known as “canonical pathway”. Smads 2 and 3 are the substrates of Type I TGF-β receptor kinases. Once the ligand binds to the receptors, Type II receptor phosphorylates Type I receptor, which then phosphorylates the Smad proteins. The activated Smad proteins bind to Smad4 (Co-Smad) and the complex translocates to the nucleus, thus, modulating gene transcription and microRNA processing. The TGF- β signal can also be transduced by intracellular Smad-independent pathways, including LIM domain kinase 1 (LIMK1)-actin depolymerizing factor (ADF)-cofilin and mitogen activated protein kinase pathways, the “noncanonical” pathways. TDP43 is one of the several genes that have been found to be mutated in ALS patients. The mutated TDP43 protein tends to aggregate and the intracytoplasmic inclusion bodies not only contain TDP43, but have also been reported to have Smurf2, an E3 ubiquitin ligase that promotes ubiquitindependent degradation of Smad 2/3 proteins, and phosphorylated Smad 2/3 proteins, the transducers of the TGF- β signaling. Thus, this gene mutation has
6
been linked with deregulation of TGF-β and BMP signaling pathways in ALS [10]. Recent studies report dysregulations of the TGF-β system as a common feature of neurodegenerative disorders. The upregulation of this system has been linked with ALS progression, leading to diminished stem cell activity, arrested neurogenesis at late stage ALS and increased proinflammatory cytokines, suggesting a shift from a neuroprotective toward a neurotoxic immune response. Thus, persistent elevated TGF-β system activity interacts with three different systems: the immune response; the activity of the adult neurogenic niche, and fibrotic scarring. ALS disease course may be divided in two distinct immunological phases: (1) a presymptomatic or stable phase, with a predominantly pronounced antiinflammatory T2 immune response and (2) a progressive phase with a tendency toward a proinflammatory T1 immunity. As previously mentioned, normal TGFβ levels enhance Treg cells maturation, therefore, diminishing the activity of effector T cells and preventing excessive inflammatory activity. A recent study has demonstrated that as ALS progresses, Treg cell levels decline, meanwhile increasing effector T cell functions (Fig. 1). High TGF-β serum levels might reflect a compensatory mechanism to fight the neurotoxic Th1-mediated immune response, inducing T cells to differentiate into non-regulatory phenotypes such as proinflammatory Th17 effectors. This way, the upregulation of TGF-β family contributes to the switch from neuroprotective to neurotoxic immune response.
7
While in normal brain tissue TGF-β favors neuronal differentiation and survival, in the damaged brain, high TGF-β levels induce cell cycle arrest and stem cell quiescence, thus preventing neurogenesis. Moreover, TGF-β indirectly affects hippocampal neurogenesis via immune interactions. Fibrosis is a process that involves the replacement of functional tissue by connective tissue mainly composed by extracellular matrix (ECM), therefore affecting normal functioning. Several factors are involved in skeletal muscle fibrosis, one of which is the up-regulation of TGF-β. A recent murine in vivo study has shown that enhanced TGF-β signaling directly enhances muscle fibrosis, through direct induction of a profibrotic phenotype on fibroblasts [10]. Other studies correlate with this, suggesting that several fibro/adipogenic progenitors, which are interstitial populations of mesenchymal cells that participate directly in fatty and fibrous tissue deposition, are more expressed in tissues with higher TGF-β levels [12]. Enhanced fibrotic processes within the central nervous system, therefore, indirectly promote the progression of ALS, by replacing areas of neuronal loss with excessive scar tissue [11]. All these findings suggest that the TGF-β system may, therefore, represent a promising target in treatment of ALS patients [4].
EVALUATION OF THE HYPOTHESIS
In order to identify a putative correlation between TGF-β plasma levels and disease progression in ALS patients, it is important to measure TGF-β plasma levels in ALS patients, in patients with mimicking disorders and in a healthy control group. Moreover, TGF-β plasma levels should be evaluated
8
longitudinally. It is essential do correlate its values with clinical markers of disease progression (FVC and ALSFRS-R rate of decay) during disease progression. Its predictive value as a marker of disease progression, in particular functional decay, respiratory function decline and survival, are crucial targets. The confirmation of its predictive value should stimulate novel compounds able to modulate its expression. In order to test our hypothesis, we have quantified TGF-β1, TGF-β2 and TGFβ3 serum levels by Multiplex Assay in a population of 25 ALS patients and 11 healthy controls. Statistical analysis was performed to test normality of data distribution (Shapiro-Wilk test), compare results in ALS patients vs controls (unpaired t-test), and Pearson correlation coefficient was applied to investigate correlation of TGF-β serum levels with age and functional impairment in the ALS population.
RESULTS ALS population was formed by 13 men and 12 women, with a mean age of 61.4 years (SD=11.2), mean disease duration 29.05 months (SD= 37.4), 18 patients had spinal-onset, 6 bulbar-onset, 1 respiratory-onset, mean ALSFRS-R was 30.45 (SD=6.7). Healthy controls group included 12 subjects, 7 women and 5 men, mean age of 50.8 years (SD=9.6). Groups were well matched for gender (p=0.55), but controls were non-significantly younger (p=0.08). TGF-β1, TGF-β2 and TGF-β3 serum levels in ALS patients and controls (Fig. 2) followed a normal distribution (p>0.1). Even though the difference of TGF- β values in the two populations did not prove to be statistically significant (Table 1), TGF-β3 levels seem to be trendily higher in ALS patients (p=0.19).
9
In control, gender and age did not influence results (p>0.3). In the ALS group, TGF-β3 levels were not dependent on gender (p=0-7) or age (p=64) (Fig 3), and levels were similar between spinal and bulbar-onset patients (p=0.5). However, as theoretically predicted, disease severity (evaluated by ALS-FRS functional scale) was inversely correlated with TGF-β levels (Fig 3). In particular regarding TGF-β1 and TGF-β3 levels, with the same results (r = -0.59, p = 0.013) - Fig. 3.
CONCLUSIONS Because TGF-β has a relevant role in motoneuron survival, as such it should be explored as a biomarker candidate, and as a treatment target. Our preliminary results suggest a dysregulation of the TGF-β system in ALS, leading to higher TGF- β serum levels in ALS patients. Moreover, higher TGF-β levels seem to correlate with disease progression, evaluated by ALSFRS. As TGF-β3 was particularly higher in ALS patients, we intend to investigate this marker in a larger population. We believe our hypothesis could not only contribute to the discovery of early disease markers, but also to new treatment strategies: TGF-β is a promising target in ALS.
10
Fig. 1 The effect of enhanced TGF-β levels in Treg cells maturation and in effector T cells activity. While normal TGF-β levels induce Treg cells maturation, diminishing effector T cells activity, the upregulation of TGF-β leads to Treg cell decline, thus, increasing effector T cells activity.
11
DECLARATION OF CONFLICTING INTERESTS The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
FUNDING This study was partially supported by a grant from GAPIC (nº 20140012) – “22º Program of Educação para a Ciência”
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REFERENCES [1] Wijesekera LC, Leigh PN. Amyotrophic lateral sclerosis. Orphanet J Rare Dis. 2009; 4:3. [2] Volk AE, Weishaupt JH, Andersen PM, Ludolph AC, Kubisch C. Current knowledge and recent insights into the genetic basis of amyotrophic lateral sclerosis. Med Genet. 2018;30(2):252-258. [3] Hulisz D. Amyotrophic lateral sclerosis: disease state overview. Am J Manag Care. 2018;24(15 Suppl): S320-S326. [4] Peters S, Zitzelsperger E, Kuespert S, et al. The TGF-β System As a Potential Pathogenic Player in Disease Modulation of Amyotrophic Lateral Sclerosis. Front Neurol. 2017; 8:669. [5] Magnus T, Beck M, Giess R, Puls I, Naumann M, Toyka KV. Disease progression in amyotrophic lateral sclerosis: predictors of survival. Muscle Nerve. 2002;25(5):709-714. [6] Stambler N, Charatan M, Cedarbaum JM. Prognostic indicators of survival in ALS. ALS CNTF Treatment Study Group. Neurology. 1998;50(1):66-72. [7] Pinto S, de Carvalho M. Correlation between Forced Vital Capacity and Slow Vital Capacity for the assessment of respiratory involvement in Amyotrophic Lateral Sclerosis: a prospective study. Amyotroph Lateral Scler Frontotemporal Degener. 2017;18(1-2):86-91. [8] Andersen PM, Abrahams S, Borasio GD, de Carvalho M, Chio A, Van Damme P, et al. EFNS guidelines on the clinical management of amyotrophic lateral sclerosis (MALS)–revised report of an EFNS task force. Eur J Neurol 2012; 19; 360-375. [9] van den Berg LH, Sorenson E, Gronseth G, Macklin EA, Andrews J, Baloh RH, Benatar M, et al. Revised Airlie House consensus guidelines for design and implementation
of
ALS
clinical
trials.
Neurology
2019
(DOI:
https://doi.org/10.1212/WNL.0000000000007242). [10] Kashima, R., Hata, A. The role of TGF-β superfamily signaling in neurological disorders. Acta Biochim Biophys Sin (Shanghai) 50, 106-120 (2018). [11] Katsuno M, Adachi H, Banno H, Suzuki K, Tanaka F, Sobue G. Transforming growth factor-β signaling in motor neuron diseases. Curr Mol Med. 2011;11(1):48-56. 13
[12] Gonzalez, D. et al. ALS skeletal muscle shows enhanced TGF-β signaling, fibrosis and induction of fibro/adipogenic progenitor markers. PLOS One 12, e0177649 (2017).
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Table 1 – Results Groups (number of subjects)
TGF-β1 level
TGF-β2 level
TGF-β3 level
(pg/ml)
(pg/ml)
(pg/ml)
(SD)
(SD)
(SD)
ALS (23)
23424.7 (12082.3)
1632.3 (725.0)
942.5 (404.1)
Healthy controls (12)
20895.4 (12254.5)
1930.0 (834.7)
800.2 (437.5)
0.44
0.71
0.19
P value (unpaired t-test)
TGF-β - Transforming growth factor-β; TGF-β1, TGF-β2, TGF-β3 level - Transforming growth factor-β isoforms Pg/ml – pictogram per milliliter; SD – standard deviation
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Fig. 1 The effect of enhanced TGF-β levels in Treg cells maturation and in effector T cells activity. While normal TGF-β levels induce Treg cells maturation, diminishing effector T cells activity, the upregulation of TGF-β leads to Treg cell decline, thus, increasing effector T cells activity.
Fig. 2 TGF-β1, TGF-β2 and TGF-β3 serum levels in ALS patients and controls, determined by Multiplex Assay.
Fig. 3 Significant negative correlation was disclosed between TGF-β1 and TGF-β3 levels and ALS severity (ALSFRS-R scale), r = -0.59, p = 0.013. This correlation was not found for TGF-β2 levels (), r = -0.1, p = 0.71).
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