Amyotrophic Lateral Sclerosis as a Non-Cell-Autonomous Disease

ALS is a disease affecting upper and lower motor neurons, with an incidence of 1–2/100,000 per year, and mean survival of 3–5 years after diagnosis [1]. It is characterized by a progressive loss of motor neurons, but the precise pathological mechanisms involved are not fully establishedas their complex interplay with neighboring and target cells. ALS is primarily caused by the death of upper and lower motor neurons. Nevertheless, in the last 15 years, besides the main classical “neuron-centric” view of ALS, a number of research studies evidenced that ALS could also be a non-cell-autonomous disease [2,3]. Data have been mostly obtained using ALS mouse models, but they may also be linked to sporadic ALS cases [4]. Glial and skeletal muscle cells demonstrated their ability to trigger or modulate ALS. The analysis of chimeric mice indicated that the restricted expression of human mutant SOD1 (mutSOD1) in motor neuron is not sufficient to induce a cell-autonomous degeneration of motor neurons [5]. Moreover, utilizing floxed mutSOD1 gene, it has been demonstrated that the damaging process starts in motor neurons and determines the disease onset, with little influence on its progression [2]. Conversely, mutSOD1 activates glial cells exacerbating the disease progression, while motor neuronal mutSOD1 has little influence on the progression of ALS [2]. Astrocytes, microglia, oligodendrocytes, and Schwann cells are all able to modulate ALS pathology, and gliosis is a hallmark of ALS (see, for review [6,7]). Activated and proliferating astrocytes may no longer provide the metabolic support to motor neurons, and also become neurotoxic by secreting cytokines or other toxic factors (among which is the TGFB) that are critical for determining the rate of disease progression [8,9]. Furthermore, activated astrocytes reduce the expression of the excitatory amino acid transporter-2 (EAAT-2), that is mandatory for glutamate re-uptake from the synaptic cleft into astrocyte, leading to excitotoxicity in motor neurons [10].The extent of microglia activation correlates with the severity of the upper motor neuron involvement [11]. Whether microglial cells are beneficial or detrimental to motor neurons is already an open question. In addition to neighboring cells, motor neurons can also be influenced by their target, the skeletal muscle cells. It has been shown, at least in familial ALS, a direct muscular toxicity and/or a functional impairment that has denervation and motor neuron death as a consequence [12,13,14]. A contribution to the initiation and progression of muscle atrophy is given by altered ALS satellite cell properties [15,16]. In addition, our previous works have indicated a dysfunctional protein quality control system in ALS muscle cells, which seem more protected than motor neurons against the presence of accumulating misfolded proteins [17,18,19].

TGFB Plasma Levels in ALS Patients

TGFB1 plasma concentration in ALS patients is significantly higher than in the healthy controls, and it positively correlates with the disease [20], but whether TGFB1 plasma level is a biomarker of ALS or not is still an open question.

TGFB and ALS-Nervous System

TGFBs have multiple functions in the CNS. They enhance synapse formation and synaptic transmission [21,22], regulate synaptic plasticity and memory [23], increase the number and length of neurites [24], control neuronal migration [25], and cerebral cortex angiogenesis [26]. CNS-TGFB1-deficient mice have reduced brain weight and loss of neurons in the CA1 hippocampal region. These mice show a reduction of dendritic spine density, impaired long-term potentiation, and facilitated long-term depression in the hippocampus, in addition to the loss of the astrocyte glutamate transporters GLT-1 (EAAT2) and GLAST (EAAT1), and decreased glutamate uptake, resulting in a higher sensibility to glutamate excitotoxicity, that is one of the possible pathogenic mechanism in ALS [27]. Even if the comparative analysis of familial ALS and sporadic ALS tissues indicates the existence of common and distinct biological mechanisms driving the different forms of the pathology, an altered regulation of the TGFB1 pathway has been reported in motor neurons of most ALS models and patients. Reduced Tgfb1 mRNA levels in the spinal cord of pre-symptomatic mutSOD1 mice could indicate a lack of the TGFB neuroprotective effect in the early stages of the disease [28]. Indeed, levels of pSMAD2 in the nuclei of lumbar motor neurons are significantly decreased at the pre-symptomatic stage, leading to the hypothesis of an aberrant nucleo/cytoplasm transport [29,30]. The role of glia-derived TGFB1 in the spinal cord of ALS patients and mice has been studied by Endo and colleagues [8]. They determined that astrocyte-derived TGFB1 accelerates disease progression in ALS mice, preventing neuroprotective responses mediated by the microglia and T cells [8].

TGFB pathway in ALS skeletal muscle

In skeletal muscle, the expression of TGFB is related to normal processes such as growth, differentiation, regeneration, and stress response. However, continuously elevated levels of TGFB are linked to impaired regeneration and atrophy. TGFB blocks myogenic responses and stimulates fibrosis [31]. It inhibits the activation of MyoD and myogenin (two transcription factors regulating muscle cell differentiation) through the signaling of SMAD3 or by inactivating cyclin-dependent kinases [32,33]. Satellite cell activation is also prevented in the presence of TGFB, and muscle overexpression of TGFB leads to muscle weakness and atrophy [34,35]. ALS muscle tissue is also characterized by alterations of the TGFB pathway. We reported increased levels of the Tgfb1 mRNA in the muscle of mice expressing mutSOD1[36]. Notably, these changes are gender-related, since male mice present an increased TGFB expression in muscle already at the pre-symptomatic stage, while in female animals, TGFB increases only at the symptomatic stage [28]. Tgfb mRNA levels are further increased with the administration of an anabolic/androgenic steroid (nandrolone decanoate), indicating that, at least at the muscular level, these molecules might exert a detrimental role in ALS, since it might exacerbate some of the alterations induced by mutSOD1 [36,37]. Evidence in human confirmed the involvement of TGFB1 since we reported an increased TGFB1 expression in muscle of female and male sporadic ALS patients with a significant gender effect [28], and other authors also reported the increase of TGFB1, 2, and 3 in ALS patient muscles [38,39]. It must also be highlighted that TGFB1 and TGFB3 mRNA show a negative correlation with muscle strength in ALS patients [39]. In the same manner, the increase of TGFB1 correlates with disease progression in mutSOD1 mice [36]. It has also been proposed that excessive oxidative processes may be a mechanism of activation of latent TGFB pool in ALS, as in other neurodegenerative diseases, leading to an increased TGFB1 release from the complex [40].

TGFB and Neuro-Muscular Junction in ALS

Since the first histological studies, recurrent denervation and reinnervation have been observed in the NMJs of ALS patients [41]. Because of that, it has been proposed to consider ALS also as a distal axonopathy, with pathological changes occurring at the NMJs prior to motor neuron degeneration and muscle fiber atrophy (see, for review [42]). TGFB pathway regulates the formation and stability of the NMJs. TGFB1 is capable of doubling the size of acetylcholine receptor clusters increasing the percentage of nerve–muscle contacts. It has also been demonstrated that this synaptogenic effect of TGFB1 might be ascribed to its ability to induce neuronal agrin expression [43]. Agrin is a proteoglycan important for the maintenance of the architecture of the postsynaptic membrane and known to be down-regulated in the muscle of ALS mice expressing mutSOD1 [13]. TGFB1 is highly concentrated at NMJs of pre-symptomatic mutSOD1 mice, and represses the expression of FGFBP1 (a factor that might potentiate the bioactivity of FGF family members during reinnervation), indicating TGFB1 pathway as a potential target for preventing NMJ dismantling in ALS mice [44].

TGFB as a target for ALS treatment

The therapeutic potential of TGFB has been investigated. SB-431542, a selective inhibitor of TGFBRI kinase activity, has been proven to extend the survival of mutSOD1 expressing mouse, even if administered after disease onset [8]. Moreover, the intraperitoneal injection of TGFB2 in the same mouse model is able to reverse initial muscle weakness, permitting a better performance at rotarod test, probably through a marked trophic action on motor neurons, as can be inferred by motor neuron nuclei and axonal enlargement. Unfortunately, this amelioration is transient, leading to an even more rapid progression of the disease [45]. Antibodies neutralizing myostatin delayed the onset and the progression of the disease in ALS mice, even if without extending their survival [46,47].

Conclusions

The imbalance of TGFB signaling has been linked to ALS progression and may have selective impact on different body districts. In the CNS, there is a lack of the neuroprotective effects of TGFB at the first stages of the disease; later, the strong increase of TGFB levels due to microglial stimulation shifts the CNS milieu toward a proinflammatory and neurotoxic environment. In the skeletal muscle, the chronically increased TGFB signaling facilitates the development of atrophy and fibrosis in skeletal muscle fiber, and the process of NMJ dismantling.  

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