Hydrogen Sulphide " a Double-faced Janus " in Amyotrophic Lateral Sclerosis (als)

We have recently published a paper in Annals of Neurology entitled " Evidence of Hydrogen Sulphide involvement in Amyotrophic Lateral Sclerosis " [1] where we reported a study performed in patients, and in a genetic model of familial ALS. The outcome of this study is an original finding: the overproduction of hydrogen sulphide (H 2 S) in the human patients and in the animal model. We also show that H 2 S is produced, mainly, by glial cells, is toxic to motor neurons and increases significantly cytosolic Ca 2+ concentration. Altogether, our data introduce H 2 S as a new contestant in the ALS-related toxic pathways, which has potential implications for innovative drug design in ALS. To cite this article: Patrizia Longone, et al. Hydrogen sulphide " a double-faced Janus " in amyotrophic lateral sclerosis Amyotrophic Lateral Sclerosis (ALS) is a fatal disease of both sporadic (sALS) and familial (fALS) origin leading to inevitable death in most cases within 5 years from diagnosis [2,3]. ALS has been identified as the motor neurons disease, since it is characterized by the death of the motor neurons in the spinal cord, brainstem and motor cortex. Although this view remains accurate, it is now clear that other neurons are involved and glial cells are crucial for the motor neuron demise [4,5]. Mutations in the gene encoding for the superoxide dismutase 1 (SOD1) protein were the first genetic link that allowed the creation of animal models [6] that helped us to understand aspects of the underlying molecular mechanisms leading to the cell death and tissue degeneration observed in this disease [7,8]. Since that first genetic link, other genes have been related to the occurrence of ALS [9] and, as a consequence, additional animal models have been created and more pathways have been drawn in, some common to other neurodegenerative diseases, few peculiar to ALS [10]. The toxic pathways traditionally linked to ALS are: excitotoxicity, oxidative stress and related protein damage,

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease of both sporadic (sALS) and familial (fALS) origin leading to inevitable death in most cases within 5 years from diagnosis [2,3] .ALS has been identified as the motor neurons disease, since it is characterized by the death of the motor neurons in the spinal cord, brainstem and motor cortex.Although this view remains accurate, it is now clear that other neurons are involved and glial cells are crucial for the motor neuron demise [4,5] .
Mutations in the gene encoding for the superoxide dismutase 1 (SOD1) protein were the first genetic link that allowed the creation of animal models [6] that helped us to understand aspects of the underlying molecular mechanisms leading to the cell death and tissue degeneration observed in this disease [7,8] .Since that first genetic link, other genes have been related to the occurrence of ALS [9] and, as a consequence, additional animal models have been created and more pathways have been drawn in, some common to other neurodegenerative diseases, few peculiar to ALS [10] .The toxic pathways traditionally linked to ALS are: excitotoxicity, oxidative stress and related protein damage,

RESEARCH HIGHLIGHT
mitochondrial dysfunction, neurofilament alterations, and neuroinflammation [11] .To this list, the role played by RNA metabolism, particularly by two proteins, the TAR DNA binding protein 43 (TDP-43) [12,13] and the fused in sarcoma/translocated in liposarcoma protein (Fus/TLS) [14,15] , has been identified as one of the key pathways leading to the ALS spectrum of disorders [16] .Yet, in spite of all the effort that the ALS research has dedicated to the understanding of this disease, many questions are still unanswered.
Hydrogen sulphide (H 2 S), a gas with a very unpleasant odor, is now considered along with carbon dioxide (CO 2 ) and nitric oxide (NO), a member of the gasotransmitters family [17]   .H 2 S is enzymatically generated by three different enzymes [18] .Cystathionine-β-synthase (CBS), a mainly cytosolic haem-containing enzyme, that accumulates in the mitochondria during hypoxia and has been, so far, identified as the major source of H2S in the mammalian brain [19,20] .Cystathionine-γ-lyase (CSE) also cytosolic and more present in the periphery then CBS [19,20] .Both enzymes metabolize cysteine and (or) homocysteine to release H 2 S. The third enzyme is 3-mercaptopyruvate sulfutransferase (3MST) that produces H 2 S from 3-mercaptopyruvate, a product of the metabolism of cysteine and α-ketoglutarate by cystein aminotransferase (CAT) [19,20] .The mitochondria are the site of H 2 S metabolism.Hydrogen sulphide has two opposite effects on the mitochondria: it is an enzymatic substrate at low concentrations and a poison at high concentrations, via the inhibition of Complex IV [18] .Increasing evidence suggests that it is a "double-faced Janus", a molecule with opposite effects (Fig. 1).It participates in the regulation of neuronal functions and signaling [20] , but it switches from neuroprotective to neurotoxic as its concentration raises [21] above a certain threshold.In fact, exposure to high µM to mM concentrations are cytotoxic (free radical generation, glutathione depletion, intracellular iron), while nM to low µM concentrations are cytoprotective (antinecrotic and antiapoptotic).Bian and co-workers have demonstrated that H 2 S suppresses oxidative stress induced by hydrogen peroxide [22] , and protects cells against the neurotoxins rotenone [23] and 6-OHDA [24] .The protective effects of H 2 S have been established also "in vivo" in animal models of Parkinson's disease [25,26] and Alzheimer's disease [27] .Slivka et al. [28] reported that memory deficiency in Alzheimer's disease may be related to reduced H 2 S.More recently Xuan et al. [29] proposed the administration of the H 2 S donor sodium hydrosulfide (NaHS) as a possible therapeutic approach for Alzheimer's disease.
On the other hand, Kurokawa et al. [30] described an H 2 S-induced NMDA-independent neuronal death, via the activation of the ERK-pathway.Moreover, the murine cortical neurons death induced by the addition of 100 µM glutamate was exacerbated by the co-incubation with the H 2 S donor NaHS [21] .The same group performing a transcriptomic profile provided evidence of an NMDAR involvement and ubiquitin-proteasome recruitment in the H 2 S-induced neuronal death [31] .In cerebella granule neurons (CGN) in culture, H 2 S raises intracellular calcium to toxic range in a dose dependent manner with a 50 % cell death within 2 hours after treatment [32] .L-type Ca2+ channels and NMDAR blockers protected CGN against H 2 S-induced death and largely attenuated the rise of cytosolic calcium.Hence, H 2 S via the modulation of the Ca2+ homeostasis, affects neuronal viability.
Staying on the issue of a biphasic biological action, the effects of H 2 S are highly divergent on the mitochondrial respiratory chain depending on its concentration [18] .Historically its toxicity has been primarily ascribed to the inhibition of cytochrome c oxidase (Complex IV) resulting in a shutdown of mitochondrial electron transport and cellular ATP generation.In striking contrast, more recent data showed that low concentrations of H 2 S serve as a stimulator of electron transport by acting as a mitochondrial electron donor [33] .
Hence, we have sought to understand if H 2 S could have a role in the ALS pathophysiology and measured its concentrations in ALS patients and in a fALS mouse model [1] .Developing a specific and sensitive HPLC test to measure H 2 S in body fluids, we found significantly higher levels of H 2 S in the CSF of male and female ALS patients, compared with the ones measured in age-matched healthy control [1] .We have also observed a remarkable relationship between H 2 S content in the CSF and the site of disease onset, with significantly higher levels in the limb onset (LO) subgroup compared to the bulbar onset (BO) subgroup [1] .A further layering of the patients population uncovered an additional caudo-cranial direction in the liquoral H 2 S concentration with the H 2 S concentration significantly elevated in the lower limb onset (LLO) compared with the upper limb onset (ULO) subgroup (BO<ULO<LLO).Furthermore, we found a relation within the ALS group between the H 2 S values and progression rate, and higher levels of liquoral H 2 S in the ALS female population compared to the male, although not significant.The H 2 S blood levels were similar in the ALS patients and in the control population.Such high liquoral levels indicate that the neuronal tissues are in a hypoxic state, which has been described in ALS [34] .
In line with these observations, we detected increased levels of this gas in the spinal cord, brain stem and cortex of the SOD1G93A mouse, a fALS murine model which over-expresses the SOD1 mutated at position 93.Similar to the findings in the human patients, we measured higher levels of H 2 S in the female mouse population.We also found a significant increased translocation of the CBS enzyme from the cytosolic to the mitochondrial fraction, a phenomenon known to occur in hypoxic conditions [35] .Furthermore, "in vitro" primary spinal cord cultures obtained from the fALS mouse model showed an increased production of H 2 S (as measured in the culture media).Remarkably, the inhibition of glial cell proliferation decreased H 2 S media concentration, but it significantly increased when inflammation was activated by Lipopolysaccharide (LPS).In addition, when H 2 S was administered to control cultures, via the H 2 S donor NaHS, we observed a dose-dependent increase in SMI-32 positive neurons death (motor neurons), while GABAergic interneurons were more resistant to the H 2 S-mediated toxicity.Finally, measuring cytosolic Ca 2+ concentration ([Ca 2+ ] i ) in response to NaHS in spinal motor neuron cultures, we found a significant rise in [Ca 2+ ] i that was strongly attenuated when the intracellular ATP concentration was increased to 2 mM.In summary our study, using patient's samples as well as pre-clinical "in vivo" and "in vitro" models identifies H 2 S as an additional pathological player in the ALS-related neurodegeneration.Our results show that H 2 S levels in the central nervous system of ALS patients reaches supra-physiological concentrations, which appear to be toxic primarily to motor neurons.We have also demonstrated that the main sources of H 2 S are glial cells, thus identifying it as a factor involved in the non-cell autonomous motor neurons death.On the other hand, it raises new and unanswered questions.Which is the primary source of H 2 S? As inhibitor of the mitochondrial respiratory chain, does it contribute to the mitochondrial distress described in ALS?Why motor neurons are more vulnerable to it than other type of neurons?Is a neuroinflammatory factor the primus movens in ALS? Future studies addressing these and other questions will help to assess if the control of H 2 S production could be a valuable approach in a multidrug therapeutic strategy aiming at slowing down and/or cure this deadly disease.

Figure 1 .
Figure 1.H2S can have a "Janus two-faced effect".Depending on its concentration it can be either protective or harmful to the cellular homeostasis.Hence it is important to clearly understand the effects that the modulation of its production/metabolism may have in brain function.