L-arginine ameliorates defective autophagy in GM2 gangliosidoses by mTOR modulation

Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause fatal and progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by β-hexosaminidase A (HexA) leads to the accumulation of its specific substrate, GM2 ganglioside, in neuronal lysosomes. Despite the development of a florid storage phenotype, the role of autophagy and its regulation by the mammalian target of rapamycin (mTOR) has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. Pathological autophagosomes, with enhanced expression of the p62/SQSTM1 protein, suggested impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed inappropriately diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities - and immediately suggests an avenue for therapeutic exploration in this cruel disease. We also contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of systemic treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy

Autophagy is a degradation and clearance function of the lysosome that is critical for cellular homeostasis 1 . Where the digestive function is defective, as in the inherited Lysosomal Storage Disorders (LSD), accumulation of undegraded substrates in the lysosomal compartment can impair this fusion process 4 . As a result, in most of these genetic diseases, autophagic flux is arrested -with the consequential accumulation of other autophagy substrates including cell debris and organelles such as mitochondria, as well as the cargo protein, sequestosome-1 -also known as the ubiquitin-binding protein p62 (SQSTM1/p62) 3,4 .
Autophagy is regulated by diverse mechanisms, each of which serves as a potential axis for therapeutic intervention. The mechanistic target of rapamycin (mTOR) is a highly conserved serine/threonine kinase that serves as a master regulator of metabolic processes centred on autophagic control. Moreover, the action of mTOR promotes lysosomal biogenesis and sustains the functional activity and integrity of this cell compartment 6 .
Modulation of mTOR activity has been reported in Pompe disease, the first LSD to be biochemically characterised, where the significant muscular atrophy can be ameliorated by experimental induction of mTOR, which leads to substantial clearance of autophagic debris 7 .
Here we have explored the role of autophagy in a class of lysosomal diseases -GM2 gangliosidosis Tay-Sachs and Sandhoff diseases -which principally affects sphingolipid recycling in the nervous system. The molecular cell pathology of these conditions reflects the striking accumulation of the primary substrate, GM2 ganglioside in neuronal lysosomes. That is due specifically to impaired hydrolysis in situ by β-hexosaminidase A (HexA). HexA is assembled as a functional heterodimer of α and β subunits. In humans, these proteins are encoded by HEXA and HEXB respectively, and the cognate subtypes are known as the genetically distinct, Tay-Sachs (Online Mendelian Inheritance in Man, OMIM, #272800) or Sandhoff (OMIM # 268800) Diseases. Since the neurodegenerative features are clinically indistinguishable, diagnosis relies primarily on enzymatic assays selective for the different β-hexosaminidase isozymes A (α/β heterodimer; HexA); B (β/β homodimer; HexB) and S (α/α homodimer; HexS).
According to our study, we report that, when compared with healthy control cells, there is an enhanced expression of p62/SQSTM1 and build-up of autophagosomes, indicating impaired autophagic flux. This abnormality in fibroblasts from patients with GM2 gangliosidosis was confirmed by biochemical analysis and electron microscopy. Further, we observed an excess release of mature cathepsin B isoform and hexosaminidase A into the cytosol, which shows that lysosomal permeability is pathologically increased. GM2 fibroblasts had inappropriately diminished mTOR signalling with reduced basal mTOR activity. It is noteworthy that L-arginine supplementation of the diseased cells partially ameliorated these cytopathological abnormalities and immediately suggests an avenue for facile therapeutic exploration in this cruel disease.

Ethical Statements
The work described was approved by the Ethics Committee of the Virgen de la Macarena  atmosphere. The medium was changed every two days to avoid changes in pH.

Statistical Analysis
Data in the figures is shown as mean ± SD. Data between different groups were analysed statistically by using ANOVA on Ranks with Sigma Plot and Sigma Stat statistical software (SPSS for Windows, 19, 2010, SPSS Inc. Chicago, IL, USA). For cell-culture studies, Student's t test was used for data analyses. A value of p<0.05 was considered significant.

Mutant fibroblasts from Tay-Sachs patients showed impaired autophagic flux
Six patients, 3 infantile and 3 juvenile, contributed to this study. The clinical characteristics of these patients are included in supplementary Table 1. With the exception of juvenile patient 1 and infantile patient 3, the point mutations identified in the HEXA and HEXB genes were predicted to destabilise the intact protein. The latter, (allele 2), was especially noteworthy since the mutation was predicted to confer greater structural stability. However, the amino acid replacement directly affects the active site region.  Table 2). Those mutations were considered to be the most destabilising and to have a more profound effect on hexosaminidase A catalysis as revealed by the impaired activities (compared with the reference values from healthy individuals ( Figure 1D)).
Here we explore the cellular pathogenesis of GM2 gangliosidosis. In culture, the skin fibroblasts obtained from patients had impaired growth rates and markedly abnormal lysosomal morphology ( Figure 1E and F). Western blotting studies showed increased abundance of the immunoreactive autophagy markers, LC3-II and p62/SQSTM1; the intracellular accumulation of lysosomal substrates as determined by p62/SQSTM1 was confirmed by immunofluorescence confocal microscopy ( Figure 1G and H).
To investigate the integrity of lysosomal maturation and the formation of autophagosomes, we used bafilomycin A1 as an inhibitor of the vacuolar H + ATPase (vATPase). The bafilomycin A1 (BafA1) assay serves as a means to explore autophagosome/autophagolysosomal formation in the living cell. As expected, its effects on vATPase caused BafA1 to abrogate lysosomal acidification and intralysosomal digestion of substrates. In control cells addition of BafA1increased cellular abundance of LC3-II but the exposure of Tay-Sachs fibroblasts to the inhibitor did not affect baseline LC3-II staining, ( Figure 1I). These findings strongly suggest that autophagosome processing is defective in these cells.

Autophagosome accumulation with arrested autophagic flux in Tay-Sachs disease
Electron microscopy of fibroblasts obtained from patients with Tay-Sachs disease revealed an extensive accumulation of autophagosomes, an abnormality not present in control cells (Figure 2A and B). The pathological changes, with abundant multilamellar bodies, closely resemble those first reported in neurons obtained from the brains of infants with Tay-Sachs disease 12 .
To distinguish between autophagosome-lysosome fusion or inefficient lysosomal degradation as factors in the accumulation of autophagosomes and altered autophagic flux, we examined lysosome-autophagosome fusion using tandem fluorescent-tagged LC3-II as an autophagosomal marker. We observed numerous yellow structures corresponding to autophagosomes in mCherry-GFP-LC3-II-expressing fibroblasts from Tay-Sachs patients, compared with those from control subjects ( Figure 2C and E). Engulfment of mitochondria by lysosomes and their digestion by mitophagy was explored by the use of high-resolution confocal microscopy to co-localise cytochrome C and LC3 (Figura 2D and F). Markedly increased abundance of engulfed, but incompletely digested mitochondria, confirmed that fusion of lysosomes with autophagosomes is impaired -an abnormality that was observed in fibroblasts from patients with Tay-Sachs disease, irrespective of their clinical severity. Given the organelle pathology that we observed in Tay-Sachs fibroblasts, we examined the subcellular distribution of the master regulator of autophagy and lysosomal biogenesis, Transcription Factor EB (TFEB). In healthy cells, TFEB is normally inactive and diffused in the cytosol in association with the surface of the lysosome, however, when the lysosomal function is inhibited, dephosphorylation of TFEB leads to its translocation to the nucleus where stimulates lysosomal biogenesis by actively upregulating the transcription of target genes harbouring the CLEAR element 13 . As predicted and illustrated in Supplementary Fig 2, immunoreactive TFEB were abundant and concentrated in nuclei of fibroblasts obtained from patients with both subtypes of GM2 gangliosidosis.

Altered mTOR pathway is associated with HexA expression
TFEB is activated under conditions of restricted nutrition and energy generation, and in authentic models of diseases in which lysosomal clearance of intracellular debris is impaired. Given as above that we found subcellular srelocalisation of TFEB from lysosomes to the nucleus in diseased fibroblasts, we investigated the potential engagement of the mechanistic target of rapamycin (mTOR), a kinase and primary regulator of autophagy and lysosomal biogenesis 7,8 in the TFEB translocation process.
The down-regulated and phosphorylated forms of p-mTOR and p-AKT were increased in fibroblasts from patients with Tay-Sachs compared with control cells (Figure 4).

Impaired mTOR/autophagy and LMP are also associated with Sandhoff disease
To test the common underlying cellular pathophysiology in GM2 gangliosidosis, we further explored the changes in Sandhoff disease, which is related to Tay-sachs disease but due to mutations in HEXB with consequential effects on the β-subunit shared by the Hex A and Hex B isoenzymes. Fibroblasts obtained from two patients with Sandhoff disease with reduced enzymatic activities and confirmed HEXB mutations were studied (Supplementary Table 1 gangliosidosis (and Tay-Sachs disease). As in the different cellular models, the analysis of brain, spinal cord, brain stem and cerebellum was consistent with marked disruption of autophagy accompanied by inhibition of p-mTOR and increased expression of active cathepsin B, CatB ( Figure 5E).

Transcriptomic analysis reveals altered molecular pathways
To better define the molecular pathophysiology of cell injury in Tay (Table S3-5 and Figure 6 and Supplementary Figure 6 and 7). Significant changes were observed in multiple genes implicated in these pathways. Of note, despite the variability observed between cells from different patients, several changes appear to be related to the pathobiological changes affecting lysosomal function that we report here. Expression of many genes of the mTOR pathway was downregulated, including ATP6V1C1 and Rictor.
At the same time, Bcl2, an inhibitor of autophagy 14 , and RAB7B, a negative regulator of autophagy flux 15 , were upregulated. Arylsulphatase G (ARSG) and aspartylglucosaminidase (AGA), lysosomal enzymes, which are mutated inlysosomal diseases both in animals and humans, were also downregulated. The former changes are compatible with the finding of increased lysosomal permeability 16,17 . Similarly reduced abundance of the LAPTM4B protein has been linked to increased membrane permeability 18 .
Of particular relevance, was the increased expression of the phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1) gene which encodes the regulatory domain (p85α) of the PI3K complex. This change was shared between both subtypes of GM2 gangliosidosis.
Accordingly, we returned to the fibroblasts and confirmed the increased abundance of the PI3K protein in the fibroblasts from the patients (Supplementary Figure 8). PI3K induces the phosphorylation of AKT and mTOR, but the phosphorylated forms of both were decreased; we propose that the enhanced expression of PI3K transcripts may reflect a compensatory change related to downregulation of the PI3K/AKT/mTOR pathway. To explore this phenomenon further, we investigated the kinase, PTEN, which canonically regulates the PI3K signalling cascade in a negative manner, thereby dampening downstream AKT/mTOR signalling 19 . According to our hypothesis, PTEN gene transcripts were overexpressed in fibroblasts from patients with GM2 gangliosidosis, compared with cells from control subjects (Supplementary Figure 8).

Arginine treatment recovers mTOR activity and lysosomal dysfunction
As a component of the master-regulator, mTOR complex 1 (mTORC1), mTOR links the availability of nutrients with cell growth and autophagy. Since mTORC1 activity is modulated by growth factors, stress, energy status and amino acids 20 , and its function is altered in fibroblasts obtained from patients with Tay-Sachs and Sandhoff Diseases, we sought to determine whether it represents a potential therapeutic target.
Accordingly, we explored the effects of modulating mTOR activity by supplementing fibroblast cultures with amino acids. First, we evaluated the effect of the natural amino acids, L-arginine and L-leucine as well as acetyl-DL-leucine, a modified, cell-penetrant amino acid which has been shown to improve clinical symptoms in Niemann-Pick type C patients 21  On the other hand, the mTOR pathway was partially rescued in patients with Tay-Sachs disease after 120 hours of exposure to L-arginine with a boost in the reduced protein synthesis of the patients ( Figure 7A and B). These observations were also mirrored in fibroblasts from patients with Sandhoff disease (Supplementary Figure 13A).
Furthermore, we examined lysosome-autophagosome fusion. A significantly reduced number of yellow structures that correspond to autophagosomes was evident in mCherry-GFP-LC3-expressing Tay-Sachs fibroblasts after L-arginine treatment ( Figure 7C). These results confirmed that mTOR activity and the fusion of autophagosomes and lysosomes could be improved by supplementation with L-arginine.
Given that altered lysosomal membrane permeability may be a critical factor in the pathophysiological alterations found in cells from patients with GM2 disease, we next evaluated the effects of L-arginine treatment on CatB release from lysosomes. Treatment with L-arginine induced a marked reduction in CatB expression levels in Tay Figure 14).
Finally, as an early exploration of proof of concept, two patients suffering from juvenile forms of Tay-Sachs and Sandhoff diseases TSD2 and Juvenile SD2) consented to take oral supplements of L-arginine (0.3g/Kg/day) for 8 months. Although it was not possible to provide an objective evaluation of the neurological outcomes, family carers and physiotherapists reported improved coordination in both patients and suggested that their rate of cognitive deterioration was partially arrested. The effects of L-arginine supplementation were explored in mononuclear blood cells obtained from these patients.
As shown in Figure 8D, administration of oral L-arginine partially restored mTOR expression. Moreover, the pathological abundance of cathepsin B was suppressed and accompanying this, the expression of HexA and arginosuccinate lyase protein in these cells was improved.

Discussion
Despite many initiatives, no treatment of proven safety and efficacy is available for patients stricken by any clinical subtype of GM2 gangliosidosis. While the genetic and biochemical basis for this disease have been well studied, an integrated description of its pathogenesis and the sequence of unitary steps that lead to its destructive neuroinflammatory effects is lacking. To explore the pathophysiological complexity of these sphingolipid diseases, we used living cells as a focus for a comprehensive molecular characterisation of their disordered cell biology. This platform facilitated corroborative investigations in the coherent model of these disorders in the genetically modified mouse, followed by early proof-of-concept studies carried out in two affected patients.
Here we describe metabolic derangements that accompany markedly impaired autophagy in human fibroblasts harbouring pathological defined HEXA and HEXB mutations. The Tay-Sachs and Sandhoff diseases 28,31,32 . , In its terminal phase, deranged autophagy is likely to contribute additionally to the pathological cascade by stimulating release of inflammatory cytokines through the agency of the p62/SQSTM1 signal 9 . As to the upstream drive to enhance autophagosome genesis in Tay-Sachs and Sandhoff diseases, we find evidence that this is due to nuclear translocation of TFEB, since it activates genes that orchestrate lysosomal biogenesis. The critical discovery of this fundamental process emerged from the brilliant realization that coordinated lysosomal expression and regulation (CLEAR) represented a gene network which could be controlled by a single major transcriptional factor that recognized a key regulatory element common to effector proteins fundamentally implicated in lysosomal pathobiology 33 . The complex disturbance of lysosomal structure and function in fibroblasts from Tay-Sachs and Sandhoff patients as a consequence of reduced activity of the lysosomal βhexosaminidases with increased autophagosome size can be attributed to TFEB activation as part of a response that will drive compensatory expansion of the lysosomal compartment. The enhanced TFEB nuclear localization, we reported here in Tay Autophagy is a constitutive but dynamically controlled process that is central to the maintenance of cellular homeostasis. Any disturbance of lysosomal function, as in the genetic disorders, Tay-Sachs and Sandhoff diseases will require compensatory adjustments to ensure, so far as possible, survival of the affected cell. These GM2 gangliosidoses, which preferentially affect the lyososomal recycling of membranederived sphingolipids abundant in neurons, provide a spectacular example of the rôle of autophagy in non-mitotic cells with a life-long dependence on mitochondrial energy generation: relentlessly progressive, these diseases cause widespread neuronal death. 39 .
Our findings provide evidence for a mechanistic link between disrupted autophagy, increased permeabiization of the lysosomal compartment and neuro-inflammatory changes 40,41 .
We contend that the pathological mTOR signalling and consequential mitochondrial and lysosomal dysfunction that we report in GM2 gangliosidosis immediately suggest avenues for therapeutic exploration. Amino acid supplementation to restore mTOR activity has been investigated in Pompe disease: arginine and leucine were found to restore mTOR signalling and partiallyrescuedthe muscle disease due to gross failure of glycogen remodeling and with the accumulation of pathological autophagosomes in the sarcoplasm 7 . In our studies with fibroblasts obtained from affected patients, improvement of the cell phenotype was found to be specific for L-arginine, rather than L-leucine, and we attribute this specificity to the consistent genetic changes in a rate-limiting enzyme of L-arginine biosynthesis (arginosuccinate lyase) prompted by gene expression studies carried out in fibroblastsfrom patients with Tay-Sachs and Sandhoff diseases. Given that the patients have neurological dysphagia and in many cases feeding-tube placement, we at first considered that the reduced serum arginine concentrations might reflect a nutritional defect, especially in the most severely affected infants. However, our studies of fibroblasts were conducted in cells obtained after prolonged outgrowth culture of skin biopsy samples. In these, microarray analysis revealed a specific alteration related directly to a rate-limiting enzyme in arginine biosynthesis. The previousfindings were corroborated by the reduced abundance of human argino-succinate synthetase protein in fibroblast extracts.

Tay-Sachs and Sandhoff diseases are paradigmatic examples of a large class of lysosomal
diseases that are scharacterised by unremitting neurodegeneration. Unfortunately, treatments can only address symptoms and management is directed towards supporting critical functions that are progressively lost. For these reasons, the conditions remain a focus of research-based principally on molecular therapies 9 . Our study considers a molecular approach that has not been explored in these diseases but is based on a fresh examination of the pathobiology of the disease. If adopted in practice, the approach is non-invasive and could be readily adapted to practical clinical care that includes a focus on nutrition. While it is an essential amino acid and natural dietary constituent, L-arginine has potent biological effects, for example on the formation of nitric oxide (for which it is the primary substrate) and in non-physiological doses has potential toxicity exerted by       between control and Tay-Sachs fibroblasts; a P < 0.05; aa P < 0.01; aaa P < 0.001 between non-treated and treated cells. (arginosuccinate synthetase) proteins was determined in peripheral blood mononuclear cells obtained from a patient with juvenile Tay-Sachs disease and a patient with juvenile Sandoff disease after oral L-arginine treatment. Data represent the mean±SD of three separate experiments.*P < 0.05; **P < 0.01; **P < 0.001 between control and Tay-Sachs patients; a P < 0.05; aa P < 0.01; aaa P < 0.001 between non-treated and treated cells.