NRF2 signaling cascade in amyotrophic lateral sclerosis: bridging the gap between promise and reality.

Amyotrophic lateral sclerosis is a very disabling disease due to the degeneration of motor neurons. Symptoms include muscle weakness and atrophy, spasticity, and progressive paralysis. Currently, there is no treatment to reverse damage to motor neurons and cure amyotrophic lateral sclerosis. The only two treatments actually approved, riluzole and edaravone, have shown mitigated beneficial effects. The difficulty to find a cure lies in the complexity and multifaceted pattern of amyotrophic lateral sclerosis pathogenesis. Among mechanisms, abnormal RNA metabolism, nucleocytoplasmic transport defects, accumulation of unfolded protein, and mitochondrial dysfunction would in fine induce oxidative damage and vice versa. A potent therapeutic strategy will be to find molecules that break this vicious circle. Sharpening the nuclear factor erythroid-2 related factor 2 signaling may fulfill this objective since nuclear factor erythroid-2 related factor 2 has a multitarget profile controlling antioxidant defense, mitochondrial functioning, and inflammation. We here discuss the interest of developing nuclear factor erythroid-2 related factor 2-based therapy in regard to the pathophysiological mechanisms and we provide a general overview of the attempted clinical assays in amyotrophic lateral sclerosis.

Sigma-1 receptor agonist PRE-084 confers protection against TAR DNA-binding protein-43 toxicity through NRF2 signalling.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting upper and lower motor neurons. As a consequence, ALS patients display a locomotor disorder related to muscle weakness and progressive paralysis. Pathological mechanisms that participate in ALS involve deficient unfolded protein response, mitochondrial dysfunction and oxidative stress, among others. Finding a therapeutic target to break the vicious circle is particularly challenging. Sigma-1 receptor (S1R) is an endoplasmic reticulum (ER) chaperone that may be one of those targets. We here address and decipher the efficiency of S1R activation on a key ALS gene, TDP43, in zebrafish vertebrate model. While expression of mutant TDP43 (TDP43G348C) led to locomotor defects, treatment with the reference S1R agonist PRE-084 rescued motor performances in a zebrafish model. Treatment with the agonist ameliorated maximal mitochondrial respiration in the TDP43 context. We observed that TDP43G348C exacerbated ER stress induced by tunicamycin, resulting in increased levels of ER stress chaperone BiP and pro-apoptotic factor CHOP. Importantly, PRE-084 treatment in the same condition further heightened BiP levels but also EIF2α/ATF4 and NRF2 signalling cascades, both known to promote antioxidant protection during ER stress. Moreover, we showed that increasing NRF2 levels directly or by sulforaphane treatment rescued locomotor defects of TDP43G348C zebrafish. For the first time, we here provide the proof of concept that PRE-084 prevents mutant TDP43 toxicity by boosting ER stress response and antioxidant cascade through NRF2 signalling.

Morphological, behavioral and cellular analyses revealed different phenotypes in Wolfram syndrome wfs1a and wfs1b zebrafish mutant lines.

Wolfram syndrome (WS) is a rare genetic disease characterized by diabetes, optic atrophy and deafness. Patients die at 35 years of age, mainly from respiratory failure or dysphagia. Unfortunately, there is no treatment to block the progression of symptoms and there is an urgent need for adequate research models. Here, we report on the phenotypical characterization of two loss-of-function zebrafish mutant lines: wfs1aC825X and wfs1bW493X. We observed that wfs1a deficiency altered the size of the ear and the retina of the fish. We also documented a decrease in the expression level of unfolded protein response (UPR) genes in basal condition and in stress condition, i.e. after tunicamycin treatment. Interestingly, both mutants lead to a decrease in their visual function measured behaviorally. These deficits were associated with a decrease in the expression level of UPR genes in basal and stress conditions. Interestingly, basal, ATP-linked and maximal mitochondrial respirations were transiently decreased in the wfs1b mutant. Taken together, these zebrafish lines highlight the critical role of wfs1a and wfs1b in UPR, mitochondrial function and visual physiology. These models will be useful tools to better understand the cellular function of Wfs1 and to develop novel therapeutic approaches for WS.

Activation of the sigma-1 receptor chaperone alleviates symptoms of Wolfram syndrome in preclinical models.

The Wolfram syndrome is a rare autosomal recessive disease affecting many organs with life-threatening consequences; currently, no treatment is available. The disease is caused by mutations in the WSF1 gene, coding for the protein wolframin, an endoplasmic reticulum (ER) transmembrane protein involved in contacts between ER and mitochondria termed as mitochondria-associated ER membranes (MAMs). Inherited mutations usually reduce the protein's stability, altering its homeostasis and ultimately reducing ER to mitochondria calcium ion transfer, leading to mitochondrial dysfunction and cell death. In this study, we found that activation of the sigma-1 receptor (S1R), an ER-resident protein involved in calcium ion transfer, could counteract the functional alterations of MAMs due to wolframin deficiency. The S1R agonist PRE-084 restored calcium ion transfer and mitochondrial respiration in vitro, corrected the associated increased autophagy and mitophagy, and was able to alleviate the behavioral symptoms observed in zebrafish and mouse models of the disease. Our findings provide a potential therapeutic strategy for treating Wolfram syndrome by efficiently boosting MAM function using the ligand-operated S1R chaperone. Moreover, such strategy might also be relevant for other degenerative and mitochondrial diseases involving MAM dysfunction.

Sigma-1 receptor chaperones rescue nucleocytoplasmic transport deficit seen in cellular and Drosophila ALS/FTD models.

In a subgroup of patients with amyotrophic lateral sclerosis (ALS)/Frontotemporal dementia (FTD), the (G4C2)-RNA repeat expansion from C9orf72 chromosome binds to the Ran-activating protein (RanGAP) at the nuclear pore, resulting in nucleocytoplasmic transport deficit and accumulation of Ran in the cytosol. Here, we found that the sigma-1 receptor (Sig-1R), a molecular chaperone, reverses the pathological effects of (G4C2)-RNA repeats in cell lines and in Drosophila. The Sig-1R colocalizes with RanGAP and nuclear pore proteins (Nups) and stabilizes the latter. Interestingly, Sig-1Rs directly bind (G4C2)-RNA repeats. Overexpression of Sig-1Rs rescues, whereas the Sig-1R knockout exacerbates, the (G4C2)-RNA repeats-induced aberrant cytoplasmic accumulation of Ran. In Drosophila, Sig-1R (but not the Sig-1R-E102Q mutant) overexpression reverses eye necrosis, climbing deficit, and firing discharge caused by (G4C2)-RNA repeats. These results on a molecular chaperone at the nuclear pore suggest that Sig-1Rs may benefit patients with C9orf72 ALS/FTD by chaperoning the nuclear pore assembly and sponging away deleterious (G4C2)-RNA repeats.

Sigma-1 receptor is a key genetic modulator in amyotrophic lateral sclerosis.

Sigma-1 receptor (S1R) is an endoplasmic reticulum (ER) chaperone that not only regulates mitochondrial respiration but also controls cellular defense against ER and oxidative stress. This makes S1R a potential therapeutic target in amyotrophic lateral sclerosis (ALS). Especially, as a missense mutation E102Q in S1R has been reported in few familial ALS cases. However, the pathogenicity of S1RE102Q and the beneficial impact of S1R in the ALS context remain to be demonstrated in vivo. To address this, we generated transgenic Drosophila that expresses human wild-type S1R or S1RE102Q. Expression of mutant S1R in fly neurons induces abnormal eye morphology and locomotor defects in a dose-dependent manner. This was accompanied by abnormal mitochondrial fragmentation, reduced adenosine triphosphate (ATP) levels and a higher fatigability at the neuromuscular junction during high energy demand. Overexpressing IP3 receptor or glucose transporter mitigates the S1RE102Q-induced eye phenotype, further highlighting the role of calcium and energy metabolism in its toxicity. More importantly, we showed that wild-type S1R rescues locomotor activity and ATP levels of flies expressing the key ALS protein, TDP43. Moreover, overexpressing wild-type S1R enhances resistance of flies to oxidative stress. Therefore, our data provide the first genetic evidence that mutant S1R recapitulates ALS pathology in vivo while increasing S1R confers neuroprotection against TDP43 toxicity.

Glycolysis upregulation is neuroprotective as a compensatory mechanism in ALS.

Amyotrophic Lateral Sclerosis (ALS), is a fatal neurodegenerative disorder, with TDP-43 inclusions as a major pathological hallmark. Using a Drosophila model of TDP-43 proteinopathy we found significant alterations in glucose metabolism including increased pyruvate, suggesting that modulating glycolysis may be neuroprotective. Indeed, a high sugar diet improves locomotor and lifespan defects caused by TDP-43 proteinopathy in motor neurons or glia, but not muscle, suggesting that metabolic dysregulation occurs in the nervous system. Overexpressing human glucose transporter GLUT-3 in motor neurons mitigates TDP-43 dependent defects in synaptic vesicle recycling and improves locomotion. Furthermore, PFK mRNA, a key indicator of glycolysis, is upregulated in flies and patient derived iPSC motor neurons with TDP-43 pathology. Surprisingly, PFK overexpression rescues TDP-43 induced locomotor deficits. These findings from multiple ALS models show that mechanistically, glycolysis is upregulated in degenerating motor neurons as a compensatory mechanism and suggest that increased glucose availability is protective.

Mitochondrial quality control in amyotrophic lateral sclerosis: towards a common pathway?

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by loss of upper and lower motor neurons. Different mechanisms contribute to the disease initiation and progression, including mitochondrial dysfunction which has been proposed to be a central determinant in ALS pathogenesis. Indeed, while mitochondrial defects have been mainly described in ALS-linked SOD1 mutants, it is now well established that mitochondria become also dysfunctional in other ALS conditions. In such context, the mitochondrial quality control system allows to restore normal functioning of mitochondria and to prevent cell death, by both eliminating and replacing damaged mitochondrial components or by degrading the entire organelle through mitophagy. Recent evidence shows that ALS-related genes interfere with the mitochondrial quality control system. This review highlights how ineffective mitochondrial quality control may render motor neurons defenseless towards the accumulating mitochondrial damage in ALS.

Enhancing Mitofusin/Marf ameliorates neuromuscular dysfunction in Drosophila models of TDP-43 proteinopathies.

Transactive response DNA-binding protein 43 kDa (TDP-43) is considered a major pathological protein in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The precise mechanisms by which TDP-43 dysregulation leads to toxicity in neurons are not fully understood. Using TDP-43-expressing Drosophila, we examined whether mitochondrial dysfunction is a central determinant in TDP-43 pathogenesis. Expression of human wild-type TDP-43 in Drosophila neurons results in abnormally small mitochondria. The mitochondrial fragmentation is correlated with a specific decrease in the mRNA and protein levels of the Drosophila profusion gene mitofusin/marf. Importantly, overexpression of Marf ameliorates defects in spontaneous walking activity and startle-induced climbing response of TDP-43-expressing flies. Partial inactivation of the mitochondrial profission factor, dynamin-related protein 1, also mitigates TDP-43-induced locomotor deficits. Expression of TDP-43 impairs neuromuscular junction transmission upon repetitive stimulation of the giant fiber circuit that controls flight muscles, which is also ameliorated by Marf overexpression. We show here for the first time that enhancing the profusion gene mitofusin/marf is beneficial in an in vivo model of TDP-43 proteinopathies, serving as a potential therapeutic target.

Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93). We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP) impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK) which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to mediate the hGluT3 protective effects, whereas, in addition, the PPP provides increased protection to oxidative stress.

The Drosophila inner-membrane protein PMI controls crista biogenesis and mitochondrial diameter.

Cristae are mitochondrial inner-membrane structures that concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling crista morphogenesis are poorly understood and few crista determinants have been identified. Among them are the Mitofilins that are required to establish crista junctions and ATP-synthase subunits that bend the membrane at the tips of the cristae. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondrial ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI overexpression is sufficient to reduce crista length in vivo. Finally, these crista defects are associated with impaired respiratory chain activity and increases in the level of reactive oxygen species. Since PMI and its human orthologue TMEM11 are regulators of mitochondrial morphology, our data suggest that, by controlling crista length, PMI influences mitochondrial diameter and tubular shape.

Reducing canonical Wingless/Wnt signaling pathway confers protection against mutant Huntingtin toxicity in Drosophila.

Huntington's disease (HD) is a genetic neurodegenerative disease characterized by movement disorders, cognitive decline and neuropsychiatric symptoms. HD is caused by expanded CAG tract within the coding region of Huntingtin protein. Despite major insights into the molecular mechanisms leading to HD, no effective cure is yet available. Mutant Huntingtin (mHtt) has been reported to alter the stability and levels of ß-Catenin, a key molecule in cell adhesion and signal transduction in Wingless (Wg)/Wnt pathway. However it remains to establish whether manipulation of Wg/Wnt signaling can impact HD pathology. We here investigated the phenotypic interactions between mHtt and Wg/Wnt signaling by using the power of Drosophila genetics. We provide compelling evidence that reducing Armadillo/ß-Catenin levels confers protection and that this beneficial effect is correlated with the inactivation of the canonical Wg/Wnt signaling pathway. Knockdowns of Wnt ligands or of the downstream transcription factor Pangolin/TCF both ameliorate the survival of HD flies. Similarly, overexpression of one Armadillo/ß-Catenin destruction complex component (Axin, APC2 or Shaggy/GSK-3ß) increases the lifespan of HD flies. Loss of functional Armadillo/ß-Catenin not only abolishes neuronal intrinsic but also glia-induced alterations in HD flies. Our findings highlight that restoring canonical Wg/Wnt signaling may be of therapeutic value.

Increased energy metabolism rescues glia-induced pathology in a Drosophila model of Huntington's disease.

Huntington's disease (HD) is a polyglutamine (polyQ) disease caused by an expanded CAG tract within the coding region of Huntingtin protein. Mutant Huntingtin (mHtt) is ubiquitously expressed, abundantly in neurons but also significantly in glial cells. Neuron-intrinsic mechanism and alterations in glia-to-neuron communication both contribute to the neuronal dysfunction and death in HD pathology. However, it remains to be determined the role of glial cells in HD pathogenesis. In recent years, development of Drosophila models facilitated the dissection of the cellular and molecular events in polyQ-related diseases. By using genetic approaches in Drosophila, we manipulated the expression levels of mitochondrial uncoupling proteins (UCPs) that regulate production of both ATP and reactive oxygen species in mitochondria. We discovered that enhanced levels of UCPs alleviated the HD phenotype when mHtt was selectively expressed in glia, including defects in locomotor behavior and early death of Drosophila. In contrast, UCPs failed to prevent the HD toxicity in neurons. Increased oxidative stress defense was found to rescue neuron but not glia-induced pathology. Evidence is now emerging that UCPs are fundamental to adapt the energy metabolism in order to meet the metabolic demand. Thus, we propose that UCPs are glioprotective by rescuing energy-dependent functions in glia that are challenged by mHtt. In support of this, increasing glucose entry in glia was found to alleviate glia-induced pathology. Altogether, our data emphasize the importance of energy metabolism in the glial alterations in HD and may lead to a new therapeutic avenue.

AKT-sensitive or insensitive pathways of toxicity in glial cells and neurons in Drosophila models of Huntington's disease.

Huntington's disease (HD) is caused by an extended polyglutamine (polyQ) tract in the Huntingtin protein. Neuronal and glial dysfunction precedes the neurodegeneration and appears to be the primary cause for the early symptoms in HD. In recent years, development of Drosophila models of polyQ-related diseases facilitated research of candidate rescuer genes. In most cases, analysis in Drosophila was performed by assessing toxicity on retinal and/or brain neurons. However, none of the potential rescuers were evaluated on glial alterations. Here we used a genetic approach in Drosophila to characterize the phenotypic effects of mutant Huntingtin (mHtt) expressed in neurons or different glia subsets and we established a sensitive assay for evaluating modifiers of glial alterations. We determined the level of cell protection ensured by activation of the AKT and ERK anti-apoptotic kinases in the retina as well as in neurons and glia of the fly brain, compared with the rescuing effects of the HSP70 chaperone. We found that both AKT and HSP70 alleviated mHtt-induced toxicity in the retina. In contrast, their protective effects differed in the brain. HSP70 rescued neurodegeneration, locomotor defects and early lethality of flies expressing mHtt in neurons or glia. AKT failed to prevent brain neuronal death and lethality of flies, but significantly improved their locomotor performance when co-expressed with mHtt in glia. ERK had no beneficial effects in the retina or brain. These results indicate that mHtt activates distinct pathways of toxicity in Drosophila, either sensitive to AKT in retinal photoreceptors and glia, or independent in brain neurons.

[Astrocytes in Huntington's chorea: accomplice or guilty of the neuronal death?]. / Les astrocytes dans la chorée de Huntington: complices ou coupables de la mort neuronale?

Huntington's disease is an hereditary dominant neurodegenerative disorder clinically characterised by progressive motor deficits, personality changes, decreased mental capacity and death after about 15-20 years. Most studies are based on the research of intrinsic mechanisms that could be responsible for dysfunction and later degeneration of neuronal subsets. It is only in the last five years that more interest has been focused on another brain cell type : the astrocytes. This review presents evidence that astroglial function is also affected in Huntington's disease. Among the possible mechanisms, Huntington's disease mutation may alter the EGF receptor signaling pathway, that regulates the astrocytic response to neuronal injuries.

Dissociation between hippocampal neuronal loss, astroglial and microglial reactivity after pharmacologically induced reverse glutamate transport.

The inflammatory central nervous system response that involves activated microglia and reactive astrocytes may both heal and harm neurons, as inflammatory mediators lead to neuroprotection or excitation at one dose but to injury at a different concentration. To investigate these complex cellular interactions, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a selective substrate inhibitor of glutamate transport, was infused during 14 days in the rat hippocampus at three different rates: 5, 10 and 25 nmol/h. A microglial reaction appeared at the 5 nmol/h PDC rate in absence of astroglial reaction and neuronal loss. Microgliosis and neuronal death were observed at PDC 10 nmol/h in absence of astrogliosis and calcium precipitation, whereas all four aspects were present at the highest rate. This dissociation between neuronal loss and astroglial reactivity took place in presence of a permanent microglial reaction. These data suggest a specific response of microglia to PDC whose neuronal effects may differ with the infused dose.

Expanded polyglutamine peptides disrupt EGF receptor signaling and glutamate transporter expression in Drosophila.

Huntington's disease (HD) is a late onset heritable neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) sequence in the protein huntingtin (Htt). Transgenic models in mice have suggested that the motor and cognitive deficits associated to this disease are triggered by extended neuronal and possibly glial dysfunction, whereas neuronal death occurs late and selectively. Here, we provide in vivo evidence that expanded polyQ peptides antagonize epidermal growth factor receptor (EGFR) signaling in Drosophila glia. We targeted the expression of the polyQ-containing domain of Htt or an extended polyQ peptide alone in a subset of Drosophila glial cells, where the only fly glutamate transporter, dEAAT1, is detected. This resulted in formation of nuclear inclusions, progressive decrease in dEAAT1 transcription and shortened adult lifespan, but no significant glial cell death. We observed that brain expression of dEAAT1 is normally sustained by the EGFR-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, suggesting that polyQ could act by antagonizing this pathway. We found that the presence of polyQ peptides indeed abolished dEAAT1 upregulation by constitutively active EGFR and potently inhibited EGFR-mediated ERK activation in fly glial cells. Long polyQ also limited the effect of activated EGFR on Drosophila eye development. Our results further indicate that the polyQ acts at an upstream step in the pathway, situated between EGFR and ERK activation. This suggests that disruption of EGFR signaling and ensuing glial cell dysfunction could play a direct role in the pathogenesis of HD and other polyQ diseases in humans.

[Huntington chorea in Drosophila and mice: toward new therapeutic steps]. / La chorée de Huntington chez la drosophile et chez la souris: vers de nouvelles pistes thérapeutiques?

Huntington's disease is an hereditary dominant neurodegenerative disorder clinically characterised by progressive dyskinesia, cognitive decline and psychiatric disturbances. One decade after the identification of the gene whose mutation is responsible for the disease, this pathology remains incurable. However, major insights into early cellular and molecular basis of Huntington's disease have arisen from transgenic models. Transcriptional dysregulation, abnormal degradation of misfolded proteins as well as excitotoxic processes and mitochondrial dysfunction are involved in Huntington's disease. The present review discusses the recent insights gained from mouse and Drosophila models towards the understanding of pathogenesis and the development of new therapeutic tools.

Abnormal phosphorylation of synapsin I predicts a neuronal transmission impairment in the R6/2 Huntington's disease transgenic mice.

Motor and cognitive deficits in Huntington's disease (HD) are likely caused by progressive neuronal dysfunction preceding neuronal cell death. Synapsin I is one of the major phosphoproteins regulating neurotransmitter release. We report here an abnormal phosphorylation state of synapsin I in the striatum and the cerebral cortex of R6/2 transgenic mice expressing the HD mutation. These changes are mostly characterized by an early overphosphorylation at sites 3-5, whereas phosphorylation at site 1 remains unchanged and at site 6 becomes reduced only close to the end stage of the disease. Such changes do not result from modification in protein expression levels. However, we show a decreased expression of the calcineurin regulatory subunit-B, which may contribute to an imbalance between kinase and phosphatase activities. Together the results suggest that an early impairment in synapsin phosphorylation-dephosphorylation may alter synaptic vesicle trafficking and lead to defective neurotransmission in HD.