Activating ATF6 in spinal muscular atrophy promotes SMN expression and motor neuron survival through the IRE1α‐XBP1 pathway

D’Amico, Domenico; Biondi, Olivier; Januel, Camille; Bezier, Cynthia; Sapaly, Delphine; Clerc, Zoé; Khoury, Mirella El; Sundaram, Venkat Krishnan; Houdebine, Léo; Josse, Thibaut; Gaspera, Bruno Della; Martinat, Cécile; Massaad, Charbel; Weill, Laure; Charbonnier, Frédéric

doi:10.1111/nan.12816

Cited by 7 publications

(4 citation statements)

References 71 publications

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“…In this respect, it is worth noticing that to observe cellular stress in hiPSC-derived MNs, it usually takes culture times longer than 25 days. Such UPR activation is rarely observed in hiPSC-derived MNs after only 25 days of culture as recently described in SMA hiPSC-derived MNs [ 8 ]. Finally, reduced MN survival in co-cultures and not in mono-cultures could be indicative of non-cell-autonomous mechanisms.…”

Section: Discussionmentioning

confidence: 71%

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons

et al. 2022

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Congenital myasthenic syndromes (CMS) are predominantly characterized by muscle weakness and fatigability and can be caused by a variety of mutations in genes required for neuromuscular junction formation and maintenance. Among them, AGRN encodes agrin, an essential synaptic protein secreted by motoneurons. We have identified severe CMS patients with uncharacterized p.R1671Q, p.R1698P and p.L1664P mutations in the LG2 domain of agrin. Overexpression in primary motoneurons cultures in vitro and in chick spinal motoneurons in vivo revealed that the mutations modified agrin trafficking, leading to its accumulation in the soma and/or in the axon. Expression of mutant agrins in cultured cells demonstrated accumulation of agrin in the endoplasmic reticulum associated with induction of unfolded protein response (UPR) and impaired secretion in the culture medium. Interestingly, evaluation of the specific activity of individual agrins on AChR cluster formation indicated that when secreted, mutant agrins retained a normal capacity to trigger the formation of AChR clusters. To confirm agrin accumulation and secretion defect, iPS cells were derived from a patient and differentiated into motoneurons. Patient iPS-derived motoneurons accumulated mutant agrin in the soma and increased XBP1 mRNA splicing, suggesting UPR activation. Moreover, co-cultures of patient iPS-derived motoneurons with myotubes confirmed the deficit in agrin secretion and revealed a reduction in motoneuron survival. Altogether, we report the first mutations in AGRN gene that specifically affect agrin secretion by motoneurons. Interestingly, the three patients carrying these mutations were initially suspected of spinal muscular atrophy (SMA). Therefore, in the presence of patients with a clinical presentation of SMA but without mutation in the SMN1 gene, it can be worth to look for mutations in AGRN.

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Section: Discussionmentioning

confidence: 71%

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons

et al. 2022

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“…3 online resource). Given that LARGE1 (acting as a bifunctional glycosyltransferase with both alpha-1,3-xylosyltransferase and beta-1,3-glucuronyltransferase activities) is localized to the ER-Golgi network and other ER-Golgi resident proteins were already unveiled as critical factors for motor neuron survival in SMA [ 5 ] and other motor neuron diseases such as ALS [ 10 ], were selected this protein as promising candidate for further analyses. This decision was also motivated by the known role of LARGE1 in the genesis of neuromuscular diseases (MIM: 608,840 & MIM:613,154).…”

Section: Resultsmentioning

confidence: 99%

Alteration of LARGE1 abundance in patients and a mouse model of 5q-associated spinal muscular atrophy

Roos,

Schmitt,

Hansmann

et al. 2024

Acta Neuropathol

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Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by recessive pathogenic variants affecting the survival of motor neuron (SMN1) gene (localized on 5q). In consequence, cells lack expression of the corresponding protein. This pathophysiological condition is clinically associated with motor neuron (MN) degeneration leading to severe muscular atrophy. Additionally, vulnerability of other cellular populations and tissues including skeletal muscle has been demonstrated. Although the therapeutic options for SMA have considerably changed, treatment responses may differ thus underlining the persistent need for validated biomarkers. To address this need and to identify novel marker proteins for SMA, we performed unbiased proteomic profiling on cerebrospinal fluid derived (CSF) from genetically proven SMA type 1–3 cases and afterwards performed ELISA studies on CSF and serum samples to validate the potential of a novel biomarker candidates in both body fluids. To further decipher the pathophysiological impact of this biomarker, immunofluorescence studies were carried out on spinal cord and skeletal muscle derived from a 5q-SMA mouse model. Proteomics revealed increase of LARGE1 in CSF derived from adult patients showing a clinical response upon treatment with nusinersen. Moreover, LARGE1 levels were validated in CSF samples of further SMA patients (type 1–3) by ELISA. These studies also unveiled a distinguishment between groups in improvement of motor skills: adult patients do present with lowered level per se at baseline visit while no elevation upon treatment in the pediatric cohort can be observed. ELISA-based studies of serum samples showed no changes in the pediatric cohort but unraveled elevated level in adult patients responding to future intervention with nusinersen, while non-responders did not show a significant increase. Additional immunofluorescence studies of LARGE1 in MN and skeletal muscle of a SMA type 3 mouse model revealed an increase of LARGE1 during disease progression. Our combined data unraveled LARGE1 as a protein dysregulated in serum and CSF of SMA-patients (and in MN and skeletal muscle of SMA mice) holding the potential to serve as a disease marker for SMA and enabling to differentiate between patients responding and non-responding to therapy with nusinersen.

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“…19 From this screen, N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (AA147; Fig 1A) emerged as a selective ATF6 activator that is able to promote adaptive ATF6 activity to mitigate pathologies associated with etiologically diverse disorders. 11,[19][20][21][22][23][24][25][26] We found that AA147 functions as a prodrug, wherein oxidative conversion of the 2-amino-p-cresol substructure by ER-resident oxidases (e.g., cytochrome P450s) leads to an electrophilic quinone methide and selective covalent engagement of a subset of ER proteins primarily consisting of protein disulfide isomerases (PDIs). 27 PDIs maintain ATF6 in disulfide-bonded structures that limit its activation.…”

Section: Introductionmentioning

confidence: 99%

Divergent Proteome Reactivity Influences Arm-Selective Activation of Pharmacological Endoplasmic Reticulum Proteostasis Regulators

Kline

Paxman

Lin

et al. 2023

Preprint

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Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the Unfolded Protein Response (UPR) has proven useful for ameliorating proteostasis deficiencies in a variety of etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino-p-cresol substructure affording a quinone methide, which then covalently modifies a subset of ER protein disulfide isomerases (PDIs). Intriguingly, another compound identified in this screen, AA132, also contains a 2-amino-p-cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent PDI modification. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. Paradoxically, activated AA132 reacts slower with PDIs, indicating it is less reactive than activated AA147. This suggests that the higher labeling of PDIs observed with activated AA132 can be attributed to its lower reactivity, which allows this activated compound to persist longer in the cellular environment prior to quenching by endogenous nucleophiles. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and allows for selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

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Activating ATF6 in spinal muscular atrophy promotes SMN expression and motor neuron survival through the IRE1α‐XBP1 pathway

Cited by 7 publications

References 71 publications

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons

Alteration of LARGE1 abundance in patients and a mouse model of 5q-associated spinal muscular atrophy

Divergent Proteome Reactivity Influences Arm-Selective Activation of Pharmacological Endoplasmic Reticulum Proteostasis Regulators

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