Background
Tuberous sclerosis complex (TSC) is an inherited multisystem disorder, comprising both TSC1 and TSC2, and involves a range of symptoms including epilepsy, autism spectrum disorder (ASD), intellectual disability (ID) and slow growing hamartomas in many organs. TSC is caused by mutations in the
TSC1 or
TSC2 genes, encoding the tumor suppressor proteins hamartin (TSC1) and tuberin (TSC2) [
1,
2]. The TSC proteins act as a central hub relaying signals from diverse cellular pathways to control mammalian/mechanistic target of rapamycin complex 1 (mTORC1) activity, which regulates cell growth and proliferation [
3,
4]. Aberrant activation of mTORC1 in TSC has led to rapamycin (Rap) analogs (“rapalogs”) emerging as a lifelong therapy for TSC hamartomas, as their discontinuation leads to resumed growth of TSC-associated lesions [
5‐
8]. Recent clinical trials revealed that rapalogs reduce epilepsy in 40% of TSC patients [
9]. In contrast, rapalogs are ineffective in treating TSC-associated neuropsychiatric defects (TAND) and autism [
10,
11]. Therefore, new treatments for TSC that are superior to rapalogs with respect to anti-proliferative effects in tumors, and efficacy toward the non-tumor CNS manifestations of the disorder are needed.
Among mTOR inhibitors, first-generation allosteric rapalogs effectively suppress phosphorylation of mTORC1 target S6K1, but not 4E-BP1 in many cell types [
12,
13]. Furthermore, allosteric rapalogs activate AKT, a downstream target of mTORC2, by negative feedback loops [
14], which prompted development of second-generation, orthosteric mTOR kinase inhibitors (active site mTOR inhibitors) including Torin 1, AZD8055 and TAK-228/MLN0128, which potently inhibit both mTORC1 and mTORC2. As mTORC2 promotes lipogenesis, glucose uptake and cell survival through downstream targets AKT and SGK, active site mTOR inhibitors appears to be more toxic than rapalogs [
12,
15]. The limited clinical benefits of first- and second-generation mTOR inhibitors led to the recent development of a third-generation mTORC1-directed inhibitor RapaLink-1. This bi-steric mTOR inhibitor links the high affinity of rapamycin for mTORC1 with the effective active site mTOR inhibition of TAK-228 [
16]. RapaLink-1 was shown to be highly potent in reducing phosphorylation of both S6K1 and 4E-BP1 while retaining approximately four-fold selectivity for mTORC1 as compared to mTORC2. More recent bi-steric compounds such as RMC-6272 show higher selectivity for mTORC1 over mTORC2 (more than 30-fold selectivity), along with potent suppression of 4E-BP1 phosphorylation [
17,
18].
Several mouse models of TSC have provided valuable clues regarding neurological symptoms, but incompletely recapitulate the human phenotypes [
19]. Recent studies examining the role of TSC1 or TSC2 have employed genetically engineered human embryonic stem cell lines with heterozygous or homozygous loss of
TSC2; TSC patient-derived induced pluripotent stem cells (iPSCs); or isogenic gene-edited iPSCs from patients with
TSC1 or
TSC2 mutations that have been differentiated into, e.g., neural progenitor cells (NPCs), forebrain neurons, cerebellar Purkinje neurons, astrocytes or oligodendrocytes [
20‐
28]. Many phenotypic alterations, including somatic hypertrophy, increased dendritic arborization, augmented proliferation rate, altered electrophysiology and hyperactivation of mTORC1, are more pronounced in
TSC1-null or
TSC2-null cells when compared with heterozygous or wild-type (WT) counterparts (reviewed in [
29,
30]). Furthermore, transcriptome analyses have revealed ample alterations when comparing isogenic gene-edited
TSC1- or
TSC2-null NPCs or neurons to heterozygous or WT cells [
21,
22,
24,
26].
Among its many activities [
14], mTORC1 plays a major role in regulating gene expression by modulating how efficiently mRNAs are translated into proteins [
31]. Consistent with a key role of mRNA translation in determining proteome composition, translatomes (commonly defined as the pool of mRNA associated with ribosomes) [
32] resemble proteomes more closely than corresponding transcriptomes [
33‐
35]. mTORC1 regulates cap-dependent translation by modulating the assembly of eukaryotic initiation factor (eIF) 4F, a complex consisting of a cap binding protein (eIF4E), a DEAD-box RNA helicase (eIF4A) and a large scaffolding protein (eIF4G). mTORC1 activation leads to direct phosphorylation of two key substrates involved in regulating translation initiation: eIF4E-binding proteins (4E-BPs) and ribosomal protein p70S6 kinases (S6Ks). 4E-BPs are a family of translation inhibitors consisting of three members, the best studied being 4E-BP1, which when de-phosphorylated competes with eIF4G for binding to eIF4E and prevents eIF4F complex formation. Once phosphorylated, 4E-BP1 dissociates from eIF4E, facilitating eIF4F complex formation [
36]. Further, activation of S6K by mTORC1 also affects translation initiation by: (1) increasing eIF4A availability through phosphorylation and degradation of a negative repressor, PDCD4, and (2) phosphorylating eIF4B which stimulates eIF4A helicase activity and promotes initiation complex formation [
37,
38]. Interestingly, a recent study reported that mTORC1-dependent translation is high in human pluripotent stem cells and is suppressed during neural differentiation [
39]. Moreover, numerous changes in mRNA translation without corresponding changes in mRNA levels have been observed across human neuronal development, highlighting the importance of translational control for developing neurons [
39].
Our comparisons of transcriptomes between isogenic NPCs revealed a quantitative genotype-dependent response whereby genes upregulated/downregulated in
TSC1-heterozygous NPCs were further increased/decreased in
TSC1-null cells when compared to genetically matched CRISPR-corrected WT cells. Interestingly, this included genes linked to ASD, epilepsy and ID [
24]. However, despite alterations in mRNA translation being a major mechanism modulating gene expression downstream of mTOR in cancer cells [
40], translatome studies are lacking in TSC stem cell models. Recent studies have documented that early neurodevelopmental events, such as NPC proliferation, neurite outgrowth and migration, that precede synaptogenesis also play a role in disease pathogenesis of ASD and other neuropsychiatric disorders [
41‐
47]. The enhanced proliferation and neurite outgrowth consistently observed in
TSC1-null NPCs when compared with isogenic WT controls suggest that this may underlie early neurodevelopmental defects in TSC [
24].
Using our isogenic NPC model generated from TSC1 patient-derived iPSCs, we identified TSC1-sensitive mRNA levels and translation. Strikingly, TSC1-sensitive mRNA translation observed in NPCs was recapitulated in human ASD brain samples from the Brodmann area 19 when contrasted to controls. Furthermore, although polysome profiling revealed a partial reversal of TSC1-sensitive translation upon rapamycin treatment, most genes related to neural activity/synaptic regulation or ASD showed rapamycin-insensitive translation. However, translation of a subset of rapamycin-insensitive genes could be reversed by the mTORC1-selective inhibitor RMC-6272, which more efficiently suppresses 4E-BP1-phosphorylation in NPCs when compared to rapamycin [
18]. This was accompanied by reversal of rapamycin-insensitive phenotypes in
TSC1-null NPCs, suggesting that more efficient targeting of mTORC1 may be an attractive treatment strategy in ASD.
Discussion
It is well established that loss of TSC1 or TSC2 results in activation of mTORC1 signaling, which has led to FDA approval for treatment of TSC-associated tumors with first-generation mTORC1 inhibitors such as everolimus/RAD-001. However, rapalogs have not been very effective for treating TSC-associated neuropsychiatric defects and autism [
10,
11]. The mTORC1 signaling pathway plays a critical role in protein synthesis in normal cells including stem cells, and in human disease through regulation of translation initiation (reviewed in [
69,
70]).The mTORC1/eIF4F axis is therefore critical in shaping the proteome. Although transcriptome-wide studies of TSC-associated mRNA translation have been performed in mouse embryonic fibroblasts [
71], the effects of TSC-loss in patient-derived NPCs have not been assessed. Here, we sought to bridge this gap in knowledge.
Here, we, for the first time, reveal the complex pattern of gene expression alterations downstream of TSC1 loss in patient-derived NPCs encompassing both changes in mRNA abundance as well as numerous alterations in translational efficiencies. Interestingly, TSC1-dependent alterations in mRNA translation observed in NPCs were largely recapitulated in human ASD brains. In addition, our study of TSC1-associated gene expression also indicated ample translational offsetting, which denotes a poorly characterized gene expression mode possibly representing adaptation [
63,
64]. Although this may be of interest to fully understand how TSC1 loss reprograms gene expression, as this mode of regulation was not observed in human ASD brains, we did not study it further herein. Furthermore, although polysome profiling revealed a partial reversal of TSC1-associated gene expression alterations following rapamycin treatment, most genes related to neural activity/synaptic regulation or ASD that showed TSC1-dependent translation were rapamycin-insensitive. Bi-steric mTORC1-selective inhibitors, including RMC-6272 and its clinical counterpart RMC-5552, show strong anti-tumor activity either alone or when combined with other treatments in several preclinical cancer models. RMC-5552 also demonstrates preliminary evidence of anti-tumor activity at tolerated doses [
18,
66]. Here, we reveal that RMC-6272 is not only more potent than rapamycin in inhibiting mTORC1, but also reverses some of the translational changes not reversed by rapamycin (Fig.
5D). These findings are consistent with previous comparisons between the effects of rapamycin and the active site mTOR inhibitor PP242 on transcriptome-wide translation in cancer cells [
65]. More importantly, unlike rapamycin, RMC-6272 can rescue early neurodevelopmental phenotypes such as proliferation and neurite outgrowth in
TSC1−/− NPCs (Fig.
6 and [
24]), raising the question whether 4E-BP1-dependent translation could be essential for some of the neurodevelopmental phenotypes in TSC and other mTORC1-activated neurodevelopmental disorders.
In addition to TSC, dysregulated mTORC1 signaling is also observed in other syndromic ASDs such as Cowden syndrome/PTEN hamartoma syndrome, Fragile X syndrome, RASopathies including NF1, Angelman syndrome and Rett syndrome, as well as idiopathic ASD [
72,
73], raising the possibility that cap-dependent translation downstream of mTORC1 could play an essential role in neurodevelopmental and neuropsychiatric disorders. Many of the recent large-scale studies have focused on the transcriptome for understanding gene expression changes in the pathophysiology of ASD and other neuropsychiatric disorders [
68,
74,
75]. Defining changes in mRNA translation in neurodevelopmental and neuropsychiatric disorders remains largely unexplored and our study here describing such changes in TSC1 patient-derived neural progenitor cells will likely open avenues for correlating transcriptional alterations with changes in mRNA translation in ASD and other neurodevelopmental disorders with dysregulated mTORC1 signaling.
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