Aurea Mediocritas: mTOR Abides the Golden Mean

Abstract

Dysregulation of mTOR signalling is a converging mechanism in lissencephaly

Zhang C, Liang D, Ercan-Sencicek AG, Bulut AS, Cortes J, Cheng IQ, Henegariu O, Nishimura S, Wang X, Peksen AB, Takeo Y, Caglar C, Lam TT, Koroglu MN, Narayanan A, Lopez-Giraldez F, Miyagishima DF, Mishra-Gorur K, Barak T, Yasuno K, Erson-Omay EZ, Yalcinkaya C, Wang G, Mane S, Kaymakcalan H, Guzel A, Caglayan AO, Tuysuz B, Sestan N, Gunel M, Louvi A, Bilguvar K (2025). Nature. 2025;638(8049):172-181. doi: 10.1038/s41586-024-08341-9. PMID: 39743596

Cerebral cortex development in humans is a highly complex and orchestrated process that is under tight genetic regulation. Rare mutations that alter gene expression or function can disrupt the structure of the cerebral cortex, resulting in a range of neurological conditions. Lissencephaly ('smooth brain”) spectrum disorders comprise a group of rare, genetically heterogeneous congenital brain malformations commonly associated with epilepsy and intellectual disability. However, the molecular mechanisms underlying disease pathogenesis remain unknown. Here we establish hypoactivity of the mTOR pathway as a clinically relevant molecular mechanism in lissencephaly spectrum disorders. We characterized 2 types of cerebral organoid derived from individuals with genetically distinct lissencephalies with a recessive mutation in p53-induced death domain protein 1 (PIDD1) or a heterozygous chromosome 17p13.3 microdeletion leading to Miller–Dieker lissencephaly syndrome (MDLS). PIDD1-mutant organoids and MDLS organoids recapitulated the thickened cortex typical of human lissencephaly and demonstrated dysregulation of protein translation, metabolism and the mTOR pathway. A brain-selective activator of mTOR complex 1 prevented and reversed cellular and molecular defects in the lissencephaly organoids. Our findings show that a converging molecular mechanism contributes to 2 genetically distinct lissencephaly spectrum disorders.

Commentary

Hyperactivating mutations in more than a dozen genes in the mTOR signaling cascade have emerged as a key cause of childhood focal epilepsy, leading to the recognition of mTORopathies as a new disease class. This class is comprised of numerous syndromes, including hemimegalencephaly, tuberous sclerosis complex and focal cortical dysplasia type II. Disease-causing mutations consistently increase mTOR signaling and can lead to benign tumors, brain overgrowth, cortical malformations, and neuronal hypertrophy, depending on the disorder and mutated gene. Clinically, patients often develop intellectual disability and focal epilepsy.

A unifying feature of mTORopathies is hyperactivation of the mTOR pathway, but is it possible that hypoactivation of the pathway is associated with a distinct class of neurological conditions? Intriguingly, a recent study by Zhang et al suggests that the answer is yes. ¹ The team used whole-exome sequencing to identify a recessive mutation in p53-induced death domain protein 1 (PIDD1) in 3 families with lissencephaly, following up on prior work that implicated the gene.^2,3 Symptoms include pachygyria, intellectual disability and epilepsy. As its name implies, PIDD1 is involved in cell cycle regulation and can eliminate unnecessary or pathogenic cells by activating caspase-2. ⁴ To characterize the patient mutations, Zhang et al created induced pluripotent stem cells (iPSCs) from an affected individual and used them to generate dorsal forebrain cerebral organoids. Organoid experiments were appropriately controlled, including an isogenic “rescue” cell line. In PIDD1-mutant organoids, ventricular zone-like progenitors exhibited reduced apoptosis, consistent with PIDD1's role in regulating cell death, and increased asymmetric divisions, suggestive of premature loss of pluripotency. The PIDD1-mutant organoids also had reduced numbers of outer radial glial (oRG) cells, a progenitor cell type linked to cortical expansion in humans. ⁵ Single-cell RNA sequencing analyses revealed dysregulation of mTOR pathway genes in PIDD1-mutant organoids, and proteomic analyses confirmed downregulation of mTOR signaling. Phosphorylated S6 (pS6) immunostaining, a marker of mTOR activation, was also reduced in PIDD1-mutant organoids. Accordingly, treatment of PIDD1-mutant organoids with NV-5138, an mTORC1 activator, enhanced pS6 levels, increased the number of oRG cells and reduced lamination defects in the organoids. Organoid results were replicated in a Miller–Dieker lissencephaly syndrome (MDLS) iPSC line with a microdeletion at chromosome 17p13.3. PIDD1 is on chromosome 11, so results in the MDLS line imply that mTOR hypoactivation is common to at least 2 distinct lissencephaly-causing mutations.

Zhang's findings align well with the known functions of mTOR in promoting cell growth. While hyperactivating mutations in mTORopathies are associated with brain overgrowth, lissencephaly is characterized by brain undergrowth. The reduction in oRG cells in PIDD1-mutant organoids is intriguing, as outer radial glia exhibit high levels of mTOR signaling, and disruption of this progenitor population is hypothesized to contribute to cortical malformations in mTORopathies. ⁶ Indeed, both hypoactivation and hyperactivation of mTOR alter the orientation of radial fibers, producing migratory deficits in oRG cells in organoids. ⁶ Thus, reduced mTOR signaling in oRG cells might impair the proliferative potential of this critical population and influence cortical migration and patterning. These data suggest that disruption of a delicate balance of mTOR signaling during development with either hypo- or hyperactivation could account for common symptoms between lissencephaly and mTORopathies, such as lamination defects and seizures. In future studies, it will be interesting to establish effects that are unique to decreased or increased signaling.

In contrast to mTORopathies, if mTOR hypoactivation is a key mechanism of lissencephaly associated with multiple genetic insults, the link must be more circuitous. Exactly how PIDD1 would act to reduce mTOR signaling is not clear, though mTOR regulation by growth hormones, metabolic factors and extensive crosstalk with other cell signaling pathways could create abundant opportunities for a connection. Alternatively, mTOR downregulation in PIDD1-mutant organoids could be a secondary effect. If mTOR pathway activation is high in oRG cells, for example, a reduction in their number (by any mechanism) would presumably produce a net reduction in mTOR signaling. Rescue experiments with the mTOR agonist favor a causal role, but more data is needed to demonstrate mTOR downregulation in human lissencephaly samples and to confirm agonist efficacy in animal models, a necessary step to extend results beyond the organoid system. While agonist treatment of organoids was able to normalize production of specific cortical cell types, studies in animal models will be needed to establish whether these neurons integrate and function correctly. The safety of mTOR agonists also needs to be validated in vivo, particularly given that increasing mTOR signaling, as exemplified by mTORopathies, has its own negative effects.

These findings raise the broader question of whether mTOR hypoactivation might be a common mediator in lissencephaly. Mechanisms of lissencephaly are heterogeneous, and include environmental factors (eg, maternal infection) and mutations in several other genes (eg, LIS1, ARX, DCX, RELN, and others). ⁷ Like PIDD1, exactly how other lissencephaly-causing genes might interact with the mTOR signaling cascade is uncertain. Given that blocking mTOR signaling produces early lethality in mouse models,^8,9 it seems more likely that these lissencephaly mutations may modulate (rather than block) mTOR function as a common disease mechanism. Additional studies in patient tissues and animal models involving other lissencephaly genes are needed to establish whether mTOR hypoactivation is a common feature of the condition or is limited to a subset of mutations.

Another area that may be worth exploring is whether, like mTORopathies, somatic mutations leading to mTOR hypoactivation could contribute to lissencephaly or related neurological diseases. Akin to the use of conditional knockouts to overcome embryonic lethality from loss of critical genes, somatic mutations restrict mutation-carrying cells to specific cell lineages, mitigating pathologies that would be lethal as germline mutations. A consequence of a somatic mechanism, however, is that the number, tissue distribution and cellular identity of mutation-carrying cells can vary dramatically among patients, depending on the development timing and potency of the affected progenitor cell. As mutations are mosaic, identifying causal mutations is particularly challenging, requiring deep-sequencing of samples from the lesion itself (eg, brain), as has been necessary to identify mTORopathies. ¹⁰ Nonetheless, this work has been incredibly instructive and raises the question as to whether somatic mTOR hypoactivating mutations contribute to lissencephaly and might also account for some subset of currently unsolved malformations. Taken together, the present work by Zhang et al implicating mTOR dysregulation in lissencephaly provides new, proof-of-principle evidence for a novel mechanism of cortical malformation, suggesting that mTORopathies may constitute a disease class in which deviation from the golden mean in either direction produces cortical malformations.