Curing a Developmental Disorder: The mTOR Pathway and TSC
The mammalian target of rapamycin (mTOR) is a protein kinase that forms two complexes, mTORC1 and mTORC2. MTORC1 integrates extracellular signals to regulate cell growth, protein translation, and autophagy. Dysregulation of mTORC1 is central to a group of neurological disorders, termed mTOR-opathies, which present with epilepsy, autism spectrum disorders (ASDs), and intellectual disability. These disorders share neuroanatomical anomalies, including cortical mislamination, neuronal dysmorphia, and cytomegally, all hallmarks of aberrant mTORC1 activity.
Tuberous Sclerosis Complex (TSC) is the prototype mTOR-opathy. TSC is a disorder caused by inactivating mutations in TSC1/TSC2. We have nearly 20 years of experience focused on understanding how unrestrained mTOR signaling leads to brain abnormalities in TSC. We use mouse models generated by in utero or neonatal electroporation of CRE plasmids into neural stem cells (NSCs) with conditional TSC alleles to test how loss of alleles of TSC1 or TSC2 results in pathogenesis. This work has provided insights into TSC’s cellular pathology, revealing that affected neurons exhibit cytomegaly, hypertrophic dendrites, and disrupted migration
Transcriptomic analyses, including RNA sequencing, translational profiling, and single-cell RNA sequencing, revealed disrupted protein translation in our TSC mouse models. We have also performed single-nucleus RNA sequencing on TSC mouse models and on TSC patient subependymal giant cell astrocytomas (SEGAs) to uncover the cellular and molecular mechanisms involved in the generation of these brain growths. Pharmacological studies from our laboratory have also demonstrated that we can rescue neuronal phenotypes and reduce SEGA burden in a mouse model of TSC.
This work has resulted in new methods to explore the mechanistic underpinnings of neurodevelopmental disorders but more importantly has made several theoretical contributions to science. The following are a few examples:
1. The timing of somatic loss of heterozygosity of TSC1 or TSC2 within different pools of neural stem cells (NSCs) causes different anatomical defects and neurological manifestations.Tsc1/2 functions are highly evolutionarily conserved in a wide range of NSCs.
2. Mutations of other genes encoding components of the mTORC1 pathway may cause neurological manifestations. For example, Aidan Sokolov, a graduate student in the laboratory discovered that excessive Rheb expression in cortical neurons, which stimulates mTORC1, can phenocopy mutations in TSC1 or TSC2. Indeed, this observation coincided with the discovery of patients with RHEB mutations having cortical malformations. He also demonstrated that loss of the amino acid transporter Slc7a5 results in loss of dendrites and neuronal death. This discovery happened around the same time that patients with Slc7a5 mutations that had microcephaly were discovered.
3. Loss of TSC genes may cause aberrant differentiation. For example, as striatal astrocytes are produced by NSCs, graduate student Victoria Riley demonstrated that the astrocytes continue to translate stem-like proteins which is associated with ectopic striatal neurogenesis.
4. Cortical tubers and subependymal giant cell astrocytomas contain mutant neurons that appear to be the primary cell type which drives pathogenesis in TSC.
Expanding mTOR Research
We have further examined the role of upstream and downstream mTOR regulators in cortical development. For instance, we demonstrated that ectopic Rheb expression induces focal cortical dysplasia-like lesions, now recognized as a rare cause of neurodevelopmental disorders. We have explored the impact of amino acid transporters, particularly Slc7a5, which was later linked to neurological disease. Our research showed that Slc7a5 is essential for dendrite maturation and neuronal survival, challenging prior assumptions that its role was restricted to the blood-brain barrier.
Our lab is currently investigating the effects of mutations in mTOR substrates, such as Unc-51-like autophagy-activating kinase 1 (Ulk1). Additionally, we are characterizing variants of unknown significance in the mTORC1 pathway, exploring how multiple seemingly benign mutations may collectively impair cortical development.
Future TSC-mTOR Research
The identification of special cell cohorts in SEGAs and additional tumors has revealed to us that regulating their signaling may be a viable target to reduce the burden of tumors. We are taking advantage of receptor signaling mechanisms to test the role of SEGA cell cohorts in regulating growth, have explored the the interactions that occur between SEGA cells and the brain, and are determining the mechanism by which these cells control tumor growth.
Do Extracellular Vesicles Regulate Brain Development?
Extracellular vesicles (EVs) are nanometer-sized particles composed of biological material, often encapsulated within lipid membranes containing proteins and RNA. These vesicles are abundant in biological fluids such as blood and cerebrospinal fluid (CSF), but their precise functions and origins remain key questions. My long-term research goal is to develop an Atlas of EV Communication in the Developing Brain, a concept I initially proposed to the Allen Institute in 2012.
Origins of My Research Interest
During my postdoctoral fellowship at Yale University, I performed in utero and neonatal electroporation by injecting plasmid DNA into the lateral ventricles of the brain. I became interested in the idea that the CSF of the ventricles might also contain endogenous molecules that control brain development. I attended a meeting on PTEN at Cold Spring Harbor Laboratory, where I first encountered extracellular vesicle biology research in the context of prostate cancer. I hypothesized that EVs could play a role in brain development and initiated studies to test this idea, starting with an analysis of EVs in the developing CSF.
Investigating EVs in Brain Development
At the time, there was significant debate about the very existence of EVs due to challenges in their isolation. Most studies focused on mRNA and proteins, though some suggested small RNAs, including miRNAs, were EV components. Given the advances in sequencing technologies, I prioritized investigating EV small RNAs. Using miRNA microarrays and qRT-PCR, I identified conserved miRNAs in rat embryonic CSF EVs and human fetal CSF EVs, discovering their role in regulating cell proliferation.
Our laboratory has analyzed CSF EVs at multiple developmental stages. By integrating size exclusion chromatography with polymeric isolation, we enhanced purification rigor and conducted miRNA microarrays and small RNA sequencing. Our findings showed that the most abundant CSF EV miRNAs remained consistent throughout life, although EV quantity decreased with age. This suggested a selective mechanism for miRNA localization to EVs. We identified a protein potentially involved in miRNA shuttling and found that choroid plexus epithelial cells, which secrete CSF, also produce EVs.
To determine additional EV sources, I proposed that neural stem cells (NSCs) in the embryonic brain’s lateral ventricles could be contributors. The Whitehall Foundation funded this hypothesis. My laboratory used CD9, a well-established EV marker, to trace EV release. Contrary to expectations, we found that postnatal NSCs in the subventricular zone (SVZ) released CD9-positive EVs. Using genetic tools, cell culture assays, and transplantation experiments, we discovered that microglia take up NSC-derived EVs, triggering NF𝑘B signaling and altering cytokine profiles, cell morphology, and chemotaxis. This ultimately suppressed SVZ NSC neurogenesis, revealing a novel neuro-immune interaction where EVs function as morphogen-associated molecular patterns (MAMPs).
Developing Tools for EV Research
To facilitate EV research, I envisioned a tool that enables cell-type-specific and temporal EV labeling, aiding the construction of an EV communication atlas. There were numerous debunked claims regarding new technologies that can magically isolate EVs. Since EV studies are predominantly in vitro due to limited in vivo tools, my lab initially employed DNA electroporation to fluorescently label EVs. However, this method had limitations in cell-type specificity and temporal control. To overcome these issues, I developed an inducible fluorescently labeled EV mouse using a Cre-recombinase system. This transgenic model enables EV imaging, flow cytometry sorting, and biochemical tracking. We validated the model through multiple approaches and are collaborating with institutions such as the Mayo Clinic, Boston Children’s Hospital, and Washington University in St. Louis to distribute this tool.
Standardizing EV isolation remains a challenge in the field. To address this, we published guidelines and a book chapter detailing rigorous and reproducible EV isolation techniques. Our transgenic inducible CD9-GFP mouse provides an additional layer of stringency for EV studies, offering an urgently needed tool for the community.
Conclusion
Our roles in the TSC and EV fields have shaped our approach to neuroscience, allowing us to apply innovative theories, transformative ideas, and rigorous methods. The privilege of contributing to the broader scientific community, from keynote lectures to collaborative research, is a testament to the collective effort that drives discovery. We remain committed to advancing our understanding of EV biology and mTOR signaling, with the ultimate goal of translating these findings into clinical applications.