A Bitopic mTORC Inhibitor Reverses Phenotypes in a Tuberous Sclerosis Complex Model

A Bitopic mTORC Inhibitor Reverses Phenotypes in a Tuberous Sclerosis Complex Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Bitopic mTORC Inhibitor Reverses Phenotypes in a Tuberous Sclerosis Complex Model Sulagna Mukherjee, Matthew Wolan, Mary Scott, Victoria Riley, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6008400/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Neural stem cells (NSCs) of the ventricular-subventricular zone (V-SVZ) generate diverse cell types including striatal glia during the neonatal period. NSC progeny uncouple stem cell-related mRNA transcripts from being translated during differentiation. We previously demonstrated that Tsc2 inactivation, which occurs in the neurodevelopmental disorder Tuberous Sclerosis Complex (TSC), prevents this from happening. Loss of Tsc2 causes hyperactivation of the protein kinase mechanistic target of rapamycin complex 1 (mTORC1), altered translation, retention of stemness in striatal glia, and the production of misplaced cytomegalic neurons having hypertrophic dendrite arbors. These phenotypes model characteristics of TSC hamartomas called subependymal giant cell astrocytomas (SEGAs). mTORC1 inhibitors called rapamycin analogs (rapalogs) are currently used to treat TSC and to assess the role of mTORC1 in regulating TSC-related phenotypes. Rapalogs are useful for treating SEGAs. However, they require lifelong application, have untoward side effects, and resistance may occur. They also incompletely inhibit mTORC1 and have limited efficacy. Rapalink-1 is a bitopic inhibitor that links rapamycin to a second-generation mTOR ATP competitive inhibitor, AZD8055. Here we explored the effect of Rapalink-1 on a TSC hamartoma model. The model is created by neonatal electroporation of mice having conditional Tsc2 genes. Prolonged Rapalink-1 treatment could be achieved with 1.5 or 3.0 mg/Kg injected intraperitoneally every five days. Rapalink-1 inhibited the mTORC1 pathway, decreased cell size, reduced neuron dendrite arbors, and reduced hamartoma size. In conclusion, these results demonstrate that cellular phenotypes in a TSC SEGA model are reversed by Rapalink-1 which may be useful to resolve TSC brain hamartomas. Biological sciences/Neuroscience/Diseases of the nervous system/Developmental disorders Biological sciences/Developmental biology/Neurogenesis/Adult neurogenesis Tsc2 Tuberous Sclerosis Complex TSC SEGA Subependymal giant cell astrocytoma Subependymal Nodule Rapalink-1 mTORC1 neurogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder affecting ~0.0166 percent of the population 1 . TSC is caused by loss of function mutations in TSC1 or TSC2 2,3 . TSC1 and TSC2 encode for the proteins hamartin and tuberin which inhibit Rheb-mTORC1 signaling 4 . TSC1/2 loss of function mutations activate the mTORC1 pathway 5–8 . mTORC1 is a protein kinase complex that promotes cell growth and proliferation 9 . TSC patients have too much mTORC1 activity and enlarged cells that create tumors called hamartomas within the heart, kidney, lung, skin, and brain 10,11 . Identifying the mechanisms that cause hamartomas to form is important for understanding the many disorders whose genetic mutations affect molecular pathways that intersect with mTORC1 and for developing therapeutic strategies to treat TSC patients. TSC patients have brain hamartomas called subependymal nodules (SENs) that invade the striatum and subependymal zone (SEZ) 12,13 . SENs are commonly detected during childhood 3,13 . Approximately a quarter of SENs are categorized as subependymal giant cell astrocytomas (SEGAs) 12–14 . SENs may transition into SEGAs 15 . SENs and SEGAs share all histopathological features 16 . Although there is no consensus, the criterion of SEGA diagnosis is >0.5-1.0 cm in size or serial growth 17 . While SEGAs occur throughout the ventricular system, cerebrospinal fluid (CSF) circulation blockade along the caudothalamic groove can cause obstructive hydrocephalus associated with migraines, seizures, and death. Unexplained changes in neurological status or TSC-associated neuropsychiatric disorders (TANDs) can also be a sign of SEGA growth 18 . The median age of SEGA diagnosis is 1 year. Only ~2.4% of SEGAs are identified after age 40 19 . SEGAs can bleed when being removed leading to non-obstructive hydrocephalus, tissue damage, and mortality 20 . Because SEGAs occur in young children and are located deep within the brain and because surgery comes with a risk, pharmacological intervention is warranted. mTORC1 inhibitors including rapamycin analogs (rapalogs) are now the standard of care except for cases of acute hydrocephalus 21 . ~57% of SEGAs are reduced by 50% volume within two years and maintenance doses are not typically associated with changed SEGA volume 18,19,22 . Thus, SEGAs do not always respond to rapalogs. Children may poorly tolerate rapalogs, and if treatments stop, SEGAs grow back 23 . Even surgical removal of SEGAs is followed by regrowth in nearly 40% of patients. The mechanisms that account for SEGA regrowth are unclear but may be related to the fact that mTORC1 allosteric inhibitors incompletely inhibit mTORC1 phosphorylation of select substrates 24,25 . Alternatively, resistance to rapalog binding may occur 26 . A novel bisteric inhibitor linking AZD8055 to rapamycin called Rapalink-1 was generated 26 . Rapalink-1 simultaneously inhibits mTORC1 through the rapamycin moiety by targeting FK506-binding protein 12 (FKBP12) and the FKBP12 rapamycin binding domain of mTOR as well as inhibiting both mTORCs through the ATP competitive inhibitor moiety, AZD8055 26 . We created a mouse model of TSC SEGAs by electroporating CRE recombinase into NSCs of mice having conditional Tsc2 genes to provide mechanistic insight into SEGA pathogenesis 27,28 . Mice developed hamartomas with SEN-like lesions that develop into SEGA-like hamartomas. We previously found that TSC mutant NSC translational programs were altered and prevented differentiation leading to the aberrant production of neurons in the striatum. These lesions were associated with ensembles of cytomegalic neurons and giant cells. We performed single nuclei RNA sequencing (snRNA-Seq) of these mice and discovered altered NSC transitional states caused by loss of Tsc2 . Moreover, neurons were a core feature in this model and had altered transcriptomes. The extent to which mTOR activity might cause these phenotypes was not assessed. What follows are the results of a study that utilizes the bisteric inhibitor Rapalink-1 on a TSC model of striatal hamartomas representing SEGA-like lesions. Results Rapalink-1 Treatment of a TSC Mouse Model. Tsc2 wt/wt and Tsc2 f/f x RFP neonatal mice were electroporated with Cre recombinase and GFP encoding DNA plasmids (Fig. 1 A). Electroporation allows for the targeting of lateral V-SVZ NSCs that generate striatal glia (Fig. 1 B). This causes recombination leading to deletion of exons 2–4 of Tsc2 and red fluorescence along the lateral ventricles that appear as SEGA-like hamartomas in postnatal day (P) 30 Tsc2 f/f mice (Fig. 1 C, D). Additionally, V-SVZ NSCs generate granule cell neurons that migrate into the olfactory bulb (OB) and are fully mature 30 days later. We previously reported hamartomas at P30 having elevated mTORC1 activity assessed by p4E-BP in glial cells 27 . We also reported increased mTORC1 as assessed by pS6 staining in neurons produced from V-SVZ NSCs 29 . We further reported measurements of P60 hamartoma and OB neuron growth. Thus, these time points helped to guide the design of our experiments. Indeed, in comparison to Tsc2 wt/wt NSCs (Fig. 1 E-G) that produced mostly glia, there were numerous Tsc2 mut/mut pS6 positive neurons and giant cells near the striatal border with the V-SVZ (Fig. 1 H-M). As confirmation of Tsc2 recombination, there was a 23.3% increase in pS6 in mutant RFP positive neurons ( Tsc2 wt/wt 1.000 ± 0.03042, n = 110 vs. Tsc2 mut/mut 1.233 ± 0.04392, n = 110) ( Supplemental Fig. 1A ). CRE electroporated V-SVZs were collected and NSCs cultured ( Supplemental Fig. 1B ). We verified high rates of Tsc2 recombination in NSCs ( Supplemental Fig. 1C, D ). We further verified that phosphorylation of the mTORC1 substrate Ulk1 was rapamycin resistant but sensitive to ATP-competitive mTORC inhibitors ( Supplemental Fig. 1E, F ). These results further confirm that CRE-mediated recombination occurs in the TSC model and that some mTORC1 substrates are resistant to rapamycin. Tsc2 f/f x RFP neonatal mice were subsequently randomized, assigned a unique identification number, and treated with Rapalink-1 or DMSO (vehicle) for 30 days or until sacrificed (Fig. 2 A). Animals were euthanized by intraperitoneal injection of euthasol or CO 2 inhalation followed by swift decapitation. No mice in the control group died and no significant changes in behavior or signs of distress were noted (Fig. 2 B). Cohorts of mice were given 1.5 mg/kg or 3.0 mg/kg Rapalink-1 once every five days. 3.0 mg/kg Raplink-1 treated mice survived on average 88.2 days (N = 8) (Fig. 2 B). Likewise, 1.5 mg/kg Rapalink-1 was well tolerated (N = 5) with all mice surviving 90 days. These doses were well tolerated with death seen at 3.0 mg/kg and having slightly lower weights at the end of treatment (Fig. 2 C). Rapalink-1 inhibits mTORC1 activity. mTORC1 activity and substrate phosphorylation in the brain is cell type dependent 29 – 31 . We noted that pS6 levels were high within RFP positive Tsc2 null cells having neuron-like morphologies (Fig. 3 A-F) 27 . pS6 was analyzed in vehicle, Rapalink-1 (1.5 mg/kg), or Rapalink-1 (3.0 mg/kg) Tsc2 mutant neurons (Fig. 3 G-L). We found that Rapalink-1 decreased pS6 (Vehcile = 1.066 ± 0.029, n = 857 vs. Rapalink-1 1.5 mg/kg = 0.9127 ± 0.065, n = 218 vs. Rapalink-1 3.0 mg/kg = 0.835 ± 0.029 n = 452, p < 0.0001) (Fig. 3 M). We confirmed the extent that mTORC1 signaling was reduced by using acute daily Rapalink-1 treatment for five days. Rapalink-1 has been used at higher doses for acute in vivo experiments 32 . We therefore tested acute efficacy of Rapalink-1 at a higher dose (6 mg/kg) but less than 10 mg/kg, which is the dose of rapamycin used to compare the two drugs 26 . Rapalink-1 reduced mTORC1 signaling as detected by examining p4EBP and pS6 staining in the brain and by immunoblotting for pS6 (Vehicle = 1.000 ± 0.04189, N = 3 vs Rapalink-1 = 0.7707 ± 0.02518, N = 3; P = 0.0094) ( Supplemental Fig. 2 ). Rapalink-1 inhibition of mTORC2 was confirmed by a reduction in pAKT as previously reported ( Supplemental Fig. 2 ). These results confirm that Rapalink-1 can indeed reduce mTOR signaling in vivo . Rapalink-1 reduces Tsc2 mutant neuron cell size. mTOR regulates OB granule cell soma growth 33 . Loss of mTOR and inhibition of mTORC1 with rapamycin decreases granule cell soma size 33 . Conversely, loss of Tsc2 increases granule cell and striatal neuron soma size 27 , 29 . We wondered to what extent soma size might be decreased in Tsc2 mutant neurons following Rapalink-1 treatment. Rapalink-1 low dose (1.5 mg/kg) and Rapalink-1 high dose (3.0 mg/kg) decreased the average soma size of mutant Tsc2 neurons (Vehicle = 1.00 ± 0.016 vs. Rapalink-1 1.5 mg/kg = 0.7463 ± 0.01 vs. Rapalink-1 3.0 mg/kg = 0.689 ± 0.015, p < 0.0001) (Fig. 4 A-H, K). We documented a population of cells appearing as giant cells having high mTORC1 activity even after 3.0 mg/kg Rapalink-1 treatment (Fig. 4 I, J). However, the relative proportion of cells that were classified as giant cells in relation to the total cell number per section, was reduced in the 3.0 mg/kg Rapalink-1 condition (Vehicle = 0.0451 ± 0.0044 vs. Rapalink-1 1.5 mg/kg = 0.0415 ± 0.0125 vs. Rapalink-1 3.0 mg/kg = 0.0274 ± 0.03623, p < 0.01). Taken together, Rapalink-1 appears to decrease the average size of Tsc2 mutant neurons. Rapalink-1 Treatment Decreases Neuron Dendrite Arbors. We previously demonstrated that loss of Tsc1, Tsc2 , or increasing Rheb, increased dendrite arbors of OB granule cell neurons produced from V-SVZ NSCs 29 , 34 , 35 . This is likely because mTOR and mTOR complex components raptor and rictor regulate granule cell dendrite arbors 33 . Neurons present in the striatum of the SEGA model also have more dendrites than wild type or Tsc2 mutant OB granule cell neurons 27 . These mutant neurons are also larger and have greater dendrite complexity in comparison to any control wild-type neurons found in the striatum. Gross reductions in dendrite arbors caused by Rapalink-1 were noted at low magnifications (Fig. 5 A, B). To confirm and quantify this observation, neurons from control and Rapalink-1 treated mice were traced (Fig. 5 C, D). Rapalink-1 substantially reduced the length and complexity of Tsc2 mut/mut neuron dendrites (Vehicle = 713.0 ± 74.53, n = 24 vs. Rapalink-1 1.5 mg/kg = 563.8 ± 46.64, n = 24 vs. Rapalink-1 3.0 mg/kg 465.5 ± 33.85, n = 50; p = 0.008) (Fig. 5 E, F). Thus, long-term Rapalink-1 treatment significantly reduces dendrite arbors in Tsc2 mutant neurons. Efficacy of Rapalink-1 for the Treatment of TSC Hamartomas. The final determinant of the efficacy of Rapalink-1 was the average lesion size at P90. Striatal hamartomas were measured by tracing lesions we previously described 27 . We noted that size continues to increase notably at P90 with some hamartomas appearing aggressive and invading different regions reaching 10 times the size of those measured at P60 (Fig. 1 , 6 ). In comparison, 1.5 mg/kg Rapalink-1 treated mice had hamartomas that were considerably (~ 68.6%) smaller and 3.0 mg/kg Rapalink-1 significantly reduced hamartoma sizes by ~ half in relation to control conditions (Vehicle = 40096 ± 8461, n = 38 vs. Rapalink-1 1.5 mg/kg = 27520 ± 2243, n = 58 vs. Rapalink-1 3.0 mg/kg 19636 ± 3266, n = 33, p = 0.0363) (Fig. 6 I). Discussion SEGAs are a significant cause of morbidity in TSC patients. Rapalogs are a front-line treatment for SEGAs 36 – 39 . Here we demonstrate that likewise, Rapalink-1 is useful for reversing neural phenotypes in a TSC mouse model of SEGA-like hamartomas (Fig. 6 J). Although, ~ 57% of TSC SEGAs are reduced by 50% within two years, continued treatment does not further reduce SEGA volume 18 , 19 , 22 . Protracted use of Rapalogs is also necessary because stopping treatment is associated with SEGA recurrence. Considering the overall moderate efficacy of Rapalogs and side effects, especially for children, warrants that new medications are deployed 23 . Studies have also demonstrated the development of resistance to rapamycin through changes in the FKB12 rapamycin binding domain of mTOR 26 . Finally, rapamycin does not fully inhibit mTORC1 phosphorylation of several substrates 25 . There is an urgent need to test new therapies that may overcome these issues. Rapalink-1 is an excellent candidate because it fulfills the need to completely inhibit mTORC1 phosphorylation of rapamycin-resistant substrates and simultaneously inhibits mTORC2 and overcomes issues with resistance 26 . It is therefore conceivable that Rapalink-1 might be useful to treat TSC. To fulfill the goal of examining additional TSC therapies, we tested Rapalink-1 in a mouse model of TSC SEGA-like hamartomas. The mechanisms that account for SEGA growth are unclear in patients and this may be related to the fact that we have little understanding of the cellular composition, regional differences in SEGAs, and whether molecular mechanisms involved in the different regions and timing of their appearance may differ. The fact that the composition of SEGAs has been debated for nearly a century is evidence that experiments must carefully examine the effect drugs have not just on SEGA size, but also on the different cell types within SEGAs 40 , 41 . SEGAs also appear to change over time. Imaging and RNA sequencing experiments suggest a stepwise change in composition with SEGAs eventually containing neurons and cytomegalic giant cells 42 – 48 . Gliotic scarring, calcification, and immune cell invasion also occur 48 . This is similar to events in TSC patient cortical tubers 49 . Although SEGAs do not frequently directly cause seizures, the presence of mutant neurons and giant cells warrants closer inspection as does determining their contributions to patient presentation. Loss of Tsc2 in V-SVZ neuroprogenitors results in the aberrant transition from quiescent to active states and during differentiation 30 , 31 , 50 , 51 . Loss of Tsc2 prevents the further downregulation of mTORC1 activity in a subset of cells within the striatum leading to growths including nodular hamartomas containing cells with heterogenous morphologies. The hamartomas are characterized by abnormal heterotopic clusters of morphologically heterogenous cells which include neurons both within and outside of the growths. Thus, the TSC SEGA-like hamartoma mouse model allowed us to examine the utility of Rapalink-1 treatment in controlling neural phenotypes and hamartoma size. Rapalink-1 intermittent dosing (1.5 mg/kg and 3.0 mg/kg every five days) was generally tolerated during the study, but preliminary experiments demonstrated acute toxicity during daily treatment (data not shown). As predicted, Rapalink-1 reduced mTORC1 activity. However, it was reduced by only ~ 15–23% as measured using pS6 as a readout and pS6 remained apparent throughout the brain. Thus, these doses might incompletely inhibit mTORC1. Indeed, a higher dose (6 mg/kg) was also tested based on efficacy in glioma and appeared more effective by immunohistochemistry and western blot but was associated with toxicity 32 . It is unclear whether extended treatments of 1.5 mg/kg and 3.0 mg/kg Rapalink-1 for more than 30 days would be accompanied by additional side effects. Additionally, 1.5 and 3.0 mg/kg Rapalink-1 treated mice were sacrificed 5 days after the last injection of Rapalink-1. Thus, mTORC1 activity could have rebound effects and our data underestimate how effective Rapalink-1 is. Nevertheless, the reduction in pS6 served as an indicator that Rapalink-1 inhibited mTORC1. We reasoned that the prolonged inhibition of mTORC1 would decrease the well-documented mTORC1-regulated phenomenon, of cell growth 52 . Previous work from our laboratory and our colleagues have demonstrated that neonatal V-SVZ NSC generated granule cells grow when Tsc1 or Tsc2 are removed 29 , 34 . Moreover, ectopic expression of wild-type or mutant Rheb can drive cell growth in granule cells 35 , 53 . Growth of granule cells is dependent on mTOR since CRE electroporation of conditional mTOR reduces soma size 33 . Moreover, rapamycin treatment reduces granule cell soma size supporting the importance of mTORC1 in this process 33 . As expected, Rapalink-1 at both doses reduced Tsc2 mutant neuron cell size by ~ 25–31%. These results further support that Rapalink-1 is likely inhibiting mTORC1-dependent cellular events. While the role of striatal neurons in the hamartoma SEGA-like model is unclear, ectopic neurons can affect a wide range of other cell types. Cortical tubers and focal malformations of cortical development appear to undergo analogous changes including astrogliosis and microglia activation 11 . And this is directly linked to neuron hyperexcitability. Thus, a major goal for TSC and related disorder research has been to reduce dendrite growth in the hopes of modulating neuronal activity. mTOR, through mTORC1 and mTORC2 regulates OB granule cell dendrite arbors 33 . While cytoskeleton regulation is most often attributed to mTORC2, rapamycin and mTORC1 have been carefully studied in relation to dendrite growth. For example, like cell growth, loss of Tsc1 and Tsc2 as well as increased Rheb activity promote dendrite growth 29 , 34 , 35 , 53 . We found that Rapalink-1 similarly reduced the dendrites of striatal Tsc2 mutant neurons. Thus, Rapalink-1, could be advantageous in that it binds both arms of mTOR signaling 32 . This is particularly important in that several groups have posited that additional pathways may regulate SEGA growth. For example, one group simultaneously removed Pten and Tsc1 in postnatal V-SVZ NSCs 16 . This resulted in the generation of SEGAs that recapitulated most aspects of those in patients. Pten/Tsc1 mutant NSCs were subsequently injected subcutaneously and generated tumors. The Pten / Tsc1 mutant NSCs had altered Erk and Akt activity too. Knockdown of the mTORC2 component rictor or combined rapamycin and PI3K-mTOR inhibition reduced tumor growth 16 . Thus, inhibitors that act on both mTORC1 and mTORC2 such as Rapalink-1 have several advantages. In line with these results, we found that Rapalink-1 produced a moderate but significant effect on the average size of SEGAs. The effect of Rapalink-1 was disproportionate to that seen on cell size or dendrites. Whether the effectiveness of Rapalink-1 is due to the effect on neuron activity or on other cell types is unclear and will require additional future experiments. A limitation of inhibiting mTORC2 is that this could lead to additional side effects not seen with rapalogs. Taken together, this study provides in vivo evidence for the utility of Rapalink-1 to control mTOR, cell size, dendrite hypertrophy, and SEGA-like hamartoma growth which may be of clinical importance. Declarations Acknowledgements This work was supported by the United States of America Department of Defense U.S. Army Medical Research Activity Award Congressionally Directed Medical Research Program Tuberous Sclerosis Complex Research Program W81XWH2010447. We acknowledge Anthony J. Minerva and Melanie Garcia for technical assistance. Author Contributions Conceptualization, DF; Methodology, SM, DF, MW, MS, AS, VR; Validation, DF, VR, MW, MS, AS; Formal Analysis, SM, MW, MS, AS, DF; Investigation, DF, SM, VR, MW, MS, AS; Resources, DF; Data Curation, DF, MW, MS; Writing-Original Draft, DF; Writing-Reviewing and Editing; DF, SM, VR, MW, MS, AS; Visualization, SM, DF; Supervision, DF; Project Administration, DF; Funding Acquisition, DF Declaration of Interests The authors declare that the researchers have no competing financial interests. Funding DMF is supported by United States of America Department of Defense U.S. Army Medical Research Activity Award Congressionally Directed Medical Research Program Tuberous Sclerosis Complex Research Program W81XWH2010447. Methods EXPERIMENTAL MODEL AND SUBJECT DETAILS; Animals. All experiments were approved by the Clemson University Institutional Animal Care and Use Committee and the Animal Care and Use Review Office (ACURO), a component of the USAMRDC Office of Research Protections (ORP) within the Department of Defense (DoD). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. Red fluorescent protein (RFP +/- , +/+ ) (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato) Hze/J ) (Strain #007909, RRID:IMSR_JAX:007909), and Tsc2 tm1.1Mjg/J (Strain #027458, RRID:IMSR_JAX:027458) were acquired from Jackson Laboratories 54 . Pharmacological treatment of cultured NSCs is on CD1 mice. Sentinel mice were free of pathogens throughout the study. Samples/subjects were allocated randomly to experimental group. Experimental manipulations were performed on mouse pups that were not involved in previous procedures and sacrificed accordingly. Both sexes were used and ages are as indicated in figures. Mice were housed under standard pathogen-free conditions in cages on racks within isolated cubicles with a 12-h light/dark cycle and fed ad libitum . Mice were injected intraperitoneally using the indicated doses and schedule. Drugs were prepared as previously described in DMSO, PEG-300, and PBS 32 . Injections were performed in biological safety cabinets. Mice were weighed 5-7 days each week and weights recorded. Animals were euthanized by intraperitoneal injection of euthasol or CO 2 inhalation followed by swift decapitation. Electroporation. B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato) Hze/J ) x Tsc2 tm1.1Mjg/J mouse pups were electroporated as previously described 55,56 . Mice were injected with equal concentrations and volumes of DNA plasmids diluted in phosphate buffered saline (PBS) with 0.1% fast green. CAG-CRE (Plasmid #13775, Addgene) and CAG-GFP (Plasmid #11150, Addgene) plasmids were used 57,58 . A borosilicate glass micropipette generated from pulled capillary tubes was loaded with DNA and injected into the lateral ventricles. Square pulse generation was performed using a pulse generator (ECM830; BTX) and tweezer electrodes (model 520; BTX) with five, 100-volt square pulses of 50 ms duration with 950-ms intervals. Polymerase Chain Reaction (PCR). Toe or tail snips were subject to modified hotshot DNA extraction and genotyped by PCR using Invitrogen TM Platinum TM Taq polymerase, mixed with nuclease-free water, magnesium free PCR buffer, MgCl 2 , dNTP mix with primers and DNA according to the manufacturers’ protocol (Invitrogen). Primers for Tsc2 were 5’-ACAATGGGAGGCACATTACC-3’ and 5-AAGCAGCAGGTCTGCAGTG-3’ and for Tomato (RFP) 5’-AAGGGAGCTGCAGTGGAGTA-3’ and 5’-CCGAAAATCTGTGGGAAGTC-3’ and 5’-GGCATTAAAGCAGCGTATCC-3’ and 5’-CTGTTCCTGTACGGCATGG-3’. Amplicons were loaded onto agarose gels with 1X Blue Juice and ran at 100 V for 20–30 mins and visualized on a BioRad Chemidoc MP. Immunohistochemistry. Brains were removed in room temperature PBS, transferred to 4% paraformaldehyde in PBS, and incubated overnight at 4°C. Brains were rinsed in PBS and mounted in 3% agarose. A Leica VTS 1000 vibratome was used to section brains coronally. Sections were blocked in 0.1% Triton X-100, 0.1% Tween-20 and 2% BSA in PBS for 1 hr at room temperature. Sections were washed in 0.1% Tween-20 in PBS. Sections were incubated in primary antibody, anti-pS6 (1:500; Cell Signaling Technology; Ser 240/244, 61H9, #4838), in 0.1% Tween-20 and 2% BSA in PBS overnight at 4°C. Sections were subjected to three additional washes in PBS containing 0.1% Tween-20. Sections were incubated with the appropriate secondary antibody (Alexa Fluor series; 1:500; Invitrogen) in 0.1% Tween-20 and 2% BSA in PBS overnight at 4°C. Sections were mounted in ProLong Antifade Mountant (ThermoFisher). Images were acquired on a spectral confocal microscope (Leica SPE) with a ×20 dry objective (N.A. 0.75). Low-magnification images were acquired with ×10 dry or a ×5 dry (N.A. 0.15) objective. Image Analysis. Images (×20) of RFP positive cells were uploaded to FIJI (ImageJ 1.5 g) and analyzed as described elsewhere 27 . The freehand selection tool was used to trace electroporated and non-electroporated cell somas in the same Z section and mean gray values for pS6 were quantified. Ratios of electroporated and non-electroporated cells were compared for RFP positive cells in Tsc2 wt/wt and Tsc2 mut/mut conditions to account for immunohistochemical variation. Soma size was simultaneously recorded for traced cells. The RFP positive hamartoma perimeter was outlined in each Z section by hand by scrolling through individual Z sections and hamartomas were subsequently traced in (×20) images. The freehand selection tool was used to trace RFP positive lesions. Images (×20) were used to measure dendrite morphology. Dendrites were traced using the simple neurite tracer plug-in. Sholl analysis was performed at 1 µm intervals to quantify dendrite arborization using the Sholl plug-in. The total number of dendritic crossings was calculated by taking the sum of crossings at all intervals for each traced neuron and averaging the total number of crossings per neuron in each condition. Western Blot. Tissue (0.1 gram) was harvested and finely minced in 2% SDS, Protease Inhibitor Cocktail (Pierce) and Phosphatase Inhibitor Cocktail, in RIPA buffer. Samples were briefly sonicated at maximum settings (100 Amplitude, QSonica) for 10 seconds three times with 30 second resting intervals. Lysate was transferred on ice to a fresh reaction tube and centrifuged at 15,000 rpm for 15 min in a tabletop Eppendorf 5415 centrifuge at 4°C. Protein concentration was quantified using the Pierce MicroBCA assay. Equal protein amounts were brought up to equal volumes with lysis buffer as described above and Laemmli buffer and heated to 95°C for 5 minutes. Proteins were resolved by 10% polyacrylamide precast mini-Protean gels (BioRad) and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were rinsed in Tris-buffered saline (TBS-T, 0.1% Tween 20) for 5 min at room temperature and blocked in 5% blotting grade block (BioRad) in TBS-T for 1 h at room temperature. Membranes were incubated for 1 h at room temperature or overnight at 4°C with the following antibodies from Cell Signaling Technology at a 1:1,000 dilution: phospho-RPS6 (D68F8, Cat# 5364) and RPS6 (5G10, Cat# 2217). Membranes were rinsed three times each for 10 min in TBS-T. Membranes were incubated for 1 h at room temperature with donkey or goat anti-rabbit antibodies in blocking buffer. Membranes were washed for 15 minutes in TBS-T and visualized using a Bio-Rad Chemidoc MP imaging system using enhanced chemiluminescence reagent (Pierce). PVDF membranes were stripped for at room temperature using Restore Western Blot Stripping Buffer according to manufacturer’s recommendations (Cat# 21059, Thermo Fisher Scientific). Primary cell culture Briefly, P0-1 CD1 mice were anesthetized on ice and decapitated and brains were removed and placed in 4°C Neurobasal A media on pre-chilled petri dishes. Micro-dissected V-SVZs were placed into 0.05% trypsin with 0.02% EDTA in Neurobasal A for 7 min in a 37°C incubator. Equal volumes of defined trypsin inhibitor (1X; Life Technologies; Lot# 1837475) were added. The samples were centrifuged at 300 x g for 5 min. The pellet was re-suspended with 1 mL Neurobasal A. The pellet was triturated with three pasture pipettes having sequentially decreased bore size to dissociate tissue. Cells were centrifuged at 300 x g for 5 min and resuspended in 200 mL Neurobasal A complete Media (1X Glutamax, 50 units/mL Penicillin/streptomycin, 20 ng/mL EGF, 20 ng/mL FGF-2 and 2% B27 Supplement). Cells were placed on laminin-coated coverslips in 24 well plates in Neurobasal A complete media for pharmacological experiments performed 24 hours later or placed into 6 well plates for recombination analysis as previously described 27,29 . Samples were harvested in 2% SDS, Protease Inhibitor Cocktail (Pierce) and Phosphatase Inhibitor Cocktail, in RIPA buffer for western blot. Alternatively, samples were subjected to DNA isolation and long range PCR for Tsc2 recombination products as previously described 27,29 . Pharmacological Treatments NSCs were treated with the 10 nM Rapamycin (#9904s, Cell Signaling Technology) or 10 nM ATP-competitive inhibitor, Torin1 (#14379, Cell Signaling Technology). Torin1 from a 1mM stock was re-constituted in DMSO (MP Biomedicals). Rapamycin was re-constituted in DMSO for a 100 mM stock. Equi-molar DMSO was used for all conditions. For the primary cell starvation experiment, NSC were placed into PBS for 2 hours prior to drug treatment and placed back into complete media to activate mTORC1 for the duration of drug treatment (2 hours). QUANTIFICATION AND STATISTICAL ANALYSIS. Measurements were graphed and statistical analysis was performed with GraphPad Prism software (Version 8.2.0, GraphPad Software Inc.). Statistical significance was determined using One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test or Student’s T-test. N (number of mice) and n (number of cells or hamartomas) are listed where applicable. Figure 3 (pS6) and Figure 4 (cell size) analysis were performed on vehicle (N=6), Rapalink-1 1.5 mg/kg (N=4), and Rapalink-1 3.0 mg/kg (N=7) mice. Sholl and hamartoma analysis are reported for vehicle (N=4), Rapalink-1 1.5 mg/kg (N=4), and Rapalink-1 3.0 mg/kg (N=6) mice in figures 5 and 6. Supplemental data were performed on N=3 mice for all immunohistochemistry analysis and western blot conditions. Error bars are reported as the standard error mean. KEY RESOURCES TABLE. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit Monoclonal pS6 Ser 240/244 Cell Signaling Technology Cat#4838; RRID: AB_659977; Clone: 61H9 Rabbit Monoclonal p4EBP Thr 37/46 Cell Signaling Technology Cat#2855; RRID: AB_560835; Clone: 236B4 Rabbit Monoclonal RPS6 Cell Signaling Technology Cat#2217; RRID: AB_331355; Clone: 5G10 Chemicals, Peptides, and Recombinant Proteins Experimental Models: Organisms/Strains Mouse: RFP +/- , RFP +/+ : B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato) Hze/J Jackson Laboratories Cat#007909; RRID: IMSR_JAX:007909 Mouse: Tsc2 wt/wt , Tsc2 mut/mut : Tsc2 tm1.1Mjg/J Jackson Laboratories Cat#027458; RRID: IMSR_JAX:027458 Oligonucleotides Primers: Tsc2: 5’-ACAATGGGAGGCACATTACC-3’, 5- AAGCAGCAGGTCTGCAGTG-3’ Integrated DNA Technologies N/A Primers: RFP: 5’-AAGGGAGCTGCAGTGGAG TA-3’, 5’-CCGAAAATCTGTGGGAAG TC-3’, 5’- GGCATTAAAGCAGCGTATCC-3’ , 5’-CTGTTCCTGTACGGCATGG-3’ Integrated DNA Technologies N/A Primers: For detecting Tsc2 recombination 5’-AAGATTCCGGCTTGAAGGAG-3’, 5’-CACTA-GTCTAGCCTGACTCT-3’, and 5’-GAGGACAAGCCAACATCCAT-3’ Integrated DNA Technologies N/A Recombinant DNA Plasmid: CAG-CRE 97 Cat#13775; RRID: Addgene_13775 Plasmid: CAG-GFP 98 Cat#11150; RRID: Addgene_11150 Software and Algorithms FIJI (ImageJ 1.5g) 109 https://imagej.net/software/fiji/downloads GraphPad Prism (v. 8.2.0) GraphPad Software Inc https://www.graphpad.com/ DATA AVAILABILITY. The datasets generated and/or analysed during the current study are available in the Mendeley Data and NeuroMorpho repository. Western blot data is deposited as Feliciano, David (2025), “A Bitopic mTORC Inhibitor Reverses Phenotypes in a Tuberous Sclerosis Complex Model Westerns”, Mendeley Data, V1, doi: 10.17632/hjt9m462d3.1 (https://data.mendeley.com/datasets/hjt9m462d3/1) to Mendeley Data. Neuron traces are available at NeuroMorpho.org under “A Bitopic mTORC Inhibitor Reverses Phenotypes in a Tuberous Sclerosis Complex Model” or navigating to neuromorpho.org/dableFiles/mukherjee_feliciano/Supplementary/Mukherjee_Feliciano.zip. Declaration of Interests. The authors declare no competing interests. References Northrup, H. et al. Updated International Tuberous Sclerosis Complex Diagnostic Criteria and Surveillance and Management Recommendations. Pediatr. Neurol. 123 . (2021). 10.1016/j.pediatrneurol.2021.07.011 The European Chromosome 16 Tuberous Sclerosis Consortium. 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(2004). 10.1073/pnas.2235688100 Additional Declarations No competing interests reported. Supplementary Files AllSupplementalRevisedReduced.pdf Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 02 Jun, 2025 Reviews received at journal 23 May, 2025 Reviews received at journal 20 May, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers agreed at journal 13 May, 2025 Reviewers invited by journal 13 May, 2025 Submission checks completed at journal 13 May, 2025 First submitted to journal 12 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6008400","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":456010542,"identity":"8a5dfb21-2ed5-464c-9d7d-158f62c90b04","order_by":0,"name":"Sulagna Mukherjee","email":"","orcid":"","institution":"Clemson University","correspondingAuthor":false,"prefix":"","firstName":"Sulagna","middleName":"","lastName":"Mukherjee","suffix":""},{"id":456010543,"identity":"bce748dd-d80e-4292-90b3-b5f8592a0d10","order_by":1,"name":"Matthew Wolan","email":"","orcid":"","institution":"Clemson University","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Wolan","suffix":""},{"id":456010544,"identity":"e5bf3c38-d225-40eb-8f1e-f7f41ff1826d","order_by":2,"name":"Mary Scott","email":"","orcid":"","institution":"Clemson University","correspondingAuthor":false,"prefix":"","firstName":"Mary","middleName":"","lastName":"Scott","suffix":""},{"id":456010545,"identity":"7a102a19-6281-48fb-8fd6-04859ab4d9bf","order_by":3,"name":"Victoria Riley","email":"","orcid":"","institution":"Clemson University","correspondingAuthor":false,"prefix":"","firstName":"Victoria","middleName":"","lastName":"Riley","suffix":""},{"id":456010546,"identity":"b65cbd3f-5491-4038-ba85-5397bdef2e4e","order_by":4,"name":"Aidan Sokolov","email":"","orcid":"","institution":"Clemson University","correspondingAuthor":false,"prefix":"","firstName":"Aidan","middleName":"","lastName":"Sokolov","suffix":""},{"id":456010547,"identity":"4314ff23-4627-4794-9b4e-0d411d1f32d9","order_by":5,"name":"David Feliciano","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDCCwwcYDyQwSMgxsMOFEghoOZbAANJizMBMkhYgldhAtBa+Y8wHDjyosUjvb2Y+uvFHxR0GfvYcA7xaJI+xJRxIOCaRO+MwW9ptnjPPGCR73uDXYnC/x+BAYoNE7gZmHrPbjG2HGQxuELDF4Bj/B5CWdANm/m83fwK12BPWwsMA0pJgwMzDdoMXZIsEYb8YgPxiCPSLGdAvh3kkzjwrwKsFGGIPH/6oqZPnb29+dvNHxWE5/vbkDXi1YAAe0pSPglEwCkbBKMAKAC4lS2em8vCGAAAAAElFTkSuQmCC","orcid":"","institution":"Clemson University","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Feliciano","suffix":""}],"badges":[],"createdAt":"2025-02-11 14:53:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6008400/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6008400/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-08345-z","type":"published","date":"2025-07-01T15:58:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82758397,"identity":"1d54029d-6643-42e9-98a7-6fac0b17ce99","added_by":"auto","created_at":"2025-05-15 02:22:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":871791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRapalink-1 Treatment in a TSC Mouse Model.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eA) Schematic diagram of conditional \u003cem\u003eTsc2 \u003c/em\u003eand inducible \u003cem\u003eRFP \u003c/em\u003egenes. CRE recombinase causes \u003cem\u003eTsc2 \u003c/em\u003eexons 2-4 to recombine, and RFP to be expressed. B) Schematic diagram of CAG-CRE and CAG-GFP (Green) plasmid electroporation. Plasmid is injected intraventricularly into P0-1 mice. Electrodes placed on the head of the pup subsequently cause plasmids to be taken up by NSCs lining the lateral ventricles when an electrical field is applied. C, D) Macroscopic images of coronal sections of P60 mice following electroporation. Black indicates RFP expression whereas blue indicates RFP saturation. E-G) Images of 20× \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003ewt/wt\u003c/em\u003e\u003c/sup\u003e coronal section demonstrating induction of RFP (red, E), pS6 staining (green, F), and composite with GFP expression (blue, G). H-J) Images of 20× \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut\u003c/em\u003e\u003c/sup\u003e coronal section demonstrating induction of RFP (red, H), pS6 staining (green, I), and composite with GFP expression (blue, J). K-L. 4× digital zoom of H-I showing cells with neuronal morphology and high pS6. Chevrons indicate macroscopic neurons, and arrowhead indicates a giant cell. E-J) Scale bar = 75 µm. K-M) Scale bar = 75 µm.\u003c/p\u003e","description":"","filename":"Figure1RevisedFlat.png","url":"https://assets-eu.researchsquare.com/files/rs-6008400/v1/5f413d18d0905ec4adafa1c0.png"},{"id":82758404,"identity":"9b1bf26f-d9f4-4adc-9223-e7aee92d57e3","added_by":"auto","created_at":"2025-05-15 02:22:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":597961,"visible":true,"origin":"","legend":"\u003cp\u003eA) Dose schedule B) Kaplan-Meier survival curve of control and Rapalink-1 treated mice. C) Weights of control and Rapalink-1 treated mice. Data is represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure2RevisedFlat.png","url":"https://assets-eu.researchsquare.com/files/rs-6008400/v1/85892bb34abca045973e56f9.png"},{"id":82759059,"identity":"5fab41e2-8165-4879-b6a3-0d94c9ecc48a","added_by":"auto","created_at":"2025-05-15 02:30:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":914659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRapalink-1 Inhibits mTORC1 Activity.\u003c/em\u003e A-C) 20× images of RFP (red, A), pS6 (green, B), and composite with GFP (blue, C) in a coronal section of a \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut \u003c/em\u003e\u003c/sup\u003eP90 vehicle treated mouse brain. Rectangle denotes magnified region in D-F. D-F) 4× digital zoomed in images of A-C demonstrating neuronal morphology. \u0026nbsp;G-I) 20× images of RFP (red, G), pS6 (green, H), and composite with GFP (blue, I) in a coronal section of a brain from a \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut \u003c/em\u003e\u003c/sup\u003eP90 Rapalink-1 (3 mg/kg) treated mouse. J-L) 4× digital zoomed in images of G-I demonstrating neuronal morphology. M) Quantification of pS6 in RFP positive cells. *=p\u0026lt;0.05, ****=p\u0026lt;0.0001. Data is represented as mean ± SEM. A-C and G-I scale bar = 75 µm. D-F and J-L scale bar = 18.75 µm.\u003c/p\u003e","description":"","filename":"Figure3RevisedFlat.png","url":"https://assets-eu.researchsquare.com/files/rs-6008400/v1/0c745bc51000242e277bb1a6.png"},{"id":82759060,"identity":"7bb70b06-81ec-4fd9-9448-8d126aab7553","added_by":"auto","created_at":"2025-05-15 02:30:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":830807,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRapalink-1 Reduces Cell Size.\u003c/em\u003e A-D) 20× images of GFP (blue, A), RFP (red, B), pS6 (green, C), and composite (D) in \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut \u003c/em\u003e\u003c/sup\u003eP90 control brains. E-H) 20× images of GFP (blue, E), RFP (red, F), pS6 (green, G), and composite (H) in \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut \u003c/em\u003e\u003c/sup\u003eP90 Rapalink-1 (3 mg/kg) brains. I, J) Images from D and H magnified 400% showing examples of giant cells. K) Quantification of neuron soma size relative to controls. Data is represented as mean ± SEM. Scale bar = 75 µm. ****=p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"Figure4RevisedFlat.png","url":"https://assets-eu.researchsquare.com/files/rs-6008400/v1/63a78a8b034df7fba3a4e5d6.png"},{"id":82758400,"identity":"5cf9889b-167c-43f7-a817-a12bedc468a3","added_by":"auto","created_at":"2025-05-15 02:22:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":955075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRapalink-1 Reduces Dendrite Arbors in a TSC Model. \u003c/em\u003eA) 20× image of RFP in a \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut \u003c/em\u003e\u003c/sup\u003eP90 control brain. B) 20× image of RFP in a \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut \u003c/em\u003e\u003c/sup\u003eP90 Rapalink-1 treated brain. C) Zoomed in image with a neuron in square of A subjected to tracing (green highlight). D) Zoomed in image with a neuron in square of B subjected to tracing (green highlight). E) Sholl Analysis demonstrating the effect of Rapalink-1 on dendrite arbors. F) The total number of dendrite crossings in each neuron of control and Rapalink-1 treated mice. Data is represented as mean ± SEM. Scale bar = 75 mm (A, B) or 37.5 mm (C, D). ***=p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Figure5RevisedFlat.png","url":"https://assets-eu.researchsquare.com/files/rs-6008400/v1/bef5dd5dc56e2b47179a15d2.png"},{"id":82758406,"identity":"50b856cb-6cb5-4a35-bf13-779f9922c4f3","added_by":"auto","created_at":"2025-05-15 02:22:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":616105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRapalink-1 Reduces Hamartoma Size.\u003c/em\u003e A-D) 20× images of GFP (blue), pS6 (green), and RFP (red) in \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut \u003c/em\u003e\u003c/sup\u003eP90 brains treated with Vehicle or E-H) 20× images of GFP (blue), pS6 (green), and RFP (red) in \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut \u003c/em\u003e\u003c/sup\u003eP90 Rapalink-1 treated brains. I) Quantification of lesion area. J) Schematic diagram of molecular pathways regulated by \u003cem\u003eTsc1/Tsc2\u003c/em\u003e in TSC model in control and Rapalink-1 treated brains. Tsc1/Tsc2 turn off mTORC1 pathway under normal conditions. However, in the TSC SEGA model (left), \u003cem\u003eTsc1/Tsc2 \u003c/em\u003eencoded protein products cannot turn mTORC1 off leading to ectopic/cytomegalic neurons with excessive mTORC1 activity, hypertrophic dendrites, giant cells, and SEGA-like lesions (i.e. striatal hamartomas). Rapalink-1 appears to partially rescue these changes with mTORC1 inhibited neuron size decreased, and reduced SEGA-like lesions. Data are represented as mean ± SEM. Scale bar = 150 mm (A, B) or 37.5 mm (C, D). *=p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"Figure6RevisedFlat.png","url":"https://assets-eu.researchsquare.com/files/rs-6008400/v1/b1fccad4c1928ce40cc2bffc.png"},{"id":86180707,"identity":"43ab8af4-5a36-4f8a-a457-1cb04196f843","added_by":"auto","created_at":"2025-07-07 16:22:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5149215,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6008400/v1/5a520f0b-df67-4957-8ff9-1c88b8b0e06f.pdf"},{"id":82758399,"identity":"1bf0f468-8b41-4904-8f44-10a776e12905","added_by":"auto","created_at":"2025-05-15 02:22:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5281249,"visible":true,"origin":"","legend":"","description":"","filename":"AllSupplementalRevisedReduced.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6008400/v1/c1df41320650293b0c83eacd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Bitopic mTORC Inhibitor Reverses Phenotypes in a Tuberous Sclerosis Complex Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTuberous Sclerosis Complex (TSC) is a multisystem genetic disorder affecting ~0.0166 percent of the population\u003csup\u003e1\u003c/sup\u003e. TSC is caused by loss of function mutations in \u003cem\u003eTSC1\u003c/em\u003e or \u003cem\u003eTSC2\u003c/em\u003e\u003csup\u003e2,3\u003c/sup\u003e. \u003cem\u003eTSC1\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;TSC2\u0026nbsp;\u003c/em\u003eencode for the proteins hamartin and tuberin which inhibit Rheb-mTORC1 signaling\u003csup\u003e4\u003c/sup\u003e. \u003cem\u003eTSC1/2\u003c/em\u003e loss of function mutations activate the mTORC1 pathway\u003csup\u003e5–8\u003c/sup\u003e. mTORC1 is a protein kinase complex that promotes cell growth and proliferation\u003csup\u003e9\u003c/sup\u003e. TSC patients have too much mTORC1 activity and enlarged cells that create tumors called hamartomas within the heart, kidney, lung, skin, and brain\u003csup\u003e10,11\u003c/sup\u003e. Identifying the mechanisms that cause hamartomas to form is important for understanding the many disorders whose genetic mutations affect molecular pathways that intersect with mTORC1 and for developing therapeutic strategies to treat TSC patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTSC patients have brain hamartomas called subependymal nodules (SENs) that invade the striatum and subependymal zone (SEZ)\u003csup\u003e12,13\u003c/sup\u003e. SENs are commonly detected during childhood\u003csup\u003e3,13\u003c/sup\u003e. Approximately a quarter of SENs are categorized as subependymal giant cell astrocytomas (SEGAs)\u003csup\u003e12–14\u003c/sup\u003e.\u0026nbsp;SENs may transition into SEGAs\u003csup\u003e15\u003c/sup\u003e. SENs and SEGAs share all histopathological features\u003csup\u003e16\u003c/sup\u003e.\u0026nbsp;Although there is no consensus, the criterion of SEGA diagnosis is \u0026gt;0.5-1.0 cm in size or serial growth\u003csup\u003e17\u003c/sup\u003e. While SEGAs occur throughout the ventricular system, cerebrospinal fluid (CSF) circulation blockade along the caudothalamic groove can cause obstructive hydrocephalus associated with migraines, seizures, and death. Unexplained changes in neurological status or TSC-associated neuropsychiatric disorders (TANDs) can also be a sign of SEGA growth\u003csup\u003e18\u003c/sup\u003e. The median age of SEGA diagnosis is 1 year. Only ~2.4% of SEGAs are identified after age 40\u003csup\u003e19\u003c/sup\u003e. SEGAs can bleed when being removed leading to non-obstructive hydrocephalus, tissue damage, and mortality\u003csup\u003e20\u003c/sup\u003e. Because SEGAs occur in young children and are located deep within the brain and because surgery comes with a risk, pharmacological intervention is warranted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003emTORC1 inhibitors including rapamycin analogs (rapalogs) are now the standard of care except for cases of acute hydrocephalus\u003csup\u003e21\u003c/sup\u003e. ~57% of SEGAs are reduced by 50% volume within two years and maintenance doses are not typically associated with changed SEGA volume\u003csup\u003e18,19,22\u003c/sup\u003e. Thus, SEGAs do not always respond to rapalogs. Children may poorly tolerate rapalogs, and if treatments stop, SEGAs grow back\u003csup\u003e23\u003c/sup\u003e. Even surgical removal of SEGAs is followed by regrowth in nearly 40% of patients. The mechanisms that account for SEGA regrowth are unclear but may be related to the fact that mTORC1 allosteric inhibitors incompletely inhibit mTORC1 phosphorylation of select substrates\u003csup\u003e24,25\u003c/sup\u003e. Alternatively, resistance to rapalog binding may occur\u003csup\u003e26\u003c/sup\u003e. A novel bisteric inhibitor linking AZD8055 to rapamycin called Rapalink-1 was generated\u003csup\u003e26\u003c/sup\u003e. Rapalink-1 simultaneously inhibits mTORC1 through the rapamycin moiety by targeting FK506-binding protein 12 (FKBP12) and the FKBP12 rapamycin binding domain of mTOR as well as inhibiting both mTORCs through the ATP competitive inhibitor moiety, AZD8055\u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe created a mouse model of TSC SEGAs by electroporating CRE recombinase into NSCs of mice having conditional \u003cem\u003eTsc2\u0026nbsp;\u003c/em\u003egenes to provide mechanistic insight into SEGA pathogenesis\u003csup\u003e27,28\u003c/sup\u003e. Mice developed hamartomas with SEN-like lesions that develop into SEGA-like hamartomas. We previously found that TSC mutant NSC translational programs were altered and prevented differentiation leading to the aberrant production of neurons in the striatum. These lesions were associated with ensembles of cytomegalic neurons and giant cells. We performed single nuclei RNA sequencing (snRNA-Seq) of these mice and discovered altered NSC transitional states caused by loss of \u003cem\u003eTsc2\u003c/em\u003e. Moreover, neurons were a core feature in this model and had altered transcriptomes. The extent to which mTOR activity might cause these phenotypes was not assessed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhat follows are the results of a study that utilizes the bisteric inhibitor Rapalink-1 on a TSC model of striatal hamartomas representing SEGA-like lesions.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eRapalink-1 Treatment of a TSC Mouse Model.\u003c/em\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eTsc2\u003c/em\u003e \u003csup\u003e \u003cem\u003ewt/wt\u003c/em\u003e \u003c/sup\u003e and \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e x \u003cem\u003eRFP\u003c/em\u003e neonatal mice were electroporated with Cre recombinase and GFP encoding DNA plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Electroporation allows for the targeting of lateral V-SVZ NSCs that generate striatal glia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This causes recombination leading to deletion of exons 2\u0026ndash;4 of \u003cem\u003eTsc2\u003c/em\u003e and red fluorescence along the lateral ventricles that appear as SEGA-like hamartomas in postnatal day (P) 30 \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). Additionally, V-SVZ NSCs generate granule cell neurons that migrate into the olfactory bulb (OB) and are fully mature 30 days later. We previously reported hamartomas at P30 having elevated mTORC1 activity assessed by p4E-BP in glial cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. We also reported increased mTORC1 as assessed by pS6 staining in neurons produced from V-SVZ NSCs\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We further reported measurements of P60 hamartoma and OB neuron growth. Thus, these time points helped to guide the design of our experiments. Indeed, in comparison to \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003ewt/wt\u003c/em\u003e\u003c/sup\u003e NSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G) that produced mostly glia, there were numerous \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut\u003c/em\u003e\u003c/sup\u003e pS6 positive neurons and giant cells near the striatal border with the V-SVZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-M).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs confirmation of \u003cem\u003eTsc2\u003c/em\u003e recombination, there was a 23.3% increase in pS6 in mutant RFP positive neurons (\u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003ewt/wt\u003c/em\u003e\u003c/sup\u003e 1.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03042, n\u0026thinsp;=\u0026thinsp;110 vs. \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut\u003c/em\u003e\u003c/sup\u003e 1.233\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04392, n\u0026thinsp;=\u0026thinsp;110) (\u003cb\u003eSupplemental Fig.\u0026nbsp;1A\u003c/b\u003e). CRE electroporated V-SVZs were collected and NSCs cultured (\u003cb\u003eSupplemental Fig.\u0026nbsp;1B\u003c/b\u003e). We verified high rates of \u003cem\u003eTsc2\u003c/em\u003e recombination in NSCs (\u003cb\u003eSupplemental Fig.\u0026nbsp;1C, D\u003c/b\u003e). We further verified that phosphorylation of the mTORC1 substrate Ulk1 was rapamycin resistant but sensitive to ATP-competitive mTORC inhibitors (\u003cb\u003eSupplemental Fig.\u0026nbsp;1E, F\u003c/b\u003e). These results further confirm that CRE-mediated recombination occurs in the TSC model and that some mTORC1 substrates are resistant to rapamycin.\u003c/p\u003e \u003cp\u003e\u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e x \u003cem\u003eRFP\u003c/em\u003e neonatal mice were subsequently randomized, assigned a unique identification number, and treated with Rapalink-1 or DMSO (vehicle) for 30 days or until sacrificed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Animals were euthanized by intraperitoneal injection of euthasol or CO\u003csub\u003e2\u003c/sub\u003e inhalation followed by swift decapitation. No mice in the control group died and no significant changes in behavior or signs of distress were noted (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Cohorts of mice were given 1.5 mg/kg or 3.0 mg/kg Rapalink-1 once every five days. 3.0 mg/kg Raplink-1 treated mice survived on average 88.2 days (N\u0026thinsp;=\u0026thinsp;8) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Likewise, 1.5 mg/kg Rapalink-1 was well tolerated (N\u0026thinsp;=\u0026thinsp;5) with all mice surviving 90 days. These doses were well tolerated with death seen at 3.0 mg/kg and having slightly lower weights at the end of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eRapalink-1 inhibits mTORC1 activity.\u003c/em\u003e \u003c/p\u003e \u003cp\u003emTORC1 activity and substrate phosphorylation in the brain is cell type dependent\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We noted that pS6 levels were high within RFP positive \u003cem\u003eTsc2\u003c/em\u003e null cells having neuron-like morphologies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-F)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. pS6 was analyzed in vehicle, Rapalink-1 (1.5 mg/kg), or Rapalink-1 (3.0 mg/kg) \u003cem\u003eTsc2\u003c/em\u003e mutant neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-L). We found that Rapalink-1 decreased pS6 (Vehcile\u0026thinsp;=\u0026thinsp;1.066\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029, n\u0026thinsp;=\u0026thinsp;857 vs. Rapalink-1 1.5 mg/kg\u0026thinsp;=\u0026thinsp;0.9127\u0026thinsp;\u0026plusmn;\u0026thinsp;0.065, n\u0026thinsp;=\u0026thinsp;218 vs. Rapalink-1 3.0 mg/kg\u0026thinsp;=\u0026thinsp;0.835\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029 n\u0026thinsp;=\u0026thinsp;452, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe confirmed the extent that mTORC1 signaling was reduced by using acute daily Rapalink-1 treatment for five days. Rapalink-1 has been used at higher doses for acute \u003cem\u003ein vivo\u003c/em\u003e experiments\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We therefore tested acute efficacy of Rapalink-1 at a higher dose (6 mg/kg) but less than 10 mg/kg, which is the dose of rapamycin used to compare the two drugs\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Rapalink-1 reduced mTORC1 signaling as detected by examining p4EBP and pS6 staining in the brain and by immunoblotting for pS6 (Vehicle\u0026thinsp;=\u0026thinsp;1.000\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04189, N\u0026thinsp;=\u0026thinsp;3 vs Rapalink-1\u0026thinsp;=\u0026thinsp;0.7707\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02518, N\u0026thinsp;=\u0026thinsp;3; P\u0026thinsp;=\u0026thinsp;0.0094) (\u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e). Rapalink-1 inhibition of mTORC2 was confirmed by a reduction in pAKT as previously reported (\u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e). These results confirm that Rapalink-1 can indeed reduce mTOR signaling \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eRapalink-1 reduces Tsc2 mutant neuron cell size.\u003c/em\u003e \u003c/p\u003e \u003cp\u003emTOR regulates OB granule cell soma growth\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Loss of mTOR and inhibition of mTORC1 with rapamycin decreases granule cell soma size\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Conversely, loss of \u003cem\u003eTsc2\u003c/em\u003e increases granule cell and striatal neuron soma size\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We wondered to what extent soma size might be decreased in \u003cem\u003eTsc2\u003c/em\u003e mutant neurons following Rapalink-1 treatment. Rapalink-1 low dose (1.5 mg/kg) and Rapalink-1 high dose (3.0 mg/kg) decreased the average soma size of mutant \u003cem\u003eTsc2\u003c/em\u003e neurons (Vehicle\u0026thinsp;=\u0026thinsp;1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016 vs. Rapalink-1 1.5 mg/kg\u0026thinsp;=\u0026thinsp;0.7463\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 vs. Rapalink-1 3.0 mg/kg\u0026thinsp;=\u0026thinsp;0.689\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-H, K). We documented a population of cells appearing as giant cells having high mTORC1 activity even after 3.0 mg/kg Rapalink-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, J). However, the relative proportion of cells that were classified as giant cells in relation to the total cell number per section, was reduced in the 3.0 mg/kg Rapalink-1 condition (Vehicle\u0026thinsp;=\u0026thinsp;0.0451\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0044 vs. Rapalink-1 1.5 mg/kg\u0026thinsp;=\u0026thinsp;0.0415\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0125 vs. Rapalink-1 3.0 mg/kg\u0026thinsp;=\u0026thinsp;0.0274\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03623, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Taken together, Rapalink-1 appears to decrease the average size of \u003cem\u003eTsc2\u003c/em\u003e mutant neurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eRapalink-1 Treatment Decreases Neuron Dendrite Arbors.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe previously demonstrated that loss of \u003cem\u003eTsc1, Tsc2\u003c/em\u003e, or increasing Rheb, increased dendrite arbors of OB granule cell neurons produced from V-SVZ NSCs\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This is likely because mTOR and mTOR complex components raptor and rictor regulate granule cell dendrite arbors\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Neurons present in the striatum of the SEGA model also have more dendrites than wild type or \u003cem\u003eTsc2\u003c/em\u003e mutant OB granule cell neurons\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. These mutant neurons are also larger and have greater dendrite complexity in comparison to any control wild-type neurons found in the striatum.\u003c/p\u003e \u003cp\u003eGross reductions in dendrite arbors caused by Rapalink-1 were noted at low magnifications (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). To confirm and quantify this observation, neurons from control and Rapalink-1 treated mice were traced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Rapalink-1 substantially reduced the length and complexity of \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003e\u003cem\u003emut/mut\u003c/em\u003e\u003c/sup\u003e neuron dendrites (Vehicle\u0026thinsp;=\u0026thinsp;713.0\u0026thinsp;\u0026plusmn;\u0026thinsp;74.53, n\u0026thinsp;=\u0026thinsp;24 vs. Rapalink-1 1.5 mg/kg\u0026thinsp;=\u0026thinsp;563.8\u0026thinsp;\u0026plusmn;\u0026thinsp;46.64, n\u0026thinsp;=\u0026thinsp;24 vs. Rapalink-1 3.0 mg/kg 465.5\u0026thinsp;\u0026plusmn;\u0026thinsp;33.85, n\u0026thinsp;=\u0026thinsp;50; p\u0026thinsp;=\u0026thinsp;0.008) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). Thus, long-term Rapalink-1 treatment significantly reduces dendrite arbors in \u003cem\u003eTsc2\u003c/em\u003e mutant neurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eEfficacy of Rapalink-1 for the Treatment of TSC Hamartomas.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe final determinant of the efficacy of Rapalink-1 was the average lesion size at P90. Striatal hamartomas were measured by tracing lesions we previously described\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. We noted that size continues to increase notably at P90 with some hamartomas appearing aggressive and invading different regions reaching 10 times the size of those measured at P60 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In comparison, 1.5 mg/kg Rapalink-1 treated mice had hamartomas that were considerably (~\u0026thinsp;68.6%) smaller and 3.0 mg/kg Rapalink-1 significantly reduced hamartoma sizes by ~\u0026thinsp;half in relation to control conditions (Vehicle\u0026thinsp;=\u0026thinsp;40096\u0026thinsp;\u0026plusmn;\u0026thinsp;8461, n\u0026thinsp;=\u0026thinsp;38 vs. Rapalink-1 1.5 mg/kg\u0026thinsp;=\u0026thinsp;27520\u0026thinsp;\u0026plusmn;\u0026thinsp;2243, n\u0026thinsp;=\u0026thinsp;58 vs. Rapalink-1 3.0 mg/kg 19636\u0026thinsp;\u0026plusmn;\u0026thinsp;3266, n\u0026thinsp;=\u0026thinsp;33, p\u0026thinsp;=\u0026thinsp;0.0363) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSEGAs are a significant cause of morbidity in TSC patients. Rapalogs are a front-line treatment for SEGAs\u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Here we demonstrate that likewise, Rapalink-1 is useful for reversing neural phenotypes in a TSC mouse model of SEGA-like hamartomas (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Although, ~\u0026thinsp;57% of TSC SEGAs are reduced by 50% within two years, continued treatment does not further reduce SEGA volume\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Protracted use of Rapalogs is also necessary because stopping treatment is associated with SEGA recurrence. Considering the overall moderate efficacy of Rapalogs and side effects, especially for children, warrants that new medications are deployed\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Studies have also demonstrated the development of resistance to rapamycin through changes in the FKB12 rapamycin binding domain of mTOR\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Finally, rapamycin does not fully inhibit mTORC1 phosphorylation of several substrates\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. There is an urgent need to test new therapies that may overcome these issues. Rapalink-1 is an excellent candidate because it fulfills the need to completely inhibit mTORC1 phosphorylation of rapamycin-resistant substrates and simultaneously inhibits mTORC2 and overcomes issues with resistance\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. It is therefore conceivable that Rapalink-1 might be useful to treat TSC. To fulfill the goal of examining additional TSC therapies, we tested Rapalink-1 in a mouse model of TSC SEGA-like hamartomas.\u003c/p\u003e \u003cp\u003eThe mechanisms that account for SEGA growth are unclear in patients and this may be related to the fact that we have little understanding of the cellular composition, regional differences in SEGAs, and whether molecular mechanisms involved in the different regions and timing of their appearance may differ. The fact that the composition of SEGAs has been debated for nearly a century is evidence that experiments must carefully examine the effect drugs have not just on SEGA size, but also on the different cell types within SEGAs\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. SEGAs also appear to change over time. Imaging and RNA sequencing experiments suggest a stepwise change in composition with SEGAs eventually containing neurons and cytomegalic giant cells\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46 CR47\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Gliotic scarring, calcification, and immune cell invasion also occur\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This is similar to events in TSC patient cortical tubers\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Although SEGAs do not frequently directly cause seizures, the presence of mutant neurons and giant cells warrants closer inspection as does determining their contributions to patient presentation.\u003c/p\u003e \u003cp\u003eLoss of \u003cem\u003eTsc2\u003c/em\u003e in V-SVZ neuroprogenitors results in the aberrant transition from quiescent to active states and during differentiation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Loss of \u003cem\u003eTsc2\u003c/em\u003e prevents the further downregulation of mTORC1 activity in a subset of cells within the striatum leading to growths including nodular hamartomas containing cells with heterogenous morphologies. The hamartomas are characterized by abnormal heterotopic clusters of morphologically heterogenous cells which include neurons both within and outside of the growths. Thus, the TSC SEGA-like hamartoma mouse model allowed us to examine the utility of Rapalink-1 treatment in controlling neural phenotypes and hamartoma size. Rapalink-1 intermittent dosing (1.5 mg/kg and 3.0 mg/kg every five days) was generally tolerated during the study, but preliminary experiments demonstrated acute toxicity during daily treatment (data not shown). As predicted, Rapalink-1 reduced mTORC1 activity. However, it was reduced by only\u0026thinsp;~\u0026thinsp;15\u0026ndash;23% as measured using pS6 as a readout and pS6 remained apparent throughout the brain. Thus, these doses might incompletely inhibit mTORC1. Indeed, a higher dose (6 mg/kg) was also tested based on efficacy in glioma and appeared more effective by immunohistochemistry and western blot but was associated with toxicity\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. It is unclear whether extended treatments of 1.5 mg/kg and 3.0 mg/kg Rapalink-1 for more than 30 days would be accompanied by additional side effects. Additionally, 1.5 and 3.0 mg/kg Rapalink-1 treated mice were sacrificed 5 days after the last injection of Rapalink-1. Thus, mTORC1 activity could have rebound effects and our data underestimate how effective Rapalink-1 is. Nevertheless, the reduction in pS6 served as an indicator that Rapalink-1 inhibited mTORC1.\u003c/p\u003e \u003cp\u003eWe reasoned that the prolonged inhibition of mTORC1 would decrease the well-documented mTORC1-regulated phenomenon, of cell growth\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Previous work from our laboratory and our colleagues have demonstrated that neonatal V-SVZ NSC generated granule cells grow when \u003cem\u003eTsc1\u003c/em\u003e or \u003cem\u003eTsc2\u003c/em\u003e are removed\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Moreover, ectopic expression of wild-type or mutant Rheb can drive cell growth in granule cells\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Growth of granule cells is dependent on mTOR since CRE electroporation of conditional mTOR reduces soma size\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Moreover, rapamycin treatment reduces granule cell soma size supporting the importance of mTORC1 in this process\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. As expected, Rapalink-1 at both doses reduced \u003cem\u003eTsc2\u003c/em\u003e mutant neuron cell size by ~\u0026thinsp;25\u0026ndash;31%. These results further support that Rapalink-1 is likely inhibiting mTORC1-dependent cellular events.\u003c/p\u003e \u003cp\u003eWhile the role of striatal neurons in the hamartoma SEGA-like model is unclear, ectopic neurons can affect a wide range of other cell types. Cortical tubers and focal malformations of cortical development appear to undergo analogous changes including astrogliosis and microglia activation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. And this is directly linked to neuron hyperexcitability. Thus, a major goal for TSC and related disorder research has been to reduce dendrite growth in the hopes of modulating neuronal activity. mTOR, through mTORC1 and mTORC2 regulates OB granule cell dendrite arbors\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. While cytoskeleton regulation is most often attributed to mTORC2, rapamycin and mTORC1 have been carefully studied in relation to dendrite growth. For example, like cell growth, loss of \u003cem\u003eTsc1\u003c/em\u003e and \u003cem\u003eTsc2\u003c/em\u003e as well as increased Rheb activity promote dendrite growth\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. We found that Rapalink-1 similarly reduced the dendrites of striatal \u003cem\u003eTsc2\u003c/em\u003e mutant neurons. Thus, Rapalink-1, could be advantageous in that it binds both arms of mTOR signaling\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis is particularly important in that several groups have posited that additional pathways may regulate SEGA growth. For example, one group simultaneously removed \u003cem\u003ePten\u003c/em\u003e and \u003cem\u003eTsc1\u003c/em\u003e in postnatal V-SVZ NSCs\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This resulted in the generation of SEGAs that recapitulated most aspects of those in patients. \u003cem\u003ePten/Tsc1\u003c/em\u003e mutant NSCs were subsequently injected subcutaneously and generated tumors. The \u003cem\u003ePten\u003c/em\u003e/\u003cem\u003eTsc1\u003c/em\u003e mutant NSCs had altered Erk and Akt activity too. Knockdown of the mTORC2 component rictor or combined rapamycin and PI3K-mTOR inhibition reduced tumor growth\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Thus, inhibitors that act on both mTORC1 and mTORC2 such as Rapalink-1 have several advantages. In line with these results, we found that Rapalink-1 produced a moderate but significant effect on the average size of SEGAs. The effect of Rapalink-1 was disproportionate to that seen on cell size or dendrites. Whether the effectiveness of Rapalink-1 is due to the effect on neuron activity or on other cell types is unclear and will require additional future experiments. A limitation of inhibiting mTORC2 is that this could lead to additional side effects not seen with rapalogs.\u003c/p\u003e \u003cp\u003eTaken together, this study provides \u003cem\u003ein vivo\u003c/em\u003e evidence for the utility of Rapalink-1 to control mTOR, cell size, dendrite hypertrophy, and SEGA-like hamartoma growth which may be of clinical importance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the United States of America Department of Defense U.S. Army Medical Research Activity Award Congressionally Directed Medical Research Program Tuberous Sclerosis Complex Research Program W81XWH2010447. We acknowledge Anthony J. Minerva and Melanie Garcia for technical assistance.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eConceptualization, DF; Methodology, SM, DF, MW, MS, AS, VR; Validation, DF, VR, MW, MS, AS; Formal Analysis, SM, MW, MS, AS, DF; Investigation, DF, SM, VR, MW, MS, AS; Resources, DF; Data Curation, DF, MW, MS; Writing-Original Draft, DF; Writing-Reviewing and Editing; DF, SM, VR, MW, MS, AS; Visualization, SM, DF; Supervision, DF; Project Administration, DF; \u0026nbsp;Funding Acquisition, DF\u003c/p\u003e\n\u003ch2\u003eDeclaration of Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that the researchers have no competing financial interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eDMF is supported by United States of America Department of Defense U.S. Army Medical Research Activity Award Congressionally Directed Medical Research Program Tuberous Sclerosis Complex Research Program W81XWH2010447.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cu\u003eEXPERIMENTAL MODEL AND SUBJECT DETAILS;\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnimals.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were approved by the Clemson University Institutional Animal Care and Use Committee and the Animal Care and Use Review Office (ACURO), a component of the USAMRDC Office of Research Protections (ORP) within the Department of Defense (DoD). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. Red fluorescent protein (RFP\u003csup\u003e+/-\u003c/sup\u003e,\u003csup\u003e+/+\u003c/sup\u003e) (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)\u003csup\u003eHze/J\u003c/sup\u003e) (Strain #007909, RRID:IMSR_JAX:007909), and\u0026nbsp;\u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003etm1.1Mjg/J\u0026nbsp;\u003c/sup\u003e(Strain #027458, RRID:IMSR_JAX:027458) were acquired from Jackson Laboratories\u003csup\u003e54\u003c/sup\u003e. Pharmacological treatment of cultured NSCs is on CD1 mice. Sentinel mice were free of pathogens throughout the study. Samples/subjects were allocated randomly to experimental group.\u0026nbsp;Experimental manipulations were performed on mouse pups that were not involved in previous procedures and sacrificed accordingly. Both sexes were used and ages are as indicated in figures. Mice were housed under standard pathogen-free conditions in cages on racks within isolated cubicles with a 12-h light/dark cycle and fed \u003cem\u003ead libitum\u003c/em\u003e. Mice were injected intraperitoneally using the indicated doses and schedule. Drugs were prepared as previously described in DMSO, PEG-300, and PBS\u003csup\u003e32\u003c/sup\u003e. Injections were performed in biological safety cabinets. Mice were weighed 5-7 days each week and weights recorded. Animals were euthanized by intraperitoneal injection of euthasol or CO\u003csub\u003e2\u003c/sub\u003e inhalation followed by swift decapitation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eElectroporation.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eB6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)\u003csup\u003eHze/J\u003c/sup\u003e) x \u003cem\u003eTsc2\u003c/em\u003e\u003csup\u003etm1.1Mjg/J\u0026nbsp;\u003c/sup\u003emouse pups were electroporated as previously described\u003csup\u003e55,56\u003c/sup\u003e. Mice were injected with equal concentrations and volumes of DNA plasmids diluted in phosphate buffered saline (PBS) with 0.1% fast green. CAG-CRE (Plasmid #13775, Addgene) and CAG-GFP (Plasmid #11150, Addgene) plasmids were used\u003csup\u003e57,58\u003c/sup\u003e. A borosilicate glass micropipette generated from pulled capillary tubes was loaded with DNA and injected into the lateral ventricles. Square pulse generation was performed using a pulse generator (ECM830; BTX) and tweezer electrodes (model 520; BTX) with five, 100-volt square pulses of 50 ms duration with 950-ms intervals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePolymerase Chain Reaction (PCR).\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eToe or tail snips were subject to modified hotshot DNA extraction and genotyped by PCR using Invitrogen\u003csup\u003eTM\u003c/sup\u003e Platinum\u003csup\u003eTM\u003c/sup\u003e Taq polymerase, mixed with nuclease-free water, magnesium free PCR buffer, MgCl\u003csub\u003e2\u003c/sub\u003e, dNTP mix with primers and DNA according to the manufacturers\u0026rsquo; protocol (Invitrogen). Primers for \u003cem\u003eTsc2\u0026nbsp;\u003c/em\u003ewere 5\u0026rsquo;-ACAATGGGAGGCACATTACC-3\u0026rsquo; and 5-AAGCAGCAGGTCTGCAGTG-3\u0026rsquo; and for Tomato (RFP) 5\u0026rsquo;-AAGGGAGCTGCAGTGGAGTA-3\u0026rsquo; and 5\u0026rsquo;-CCGAAAATCTGTGGGAAGTC-3\u0026rsquo; and 5\u0026rsquo;-GGCATTAAAGCAGCGTATCC-3\u0026rsquo; and 5\u0026rsquo;-CTGTTCCTGTACGGCATGG-3\u0026rsquo;. Amplicons were loaded onto agarose gels with 1X Blue Juice and ran at 100\u0026thinsp;V for 20\u0026ndash;30\u0026thinsp;mins and visualized on a BioRad Chemidoc MP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunohistochemistry.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBrains were removed in room temperature PBS, transferred to 4% paraformaldehyde in PBS, and incubated overnight at 4\u0026deg;C. Brains were rinsed in PBS and mounted in 3% agarose. A Leica VTS 1000 vibratome was used to section brains coronally. Sections were blocked in 0.1% Triton X-100, 0.1% Tween-20 and 2% BSA in PBS for 1 hr at room temperature. Sections were washed in 0.1% Tween-20 in PBS. Sections were incubated in primary antibody, anti-pS6 (1:500; Cell Signaling Technology; Ser 240/244, 61H9, #4838), in 0.1% Tween-20 and 2% BSA in PBS overnight at 4\u0026deg;C. Sections were subjected to three additional washes in PBS containing 0.1% Tween-20. Sections were incubated with the appropriate secondary antibody (Alexa Fluor series; 1:500; Invitrogen) in 0.1% Tween-20 and 2% BSA in PBS overnight at 4\u0026deg;C. Sections were mounted in ProLong Antifade Mountant (ThermoFisher). Images were acquired on a spectral confocal microscope (Leica SPE) with a \u0026times;20 dry objective (N.A. 0.75). Low-magnification images were acquired with \u0026times;10 dry or a \u0026times;5 dry (N.A. 0.15) objective.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImage Analysis.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eImages (\u0026times;20) of RFP positive cells were uploaded to FIJI (ImageJ 1.5 g) and analyzed as described elsewhere\u003csup\u003e27\u003c/sup\u003e. The freehand selection tool was used to trace electroporated and non-electroporated cell somas in the same Z section and mean gray values for pS6 were quantified. Ratios of electroporated and non-electroporated cells were compared for RFP positive cells in \u003cem\u003eTsc2\u003csup\u003ewt/wt\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003eTsc2\u003csup\u003emut/mut\u003c/sup\u003e\u003c/em\u003econditions to account for immunohistochemical variation. Soma size was simultaneously recorded for traced cells. The RFP positive hamartoma perimeter was outlined in each Z section by hand by scrolling through individual Z sections and hamartomas were subsequently traced in (\u0026times;20) images. The freehand selection tool was used to trace RFP positive lesions. Images (\u0026times;20) were used to measure dendrite morphology. Dendrites were traced using the simple neurite tracer plug-in. Sholl analysis was performed at 1 \u0026micro;m intervals to quantify dendrite arborization using the Sholl plug-in. The total number of dendritic crossings was calculated by taking the sum of crossings at all intervals for each traced neuron and averaging the total number of crossings per neuron in each condition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWestern Blot.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTissue (0.1 gram) was harvested and finely minced in 2% SDS, Protease Inhibitor Cocktail (Pierce) and Phosphatase Inhibitor Cocktail, in RIPA buffer. Samples were briefly sonicated at maximum settings (100 Amplitude, QSonica) for 10 seconds three times with 30 second resting intervals. Lysate was transferred on ice to a fresh reaction tube and centrifuged at 15,000 rpm for 15 min in a tabletop Eppendorf 5415 centrifuge at 4\u0026deg;C. Protein concentration was quantified using the Pierce MicroBCA assay. Equal protein amounts were brought up to equal volumes with lysis buffer as described above and Laemmli buffer and heated to 95\u0026deg;C for 5 minutes. Proteins were resolved by 10% polyacrylamide precast mini-Protean gels (BioRad) and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were rinsed in Tris-buffered saline (TBS-T, 0.1% Tween 20) for 5 min at room temperature and blocked in 5% blotting grade block (BioRad) in TBS-T for 1 h at room temperature. Membranes were incubated for 1 h at room temperature or overnight at 4\u0026deg;C with the following antibodies from Cell Signaling Technology at a 1:1,000 dilution: phospho-RPS6 (D68F8, Cat# 5364) and RPS6 (5G10, Cat# 2217). Membranes were rinsed three times each for 10 min in TBS-T. Membranes were incubated for 1 h at room temperature with donkey or goat anti-rabbit antibodies in blocking buffer. Membranes were washed for 15 minutes in TBS-T and visualized using a Bio-Rad Chemidoc MP imaging system using enhanced chemiluminescence reagent (Pierce). PVDF membranes were stripped for at room temperature using Restore Western Blot Stripping Buffer according to manufacturer\u0026rsquo;s recommendations (Cat# 21059, Thermo Fisher Scientific).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePrimary cell culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBriefly, P0-1 CD1 mice were anesthetized on ice and decapitated and brains were removed and placed in 4\u0026deg;C Neurobasal A media on pre-chilled petri dishes. Micro-dissected V-SVZs were placed into 0.05% trypsin with 0.02% EDTA in Neurobasal A for 7 min in a 37\u0026deg;C incubator. Equal volumes of defined trypsin inhibitor (1X; Life Technologies; Lot# 1837475) were added. The samples were centrifuged at 300 x g for 5 min. The pellet was re-suspended with 1 mL Neurobasal A. The pellet was triturated with three pasture pipettes having sequentially decreased bore size to dissociate tissue. Cells were centrifuged at 300 x g for 5 min and resuspended in 200\u0026nbsp;mL Neurobasal A complete Media (1X Glutamax, 50 units/mL Penicillin/streptomycin, 20 ng/mL EGF, 20 ng/mL FGF-2 and 2% B27 Supplement). Cells were placed on laminin-coated coverslips in 24 well plates in Neurobasal A complete media for pharmacological experiments performed 24 hours later or placed into 6 well plates for recombination analysis as previously described\u003csup\u003e27,29\u003c/sup\u003e. Samples were harvested in 2% SDS, Protease Inhibitor Cocktail (Pierce) and Phosphatase Inhibitor Cocktail, in RIPA buffer for western blot. Alternatively, samples were subjected to DNA isolation and long range PCR for \u003cem\u003eTsc2\u0026nbsp;\u003c/em\u003erecombination products as previously described\u003csup\u003e27,29\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePharmacological Treatments\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNSCs were treated with the 10 nM Rapamycin (#9904s, Cell Signaling Technology) or 10 nM ATP-competitive inhibitor, Torin1 (#14379, Cell Signaling Technology). Torin1 from a 1mM stock was re-constituted in DMSO (MP Biomedicals). Rapamycin was re-constituted in DMSO for a 100 mM stock. Equi-molar DMSO was used for all conditions. For the primary cell starvation experiment, NSC were placed into PBS for 2 hours prior to drug treatment and placed back into complete media to activate mTORC1 for the duration of drug treatment (2 hours).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eQUANTIFICATION AND STATISTICAL ANALYSIS.\u003c/u\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMeasurements were graphed and statistical analysis was performed with GraphPad Prism software (Version 8.2.0, GraphPad Software Inc.). Statistical significance was determined using One-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s multiple comparisons test or Student\u0026rsquo;s T-test. N (number of mice) and n (number of cells or hamartomas) are listed where applicable. Figure 3 (pS6) and Figure 4 (cell size) analysis were performed on vehicle (N=6), Rapalink-1 1.5 mg/kg (N=4), and Rapalink-1 3.0 mg/kg (N=7) mice. Sholl and hamartoma analysis are reported for vehicle (N=4), Rapalink-1 1.5 mg/kg (N=4), and Rapalink-1 3.0 mg/kg (N=6) mice in figures 5 and 6. Supplemental data were performed on N=3 mice for all immunohistochemistry analysis and western blot conditions. Error bars are reported as the standard error mean.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eKEY RESOURCES TABLE.\u0026nbsp;\u003c/u\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"625\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eREAGENT or RESOURCE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSOURCE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eIDENTIFIER\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003eAntibodies\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRabbit Monoclonal pS6 Ser 240/244\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCell Signaling Technology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat#4838; RRID: AB_659977; Clone: 61H9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRabbit Monoclonal p4EBP Thr 37/46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCell Signaling Technology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat#2855; RRID: AB_560835; Clone: 236B4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRabbit Monoclonal RPS6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCell Signaling Technology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat#2217; RRID: AB_331355; Clone: 5G10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003eChemicals, Peptides, and Recombinant Proteins\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003eExperimental Models: Organisms/Strains\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMouse: RFP\u003csup\u003e+/-\u003c/sup\u003e, RFP\u003csup\u003e+/+\u003c/sup\u003e: B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)\u003csup\u003eHze/J\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eJackson Laboratories\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat#007909; RRID: IMSR_JAX:007909\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMouse: Tsc2\u003csup\u003ewt/wt\u003c/sup\u003e, Tsc2\u003csup\u003emut/mut\u003c/sup\u003e:\u0026nbsp;Tsc2\u003csup\u003etm1.1Mjg/J\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eJackson Laboratories\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat#027458; RRID: IMSR_JAX:027458\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003eOligonucleotides\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrimers: \u003cem\u003eTsc2:\u0026nbsp;\u003c/em\u003e5\u0026rsquo;-ACAATGGGAGGCACATTACC-3\u0026rsquo;, 5- AAGCAGCAGGTCTGCAGTG-3\u0026rsquo;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIntegrated DNA Technologies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrimers: RFP: 5\u0026rsquo;-AAGGGAGCTGCAGTGGAG TA-3\u0026rsquo;, 5\u0026rsquo;-CCGAAAATCTGTGGGAAG TC-3\u0026rsquo;, 5\u0026rsquo;- GGCATTAAAGCAGCGTATCC-3\u0026rsquo; , 5\u0026rsquo;-CTGTTCCTGTACGGCATGG-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIntegrated DNA Technologies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePrimers: For detecting \u003cem\u003eTsc2\u0026nbsp;\u003c/em\u003erecombination 5\u0026rsquo;-AAGATTCCGGCTTGAAGGAG-3\u0026rsquo;, 5\u0026rsquo;-CACTA-GTCTAGCCTGACTCT-3\u0026rsquo;, and 5\u0026rsquo;-GAGGACAAGCCAACATCCAT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIntegrated DNA Technologies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003eRecombinant DNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePlasmid: CAG-CRE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat#13775; RRID: Addgene_13775\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePlasmid: CAG-GFP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCat#11150; RRID: Addgene_11150\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003eSoftware and Algorithms\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFIJI (ImageJ 1.5g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ehttps://imagej.net/software/fiji/downloads\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGraphPad Prism (v. 8.2.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGraphPad Software Inc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ehttps://www.graphpad.com/\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cu\u003eDATA AVAILABILITY.\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available in the Mendeley Data and NeuroMorpho repository. Western blot data is deposited as Feliciano, David (2025), \u0026ldquo;A Bitopic mTORC Inhibitor Reverses Phenotypes in a Tuberous Sclerosis Complex Model Westerns\u0026rdquo;, Mendeley Data, V1, doi: 10.17632/hjt9m462d3.1 (https://data.mendeley.com/datasets/hjt9m462d3/1) to Mendeley Data. Neuron traces are available at NeuroMorpho.org under \u0026ldquo;A Bitopic mTORC Inhibitor Reverses Phenotypes in a Tuberous Sclerosis Complex Model\u0026rdquo; or navigating to neuromorpho.org/dableFiles/mukherjee_feliciano/Supplementary/Mukherjee_Feliciano.zip.\u003c/p\u003e\n\u003cp\u003eDeclaration of Interests. The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNorthrup, H. et al. Updated International Tuberous Sclerosis Complex Diagnostic Criteria and Surveillance and Management Recommendations. Pediatr. Neurol. \u003cem\u003e123\u003c/em\u003e. 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(2004). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.2235688100\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2235688100\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tsc2, Tuberous Sclerosis Complex, TSC, SEGA, Subependymal giant cell astrocytoma, Subependymal Nodule, Rapalink-1, mTORC1, neurogenesis","lastPublishedDoi":"10.21203/rs.3.rs-6008400/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6008400/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeural stem cells (NSCs) of the ventricular-subventricular zone (V-SVZ) generate diverse cell types including striatal glia during the neonatal period. NSC progeny uncouple stem cell-related mRNA transcripts from being translated during differentiation. We previously demonstrated that \u003cem\u003eTsc2 \u003c/em\u003einactivation, which occurs in the neurodevelopmental disorder Tuberous Sclerosis Complex (TSC), prevents this from happening. Loss of \u003cem\u003eTsc2 \u003c/em\u003ecauses hyperactivation of the protein kinase mechanistic target of rapamycin complex 1 (mTORC1), altered translation, retention of stemness in striatal glia, and the production of misplaced cytomegalic neurons having hypertrophic dendrite arbors. These phenotypes model characteristics of TSC hamartomas called subependymal giant cell astrocytomas (SEGAs). mTORC1 inhibitors called rapamycin analogs (rapalogs) are currently used to treat TSC and to assess the role of mTORC1 in regulating TSC-related phenotypes. Rapalogs are useful for treating SEGAs. However, they require lifelong application, have untoward side effects, and resistance may occur. They also incompletely inhibit mTORC1 and have limited efficacy. Rapalink-1 is a bitopic inhibitor that links rapamycin to a second-generation mTOR ATP competitive inhibitor, AZD8055. Here we explored the effect of Rapalink-1 on a TSC hamartoma model. The model is created by neonatal electroporation of mice having conditional \u003cem\u003eTsc2\u003c/em\u003egenes. Prolonged Rapalink-1 treatment could be achieved with 1.5 or 3.0 mg/Kg injected intraperitoneally every five days. Rapalink-1 inhibited the mTORC1 pathway, decreased cell size, reduced neuron dendrite arbors, and reduced hamartoma size. In conclusion, these results demonstrate that cellular phenotypes in a TSC SEGA model are reversed by Rapalink-1 which may be useful to resolve TSC brain hamartomas.\u003c/p\u003e","manuscriptTitle":"A Bitopic mTORC Inhibitor Reverses Phenotypes in a Tuberous Sclerosis Complex Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-15 02:22:06","doi":"10.21203/rs.3.rs-6008400/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-02T18:31:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-23T09:57:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-20T09:10:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79940387379874465322883161147093284604","date":"2025-05-14T05:32:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196371531103337673514488994176574665753","date":"2025-05-13T13:47:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-13T13:37:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-13T04:18:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-12T13:43:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d074be34-bf48-4234-981d-7d2fa2c11d39","owner":[],"postedDate":"May 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48472337,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Developmental disorders"},{"id":48472338,"name":"Biological sciences/Developmental biology/Neurogenesis/Adult neurogenesis"}],"tags":[],"updatedAt":"2025-07-07T16:18:23+00:00","versionOfRecord":{"articleIdentity":"rs-6008400","link":"https://doi.org/10.1038/s41598-025-08345-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 15:58:49","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-05-15 02:22:06","video":"","vorDoi":"10.1038/s41598-025-08345-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-08345-z","workflowStages":[]},"version":"v1","identity":"rs-6008400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6008400","identity":"rs-6008400","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}