The Society for Neuroscience (SfN) hosted its 53rd Annual Meeting, Neuroscience 2024, from October 5 to 9, 2024, at McCormick Place Convention Center in Chicago, Illinois. This premier event brings together neuroscientists from around the world to share groundbreaking research and advancements in the field.
Victoria K. Alstat1, Nicholas S. Coungeris1, Andrew S. LaCroix1
1 AxoSim Inc., Maple Grove, MN, USA
Rett syndrome (RTT) and CDKL5 deficiency disorder (CDD) are rare X-linked neurodevelopmental disorders with overlapping phenotypic features including cognitive deficits, developmental delays, and seizures. There are currently no disease-modifying treatments available for either disorder. Furthermore, recapitulating pathophysiology in vitro has proven difficult for complex disorders like RTT and CDD, as there is a lack of scalable, physiologically relevant screening platforms on the market.
We address this need by utilizing our high-throughput screening (HTS)-capable microBrain™ organoid platform to build disease models from CDD and RTT patient-derived induced pluripotent stem cells (iPSCs). We observed striking differences in each disease model in terms of their spontaneous calcium bursting activity profiles; CDD organoids show a hyperexcitability phenotype (increase in calcium peak frequency), while RTT organoids exhibit aberrant calcium oscillations (non-uniform peak heights and peak shape). We verified the reproducibility of these distinct phenotypes across independent batches of organoid plates, showing consistency in the phenotypic fingerprint and strength across organoids, plates, and batches.
On this strong foundation, we developed an approach to identifying disease-modifying therapeutic candidates using the high throughput calcium imaging FLIPR assay. Through this work, we screened over 5000 compounds and identified promising biological targets and molecules that rescued the functional phenotypes in each model through unique mechanisms of action. Intriguingly, we saw no overlap in the molecules that rescued each disease phenotype, indicating that our organoid model reflects the clinical independence of the two conditions. Further screening was used to narrow down the top targets and drug candidates and assess any adverse effects in control organoids. Together, this data not only indicates that our microBrain™ cortical organoid platform is amenable to modeling neurodevelopmental diseases, but also suggests the capacity for this organoid technology to accelerate drug discovery and generate compelling preclinical human efficacy data.
Harbom, Lise1; Terral, Megan1; Anderson, Wesley; Schmidt, Eva1; Schwarzbach, Erin1; Dillon, Sabrina1; Brengartner, Ally1; Spack, Edward1; Curley, Lowry1
1 AxoSim, Inc.; New Orleans, LA
Progress in neurological drug discovery has been hindered by lack of effective models for drug testing. The development of human induced pluripotent stem cell- (iPSC-) derived neurons has been integral in bridging the translational gap between animal models and clinical trials. However, accurate modeling of the central nervous system is dependent upon a cellular landscape comprised of both neuronal and glial cells, including astrocytes and the oligodendrocyte lineage pathway consisting of oligodendrocyte precursor cells (OPCs), oligodendrocytes (OLs), and myelinating OLs. Astrocytes are integral in maintaining neuronal homeostasis and network function, while myelin sheaths formed by OLs enable efficient neuronal conduction.
To optimize AxoSim’s proprietary iPSC-derived BrainSim platform for glial differentiation, spheroids were differentiated for 12 weeks in two different media formulations, with collection points at DIV42, DIV56, DIV70, and DIV84. Transcript and protein expression for markers associated with astrocytes, OPCs, and OLs were assessed via immunohistochemistry (IHC), Western blot, and qPCR. These endpoints confirmed the presence of all three cell types within the spheroids in both test groups.
Additionally, one test group was further supplemented with clemastine, a pro-myelinating compound known to target OL differentiation, for a 4-week and a 2-week treatment period starting at DIV56 and DIV70 respectively. Clemastine treatment resulted in an increase in OPC and OL markers, including myelin basic protein (MBP) at DIV70 and DIV84. This demonstrates that the OPC/OL differentiation pathway is both present and modulable in BrainSim, making it an ideal candidate for testing compounds affecting the myelination or re-myelination process, including those targeting demyelinating diseases such as multiple sclerosis.
Authors: C. Ethan Byrne1, Tyler Rodriguez1, Megan Terral1, Eva Schmidt1, Lowry Curley1, Michale J. Moore1, 2, 3, Edward Spack1, and Corey Rountree1
1 AxoSim Inc., New Orleans, LA, USA
2 Department of Biomedical Engineering, Tulane University, New Orleans, LA, USA
3 Brain Institute, Tulane University, New Orleans, LA, USA
Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating roadblock to lifesaving medications for critically ill cancer patients. In vitro and animal models are poor predictors of CIPN due to incongruencies of the underlying biology. We have developed the human NerveSim (hNS) model, the first physiologically-relevant, preclinical model of myelinated peripheral nerve tissue, ideal for investigating CIPN and preventative therapeutics.
The hNS combines cell culture, tissue engineering, and neuroscience to produce an in vitro neurobiology platform. Human iPSC-derived neuron and primary Schwann cell coculture spheroids are placed in our custom 24-well embedded electrode array culture plate and cultured for 42 days prior to a 7-day dosing period. Axons extend down the growth channel and over the electrodes; Schwann cells align with the axons and myelinate them. hNS is powered by the Intan electrophysiology (Ephys) data acquisition system allowing stimulation and recording from each electrode longitudinally.
We developed advanced metrics that utilize machine learning algorithms for in-depth Ephys analysis. These metrics include velocity density index (VDI), stimulus threshold, nerve-conduction velocity, and response amplitude. Multiplexing allows other endpoints, in addition to functional Ephys analysis, including transcriptomics, western blot, PCR, image-based neurodegeneration analysis, IHC, LDH, and more.
We demonstrated the ability to induce peripheral neuropathy in vitro with the chemotherapeutic vincristine (VinC) and to delay CIPN onset through SARM1 inhibition with compounds NB-7, DSRM-3716, and WX-02-37. First, we validated our hNS CIPN model using VinC; we established the IC50 via multiple functional and morphological metrics and also identified a CIPN transcriptional profile. Next, co-administration of VinC with the SARM1 inhibitors revealed functional and morphological protection against CIPN over the dosing period. NB-7 and WX-02-37 preserved some neuronal functionality as assessed by VDI for up to 5 days. They also showed morphological protection for the entire 7-day dosing period which implies the potential for recovery. Ephys proved to be the most sensitive of the assays by repeatedly detecting functional dysregulation before neurodegeneration analysis detected morphological dysfunction. The ability to multiplex longitudinal Ephys data with morphological, molecular biology, and next-generation sequencing data in hNS makes it a powerful platform for neurotoxicity screening and drug discovery.