Accumulating evidence suggests that cerebellar dysfunction early in life is associated with autism spectrum disorder (ASD), but the molecular mechanisms underlying the cerebellar deficits at the cellular level are unclear. Tuberous sclerosis complex (TSC) is a neurocutaneous disorder that often presents with ASD. Here, we developed a cerebellar Purkinje cell (PC) model of TSC with patient derived human induced pluripotent stem cells (hiPSCs) to characterize the molecular mechanism underlying cerebellar abnormalities in ASD and TSC. Our results show that hiPSCs-derived PCs from patients with pathogenic TSC2 mutations displayed mTORC1-pathway hyperactivation, defects in neuronal differentiation and RNA regulation, hypoexcitability and reduced synaptic activity when compared to those derived from controls. Our gene expression analyses revealed downregulation of several components of fragile-X mental retardation protein (FMRP) targets in TSC2-deficient hiPSC-PCs. We detected decreased expression of FMRP, glutamate receptor δ2 (GRID2) and pre- and post-synaptic markers such as synaptophysin and PSD95 in the TSC2-deficient hiPSC-PCs. The mTOR-inhibitor rapamycin rescued the deficits in differentiation, synaptic dysfunction and hypoexcitability of TSC2-mutant hiPSC-PCs in vitro. Our findings suggest that these gene expression changes and cellular abnormalities contribute to aberrant PC function during development in TSC affected individuals.

SUMMARY Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disease caused by TSC1 or TSC2 mutations and subsequent activation of the mTORC1 kinase. Upon mTORC1 activation, anabolic metabolism, which requires mitochondria, is induced, yet at the same time the principal pathway for mitochondrial turnover, autophagy, is compromised. How mTORC1 activation impacts mitochondrial turnover in neurons remains unknown. Here we demonstrate impaired mitochondrial homeostasis in neuronal in vitro and in vivo models of TSC. We find that Tsc1/2-deficient neurons accumulate mitochondria in cell bodies but are depleted of axonal mitochondria, including those supporting presynaptic sites. Axonal and global mitophagy of damaged mitochondria is impaired, suggesting that decreased turnover may act upstream of impaired mitochondrial metabolism. Importantly, blocking mTORC1 or inducing mTOR-independent autophagy restores mitochondrial homeostasis. Our study clarifies the complex relationship between the TSC-mTORC1 pathway, autophagy and mitophagy, and defines mitochondrial homeostasis as a therapeutic target for TSC and related diseases.

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young's elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.