Genetically engineered human cortical spheroid models of tuberous sclerosis.

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder caused by mutations in the TSC1 or TSC2 genes, whose protein products are negative regulators of mechanistic target of rapamycin complex 1 signaling. Hallmark pathologies of TSC are cortical tubers-regions of dysmorphic, disorganized neurons and glia in the cortex that are linked to epileptogenesis. To determine the developmental origin of tuber cells, we established human cellular models of TSC by CRISPR-Cas9-mediated gene editing of TSC1 or TSC2 in human pluripotent stem cells (hPSCs). Using heterozygous TSC2 hPSCs with a conditional mutation in the functional allele, we show that mosaic biallelic inactivation during neural progenitor expansion is necessary for the formation of dysplastic cells and increased glia production in three-dimensional cortical spheroids. Our findings provide support for the second-hit model of cortical tuber formation and suggest that variable developmental timing of somatic mutations could contribute to the heterogeneity in the neurological presentation of TSC

Corticostriatal Transmission Is Selectively Enhanced in Striatonigral Neurons with Postnatal Loss of Tsc1.

mTORC1 is a central signaling hub that integrates intra- and extracellular signals to regulate a variety of cellular metabolic processes. Mutations in regulators of mTORC1 lead to neurodevelopmental disorders associated with autism, which is characterized by repetitive, inflexible behaviors. These behaviors may result from alterations in striatal circuits that control motor learning and habit formation. However, the consequences of mTORC1 dysregulation on striatal neuron function are largely unknown. To investigate this, we deleted the mTORC1 negative regulator Tsc1 from identified striatonigral and striatopallidal neurons and examined how cell-autonomous upregulation of mTORC1 activity affects their morphology and physiology. We find that loss of Tsc1 increases the excitability of striatonigral, but not striatopallidal, neurons and selectively enhances corticostriatal synaptic transmission. These findings highlight the critical role of mTORC1 in regulating striatal activity in a cell type- and input-specific manner, with implications for striatonigral pathway dysfunction in neuropsychiatric disease

The Differential Contribution of Striatonigral and Striatopallidal Neurons in Mediating Responses to Therapeutic Agents and Drugs of Abuse: A Dual Role for DARPP-32

The basal ganglia are a set of subcortical structures which integrate information from diverse brain areas to coordinate vital behaviors including movement, reward, and motivational processes. The striatum is the main input center of the basal ganglia which sends projections to the output nuclei via two pathways, the direct striatonigral pathway and the indirect striatopallidal pathway. These two pathways work together to modulate behavior and imbalance of these pathways can have profound physiological consequences. DARPP-32 is a dual function kinase/phosphatase inhibitor which has been shown to be a key mediator of signaling in both striatonigral and triatopallidal neurons. A variety of therapeutic agents and drugs of abuse can affect the phosphorylation of DARPP-32. Psychostimulants such as cocaine increase DARPP-32 phosphorylation at its main regulatory site, T34. Paradoxically, antipsychotics such as haloperidol also increase T34 phosphorylation to a similar degree. Despite this similar biochemical regulation, psychostimulants and antipsychotics have opposing behavioral and clinical effects. We hypothesized that these drugs act via the same biochemical pathway but in distinct populations of striatal neurons. To directly test this idea, we generated BAC transgenic mice which express epitope tagged DARPP-32 selectively in striatonigral and striatopallidal neurons using the D1 and D2 receptor promoters. We developed a protocol to immunoprecipitate DARPP-32 from drug treated mice and study phosphorylation in a cell-type specific manner. Using this new methodology we demonstrate that the increases in T34 phosphorylation with acute cocaine and haloperidol are restricted to striatonigral and striatopallidal neurons, respectively. Additionally, we show that the changes in DARPP-32 phosphorylation induced by a variety of drugs targeting the striatum have cell-type specific patterns. In a complimentary approach, we generated conditional knock-out mice in which DARPP-32 is selectively deleted in striatonigral or striatopallidal neurons. This allowed us to study the behavioral consequences of alteration in the direct and indirect pathways on psychostimulant and antipsychotic mediated locomotor behavior. These studies provided direct evidence for the theory that the direct and indirect pathways exert opposing influences on locomotor behavior. Additionally, we showed that dopamine can differentially modulate activity in these pathways resulting in a synergistic stimulation of locomotor activity

Ignition length study of jp-8 + 100 in a supersonic duct

In scramjets, hydrocarbon fuels are being considered for their endothermic potential and for use in flights where compact, volume critical designs are required at flight speeds at Mach 5+. Long-chain hydrocarbon (LCHC) fuels, like aviation kerosene, have handling and storage advantages over hazardous and volatile fuels, like hydrogen, that are more aligned with current flight systems. This research investigates the conditions under which kerosene fuel (JP-8 + 100) may be used in a supersonic duct at a hypervelocity impulse facility. Experimental data on kerosene ignition lengths for temperatures in the range 1100-1550 K, pressure of 1 atm, and equivalence ratios of 0.2-2.5 are compared with ignition delay correlations from literature