Interindividual variation in human cortical cell type abundance and expression.

Single-cell transcriptomic studies have identified a conserved set of neocortical cell types from small postmortem cohorts. We extended these efforts by assessing cell type variation across 75 adult individuals undergoing epilepsy and tumor surgeries. Nearly all nuclei map to one of 125 robust cell types identified in the middle temporal gyrus. However, we found interindividual variance in abundances and gene expression signatures, particularly in deep-layer glutamatergic neurons and microglia. A minority of donor variance is explainable by age, sex, ancestry, disease state, and cell state. Genomic variation was associated with expression of 150 to 250 genes for most cell types. This characterization of cellular variation provides a baseline for cell typing in health and disease.

Protein interaction network analysis of mTOR signaling reveals modular organization.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts as a central mediator of translation and plays important roles in cell growth, synaptic plasticity, cancer, and a wide range of developmental disorders. The signaling cascade linking lipid kinases (phosphoinositide 3-kinases), protein kinases (AKT), and translation initiation complexes (EIFs) to mTOR has been extensively modeled, but does not fully describe mTOR system behavior. Here, we use quantitative multiplex coimmunoprecipitation to monitor a protein interaction network (PIN) composed of 300+ binary interactions among mTOR-related proteins. Using a simple model system of serum-deprived or fresh-media-fed mouse 3T3 fibroblasts, we observed extensive PIN remodeling involving 27+ individual protein interactions after 1 h, despite phosphorylation changes observed after only 5 min. Using small molecule inhibitors of phosphoinositide 3-kinase, AKT, mTOR, MEK and ERK, we define subsets of the PIN, termed "modules", that respond differently to each inhibitor. Using primary fibroblasts from individuals with overgrowth disorders caused by pathogenic PIK3CA or MTOR variants, we find that hyperactivation of mTOR pathway components is reflected in a hyperactive PIN. Our data define a "modular" organization of the mTOR PIN in which coordinated groups of interactions respond to the activation or inhibition of distinct nodes, and demonstrate that kinase inhibitors affect the modular network architecture in a complex manner, inconsistent with simple linear models of signal transduction.

Protein interaction network analysis of mTOR signaling reveals modular organization.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts as a central mediator of translation, and plays important roles in cell growth, synaptic plasticity, cancer, and a wide range of developmental disorders. The signaling cascade linking lipid kinases (PI3Ks), protein kinases (AKT) and translation initiation complexes (EIFs) to mTOR has been extensively modeled, but does not fully describe mTOR system behavior. Here, we use quantitative multiplex co-immunoprecipitation to monitor a protein interaction network (PIN) composed of 300+ binary interactions among mTOR-related proteins. Using a simple model system of serum deprived or fresh-media-fed mouse 3T3 fibroblasts, we observed extensive PIN remodeling involving 27+ individual protein interactions after one hour, despite phosphorylation changes observed after only five minutes. Using small molecule inhibitors of PI3K, AKT, mTOR, MEK and ERK, we define subsets of the PIN, termed 'modules', that respond differently to each inhibitor. Using primary fibroblasts from individuals with overgrowth disorders caused by pathogenic PIK3CA or MTOR variants, we find that hyperactivation of mTOR pathway components is reflected in a hyperactive PIN. Our data define a "modular" organization of the mTOR PIN in which coordinated groups of interactions respond to activation or inhibition of distinct nodes, and demonstrate that kinase inhibitors affect the modular network architecture in a complex manner, inconsistent with simple linear models of signal transduction.

Profiling PI3K-AKT-MTOR variants in focal brain malformations reveals new insights for diagnostic care.

Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.