Tom Dunkley

mTORC1 Inhibition Corrects Neurodevelopmental and Synaptic Alterations in a Human Stem Cell Model of Tuberous Sclerosis

Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD), including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2(+/-) and TSC2(-/-) neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis.

Quantitative ADME Proteomics – CYP and UGT Enzymes in the Beagle Dog Liver and Intestine

PurposeBeagle dogs are used to study oral pharmacokinetics and guide development of drug formulations for human use. Since mechanistic insight into species differences is needed to translate findings in this species to human, abundances of cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) drug metabolizing enzymes have been quantified in dog liver and intestine.MethodsAbundances of enzymes were measured in Beagle dog intestine and liver using selected reaction monitoring mass spectrometry.ResultsSeven and two CYPs were present in the liver and intestine, respectively. CYP3A12 was the most abundant CYP in both tissues. Seven UGT enzymes were quantified in the liver and seven in the intestine although UGT1A11 and UGT1A9 were present only in the intestine and UGT1A7 and UGT2B31 were found only in the liver. UGT1A11 and UGT1A2 were the most abundant UGTs in the intestine and UGT2B31 was the most abundant UGT in the liver. Summed abundance of UGT enzymes was similar to the sum of CYP enzymes in the liver whereas intestinal UGTs were up to four times more abundant than CYPs. The estimated coefficients of variation of abundance estimates in the livers of 14 donors were separated into biological and technical components which ranged from 14 to 49% and 20 to 39%, respectively.ConclusionsAbundances of canine CYP enzymes in liver and intestine have been confirmed in a larger number of dogs and UGT abundances have been quantified for the first time. The biological variability in hepatic CYPs and UGTs has also been estimated.

Characterization of a human pluripotent stem cell-derived model of neuronal development using multiplexed targeted proteomics

Human pluripotent stem cell (hPSC)‐derived cellular models have great potential to enable drug discovery and improve translation of preclinical insights to the clinic. We have developed a hPSC‐derived neural precursor cell model for studying early events in human brain development. We present protein‐level characterization of this model, using a multiplexed SRM approach, to establish reproducibility and physiological relevance; essential prerequisites for utilization of the neuronal development model in phenotypic screening‐based drug discovery.

HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment.

Chapter 3:Targeted Proteomics to Support Transporter IVIVE and PBPK

Determining the activity of transporter proteins expressed in tissues and in in vitro models is a key component for building a quantitative understanding of the impact of drug transporters on the pharmacokinetics of their substrate compounds. The abundance of a transporter protein in vivo is an important determinant of its activity. Gene expression is not always a good surrogate for corresponding protein abundance in tissues, necessitating absolute quantification of proteins to understand transporter abundance. Immunochemical methods of protein quantification are limited by the availability of specific antibodies and protein standards, thus restricting absolute quantification of drug transporters. Recent advances in MS based proteomics have, however, resulted in a rapid increase in transporter protein quantification data in the literature, which have the potential to contribute considerably to our ability to account for active transport in in vitro to in vivo extrapolation and physiologically based pharmacokinetic modelling. The aim of this chapter is to give an overview of targeted mass spectrometry proteomics methods applied to drug transporters, and to discuss the utility and limitations of the absolute protein quantification data obtained.

N-Terminomics identifies HtrA1 cleavage of thrombospondin-1 with generation of a proangiogenic fragment in the polarized retinal pigment epithelial cell model of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population. Variants in the HTRA1-ARMS2 locus have been linked to increased AMD risk. In the present study we investigated the impact of elevated HtrA1 levels on the retina pigment epithelial (RPE) secretome using a polarized culture system. Upregulation of HtrA1 alters the abundance of key proteins involved in angiogenesis and extracellular matrix remodeling. Thrombospondin-1, an angiogenesis modulator, was identified as a substrate for HtrA1 using terminal amine isotope labeling of substrates in conjunction with HtrA1 specificity profiling. HtrA1 cleavage of thrombospondin-1 was further corroborated by in vitro cleavage assays and targeted proteomics together with small molecule inhibition of HtrA1. While thrombospondin-1 is anti-angiogenic, the proteolytically released N-terminal fragment promotes the formation of tube-like structure by endothelial cells. Taken together, our findings suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier. For quantitative secretome analysis, project accession: PXD007691, username: reviewer45093@ebi.ac.uk, password: 1FUpS6Yq. For TAILS analysis, project accession: PXD007139, username: reviewer76731@ebi.ac.uk, password: sNbMp7xK.

HtrA1 activation is driven by an allosteric mechanism of inter-monomer communication

The human protease family HtrA is responsible for preventing protein misfolding and mislocalization, and a key player in several cellular processes. Among these, HtrA1 is implicated in several cancers, cerebrovascular disease and age-related macular degeneration. Currently, HtrA1 activation is not fully characterized and relevant for drug-targeting this protease. Our work provides a mechanistic step-by-step description of HtrA1 activation and regulation. We report that the HtrA1 trimer is regulated by an allosteric mechanism by which monomers relay the activation signal to each other, in a PDZ-domain independent fashion. Notably, we show that inhibitor binding is precluded if HtrA1 monomers cannot communicate with each other. Our study establishes how HtrA1 trimerization plays a fundamental role in proteolytic activity. Moreover, it offers a structural explanation for HtrA1-defective pathologies as well as mechanistic insights into the degradation of complex extracellular fibrils such as tubulin, amyloid beta and tau that belong to the repertoire of HtrA1.