Geoff Clarke

A one-hit model of cell death in inherited neuronal degenerations

In genetic disorders associated with premature neuronal death, symptoms may not appear for years or decades. This delay in clinical onset is often assumed to reflect the occurrence of age-dependent cumulative damage. For example, it has been suggested that oxidative stress disrupts metabolism in neurological degenerative disorders by the cumulative damage of essential macromolecules. A prediction of the cumulative damage hypothesis is that the probability of cell death will increase over time. Here we show in contrast that the kinetics of neuronal death in 12 models of photoreceptor degeneration, hippocampal neurons undergoing excitotoxic cell death, a mouse model of cerebellar degeneration and Parkinsons and Huntingtons diseases are all exponential and better explained by mathematical models in which the risk of cell death remains constant or decreases exponentially with age. These kinetics argue against the cumulative damage hypothesis; instead, the time of death of any neuron is random. Our findings are most simply accommodated by a ‘one-hit’ biochemical model in which mutation imposes a mutant steady state on the neuron and a single event randomly initiates cell death. This model appears to be common to many forms of neurodegeneration and has implications for therapeutic strategies.

Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis.

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2−/− mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1−/− mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1−/− mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1−/− rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is ≈60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.

Predictive microfluidic control of regulatory ligand trajectories in individual pluripotent cells.

Local (cell-level) signaling environments, regulated by autocrine and paracrine signaling, and modulated by cell organization, are hypothesized to be fundamental stem cell fate control mechanisms used during development. It has, however, been challenging to demonstrate the impact of cell-level organization on stem cell fate control and to relate stem cell fate outcomes to autocrine and paracrine signaling. We address this fundamental problem using a combined in silico and experimental approach in which we directly manipulate, using laminar fluid flow, the local impact of endogenously secreted gp130-activating ligands and their activation of signal transducer and activator of transcription3 (STAT3) signaling in mouse embryonic stem cells (mESC). Our model analysis predicted that flow-dependent changes in autocrine and paracrine ligand binding would impact heterogeneity in cell- and colony-level STAT3 signaling activation and cause a gradient of cell fate determination along the direction of flow. Interestingly, analysis also predicted that local cell density would be inversely proportional to the degree to which endogenous secretion contributed to cell fate determination. Experimental validation using functional activation of STAT3 by secreted factors under microfluidic perfusion culture demonstrated that STAT3 activation and consequently mESC fate were manipulable by flow rate, position in the flow field, and local cell organization. As a unique demonstration of how quantitative control of autocrine and paracrine signaling can be integrated with spatial organization to elicit higher order cell fate effects, this work provides a general template to investigate organizing principles due to secreted factors.

Rational bioprocess design for human pluripotent stem cell expansion and endoderm differentiation based on cellular dynamics

We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation, and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and, with quantitative cell division tracking and fate monitoring, identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion, during directed differentiation, to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system.

Recent advances in the molecular basis of inherited photoreceptor degeneration.

To date, 118 loci have been associated with photoreceptor degenerative disease. In this review, we will discuss recent advances in the identification of genes that cause progressive photoreceptor cell death when mutated. We will focus on 12 genes isolated within the last two years that have been shown to be photoreceptor‐specific, or that have provided insight into photoreceptor biology and the mechanisms of photoreceptor cell death. To aid in understanding the biologic basis for these diseases, we also briefly review photoreceptor biology. Finally, we report on recent advances towards the treatment of these disorders.

Heterogeneous cellular environments modulate one-hit neuronal death kinetics

We recently demonstrated that cell loss kinetics in diverse forms of neurodegeneration (ND) suggests a universal death switch mechanism in which each cell is at a constant risk to initiate apoptosis. We proposed that mutant and injured neurons exist in a viable state typified by an increased risk of initiating death processes [Clarke, Collins, Leavitt, Andrews, Hayden, Lumsden, McInnes, A one-hit model of cell death in inherited neuronal degenerations, Nature 406 (2000) 195-199]. To date, however, measurements of cell death risk have been available only as averages across the affected cell population. Here we develop and apply a method of death kinetic analysis in which the risk factors vary across the neuronal population, as for example due to regional heterogeneities in the cellular microenvironment. We find that most cases of ND for which cell loss data has been obtained are better explained by death risks that vary from cell to cell, compared to death risk that is constant across the neuronal population. Strikingly, a common form of the frequency distribution defining the death risk heterogeneity is shared across most of these cases. This first characterization of the kinetic heterogeneity in one-hit neuronal death, therefore, suggests that the wide variety of ND now known may share mechanisms through which regional differences in the cellular microenvironment modulate the kinetics of cell loss.

Computational Modeling and Stem Cell Engineering

A key goal of regenerative medicine and bioengineering is the quantitative and robust control over the fate and behavior of individual cells and their populations, both in vitro and in vivo. Central to this endeavor are stem cells (SCs), which can be functionally defined as undifferentiated cells of a multicellular organism that balance the capacity for sustained self-renewal with the potential to differentiate into specialized cell types. The biology of multicellular organisms necessitates the existence and precise control of SCs to facilitate development from a single cell during embryogenesis, and tissue homeostasis in the face of continual loss of terminally differentiated cells. It is therefore not surprising that SCs have been identified and isolated from numerous adult human tissues, as well as more recently, the inner cell mass of the preimplantation human blastocyst. SCs promise a renewable source of human tissue for research, pharmaceutical testing, and cell-based therapies. Fulfilling this promise will require not only the precise control of SC self-renewal and differentiation, but also imposing this control on the formation of more functionally complex tissue-like structures.