Directing LRRK2 to membranes of the endolysosomal pathway triggers RAB phosphorylation and JIP4 recruitment

Coding mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene, which are associated with dominantly inherited Parkinson's disease (PD), lead to an increased activity of the encoded LRRK2 protein kinase. As such, kinase inhibitors are being considered as therapeutic agents for PD. It is therefore of interest to understand the mechanism(s) by which LRRK2 is activated during cellular signaling. Lysosomal membrane damage represents one way of activating LRRK2 and leads to phosphorylation of downstream RAB substrates and recruitment of the motor adaptor protein JIP4. However, it is unclear whether the activation of LRRK2 would be seen at other membranes of the endolysosomal system, where LRRK2 has also shown to be localized, or whether these signaling events can be induced without membrane damage. Here, we use a rapamycin-dependent oligomerization system to direct LRRK2 to various endomembranes including the Golgi apparatus, lysosomes, the plasma membrane, recycling, early, and late endosomes. Irrespective of membrane location, the recruitment of LRRK2 to membranes results in local accumulation of phosphorylated RAB10, RAB12, and JIP4. We also show that endogenous RAB29, previously nominated as an activator of LRRK2 based on overexpression, is not required for activation of LRRK2 at the Golgi nor lysosome. We therefore conclude that LRRK2 signaling to RAB10, RAB12, and JIP4 can be activated once LRRK2 is accumulated at any cellular organelle along the endolysosomal pathway

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes

Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson’s disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments

Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field still needs to rely on a 50-y-old laborious and time-consuming method to assess the lifespan of yeast cells and to isolate differentially aged cells for microscopic snapshots via manual dissection of daughter cells from the larger mother cell. Here, we are unique in achieving continuous and high-resolution microscopic imaging of the entire replicative lifespan of single yeast cells. Our microfluidic dissection platform features an optically prealigned single focal plane and an integrated array of soft elastomer-based micropads, used together to allow for trapping of mother cells, removal of daughter cells, monitoring gradual changes in aging, and unprecedented microscopic imaging of the whole aging process. Using the platform, we found remarkable age-associated changes in phenotypes (e.g., that cells can show strikingly differential cell and vacuole morphologies at the moment of their deaths), indicating substantial heterogeneity in cell aging and death. We envision the microfluidic dissection platform to become a major tool in aging research.

Economic Values for Perennial Ryegrass Traits in New Zealand Dairy Farm Systems

Perennial ryegrass (Lolium perenne L.) is the main species used in dairy pastures throughout New Zealand. There are approximately 30 perennial ryegrass cultivars sold commercially in New Zealand, but currently there is no evaluation system which allows farmers to compare the potential impact of different cultivars on the profitability of their farm business. Such an economic evaluation system requires information on performance values (PV) for cultivars which quantifies their performance with respect to the major productivity traits (herbage accumulation (HA, kg DM/ha), nutritive value and persistence) relative to a genetic base, and economic values (EV, Doyle and Elliott 1983) which estimate the additional profit resulting from each unit change in the trait of interest (Equation 1). Economic value = Δ operating profit/Δ trait of interest (1) This paper describes a system modelling approach developed to estimate EV for seasonal HA of pasture in the major dairying regions of New Zealand. This information is used in the DairyNZ Forage Value Index system (www.dairynzfvi.co.nz) which is being developed to include information on all three productivity traits for commercially available ryegrass cultivars

Balancing equity and efficiency in the Dutch basic benefits package using the principle of proportional shortfall

Economic evaluations are increasingly used to inform decisions regarding the allocation of scarce health care resources. To systematically incorporate societal preferences into these evaluations, quality-adjusted life year gains could be weighted according to some equity principle, the most suitable of which is a matter of frequent debate. While many countries still struggle with equity concerns for priority setting in health care, the Netherlands has reached a broad consensus to use the concept of proportional shortfall. Our study evaluates the concept and its support in the Dutch health care context. We discuss arguments in the Netherlands for using proportional shortfall and difficulties in transitioning from principle to practice. In doing so, we address universal issues leading to a systematic consideration of equity concerns for priority setting in health care. The article thus has relevance to all countries struggling with the formalization of equity concerns for priority setting

Development of a Forage Evaluation System for Perennial Ryegrass Cultivar and Endophyte Combinations in New Zealand Dairy Systems

An economic index for perennial ryegrass (Lolium perenne L.) cultivars is a relatively new concept, although recently introduced in Ireland (McEvoy et al. 2011). By contrast, in dairy cattle breeding, the concept of an economic index rating animals and economic values underlying that index is well entrenched (Philipson et al. 1994; Veerkamp, 1998). Historically, forage evaluation data for individual cultivars were either displayed using absolute numbers for seasonal dry matter production within a season or across all seasons with a notation to indicate statistical differences, or percentage values where a reference cultivar is 100. The adoption of an economic index and routine evaluation approach for perennial ryegrass provides a method to identify traits of economic importance to focus plant breeding efforts better and to provide clarity for farmers around predicting cultivars that will maximise farm profit. It also allows for routine tracking of genetic gain of individual traits and the economic index. In this paper, the economic based forage evaluation techniques now used in New Zealand for perennial ryegrass cultivar/endophyte combinations are presented

MesoGraph: automatic profiling of mesothelioma subtypes from histological images

Mesothelioma is classified into three histological subtypes, epithelioid, sarcomatoid, and biphasic, according to the relative proportions of epithelioid and sarcomatoid tumor cells present. Current guidelines recommend that the sarcomatoid component of each mesothelioma is quantified, as a higher percentage of sarcomatoid pattern in biphasic mesothelioma shows poorer prognosis. In this work, we develop a dual-task graph neural network (GNN) architecture with ranking loss to learn a model capable of scoring regions of tissue down to cellular resolution. This allows quantitative profiling of a tumor sample according to the aggregate sarcomatoid association score. Tissue is represented by a cell graph with both cell-level morphological and regional features. We use an external multicentric test set from Mesobank, on which we demonstrate the predictive performance of our model. We additionally validate our model predictions through an analysis of the typical morphological features of cells according to their predicted score

Malignant Mesothelioma subtyping via sampling driven multiple instance prediction on tissue image and cell morphology data

Malignant Mesothelioma is a difficult to diagnose and highly lethal cancer usually associated with asbestos exposure. It can be broadly classified into three subtypes: Epithelioid, Sarcomatoid, and a hybrid Biphasic subtype in which significant components of both of the previous subtypes are present. Early diagnosis and identification of the subtype informs treatment and can help improve patient outcome. However, the subtyping of malignant mesothelioma, and specifically the recognition of transitional features from routine histology slides has a high level of inter-observer variability. In this work, we propose an end-to-end multiple instance learning (MIL) approach for malignant mesothelioma subtyping. This uses an adaptive instance-based sampling scheme for training deep convolutional neural networks on bags of image patches that allows learning on a wider range of relevant instances compared to max or top-N based MIL approaches. We also investigate augmenting the instance representation to include aggregate cellular morphology features from cell segmentation. The proposed MIL approach enables identification of malignant mesothelial subtypes of specific tissue regions. From this a continuous characterisation of a sample according to predominance of sarcomatoid vs epithelioid regions is possible, thus avoiding the arbitrary and highly subjective categorisation by currently used subtypes. Instance scoring also enables studying tumor heterogeneity and identifying patterns associated with different subtypes. We have evaluated the proposed method on a dataset of 234 tissue micro-array cores with an AUROC of 0.89±0.05 for this task. The dataset and developed methodology is available for the community at: https://github.com/measty/PINS

mTOR independent alteration in ULK1 Ser758 phosphorylation following chronic LRRK2 kinase inhibition

Unc-51 Like Kinase 1 (ULK1) is a critical regulator of the biogenesis of autophagosomes, the central component of the catabolic macroautophagy pathway. Regulation of ULK1 activity is dependent upon several phosphorylation events acting to repress or activate the enzymatic function of this protein. Phosphorylation of Ser758 ULK1 has been linked to repression of autophagosome biogenesis and was thought to be exclusively dependent upon mTOR complex 1 kinase activity. In this study, a novel regulation of Ser758 ULK1 phosphorylation is reported following prolonged inhibition of the Parkinson's disease linked protein Leucine Rich Repeat Kinase 2 (LRRK2). Here, modulation of Ser758 ULK1 phosphorylation following LRRK2 inhibition is decoupled from the repression of autophagosome biogenesis and independent of mTOR complex 1 activity