Deicing Impacts on the Danforth Campus, Fall 2020

De-Icing Impacts on the Danforth Campus, Sustainability Exchange, Washington University in St. Louis, Fall 2020

Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity

Background: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson’s disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10. Objective: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols. Methods: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers. Results: Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated. Conclusion: Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution

Inhibition of LRRK2 kinase activity promotes anterograde axonal transport and presynaptic targeting of α-synuclein

Abstract Pathologic inclusions composed of α-synuclein called Lewy pathology are hallmarks of Parkinson’s Disease (PD). Dominant inherited mutations in leucine rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. Lewy pathology is found in the majority of individuals with LRRK2-PD, particularly those with the G2019S-LRRK2 mutation. Lewy pathology in LRRK2-PD associates with increased non-motor symptoms such as cognitive deficits, anxiety, and orthostatic hypotension. Thus, understanding the relationship between LRRK2 and α-synuclein could be important for determining the mechanisms of non-motor symptoms. In PD models, expression of mutant LRRK2 reduces membrane localization of α-synuclein, and enhances formation of pathologic α-synuclein, particularly when synaptic activity is increased. α-Synuclein and LRRK2 both localize to the presynaptic terminal. LRRK2 plays a role in membrane traffic, including axonal transport, and therefore may influence α-synuclein synaptic localization. This study shows that LRRK2 kinase activity influences α-synuclein targeting to the presynaptic terminal. We used the selective LRRK2 kinase inhibitors, MLi-2 and PF-06685360 (PF-360) to determine the impact of reduced LRRK2 kinase activity on presynaptic localization of α-synuclein. Expansion microscopy (ExM) in primary hippocampal cultures and the mouse striatum, in vivo, was used to more precisely resolve the presynaptic localization of α-synuclein. Live imaging of axonal transport of α-synuclein-GFP was used to investigate the impact of LRRK2 kinase inhibition on α-synuclein axonal transport towards the presynaptic terminal. Reduced LRRK2 kinase activity increases α-synuclein overlap with presynaptic markers in primary neurons, and increases anterograde axonal transport of α-synuclein-GFP. In vivo, LRRK2 inhibition increases α-synuclein overlap with glutamatergic, cortico-striatal terminals, and dopaminergic nigral-striatal presynaptic terminals. The findings suggest that LRRK2 kinase activity plays a role in axonal transport, and presynaptic targeting of α-synuclein. These data provide potential mechanisms by which LRRK2-mediated perturbations of α-synuclein localization could cause pathology in both LRRK2-PD, and idiopathic PD

Associations between inhibitory control, eating behaviours and adiposity in 6 year-old children

BackgroundLower inhibitory control has been associated with obesity. One prediction is that lower inhibitory control underlies eating behaviours that promote increased energy intakes. This study examined the relationships between children’s inhibitory control measured using the Stop Signal Task (SST), body composition and eating behaviours, which included self-served portion size, number of servings, eating rate, and energy intake at lunch and in an eating in the absence of hunger (EAH) task.MethodsThe sample included 255 6-year-old children from an Asian cohort. Stop-signal reaction time (SSRT) was used as an index of inhibitory control. Children participated in a recorded self-served lunchtime meal, followed by the EAH task where they were exposed to energy-dense snacks. Behavioural coding of oral processing was used to estimate eating rates (g/min). BMI, waist circumference and skinfolds were used as indices of adiposity.ResultsChildren with lower inhibitory control tended to self-serve larger food portions (p = 0.054), had multiple food servings (p = 0.006) and significantly faster eating rates (p = 0.041). Inhibitory control did not predict energy intake at lunch (p = 0.17) or during the EAH task (p = 0.45), and was unrelated to measures of adiposity (p &gt; 0.32). Twenty percent of the children in the sample had problems focusing on the SST and were described as ‘restless’. Post-hoc analysis revealed that these children had lower inhibitory control (p &lt; 0.001) and consumed more energy during the EAH task (p = 0.01), but did not differ in any other key outcomes from the rest of the sample (p &gt; 0.1).ConclusionsChildren with lower inhibitory control showed a trend to select larger food portions, had multiple food servings and faster eating rates, but were equally as responsive to snacks served in the absence of hunger as children with better inhibitory control. Inhibitory control may impact a number of eating behaviours, not limited to energy-dense snacks.</p