Endocytic pathways of pathogenic protein aggregates in neurodegenerative diseases

Endocytosis is the fundamental uptake process through which cells internalize extracellular materials and species. Neurodegenerative diseases (NDs) are characterized by a progressive accumulation of intrinsically disordered protein species, leading to neuronal death. Misfolding in many proteins leads to various NDs such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and other disorders. Despite the significance of disordered protein species in neurodegeneration, their spread between cells and the cellular uptake of extracellular species is not entirely understood. This review discusses the major internalization mechanisms of the different conformer species of these proteins and their endocytic mechanisms. We briefly introduce the broad types of endocytic mechanisms found in cells and then summarize what is known about the endocytosis of monomeric, oligomeric and aggregated conformations of tau, Aβ, α-Syn, Huntingtin, Prions, SOD1, TDP-43 and other proteins associated with neurodegeneration. We also highlight the key players involved in internalizing these disordered proteins and the several techniques and approaches to identify their endocytic mechanisms. Finally, we discuss the obstacles involved in studying the endocytosis of these protein species and the need to develop better techniques to elucidate the uptake mechanisms of a particular disordered protein species.     

Enhancing Data Reliability in TOMAHAQ for Large‐Scale Protein Quantification

The analytical scale of most mass‐spectrometry‐based targeted proteomics assays is usually limited by assay performance and instrument utilization. A recently introduced method, called triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantitation (TOMAHAQ), combines both peptide and sample multiplexing to simultaneously improve analytical scale and quantitative performance. In the present work, critical technical requirements and data analysis considerations for successful implementation of the TOMAHAQ technique based on the study of a total of 185 target peptides across over 200 clinical plasma samples are discussed. Importantly, it is observed that significant interference originate from the TMTzero reporter ion used for the synthetic trigger peptides. This interference is not expected because only TMT10plex reporter ions from the target peptides should be observed under typical TOMAHAQ conditions. In order to unlock the great promise of the technique for high throughput quantification, here a post‐acquisition data correction strategy to deconvolute the reporter ion superposition and recover reliable data is proposed.     

Recognition of Childhood Overweight during Health Supervision Visits: Does BMI Help Pediatricians?

Objective: To assess, in diverse pediatric practices, the frequency of overweight/obesity (OW/OB) identification during health supervision visits and its association with BMI curve use. Research Methods and Procedures: Pediatricians in public and private practice in St. Louis, MO, participated in a study of the care of chronic conditions during health supervision visits. Requested information from 30 visits per pediatrician of children 6 to 17 years of age included the visit note, the growth chart, and a one‐page questionnaire about patient demographics and visit content. Pediatricians indicated the presence and discussion of common chronic conditions, including OW/OB. Identification was compared with patient BMI category, and associations between identification and patient and visit characteristics, including BMI curve use, were examined. Results: Twenty‐one (40%) of contacted pediatricians returned information from 557 visits. Pediatricians identified OW/OB in 27% of children with a BMI at the 85th to 94th percentile and 86% of children with a BMI at or above the 95th percentile. Identification was higher in adolescents but was not associated with patient sex or race, practice setting, insurance type, or visit length. Only 41% of growth charts were current, and 6.1% had BMI plotted. BMI plotting was associated with OW/OB identification when the BMI was at the 85th to 94th percentile but not when the BMI was at or above the 95th percentile. After controlling for BMI percentile, OW/OB identification was significantly associated with diet counseling (odds ratio, 7.46; 95% confidence interval, 3.42 to 16.24) and exercise counseling (odds ratio, 5.57; 95% confidence interval, 2.61 to 11.90). Discussion: Despite low BMI curve use, pediatricians recognized most overweight/obese children with a BMI at or above the 95th percentile. BMI plotting may increase recognition in mildly overweight children.     

The C‐terminal tails of heterotrimeric kinesin‐2 motor subunits directly bind to α‐tubulin1: Possible implications for cilia‐specific tubulin entry

The assembly of microtubule‐based cytoskeleton propels the cilia and flagella growth. Previous studies have indicated that the kinesin‐2 family motors transport tubulin into the cilia through intraflagellar transport. Here, we report a direct interaction between the C‐terminal tail fragments of heterotrimeric kinesin‐2 and α‐tubulin1 isoforms in vitro. Blot overlay screen, affinity purification from tissue extracts, cosedimentation with subtilisin‐treated microtubule and LC‐ESI‐MS/MS characterization of the tail‐fragment‐associated tubulin identified an association between the tail domains and α‐tubulin1A/D isotype. The interaction was confirmed by Forster's resonance energy transfer assay in tissue‐cultured cells. The overexpression of the recombinant tails in NIH3T3 cells affected the primary cilia growth, which was rescued by coexpression of a α‐tubulin1 transgene. Furthermore, fluorescent recovery after photobleach analysis in the olfactory cilia of Drosophila indicated that tubulin is transported in a non‐particulate form requiring kinesin‐2. These results provide additional new insight into the mechanisms underlying selective tubulin isoform enrichment in the cilia.