The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal sub-domains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.

Background:The effect of HDAC inhibitor kinetic properties on biological function is currently unknown. Results:The kinetic rate constants of HDAC inhibitors differentially affect histone acetylation, cell viability, and gene expression. Conclusion: Evaluating HDAC inhibitor properties using histone acetylation is not predictive of their function on cellular activity. Significance: Characterizing the biological effect of different HDAC inhibitors will help to evaluate their clinical utility.

A common approach to understanding neurodegenerative disease is comparing gene expression in diseased versus healthy tissues. We illustrate that expression profiles derived from whole tissue RNA highly reflect the degenerating tissues' altered cellular composition, not necessarily transcriptional regulation. To accurately understand transcriptional changes that accompany neuropathology, we acutely purify neurons, astrocytes and microglia from single adult mouse brains and analyse their transcriptomes by RNA sequencing. Using peripheral endotoxemia to establish the method, we reveal highly specific transcriptional responses and altered RNA processing in each cell type, with Tnfr1 required for the astrocytic response. Extending the method to an Alzheimer's disease model, we confirm that transcriptomic changes observed in whole tissue are driven primarily by cell type composition, not transcriptional regulation, and identify hundreds of cell type-specific changes undetected in whole tissue RNA. Applying similar methods to additional models and patient tissues will transform our understanding of aberrant gene expression in neurological disease.

G protein–coupled receptors rely on the PDZ domain of SNX27 for endosomal recycling.

Age is the main risk factor for sporadic Alzheimer’s disease. Yet, cognitive decline in aged rodents has been less well studied, possibly due to concomitant changes in sensory or locomotor function that can complicate cognitive tests. We tested mice that were 3, 11, and 23 months old in cognitive, sensory, and motor measures, and postmortem measures of gliosis and neural activity (c-Fos). Hippocampal synaptic function was also examined. While age-related impairments were detectable in tests of spatial memory, greater age-dependent effects were observed in tests of associative learning [active avoidance (AA)]. Gross visual function was largely normal, but startle responses to acoustic stimuli decreased with increased age, possibly due to hearing impairments. Therefore, a novel AA variant in which light alone served as the conditioning stimuli was used. Age-related deficits were again observed. Mild changes in vision, as measured by optokinetic responses, were detected in 19- versus 4-month-old mice, but these were not correlated to AA performance. Thus, deficits in hearing or vision are unlikely to account for the observed deficits in cognitive measures. Increased gliosis was observed in the hippocampal formation at older ages. Age-related changes in neural function and plasticity were observed with decreased c-Fos in the dentate gyrus, and decreased synaptic strength and paired-pulse facilitation in CA1 slices. This work, which carefully outlines age-dependent impairments in cognitive and synaptic function, c-Fos activity, and gliosis during normal aging in the mouse, suggests robust translational measures that will facilitate further study of the biology of aging.