Shared as well as distinct roles of EHD proteins revealed by biochemical and functional comparisons in mammalian cells and C. elegans.

BACKGROUND: The four highly homologous human EHD proteins (EHD1-4) form a distinct subfamily of the Eps15 homology domain-containing protein family and are thought to regulate endocytic recycling. Certain members of this family have been studied in different cellular contexts; however, a lack of concurrent analyses of all four proteins has impeded an appreciation of their redundant versus distinct functions. RESULTS: Here, we analyzed the four EHD proteins both in mammalian cells and in a cross-species complementation assay using a C. elegans mutant lacking the EHD ortholog RME-1. We show that all human EHD proteins rescue the vacuolated intestinal phenotype of C. elegans rme-1 mutant, are simultaneously expressed in a panel of mammalian cell lines and tissues tested, and variably homo- and hetero-oligomerize and colocalize with each other and Rab11, a recycling endosome marker. Small interfering RNA (siRNA) knock-down of EHD1, 2 and 4, and expression of dominant-negative EH domain deletion mutants showed that loss of EHD1 and 3 (and to a lesser extent EHD4) but not EHD2 function retarded transferrin exit from the endocytic recycling compartment. EH domain deletion mutants of EHD1 and 3 but not 2 or 4, induced a striking perinuclear clustering of co-transfected Rab11. Knock-down analyses indicated that EHD1 and 2 regulate the exit of cargo from the recycling endosome while EHD4, similar to that reported for EHD3 (Naslavsky et al. (2006) Mol. Biol. Cell 17, 163), regulates transport from the early endosome to the recycling endosome. CONCLUSION: Altogether, our studies suggest that concurrently expressed human EHD proteins perform shared as well as discrete functions in the endocytic recycling pathway and lay a foundation for future studies to identify and characterize the molecular pathways involved.

Outcomes of intra-arterial thrombolytic treatment in acute ischemic stroke patients with a matched defect on diffusion and perfusion MR images.

BACKGROUND: For acute ischemic stroke patients with matched defects on diffusion-perfusion imaging, the effects of reperfusion therapy remain poorly documented. The outcomes in a rare series of patients who had a matched defect and then underwent intra-arterial thrombolytic treatment (IAT) are reported. METHODS: Medical record and MR image review between 1 January 1998 and 15 October 2008 revealed only eight acute ischemic stroke patients satisfying the atypical combination of both matched defect and IAT. Successful recanalization (SR), favorable clinical response (FCR) and symptomatic intracranial hemorrhage (SICH) were defined respectively as thrombolysis in cerebral infarction score ≥2 after IAT, discharge National Institutes of Health Stroke Scale (NIHSS) 0-1/≥8 point decrease from baseline and intracranial hemorrhage in infarct zone with ≥4 point increase in NIHSS Score within 24 h of IAT. RESULTS: Median (range) baseline NIHSS score was 16.5 (6-22). Median (range) time delays from symptom onset to MRI and to IAT initiation were 200 (83-240) and 267.5 (160-360) min, respectively. Median (range) values of diffusion and perfusion lesion volumes were 119.5 (24-205) and 118 (18-207) ml. Out of eight patients, one (12.5%) achieved FCR, four (50%) had SICH and five (62.5%) died. Out of six patients with SR, one achieved FCR and four had SICH and died, and of two patients without SR, none had FCR or SICH and one died. CONCLUSION: Our data on rare patients with matched defects who nevertheless had attempted rescue with IAT confirm a poor risk-benefit ratio generated by low favorable responses and high mortality rates, especially in large ischemic lesions.