Advanced Microscopy for Liver and Gut Ultrastructural Pathology in Patients with MVID and PFIC Caused by MYO5B Mutations.

Michael W Hess,Iris M Krainer,Andreas R Janecke,Lukas A Huber,Georg F Vogel,Heinz Zoller,Denise Aldrian,James R Goldenring,Ekkehard Sturm,Thomas Müller,Ilja Vietor,Barbara Witting,Karin Gutleben,Przemyslaw A Filipek

doi:10.3390/jcm10091901

Abstract

Mutations in the actin motor protein myosinVb (myo5b) cause aberrant apical cargo transport and the congenital enteropathy microvillus inclusion disease (MVID). Recently, missense mutations in myo5b were also associated with progressive familial intrahepatic cholestasis (MYO5B-PFIC). Here, we thoroughly characterized the ultrastructural and immuno-cytochemical phenotype of hepatocytes and duodenal enterocytes from a unique case of an adult MYO5B-PFIC patient who showed constant hepatopathy but only periodic enteric symptoms. Selected data from two other patients supported the findings. Advanced methods such as cryo-fixation, freeze-substitution, immuno-gold labeling, electron tomography and immuno-fluorescence microscopy complemented the standard procedures. Liver biopsies showed mislocalization of Rab11 and bile canalicular membrane proteins. Rab11-positive vesicles clustered around bile canaliculi and resembled subapical clusters of aberrant recycling endosomes in enterocytes from MVID patients. The adult patient studied in detail showed a severe, MVID-specific enterocyte phenotype, despite only a mild clinical intestinal presentation. This included mislocalization of numerous proteins essential for apical cargo transport and morphological alterations. We characterized the heterogeneous population of large catabolic organelles regarding their complex ultrastructure and differential distribution of autophagic and lysosomal marker proteins. Finally, we generated duodenal organoids/enteroids from biopsies that recapitulated all MVID hallmarks, demonstrating the potential of this disease model for personalized medicine.

Highlights

For EM we analyzed duodenal samples from 2 patients with functional dyspepsia and liver samples from one patient with Friedreich’s ataxia, from one patient with alpha-1 antitrypsin deficiency, and from a healthy donor liver
Complementary sample preparation and microscopy techniques we comparatively documented here the ultrastructural and immuno-cytochemical phenotypes of a MYO5B-PFIC patient in great detail, who present besides a constant hepatopathy only periodically with clinical intestinal symptoms. Those findings were supplemented by selected data from two other relevant cases
Both in hepatocytes and enterocytes from patient #1 the biosynthetic compartments appear functional and even hypertrophic. This is deduced from the constant presence of dilated rough endoplasmic reticulum (rER) with numerous ER-exit buds, enlarged Trans Golgi Network (TGN) and/or Golgi cisternae, as well abundant smooth ER (sER) in hepatocytes—and inflated TGN in enterocytes

Summary

Introduction

High-resolution imaging and preparation techniques for cell and tissue samples have considerably improved during the past decades. Biopsies from patients must be quickly processed and analyzed for diagnosis and treatment by using standard methods and instrumentation [1,2]. Research laboratories frequently benefit of amenable disease models and more complex instrumentation, as well as a wider range of analytical techniques. Animal or cell models and organoid cultures serve as valuable complement, overcoming the always limited availability of fresh patient samples [3,4,5,6,7,8,9,10,11,12,13,14,15,16]

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Results

Conclusion