Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

Summary

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

Abstract

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from α-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Introduction

Amyloid-related disorders, known as amyloidoses, constitute a large group of diseases defined by the appearance of insoluble protein deposits in different tissues and organs^1,2. Among them, neurodegenerative disorders are the most frequently forms in which protein aggregates appear in the central or peripheral nervous system². Although a number of mechanisms have been proposed to be involved in the toxicity of amyloid aggregates³, a growing body of evidence points to cell membrane disruption and permeabilization as the primary mechanism of amyloid pathology

Protocol

All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of Medical Sciences of Tehran University. Maximal efforts were made to minimize suffering and detrimental effects to the rats by sharpening the guillotine blades and applying resolute and swift movements of the blade.

1. Brain homogenization and mitochondrial isolation

NOTE: All reagents for mitochondrial isolation were prepared according to Sims and Anders.......

Discussion

A wealth of experimental results supports the hypothesis that the cytotoxicity of fibrillar aggregates is significantly associated with their ability to interact with and permeabilize biological membranes^4,5. However, most of the data are based on artificial lipid bilayers that do not necessarily reflect the intrinsic properties of biological membranes, which are heterogeneous structures with a wide variety of phospholipids and proteins. Here, using brain mitocho.......

Materials

Name	Company	Catalog Number	Comments
2′,7′-Dichlorodihydrofluorescein diacetate	Sigma	35845
Ammonium sulfate	Merck	1012171000
Black 96-well plate	Corning
Black Clear-bottomed 96-well plate	Corning
Bovine insulin	Sigma	I6634
Bovine Serum Albumin (BSA)	Sigma	A2153
BSA essentially fatty acid-free	Sigma	A6003
Centrifuge	Sigma
Crystal clear sealing tape	Corning
CuSO4	Sigma	451657
Dialysis bag (cut off 2 KDa)	Sigma	D2272
Dounce homogenizer	Potter Elvehjem
EDTA	Sigma	E9884
Fluorescence plate reader	BioTek
Fluorescence spectrophotometer	Cary Eclipse VARIAN
Folin	Merck	F9252
Glycine	Sigma	G7126
Guillotine	Made in Iran
HCl	Merck	H1758
Hen Egg White Lysozyme (HEWL)	Sigma	L6876
Na2CO3	Sigma	S7795
NaH2PO4	Sigma	S7907
NaOH	Merck	S8045
Oxaloacetate	Sigma	O4126
Percoll	GE Healthcare
Phosphate Buffer Saline (PBS)	Sigma	CS0030
PMSF	Sigma	P7626
Potassium sodium tartrate	Sigma	217255
Quartz cuvette	Sigma
Spectrophotometer	analytik jena	SPEKOL 2000 model
Succinate	Sigma	S2378
Sucrose	Merck	1076871000
Thermomixer	Eppendorph
Thioflavin T	Sigma	T3516
Tris-HCl	Merck	1082191000
Triton X-100	Sigma	T9284
Tryptone	QUELAB
Water bath	Memmert
Yeast Extract	QUELAB
β-NADH	Sigma	N8129