Influence of iron overload on neurons in artificially induced Alzheimer"s disease

Abstract text (incl. figure legends and references)

Introduction:

Organotypic cultures (OTC) represent a promising method to study neurodegenerative diseases with the advantage of preservation of original architecture and connections. We established Alzheimer"s disease (AD) condition ex-vivo in the hippocampus region of the porcine brain and the human brain. We designed a simple protocol to generate the fibrils from the amyloid beta (Aβ) peptide prior to our treatments. We used this as our model to study the influence of iron accumulation in AD pathology. Iron is an essential and most abundant trace element in the human body. Iron deposition in AD is correlated with free radical damage, oxidative stress, amyloid beta plaques, etc1,2. Several studies report the increase in toxicity of AD due to iron overload3. Hence, we hypothesize that the unexplained toxicity due to the accumulation of iron in the brain could be due to its effect on the structure of the Aβ fibrils. To understand the same, we plan to mimic the iron accumulation in the brain using ferric citrate as the iron (Fe+3) donor in our ex-vivo 3D model - organotypic culture induced with Aβ.

Objectives:

To establish an authentic ex-vivo Alzheimer"s model in the human brain to enable the closer study of AD pathology. To establish iron overload in the brain using ferric citrate as the iron donor. To study the effect of iron overload on the structure of Aβ fibrils.

Materials and methods:

Human brain specimen was obtained either from the glioma surgery or from the post-autopsy routine of patients. The specimen was sectioned into 160µm thickness using vibratome. The slices were immediately shifted onto a porous membrane over neurobasal media and maintained for several days. To induce artificial AD and iron overload conditions in the culture ex-vivo, the exogenous Aβ1-42 peptides and ferric citrate were added respectively to the serum-free neurobasal media. Harvested sections were processed accordingly for immunofluorescent staining and electron microscopy imaging. The embedded tissue was sectioned into 70nm thickness and used for routine electron microscopy and negative contrasting. We plan to use ATUMtome to produce serial sections for electron tomography to study the effect of iron on the fibrils" structure in detail.

Results:

We compared the morphology of the samples obtained from post-mortem porcine samples with OTC samples and found that the cell integrity and the neuronal connections are viable and the cells are in an active state. The slices exposed to Aβ peptides polymerized and formed aggregates and displayed a mesh-like appearance in the extracellular matrix when observed in the electron microscope. Having established this model, we now plan to induce iron accumulation in the brain to characterize the structure of Aβ fibrils in former condition using 2D electron micrographs and perform electron tomography to understand the structural changes in the fibrils in 3D.

Conclusion:

Organotypic culture systems have been a long-standing tool in neuroscience. However, in neuropathological conditions like AD, most of the studies opt for transgenic mice models to study the disease. We hope that this ex-vivo model system of AD from the human brain with slight modifications serves as an excellent alternative to studying the AD pathological conditions and the factors contributing to it. (The project is funded by FWF – Austrian Science fund - P 29370).