NADH Fluorescence Lifetime Imaging Microscopy Reveals Selective Mitochondrial Dysfunction in Neurons Overexpressing Alzheimer's Disease-Related Proteins.

Moritz A Niederschweiberer,Devyani Bhosale,Douglas C Wallace,Raphael Hesse,Hans A Kestler,Christine A F Von Arnim,Patrick M Schaefer,Enrico Calzia,Angelika Rueck,Bjoern Von Einem,Larry N Singh,Ludwig Lausser

doi:10.3389/fmolb.2021.671274

Abstract

Alzheimer’s disease (AD), the most prevalent form of dementia, affects globally more than 30 million people suffering from cognitive deficits and neuropsychiatric symptoms. Substantial evidence for the involvement of mitochondrial dysfunction in the development and/or progression of AD has been shown in addition to the pathological hallmarks amyloid beta (Aβ) and tau. Still, the selective vulnerability and associated selective mitochondrial dysfunction cannot even be resolved to date. We aimed at optically quantifying mitochondrial function on a single-cell level in primary hippocampal neuron models of AD, unraveling differential involvement of cell and mitochondrial populations in amyloid precursor protein (APP)-associated mitochondrial dysfunction. NADH lifetime imaging is a highly sensitive marker-free method with high spatial resolution. However, deciphering cellular bioenergetics of complex cells like primary neurons has still not succeeded yet. To achieve this, we combined highly sensitive NADH lifetime imaging with respiratory inhibitor treatment, allowing characterization of mitochondrial function down to even the subcellular level in primary neurons. Measuring NADH lifetime of the same neuron before and after respiratory treatment reveals the metabolic delta, which can be taken as a surrogate for cellular redox capacity. Correlating NADH lifetime delta with overexpression strength of Aβ-related proteins on the single-cell level, we could verify the important role of intracellular Aβ-mediated mitochondrial toxicity. Subcellularly, we could demonstrate a higher respiration in neuronal somata in general than dendrites, but a similar impairment of somatic and dendritic mitochondria in our AD models. This illustrates the power of NADH lifetime imaging in revealing mitochondrial function on a single and even subcellular level and its potential to shed light into bioenergetic alterations in neuropsychiatric diseases and beyond.

Highlights

Neurons are highly complex and specific cells with synapses as their key structures, requiring a high energy supply to ensure neurointegrity and fast neurotransmission in the central nervous system
Aβ derives from proteolytic processing of the amyloid precursor protein (APP) by beta-secretase (BACE1) and gamma-secretase
This so-called APP Swedish mutation (APPswe) increases the APP cleavage by BACE1, which results in 6- to 8-fold level of Aβ compared to the normal wild-type APP (Citron et al, 1992)

Summary

INTRODUCTION

Neurons are highly complex and specific cells with synapses as their key structures, requiring a high energy supply to ensure neurointegrity and fast neurotransmission in the central nervous system. We combine NADH FLIM and respiratory inhibitor treatment to introduce the metabolic delta, a robust parameter that allows differentiating mitochondrial function in primary hippocampal neurons on the single-cell level and between the somatic and neuritic mitochondrial pool. We apply NADH FLIM to primary hippocampal neurons overexpressing APP, APPswe, and BACE1 proteins, in order to reveal their effect on mitochondrial function down to the subcellular level. This enables uncovering crucial mechanisms of selective mitochondrial dysfunction in neuropsychiatric diseases such as Alzheimer’s disease

METHODS

RESULTS AND DISCUSSION

ETHICS STATEMENT