Assessing the Impact of Aggregation-Prone Proteins on the Mitochondrial-Metallome Interplay in a Novel 3D Spheroid Cholinergic Cell Model of Neurodegenerative Disease

Although showing diverse symptoms affecting cognition and movement, the brains of patients with neurodegenerative disorders (NDs) display similar neuropathological features namely protein aggregation within the brain. The aggregates and/or oligomers appear to be toxic, causing injury or death to brain cells. In general, the greater the degree of aggregation, the greater the disease severity. Another common feature is that neurons producing the neurotransmitter acetylcholine (ACh), called cholinergic neurons, progressively degenerate. However, a major hurdle for interrogating neuropathological processes linked to oligomer toxicity affecting the cholinergic system has been lack of a reliable cholinergic cell model. We will differentiate neuroblastoma ‘LA-N’ cells into cholinergic neurons using published protocols; this cell line is the closest immortalized semblance of human cholinergic neurons currently available. Cholinergic neurons will then be challenged with aggregated Aβ/tau/α-synuclein, found within Alzheimer’s disease and Parkinson’s disease brains, with effects on neuronal function evaluated. The cell model will be generated as 3D spheroids to reflect cholinergic neurons in the brain’s microenvironment. Gender is an ND risk factor; we will consider gender-specific effects by comparing measures from the female-originating LA-N cell line vs male-originating one. The dose-response impact of various oligomeric species on cholinergic neurons will be characterised at various levels: Neuronal loss will be assessed using colorimetric assays. Neuronal morphology and neurite formation will be measured with image analysis software applied to whole-well scanned images of neurons stained with a lipophilic membrane tracer. For functional cholinergic assessment, we will quantify protein expression levels of ACh metabolism enzymes. Also, ACh levels will be measured in real-time with a genetically encoded G-protein-coupled receptor-based ‘GRAB’ sensor.

ND-linked aggregated proteins exert neurotoxic effects on mitochondria i.e. by deregulating mitochondrial calcium handling and high oxidative stress. We will profile the mitochondrial DNA response to the various proteins i.e. by measuring mitochondrial DNA copy number (a marker of the cell’s energy needs), levels of mitochondrial DNA damage and mitochondrial respiration.

Metals can cause NDs by disrupting mitochondrial function i.e. depleting ATP and inducing ROS production. However, the effects of various metals on different NDs are not identical, and their specific mechanisms of damage requires clarification. The ‘metallome’ (distribution of free metal ions in a cellular compartment) is central in many NDs. Redox-active transition metals e.g. iron and copper, and other biometals e.g. zinc are essential for brain function but are present in amyloid-β plaques in ND patient’s brain tissue. To provide insight into toxic metal-related pathways activated by accumulation of different ND-associated protein species, we will use label-free synchrotron x-ray fluorescence metal-imaging to map and quantify metal element distribution in individual cells.

Whole-cell patch clamp recordings will be combined with computational modelling to explore effects on the passive and active properties of the neurons and evaluate if they differ between different protein species and/or the male vs female lines. Finally, having characterised the cellular disease platform, we will test if synergistic treatment with antioxidants and metal chelators could reverse the effects induced by the specific protein aggregates.

The work will give insight as to gender-related neuromodulating effects by cholinergic neurons in response to increasing oligomeric burden, and how cytotoxic effects result from interaction with metals at the mitochondrial interface. A reliable cell culture in vitro system reflecting ND’s cholinergic pathology will provide a platform by which to assess promising pharmacological agents against disease relevant outcomes.