Copper & protein aggregation in neurodegenerative diseases

Neurodegenerative diseases such as Alzheimer's disease (AD), Amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) are on the rise and so far, only disease modifying treatments are clinically available and there is no established cure. There is a pressing need to further understand the exact causes of these diseases from which we can develop methods to treat them. One interesting set of features that connects these neurodegenerative diseases are neuroinflammation, protein aggregation and copper dyshomeostasis. For instance, Amyloid beta (AB), and Tubulin associated unit (Tau), which are important factors associated with AD that when aggregated can be sources of inflammation for the central nervous system (CNS). Interestingly both bind copper.

Copper is a trace element that is essential for life with unique redox properties which allows it to form the catalytic domain for a number of enzymes including superoxide dismutase 1 (SOD1) for cellular antioxidant defence, mitochondrial cytochrome C oxidase (CcO) complex IV for oxidative respiration, ceruloplasmin (CP) for ferroxidase activity governing iron export from cells, dopamine beta hydroxylase (DBH) for noradrenaline (NA) synthesis. It also plays a role in protein aggregation either by stabilizing the protein structure in specific circumstances or more commonly induce protein aggregation due to its affinity to amino acids such as cysteine, histidine and tyrosine. Under oxidative conditions, this redox capacity can generate reactive oxygen species (ROS) if uncontrolled. Under reducing conditions or hypoxia, the Cult state is favoured that can accelerate protein aggregation. Due to this aggregation inducing nature we decided to look at methods of inhibiting this effect by using an endogenous copper binding tripeptide Gly-His-Lys (GHK) that can reduce the redox activity of copper once bound in STUDY I. Here we demonstrate that GHK can attenuate the redox activity of copper and prevent copper induced cell death in vitro and prevent copper- induced protein aggregation in a model using BSA. We continue with some further experiments outside of the published study using either GHK or GHK-Cu in a mouse model of multiple sclerosis called experimental autoimmune encephalomyelitis (EAE) which models aspects of autoimmunity and neuroinflammation, although we did not have success in attenuating the disease.

However, although copper can cause protein aggregation, it is tempting to associate it with a negative light in the context of neurodegeneration. STUDY II is a literature review regarding copper, protein aggregation and ALS whose aim was to broadly survey the various research fields from the molecular scale involving the aggregation of proteins such as SOD1, to the higher order observations such as mitochondrial dysfunction, vascular dysfunction and to connect them with epidemiological evidence and disparate clinical observations from cases that bear very similar features as ALS. Intriguingly, copper deficiency seems likely as it underpins these functions. Furthermore, environmental factors are investigated which points to heavy metals, algal neurotoxins such as ß-Methylamino-L-alanine, (BMAA), formaldehyde and herbicides such as paraquat being strong risk factors. I hypothesize that the link between these factors coalesces in the disruption of copper homeostasis and cuproenzyme function that is central to ALS that reveals itself in a copper mis-partitioning whereby copper is shunted away from the neurons which suffer from deficiency, into microglia which require it for an elevated immune response. The conclusion from this study is a mis-partitioning of copper with functional deficiency and excess in other areas.

In STUDY III we focus our attention on AD. In this review we aimed at unifying the roles of major AD risk factors into a comprehensive understanding. Amyloid beta (AB), tubulin associated unit (Tau) and apolipoprotein E (ApoE) all have alternative functions as antimicrobial peptides that are modulated by their aggregation propensity. Rather than aggregation being a fault, it appears to be a function to be called upon as a primitive arm of the innate immune response available to many cells including non-immune specialized cells such as neurons as a factor capable of neutralizing viruses, bacteria and fungi, both intracellularly and extracellularly. This appears to answer in part how non-immune specialized cells can defend themselves from invasion. AB, Tau and ApoE can all bind copper, and it may be possible that copper could be used to accelerate the aggregation of these proteins, for instance in the lysosomes. Copper also accumulates inside microglia, especially inside lysosomes for immune function and could represent a copper sink. Furthermore, AB may also act as a copper sink especially since copper accumulates in the AB rich plaques. As in ALS, clinical data reveal that AD bears signs of copper deficiency (brain-wide) which would in turn reflect in the loss of cuproprotein function such as mitochondrial deficiencies and iron accumulation. Furthermore, I present a compilation of evidence that links microbial infection in the CNS of AD patients that suggests that chronic parasitization of specially adapted pathogens such as herpes viruses, anaerobic spirochetes and biofilm- forming bacteria can insidiously enter the brain and trigger a long-lasting immune response that diverts AB, Tau and ApoE to a defensive aggregative role that over time leads to progressive loss of copper homeostasis and neurodegeneration. Overall, I provide evidence from STUDIES II & III to support the role of copper dyshomeostasis being key to neurodegeneration that can be induced by a variety of factors.

STUDY IV characterizes a novel use of the AB stain thioflavin-T (THT) that describes use as a rapid and easy fluorescent Nissl stain allowing detection of brain structures, neuronal populations and nucleoli that can also be used in vitro in live cells. We also characterize the photochemical properties upon blue light exposure that leads to a photo-enhancement of areas stimulated by this light. This allowed us to develop a protocol that is compatible with fluorescent immunohistochemistry that also avoids the complications of this effect. Furthermore, we refine the protocol into two variants that allows for THT to be used as a Nissl stain or an amyloid stain.

Preliminary Studies have also been included as a record of other work I have spent significant time on during my PhD. They mainly focus on microglial depletion and repopulation with anti-inflammatory microglial like cells (MLCs) as a method to target neuroinflammation. We characterize the effect of old age on the depletion and repopulation kinetics of microglia using the colony stimulating factor receptor (CSF-1R) inhibitor Plexidartinib (PLX3397) and investigate microglial depletion in the spinal cords. This basic work indicated that microglial depletion is not complete, which represents a challenge as replacement of the microglial with external microglia-like cells (MLCs) requires additional factors to facilitate successful engraftment of donor cells. To do this we attempted to load exosomes with the myeloablative drug busulfan that could hopefully attenuate the resident microglial repopulation long enough to give MLCs a better engraftment rate, but this experiment failed in the developmental stage. We also investigated the effects of different activation states of microglia and astrocytes on the phagocytosis of microglial debris that will be inevitably left over after depletion.

One aspect that was recognized through these studies was the autofluorescence of CNS structures. This led to testing whether autofluorescence could be used as a label-free marker for inflammatory changes in fresh blood using flow cytometry, which provided interesting preliminary results. Finally, I document the preliminary work regarding the role of transforming growth factor beta 1 (TGF-β1) depletion in microglia by breeding of a transgenic strain and characterizing the basic pathological phenotype.

List of scientific papers

I. Min J, Sarlus H, Harris RA. Glycyl-l-histidyl-l-lysine prevents copper- and zinc-induced protein aggregation and central nervous system cell death in vitro. Metallomics 2024 2;16(5):mfae019. https://doi.org/10.1093/mtomcs/mfae019

II. Min J, Sarlus H, Harris RA. Copper toxicity and deficiency: the vicious cycle at the core of protein aggregation in ALS Front. Mol. Neurosci Volume 17 - 2024. https://doi.org/10.3389/fnmol.2024.1408159

III. Min J, Sarlus H, Harris RA. MAD-microbial (origin of) Alzheimer's disease hypothesis: from infection and the antimicrobial response to disruption of key copper-based systems. Front. Neurosci., Volume 18 - 2024.
https://doi.org/10.3389/fnins.2024.1467333

IV. Min J, Sarlus H, Sho Oasa, Robert A Harris, Thioflavin-T: application as a neuronal body and nucleolar stain and the blue light photo enhancement effect. Sci Rep. 22;14(1):24846. https://doi.org/10.1038/s41598-024-74359-8