Role and effect of amyloid proteins on NET formation in the CNS

Neutrophils are actively recruited first responders to sites of infections and
protect our bodies from infiltrating harmful pathogens. Upon stimulation,
neutrophils undergo a specific cell death mechanism called NETosis, which is a
critical innate immune response characterized by ROS production and the release
of chromatin decorated with antimicrobial peptides, and serine proteases,
accompanied by various pro-inflammatory and immunostimulatory molecules. While
up to 60% of our circulating blood consists of neutrophils, they are virtually
absent from the central nervous system in healthy individuals. It was recently
found, however, that neutrophils can pass the otherwise protective blood-brain
barrier and enter our brain under specific neuropathological conditions such as
Alzheimer's disease (AD), Multiple Sclerosis (MS), amyotrophic lateral
sclerosis (ALS), and Parkinson*s disease (PD).

In line with the presence of neutrophils in the diseased brain, it is becoming
increasingly more evident that bacterial infections may contribute to the
developmental onset of several neurological pathologies. For example, chronic
periodontal infection was linked as a possible cause of Alzheimer*s disease,
and Porphyromonas gingivalis was recently identified as a keystone pathogen.
Here, a positive correlation was found between bacterial infection and the
formation of Aβ plaques, a characteristic feature of AD and recently assigned
to have antimicrobial properties. In line with their role in innate immunity,
neutrophils were also found in the surroundings of Aβ deposits and the reduced
cortical blood flow commonly observed in AD patients was assigned to neutrophil
adhesion in brain capillaries. On a similar note, neutrophils and neutrophil
extracellular traps (NETs) were also found in the cerebrospinal fluid of
patients with other neurodegenerative diseases such as MS.

Besides the currently accepted mechanism of microbial-induced neutrophil
activation and NET formation, it was recently shown that amyloid fibrils
themselves from three different protein sources (e.g. α-synuclein, Sup35, and
transthyretin) also induced NET formation in vitro. The accompanying release of
NET-associated proteases further degraded the amyloid fibrils into shorter
peptide products that showed increased toxicity on liver and kidney cells.
Because of the cell-damaging processes involved, it is tempting to speculate
that amyloid-induced NETosis contribute to neurotoxicity. The in vivo
pathogenic relevance of the presence of neutrophils in brain tissue and the
effect of the accompanying NETs on neuronal cells and disease progression,
however, has not yet been systematically investigated.

In this research proposal, we aim to develop chemical reporter molecules to
study neutrophil activity and NET formation in real-time. We will
systematically study the effect of brain-associated amyloid fibrils on NET
formation and investigate the effect of the accompanying secretion molecules on
neuronal activation and synapse growth in vitro and in vivo. The specific
objectives are as follows:

1. Development of molecular reporters and activity-based probes to study and
identify NETs in vitro and in vivo.
2. Evaluate neutrophil infiltration and activation in neurodegenerative
disorders using immunostaining and activity-based probes.
3. Evaluate the effect of amyloids and aggregates on NET formation on isolated
neutrophils in vitro.
4. Evaluate the effect and toxicity of NETs on neuron cells.

In order to study neutrophils and NET formation in vitro and in vivo, we will
develop activity-based probes (ABP) for a set of proteins that are specifically
secreted by neutrophils during NETosis namely Neutrophil Elastase (NE),
Cathepsin G (CatG), Peptidyl Arginine Deiminase (PAD) and Myeloperoxidase
(MPO). The prepared probes will first be evaluated for their suitability to
detect enzyme activity during NETosis on neutrophils.

The process of NETosis can be initiated by several triggers and proceed via a
NADPH oxidase-dependent as well as an NADPH oxidase-independent pathway. Both
pathways result in NETs with unique molecular compositions and it is therefore
interesting to investigate the effect of amyloid-induced NETosis in more
detail. Here, we aim to examine whether amyloid proteins of different structure
and size activates neutrophils and induces NETosis. We will generate different
conformations of amyloid ranging from monomers to oligomers and fibrils using
established protocols, and incubate them with neutrophils in vitro. Activation
will be quantified using the developed activity-based probes and by NETosis.

Neutrophils are very short-lived (roughly 1 day) and thus, will need to be
isolated from fresh blood samples. Blood samples (10-40 mL) will be collected
via venapuction at the RadboudUMC in EDTA anti-coagulation tubes after signed
informed consent. The exact amount depends on the nature of the planned
experiment and the number of neutrophils that is needed to perform it. Donors
will not be followed over time or subjected to other procedures than donating
blood. This research will not yield findings that may be stressful or
clinically relevant to the donors, since the only clinical parameter that will
be checked are cell counts.

Neutrophils will be isolated from the donated blood according to standard
operating procedures (SOP) and subsequent in vitro experiments will be
performed on the day of donation. After the experiments are finished, all
materials will be discarded; biomaterials involved in this study will not be
stored.

Risk and burdens: risk is negligible and the burden is minimal. The most common
risks related to blood donation from the subject's arm are brief pain and/or
bruising. Infection, excess bleeding, clotting, or fainting are also possible
but unlikely.

Benefits: there will be no direct benefit for the donor other than a 15¤
Bol.com voucher. However, participation of subject will help the researchers to
gain knowledge.