Scientific approach

Background

 
NADPH oxidases (NOX) generate reactive oxygen species

Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen. At low levels, they are involved in the normal function of the cells, but at high levels, they are toxic and lead to tissue destruction. NOX enzymes are a group of proteins, which have as a function to specifically catalyze the transfer of electron from intracellular to extracellular space, thereby generating ROS by specific reduction of molecular oxygen (figure 1). In normal conditions, depending on their localization, NOX enzymes generate low or high levels of ROS:  high levels to defend the organism by killing microbes and low levels to control a numerous function, such as immune response, hormone synthesis, and neurotransmitter release among others. However, in pathological state and especially in disorders affecting the central nervous system (CNS), increased ROS is a known feature of neurodegeneration and NOX enzymes have recently emerged as key effectors of CNS disorders.

NOX enzymes known to be present in the CNS are the isoforms 1, 2 and 4. They share functional (transferring electrons leading to generation of ROS) and structural characteristics (six transmembrane domains, intracellular NADPH binding site and stabilization in the membrane by another transmembrane protein (p22phox). NOX4 activity is constitutive, NOX1 and NOX2 require activation (phosphorylation) by other cytosolic proteins (not shown in this scheme).
Figure 1: NOX enzymes known to be present in the CNS are the isoforms 1, 2 and 4. They share functional (transferring electrons leading to generation of ROS) and structural characteristics (six transmembrane domains, intracellular NADPH binding site and stabilization in the membrane by another transmembrane protein (p22phox). NOX4 activity is constitutive, NOX1 and NOX2 require activation (phosphorylation) by other cytosolic proteins (not shown in this scheme).


A strong need for novel treatments of neurodegenerative diseases

Neurodegenerative diseases (ND) represent a major healthcare problem worldwide and in Europe. ND affect mostly adults and can last for decades, causing long term suffering to patients and their families. There are more than 600 different ND, affecting millions of people.
Current treatments for ND are mostly palliative or only treat the symptoms rather than the cause, hence provide only slight or temporary relief instead of cure. The understanding of the biological basis of ND has increased, and numerous pathological mediators of ND have been identified. However, so far, drugs used to treat ND have been largely inefficient and clinical trials undertaken have often failed. Some specific ND drugs have been approved, such as recombinant interferons for the treatment of relapsing-remitting MS, or Riluzole™ for the treatment of ALS, but their ability to modify the disease course remains clearly insufficient.

NOX as novel therapeutic targets

An emerging aspect in neuropathologies is the role played by neuroinflammation in the development of neurodegeneration. Modulating this phenomenon may have strong therapeutic benefit. NEURINOX aims at identifying novel therapeutic targets for neuroinflammatory diseases, by focusing on NADPH oxidases (NOX). NOX enzymes catalyse the formation of ROS and are key regulators of neuroinflammation: establishment of chronic neuroinflammation is characterized by either increased or decreased NOX activity. Therefore, NOX enzymes regulate inflammatory pathways shared by many CNS disorders. The NEURINOX project aims at elucidating the links between neuroinflammation, NOX enzyme activity and neurodegenerative diseases.

What will NEURINOX researchers study?


The NEURINOX consortium puts together different competencies for a common goal: understand the role of NOX enzymes in neuroinflammation and evaluate their potential as drug targets for the treatment of neurodegenerative disease. The underlying concept is to use small molecules and genetic tools (knock-out, transgenic and mutant mice) to evaluate the effect of NOX inhibition and/or activation on neurodegenerative processes.
The project has 3 main axes: i) basic research using in vitro models and animal models of neuroinflammation, ii) clinical research using samples (blood, DNA) from patient affected by neurodegenerative disease and iii) discovery of small molecule drugs targeting NOX enzymes.
The NEURINOX consortium will try to answer the following questions:

  • What is the role of NOX enzymes in neurodegenerative diseases (ND)?
  • What is the link between NOX and neuroinflammation?
  • Are NOX enzymes efficient targets for treating ND?
  • What are the mechanisms of neuroinflammation controlled by NOX activity?

In order to address these questions, NEURINOX will carry out the following four main activities focusing on several diseases with strong neuroinflammatory components (ALS, MTLE-HS, MS  and other autoimmune neuropathies):

1) Animal models of the above-mentioned diseases will be studied in mice carrying mutation in NOX and knock-out animals of different NOX isoforms.

2) Although in vivo models are the most representative system to assess NOX-mediated neuroinflammation their complexity reduces screening capacity. An alternative method is the use of organotypic slice cultures. Slices can be prepared from essentially any region of the brain and maintained in culture with various treatments of interest for several months. The cultured slices maintain the 3D organisation of neuronal and glial subpopulations needed for normal brain anatomy and function.

 

Representative images of cerebellar (1) and hippocampal (2) slices in culture: neurons are stained with NeuN (red), astrocytes are stained with GFAP (green), nuclei are stained with DAPI (blue)
Figure 2: Representative images of cerebellar and hippocampal slices in culture: neurons are stained with NeuN (red), astrocytes are stained with GFAP (green), cell nuclei are stained with DAPI (blue). Image provided by UZH.

 

3) Drug discovery: development of small molecules targeting NOX enzymes (inhibitors and enhancers) will be tested in animal models and organotypic brain slices.


4) Patient studies:

  • DNA, plasma, cerebrospinal fluid and peripheral blood leukocytes (PBL) from ALS and MS patients will be used to measure NOX expression and activity as well as indirect markers of oxidative stress.
  • NOX expression in brain samples from resective surgery performed in epileptic patients will be analyzed.
  • B lymphocytes from patients will be isolated and immortalized with the Epstein Barr virus (EBV). A potential correlation between NOX2 activity and disease will be evaluated.
Figure 3: Increased NOX2 expression (red staining in microglia) in the hippocampus of an epileptic patient. Neurons are immunostained usong a Map2 antibody (green) and nuclei using DAPI (blue). Image provided by USZ.
Figure 3: Increased NOX2 expression (red staining in microglia) in the hippocampus of an epileptic patient. Neurons are immunostained usong a Map2 antibody (green) and nuclei using DAPI (blue). Image provided by UZH.

 

In addition to study expression, localization and activity of NOX enzymes, analysis of molecular pathways regulated by NOX enzymes will be studied by global Proteome and Transcriptome analysis. Moreover, a platform will be dedicated to study ROS generation in tissues using specific probes.

Altogether, this approach should lead to identification and validation of novel drugs for the treatment of the neuroinflammatory aspect of ND and to slow down their development.