AMIGO3 gene expression and function during oligodendrocyte differentiation in the central nervous system

Almutairi, Majed (2024). AMIGO3 gene expression and function during oligodendrocyte differentiation in the central nervous system. University of Birmingham. Ph.D.

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Abstract

Demyelination causes disruption to neuronal signalling and occurs in many neurodegenerative diseases or in response to brain injury. There are no cures available for demyelinating diseases, including multiple sclerosis (MS), the most common autoimmune inflammatory disease in the central nervous system (CNS). Current approaches to treat MS focus on anti-inflammatory drugs, which prevent demyelination progression and reduce clinical relapses, but do not allow repair or replacement myelin. A natural repair process called remyelination can occur following demyelination. Remyelination can help to repair demyelinating plaques and protect axons from degeneration, but endogenous remyelination is inefficient and fails with ageing and disease progression, Improving remyelination is therefore an important for the focus for the development of MS therapies. Studies of animal models have to led to the discovery of new therapeutic targets which can promote endogenous remyelination. An example is the leucine rich repeat protein (LRRP) leucine rich repeat and immunoglobulin-like domain containing protein 1 (LINGO1). Inhibition of LINGO1 promoted remyelination in several animal models of demyelination, but clinical inhabitation of LINGO1 showed unsuccessful results in clinical trials. We considered the idea that the LINGO1 clinical trials failed due to compensation from another LRRP, amphoterin-induced gene and open reading fram-3 (AMIGO3). AMIGO3 is upregulated more rapidly than LINGO1 after spinal cord injury (SCI) in animal models, and also increases in expression rapidly after axotomy in culture of dorsal root ganglion neurons and retinal ganglion cells. Importantly, AMIGO3 expression is rapidly increased in the CNS during postnatal development when myelinating oligodendrocyte (OL) are generated, and decreases again quickly after myelination begins. AMIGO3 may therefore act a regulator of OL differentiation, and its inhibition could be an alternative therapeutic target for demyelinating diseases.

Considering the above, the first aim of this project was to study the function of AMIGO3 using in vitro models of OL differentiation. For this work, a primary mixed glial culture system was developed to study OL differentiation during early stage of CNS development. Using this system we found that AMIGO3 is downregulated in mixed glial cultures during OL differentiation. We also found that downregulation of AMIGO3 expression by siRNA resulted in the upregulation of OL maturation. On the other hand, increased AMIGO3 levels produced by treatment with recombinant AMIGO3 reduced OL maturation, and activated RhoA GTP activity, a molecule involved in AMIGO3 intracellular signalling. These findings suggest that AMIGO3 downregulates OL differentiation via RhoA GTP signalling.

AMIGO3 is known to engage in homotypic interactions, thus expression on different glial cells could allow cell to cell signalling. Also, AMIGO3 is expressed in cells of the OL lineage and astrocytes, thus AMIGO3 expression in non-OL glia could provide a signal to regulate OL differentiation. Considering this idea, the second aim in this thesis was to compare the expression of AMIGO3 and LINGO1 in primary cultures of astrocytes and microglia. Using Western blot and immunocytochemistry we found strong expression of AMIGO3 proteins in astrocytes, but not in microglia. Surprisingly, we also found that astrocytes express LINGO1 proteins. This work shows that astrocytes, but not microglia, are an abundant source of both AMIGO3 and LINGO1, which together could influence OL differentiation and myelin formation and repair.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):