Background: Amyotrophic lateral sclerosis is a devastating neurodegenerative disorder that affects motor neurons in the brain and spinal cord. A (GGGGCC)n hexanucleotide repeat expansion in the gene C9orf72 is the most common genetic cause of ALS. Although ALS is characterised by the selective degeneration of motor neurons, a large body of evidence implicates other cell types such as astrocytes, microglia and oligodendrocytes in disease progression. Astrocytes are the most abundant cell type in the central nervous system and play a key role in neuronal maintenance. However, during disease progression mutant astrocytes assume an unsupportive role and selectively induce motor neuronal death. Our data shows that C9orf72 patient-derived astrocytes secrete significantly less extracellular vesicles compared to healthy control astrocytes and motor neurons treated with C9orf72 astrocyte derived EVs generate shorter axons and neurites and ultimately have reduced survival. This project will focus on identifying the point within the extracellular vesicle formation pathway that contributes to reduced extracellular vesicle secretion from C9orf72 patient derived astrocytes.
Aims and Objectives: 1). Validate a fluorescent reporter probe that will later be employed to assess extracellular vesicle release. 2). Identify the point in the endosomal trafficking network that is impaired and mediates the impaired secretion of extracellular vesicles. 3). Identify which pathogenic mechanism, haploinsufficiency, RNA toxicity and dipeptide repeat protein toxicity caused by the C9orf72 mutation is responsible for impaired extracellular vesicle secretion.
Results: A C9orf72-specific increase in transferrin uptake was observed in patient astrocytes, that was associated with a significant increase in transferrin receptor expression on the membrane of C9orf72 astrocytes. Early to late endosomal maturation represents a critical step in production of extracellular vesicles. This increase in endocytic uptake did not result in a significant increase in early or late endosomes measured by EEA1, Rab5 and Rab7 expression and organelle size.
Conclusion: C9orf72 astrocytes exhibited increased transferrin uptake indicative of enhanced endocytosis. The increased endocytic uptake did not disrupt the early endosomal compartment, and these findings do not suggest that early to late endosomal maturation is impaired at the level of the Rab switch. Furthermore, C9orf72-patient derived astrocytes did not display morphological alterations consistent with previously reported phenotypes associated with impaired endosomal maturation. Multivesicular body-plasma membrane fusion represents the following stage of extracellular vesicle formation and the CD63-pHluorin reporter probe that was validated during this research should be utilised to investigate whether disrupted multivesicular body-plasma membrane fusion is responsible for the reduced secretion of extracellular vesicles.
