A Dictyostelium model for neuronal ceroid lipofuscinoses (Batten disease)

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Microbiology, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

Batten Disease or Neuronal Ceroid Lipofuscinosis (NCL) is a neurodegenerative disease of a childhood that arises from mutations affecting one of 14 lysosomal or endoplasmic reticulum (ER) or plasma membrane proteins. Deficiencies in CLN5 and CLN7 cause the Finnish and Turkish variant forms of late infantile NCL respectively. A D. discoideum model was created successfully for studying these forms of Batten Disease by altering CLN5 and CLN7 protein levels using antisense inhibition and overexpression approaches. CLN5 deficiency caused abnormalities in cell growth, endocytosis, autophagy and lysosomal pH regulation. CLN7 has been hypothesized to function as a lysosomal transport protein and I found direct evidence for this in the form of altered lysosomal uptake of Neutral Red in our CLN7 transformants. In vitro lysosomal uptake assays showed that Neutral Red uptake could be inhibited, presumably competitively, by a variety of but not all potential substrates. The uptake was independent of ionophores that abolish proton, potassium and charge gradients, suggesting that CLN7 mediates the passive transport of its substrates. When CLN7 expression was altered in D. discoideum, defects in cell growth, endocytosis, autophagic cell death, lysosomal pH, volume and osmoregulation were observed. Cotransformation experiments to assess genetic interactions showed that the Rheb signalling pathway mediates the defects in phagocytosis observed when CLN7 expression levels are altered. However, the elevated rates of pinocytosis caused by CLN7 deficiency appear not to be mediated by inhibition of Rheb signalling. Thus my results suggest that simulating Batten Disease in D. discoideum by altering CLN5 and CLN7 protein expression affects cell signalling pathways that regulate vesicle trafficking in autophagy and endocytosis.