|dc.description.abstract||Batten disease (Neuronal ceroid lipofuscinosis, NCL) is a group of devastating neurodegenerative diseases that affect children, caused by mutations in a number of genes, but the underlying pathogenic mechanisms remain unclear. However, the remarkable similarity of pathological features between classical forms of NCL indicates that there may be a unique pathway in which NCL proteins play a role. Immunohistochemical investigations of ovine Batten disease revealed neuroinflammation preceded neurodegeneration in a regionally specific manner. A previous study showed that chronic treatment with an anti-inflammatory compound minocycline did not inhibit the neuroinflammation, indicating the need to understand the specific neuroinflammatory cascade in Batten disease to identify likely druggable targets before embarking on drug therapies. A drug screening method is not practical in large animals. Thus, a proposed neuroinflammatory cascade was investigated in a longitudinal study of CLN6 affected sheep brains. This thesis describes the changes in selective neuroinflammatory modulators in this proposed neuroinflammatory cascade during the disease progression.
Selected neuroinflammatory modulators (TNF-α, TGF-β, IL-1β, IL-10, NF-ᴋB, MAPK14, JAK/STAT, SOCS3, MnSOD, iNOS, Trk B and BDNF) were investigated by quantitative PCR on RNA extracted from different brain regions across ages of disease development (2, 6, 9, 18 and 24 months). Longitudinal expression of MnSOD and mitochondrial marker (COX IV) was also studied by immunohistochemistry on perfusion-fixed brain sections.
The distribution of MnSOD through the cortical grey matter co-localised to mitochondria and became compressed to the boundary between layers I and II and between layers IV and V in the affected cortices at 18 and 24 months, following severe neurodegeneration. There was no such compression in the non-degenerating cerebellum and brain stem. Quantification of MnSOD and COX IV across the cortical grey matter showed that the expressions were reduced at 18 and more so at 24 months, indicating that previous reports of enhanced activity are likely to have arisen from unmatched sampling.
Anti-inflammatory SOCS3 mRNA expression was up-regulated significantly prior to neurodegeneration and concurrent to the neuroinflammation, and was accompanied by up-regulation of its protein expression. It however did not suppress the neuroinflammation. Both pro- and anti -inflammatory cytokines (TNF-α, IL-1β, TGF-β, IL-10) were upregulated at the initiation of neurodegeneration, at 4-6 months of age, prior to the onset of clinical disease and cortical atrophy evident at 10-14 months, whereas the oxidative responsive genes SOD2/MnSOD and HO-1 were not. iNOS expression was not found in sheep. NF-ᴋB, MAPK14 and JAK/STAT pathway activation followed elevation of cytokines. TrkB expression was increased in the advanced disease while BDNF expression remained unchanged. Other recently proposed neuroinflammatory modulators (PI3Ks, MT I/II/III, GRN) were also investigated. The up-regulation of MT I/II/III followed the increase of cytokines and PI3Ks were not changed by the disease. GRN, a mutation in which was thought to cause a new form of NCL, showed no causative role in CLN6.
These results discount the oxidative stress and disruption of blood brain barrier, and reveal an uncontrolled neuroinflammatory pathway mediated by irregular cytokine signalling. This study highlights the weakness of building scenarios based on observed changes in single gene expressions in isolation. Also discussed are the problems associated with the newly assigned NCLs that lack a thorough biochemical analyses. A similar study of CLN5 ovine Batten disease underway will indicate the generality of these findings. A change of strategy is planned to continue these studies. Given the large number of changes observed, a transcriptomic approach is probably more appropriate.||en