Role of neurofilaments in ageing and neurodegenerative disease

Cytoskeletal protein abnormalities are common features of a number of neurodegenerative conditions. However, the potential roles of neurofilaments (NFs) in neurodegenerative disease and in ageing-related cellular changes are poorly understood. Many models of NF abnormalities in mice have been reported, but how ageing affects the NF pathology and the role of ageing in neurodegenerative diseases are still unknown. This thesis, therefore, sought to examine the effects of the absence of a major class of NF proteins, in NF light (NF-L) knockout (KO) mice, on the ageing-related adaptive capacity of other cytoskeletal elements in neurons, and also on neurodegeneration-linked alteration of cytoskeletal proteins, cytoskeletal RNAbinding proteins (TDP-43) and myelin proteins. The NF-L subunit is considered as an obligate subunit polymer for the assembly of NF triplet proteins into neuronal intermediate filaments (IFs). Previous studies have shown that NF-L KO mice have substantially reduced axonal intermediate filaments. NF abnormalities such as NF accumulation, reduced NF-L mRNA, and NF mutations have been described in human neurodegenerative disease. This thesis has used immunohistochemical and quantitative Western blot techniques to examine cytoskeletal changes in the brains of NF-L KO and wild-type (WT) mice at different ages. It was demonstrated that depletion of NF-L protein resulted in alteration of the expression of a range of cytoskeletal proteins, and disrupts other neuronal IF proteins, through ageing. However, there were no gross structural changes in neurons or cytoarchitecture in regions such as the cerebral cortex. In this regard, alterations in the expression of other cytoskeletal proteins may compensate for decreased NFs. TDP-43, an NF-L mRNA binding protein, has recently been pathologically associated with a number of neurodegenerative diseases of ageing. However, mechanisms of TDP-43 pathogenesis are unknown. As TDP-43 is known to bind to NF-L mRNA, this thesis used immunohistochemical and quantitative Western blot approaches to characterize and quantify the changes of TDP-43 expression and distribution in brain and spinal cord of ageing NF-L KO mice as compared to WT animals. It was found that a significant increase of TDP-43 protein levels occurred in the cortex and lumbar spinal cord in NF-L KO mice at 12 months of age, compared to WT mice. Moreover, an increase of phosphorylated TDP-43 was found in the cervical spinal cord of NF-L KO mice at 12 months, compared to WT controls. NFs have a critical role in myelination by regulating axonal diameter. To investigate whether deficiency of NF-L protein results in myelin alteration during ageing, this thesis measured the relative protein level of myelin basic protein (MBP) by using quantitative Western blot. It was demonstrated that the level of MBP was significantly reduced in the cortex of NF-L KO mice at 12 months, compared to WT mice. In summary, these findings indicate that pathological and compensatory neuronal effects of NF-L KO and reduced NFs are affected by ageing. The absence of NF-L protein results in the alteration of NFs and other cytosekeletal, mRNA-binding and myelin proteins during ageing. These results indicate that the absence of NF proteins can lead to abnormalities in neurodegenerative disease-related proteins, but also that the central nervous system (CNS) is capable of adaptive alterations in the absence of NFs