Proteomic analysis of brain tissue from novel transgenic mouse models for Alzheimer’s disease.
4.3 Mouse brain protein identification by 2D gel electrophoresis from mature (8-12 months) transgenic AD models.
4.3.4 Characterisation of the four proteins chosen for further study
It was interesting to note that all of these proteins are reported to be active in pathways involved with neuronal growth, lipid transport or cellular stress responses. The identification in a change in these proteins’ expression in this AD model, suggests either a protective or toxic effect in the developing pathology of AD. Some of the proteins identified are involved with energy supplying pathways. Of the identified proteins, their potential significance is briefly described below, and for some they are discussed further in the discussion.
4.3.4.1 Peroxiredoxin II
Peroxiredoxin II (PrxII) is a cytosolic member of a family of peroxidases with high antioxidant efficiency and regulates H2O2 mediated signaling (Wood et al., 2003). Mammalian peroxiredoxins have six distinct members located in subcellular compartments, mainly where oxidative stress occurs (Table 4.2).
Prx Subtype
PrxI PrxII PrxIII PrxIV PrxV PrxVI
Cellular
location Cytosol, Nucleus Membrane Cytosol, Mitochondria Cytosol, Golgi. Secreted
Mitochondria, Peroxisome,
Cytosol
Cytosol
Table 4.2 Cellular locations of peroxiredoxin subgroups.
Induction of peroxiredoxins is observed as a response to increased production of reactive oxygen species (Kang et al., 1998). The increased expression of PrxII is linked to oxidative stress that correlates well with the development of AD pathology. Increased protein levels of PrxII could provide protection against neuronal cell death induced by hydrogen peroxide (Kim et al., 2001). PrxII has also been reported to have increased expression in the frontal cortex of patients with AD, Parkinson’s disease, Pick’s disease and Down’s syndrome (Kim et al., 2001; Krapfenbauer et al., 2003), and so its identification as being up-regulated in these transgenic animals provided confidence in them being a relevant AD animal model.
4.3.2.2 Endophilin 1
Endophilin 1 is a presynaptic protein that binds to dynamin, a GTPase involved with endocytosis and recycling of synaptic vesicles (Schmidt et al., 1999). Endophilins contain an SH3 domain at their C-terminus and are involved in membrane trafficking in the endocytic pathway. Endophilin 1 also acts as a substrate for the endosome-localised ubiquitin ligase, Itch and this interaction may be involved in ubiquitin-mediated sorting mechanisms operating at the level of endosomes (Angers et al., 2004). Endophilin binds other endocytic proteins, synaptojanin and dynamin (Reutens and Begley, 2002) and is known to have a
role in vesicle formation. The binding of endophilin 1 with dynamin mediates synaptic vesicle invagination from the plasma membrane, to create channels in the membrane, as such this may be linked to abnormal transition of peptides or lipids through the plasma membrane in AD (Sundborger et al., 2011). In addition, it is known that phospholipid metabolites can accumulate in cell membranes that contribute to Aβ deposition (Breteler, 2000) and potentially endophilin I may contribute to this as well.
4.3.4.3 ATP synthase β subunit
This is part of the intermembranous, five protein complex in the mitochondria which drives protons between the intermembrane space and the matrix. This complex catalyses ATP synthesis, and is essential for cell energy and survival. The identified β chain is the catalytic subunit (Ackrell, 2000). CNS tissue uses ATP rapidly and requires a constant energy reservoir for rapid regeneration of ATP. There is evidence that suggests expression of ATP synthase β subunit is tissue specific, the highest levels in the heart and the lowest in liver and kidney (Neckelmann et al., 1989). ABAD and Aβ disrupt mitochondrial function in AD and so it is probable that this relationship alters the regulation of this protein during the progression of AD.
4.3.4.4 Creatine kinase β subunit
Creatine kinases (CK) are a family of enzymes that regulate ATP levels and as such are central in energy homeostasis in tissues with large fluctuating energy changes. This is particularly so in muscle and the brain (Monge et al., 2008). CK
is elevated in serum after injury and is a significant marker for myocardial infarction (Alpert et al., 2000). It is also known to be elevated in fatty acid and glycerol metabolism disorders: medium-chain acyl dehydrogenase deficiency (MCADD) and long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) (Lund et al., 2010). Therefore an abnormal increase in expression of this protein could be related to the over-expression of ABAD and its catabolism of fatty acids. Other studies have noted an increase in CK expression in early stages of AD with a significant decrease in CK in the late stages in human AD brain (Lynn et al., 2010).
4.3.4.5 ApoE
The protein ApoE transports fat-soluble proteins, cholesterol and lipoproteins into the lymphatic system. ApoE generally aids proteolytic disruption of the Aβ peptide. However the ApoE4 isoform has been linked with AD as it is less efficient at this proteolysis (Deane et al., 2008). Individuals with the ApoE4 isoform gene are more susceptible to late stage AD. Cerebrospinal fluid (CSF) contains only high-density lipoproteins composed of ApoE and ApoJ secreted from astrocytes and of ApoA-I and ApoA-II transported via the blood brain barrier (Suzuki et al., 2002). These apolipoproteins can bind to Aβ and possibly relate to its clearance. In this study, the 2D gel analysis on 8-12 month mice showed increase expression of ApoE in 2 x Tg mAPP/ABAD, and therefore indicates that ABAD and APP expression levels may affect ApoE expression, but due to commitments, further study was not pursued in this thesis.