Abstract:
Creatine is a one molecule in a class of phosphagens which can carry the high-energy phosphate molecule derived from adenosine triphosphate. It exchanges this phosphate molecule through a kinase reaction catalysed either by a creatine kinase isozyme localised in the cytosol or mitochondrion. In the brain these are known as brain-type creatine kinase (BCK) and ubiquitous mitochondrial creatine kinase (uMtCK) respectively. Creatine is synthesised from amino acids via a two-step reaction catalysed by arginine:glycine amidinotransferase (AGAT) and then guanidinoacetate methyltransferase (GAMT) by a limited number of tissues. It is then taken up into cells requiring creatine through a secondary-active creatine transporter (CrT). Together these five proteins define what is known as the creatine system. Similar neurological disorders result from mutations in AGAT, GAMT, and CrT. Studies have also shown that creatine has neuroprotective effects in models of several neurodegenerative diseases, including Parkinson’s disease (PD) and Huntington’s disease (HD). While the creatine system has been well-defined in skeletal and cardiac muscle tissue, much less is known about its organisation in the brain. This is especially so in the human, where most studies have focused on rodents. Given the relevance of the creatine system today in several human diseases, a sound description of the anatomical basis of the creatine system in the human brain is warranted. Moreover, so is an assessment of changes in the system which occurs in diseases such as PD and HD, especially given that creatine is being trialled in phase III studies for both these conditions. This thesis presents localization data for each of the five creatine system proteins using an immunohistochemical technique on fixed human brain tissue. Further, it characterises the changes which occur in the HD motor cortex to the creatine system proteins using Western blotting, qPCR, and stereological cell counting. These results demonstrate that the creatine system has a remarkable heterogeneity of expression in the neurologically-normal human brain. The cells capable of creatine uptake via CrT appear to be predominantly neurons, with large excitatory projection neurons having the highest level of expression, and local circuit inhibitory neurons less. Oligodendrocytes and endothelial cells had detectable-levels of CrT expression but far less than that seen in neurons. Notably, astrocytes have no CrT expression. In contrast, the expression of AGAT and GAMT was found in all cell types in the brain. GAMT was most obviously expressed in neurons. AGAT was also expressed in neurons but tended to show more apparent glial staining. Globally these results suggest that the human brain has a widespread capacity to synthesise creatine, though its uptake appears to be required mostly by neurons. The expression pattern of the creatine kinases was found to be dissociated. BCK was found to be expressed strongly in astrocytes, whereas uMtCK had an exclusive neuronal expression pattern. BCK was also expressed in some neurons, though inversely to uMtCK. Neurons with high BCK expression tended to include those expressing parvalbumin, including Purkinje cells and many cortical interneurons. But the highest level of BCK expression was found in medium spiny neurons of the striatum. This opposing pattern of expression suggests that the creatine kinases are used in functions which are opposing. It is argued in this thesis that it probably represents differences in the utilisation of energy between cells: those with high uMtCK (all neurons) tend to acquire their energy from oxidative phosphorylation; and those with high BCK (some neurons and astrocytes) tend to acquire more of their energy from aerobic glycolysis. Similarities between the expression pattern of the creatine kinases in muscle tissue and brain tissue are examined. Finally, in the HD motor cortex, a significant decrease in the protein levels of BCK was detected. This was not paralleled with a similar change in transcription. It is possible that this is due to oxidative attack on the BCK protein, which it is known to be vulnerable to, which may cause a preferential decrease in BCK protein levels. A small but significant decrease in CrT transcription was also observed. No significant change in protein levels was observed; but there was a decrease in the number of CrT-expressing neurons in the motor cortex. Together these results show that changes in the creatine system do occur in HD. Moreover, these differences should be taken into account when considering the effects of creatine supplementation in this disease. It is anticipated that the description of the creatine system presented in this thesis will provide a good neuroanatomical basis on which to support future studies elucidating the operation of the creatine system in the human brain. It is also hoped that this data will provide a unique insight into the energetics of the brain from the perspective of a singular and well-defined system.