The investigation of the hBCAT proteins in control and diseased human brains: Implications for glutamate toxicity in Alzheimer’s disease

Hull, Jonathan

Abstract

Introduction & Aims: The distribution of the BCAT proteins has been extensively mapped in rodent models, and metabolic studies have established that BCAT transamination in the rodent brain is responsible for 30% of de novo glutamate synthesis. However, to date the BCAT proteins have not been mapped to the human brain and their role in pathogenic conditions where glutamate toxicity features has not been investigated. To this end, this study aimed to map the hBCAT proteins to several brain regions. Furthermore, the expression of hBCAT in AD relative to matched controls was investigated and correlated with both physiological and pathological features of AD. Finally, metabolic and inflammatory stimuli were examined for their effect on neuronal expression of hBCATc.
Methods: Distribution of the hBCAT proteins were assessed utilising immunohistochemistry and imaged utilising a 12-bit camera mounted on a Leica DM microscope. Western blot analysis and microscopy determined the expressional difference in AD compared to age and gender matched controls in addition to cell types responsible for the increased expression. Further investigation of neuronal hBCATc expression was examined in the immortal cell line IMR32 utilising Western blot analysis, phase contrast microscopy, flow cytometry and 14C radiolabelled activity assay.
Results & Discussion: For the first time this work demonstrates key differences between the animal model of BCAA metabolism and humans. All brain regions contained cell types labelled for hBCATc and hBCATm. However, while this work mirrored animal models in that hBCATc was localised specifically to neurons, hBCATm was absent from astrocytes and instead labelled the vasculature – contrary to animal models. Another novel finding of this work links altered aminotransferase expression to AD pathology. An increase of hBCATm expression of +117% (p = 2.29 x 10-4) and +143% (p = 7.70 x 10-5) in the frontal and temporal cortex of AD subjects relative to matched controls demonstrates the disease association of hBCATm. A non-significant increase of 32% was observed for hBCATc in the frontal region. With hBCATm expression correlating with Braak stage in both the frontal (p = 1.2 x 10-5, ρ+0.468) and temporal (p = 3.4 x 10-4, ρ+0.391) cortex this work posits that altered BCAA metabolism is occurring simultaneously with AD progression and may be a novel therapeutic target for the treatment of dementia. Another novel aspect of this work also demonstrates cell surface expression of hBCATc and relates this to mTOR signalling. Altered cell surface and protein expression was investigated with functional activity. Together this data demonstrated expressional, functional or activity changes in hBCATc due to glutamate, insulin, leucine, TNFα and IL1α.

• Thesis Type: Thesis
• Publicly Available Date: Jun 6, 2019
• Keywords: BCAT glutamate, Alzheimer's
• Public URL: https://uwe-repository.worktribe.com/output/824745
• Award Date: Jan 1, 2014