Stroke is one of the most common cardiovascular diseases and is the third main cause of death following heart disease and cancer. Previous studies have documented the presence of autophagy in the brain which is activated as a result of cell stress following stroke. Aside from intravenous (i.v.) tissue plasminogen activator (tPA), there is no current cure or treatment for stroke. As a result, this study aims to target autophagy in neural stem cells to identify if stem cell functionality can be improved in in vivo models of ischaemic brain injury. The initial objective of the study was to identify if autophagy could be altered in vitro in neural stem cells, more specifically, Maudsley Hippocampal Cell Line-Clone 36 (MHP-36 cells). Viral transduction was used to alter autophagy in the cells by over-expressing ULK1 or knocking down Atg7. MHP-36 cells showed some markers of increased autophagy in vitro following ULK1 over-expression, but effects were not robust, as compared to parallel tests with other model cell lines (293A HEK). As such, we relied more on the alternate strategy of Atg7 knockdown as a means to investigate more generally the requirement for autophagy in neural stem cell functionality. Experiments in this thesis were able to thoroughly characterise inhibition of basal autophagy in vitro following Atg7 knockdown in MHP-36 cells and these autophagy-deficient neural stem cells were transplanted into the brain after experimental stroke using a mouse model of middle cerebral artery occlusion (MCAO). Animals were split into 3 transplantation groups: vehicle control; MHP/shRNA Scramble control; and MHP/Atg7-KD; and stem cell functionality in the ischaemic brain was measured using functional behavioural testing by the ladder crossing test. Animals were assessed pre-MCAO, post-MCAO and post stem cell treatment for 4 weeks. The motor function data set so far suggest that both stem cell treatments (shScrambled and shAtg7) can provide some additional functional recovery above levels observed in the vehicle control group, most clearly at the end of the 4 week post-injection period. Surprisingly, MHP-36 cells with autophagy knockdown did not show any significant differences as compared to the shScrambled non- targeted stem cells, based on the motor functional recovery measurements. In conclusion, our results so far suggest that autophagy may not be absolutely critical for the functionality of therapeutic MHP-36 neural stem cells, contrary to current models, although additional evidence is needed with larger group sizes in order to improve our power to detect these effects.
|Date of Award||29 May 2015|
- University Of Strathclyde
|Supervisor||Edmond Chan (Supervisor) & Hilary Carswell (Supervisor)|