2015 Progress Reports Brain Foundation brings you the latest research findings from 2013 Grants. Two of these projects have applied for NHMRC (government) funding to further progress the initial results towards a better understanding for treatments in Stroke and Frontotemporal dementia.
Dendric spine alterations in TDP-43 aggregated Frontotemporal dementia: a novel therapeutic target 2013 Brain Foundation Grant
Chief Investigator: Dr Catherine Blizzard Frontotemporal dementia (FTD) has a prevalence second only to Alzheimer’s disease persons under the age of 65. And yet, relatively little is known about how pathogenic events lead to cognitive decline. FTD is caused by frontotemporal lobar degeneration (FTLD) that can be pathologically characterised by the cytoplasmic accumulation of aggregated proteins. One such protein is the transactive response DNA-binding protein 43 (TDP-43). There is increasing evidence that early synaptic alterations play a key role in many neurodegenerative diseases. Recent studies have shown that, in addition to its’ localisation to the nucleus and pathologically in the cytoplasm, TDP-43 is also localised within somatodendritic spines of hippocampal neurons, where it has been shown to act as an upstream regulator of spine turnover and homeostasis. These observations, in conjunction with our preliminary evidence of synaptic scaffolding proteins being misprocessed
in the TDP-43 A315T biological model of FTLD, open up a new potential route by which TDP-43 can affect neuronal function and conversely be implicated in the pathogenesis of neurodegenerative diseases. Therefore we are investigating the novel hypothesis ‘mutant TDP-43 results in synaptic pathology, leading to impaired neuroplasticity and ALS’. To achieve this we have created a novel transgenic biological model cross, using the TDP-43A315T biological model of FTD, and yellow fluorescent protein (YFP) models. These models ubiquitously express YFP in subsets of motor neurons in the cortex, making them an ideal model to study synapse morphology. With this transgenic biological model we have created a complete tissue bank of YFP crossed TDP-43A315T positive and control brains over a time-course of disease and are currently characterising levels of synaptic proteins and postsynaptic spine densities and morphology. Immunohistochemistry directed at endogenous YFP and synaptic markers
was performed on tissue from TDP43A315T models crossed with YFP-H models (P30 n = 5, P90 n =5) and in YFP-H litter matched controls (P30 n = 5, P90 n = 5). Confocal microscopy with subsequent image analysis was used to quantify YFP-H expressing layer V pyramidal neurons, pre-synaptic punctum and basal and apical dendritic spines in the motor and somatosensory cortices. There was no significant difference in the number of YFP-expressing pyramidal neurons in either the motor or somatosensory cortices between TDP43A315T x YFP-H models and YFP-H controls at 30 days (p > 0.05, twoway ANOVA with Bonferroni post-hoc test). There was however, a significant reduction in pyramidal neurons of the motor cortex in TDP-43A315T x YFP-H models compared to controls at 90 days, and in comparison to the motor cortex of TDP-43A315T x YFP-H models at 30 days. No significant changes were identified in pre-synaptic punctum levels at either time point (p > 0.05, two ANOVA
with Bonferroni post-hoc text). At 30 days, there were no significant differences in spine density between TDP-43A315T x YFP-H models and controls; however, in TDP-43A315T x YFP-H models at 90 days there were significant reductions in the spine density of motor basal and apical dendrites, and of somatosensory apical dendrites, in comparison to YFP-H controls. Furthermore we are currently in the process of establishing the 2PLSM live imaging platform in this novel mouse cross that will enable us to determine a precise time-course of synaptic alterations, pin-pointing the earliest synaptic events occurring prior to cell loss. Our findings, which form the backbone of a 2016 NHMRC project grant application, suggest post-synaptic dysfunction may be an early-occurring event in mutated TDP-43 pathology, occurring prior to neuronal loss. Understanding the role that TDP-43 plays in synaptic dysfunction may reveal new therapeutic windows for intervention in TDP-43 proteinopathies.
Targeting astrogliosis and brain stimulation after stroke to promote plasticity and functional recovery 2013 Brain Foundation Grant
Chief Investigator: Dr Carli Roulston Stroke often results in permanent brain damage which is ultimately due to a failure of nerve cells to re-grow across the injury site. Although the brain generates new stem cells in response to injury very few of these cells end up replacing damaged circuitry. Indeed, studies now show that the majority of migrating stem cells after stroke differentiate into astrocytes that become rapidly activated and contribute further to glial scar formation. Over activation of pre-existing astrocytes also disrupts neuronal signalling through reduced neurotransmitter turnover and energy transfer. We therefore hypothesised that targeting over-activation of astrocytes to reduce the glial scar would facilitate recovery of neuronal pathways affected by stroke.
Our first study explored the potential effect of delayed treatment with the Rho-kinase inhibitor, fasudil, on reducing astrocyte reactivity after stroke. These studies were performed by an honours student, Ms Ellie Phillips, supervised by Dr Carli Roulston, as part of the Biomedical Science degree at the University of Melbourne. Stroke was induced through application of the potent vasoconstrictor Endothelin-1 (60mol in 3µL saline) to constrict the middle cerebral artery in conscious male hooded Wistar models. Fasudil treatment was delayed until 3 days after stroke (50 mg/kg, intraperitoneal injections, daily) with vehicle (saline) and sham controls. Models were treated for 28 days (n=9 per treatment group), over which time functional outcomes were assessed
using various tests including the neurological deficit score, cylinder test, tape test and staircase test. After 28 days histological analysis of brain sections were conducted to observe the effect of fasudil treatment on astrocytes and other brain remodeling events. A smaller cohort (n=5 per treatment group) was recovered to 14 days and quantitative proteomics conducted using stable isotope dimethyl labelling in order to assess early changes in protein expression that may lead to functional recovery. Stroke resulted in observable brain injury in all models with no difference in infarct size detected between treatment groups. Neurological deficits were detected in all groups after stroke and were significantly recovered following treatment with
Fasudil, in comparison to vehicle controls. Given that treatment with Fasudil was delayed until after damage had occurred, the results of this study suggest that Fasudil can promote functional recovery in the absence of neuroprotection. Further histological analysis confirmed that fasudil treatment significantly reduced the number of astrocytes present in the damaged striatum and cortex and astrocytes that were detected showed a less reactive morphology similar to that of trophic astrocytes. Since recovery to the contralateral forepaw was rapidly observed upon treatment, we suggest that the effects of Fasudil are likely mediated through restored neurotransmission in surviving pathways, rather than axonal regeneration or remodelling. As such we have identified a new target for Fasudil, To top of page 5
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