| abstract |
Past research into therapeutic compounds for Alzheimer's disease which have
often focused on targeting amyloid beta, tau, and post-synaptic NMDA receptors, have
shown largely underwhelming results. An alternative approach is targeting pre-synaptic
glutamate release, achieved by modulating pre-synaptic metabotropic glutamate
receptors. Group III mGluRs, mGluR4 in particular, through inhibition of second
messenger pathways such as adenylyl cyclase, regulate the release of the glutamate from
the pre-synaptic neuron. This activity may mitigate excitotoxic glutamatergic
transmission and provide neuroprotection, as seen in studies using the mGluR4 positive
allosteric modulator, PHCCC, and the mGluR4 agonist, L-AP4. The present pilot study
seeks to investigate a potential mGluR4 PAM, known as RD100, in a FAB/NMDA
primary rat cell culture model of AD. The goals of this study are to identify the effects of
RD100 on cell survival and oxidative stress and identify its mechanism of action.
Elucidation of RD100's mechanistic pathway will be conducted with co-application of
MSOP, a broad group III mGluR antagonist. This study also seeks to optimize use of the
FAB/NMDA model system by identifying appropriately neurotoxic concentrations, and
assess its effects on oxidative stress. To assess oxidative stress, MitoSOX red
mitochondrial superoxide indicator will be utilized as a marker of superoxide. The most
useful and reflective measurements of MitoSOX fluorescence under the compound
microscope will also be determined.
At 5uM, RD100 does not have a significant effect on cellular survival. FAB/100
uM NMDA elicits a concentration dependent negative effect on cellular survival, with at
least 33.3% FAB significantly reducing survival. For assessment of superoxide levels,
correction of relative intensity by binary area (intensity/area) should be conducted as the
most reflective measure of MitoSOX fluorescence. Neither RD100 nor FAB/NMDA
have any significant effect on superoxide at 24 hrs after application. It was unable to be
determined whether RD100 acts as a positive modulator of mGluR4 due to RD100's and
MSOP's lack of significant effects on cell survival, though joint toxicity of RD100 and
MSOP suggests so. Recommendations for further studies include determining the effects
of PHCCC in the FAB/NMDA model, investigating the neuroprotection of other RD
compounds, and further elucidation of the timing of ROS influenced by FAB/100 uM
NMDA.
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