Investigation of Two Potential Treatments for Frontotemporal Dementias by Mass Spectrometry

Intraneuronal tangles of the microtubule associated protein tau are a hallmark of frontotemporal dementia and other tauopathies. Tau stabilizes tubulin subunits to form microtubules in neuronal projections, but must be removed to allow transport vesicles to pass. This dynamic process is controlled by the phosphorylation state of tau; increased phosphorylation removes tau from the microtubule. Unbound tau aggregates and is unable to be cleared, so a small molecule inhibitor of aggregation could stop the progression of the pathology. The van Breemen lab has access to various natural product fractions prepared by collaborating research groups and are not widely available. The previously published tau aggregation inhibitions screening assay utilized a fluorescence detector, which can only screen single molecule samples. A mass spectrometry based screen was developed to be able to screen natural product fractions, which is described in Chapter 2. Natural product fractions obtained from marine actinomycetes were screened in Chapter 3 and hit fractions were identified. The three fractions which inhibited aggregation were profiled using a metabolomics approach, which is described in Chapter 4. The identification and structure elucidation of a small amount of natural product is the most difficult step, so this approach moves it to the end to minimize the number of compounds that need to be identified. The actinomycetes need to be recultured, taking several months, so the profiles of the new and old fractions can be compared to confirm the same compounds are present. The 5-HT6 receptor antagonist, PRX-07034 is a potential treatment for memory issues related to frontotemporal dementias. Chapter 5 outlines a validated UHPLC-MS-MS method to quantitate the compound in both rat serum and brain. The behavioral effects of the treatment have been studied for nearly 10 years; however the compound was never analytically determined to reach the brai

ISSLS PRIZE IN BASIC SCIENCE 2018: Growth differentiation factor-6 attenuated pro-inflammatory molecular changes in the rabbit anular-puncture model and degenerated disc-induced pain generation in the rat xenograft radiculopathy model

PURPOSE:To elucidate the effects of growth differentiation factor-6 (GDF6) on: (i) gene expression of inflammatory/pain-related molecules and structural integrity in the rabbit intervertebral disc (IVD) degeneration model, and (ii) sensory dysfunction and changes in pain-marker expression in dorsal nerve ganglia (DRGs) in the rat xenograft radiculopathy model. METHODS:Forty-six adolescent rabbits received anular-puncture in two non-consecutive lumbar IVDs. Four weeks later, phosphate-buffered saline (PBS) or GDF6 (1, 10 or 100 µg) was injected into the nucleus pulposus (NP) of punctured discs and followed for 4 weeks for gene expression analysis and 12 weeks for structural analyses. For pain assessment, eight rabbits were sacrificed at 4 weeks post-injection and NP tissues of injected discs were transplanted onto L5 DRGs of 16 nude rats to examine mechanical allodynia. The rat DRGs were analyzed immunohistochemically. RESULTS:In GDF6-treated rabbit NPs, gene expressions of interleukin-6, tumor necrosis factor-α, vascular endothelial growth factor, prostaglandin-endoperoxide synthase 2, and nerve growth factor were significantly lower than those in the PBS group. GDF6 injections resulted in partial restoration of disc height and improvement of MRI disc degeneration grades with statistical significance in rabbit structural analyses. Allodynia induced by xenograft transplantation of rabbit degenerated NPs onto rat DRGs was significantly reduced by GDF6 injection. Staining intensities for ionized calcium-binding adaptor molecule-1 and calcitonin gene-related peptide in rat DRGs of the GDF6 group were significantly lower than those of the PBS group. CONCLUSION:GDF6 injection may change the pathological status of degenerative discs and attenuate degenerated IVD-induced pain