Traumatic brain injury (TBI) and Alzheimer's disease (AD) result in long-term behavioral deficits and brain
neurodegeneration. There are no effective therapeutic agents for TBI or AD; therefore, advances in mechanism-
based understanding of these brain disorders can identify new drug targeting approaches. Significantly,
cathepsin B has been shown as a novel mechanism participating in behavioral dysfunctions and neuropathology
of TBI and AD. The premise for the cathepsin B mechanism is that (a) cathepsin B is elevated in TBI and AD
patients, (b) knockout (KO) of the cathepsin B gene in TBI and AD mouse models improves deficits in behavioral
dysfunctions, respectively, and (c) cathepsin B KO reduces brain neuropathology.
TBI and AD results in lysosomal leakage and redistribution of cathepsin B from lysosomes to the cytosol to
result in cell death and activation of inflammatory IL-1ß in brain. These findings lead to the hypothesis that
cytosolic cathepsin B participates in the pathogenesis of TBI and AD. To test this hypothesis, it will be ideal to
inhibit the pathogenic cytosolic cathepsin, without affecting its normal lysosomal function, with pH selective
inhibitors as molecular probes. Our data shows that cathepsin B displays different peptide cleavage properties
at neutral cytosolic pH compared to lysosomal acidic pH. These differential cleavage properties support the
development of selective substrates and peptide inhibitors of cytosolic compared to lysosomal cathepsin B. The
goal of this project will be to develop pH selective inhibitors of neutral cytosolic cathepsin B, compared
to acidic lysosomal cathepsin B, as molecular probes for evaluation of the hypothesized pathogenic role
of cytosolic cathepsin B during cellular lysosomal leakage which leads to neurodegeneration and
behavioral deficits of TBI and AD. Aim 1 will assess the selective cleavage properties of cathepsin B at neutral
and acidic pHs, achieved by global 'Multiplex Substrate Profiling Mass Spectrometry (MSP-MS) and positional
scanning using a synthetic combinatorial library (PSSCL), for design and testing of pH selective peptide
substrates. Aim 2 will utilize pH selective assays of cathepsin B to identify natural product inhibitors, achieved
by screening collections of marine and terrestrial natural products, and assessing selectivity and potency. Aim
3 will develop peptidic inhibitors of cathepsin B, achieved by modifying pH selective peptide substrates with
AOMK, CMK, or VS groups; selectivity and potency will be assessed. Peptidic and natural product inhibitors will
be assessed for effects on cell death and IL-1ß levels during Aß- and H2O2-induced lysosomal leakage in neurons
and glial cells. Aim 4 will characterize in vivo lysosomal leakage of cathepsin B in TBI and AD mouse models
with respect to time-course, brain regions, and neuronal and glial cells. Cathepsin B inhibitors, known and newly
developed inhibitors, will be given before and during lysosomal leakage for evaluation of improvements in
behavioral deficits and neuropathology. The novel inhibitors will advantageously target pathogenic cytosolic
cathepsin B, rather than normal lysosomal cathepsin B, for future development of TBI and AD therapeutics.