Multitargeted Imidazoles: Potential Therapeutic Leads for Alzheimer's and Other Neurodegenerative Diseases

Alzheimer’s disease (AD) is a complex, multifactorial disease in which different neuropathological mechanisms are likely involved, including those associated with pathological tau and Aβ species as well as neuroinflammation. In this context, the development of single multitargeted therapeutics directed against two or more disease mechanisms could be advantageous. Starting from a series of 1,5-diarylimidazoles with microtubule (MT)-stabilizing activity and structural similarities with known NSAIDs, we conducted structure−activity relationship studies that led to the identification of multitargeted prototypes with activities as MT-stabilizing agents and/or inhibitors of the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways. Several examples are brain-penetrant and exhibit balanced multitargeted in vitro activity in the low μM range. As brain-penetrant MT-stabilizing agents have proven effective against tau-mediated neurodegeneration in animal models, and because COX- and 5-LOX-derived eicosanoids are thought to contribute to Aβ plaque deposition, these 1,5-diarylimidazoles provide tools to explore novel multitargeted strategies for AD and other neurodegenerative diseases

PINCH in the cellular stress response to tau-hyperphosphorylation.

Particularly interesting new cysteine- histidine- rich protein (PINCH) is an adaptor protein that our data have shown is required for neurite extension under stressful conditions. Our previous studies also report that PINCH is recalled by neurons showing decreased levels of synaptodendritic signaling proteins such as MAP2 or synaptophysin in the brains of human immunodeficiency virus (HIV) patients. The current study addressed potential role(s) for PINCH in neurodegenerative diseases. Mass spectrometry predicted the interaction of PINCH with Tau and with members of the heat shock response. Our in vitro data confirmed that PINCH binds to hyperphosphorylated (hp) Tau and to E3 ubiquitin ligase, carboxy-terminus of heat shock-70 interacting protein. Silencing PINCH prior to induction of hp-Tau resulted in more efficient clearance of accumulating hp-Tau, suggesting that PINCH may play a role in stabilizing hp-Tau. Accumulation of hp-Tau is implicated in more than 20 neuropathological diseases including Alzheimer's disease (AD), frontotemporal dementia (FTD), and human immunodeficiency virus encephalitis (HIVE). Analyses of brain tissues from HIVE, AD and FTD patients showed that PINCH is increased and binds to hp-Tau. These studies address a new mechanism by which AD and HIV may intersect and identify PINCH as a contributing factor to the accumulation of hyperphosphorylated Tau

PINCH deletion mutants and sequences.

<p>PINCH deletion mutants and sequences.</p

PINCH levels in relation to changes in Tau solubility.

<p>The proteins from frontal cortex homogenates from normal, different Braak stages (1, 3, 5) of Alzheimer's disease (AD), HIV encephalitis (HIVE), and frontotemporal dementia (FTD) patients were fractionated into RAB (most soluble, RB), RIPA (less soluble, RB) and formic acid (FA) (least soluble). Representative Western blots from A) normal control, AD, HIVE, and FTD showing levels of PINCH, hpTau (s396) and total Tau (HT7). B) Coomassie stained gel of the same cases indicating total protein in each fraction. Compared to the control, increased levels of hp-Tau and PINCH are observed in disease cases. Loss of Tau and PINCH solubility are apparent in AD, HIVE and FTD, as well. Arrow in the FTD case indicates a blank lane in the gel.</p

Exposure of neurons to supernatant from HIV-infected PBMCs results in increased PINCH, hp-Tau and CHIP.

<p>A) Representative Western blot showing levels of PINCH1, hpTau, total Tau, Hsp90, Hsp70 and CHIP from neurons grown in media without PBMCs (con), neurons exposed to supernatant from control PBMCs without HIV infection (PBMC), supernatant from PBMCs infected with HIV (PBMC+HIV), or neurons exposed to cell-free virus (HIV). Grb-2 was used as the loading control. B) Fold change in hpTau/Total Tau.</p

Reciprocal immunoprecipitations indicate PINCH interacts with hp-Tau and CHIP.

<p>Neuronal lysate was immunoprecipitated with anti PINCH and reacted with antibodies against hp-Tau A) AT100 and B) AT8. C) Neuronal lysate was immunoprecipitated with anti-AT8 antibody and reacted with anti-PINCH, anti-Hsp90, anti-Hsp70 and CHIP. D) Neuronal lysate was immunoprecipitated with anti-PINCH antibody and reacted with anti-Hsp90, anti-Hsp70, anti-CHIP and anti-ILK. E) Reciprocal IP with anti-CHIP and reaction with anti-PINCH. F) Neuronal lysate was treated with (+) or without (−) phosphatase and immunoprecipitated with anti-HT7 antibody against total Tau and reacted with anti-PINCH antibody. G) The same neuronal lysate with (+) and without (−) phosphatase was immunoblotted with anti-HT7 and anti-PINCH antibodies without immunoprecipitation. Arrows indicate immunoreactive bands. Hc, heavy chain; lc, light chain; No IP (lysate), beads only (IgG); immunoprecipitation (IP).</p

Exposure of neurons to TNF-α results in increased levels of PINCH, hp-Tau and CHIP.

<p>Representative Western blot of A) neurons exposed to TNF-α; B) graphic representation of fold change of PINCH1, hp-Tau, total Tau and CHIP levels in TNF-α treated neurons over control. Results are from 4 separate experiments and are expressed as fold change over control. * p<0.005, **p<0.001 by one-way ANOVA with Tukey-Kramer post-hoc analyses. Grb-2 was used as the loading control. C) Fold change in hp-Tau/Total Tau * p = 0.0293 by one-tailed paired T-test.</p

Expression levels of hp-Tau and PINCH in brain tissue from human Tau transgenic mouse.

<p>A) Western analyses of anterior frontal cortex (Ant-Fc), ventro-lateral posterior cortex (V-L-post-FC), posterior frontal cortex (post-FC), cerebellum (CB). B) Double immunofluorescence labeling of PINCH (green) and hp-Tau (red), co-localization (yellow) in hippocampal tissue from a wild-type mouse and the Tau-Tg mouse and respectively.</p

shRNA sequence data for human PINCH1 and 2.

<p>shRNA sequence data for human PINCH1 and 2.</p