Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease.

Huntington's disease (HD) is caused by an expanded CAG tract in the Interesting transcript 15 (IT15) gene encoding the 350 kDa huntingtin protein. Cellular stresses can trigger the release of huntingtin from the endoplasmic reticulum, allowing huntingtin nuclear entry. Here, we show that endogenous, full-length huntingtin localizes to nuclear cofilin-actin rods during stress and is required for the proper stress response involving actin remodeling. Mutant huntingtin induces a dominant, persistent nuclear rod phenotype similar to that described in Alzheimer's disease for cytoplasmic cofilin-actin rods. Using live cell temporal studies, we show that this stress response is similarly impaired when mutant huntingtin is present, or when normal huntingtin levels are reduced. In clinical lymphocyte samples from HD patients, we have quantitatively detected cross-linked complexes of actin and cofilin with complex formation varying in correlation with disease progression. By live cell fluorescence lifetime imaging measurement-Förster resonant energy transfer studies and western blot assays, we quantitatively observed that stress-activated tissue transglutaminase 2 (TG2) is responsible for the actin-cofilin covalent cross-linking observed in HD. These data support a direct role for huntingtin in nuclear actin re-organization, and describe a new pathogenic mechanism for aberrant TG2 enzymatic hyperactivity in neurodegenerative diseases.

Amyloid-ß-induced amyloid-ß secretion: a possible feed-forward mechanism in Alzheimer's Disease.

Amyloid-ß (Aß) peptides, 36-43 amino acids in length, are produced from ß- and γ-secretase cleavage of the amyloid-ß protein precursor (AßPP), and are one of the causative agents of Alzheimer's disease (AD). Here we show that an ELISA can detect total rodent Aß without interference from physiological concentrations of human Aß. In cultured dissociated rat cortical neurons and rat and mouse hippocampal organotypic slices, we apply the assay to measure the production of Aß in response to treatment with hydrogen peroxide, a known stimulator of Aß secretion, or human Aß dimer/trimer (Aßd/t), fractionated from the culture medium of 7PA2 cells. Peroxide increases Aß secretion by about 2 fold, similar to results from previous reports that used a different assay. Of greater significance is that physiologically relevant concentrations (~250 pM) of human Aßd/t increase rodent Aß secretion from cultured rat cortical neurons by >3 fold over 4 days. Surprisingly, neither treatment with peroxide nor human Aßd/t leads to accumulation of intracellular Aß. Human Aßd/t increased >2 fold the Aß secreted by organotypic hippocampal slices from tau knock-out mice whether or not they expressed a human tau transgene, suggesting tau plays no role in enhanced Aß secretion. Together, these results support an Aß-mediated feed-forward mechanism in AD progression.

Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin-phosphorylation.

BACKGROUND: Previously we reported 1 µM synthetic human amyloid beta1-42 oligomers induced cofilin dephosphorylation (activation) and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus. RESULTS: Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aß dimers and trimers (Aßd/t) induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aß oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aß1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC50) in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aßd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state), lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aß monomers are not active, suggesting oxidized SDS-stable Aß1-42 dimers in a low-n state are the most active rod-inducing species. Aßd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aßd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aßd/t, whereas overexpression of a cofilin kinase inhibits Aßd/t-induced rod formation. CONCLUSIONS: Together these data support a mechanism by which Aßd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

Validation of pharmaceutical potency determinations by quantitative nuclear magnetic resonance spectrometry.

With the changing development paradigms in the pharmaceutical industry, laboratories are challenged to release materials for clinical studies with rapid turnaround times. To minimize cost demands, many businesses are looking to develop ways of using early Good Manufacturing Practice (GMP) materials of active pharmaceutical ingredients (API) for Good Laboratory Practice (GLP) toxicology studies. To make this happen, the analytical laboratory releases the material by one of three scenarios: (1) holding the GLP release until full GMP testing is ready, (2) issuing a separate lot number for a portion of the GMP material and releasing the material for GLP use, or (3) releasing the lot of material for GLP using alternate (equivalent) method(s) not specified for GMP release testing. Many companies are finding the third scenario to be advantageous in terms of cost and efficiency through the use of quantitative nuclear magnetic resonance (q-NMR). The use of q-NMR has proved to be a single-point replacement for routine early development testing that previously combined elements of identity testing, chromatographic assay, moisture analysis, residual solvent analysis, and elemental analysis. This study highlights that q-NMR can be validated to meet current regulatory analytical method guidelines for routine pharmaceutical analysis.

Determination of relative response factors for chromatographic investigations using NMR spectrometry.

In the absence of suitable reference materials for impurity quantitation, laboratories have developed techniques using mass detectors such as the chemical luminescence detector (CLND) and the charged aerosol detector (CAD) to normalize the UV response of each impurity of interest by their molar ratios and thus generate relative response factors without requiring isolated and purified compound-specific standards. While effective, these detectors are limited in response and are effective only with specific mobile phase requirements. Nuclear magnetic resonance (NMR) spectrometry has the advantage of allowing the universal detection of protons while not suffering from the limitations observed for CLND, CAD, and other common detectors. The determination of relative response factors using NMR has been successfully applied to several LC methods. An overview of this technique and representative results are presented.

An efficient, stereoselective synthesis of the hydroxyethylene dipeptide isostere core for the HIV protease inhibitor A-792611.

A stereoselective synthesis of the hydroxyethylene dipeptide isostere 1 is described. The route employs a substrate-directed kinetic protonation of an alpha/gamma-substituted lactone to afford the desired stereochemistry. A method for converting the diastereomerically enriched intermediate lactone to the ring-open form with retention of stereochemistry is demonstrated. A novel procedure for utilizing N,N-dibromo-5,5-dimethylhydantoin in Hofmann rearrangements is disclosed. This route was used to prepare amino alcohol 1, the core portion of the HIV protease inhibitor A-792611, in 46% yield from phenylalanine-derived epoxide 2.

An unusual intramolecular hetero-Diels-Alder cycloaddition.

A new reaction of erythronolides, an intramolecular hetero-Diels-Alder, has been discovered. Heated aqueous alcoholic solutions of ABT-773 (1) and its cis isomer (3) convert slowly to cycloadducts 2 and 4, respectively. Optimal reaction conditions, mechanistic studies supported by molecular modeling, and biological activity data are reported. Single-crystal X-ray structures for both adducts 2 and 4 have been obtained.