Aida Attar

Induction of methionine-sulfoxide reductases protects neurons from amyloid β-protein insults in vitro and in vivo.

Self-assembly of amyloid β-protein (Aβ) into toxic oligomers and fibrillar polymers is believed to cause Alzheimers disease (AD). In the AD brain, a high percentage of Aβ contains Met-sulfoxide at position 35, though the role this modification plays in AD is not clear. Oxidation of Met(35) to sulfoxide has been reported to decrease the extent of Aβ assembly and neurotoxicity, whereas surprisingly, oxidation of Met(35) to sulfone yields a toxicity similar to that of unoxidized Aβ. We hypothesized that the lower toxicity of Aβ-sulfoxide might result not only from structural alteration of the C-terminal region but also from activation of methionine-sulfoxide reductase (Msr), an important component of the cellular antioxidant system. Supporting this hypothesis, we found that the low toxicity of Aβ-sulfoxide correlated with induction of Msr activity. In agreement with these observations, in MsrA(-/-) mice the difference in toxicity between native Aβ and Aβ-sulfoxide was essentially eliminated. Subsequently, we found that treatment with N-acetyl-Met-sulfoxide could induce Msr activity and protect neuronal cells from Aβ toxicity. In addition, we measured Msr activity in a double-transgenic mouse model of AD and found that it was increased significantly relative to that of nontransgenic mice. Immunization with a novel Met-sulfoxide-rich antigen for 6 months led to antibody production, decreased Msr activity, and lowered hippocampal plaque burden. The data suggest an important neuroprotective role for the Msr system in the AD brain, which may lead to development of new therapeutic approaches for AD.

Molecular Basis for Preventing α-Synuclein Aggregation by a Molecular Tweezer

Background: The molecular tweezer, CLR01, binds to Lys and prevents aggregation of α-synuclein. Results: CLR01 binds directly to monomeric α-synuclein near the N terminus and changes the charge distribution in the sequence, swelling the chain, and increasing the protein reconfiguration rate. Conclusion: Aggregation is inhibited by making the protein more diffusive. Significance: The most effective aggregation inhibitors may change monomer dynamics rather than structure. Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examining α-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 to α-synuclein occurs at the N-terminal Lys-10/Lys-12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis, α-synuclein is predominantly monomeric. The results can be successfully modeled using a kinetic scheme in which two aggregation-prone monomers can form an encounter complex that leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high. Taken together, the data provide important insights into the preferred binding site of CLR01 on α-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins.

A shortened barnes maze protocol reveals memory deficits at 4-months of age in the triple-transgenic mouse model of Alzheimer's disease

Alzheimers disease is a progressive neurodegenerative disease that manifests as memory loss, cognitive dysfunction, and dementia. Animal models of Alzheimers disease have been instrumental in understanding the underlying pathological mechanism and in evaluation of potential therapies. The triple transgenic (3×Tg) mouse model of AD is unique because it recapitulates both pathologic hallmarks of Alzheimers disease - amyloid plaques and neurofibrillary tangles. The earliest cognitive deficits in this model have been shown at 6-m of age by most groups, necessitating aging of the mice to this age before initiating evaluation of the cognitive effects of therapies. To assess cognitive deficits in the 3×Tg mice, originally we employed a typical Barnes maze protocol of 15 training trials, but found no significant deficits in aged mice. Therefore, we shortened the protocol to include only 5 training trials to increase difficulty. We found cognitive deficits using this protocol using mainly measures from the probe day, rather than the training trials. This also decreased the effort involved with data analysis. We compared 3×Tg and wild-type mice at 4-m- and 15-m of age using both the original, long training, and the short training paradigms. We found that differences in learning between 3×Tg and wild-type mice disappeared after the 4th training trial. Measures of learning and memory on the probe day showed significant differences between 3×Tg and wild-type mice following the short, 5-training trial protocol but not the long, 15-training trial protocol. Importantly, we detected cognitive dysfunction already at 4-m of age in 3×Tg mice using the short Barnes-maze protocol. The ability to test learning and memory in 4-m old 3×Tg mice using a shortened Barnes maze protocol offers considerable time and cost savings and provides support for the utilization of this model at pre-pathology stages for therapeutic studies.

Molecular Tweezers Targeting Transthyretin Amyloidosis

Transthyretin (TTR) amyloidoses comprise a wide spectrum of acquired and hereditary diseases triggered by extracellular deposition of toxic TTR aggregates in various organs. Despite recent advances regarding the elucidation of the molecular mechanisms underlying TTR misfolding and pathogenic self-assembly, there is still no effective therapy for treatment of these fatal disorders. Recently, the “molecular tweezers”, CLR01, has been reported to inhibit self-assembly and toxicity of different amyloidogenic proteins in vitro, including TTR, by interfering with hydrophobic and electrostatic interactions known to play an important role in the aggregation process. In addition, CLR01 showed therapeutic effects in animal models of Alzheimer’s disease and Parkinson’s disease. Here, we assessed the ability of CLR01 to modulate TTR misfolding and aggregation in cell culture and in an animal model. In cell culture assays we found that CLR01 inhibited TTR oligomerization in the conditioned medium and alleviated TTR-induced neurotoxicity by redirecting TTR aggregation into the formation of innocuous assemblies. To determine whether CLR01 was effective in vivo, we tested the compound in mice expressing TTR V30M, a model of familial amyloidotic polyneuropathy, which recapitulates the main pathological features of the human disease. Immunohistochemical and Western blot analyses showed a significant decrease in TTR burden in the gastrointestinal tract and the peripheral nervous system in mice treated with CLR01, with a concomitant reduction in aggregate-induced endoplasmic reticulum stress response, protein oxidation, and apoptosis. Taken together, our preclinical data suggest that CLR01 is a promising lead compound for development of innovative, disease-modifying therapy for TTR amyloidosis.

Modulators of amyloid protein aggregation and toxicity: EGCG and CLR01

Abnormal protein folding and self-assembly causes over 30 cureless human diseases for which no disease-modifying therapies are available. The common side to all these diseases is formation of aberrant toxic protein oligomers and amyloid fibrils. Both types of assemblies are drug targets, yet each presents major challenges to drug design, discovery, and development. In this review, we focus on two small molecules that inhibit formation of toxic amyloid protein assemblies — the green-tea derivative (−)-epigallocatechin-3-gallate (EGCG), which was identified through a combination of epidemiologic data and a compound library screen, and the molecular tweezer CLR01, whose inhibitory activity was discovered in our group based on rational reasoning, and subsequently confirmed experimentally. Both compounds act in a manner that is not specific to one particular protein and thus are useful against a multitude of amyloidogenic proteins, yet they act via distinct putative mechanisms. CLR01 disrupts protein aggregation through specific binding to lysine residues, whereas the mechanisms underlying the activity of EGCG are only recently beginning to unveil. We discuss current in vitro and, where available, in vivo literature related to EGCG and CLR01’s effects on amyloid β-protein, α-synuclein, transthyretin, islet amyloid polypeptide, and calcitonin. We also describe the toxicity, pharmacokinetics, and mechanism of action of each compound.

Assembly of Amyloid β-Protein Variants Containing Familial Alzheimer's Disease- Linked Amino Acid Substitutions

Amyloid β-protein (Aβ) is believed to cause Alzheimer’s disease (AD); this belief is based largely on studies showing autosomal dominant inheritance of familial AD (FAD) due to mutations that increase brain concentration levels of Aβ or of particular forms of Aβ. However, how specifically this leads to AD is not clear. Several mutations have been identified inside the Aβ-coding region of the amyloid β protein precursor (APP); these mutations change both the biophysical characteristics of Aβ and the disease phenotype, such as age of onset of dementia and location and morphology of amyloid deposits. Characterizing the effects of the amino acid changes in Aβ on the protein’s assembly and toxicity may help to identify Aβ regions particularly useful as drug targets. We summarize here key findings related to substitutions at positions 2, 6, 7, 16, 21–23, and 34 in Aβ and their relation to FAD.

Project brainstorm: using neuroscience to connect college students with local schools.

Neuroscience can be used as a tool to inspire an interest in science in school children as well as to provide teaching experience to college students.

Using Molecular Tweezers to Remodel Abnormal Protein Self-Assembly and Inhibit the Toxicity of Amyloidogenic Proteins

Molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein self-assembly, which act by binding selectively to lysine and arginine residues. Through this unique mechanism of action, MTs inhibit formation of toxic oligomers and aggregates. Their efficacy and safety have been demonstrated in vitro, in cell culture, and in animal models. Here, we discuss the application of MTs in diverse in vitro and in vivo systems, the experimental details, the scope of their use, and the limitations of the approach. We also consider methods for administration of MTs in animal models to measure efficacy, pharmacokinetic, and pharmacodynamic parameters in proteinopathies.

Erratum to: A Novel “Molecular Tweezer” Inhibitor of α-Synuclein Neurotoxicity In Vitro and In Vivo

Erratum to: Neurotherapeutics DOI 10.1007/s13311-012-0105-1 Rajeswari Lakshmanan was listed incorrectly on the original publication of this article. The author list is corrected here.

Novel, small molecule inhibitors of protein aggregation for treatment of amyloid-related diseases

Ab plaque burden. Catalytically incompetent 2E6 treated with a protease inhibitor and a non-proteolytic control IgVL2 did not express these activities. The major 2E6-cleavage site in Ab was the His14-Gln15 peptide bond. Epitope mapping indicated competitive inhibition of 2E6-catalyzed I-Ab degradation by the remote Ab29-40 peptide, identifying this region as the noncovalent recognition epitope. 2E6-Ab immune complexes were undetectable by ELISA, consistent with rapid progress of the reaction to the catalysis step. 2E6 did not cleave His-Gln containing proteins, indicating that specificity for Ab derives from the noncovalent binding step. Conclusions: The Ab fragment targets monomer and aggregate forms of Ab and clears brain Ab plaques by initial noncovalent recognition of the amyloidogenic C terminal region followed by cleavage at remote peptide bonds. Catalysis is rapid and no immune complexes are detectable. This catalytic antibody fragment can be developed for more effective and safe AD immunotherapy.