The aggregation of the amyloid beta (Aβ) protein into plaques is a pathological feature of Alzheimer's disease (AD). While amyloid aggregates have been extensively studied in vitro, their structural aspects and associated chemistry in the brain are not fully understood. In this report, we demonstrate, using infrared spectroscopic imaging, that Aβ plaques exhibit significant heterogeneities in terms of their secondary structure and phospholipid content. We show that the capabilities of discrete frequency infrared imaging (DFIR) are ideally suited for characterization of amyloid deposits in brain tissues and employ DFIR to identify nonplaque β-sheet aggregates distributed throughout brain tissues. We further demonstrate that phospholipid-rich β-sheet deposits exist outside of plaques in all diseased tissues, indicating their potential clinical significance. This is the very first application of DFIR toward a characterization of protein aggregates in an AD brain and provides a rapid, label-free approach that allows us to uncover β-sheet heterogeneities in the AD, which may be significant for targeted therapeutic strategies in the future.

Amyloid plaques are one of the central manifestations of Alzheimer’s disease pathology. Aggregation of the amyloid beta (Aβ) protein from amorphous oligomeric species to mature fibrils has been extensively studied. However, structural heterogeneities in prefibrillar species, and how that affects the structure of later-stage aggregates are not yet well understood. The integration of infrared spectroscopy with atomic force microscopy (AFM-IR) allows for identifying the signatures of individual nanoscale aggregates by spatially resolving spectra. We use AFM-IR to demonstrate that amyloid oligomers exhibit significant structural variations as evidenced in their infrared spectra. This heterogeneity is transmitted to and retained in protofibrils and fibrils. We show that amyloid fibrils do not always conform to their putative ordered structure and structurally different domains exist in the same fibril. We further demonstrate that these structural heterogeneities manifest themselves as a lack of β sheet structure in amyloid plaques in Alzheimer’s tissue using infrared imaging.

Prostate cancer (PCa) is the second most commonly occurring cancer among men and the fourth most commonly occurring cancer overall. According to American Cancer Society, in the year 2018, it was estimated that there will be 164,690 new cases and 29,430 deaths from PCa. The treatment options currently available for PCa are found to be ineffective with varied side effects and complications associated with the development of resistance among patients. Therefore, there is an unmet need to find a safe and potent agent to treat PCa. In our study, we focused on studying the anticancer potential of Methylene Blue (MB) which belongs to the class of phenothiazinium salt. MB has been widely used to treat the condition of methemoglobinemia, emerging studies have shown that it has been effectively used as a photosensitizer in the treatment of cancer by means of photodynamic therapy (PDT). Our initial analysis showed that MB effectively reduced the viability of androgen-dependent (LNCaP) and androgen-independent (PC3 and DU145) PCa cells. Further experimental evaluations showed that MB inhibited the colony forming ability of PCa cells in-vitro suggesting its tumor suppressive potential. In addition, our studies showed that MB treatment disrupted the migration potential of PCa cells in a wound healing assay indicating the anti-metastatic function of MB. Moreover, confocal and FACS analysis using Annexin V FITC and propidium iodide staining revealed that MB effectively targeted the PCa cell lines by inducing apoptotic cell death. To delineate the underlying anticancer mechanism of MB, apoptosis protein array was performed employing LNCaP cells, and the results of which showed that key apoptotic molecules such as Bax, TRAIL R2/D5, and phospho p53 (Serine 15, Serine 46, Serine 392) were robustly upregulated in LNCaP cells following MB treatment. In conclusion, our findings suggest that MB induces apoptosis in PCa cells and thus could serve as a potential anticancer agent for treating both hormone-dependent and -independent PCa. Citation Format: Priyadarshini Thiruvalluvan Shanthi, Abigail Foes, Gnanasekar Munirathinam. Evaluating the therapeutic effects of methylene blue against prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2958.

Aggregation of amyloid beta (Aβ) peptides into extracellular plaques is a hallmark of the molecular pathology of Alzheimers disease (AD). Amyloid aggregates have been extensively studied in-vitro, and it is well known that mature amyloid fibrils contain an ordered parallel β structure. The structural evolution from unaggregated peptide to fibrils can be mediated through intermediate structures that deviate significantly from mature fibrils, such as antiparallel β-sheets. However, it is currently unknown if these intermediate structures exist in plaques, which limits the translation of findings from in-vitro structural characterizations of amyloid aggregates to AD. This arises from the inability to extend common structural biology techniques to ex-vivo tissue measurements. Here we report the use of infrared (IR) imaging, wherein we can spatially localize plaques and probe their protein structural distributions with the molecular sensitivity of IR spectroscopy. Analyzing individual plaques in AD tissues, we demonstrate that fibrillar amyloid plaques exhibit antiparallel β-sheet signatures, thus providing a direct connection between in-vitro structures and amyloid aggregates in AD brain. We further validate results with IR imaging of in-vitro aggregates and show that antiparallel β-sheet structure is a distinct structural facet of amyloid fibrils.

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