Synthesis and Evaluation of a Novel PET Radioligand for Imaging Glutaminyl Cyclase Activity as a Biomarker for Detecting Alzheimer's Disease.

Several new lines of research have demonstrated that a significant number of amyloid-ß peptides found in Alzheimer's disease (AD) are truncated and undergo post-translational modification by glutaminyl cyclase (QC) at the N-terminal. Notably, QC's products of Abeta-pE3 and Abeta-pE11 have been active targets for investigational drug development. This work describes the design, synthesis, characterization, and in vivo validation of a novel PET radioligand, [18F]PB0822, for targeted imaging of QC. We report herein a simplified and robust chemistry for the synthesis of the standard compound, [19F]PB0822, and the corresponding [18F]PB0822 radioligand. The PET probe was developed with 99.9% radiochemical purity, a molar activity of 965 Ci.mmol-1, and an IC50 of 56.3 nM, comparable to those of the parent PQ912 inhibitor (62.5 nM). Noninvasive PET imaging showed that the probe is distributed in the brain 5 min after intravenous injection. Further, in vivo PET imaging with [18F]PB0822 revealed that AD 5XFAD mice harbor significantly higher QC activity than WT counterparts. The data also suggested that QC activity is found across different brain regions of the tested animals.

TARGETING SOLUBLE AMYLOID-BETA OLIGOMERS WITH A NOVEL NANOBODY.

The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-ß oligomers (SAßOs) accumulate early, prior to amyloid plaque formation. SAßOs induce memory impairment and disrupt cognitive function independent of amyloid-ß plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAßO (E3) nanobody generated from an alpaca immunized with SAßO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAßOs and amyloid-ß plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAßO and amyloid-ß plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAßOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAßO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.

Ergothioneine, a dietary antioxidant improves amyloid beta clearance in the neuroretina of a mouse model of Alzheimer's disease.

Introduction: Ergothioneine (Ergo) is a naturally occurring dietary antioxidant. Ergo uptake is dependent on the transporter, organic cation transporter novel-type 1 (OCTN1) distribution. OCTN1 is highly expressed in blood cells (myeloid lineage cells), brain and ocular tissues that are likely predisposed to oxidative stress. Ergo may protect the brain and eye against oxidative damage and inflammation, however, the underlying mechanism remains unclear. Amyloid beta (Aß) clearance is a complex process mediated by various systems and cell types including vascular transport across the blood-brain barrier, glymphatic drainage, and engulfment and degradation by resident microglia and infiltrating innate immune cells. Impaired Aß clearance is a major cause for Alzheimer's disease (AD). Here we investigated neuroretinas to explore the neuroprotective effect of Ergo in a transgenic AD mouse model. Methods: Age-matched groups of Ergo-treated 5XFAD, non-treated 5XFAD, and C57BL/6J wildtype (WT controls) were used to assess Ergo transporter OCTN1 expression and Aß load along with microglia/macrophage (IBA1) and astrocyte (GFAP) markers in wholemount neuroretinas (n = 26) and eye cross-sections (n = 18). Immunoreactivity was quantified by fluorescence or by semi-quantitative assessments. Results and discussion: OCTN1 immunoreactivity was significantly low in the eye cross-sections of Ergo-treated and non-treated 5XFAD vs. WT controls. Strong Aß labeling, detected in the superficial layers in the wholemounts of Ergo-treated 5XFAD vs. non-treated 5XFAD reflects the existence of an effective Aß clearance system. This was supported by imaging of cross-sections where Aß immunoreactivity was significantly low in the neuroretina of Ergo-treated 5XFAD vs. non-treated 5XFAD. Moreover, semi-quantitative analysis in wholemounts identified a significantly reduced number of large Aß deposits or plaques, and a significantly increased number of IBA1(+)ve blood-derived phagocytic macrophages in Ergo-treated 5XFAD vs. non-treated 5XFAD. In sum, enhanced Aß clearance in Ergo-treated 5XFAD suggests that Ergo uptake may promote Aß clearance possibly by blood-derived phagocytic macrophages and via perivascular drainage.

Longitudinal Consumption of Ergothioneine Reduces Oxidative Stress and Amyloid Plaques and Restores Glucose Metabolism in the 5XFAD Mouse Model of Alzheimer's Disease.

Background: Ergothioneine (ERGO) is a unique antioxidant and a rare amino acid available in fungi and various bacteria but not in higher plants or animals. Substantial research data indicate that ERGO is a physiological antioxidant cytoprotectant. Different from other antioxidants that need to breach the blood-brain barrier to enter the brain parenchyma, a specialized transporter called OCTN1 has been identified for transporting ERGO to the brain. Purpose: To assess whether consumption of ERGO can prevent the progress of Alzheimer's disease (AD) on young (4-month-old) 5XFAD mice. Methods and materials: Three cohorts of mice were tested in this study, including ERGO-treated 5XFAD, non-treated 5XFAD, and WT mice. After the therapy, the animals went through various behavioral experiments to assess cognition. Then, mice were scanned with PET imaging to evaluate the biomarkers associated with AD using [11C]PIB, [11C]ERGO, and [18F]FDG radioligands. At the end of imaging, the animals went through cardiac perfusion, and the brains were isolated for immunohistology. Results: Young (4-month-old) 5XFAD mice did not show a cognitive deficit, and thus, we observed modest improvement in the treated counterparts. In contrast, the response to therapy was clearly detected at the molecular level. Treating 5XFAD mice with ERGO resulted in reduced amyloid plaques, oxidative stress, and rescued glucose metabolism. Conclusions: Consumption of high amounts of ERGO benefits the brain. ERGO has the potential to prevent AD. This work also demonstrates the power of imaging technology to assess response during therapy.

Improved synthesis of an ergothioneine PET radioligand for imaging oxidative stress in Alzheimer's disease.

L-ergothioneine (ERGO) is a potent antioxidant with cytoprotective effects. To study ERGO biodistribution and detect oxidative stress in vivo, we report an efficient and reproducible preparation of [11 C]-labeled ERGO PET radioligand based on protecting the histidine carboxylic group with a methyl ester. Overall, this new protection approach using methyl ester improved the chemical yield of a 4-step reaction from 14% to 24% compared to the previous report using t-butyl ester. The [11 C]CH3 methylation of the precursor provided the desired product with 55 ± 10% radiochemical purity and a molar activity of 450 ± 200 TBq·mmol-1 . The [11 C]ERGO radioligand was able to detect threshold levels of oxidative stress in a preclinical animal model of Alzheimer's disease.

A novel antioxidant ergothioneine PET radioligand for in vivo imaging applications.

Ergothioneine (ERGO) is a rare amino acid mostly found in fungi, including mushrooms, with recognized antioxidant activity to protect tissues from damage by reactive oxygen species (ROS) components. Prior to this publication, the biodistribution of ERGO has been performed solely in vitro using extracted tissues. The aim of this study was to develop a feasible chemistry for the synthesis of an ERGO PET radioligand, [11C]ERGO, to facilitate in vivo study. The radioligand probe was synthesized with identical structure to ERGO by employing an orthogonal protection/deprotection approach. [11C]methylation of the precursor was performed via [11C]CH3OTf to provide [11C]ERGO radioligand. The [11C]ERGO was isolated by RP-HPLC with a molar activity of 690 TBq/mmol. To demonstrate the biodistribution of the radioligand, we administered approximately 37 MBq/0.1 mL in 5XFAD mice, a mouse model of Alzheimer's disease via the tail vein. The distribution of ERGO in the brain was monitored using 90-min dynamic PET scans. The delivery and specific retention of [11C]ERGO in an LPS-mediated neuroinflammation mouse model was also demonstrated. For the pharmacokinetic study, the concentration of the compound in the serum started to decrease 10 min after injection while starting to distribute in other peripheral tissues. In particular, a significant amount of the compound was found in the eyes and small intestine. The radioligand was also distributed in several regions of the brain of 5XFAD mice, and the signal remained strong 30 min post-injection. This is the first time the biodistribution of this antioxidant and rare amino acid has been demonstrated in a preclinical mouse model in a highly sensitive and non-invasive manner.

Design, Synthesis, and Validation of a Novel [11C]Promethazine PET Probe for Imaging Abeta Using Autoradiography.

Promethazine, an antihistamine drug used in the clinical treatment of nausea, has been demonstrated the ability to bind Abeta in a transgenic mouse model of Alzheimer's disease. However, so far, all of the studies were performed in vitro using extracted tissues. In this work, we report the design and synthesis of a novel [11C]promethazine PET radioligand for future in vivo studies. The [11C]promethazine was isolated by RP-HPLC with radiochemical purity >95% and molar activity of 48 TBq/mmol. The specificity of the probe was demonstrated using human hippocampal tissues via autoradiography.

Inhalable Thioflavin S for the Detection of Amyloid Beta Deposits in the Retina.

We present an integrated delivery technology herein employing the aerosolized method to repurpose thioflavin S for imaging amyloid beta (Abeta) deposits in the retina as a surrogate of Abeta in the brain for early detection of Alzheimer's disease. The data showed that wild type (WT) mice also have Abeta deposits in the retinae, albeit much less than 5XFAD mice. Further, only in 5XFAD mice, significant Abeta deposits were found associated with retinal ganglion cells (RGCs) in whole-mount and cross-section data. Furthermore, the fluorescent signal depicted from thioflavin S corroborates with Abeta immunohistochemistry staining information. Overall, this probe delivery via inhalation method is also applicable to other Abeta-binding molecules, such as Congo red, curcumin, and thioflavin T. The advantage of imaging retinal amyloid deposits compared to the brain counterparts is that the eye is easily accessible by in vivo imaging and it reduces the effort to design a probe that must cross the formidable blood-brain barrier.

The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells.

BACKGROUND: Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K+ channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells. METHODS: We transduced C2C12 myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student's t test or two-way ANOVAs and SAS software as indicated. RESULTS: Expression of human ERG1A in C2C12 myotubes increased basal intracellular calcium concentration 51.7% (p < 0.0001; n = 177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p < 0.08; n = 24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p < 0.05; n = 6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p < 0.05; n = 6) in calpastatin which could contribute to enhanced calpain activity. CONCLUSIONS: Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.