Formation Of Amyloid Oligomers Research Articles

Disulfide-Driven Charge and Hydrophobicity Rearrangement of Remodeled Membrane Proteins toward Amyloid-Type Aggregation.

As a common pathological hallmark, protein aggregation into amyloids is a highly complicated phenomenon, attracting extensive research interest for elucidating its structural details and formation mechanisms. Membrane deposition and disulfide-driven protein misfolding play critical roles in amyloid-type aggregation, yet the underlying molecular process remains unclear. Here, we employed sum frequency generation (SFG) vibrational spectroscopy to comprehensively investigate the remodeling process of lysozyme, as the model protein, into amyloid-type aggregates at the cell membrane interface. It was discovered that disulfide reduction concurrently induced the transition of membrane-bound lysozyme from predominantly α-helical to antiparallel β-sheet structures, under a mode switch of membrane interaction from electrostatic to hydrophobic, and subsequent oligomeric aggregation. These findings shed light on the systematic understanding of dynamic molecular mechanisms underlying membrane-interactive amyloid oligomer formation.

Reduction of Hippocampal Atrophy and Lateral Ventricle Enlargement Correlates with Clinical Benefit in APOE4 Carriers with Early AD: 1‐Year Results from a Phase 2 Biomarker Study with Oral Anti‐Amyloid Oligomer Inhibitor ALZ‐801

AbstractBackgroundALZ‐801 is an oral inhibitor of amyloid oligomer formation in development as a disease‐modifying Alzheimer’s treatment, including a Phase 3 trial in APOE4/4 homozygotes and a Phase 2 biomarker study in APOE4 carriers. We analyzed correlations between brain volume and cognitive effects of ALZ‐801 after 1 year in the Phase 2 study. We previously reported that at 1‐year timepoint, plasma p‐tau181 (primary outcome) was significantly reduced by 41% from baseline, and hippocampal volume (HV) showed ∼20% less atrophy compared to external matched controls.MethodThe study is conducted at 7 European sites and enrolled 84 APOE4 carriers (MMSE 22‐30) with positive amyloid/tau biomarkers who receive ALZ‐801 265 mg BID for 104 weeks. Primary outcome is plasma p‐tau181 at 104 weeks, and primary imaging outcome is hippocampal volume (HV) atrophy. Volumetric vMRI measures (baseline, 52 and 104 weeks) are performed by Clario, and MMSE conducted. A PK sub‐study was conducted at 65 weeks (n = 23) with bioanalysis at DDS (UK) and non‐compartmental PK analyzed by WinNonlin. Pearson correlations of MMSE changes to BSI changes of HV and lateral ventricle volume (LVV) were conducted, with two‐sided p‐values reported.ResultAt 52 and 65 weeks, changes of MMSE from baseline showed significant correlations to HV (r = 0.32 and 0.34; p<0.01, n = 66) and LVV changes (r = ‐0.42 and ‐0.44; p<0.001, n = 65). Correlations between DMMSE and DLVV were significant for both genders (range r = ‐0.54 to ‐0.42, p<0.02); correlations were observed for DHV in females (range r = 0.39‐0.47; p<0.05) and males (range r = 0.26‐0.23; p<0.18, ns). PK/PD analysis in 23 subjects showed trends (r = 0.21‐0.16, ns) between decreased HV atrophy/LVV expansion and plasma AUC24h of tramiprosate+3‐SPA (active and primary metabolite). ALZ‐801 exhibited favorable safety; main treatment‐related adverse event was mild nausea. No ARIA‐E was observed.ConclusionEarly AD subjects treated with ALZ‐801 for 65 weeks in the Phase 2 biomarker study showed strong correlations between MMSE stability, HV reduction, and decreased ventricular enlargement in overall group, and by genders. The significant correlations between cognitive and brain volume effects support the clinical benefits of ALZ‐801 in APOE4 carriers with early AD. Oral ALZ‐801 showed favorable safety and no brain edema/ARIA‐E.

Effects of ALZ-801, an Oral Amyloid Oligomer Inhibitor, on Biomarkers of Alzheimer’s Disease (AD): 12-Month Results of Phase 2 Biomarker Study in Early AD (S26.007)

<h3>Objective:</h3> To evaluate effects of ALZ-801 on plasma and imaging biomarkers of AD pathology. <h3>Background:</h3> ALZ-801 (valiltramiprosate) is an oral small molecule inhibitor of amyloid oligomer formation. Fully enrolled Phase 2 study in APOE4 carriers is evaluating effects on plasma biomarkers, including hyperphosphorylated tau (p-tau₁₈₁) and beta amyloid (Aβ42 and Aβ40). Plasma p-tau₁₈₁ is elevated in AD and reduced by efficacious doses of lecanemab and aducanumab. APOLLOE4 Phase 3 trial is ongoing in Early AD patients with APOE4/4 genotype. <h3>Design/Methods:</h3> Phase 2 study enrolled subjects with MMSE ≥ 22 and CDR-G 0.5/1, and amyloid positive by CSF or PET scans who received ALZ-801 265 mg BID over 2 years (7 European sites). Biomarker analyses were conducted with Lumipulse (CSF) and Simoa (plasma) assays at Dr. Kaj Blennow’s laboratory. Hippocampal volume (HV) atrophy was assessed by Clario. Changes from baseline were analyzed in mITT population on observed data with paired t-tests and 2-sided p-values. <h3>Results:</h3> mITT population included 84 subjects with 75 completing 52 weeks. Mean age was 69 years, 51% female, MMSE 26.0, 70/30% MCI/Mild AD. Significant plasma p-tau₁₈₁ reduction started at 13 weeks and reached −41% at 52 weeks (p=0.016), with significant reduction in plasma Aβ42 and Aβ40 at 52 weeks (−5%, p=0.002 &amp; p=0.005). Hippocampal atrophy was reduced by 25% compared to matched ADNI controls. Composite cognitive Z-score improved significantly at 13 and 26 weeks and remained above baseline at 52 weeks. Common adverse events were mild nausea and COVID infection, with no drug-related serious events or ARIA-E. <h3>Conclusions:</h3> ALZ-801 produced a significant reduction of plasma p-tau₁₈₁, a marker of amyloid-induced neuronal injury in AD, as well as slowing of hippocampal atrophy at 1 year and cognitive stabilization, suggesting a disease modifying effect in AD patients. These biomarker and clinical effects are being confirmed in the Phase 3 trial. <b>Disclosure:</b> Dr. Hey has received personal compensation for serving as an employee of Alzheon. Dr. Hey has stock in Alzheon. Dr. Hey has stock in Alzheon. Dr. Hey has received intellectual property interests from a discovery or technology relating to health care. Dr. Abushakra has received personal compensation for serving as an employee of Alzheon Inc. Dr. Abushakra has stock in Alzheon Inc. The institution of Dr. Abushakra has received research support from NIA. Dr. Abushakra has received intellectual property interests from a discovery or technology relating to health care. Kaj Blennow has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Biogen, Axon, JOMDD, Roche, Siemens. Kaj Blennow has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Novartis, Julius Clinical. Kaj Blennow has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for MagQu. Dr. Scheltens has nothing to disclose. Dr. Hort has received personal compensation in the range of $0-$499 for serving as a Consultant for Alzheimerchain. Dr. Hort has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Biogen. Dr. Hort has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Schwabe. Dr. Hort has stock in Alzheon. Dr. Sheardova has stock in Alzheon. Niels Prins has nothing to disclose. Mrs. Rutgers has received personal compensation for serving as an employee of Brain Research Center Amsterdam B.V.. Dr. Dautzenberg has nothing to disclose. Dr. Pazdera has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Biogen. Dr. Pazdera has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Teva. Patrick Kesslak has nothing to disclose. Rosalind Mandelbaum has nothing to disclose. Dr. Power has received personal compensation for serving as an employee of Alzheon. Dr. Power has stock in Pfizer. Dr. Power has stock in Alzheon. Dr. Tolar has received personal compensation for serving as an employee of Alzheon. Dr. Tolar has received intellectual property interests from a discovery or technology relating to health care.

A protein aggregation platform that distinguishes oligomers from amyloid fibrils.

Deposition of aggregated proteins is a pathological feature in many neurodegenerative disorders such as Alzheimer's and Parkinson's. In addition to insoluble amyloid fibrils, protein aggregation leads to the formation of soluble oligomers, which are more toxic and pathogenic than fibrils. However, it is challenging to screen for inhibitors targeting oligomers due to the overlapping processes of oligomerization and fibrillization. Here we report a protein aggregation platform that uses intact and split TEM-1 β-lactamase proteins as reporters of protein aggregation. The intact β-lactamase fused with an amyloid protein can report the overall protein aggregation, which leads to loss of lactamase activity. On the other hand, reconstitution of active β-lactamase from the split lactamase construct requires the formation of amyloid oligomers, making the split lactamase system sensitive to oligomerization. Using Aβ, a protein that forms amyloid plaques in Alzheimer's disease, we show that the growth curves of bacterial cells expressing either intact or split lactamase-Aβ fusion proteins can report changes in the Aβ aggregation. The cell lysate lactamase activity assays show that the oligomer fraction accounts for 20% of total activity for the split lactamase-Aβ construct, but only 3% of total activity for the intact lactamase-Aβ construct, confirming the sensitivity of the split lactamase to oligomerization. The combination of the intact and split lactamase constructs allows the distinction of aggregation modulators targeting oligomerization from those targeting overall aggregation. These low-cost bacterial cell-based and biochemical assays are suitable for high-throughput screening of aggregation inhibitors targeting oligomers of various amyloid proteins.

Effects of Oral ALZ‐801, an Amyloid Oligomer Inhibitor, on Plasma Biomarkers in APOE4 Carriers with Early Alzheimer’s Disease: Results of Six‐month Interim Analysis from a Phase 2 Biomarker Study

AbstractBackgroundALZ‐801, an oral, brain‐penetrant small molecule inhibitor of amyloid oligomer formation is being evaluated as a disease‐modifying treatment in APOLLOE4, a Phase 3 trial in APOE4/4 homozygotes with Early Alzheimer’s disease (AD). A concurrent Phase 2 study is in progress to study its effects on core AD biomarkers, beta‐amyloid (Aβ) and hyperphosphorylated tau (p‐tau) in APOE4 carriers with Early AD.MethodThe Phase 2 study in Czech Republic and Netherlands enrolled APOE4/4 and APOE3/4 subjects with MMSE 22‐30 (CDR‐G 0.5 or 1), who receive ALZ‐801 265 mg tablets BID for 104 weeks. Subjects were amyloid‐PET positive or A+/T+ on CSF assays (ratio of Aβ42/40×10 &lt; 0.61, and p‐tau181 &gt; 61 pg/ml). CSF is evaluated at 52 and 104 weeks, and plasma biomarkers at every visit. Memory tests include the Rey Auditory Verbal Learning Test (RAVLT). Primary outcome is CSF p‐tau181 reduction at 104 weeks; interim analysis on plasma was conducted when all subjects completed 26 weeks. Dr. Blennow’s laboratory (Mölndal, Sweden) performed biomarker analyses blinded to clinical data, using Lumipulse (Fujirebio) for CSF and Simoa assays for plasma. Change from baseline in observed biomarker values at each visit were analyzed with paired t‐tests (2‐sided p‐values).ResultOf 84 enrolled subjects (mean 69 years, 51% female, MMSE 25.6), 80 completed 26 weeks. Plasma p‐tau181, the interim primary outcome, showed significant 18% and 29% reductions at 13 and 26 weeks (p = 0.038 and p = 0.028). Reductions of p‐tau181/Aβ42 were also significant 21% and 30% at 13 and 26 weeks (p = 0.018 and p = 0.022). RAVLT memory scores (immediate + delayed) improved significantly at 26 weeks (p = 0.002). Most common adverse events were mild nausea with no drug‐ related serious events and no ARIA‐E at 52 weeks.ConclusionInterim Phase 2 plasma biomarker results show significant and robust reductions in plasma p‐tau181 and p‐tau181/Aβ42 at 26 weeks with memory improvement at 26 weeks. These ALZ‐801 effects on core biomarkers confirm target engagement in AD brain, and support disease modification potential of ALZ‐801. The biomarker effects, oral formulation, and favorable safety without ARIA‐E, make ALZ‐801 an attractive potential treatment for APOE4 carriers with AD.

Kinetic profiling of therapeutic strategies for inhibiting the formation of amyloid oligomers.

Protein self-assembly into amyloid fibrils underlies several neurodegenerative conditions, including Alzheimer's and Parkinson's diseases. It has become apparent that the small oligomers formed during this process constitute neurotoxic molecular species associated with amyloid aggregation. Targeting the formation of oligomers represents, therefore, a possible therapeutic avenue to combat these diseases. However, it remains challenging to establish which microscopic steps should be targeted to suppress most effectively the generation of oligomeric aggregates. Recently, we have developed a kinetic model of oligomer dynamics during amyloid aggregation. Here, we use this approach to derive explicit scaling relationships that reveal how key features of the time evolution of oligomers, including oligomer peak concentration and lifetime, are controlled by the different rate parameters. We discuss the therapeutic implications of our framework by predicting changes in oligomer concentrations when the rates of the individual microscopic events are varied. Our results identify the kinetic parameters that control most effectively the generation of oligomers, thus opening a new path for the systematic rational design of therapeutic strategies against amyloid-related diseases.

Molecular Dynamics Simulations of Protein Aggregation: Protocols for Simulation Setup and Analysis with Markov State Models and Transition Networks.

Protein disorder and aggregation play significant roles in the pathogenesis of numerous neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. The end products of the aggregation process in these diseases are highly structured amyloid fibrils. Though in most cases, small, soluble oligomers formed during amyloid aggregation are the toxic species. A full understanding of the physicochemical forces that drive protein aggregation is thus required if one aims for the rational design of drugs targeting the formation of amyloid oligomers. Among a multitude of biophysical and biochemical techniques that are employed for studying protein aggregation, molecular dynamics (MD) simulations at the atomic level provide the highest temporal and spatial resolution of this process, capturing key steps during the formation of amyloid oligomers. Here we provide a step-by-step guide for setting up, running, and analyzing MD simulations of aggregating peptides using GROMACS. For the analysis, we provide the scripts that were developed in our lab, which allow to determine the oligomer size and inter-peptide contacts that drive the aggregation process. Moreover, we explain and provide the tools to derive Markov state models and transition networks from MD data of peptide aggregation.

Lipid membranes induce structural conversion from amyloid oligomers to fibrils

Formation of amyloid oligomers and fibrils underlies the pathogenesis of a number of neurodegenerative diseases such as Alzheimer’s. One mechanism of action by which Aβ aggregates cause neuronal toxicity is through interactions with cellular membranes. Aβ aggregates have been shown to disrupt membrane integrity via pore formation, membrane thinning, or lipid extraction. At the same time, lipid membranes also affect the rate of Aβ aggregation and remodel pre-formed Aβ fibrils. Here we show that Aβ42 globulomers, a type of well-characterized and stable Aβ oligomers, convert to amyloid fibrils in the presence of DOPC liposomes. Electron paramagnetic resonance studies show that the fibrils converted from Aβ42 globulomers adopt the same structure as fibrils formed directly from monomers. Our results suggest that the interactions between Aβ oligomers and cellular membranes are dynamic. By converting Aβ oligomers to fibrils, the lipid membrane can reduce the membrane-disrupting activities caused by these oligomers. Modulation of Aβ-membrane interactions as a therapeutic strategy should take into account the dynamic nature of these interactions.

Unpacking the aggregation-oligomerization-fibrillization process of naturally-occurring hIAPP amyloid oligomers isolated directly from sera of children with obesity or diabetes mellitus

The formation of amyloid oligomers and fibrils of the human islet amyloid polypeptide (hIAPP) has been linked with β- cell failure and death which causes the onset, progression, and comorbidities of diabetes. We begin to unpack the aggregation-oligomerization-fibrillization process of these oligomers taken from sera of pediatric patients. The naturally occurring or real hIAPP (not synthetic) amyloid oligomers (RIAO) were successfully isolated, we demonstrated the presence of homo (dodecamers, hexamers, and trimers) and hetero-RIAO, as well as several biophysical characterizations which allow us to learn from the real phenomenon taking place. We found that the aggregation/oligomerization process is active in the sera and showed that it happens very fast. The RIAO can form fibers and react with anti-hIAPP and anti-amyloid oligomers antibodies. Our results opens the epistemic horizon and reveal real differences between the four groups (Controls vs obesity, T1DM or T2DM) accelerating the process of understanding and discovering novel and more efficient prevention, diagnostic, transmission and therapeutic pathways.

Monitoring the Formation of Amyloid Oligomers Using Photoluminescence Anisotropy.

The formation of oligomeric soluble aggregates is related to the toxicity of amyloid peptides and proteins. In this manuscript, we report the use of a ruthenium polypyridyl complex ([Ru(bpy)2(dpqp)]2+) to track the formation of amyloid oligomers at different times using photoluminescence anisotropy. This technique is sensitive to the rotational correlation time of the molecule under study, which is consequently related to the size of the molecule. [Ru(bpy)2(dpqp)]2+ presents anisotropy values of zero when free in solution (due to its rapid rotation and long lifetime) but larger values as the size and concentration of amyloid-β (Aβ) oligomers increase. Our assays show that Aβ forms oligomers immediately after the assay is started, reaching a steady state at ∼48 h. SDS-PAGE, DLS, and TEM were used to confirm and characterize the formation of oligomers. Our experiments show that the rate of formation for Aβ oligomers is temperature dependent, with faster rates as the temperature of the assay is increased. The probe was also effective in monitoring the formation of α-synuclein oligomers at different times.

Targeting the Formation of Amyloid Oligomers using Rationally Designed Antibodies

Targeting the Formation of Amyloid Oligomers using Rationally Designed Antibodies

A Free Radical-Generating System Regulates Amyloid Oligomers: Involvement of Cathepsin B.

Amyloid-β (Aβ), a major component of senile plaques, is generated via the proteolysis of amyloid-β protein precursor (AβPP). This cleavage also produces AβPP fragment-derived oligomers which can be highly neurotoxic. AβPP metabolism/processing is affected by many factors, one of which is oxidative stress (OS). Associated with aging, OS is an important risk factor for Alzheimer's disease. In addition, the protein degradation systems, especially those involving cathepsins, are impaired in aging brains. Moreover, cathepsin B (CTSB) is a cysteine protease with potentially specific roles in AβPP proteolysis (β-secretase activity) and Aβ clearance (Aβ degradative activity). The present work examines the effect of OS and the involvement of CTSB in amyloid oligomer formation. The xanthine/xanthine oxidase (X-XOD) free radical generating system induced the partial inhibition of CTSB activity, which was accompanied by an increase in large amyloid oligomers. These were located throughout the cytosol and in endo-lysosomal vesicles. Cells treated with the CTSB inhibitor CA-074Me also showed increased amyloid oligomer levels, whereas those subjected to OS in the presence of the inhibitor showed no such increase. However, CTSB inhibition clearly modulated the AβPP metabolism/processing induced by X-XOD, as revealed by the increase in intracellular AβPP and secreted α-secretase-cleaved soluble AβPP. The present results suggest that CTSB participates in the changes of amyloid oligomer induced by mild OS.

Correction to: Discovery and Identification of an Endogenous Metabolite of Tramiprosate and Its Prodrug ALZ-801 that Inhibits Beta Amyloid Oligomer Formation in the Human Brain.

Page 856: Fig. 6 was an incorrect duplication of Fig. 5. The correct figure is given below.

On the Applicability of Force Fields To Study the Aggregation of Amyloidogenic Peptides Using Molecular Dynamics Simulations.

Molecular dynamics simulations play an essential role in understanding biomolecular processes such as protein aggregation at temporal and spatial resolutions which are not attainable by experimental methods. For a correct modeling of protein aggregation, force fields must accurately represent molecular interactions. Here, we study the effect of five different force fields on the oligomer formation of Alzheimer's Aβ16-22 peptide and two of its mutants: Aβ16-22(F19V,F20V), which does not form fibrils, and Aβ16-22(F19L) which forms fibrils faster than the wild type. We observe that while oligomer formation kinetics depends strongly on the force field, structural properties, such as the most relevant protein-protein contacts, are similar between them. The oligomer formation kinetics obtained with different force fields differ more from each other than the kinetics between aggregating and nonaggregating peptides simulated with a single force field. We discuss the difficulties in comparing atomistic simulations of amyloid oligomer formation with experimental observables.

Esterification of PQQ Enhances Blood-Brain Barrier Permeability and Inhibitory Activity against Amyloidogenic Protein Fibril Formation.

Several neurodegenerative diseases have a common pathophysiology where selective damage to neurons results from the accumulation of amyloid oligomer proteins formed via fibrilization. Considering that the formation of amyloid oligomers leads to cytotoxicity, the development of chemical compounds that are able to effectively cross the blood-brain barrier (BBB) and inhibit this conversion to oligomers and/or fibrils is essential for neurodegenerative disease therapy. We previously reported that pyrroloquinoline quinone (PQQ) prevented aggregation and fibrillation of α-synuclein, amyloid β1-42 (Aβ1-42), and mouse prion protein. To develop a novel drug against neurodegenerative diseases based on PQQ, it is necessary to improve the insufficient BBB permeability of PQQ. Here, we show that an esterified compound of PQQ, PQQ-trimethylester (PQQ-TME), has twice the BBB permeability than PQQ in vitro. Moreover, PQQ-TME exhibited greater inhibitory activity against fibrillation of α-synuclein, Aβ1-42, and prion protein. These results indicated that esterification of PQQ could be a useful approach in developing a novel PQQ-based amyloid inhibitor.

Discovery and Identification of an Endogenous Metabolite of Tramiprosate and Its Prodrug ALZ-801 that Inhibits Beta Amyloid Oligomer Formation in the Human Brain

BackgroundALZ-801 is an oral, small-molecule inhibitor of beta amyloid (Aβ) oligomer formation in clinical development for Alzheimer’s disease (AD). ALZ-801 is a prodrug of tramiprosate with improved pharmacokinetic properties and gastrointestinal tolerability. During clinical studies, we discovered that the primary metabolite of tramiprosate and its prodrug ALZ-801, 3-sulfopropanoic acid (3-SPA), is an endogenous molecule in the human brain and present in the cerebrospinal fluid (CSF) of patients with AD and other neurodegenerative brain diseases.ObjectiveThe objectives of this research were to (1) identify and confirm the presence of 3-SPA in CSF samples from elderly, drug-naïve patients with memory deficits; (2) quantify the levels of 3-SPA in the CSF of patients with AD from tramiprosate phase III North American (NA) trial; (3) evaluate the in vitro anti-Aβ42 oligomer activity of 3-SPA; and (4) characterize the pharmacokinetics and brain-penetration properties of 3-SPA.MethodsLumbar CSF samples from 64 drug-naïve patients with cognitive deficits (Mini-Mental State Examination [MMSE] score range 15–30) and six patients with AD treated with tramiprosate 150 mg twice daily in the phase III trial, at week 78, were analyzed. We used liquid chromatography–tandem mass spectrometry to confirm the structural molecular identity of endogenous 3-SPA with a 3-SPA reference standard and ion-mobility spectrometry–mass spectrometry with molecular dynamics to characterize interactions of 3-SPA with Aβ42 monomers, and the resultant conformational alterations. Rat studies using oral (30 mg/kg) and intravenous (10 mg/kg) doses were conducted to characterize the pharmacokinetic properties and brain penetration of 3-SPA.ResultsWe confirmed the presence of 3-SPA in the CSF of drug-naïve patients with cognitive deficits (mean concentration 11.7 ± 4.3 nM). The mean concentration of 3-SPA in patients with AD treated with tramiprosate was 135 ± 51 nM. In vitro studies revealed a multi-ligand interaction of 3-SPA with monomeric Aβ42 that inhibits the aggregation of Aβ42 into small oligomers. Comparisons of the molecular interactions of tramiprosate and 3-SPA with Aβ42 are also presented. Furthermore, in rat preclinical studies, 3-SPA displayed 100% oral bioavailability and 25% brain penetration, indicating that the metabolite is well absorbed and crosses the blood–brain barrier.ConclusionsWe confirmed the endogenous presence of 3-SPA, the major metabolite of tramiprosate, in the CSF of drug-naïve elderly patients with memory deficits due to AD and a variety of other neurodegenerative disorders. The levels of 3-SPA were up to 12.6-fold greater in patients with AD receiving tramiprosate than in drug-naïve patients. In addition, we showed that 3-SPA has potent anti-Aβ oligomer activity, inhibiting aggregation of Aβ42 into small oligomers with efficacy comparable to that of tramiprosate. 3-SPA displays excellent oral availability and brain penetration in rats, suggesting that the higher CSF concentrations of 3-SPA in the human brain after oral administration of ALZ-801 or tramiprosate (and subsequent conversion to 3-SPA) result from the penetration of the metabolite into the central nervous system. These data suggest that 3-SPA is an endogenous agent with potential activity stabilizing the conformational flexibility of Aβ monomers that, in turn, inhibit Aβ misfolding and formation of soluble toxic Aβ oligomers in humans, thereby preventing the initial pathogenic step in the progression of AD. Clinical improvements observed in patients with AD carrying the ε4 allele of the apolipoprotein E gene in tramiprosate phase III studies may in part be explained by the therapeutic effects of excess levels of the metabolite in the brains of these patients. The potential protective role of 3-SPA in AD pathogenesis, as well as its therapeutic role in AD and other neurodegenerative disorders, warrants further investigation.

Novel insight into streptozotocin-induced diabetic rats from the protein misfolding perspective

Protein folding is a process of self-assembly defined by the sequence of the amino acids of the protein involved. Additionally, proteins tend to unfold, misfold and aggregate due to both intrinsic and extrinsic causes. Human islet amyloid polypeptide (hIAPP) aggregation is an early step in diabetes mellitus. However, the aggregation of rat IAPP (rIAPP) remains an open question. Adult female Sprague-Dawley rats weighing 150–250 g were divided into two groups. The experimental group (streptozotocin [STZ]) (n = 21) received an intraperitoneal injection of a single dose of 40 mg/kg STZ. We used the mouse anti-IAPP antibody and the anti-amyloid oligomer antibody to study the temporal course of rIAPP oligomerization during STZ-induced diabetes using a wide array of methods, strategies and ideas derived from biochemistry, cell biology, and proteomic medicine. Here, we demonstrated the tendency of rIAPP to aggregate and trigger cooperative processes of self-association or hetero-assembly that lead to the formation of amyloid oligomers (trimers and hexamers). Our results are the first to demonstrate the role of rIAPP amyloid oligomers in the development of STZ-induced diabetes in rats. The IAPP amyloid oligomers are biomarkers of the onset and progression of diabetes and could play a role as therapeutic targets.

Pseudo-peptide amyloid-β blocking inhibitors: molecular dynamics and single molecule force spectroscopy study

By combining MD simulations and AFS experimental technique, we demonstrated a powerful approach for rational design and single molecule testing of novel inhibitor molecules which can block amyloid-amyloid binding – the first step of toxic amyloid oligomer formation. We designed and tested novel pseudo-peptide amyloid-β (Aβ) inhibitors that bind to the Aβ peptide and effectively prevent amyloid-amyloid binding. First, molecular dynamics (MD) simulations have provided information on the structures and binding characteristics of the designed pseudo-peptides targeting amyloid fragment Aβ (13−23). The binding affinities between the inhibitor and Aβ as well as the inhibitor to itself have been estimated using Umbrella Sampling calculations. Atomic Force Spectroscopy (AFS) was used to experimentally test several proposed inhibitors in their ability to block amyloid-amyloid binding – the first step of toxic amyloid oligomer formation. The experimental AFS data are in a good agreement with theoretical MD calculations and demonstrate that three proposed pseudo-peptides bind to amyloid fragment with different affinities and all effectively prevent Aβ-Aβ binding in similar way. We propose that the designed pseudo-peptides can be used as potential drug candidates to prevent Aβ toxicity in Alzheimer's disease.

Nucleation of Amyloid Oligomers by RepA-WH1-Prionoid-Functionalized Gold Nanorods.

Understanding protein amyloidogenesis is an important topic in protein science, fueled by the role of amyloid aggregates, especially oligomers, in the etiology of a number of devastating human degenerative diseases. However, the mechanisms that determine the formation of amyloid oligomers remain elusive due to the high complexity of the amyloidogenesis process. For instance, gold nanoparticles promote or inhibit amyloid fibrillation. We have functionalized gold nanorods with a metal-chelating group to selectively immobilize soluble RepA-WH1, a model synthetic bacterial prionoid, using a hexa-histidine tag (H6). H6-RepA-WH1 undergoes stable amyloid oligomerization in the presence of catalytic concentrations of anisotropic nanoparticles. Then, in a physically separated event, such oligomers promote the growth of amyloid fibers of untagged RepA-WH1. SERS spectral changes of H6-RepA-WH1 on spherical citrate-AuNP substrates provide evidence for structural modifications in the protein, which are compatible with a gradual increase in β-sheet structure, as expected in amyloid oligomerization.

Nucleation of Amyloid Oligomers by RepA‐WH1‐Prionoid‐Functionalized Gold Nanorods

AbstractUnderstanding protein amyloidogenesis is an important topic in protein science, fueled by the role of amyloid aggregates, especially oligomers, in the etiology of a number of devastating human degenerative diseases. However, the mechanisms that determine the formation of amyloid oligomers remain elusive due to the high complexity of the amyloidogenesis process. For instance, gold nanoparticles promote or inhibit amyloid fibrillation. We have functionalized gold nanorods with a metal‐chelating group to selectively immobilize soluble RepA‐WH1, a model synthetic bacterial prionoid, using a hexa‐histidine tag (H6). H6‐RepA‐WH1 undergoes stable amyloid oligomerization in the presence of catalytic concentrations of anisotropic nanoparticles. Then, in a physically separated event, such oligomers promote the growth of amyloid fibers of untagged RepA‐WH1. SERS spectral changes of H6‐RepA‐WH1 on spherical citrate‐AuNP substrates provide evidence for structural modifications in the protein, which are compatible with a gradual increase in β‐sheet structure, as expected in amyloid oligomerization.