The amyloid-β1-42-oligomer interacting peptide D-AIP possesses favorable biostability, pharmacokinetics, and brain region distribution.

We have previously developed a unique 8-amino acid Aβ42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer's disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aβ42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid "seeding" in vitro (i.e., freshly monomerized versus preaggregated Aβ42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aβ42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer's disease

Biophysical characterization as a tool to predict amyloidogenic and toxic properties of amyloid-beta 42 peptides

Amyloid-beta 42 (A beta 42) peptides are central to the amyloid pathology in Alzheimer's disease (AD). As biological mimetics, properties of synthetic A beta peptides usually vary between vendors and batches, thus impacting the reproducibility of experimental studies. Here, we tested recombinantly expressed A beta 42 (Aspl to A1a42) against synthetic A beta 42 from different suppliers using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), circular dichroism (CD) spectroscopy, thioflavin T aggregation, surface plasmon resonance, and MTT cell viability assays. Overall, our recombinant A beta 42 provided a reproducible mimetic of desired properties. Across experimental approaches, the combined detection of A beta 42 dimers and random coil to beta-sheet transition only correlated with aggregation-prone and cytotoxic peptides. Conclusively, combining MALDI-MS with CD appears to provide a rapid, reliable means to predict the 'bioactivity' of A beta 42

The amyloid-beta degradation intermediate A beta 34 is pericyte-associated and reduced in brain capillaries of patients with Alzheimer's disease

An impairment of amyloid beta-peptide (A beta) clearance is suggested to play a key role in the pathogenesis of sporadic Alzheimer's disease (AD). Amyloid degradation is mediated by various mechanisms including fragmentation by enzymes like neprilysin, matrix metalloproteinases (MMPs) and a recently identified amyloidolytic activity of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 cleavage of A beta 40 and A beta 42 results in the formation of a common A beta 34 intermediate which was found elevated in cerebrospinal fluid levels of patients at the earliest disease stages. To further investigate the role of A beta 34 as a marker for amyloid clearance in AD, we performed a systematic and comprehensive analysis of A beta 34 immunoreactivity in hippocampal and cortical post-mortem brain tissue from AD patients and non-demented elderly individuals. In early Braak stages, A beta 34 was predominantly detectable in a subset of brain capillaries associated with pericytes, while in later disease stages, in clinically diagnosed AD, this pericyte-associated A beta 34 immunoreactivity was largely lost. A beta 34 was also detected in isolated human cortical microvessels associated with brain pericytes and its levels correlated with A beta 40, but not with A beta 42 levels. Moreover, a significantly decreased A beta 34/A beta 40 ratio was observed in microvessels from AD patients in comparison to non-demented controls suggesting a reduced proteolytic degradation of A beta 40 to A beta 34 in AD. In line with the hypothesis that pericytes at the neurovascular unit are major producers of A beta 34, biochemical studies in cultured human primary pericytes revealed a time and dose dependent increase of A beta 34 levels upon treatment with recombinant A beta 40 peptides while A beta 34 production was impaired when A beta 40 uptake was reduced or BACE1 activity was inhibited. Collectively, our findings indicate that A beta 34 is generated by a novel BACE1-mediated A beta clearance pathway in pericytes of brain capillaries. As amyloid clearance is significantly reduced in AD, impairment of this pathway might be a major driver of the pathogenesis in sporadic AD

The downfall of TBA-354 – a possible explanation for its neurotoxicity <i>via</i> mass spectrometric imaging

<p>1. TBA-354 was a promising antitubercular compound with activity against both replicating and static <i>Mycobacterium tuberculosis</i> (<i>M.tb</i>), making it the focal point of many clinical trials conducted by the TB Alliance. However, findings from these trials have shown that TBA-354 results in mild signs of reversible neurotoxicity; this left the TB Alliance with no other choice but to stop the research.</p> <p>2. In this study, mass spectrometric methods were used to evaluate the pharmacokinetics and spatial distribution of TBA-354 in the brain using a validated liquid chromatography tandem-mass spectrometry (LCMS/MS) and mass spectrometric imaging (MSI), respectively. Healthy female Sprague-Dawley rats received intraperitoneal (i.p.) doses of TBA-354 (20 mg/kg bw).</p> <p>3. The concentrationtime profiles showed a gradual absorption and tissue penetration of TBA-354 reaching the <i>C</i>_max at 6 h post dose, followed by a rapid elimination. MSI analysis showed a time-dependent drug distribution, with highest drug concentration mainly in the neocortical regions of the brain.</p> <p>4. The distribution of TBA-354 provides a possible explanation for the motor dysfunction observed in clinical trials. These results prove the importance of MSI as a potential tool in preclinical evaluations of suspected neurotoxic compounds.</p

Aβ34 is a BACE1-derived degradation intermediate associated with amyloid clearance and Alzheimer’s disease progression

The beta-site APP cleaving enzyme 1 (BACE1) is known primarily for its initial cleavage of the amyloid precursor protein (APP), which ultimately leads to the generation of Aβ peptides. Here, we provide evidence that altered BACE1 levels and activity impact the degradation of Aβ40 and Aβ42 into a common Aβ34 intermediate. Using human cerebrospinal fluid (CSF) samples from the Amsterdam Dementia Cohort, we show that Aβ34 is elevated in individuals with mild cognitive impairment who later progressed to dementia. Furthermore, Aβ34 levels correlate with the overall Aβ clearance rates in amyloid positive individuals. Using CSF samples from the PREVENT-AD cohort (cognitively normal individuals at risk for Alzheimer’s disease), we further demonstrate that the Aβ34/Aβ42 ratio, representing Aβ degradation and cortical deposition, associates with pre-clinical markers of neurodegeneration. We propose that Aβ34 represents a marker of amyloid clearance and may be helpful for the characterization of Aβ turnover in clinical samples

Aβ34 is a BACE1-derived degradation intermediate associated with amyloid clearance and Alzheimer's disease progression

The beta-site APP cleaving enzyme 1 (BACE1) is known primarily for its initial cleavage of the amyloid precursor protein (APP), which ultimately leads to the generation of Aβ peptides. Here, we provide evidence that altered BACE1 levels and activity impact the degradation of Aβ40 and Aβ42 into a common Aβ34 intermediate. Using human cerebrospinal fluid (CSF) samples from the Amsterdam Dementia Cohort, we show that Aβ34 is elevated in individuals with mild cognitive impairment who later progressed to dementia. Furthermore, Aβ34 levels correlate with the overall Aβ clearance rates in amyloid positive individuals. Using CSF samples from the PREVENT-AD cohort (cognitively normal individuals at risk for Alzheimer's disease), we further demonstrate that the Aβ34/Aβ42 ratio, representing Aβ degradation and cortical deposition, associates with pre-clinical markers of neurodegeneration. We propose that Aβ34 represents a marker of amyloid clearance and may be helpful for the characterization of Aβ turnover in clinical samples

Sulfated Hyperbranched and Linear Polyglycerols Modulate HMGB1 and Morphological Plasticity in Neural Cells

The objective of this study was to establish if polyglycerols with sulfate or sialic acid functional groups interact with high mobility group box 1 (HMGB1), and if so, which polyglycerol could prevent loss of morphological plasticity in excitatory neurons in the hippocampus. Considering that HMGB1 binds to heparan sulfate and that heparan sulfate has structural similarities with dendritic polyglycerol sulfates (dPGS), we performed the experiments to show if polyglycerols can mimic heparin functions by addressing the following questions: (1) do dendritic and linear polyglycerols interact with the alarmin molecule HMGB1? (2) Does dPGS interaction with HMGB1 influence the redox status of HMGB1? (3) Can dPGS prevent the loss of dendritic spines in organotypic cultures challenged with lipopolysaccharide (LPS)? LPS plays a critical role in infections with Gram-negative bacteria and is commonly used to test candidate therapeutic agents for inflammation and endotoxemia. Pathologically high LPS concentrations and other stressful stimuli cause HMGB1 release and post-translational modifications. We hypothesized that (i) electrostatic interactions of hyperbranched and linear polysulfated polyglycerols with HMGB1 will likely involve sites similar to those of heparan sulfate. (ii) dPGS can normalize HMGB1 compartmentalization in microglia exposed to LPS and prevent dendritic spine loss in the excitatory hippocampal neurons. We performed immunocytochemistry and biochemical analyses combined with confocal microscopy to determine cellular and extracellular locations of HMGB1 and morphological plasticity. Our results suggest that dPGS interacts with HMGB1 similarly to heparan sulfate. Hyperbranched dPGS and linear sulfated polymers prevent dendritic spine loss in hippocampal excitatory neurons. MS/MS analyses reveal that dPGS-HMGB1 interactions result in fully oxidized HMGB1 at critical cysteine residues (Cys23, Cys45, and Cys106). Triply oxidized HMGB1 leads to the loss of its pro-inflammatory action and could participate in dPGS-mediated spine loss prevention. LPG-Sia exposure to HMGB1 results in the oxidation of Cys23 and Cys106 but does not normalize spine density