Adam P. Gunn

PBT2 rapidly improves cognition in Alzheimer's Disease: additional phase II analyses.

PBT2 is a copper/zinc ionophore that rapidly restores cognition in mouse models of Alzheimers disease (AD). A recent Phase IIa double-blind, randomized, placebo-controlled trial found that the 250 mg dose of PBT2 was well-tolerated, significantly lowered cerebrospinal fluid (CSF) levels of amyloid-beta42, and significantly improved executive function on a Neuro-psychological Test Battery (NTB) within 12 weeks of treatment in patients with AD. In the post-hoc analysis reported here, the cognitive, blood marker, and CSF neurochemistry outcomes from the trial were subjected to further analysis. Ranking the responses to treatment after 12 weeks with placebo, PBT2 50 mg, and PBT2 250 mg revealed that the proportions of patients showing improvement on NTB Composite or Executive Factor z-scores were significantly greater in the PBT2 250 mg group than in the placebo group. Receiver-operator characteristic analyses revealed that the probability of an improver at any level coming from the PBT2 250 mg group was significantly greater, compared to placebo, for Composite z-scores (Area Under the Curve [AUC] =0.76, p=0.0007), Executive Factor z-scores (AUC =0.93, p=1.3 x 10(-9)), and near-significant for the ADAS-cog (AUC =0.72, p=0.056). There were no correlations between changes in CSF amyloid-beta or tau species and cognitive changes. These findings further encourage larger-scale testing of PBT2 for AD.

Sequestration of Copper from β-Amyloid Promotes Selective Lysis by Cyclen-Hybrid Cleavage Agents

Decelerated degradation of β-amyloid (Aβ) and its interaction with synaptic copper may be pathogenic in Alzheimer disease. Recently, Co(III)-cyclen tagged to an aromatic recognition motif was shown to degrade Aβ in vitro. Here, we report that apocyclen attached to selective Aβ recognition motifs (KLVFF or curcumin) can capture copper bound to Aβ and use the Cu(II) in place of Co(III) to become proteolytically active. The resultant complexes interfere with Aβ aggregation, degrade Aβ into fragments, preventing H2O2 formation and toxicity in neuronal cell culture. Because Aβ binds Cu in amyloid plaques, apocyclen-tagged targeting molecules may be a promising approach to the selective degradation of Aβ in Alzheimer disease. The principle of copper capture could generalize to other amyloidoses where copper is implicated.

The Caenorhabditis elegans Aβ1–42 Model of Alzheimer Disease Predominantly Expresses Aβ3–42

Transgenic expression of human amyloid β (Aβ) peptide in body wall muscle cells of Caenorhabditis elegans has been used to better understand aspects of Alzheimer disease (AD). In human aging and AD, Aβ undergoes post-translational changes including covalent modifications, truncations, and oligomerization. Amino truncated Aβ is increasingly recognized as potentially contributing to AD pathogenesis. Here we describe surface-enhanced laser desorption ionization-time of flight mass spectrometry mass spectrometry of Aβ peptide in established transgenic C. elegans lines. Surprisingly, the Aβ being expressed is not full-length 1–42 (amino acids) as expected but rather a 3–42 truncation product. In vitro analysis demonstrates that Aβ3–42 self-aggregates like Aβ1–42, but more rapidly, and forms fibrillar structures. Similarly, Aβ3–42 is also the more potent initiator of Aβ1–40 aggregation. Seeded aggregation via Aβ3–42 is further enhanced via co-incubation with the transition metal Cu(II). Although unexpected, the C. elegans model of Aβ expression can now be co-opted to study the proteotoxic effects and processing of Aβ3–42.

Pyroglutamate-Aβ: role in the natural history of Alzheimer's disease.

The accumulation of amyloid-beta (Aβ) peptides is believed to be a central contributor to the neurodegeneration typically seen in Alzheimers disease (AD) brain. Aβ extracted from AD brains invariably possesses extensive truncations, yielding peptides of differing N- and C-terminal composition. Whilst Aβ is often abundant in the brains of cognitively normal elderly people, the brains of AD patients are highly enriched for N-terminally truncated Aβ bearing the pyroglutamate modification. Pyroglutamate-Aβ (pE-Aβ) has a higher propensity for oligomerisation and aggregation than full-length Aβ, potentially seeding the accumulation of neurotoxic Aβ oligomers and amyloid deposits. In addition, pE-Aβ has increased resistance to clearance by peptidases, causing these peptides to persist in biological fluids and tissues. The extensive deposition of pE-Aβ in human AD brain is under-represented in many transgenic mouse models of AD, reflecting major differences in the production and processing of Aβ peptides in these models compared to the human disease state.

Amyloid-β Peptide Aβ3pE-42 Induces Lipid Peroxidation, Membrane Permeabilization, and Calcium Influx in Neurons

Pyroglutamate-modified amyloid-β (pE-Aβ) is a highly neurotoxic amyloid-β (Aβ) isoform and is enriched in the brains of individuals with Alzheimer disease compared with healthy aged controls. Pyroglutamate formation increases the rate of Aβ oligomerization and alters the interactions of Aβ with Cu2+ and lipids; however, a link between these properties and the toxicity of pE-Aβ peptides has not been established. We report here that Aβ3pE-42 has an enhanced capacity to cause lipid peroxidation in primary cortical mouse neurons compared with the full-length isoform (Aβ(1–42)). In contrast, Aβ(1–42) caused a significant elevation in cytosolic reactive oxygen species, whereas Aβ3pE-42 did not. We also report that Aβ3pE-42 preferentially associates with neuronal membranes and triggers Ca2+ influx that can be partially blocked by the N-methyl-d-aspartate receptor antagonist MK-801. Aβ3pE-42 further caused a loss of plasma membrane integrity and remained bound to neurons at significantly higher levels than Aβ(1–42) over extended incubations. Pyroglutamate formation was additionally found to increase the relative efficiency of Aβ-dityrosine oligomer formation mediated by copper-redox cycling.

Rapid Generation of Dityrosine Cross-linked Aβ Oligomers via Cu-Redox Cycling

There is a great interest in the role of free radicals and oxidative stress in Alzheimers disease and for the role of transition metals in the generation of oligomers of Aβ peptides. In the literature, there are a multitude of varying methods that can be used to create soluble oligomers of Aβ, however, the processes that create these oligomers are often stochastic by nature and thus reproducibility is an issue. Here we report a simple and reproducible method for the production of radically derived dityrosine cross-linked oligomers of Aβ, through reaction with copper and ascorbic acid.

Anti-Aβ antibody target engagement: a response to Siemers et al.

engagement with brain Aβ, consistent with published clinical data”. To reiterate, we reported data showing that bapineuzumab was capable of binding soluble Aβ with a low nanomolar affinity and demonstrated that the antibody could detect Aβ species in buffer and in brain homogenate and plasma from transgenic animal models of AD. Furthermore, in agreement with the phase 3 clinical data [23], we demonstrated target engagement for bapineuzumab, where its target is brain-derived amyloid-β peptides associated with Alzheimer’s pathology (see, Fig. 3a [28]). Our data also showed that neither solanezumab nor crenezumab effectively engaged this pool of Aβ, again consistent with the publicly available clinical data [9, 10]. We have read with interest the commentary by siemers et al. [28] regarding our paper describing the ability of the three anti-Aβ antibodies, bapineuzumab, crenezumab and solanezumab to engage Aβ in both a synthetic and a biological setting. We appreciate the opportunity to clarify any misunderstandings and here provide a brief response to their concerns. siemers et al. [28] begin their commentary by stating that our findings led to the conclusion “that all three antibodies failed to engage the intended molecular targets”. This statement is wrong; as clearly stated in the abstract of our paper, “Bapineuzumab demonstrated target

The influence of the ethane-1,2-diamine ligand on the activity of a monofunctional platinum complex

The continued use of platinum-based chemotherapeutic drugs in the clinic mandates the need for further investigation of the biological activity of structural analogues of the clinically approved complexes. Of interest are monofunctional platinum(II) complexes, which bear only one labile ligand, for which it is believed that each complex binds to DNA only once. Pyriplatin ([PtCl(NH3)2(py)]+) and enpyriplatin ([PtCl(en)(py)]+) are both monofunctional platinum(II) complexes that bear a pyridine ligand and a labile chlorido ligand, differing in their cis‑ammine and ethane-1,2-diamine (en) ligands respectively. Despite their similar structure, the complexes exhibit dramatically different cytotoxicities. In this study, we synthesized and characterized both complexes in terms of their cytotoxicity, lipophilicity, DNA binding and cellular accumulation. There was no significant difference between the lipophilicities of the complexes and both complexes exhibited monofunctional type binding, but it was the temporal accumulation profiles of the two complexes which differed greatly. The complexes were further analyzed with size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS) to determine the platination state of the proteins. Consistent with the accumulation studies, pyriplatin bound to proteins in far greater amounts than enpyriplatin, and this study also revealed some different protein targets between the bifunctional cisplatin and monofunctional pyriplatin. This study highlights the need for more sophisticated techniques, such as SEC-ICP-MS, to determine not only how much of a platinum complex accumulates in cells, but also the speciation and metabolites of platinum anticancer drugs.

THE INFLUENCE OF AMYLOID-B PRECURSOR PROTEIN PROTEOLYTIC PROCESSING ON NEURONAL IRON HOMEOSTASIS

Background: Reduced plasma 25-hydroxyvitamin D (25-OH-D) was proved to be associated with poorer cognitive function and a higher risk of developingAlzheimer’s disease (AD), but the specific mechanisms were unknown. Increased vascular injury due to activation of renin-angiotensin-system is one of the hypotheses about the relationship between vitamin D and AD.Methods:We recruited patients with early AD from two teaching hospitals in Taiwan. All patients underwent clinically functional assessment and a neuropsychological test battery. Plasma 25-OH-D level was checked by radioimmunoassay. Brain magnetic resonance imaging (MRI) was used to determine the volume of white matter hyperintensities (WMH), a surrogate of cerebral vascular injury. We analyzed the relationship between cognitive function, plasma level of 25-OHD, and WMH volume in early AD patients. Results: In total, 146 early AD patients (68 males/78 females; mean age 79.1 6 7.0 years; mean education 10.2 6 4.3 years) were recruited. Their mean Mini-Mental State Examination (MMSE) was 21.0 6 3.8. The clinical dementia rating (CDR) score was 0.5 in 24 patients and 1.0 in 123 patients. The percentage of APOE ε4 carrier was 35.4 %. The correlation analysis between WMH volume and 25OH-D level showed significant negative correlation (R 1⁄4 0.086, adjusted p 1⁄4 0.003, adjusting age, sex, hypertension, diabetes mellitus, hyperlipidemia, and coronary heart disease). The correlation analysis between MMSE and 25-OH-D level showed significant positive correlation (R 1⁄4 0.067, adjusted p 1⁄4 0.002, adjusting age, sex, and education years). Multivariate regression analysis, performed by using MMSE as the dependent variable and adjusting age, sex, and education years, showed 25-OH-D level was an independent predictor for MMSE score (b 1⁄4 0.25, p 1⁄4 0.003), but WMH volume was not (b1⁄4 -0.02, p1⁄4 0.82). Conclusions:Reduced plasma 25-OH-D was associated with low MMSE scores in early AD patients. The underlying mechanisms were partially attributed to cerebral vascular injury, and it also suggested the presence of other different mechanisms.