Amanda Gonzales

Can platelet BACE1 levels be used as a biomarker for Alzheimer's disease? Proof-of-concept study

To date there is no validated peripheral biomarker to assist with the clinical diagnosis of Alzheimers disease (AD). Platelet proteins have been studied as AD biomarkers with relative success. In this study, we investigated whether platelet BACE1 levels differ between AD and cognitively normal (CN) control patients. Using a newly developed ELISA method, we found that BACE1 levels were significantly lower in AD compared to CN subjects. These data were supported by the observation that several BACE1 isoforms, identified by Western blotting, were also lower in AD platelets. This proof-of-concept study provides evidence for testing platelet BACE1 levels as a peripheral AD biomarker using a novel, sensitive and inexpensive method.

Development of a specific ELISA to measure BACE1 levels in human tissues.

The aspartyl protease BACE1 is the rate limiting enzyme in the synthesis of amyloid beta, which accumulation in the human brain is a hallmark of Alzheimers disease (AD). BACE1 has been proposed as a surrogate marker of AD; however, very few BACE1 immunoassays have been reported. In the present study we have screened ten BACE1 antibodies by Western blot and several antibody pairs to develop a new BACE1 sandwich ELISA procedure. We identified one pair that showed little background and good reproducibility. Several dilution buffers and sample denaturation methods were tried to partially unfold BACE1 before capture. We found that dilution in PBS followed by 10 min incubation at 50°C critically improves the performance of the assay. Finally, we successfully measured BACE1 levels in a few human brain and platelet lysates as well as in plasma and AD CSF. We anticipate that this assay will lay the ground to accurately measure BACE1 levels in human tissues, which could facilitate the molecular diagnosis of AD in the near future.

A cellular model of amyloid precursor protein processing and amyloid-β peptide production☆

BACKGROUND A hallmark pathologic feature of Alzheimers disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-β (Aβ) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by β-, and γ-secretases. There is a great interest in approaches to modulate Aβ peptide production and develop therapeutic interventions to reduce Aβ levels to halt or slow the progression of neurodegeneration. NEW METHOD We characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future (1) functional studies of molecular regulators in Aβ production, and (2) high-throughput screening assays of new pharmacotherapeutics. RESULTS In BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products Aβ40 and Aβ42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line. COMPARISON WITH EXISTING METHOD(S) In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations. CONCLUSIONS Our evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of Aβ production.

BACE1 levels by APOE genotype in non-demented and Alzheimer's post-mortem brains.

The APOE genotype is a known susceptibility factor for Alzheimers disease (AD). It is apparent that the presence of the APOE ε40 allele increases the risk for developing AD, lowers the age of onset in AD, and may influence the pathological burden seen in AD. In this study, we asked whether BACE1 levels differ by APOE genotype in the AD and non-demented (ND) brain. We isolated mid-frontal cortex (MFC) and mid-temporal cortex (MTC) from post-mortem ND and AD subjects that were APOE ε3/3, ε3/4, ε4/4 carriers. All AD subjects met NINDS-ADRDA and NIA-Reagan criteria for a diagnosis of AD. The MFC and MTC were homogenized and the lysates underwent ELISA and Western blotting for BACE1. The ELISA revealed that total BACE1 levels were lower in the MFC of AD compared to ND subjects. Furthermore, in APOE ε4 carriers BACE1 levels were lower than ε3/3 carriers in the ND frontal cortex. No difference in BACE1 levels was observed in AD MFC and in ND and AD MTC tissues. The ELISA results were confirmed by Western blotting. Our data suggest that brain BACEl levels may be influenced by the apolipoprotein E genotype before the onset of AD, providing an alternative explanation for the lower amyloid beta 42 levels in CSF in ND and AD subjects.

Poor Safety and Tolerability Hamper Reaching a Potentially Therapeutic Dose in the Use of Thalidomide for Alzheimer’s Disease: Results from a Double-Blind, Placebo-Controlled Trial

INTRODUCTION To date there is no cure for Alzheimers disease (AD). After amyloid beta immunotherapies have failed to meet primary endpoints of slowing cognitive decline in AD subjects, the inhibition of the beta-secretase BACE1 appears as a promising therapeutic approach. Pre-clinical data obtained in APP23 mice suggested that the anti-cancer drug thalidomide decreases brainBACE1 and Aβ levels. This prompted us to develop an NIH-supported Phase IIa clinical trial to test the potential of thalidomide for AD. We hypothesized that thalidomide can decrease or stabilize brain amyloid deposits, which would result in slower cognitive decline in drug- versus placebo-treated subjects. METHODS This was a 24-week, randomized, double-blind, placebo-controlled, parallel group study with escalating dose regimen of thalidomide with a target dose of 400mg daily in patients with mild to moderate AD. The primary outcome measures were tolerability and cognitive performance assessed by a battery of tests. RESULTS A total of 185 subjects have been pre-screened, out of which25 were randomized. Mean age of the sample at baseline was 73.64 (±7.20) years; mean education was 14.24 (±2.3) years; mean MMSE score was 21.00 (±5.32); and mean GDS score was 2.76 (±2.28).Among the 25 participants, 14 (56%) terminated early due to adverse events, dramatically decreasing the power of the study. In addition, those who completed the study (44%) never reached the estimated therapeutic dose of 400 mg/day thalidomide because of reported adverse events. The cognitive data showed no difference between the treated and placebo groups at the end of the trial. CONCLUSION This study demonstrates AD patients have poor tolerability for thalidomide, and are unable to reach a therapeutic dose felt to be sufficient to have effects on BACE1. Because of poor tolerability, this study failed to demonstrate a beneficial effect on cognition.

Platelet BACE1 levels as a possible biomarker for Alzheimer’s disease

Materials and methods We collected blood samples from 12 CN and 15 AD subjects using a standardized procedure. All samples were processed within 5-60 min after collection. Blood fractions were separated by successive centrifugations: red blood cells, platelet-rich fraction, and platelet-poor plasma. Both platelet and plasma samples were analyzed using a newly developed BACE1 ELISA method and Western blotting.

A phase II double-blind, placebo-controlled randomized trial of an escalating dose of thalidomide in the treatment of Alzheimer's disease: Baseline characteristics

Background:A phase II 12-month trial of resveratrol for mild-moderate dementia due to Alzheimer’s disease (AD) will determine its safety and tolerability and putative effects on clinical outcomes, volumetric MRI, Aband tau levels in cerebrospinal fluid, and metabolic profiles. A history of metabolic syndrome or type 2 diabetes mellitus (T2D), particularly during midlife, is a well-known risk factor for AD in late life but underlying mechanisms are unknown.Methods: Exclusionary criteria include individuals consuming any resveratrol-containing supplement and subjects treated for T2D. Eligible subjects (50-90 year old men and women recruited from and/or referred to specialty clinics) with mild-moderate AD (MMSE 1226) were administered a baseline oral glucose tolerance test (OGTT, 75 g) after an overnight fast. Results will be compared to a repeat OGTT on the maximally tolerated resveratrol dose after 12 months. Resveratrol was synthesized, encapsulated, and packaged by Aptuit Laurus (now Catalent), initiated at 500 mg bymouth once daily, and will end with 1 g twice daily (with 500 mg increments every 3 months). Results: Five subjects (5/128 or 4%) revealed impaired fasting glycemia (110-125 mg/dl) and 38 subjects (38/ 125 or 30%) revealed impaired glucose tolerance at 2 hours (140-200 mg/ dl). Three subjects (3/128 or 2%) had findings consistent with a diagnosis of T2D (fasting glucose > 125 mg/dl) and 16 subjects (16/125 or 13%) had results consistent with T2D (glucose > 200 mg/dl) at 2 hours. The mean fasting glucose (+/S.E.) was 92 +/1 mg/dl. The mean glucose at 2 hours was 143 +/5 mg/dl. These data revealed a prevalence of impaired fasting glycemia or T2D at baseline of 6%. The prevalence of impaired glucose tolerance or T2D at 2 hours was 43%. Six subjects screen-failed due to newly-diagnosed or treated T2D.Conclusions:Due to this high prevalence, the OGTT may be considered for individuals with AD in order to optimize medical management of borderline or undiagnosed T2D. AD, a chronic inflammatory disorder may increase risk of co-incident impaired fasting glycemia, glucose intolerance, and T2D. Conversely, a glucoregulatory disturbance may promote AD. Treatment of this study cohort with resveratrol or placebo is in progress.

Lenalidomide as an antineuroinflammatory and BACE1 inhibitor: Pilot study on APP23 mice

Background: Oxidative damage in Alzheimer disease (AD) is strongly associated with amyloid-b (Ab), a pathological hallmark of the disorder. As treatment and prevention options for dementia reach the forefront of the neuro-medical community, the benefits of antioxidant therapy are becoming increasingly evident. Sulforaphane (SFN), well-known antioxidants, found in broccoli and other cruciferous vegetables, is one of the most biologically active phytochemicals in the human diet. We hypothesized that the antioxidative activity of SFN would help protect the AD brain from brain damages induced by Ab and have significant potency as an AD therapeutic candidate. Administration of SFN ameliorated cognitive function of Ab-induced AD acute mouse models in Y-maze and passive avoidance behavior tests. Interestingly, we found that the therapeutic effect of SFN did not involve inhibition of Ab aggregation. While the exact mechanism of interaction of SFN in AD has not yet been ascertained, our results suggest that SFN can aid in cognitive impairment and may protect the brain from amyloidogenic damages. Methods: 1) Transmission electron microscopy (TEM)2) Cytotoxicity test 3) Amyloidogenesis test 4) Acute AD model (male ICR, 5.5 weeks): single intracerebroventricular (i.c.v.) injection of Ab aggregates 5) Administration of SFN administration via intraperitoneal (i.p.) injection 6) Behavior tests: Y-maze and passive avoidance (PA) tests. Results: From Y-maze and PA tests, SFN significantly improved cognitive, working memory and contextual memory of acute AD model increasing alternation and latency (Figure A). However, the ThT assay and TEM imaging indicated that SFN did not directly interact with Ab in its neuroprotective action (Figure B). Rather, we expect that SFN acted as a downstream protectant against Ab-induced oxidative stress in the AD brain. Conclusions: Administration of SFN ameliorated cognitive dysfunction ofAb-inducedADacutemice.Since aggregationofAb is oneof the principle causes of neuronal dysfunction in the disorder, therapeutic strategies for AD have focused on development of Ab modulators affecting on abnormal production and accumulation of Ab. Although SFN did not regulate amyloidogenesis, the downstream protective role was noteworthy against Ab-induced oxidative stress. Thus, we suggest that antioxidants such as SFN can be a complementary but promising strategy to approach AD treatment.

Preclinical testing of lenalidomide as anti-amyloid treatment for Alzheimer's disease

Background: Alzheimer’s disease (AD) is characterized by deposition of Ab plaques and neuroinflammation leading to neuronal loss. Ab is generated by enzymatic cleavage of amyloid precursor protein (APP) by b and g secretase enzymes. APP is also processed by non-amyloidogenic6 -secretase, which precludes Ab production. We have previously demonstrated that some cholinesterase inhibitors (ChEIs) alter APP processing (Lahiri et al., JPET, 2007). In this context, we report that rivastigmine, a ChEI used for mild-moderate AD also modulates APP processing in neuron culture, animal model and human subjects.Methods:We have treated primary human neurons with 100nM, 1 mM and 10 mM rivastigmine6 TAPI 2 for 4 days. APP transgenic mice were treated with rivastigmine for three weeks and postmortem brain bank tissues of AD patients who received only rivastigmine, non-medicated AD patients, and non AD controls were also analyzed Results: Analyses of conditioned media (CM) samples from neuron culture showed a significant dose-dependent increase in levels of sAPP 6 with rivastigmine treatments versus vehicle-treated samples. Furthermore, both Ab (1-40) and (1-42) species and sAPPb were significantly decreased by rivastigmine treatments. Analyses of intracellular proteins revealed a significant increase in levels of intracellular ADAM10, which is an6 -secreatse by all the doses of rivastigmine versus controls. We also observed increased sAPP6 and decreased soluble Ab in the brains of Tg mice treated with rivastigmine versus controls. Analyses of postmortem brain samples revealed an increasing trend in the levels of brain sAPP6 in subjects who had received rivastigmine versus non-medicated subjects. Ab (1-42) was also significantly decreased in the brain of rivastigmine-treated patientsConclusions: Increase sAPP6 , decreased sAPPb and Ab peptides by rivastigmine treatments indicate a shift in APP processing towards the6 -secreatse pathway, which was blocked by co-treatment of a non-selective ADAM inhibitor, TAPI2. This effect of rivastigmine was translated to animal model as well as in human subjects. Taken together, these results suggest that rivastigmine promotes the 6 -secretase pathway by up-regulating or preserving ADAM10. Rivastigmine’s dual functions, anticholinesterase and Ab lowering properties, warrant further investigation in larger clinical settings.