When the dust settles: what did we learn from the bexarotene discussion?

With 27 million people affected by Alzheimer's disease (AD), any proposal of a novel avenue for drug development is hot news. When Cramer and colleagues proposed last year that they could tackle AD pathology in an AD mouse model with bexarotene, a drug already in use in the clinic for other diseases, the news was covered worldwide by the popular press. Apolipoprotein E4 is the strongest genetic risk factor for AD and bexarotene appeared to exert spectacular effects on AD pathology when tested in APP/PS1 transgenic mice. One year later the slumbering discussion on the use of bexarotene in AD exploded in a flurry of papers. Four papers question the initial optimistic claims, while two others can only partially support the original work. We summarize here the available data and try to make sense out of the controversy. The major question is what we can learn from the experiments and what these studies imply for the further development of bexarotene in the clinic.status: publishe

Concordance of Alzheimer's Disease-Related Biomarkers Between Intraventricular and Lumbar Cerebrospinal Fluid in Idiopathic Normal Pressure Hydrocephalus

BACKGROUND: Alzheimer's disease cerebrospinal fluid (CSF) biomarkers amyloid-β 1-42 (Aβ42), total tau (T-tau), and phosphorylated tau 181 (P-tau181) are widely used. However, concentration gradient of these biomarkers between intraventricular (V-CSF) and lumbar CSF (L-CSF) has been demonstrated in idiopathic normal pressure hydrocephalus (iNPH), potentially affecting clinical utility. OBJECTIVE: Here we aim to provide conversion factors for clinical and research use between V-CSF and L-CSF. METHODS: Altogether 138 iNPH patients participated. L-CSF samples were obtained prior to shunt surgery. Intraoperative V-CSF samples were obtained from 97 patients. Post-operative follow-up L- and V-CSF (shunt reservoir) samples of 41 patients were obtained 1-73 months after surgery and then after 3, 6, and 18 months. CSF concentrations of Aβ42, T-tau, and P-tau181 were analyzed using commercial ELISA assays. RESULTS: Preoperative L-CSF Aβ42, T-tau, and P-tau181 correlated to intraoperative V-CSF (ρ= 0.34-0.55, p < 0.001). Strong correlations were seen between postoperative L- and V-CSF for all biomarkers in every follow-up sampling point (ρs Aβ 42: 0.77-0.88, T-tau: 0.91-0.94, P-tau181: 0.94-0.96, p < 0.0001). Regression equations were determined for intraoperative V- and preoperative L-CSF (Aβ 42: V-CSF = 185+0.34*L-CSF, T-tau: Ln(V-CSF) = 3.11+0.49*Ln(L-CSF), P-tau181: V-CSF = 8.2+0.51*L-CSF), and for postoperative V- and L-CSF (Aβ 42: V-CSF = 86.7+0.75*L-CSF, T-tau: V-CSF = 86.9+0.62*L-CSF, P-tau181: V-CSF = 2.6+0.74*L-CSF). CONCLUSION: Aβ 42, T-tau, and P-tau181 correlate linearly in-between V- and L-CSF, even stronger after CSF shunt surgery. Equations presented here, provide a novel tool to use V-CSF for diagnostic and prognostic entities relying on the L-CSF concentrations and can be applicable to clinical use when L-CSF samples are not available or less invasively obtained shunt reservoir samples should be interpreted

Time Trends of Cerebrospinal Fluid Biomarkers of Neurodegeneration in Idiopathic Normal Pressure Hydrocephalus

Background: Longitudinal changes in cerebrospinal fluid (CSF) biomarkers are seldom studied. Furthermore, data on biomarker gradient between lumbar (L-) and ventricular (V-) compartments seems to be discordant. Objective: To examine alteration of CSF biomarkers reflecting Alzheimer's disease (AD)-related amyloid-beta (A beta) aggregation, tau pathology, neurodegeneration, and early synaptic degeneration by CSF shunt surgery in idiopathic normal pressure hydrocephalus (iNPH) in relation to AD-related changes in brain biopsy. In addition, biomarker levels in L- and V-CSF were compared. Methods: L-CSF was collected prior to shunt placement and, together with V-CSF, 3-73 months after surgery. Thereafter, additional CSF sampling took place at 3, 6, and 18 months after the baseline sample from 26 iNPH patients with confirmed A beta plaques in frontal cortical brain biopsy and 13 iNPH patients without A beta pathology. CSF Amyloid-beta(42) (A beta(42)), total tau (T-tau), phosphorylated tau (P-tau(181)), neurofilament light (NFL), and neurogranin (NRGN) were analyzed with customized ELISAs. Results: All biomarkers but A beta(42) increased notably by 140-810% in L-CSF after CSF diversion and then stabilized. A beta(42) instead showed divergent longitudinal decrease between A beta-positive and -negative patients in L-CSF, and thereafter increase in A beta-negative iNPH patients in both L- and V-CSF. All five biomarkers correlated highly between V-CSF and L-CSF (A beta(42) R = 0.87, T-tau R = 0.83, P-tau R = 0.92, NFL R = 0.94, NRGN R = 0.9; all p < 0.0001) but were systematically lower in V-CSF (A beta(42) 14 %, T-tau 22%, P-tau 20%, NFL 32%, NRGN 19%). With APOE genotype-grouping, only A beta(42) showed higher concentration in non-carriers of allele epsilon 4. Conclusion: Longitudinal follow up shows that after an initial post-surgery increase, T-tau, P-tau, and NRGN are stable in iNPH patients regardless of brain biopsy A beta pathology, while NFL normalized toward its pre-shunt levels. A beta(42) as biomarker seems to be the least affected by the surgical procedure or shunt and may be the best predictor of AD risk in iNPH patients. All biomarker concentrations were lower in V-than L-CSF yet showing strong correlations.Peer reviewe

Regulation of Amyloid Precursor Protein Processing by Serotonin Signaling

<div><p>Proteolytic processing of the amyloid precursor protein (APP) by the β- and γ-secretases releases the amyloid-β peptide (Aβ), which deposits in senile plaques and contributes to the etiology of Alzheimer's disease (AD). The α-secretase cleaves APP in the Aβ peptide sequence to generate soluble APPα (sAPPα). Upregulation of α-secretase activity through the 5-hydroxytryptamine 4 (5-HT₄) receptor has been shown to reduce Aβ production, amyloid plaque load and to improve cognitive impairment in transgenic mouse models of AD. Consequently, activation of 5-HT₄ receptors following agonist stimulation is considered to be a therapeutic strategy for AD treatment; however, the signaling cascade involved in 5-HT₄ receptor-stimulated proteolysis of APP remains to be determined. Here we used chemical and siRNA inhibition to identify the proteins which mediate 5-HT_4d receptor-stimulated α-secretase activity in the SH-SY5Y human neuronal cell line. We show that G protein and Src dependent activation of phospholipase C are required for α-secretase activity, while, unexpectedly, adenylyl cyclase and cAMP are not involved. Further elucidation of the signaling pathway indicates that inositol triphosphate phosphorylation and casein kinase 2 activation is also a prerequisite for α-secretase activity. Our findings provide a novel route to explore the treatment of AD through 5-HT₄ receptor-induced α-secretase activation.</p></div

Single knock-down of ADAM9, ADAM10, ADAM17 and MMP9 does not affect 5-HT_4d receptor-stimulated APP shedding.

<p>(A) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d, were treated with 1 µM prucalopride or 5-HT (5-HT₄ receptor agonists) in the absence or presence of 80 µM GM6001 (metalloproteinases inhibitor) and secretion of sAPPα was analyzed via measuring SEAP. (B) SEAP levels were measured in supernatants of SH-SY5Y cells, co-transfected with pEAK12-AP-APP, pcDNA3.1-5-HT_4d and 3 nM siRNA for knock-down of ADAM9 (A9), ADAM10 (A10), ADAM17 (A17) and MMP9 and treated with 1 µM prucalopride. (C) Cell lysates of (B) were analyzed for protein expression of ADAM9, 10, 17 and MMP9 by western blotting. The ADAM10 and ADAM17 immature precursor proteins are indicated by an x, whereas the mature catalytically active forms are indicated by an xx for ADAM9, 17 and MMP9. The immature ADAM9 and the mature ADAM10 proteins were not visible. (D) Quantification of experiments in (C). Values shown are mean ± SEM of 6 individual wells and were normalized to vehicle control. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.</p

Overview of agonists, antagonists and inhibitors used to investigate the proteins contributing to the induction of sAPPα after 5-HT_4d receptor stimulation.

<p>Ag = agonist; antag = antagonist; inh = inhibitor.</p

Schematic representation of the proposed 5-HT_4d receptor-stimulated signaling pathway leading to increased sAPPα production.

<p>The proteins involved in 5-HT_4d receptor-mediated non-amyloidogenic APP shedding are shown with green circles while orange circles and red characters indicate proteins or second messengers that were tested but were ineffective in modulating 5-HT_4d receptor-stimulated sAPPα release. The dotted lines with the question marks indicate remaining areas of investigation for further elucidation of the molecular mechanism of α-secretase activation. cAMP-dependent pathway of α-secretase induction was previously reported and is depicted as a plausible way for 5-HT_4d receptor-mediated sAPPα release <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087014#pone.0087014-Maillet1" target="_blank">[15]</a>.</p

5-HT_4d receptor-stimulated APP shedding requires inositol polyphosphates and casein kinase 2.

<p>(A), (B) and (C) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d, were treated with 1 µM prucalopride or 5-HT (5-HT₄ receptor agonists) in the absence or presence of 20 µM IP3K inhibitor (A), 80 µM CGA (IPMK inhibitor) (B) or 2.5 µM TBB (CK2 inhibitor) (C) and secretion of sAPPα was analyzed via measuring SEAP. (D) SEAP levels were measured in supernatants of SH-SY5Y cells, co-transfected with pEAK12-AP-APP, pcDNA3.1-5-HT_4d and 3 nM siRNA for knock-down of CK2 and treated with 1 µM prucalopride. (E) Cell lysates of (D) were analyzed for CK2 expression levels by western blotting. (F) Quantification of experiments in (E). Values shown are mean ± SEM of 6 individual wells and were normalized to vehicle control. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.</p

5-HT_4d receptor-stimulated APP shedding requires Src and phospholipase C, but not PKC or calcium.

<p>(A), (B) and (C) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d, were treated with 1 µM prucalopride or 5-HT (5-HT₄ receptor agonists) and PMA in the absence or presence of 50 µM Bosutinib (Src inhibitor) (A), 30 µM D609 (PLC inhibitor) (B) or 2 µM GF109203X (PKC inhibitor) (C) and secretion of sAPPα was analyzed via measuring SEAP. Values shown are mean ± SEM of 6 individual wells and were normalized towards vehicle control. (D) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d, were loaded with Fluo-4 NW mix and fluorescence of the calcium-sensitive dye in each well was recorded at the baseline (F₀) and after stimulation with 1 µM prucalopride, 30 µM ATP (purinergic ionotropic receptors agonist), 20 µM Ionomycin (calcium ionophore) or DMSO (F). Calcium response shown is a ratio of maximum fluorescence intensity at 40 sec to baseline fluorescence (F/F₀). Values shown are mean ± SEM of 2 individual wells and were normalized to vehicle control. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.</p