Liang Ye

PPARγ activation enhances cell surface ENaCα via up-regulation of SGK1 in human collecting duct cells

Peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand‐dependent transcription factor that belongs to the nuclear receptor family that plays a critical role in adipocyte differentiation and lipid metabolism. Here we report for the first time that PPARγ is expressed in human renal cortical collecting ducts (CCD), segments of the nephor involved in regulation of sodium and water homeostasis via action of the epithelial sodium channel (ENaC). ENaC activity is regulated by the hormones aldosterone and insulin, primarily through co‐ordinate actions on serum and glucocorticoid regulated kinase 1 (SGK1). We show that SGK1 activity is stimulated by treatment of a human CCD cell line with PPARγ agonists, paralleled by an increase in SGK1 mRNA that is abolished by pretreatment with a specific PPARγ antagonist, and that this leads to increased levels of cell surface ENaCα. Electrophoretic mobility shift assays suggest that these effects are caused by binding of PPARγ to a specific response element in the SGK1 promoter. Our results identify SGK1 as a target for PPARγ and suggest a novel role for PPARγ in regulation of sodium re‐absorption in the CCD via stimulation of ENaC activity. This pathway may play a role in sodium retention caused by activation of PPARγ in man.

Interaction of the amyloid imaging tracer FDDNP with hallmark Alzheimer’s disease pathologies

The distinctive cortical uptake of the tracer 18F‐FDDNP (2‐(1‐{6‐[(2‐fluoroethyl(methyl)amino]‐2‐naphthyl}ethylidene)malononitrile) in Alzheimer’s disease (AD) is believed to be because of its binding to both neurofibrillary tangles (NFTs) and highly fibrillar senile plaques. We therefore investigated the binding of a tracer concentration of 3H‐FDDNP to brain sections containing AD hallmark pathologies. Semi‐adjacent sections were labelled with 3H‐PIB (Pittsburgh compound‐B, 2‐[4′‐(methylamino)phenyl]‐6‐hydroxybenzothiazole) and 14C‐SB13 (4‐N‐methylamino‐4′‐hydroxystilbene) for comparison. Neocortical sections containing widespread senile plaques and cerebrovascular amyloid angiopathy, produced a sparse and weak labelling following incubation with 3H‐FDDNP. Furthermore, in sections containing NFTs, there was no overt labelling of the pathology by 3H‐FDDNP. In contrast, sections labelled with 3H‐PIB displayed extensive labelling of diffuse plaques, classical plaques, cerebrovascular amyloid angiopathy and NFTs. 14C‐SB13 produced a broadly similar binding pattern to PIB. Radioligand binding assays employing in vitro generated amyloid‐β peptide fibrils demonstrated a ∼10‐fold reduced affinity for 3H‐FDDNP (85.0 ± 2.0 nM) compared with 3H‐PIB (8.5 ± 1.3 nM). These data provide an alternative mechanistic explanation for the observed low cortical uptake of 18F‐FDDNP in AD; in that the ligand is only weakly retained by the hallmark neuropathology because of its low affinity for amyloid structures.

Delineation of positron emission tomography imaging agent binding sites on β-amyloid peptide fibrils

A range of imaging agents for use in the positron emission tomography of Alzheimers disease is currently under development. Each of the main compound classes, derived from thioflavin T (PIB), Congo Red (BSB), and aminonaphthalene (FDDNP) are believed to bind to mutually exclusive sites on the β-amyloid (Aβ) peptide fibrils. We recently reported the presence of three classes of binding sites (BS1, BS2, BS3) on the Aβ fibrils for thioflavin T derivatives and now extend these findings to demonstrate that these sites are also able to accommodate ligands from the other chemotype classes. The results from competition assays using [3H]Me-BTA-1 (BS3 probe) indicated that both PIB and FDDNP were able to displace the radioligand with Ki values of 25 and 42 nm, respectively. BSB was unable to displace the radioligand tracer from the Aβ fibrils. In contrast, each of the compounds examined were able to displace thioflavin T (BS1 probe) from the Aβ fibrils when evaluated in a fluorescence competition assay with Ki values for PIB, FDDNP, and BSB of 1865, 335, and 600 nm, respectively. Finally, the Kd values for FDDNP and BSB binding to Aβ fibrils were directly determined by monitoring the increases in the ligand intrinsic fluorescence, which were 290 and 104 nm, respectively. The results from these assays indicate that (i) the three classes of thioflavin T binding sites are able to accommodate a wide range of chemotype structures, (ii) BSB binds to two sites on the Aβ fibrils, one of which is BS2, and the other is distinct from the thioflavin T derivative binding sites, and (iii) there is no independent binding site on the fibrils for FDDNP, and the ligand binds to both the BS1 and BS3 sites with significantly lower affinities than previously reported.

In vitro high affinity α-synuclein binding sites for the amyloid imaging agent PIB are not matched by binding to Lewy bodies in postmortem human brain

Amyloid containing deposits are a defining neuropathological feature of a wide range of dementias and movement disorders. The positron emission tomography tracer PIB (Pittsburgh Compound‐B, 2‐[4′‐(methylamino)phenyl]‐6‐hydroxybenzothiazole) was developed to target senile plaques, an amyloid containing pathological hallmark of Alzheimer’s disease, formed from the amyloid‐β peptide. Despite the fact that PIB was developed from the pan‐amyloid staining dye thioflavin T, no detailed characterisation of its interaction with other amyloid structures has been reported. In this study, we demonstrate the presence of a high affinity binding site (Kd∼4 nM) for benzothiazole derivatives, including [3H]‐PIB, on α‐synuclein (AS) filaments generated in vitro, and further characterise this binding site through the use of radioligand displacement assays employing 4‐N‐methylamino‐4′‐hydroxystilbene (SB13) (Ki = 87 nM) and 2‐(1‐{6‐[(2‐fluoroethyl(methyl)amino]‐2‐naphthyl}ethylidene)malononitrile (FDDNP) (Ki = 210 nM). Despite the presence of a high‐affinity binding site on AS filaments, no discernible interaction of [3H]‐PIB was detected with amygdala sections from Parkinson’s disease cases containing frequent AS‐immunoreactive Lewy bodies and related neurities. These findings suggest that the density and/or accessibility of AS binding sites in vivo are significantly less than those associated with amyloid‐β peptide lesions. Lewy bodies pathology is therefore unlikely to contribute significantly to the retention of PIB in positron emission tomography imaging studies.

Pyridazine-derived γ-secretase modulators

SAR of a novel series of pyridine-derived γ-secretase modulators is described. Compound 5 was found to be a potent modulator in vitro, which on further profiling, was found to decrease Aβ42 and Aβ40, and maintain (or increase) the levels of total Aβ. Furthermore, representative compounds 1 and 5 demonstrated in vivo efficacy to lower Aβ42 in the brain without altering Notch processing in the peripheral.

γ-Secretase modulators do not induce Aβ-rebound and accumulation of β-C-terminal fragment

J. Neurochem. (2012) 121, 277–286.

Orally bioavailable and brain-penetrant pyridazine and pyridine-derived γ-secretase modulators reduced amyloidogenic Aβ peptides in vivo

Accumulation of amyloid β (Aβ) in brain is a pathological hallmark of Alzheimers disease (AD). Aβ is generated after sequential cleavage of its parental molecule, amyloid precursor protein (APP), by β- and γ-secretases. Inhibition of γ-secretase activity is an effective approach for the reduction of Aβ levels. Since γ-secretase targets many different substrates, selective inhibition of its cleavage of APP is believed to be critical in order to avoid undesirable side effects. γ-Secretase modulator (GSM) shifts the cleavage site on APP and production of amyloidogenic to non-amyloidogenic Aβ fragments. Since GSMs only modulate and do not block cleavage of γ-secretase substrates, they are believed less likely to produce untoward adverse reactions. Here, we report in vivo Aβ-lowering profiles of a pyridazine and a pyridine-derived GSM: GSM-C (Wan et al., 2011a) and GSM-D (Wan et al., 2011b). Both compounds reduced Aβ40 and Aβ42 productions, increased shorter Aβ fragments, and had little effect on Notch signaling (∼100-fold selective). They had excellent oral bioavailability (97.8% for GSM-C, ∼100% for GSM-D) and good brain permeability (free brain to free blood AUC ratio of 0.41 and 1.10 for GSM-C and GSM-D, respectively). Oral administration of these compounds in both acute and sub-chronic conditions reduced Aβ levels in plasma and brain in rats in a dose- and time-dependent manner. Therefore, GSM-C and GSM-D represent two GSMs that are orally bioavailable and brain-permeable. They could serve as excellent tools in the investigation of the role of Aβ peptides in AD pathogenesis.

γ-secretase inhibitor, but not modulator, causes Aβ overshoot and accumulation of β-CTF

peptides mainly generate two isoform, Aß40 and Aß42 by enzymatic proteolysis of amyloid precursor protein (APP). In particular, the Aß42 is believed to be the major etiologic agent in pathogenesis of AD due to its higher fibrillation or oligomerization properties than that of Aß40. Recently we have established conformation dependent human antibody, B6, which binds to Aß42 fibril, but not to soluble form of Aß42, inhibiting Aß42 fibril formation. Concurrently, we have identified a mimotope of B6, B6-C15, using the PhD.C7C phage library. We chemically synthesized TAT-conjugated B6-C15 peptide, TATB6-C15. This synthetic peptide has inhibitory activity on Aß42 fibrillation. Furthermore, TAT-B6-C15 specifically binds to the oligomer form of Aß42, but not to freshly prepared monomer Aß42 nor its fibril form. In this study, we investigated the effect of this TAT-B6-C15 peptide on Aß40 assembly. Methods: Aß42 or Aß40 was incubated at 37 C in the presence or absence of TAT-B6-C15 peptide. Aß fibrillation was monitored by amyloid specific fluorescence dye, Thioflavin T. To identify the Aß conformers which specifically bound to TAT-B6-C15 peptide, we performed dot blot analysis. Aß conformers were periodically sampled after the onset of Aß40 or 42 fibrillation assay and hand-spotted onto nitrocellulose membrane, followed by incubation with detection probes such as TAT-B6-C15 or anti-Aß antibody. Results: The TAT-B6-C15 peptide exhibited inhibitory effect on Aß42, but not Aß40 fibrillation. Furthermore, the TAT-B6-C15 showed binding activity to the Aß42 prefibrillar oligomer, but not any Aß40 conformers. Conclusions: The mimotope peptide which identified as conformation dependent antibody epitope, specifically binds to prefibrillar oligomers of Aß42, inhibiting Aß42 but not Aß40 fibril formation.

P2-275: A clue to the lack of success of amyloid imaging agents in transgenic mouse models of Alzheimer’s disease

amyloid senile plaques and tau neurofibrillary tangles, also accumulate in cortical brain regions in patients with mild cognitive impairment (MCI) who are at risk for Alzheimer’s disease. A non-invasive method to determine regional cerebral patterns of these abnormal protein aggregates would facilitate early diagnosis and monitoring of novel treatments to prevent and eliminate their accumulation. Methods: We performed positron emission tomography (PET) scans on 60 subjects (20 cognitively intact controls, 20 with MCI, 20 with Alzheimer’s disease) after intravenous injections of 2-(1-{6-[(2-[F-18]fluoroethyl)(methyl) amino]-2-naphthyl} ethylidene)malononitrile (FDDNP), a molecule that binds to plaques and tangles in vitro. Subject groups were age-matched, and clinical assessments and FDDNPPET scans were repeated for 9 subjects (5 controls, 4 MCI subjects) after approximately two years (mean SD 24 5 months). Autopsy follow-up was available on 1 patient with Alzheimer’s disease. Results: Global FDDNP-PET binding (temporal, parietal, posterior cingulate, and frontal average) was lower for the control group compared with the MCI group (P 0.001), which showed lower binding compared with the Alzheimer’s disease group (P 0.001). Higher cognitive test scores correlated inversely with lower FDDNP binding values (P 0.001). At follow-up, subjects who converted from normal cognitive status to MCI or from MCI to Alzheimer’s disease showed regional FDDNP binding increases ranging from 5 to 11 percent, and autopsy follow-up demonstrated high concentrations of plaques and tangles in brain regions with high FDDNP binding. Conclusions: Although previous PET studies have found differences in cerebral amyloid measures when small groups of dementia patients and controls are compared, the present study is the first to describe in vivo cerebral imaging of amyloid and tau deposits in larger subject groups, including patients with MCI, as well as longitudinal clinical and autopsy follow-up. FDDNP-PET differentiates MCI from Alzheimer’s disease and controls and demonstrates increased binding as clinical neurodegeneration progresses. These results point to the potential utility of FDDNP-PET in early, “preclinical” diagnosis, as well as in monitoring interventions designed to prevent or reduce brain amyloid or tau accumulation.