Reducing amyloid-related Alzheimer's disease pathogenesis by a small molecule targeting filamin A

Abstract

PTI-125 is a novel compound demonstrating a promising new approach to treating Alzheimer's disease (AD), characterized by neurodegeneration and amyloid plaque and neurofibrillary pathologies. We show that the toxic signaling of amyloid-β(42) (Aβ(42)) by the α7-nicotinic acetylcholine receptor (α7nAChR), which results in tau phosphorylation and formation of neurofibrillary tangles, requires the recruitment of the scaffolding protein filamin A (FLNA). By binding FLNA with high affinity, PTI-125 prevents Aβ(42)'s toxic cascade, decreasing phospho-tau and Aβ aggregates and reducing the dysfunction of α7nAChRs, NMDARs, and insulin receptors. PTI-125 prevents Aβ(42) signaling by drastically reducing its affinity for α7nAChRs and can even dissociate existing Aβ(42)-α7nAChR complexes. Additionally, PTI-125 prevents Aβ-induced inflammatory cytokine release by blocking FLNA recruitment to toll-like receptor 4, illustrating an anti-inflammatory effect. PTI-125's broad spectrum of beneficial effects is demonstrated here in an intracerebroventricular Aβ(42) infusion mouse model of AD and in human postmortem AD brain tissue.

Figures

Figure 1.

A, Continuous ICV Aβ42 infusion for 1 week increased FLNA association with α7nAChR, and twice-daily intraperitoneal injections of PTI-125 for 2 weeks significantly reduced this interaction. Synaptosomes prepared from prefrontal cortex and hippocampus of treated mice were immunoprecipitated with immobilized anti-FLNA, and α7nAChR levels in the immunoprecipitates were detected by Western blot (WB) using an α7nAChR-specific antibody. B, ICV Aβ42 infusion also increased FLNA association with TLR4, detected in the same anti-FLNA immunoprecipitates with a TLR4-specific antibody. C, Aβ42 strongly promoted tau phosphorylation at Ser202, Thr231, and Thr181, and PTI-125 significantly reduced phosphorylation at all three sites. Levels of tau protein phosphorylated at each site were measured in the immunoprecipitates of an anti-tau antibody that did not distinguish its phosphorylation state by Western blotting with specific antibodies separately recognizing each tau phosphoepitope. Blots (inset) were analyzed by densitometric quantitation. n = 7 or 8. *p < 0.01 vs sham, vehicle; #p < 0.01 vs Aβ42, vehicle.

Figure 2.

A–D, Incubation for 1 h with 1 nm PTI-125 reduced FLNA–α7nAChR/TLR4 associations shown by Western blot detection of α7nAChR (A) or TLR4 (B) in anti-FLNA immunoprecipitates from AD and Aβ42-treated control FCX slices or by Western detection of FLNA in anti-α7nAChR (C) or anti-TLR4 (D) immunoprecipitates. Western blots (inset) were analyzed by densitometric quantitation. n = 11. *p < 0.01 vs vehicle-treated control; #p < 0.01 vs Aβ42-treated control or vehicle-treated AD.

Figure 3.

PTI-125 treatment of ICV Aβ42-infused mice normalized the Aβ42-induced α7nAChR (A) and NMDAR (B) dysfunction, realized by reduced calcium influx in response to varying doses of the full α7nAChR agonist PNU282987 and by the NMDAR co-agonists NMDA and glycine. n = 7 or 8. *p < 0.01 vs sham, vehicle; #p < 0.01 vs Aβ42, vehicle; +p < 0.01 vs vehicle-treated and PTI-125-treated sham groups. Similarly, 1 h incubation with PTI-125 of postmortem AD or Aβ42-treated control tissue restored α7nAChR (C) and NMDAR (D) function. n = 11. *p < 0.01 vs vehicle-treated control; #p < 0.01 vs vehicle-treated AD group; +p < 0.01 vs vehicle-treated and PTI-125-treated control groups.

Figure 4.

A, Aβ42 reduced K+-evoked Ca+2 influx, indicating dead, dying, or dysfunctional cells, and PTI-125 attenuated this Aβ42-induced deleterious effect. n = 7 or 8. *p < 0.01 vs sham, vehicle; #p < 0.01 vs Aβ42, vehicle; ++p < 0.05 vs vehicle-treated and PTI-125-treated sham groups. B, Similarly, PTI-125 incubation partially normalized the drastic reduction in depolarization-induced calcium influx in postmortem AD or Aβ42-treated control tissue. n = 11. *p < 0.01 vs vehicle-treated control; #p < 0.01 vs vehicle-treated AD group; +p < 0.01 vs vehicle-treated and PTI-125-treated control groups.

Figure 5.

Incubation for 1 h with PTI-125 normalized NMDAR signaling impairment in postmortem AD brain slices and Aβ42-treated control brain slices, evidenced by reductions in linkages of several signaling molecules to NR1, the obligatory NMDAR subunit. Western blots (inset) were analyzed by densitometric quantitation. n = 11. *p < 0.01 vs vehicle-treated control group; #p < 0.01 vs vehicle-treated AD group; +p < 0.01 compared with PTI-125-treated control group.

Figure 6.

A, PTI-125 treatment ameliorated the Aβ42-induced impairment in IR signaling in ICV Aβ42-infused mice, as measured by the phosphorylation of ΙRβ and its association with the signaling adaptor molecule IRS-1. n = 7 or 8. *p < 0.01 vs sham, vehicle; #p < 0.01 vs Aβ42, vehicle. B, Similarly, the deficit in IR signaling seen in postmortem AD and Aβ42-treated control brain slices was lessened by 1 h PTI-125 incubation. n = 11. *p < 0.01 vs vehicle-treated control group; #p < 0.01 vs vehicle-treated AD group.

Figure 7.

PTI-125 treatment of ICV Aβ42-infused mice nearly abolished Aβ42-induced production of inflammatory cytokines IL-6, TNF-α, and IL-1β in parietal cortex, measured by a fluorescence ELISA assay. n = 7 or 8. *p < 0.01 vs respective cytokine level in sham, vehicle group; #p < 0.01 vs respective cytokine level in ICV Aβ42, vehicle group.

Figure 8.

A, Representative sections immunostained with an anti-NFT (phospho-tau) antibody show that PTI-125 treatment dramatically reduced NFT immunoreactivity in both prefrontal cortex and hippocampus of mice receiving ICV Aβ42 infusions. B, Representative sections immunostained for Aβ42 aggregates show that PTI-125 treatment greatly reduced Aβ42 deposits in both brain regions of Aβ42-infused mice. Immunostaining was quantified by image analysis software. n = 7 or 8. *p < 0.01 vs sham, vehicle; #p < 0.01 vs Aβ42, vehicle.

Figure 9.

A, Aβ42–α7nAChR complexes were elevated in both Aβ42-treated control tissue and AD tissue, and PTI-125 incubation reduced this interaction in each. Blots (inset) of anti-Aβ42 immunoprecipitates were probed with anti-α7nAChR and analyzed by densitometric quantitation. B, PTI-125 incubation reduced Aβ42 binding affinity for α7nAChR 1000-fold from 100 fm to 16 nm (dotted line) in biotinylated synaptic membranes from postmortem frontal cortices of controls without dementia. In the K-R control condition (solid line), there were two binding sites, a high-affinity (EC50_1) and a lower-affinity (EC50_2) binding site, which were also reduced by PTI-125 incubation. C, In fresh SK-N-MC cells, PTI-125 reduced this binding affinity 10,000-fold from 770 fm, the high-affinity binding site in the K-R control condition (solid line), to 1 nm (dotted line). n = 11 for postmortem control tissue; n = 6 for SK-N-MC cells. *p < 0.01 vs vehicle-treated control; #p < 0.01 vs Aβ42-treated control or vehicle-treated AD.

Figure 10.

A, Acting as a decoy for the FLNA protein, the pentapeptide binding site of PTI-125 on FLNA (VAKGL) blocked the reduction by PTI-125 in Aβ42-induced FLNA–α7nAChR/TLR4 association in postmortem frontal cortical synaptosomes. The inactive control pentapeptide (VAAGL) did not disrupt PTI-125 reductions of these Aβ42-induced FLNA associations. Western blots (A) were analyzed by densitometric quantification (B). n = 3. *p < 0.01 vs respective basal level; #p < 0.01 vs Aβ42-exposed tissues.

Figure 11.

The VAKGL pentapeptide also blocked PTI-125 reduction in Aβ42-induced tau phosphorylation at three phosphorylation sites in postmortem frontal cortical synaptosomes. Again, the inactive control pentapeptide (VAAGL) did not disrupt PTI-125 reduction of Aβ42-induced tau phosphorylation. Western blots (A) were analyzed by densitometric quantification (B). n = 3. *p < 0.01 vs respective basal level; #p < 0.01 vs Aβ42-exposed tissues.

Figure 12.

FLNA associations with α7nAChR and TLR4 were markedly increased in AD and Aβ42-treated lymphocytes, and 30 min incubation with PTI-125 reduced these associations. Western blots (A) were analyzed by densitometric quantification (B). n = 2. **p < 0.05, *p < 0.01 vs vehicle-treated control; ##p < 0.05, #p < 0.01 vs Aβ42-treated control or vehicle-treated AD.

Figure 13.

Proposed model of PTI-125 normalizing Aβ42-induced synaptic dysfunction. Soluble Aβ42 or Aβ42 oligomers bind to α7nAChRs in AD brains to induce synaptic dysfunction by first activating α7nAChRs (Dougherty et al., 2003) and TLR4s (Lotz et al., 2005) (by CD14) to recruit FLNA, which enables a high-affinity interaction of Aβ42 with these receptors. The result of FLNA recruitment to TLR4 is enhanced signaling and cytokine release. With FLNA recruited to α7nAChR, Aβ42 binding activates kinases that leads to rapid tau phosphorylation and neurofibrillary lesions (Wang et al., 2003) and causes desensitization, restricting Ca2+ influx through α7nAChRs and impairing downstream NMDAR activity and signaling. In addition, Aβ42 oligomers acting through multiple pathways impair IR function, leading to insulin resistance in neuronal cells (Zhao et al., 2008). Similar to a partial pharmacological blockade of NMDARs, Aβ42-induced hypofunctioning NMDARs are expected to reduce NMDAR-dependent LTP and to increase LTD, leading to dendritic spine shrinkage and retraction (Shankar et al., 2007) that further dampens excitatory neurotransmission. By binding to a defined pentapeptide binding domain with high affinity, PTI-125 may elicit conformational changes in FLNA to prevent its recruitment to α7nAChRs and TLR4s, thereby reducing Aβ42 affinity and facilitating the dissociation of Aβ42 from these receptors. The normalized α7nAChR and TLR4 activities improve NMDAR activation and reduce cytokine release (inflammation). Although neither Aβ42 nor PTI-125 alters FLNA recruitment to IRs, the PTI-125-induced conformational change in FLNA restores Aβ42-suppressed IR function and lessens insulin resistance. The augmented NMDAR activation favors LTP induction and healthier dendritic spines, resulting in more normal excitatory neurotransmission and cognitive processing. The improved IR function leads to healthier cells. Together, PTI-125 treatment lessens neurodegeneration and reduces the burdens of neuritic plaques and neurofibrillary tangles. Changes in color and shape of receptors and FLNA represent conformational or affinity changes.