Growing evidence indicates that insulin signaling may be disrupted in AD. In addition to epidemiology linking diabetes with AD (see ARF related news story), researchers led by Konrad Talbot at the University of Pennsylvania, Philadelphia, have shown that insulin receptor (IR) activation gets lost as patients progress from a diagnosis of MCI to one of AD (see ARF related news story). More directly, work from Dennis Selkoe’s group at Brigham and Women’s Hospital, Boston, and previous work from Klein’s group has tied Aβ oligomers to IR dynamics. Selkoe’s group showed that Aβ inhibits autocatalytic activation of the receptors, while Klein’s group showed that the receptors are lost from neuronal dendrites under an Aβ onslaught. Now, Klein and colleagues extend those observations by demonstrating a tit-for-tat: while small, soluble Aβ oligomers (which they call ADDLs, or Aβ-derived diffusible ligands) drive uptake of insulin receptors, insulin signaling drives loss of Aβ oligomer binding sites. What those binding sites are is still not clear, but the evidence suggests that they are not the insulin receptors themselves.

“To us, it was a total surprise that insulin was doing to the ADDL binding sites what ADDLs were doing to the insulin binding sites,” Klein told ARF in an interview. Using hippocampal neuron cultures, first author Fernanda De Felice and colleagues found that not only did insulin completely block the ADDL-induced uptake of insulin receptors from dendrites, but it also prevented the oligomers from binding to neurons. In so doing, insulin reduced Aβ oligomer-induced loss of dendritic spines and oligomer-associated oxidative stress. The simplest explanation for these protective effects might be that insulin and the Aβ oligomers compete for the same binding site, namely the insulin receptors. “But of course the simplest explanation was too simple,” said Klein. “If we blocked insulin receptor activity, then insulin was totally without effect.”

The findings suggest that insulin signaling somehow downregulates surface ADDL binding sites. In support of this, rosiglitazone, a PPAR-γ (peroxysome proliferator-activating receptor-γ) agonist and activator of IR kinase activity, potentiated insulin’s effects, whereas AG1024, an IR protein tyrosine kinase inhibitor, eliminated the ability of insulin to reduce Aβ binding. Klein said it is not clear what the surface ADDL binding sites are, “but this work gives us a handle on that, potentially, because if the insulin is causing them to go down, we can use that to look for the missing smoking gun.”

Interestingly, the researchers found that the oligomer-induced loss of IRs on the neurons’ surface did not occur in the presence of inhibitors of casein kinase 2 (CK2) and calcium/calmodulin-dependent kinase II (CaMKII). Both enzymes are involved in activity-dependent uptake of NMDA receptors. Blocking CK2 completely prevented ADDL-induced loss of both IRs and NMDARs from hippocampal neuron dendrites, suggesting that the mechanism for receptor loss may be the same in both cases. Insulin has been shown to affect synaptic plasticity in an NMDAR-dependent manner (see van der Heide et al., 2005), but whether the NMDARs represent the Aβ binding sites that are sensitive to insulin signaling is not clear.

“Results strongly support the hypothesis that insulin signaling plays a role in defending CNS neurons against AD and provides a disease-specific basis for treatments based on stimulating CNS insulin pathways,” the authors write. Interestingly, Talbot’s work showing increased activation of IR downstream kinases, despite loss of IR in AD (see ARF related news story), might suggest that some kind of compensation is already going on in the brain. Insulin-based strategies, including intranasal administration of the hormone itself, and PPAR-γ agonists, have shown some efficacy in clinical trials (see ARF related news story), and GlaxoSmithKline is currently testing rosiglitazone in several Phase 3 clinical trials (see REFLECT-1, REFLECT 3, REFLECT-4, and REFLECT-5).

Intranasal administration to the brain is being tested for various therapeutic agents; it is one potential route for the neuropeptide Y fragments that Masliah’s group has identified. Consisting of a 36-amino acid peptide, NPY is a neurotransmitter released by inhibitory interneurons. It is highly expressed in the hippocampus, a site of early AD pathology. The peptide draws researchers’ curiosity mostly for its role in controlling appetite and circadian rhythms, but according to Masliah also has proliferative and neuroprotective activity and has been linked to memory function. A variety of proteases besides neprilysin degrade NPY, but the breakdown products they generate were not considered active. “Most people thought of this degradation as a terminal event,” said Masliah.

First author John Rose and colleagues tested the effects of increased neprilysin activity in the mouse brain. Boosting the protease is one potential mechanism of clearing Aβ from the brain (see ARF related news story), and the researchers wanted to see if doing so might have untoward consequences. They analyzed a number of neuropeptides (other than Aβ) in the hippocampus of mice expressing ~1.5-fold normal levels of neprilysin and found that only levels of full-length NPY were lower than normal. Using an antibody to the C-terminal of NPY and also mass spectrometry, the researchers found two major NPY fragments were generated, a piece corresponding to amino acids 21-36, and a smaller 31-36-amino acid fragment.

To see if these fragments are biologically active, the researchers infused them into the brains of normal and APP transgenic mice. After four weeks, sham-infused mice had lost dendrites (as judged by levels of neuronal marker MAP-2), while the dendritic arbor in mice infused with the peptides was not statistically different from that of non-transgenic animals. The findings suggested that the NPY fragments were neuroprotective, which the authors confirmed using human primary neuronal cultures. In those cells, pretreatment with the NPY fragments prevented synapse loss (as per synaptophysin staining) in response to Aβ42.

How these fragments protect against Aβ is not entirely clear, but they may work by binding to NPY receptors. There are six different NPY receptors (Y1 to Y6) with different affinities for full-length NPY and its fragments. That neprilysin can generate C-terminal fragments of NPY in vitro has been documented previously (see Medeiros Mdos et al., 1996), leading to the suggestion that they may be active. In support of that idea, Rose and colleagues showed that the protective effects of the C-terminal fragments of NPY are blocked by a Y2 inhibitor—a Y1 inhibitor had no effect.

These findings add to a growing number of studies that cast NPY in the umbra of AD research. The Y2 receptor has recently been implicated in learning and memory (see Redrobe et al., 2004) and NPY upregulation has been seen in mouse models of AD (see Diez et al., 2003) and may be related to epileptic seizures seen in animal models (see ARF related news story).

Whether these fragments could become useful therapeutically is another question. Their in vivo half-lives are unknown at present, and they would require viruses or intranasal delivery to get them past the blood-brain barrier.—Tom Fagan.

References:
De Felice FG, Vieira MNN, Bomfim TR, Decker H, Velasco PT, Lambert MP, Viola KL, Zhao W-Q, Ferreira ST, Klein WL. Protection of synapses against Alzheimer’s-linked toxins: Insulin signaling prevents the pathogenic binding of Abeta oligomers. PNAS early edition 2009, February 2. Abstract

Rose JB, Crews L, Rockenstein E, Adame A, Mante M, Hersh LB, Gage FH, Spencer B, Potkar R, Marr RA, Masliah E. Neuropeptide Y fragments derived from neprilysin processing are neuroprotective in a transgenic model of Alzheimer’s disease. J. Neuroscience. 2009, January 28; 29:1115-1125. Abstract