Two years after discovering a way to neutralize a rogue protein linked to Alzheimer’s disease, University of Alberta Distinguished University Professor and neurologist Jack Jhamandas has found a new piece of the Alzheimer’s puzzle, bringing him closer to a treatment for the disease.
In a study published in Scientific Reports, Jhamandas and his team found two short peptides, or strings of amino acids, that when injected into mice with Alzheimer’s disease daily for five weeks, significantly improved the mice’s memory.
The treatment also reduced some of the harmful physical changes in the brain that are associated with the disease.
“In the mice that received the drugs, we found less amyloid plaque buildup and a reduction in brain inflammation,” said Jhamandas, who is also a member of the Neuroscience and Mental Health Institute.
“So this was very interesting and exciting because it showed us that not only was memory being improved in the mice, but signs of brain pathology in Alzheimer’s disease were also greatly improved. That was a bit of a surprise for us.”
Building on previous research
This discovery builds on previous findings of a compound called AC253 that can block the toxic effects of a protein called amyloid beta, which is believed to be a major contributor to Alzheimer’s because it is often found in large quantities in the brains of patients with the disease. AC253 blocks amyloid beta from attaching to certain receptors in brain cells – a process Jhamandas likens to plugging a keyhole.
However, while AC253 was shown to prevent a buildup of amyloid beta, it isn’t very effective at reaching the brain and is quickly metabolized in the bloodstream.
As a result, treatment using AC253 requires large amounts of the compound to be effective, which is impractical and increases the chances of the body developing an immune reaction to treatment.
Transforming AC253 from an injectable drug into a pill would address the metabolism issues and increase efficacy, but AC253 was too complex to be able to make an effective oral drug.
Jhamandas’ solution was to chop AC253 into pieces to see whether he could create smaller peptide strings that blocked amyloid beta in the same way AC253 did.
Through a series of tests using mice genetically modified to carry Alzheimer’s disease, Jhamandas’ team found two shorter pieces of AC253 that replicated the preventative and restorative abilities of the larger peptide.
New drug being developed
With the short peptides identified, Jhamandas and his team, which includes renowned virologists Lorne Tyrell and Michael Houghton, used a process of computer modelling and artificial intelligence to discover a small-molecule drug – similar to medications used to treat high blood pressure or cholesterol—it’s now developing.
This discovery builds on previous findings of a compound called AC253 that can block the toxic effects of a protein called amyloid beta, which is believed to be a major contributor to Alzheimer’s because it is often found in large quantities in the brains of patients with the disease.
AC253 blocks amyloid beta from attaching to certain receptors in brain cells—a process Jhamandas likens to plugging a keyhole.
The team is focused on manufacturing an optimized and oral version of the drug so human clinical trials can begin, said Jhamandas, who added small-molecule drugs are preferable for treatments, particularly for drug companies, because they are cheaper to make, can be taken orally and can more easily reach the brain through the blood, said Jhamandas.
While Jhamandas is optimistic about the potential of his new drug to change the way Alzheimer’s is managed, he is quick to point out the years of research he and other researchers have done to get to this point.
“This has been 15, 20 years of painstaking and incremental work,” he said. “And it’s like building a house: you put one brick down, then you put another brick on top of that, and pretty soon you have a foundation and then you have a house.
“Occasionally you come across a discovery that has the potential to change the game in a very fundamental way, like hitting a home run, and I’m very excited that we are really on to something here.”
Funding: Jhamandas’ research was supported by grants from the Canadian Institutes of Health Research, Alberta Innovates (Alberta Prion Research Institute), Alzheimer’s Society of Alberta and Northwest Territories, and University Hospital Foundation. The research was also supported by the Centre for Prion & Protein Folding Diseases, the Li Ka Shing Institute of Virology and the Applied Virology Institute.
Introduction
Alzheimer’s disease (AD) is the most common form of dementia that affects over 44 million individuals worldwide, and its prevalence of this condition continues to rise1.
One of the defining features of AD is the presence of soluble oligomers of amyloid beta (Aβ) protein that aggregate into extracellular fibrillary deposits known as amyloid plaques.
Progressive accumulation of intra- and extracellular Aβ within brain regions, that are critical for memory and cognitive functions, is linked to the neurodegeneration observed in this condition2–4.
Progressive accumulation of Aβ is an early pathological event in AD and may precede clinical symptom onset by 15–25 years5.
Recent clinical trials aimed at reducing the levels of Aβ, either through increasing its brain clearance using Aβ vaccine-based therapies, or inhibiting its generation by blocking the involved secretase enzymes, have been largely unsuccessful6,7.
Thus as yet, of the four FDA-approved therapies for AD, none are disease-modifying.
One potentially promising approach for the treatment of AD includes targeting specific receptors that serve as mediators of the toxic effects of Aβ oligomers.
Multiple receptors (the p75NTR receptor, scavenger receptors such as SCARA1/2, neuronal nicotinic acetylcholine receptors), have been implicated in mediating Aβ-induced disruption of neuronal and synaptic processes in AD and thus identified as potential drug targets for developing anti-Aβ therapies, although as yet none have fulfilled this goal8,9.
Nonetheless, identification of a target that is implicated in the three key aspects of Alzheimer disease pathogenesis, i.e. neuronal loss, inflammation and vasculopathy, could offer a promising avenue for the development of therapeutics aimed at mitigating disease progression.
Emerging lines of evidence have highlighted the role of amylin receptor (AMY) as a putative target for the deleterious effects of Aβ in the context of AD10.
Amylin receptor is a Class B G-protein-coupled receptor comprised of heterodimers of calcitonin receptor (CTR) and one of three receptor activity modifying proteins (RAMP1-3) that generate multiple subtypes of amylin receptors, AMY1-311,12.
Amylin receptor antagonist, AC253, is a 24-amino acid peptide, originally derived from 8-32 fragment of salmon calcitonin hormone13. Data from our group and others demonstrates that amylin receptors are abundantly expressed on neurons, microglia, and vasculature, three core elements implicated in the AD pathology10,14–16.
Furthermore, Aβ-induced dysfunction and death of neurons that are preferentially affected in AD is attenuated by administration of amylin receptor antagonists, AC253 and AC18717–19.
Interestingly, AC253 also effectively reverses the impairment of Aβ- or human amylin (hAmylin)-induced depression of hippocampal long-term potentiation (LTP), a recognized cellular surrogate of memory20.
Most importantly, a recent study demonstrated that intracerebroventricular (icv) infusions of AC253 or intraperitoneal administration of the brain penetrant cyclized AC253 (cAC253), improved age-dependent deficits in spatial memory and learning in transgenic AD mice without gross adverse effects21.
These antagonists improved synaptic markers along with suppression of microglial activation and neuroinflammation21.
Other studies have reported that peripheral administration of amylin or its synthetic analog, pramlintide, resulted in improved spatial memory in mouse models of AD22,23.
The improvement in behavioral measures and accompanying reduction in amyloid burden in the brain in these studies was attributed to an efflux of brain Aβ (including monomers and small oligomers) into the blood.
Thus, the presence of amylin peptides in the circulation was postulated to serve as a “peripheral sink” for the egress of amyloid across the blood brain barrier and deemed to involve amylin receptors located on endothelial cells23.
Collectively, these studies identify the amylin receptor as a viable and potentially promising target for the development of AD therapeutics.
In order to optimize AC253 based peptides for AD therapy, we generated shorter peptide fragments based on the AC253 sequence for additional translational studies. Shorter peptides offer several advantages over longer sequences: higher stability and selectivity, better toxicity profile, significant brain penetration when administered systemically and a lower cost for both small- and large-scale synthesis and purification24–26.
Hence, we screened an AC253-based peptide fragment library and identified two promising shorter peptides, R5 (SQELHRLQTYPR), and R14 (LGRLSQELHRLQTY), which demonstrate high affinity binding to the amylin receptor subtype 3 (AMY3) and also recapitulate neuroprotective properties of the full length AC253.
In experimental in vitro and in vivo transgenic AD models, these peptide fragments show a significant improvement in memory and learning, and an attenuation of some characteristic features of AD pathology.
Results
Identification of short peptide fragments that selectively bind to amylin receptor
In order to identify shorter peptides with selective recognition and binding to amylin receptor, enhanced metabolic stability and brain penetrability than the linear full length peptide, we designed a peptide library comprised of 14 different sequences, namely, R1-R14.
Fragments (R1−R13) are 12 amino acids in length and peptide R14 is 14 amino acids. The initial fragment comprised the first 12 amino acids from the N-terminus of AC253 sequence, and subsequent fragments derived from shifting one amino acid at a time, as depicted in Supplemental Fig. S1A.
The library was synthesized on non-cleavable cellulose membrane (aminoPEG500) using SPOT synthesis, where the C-terminus of the peptide was attached to the surface of the amino-PEG500 cellulose membrane through β-ala spacer as described previously27.
Each amino acid was added to the free amino functional group using a stepwise Fmoc-SPPS procedure.
Each peptide was synthesized in duplicate at approximately 50 nmol on a spot on the membrane with a diameter of 4 mm (Supplemental Fig. S1B). Since our previous studies identified AMY3 receptor subtype as the preferential target for the direct actions of Aβ (and hAmylin) at the level of the cell membrane10, we targeted this receptor isoform in the current study.
A peptide library membrane was incubated with green fluorescent protein (GFP) labeled AMY3-expressing HEK293 cells (HEK-AMY3) to identify the highly binding sequences (Supplemental Fig. S1B).
The relative binding affinities of peptide fragments were determined through measuring and plotting the net fluorescence intensity of the bound GFP labeled live cells on each spot as measured with a fluorescence Kodak imager (Supplemental Fig. S1C).
Furthermore, to evaluate amylin receptor specificity of binding, the library was further screened against transfected CTR, and Wild-type HEK293 cells (HEK-WT). (Supplemental Fig. S1C).
The screening identified several peptide fragments that demonstrated significant specific binding to HEK-AMY3 cells compared to either HEK-WT or HEK-CTR cells (Supplemental Fig. S1C,D).
Fragments from the N-terminus domain showed higher affinity binding to the AMY3 receptor compared to those generated from the C-terminal region. Among the array of peptide fragments, peptides R5, and R14 were selected as demonstrating the highest specific binding to HEK-AMY3 expressing cells (Supplemental Fig. S1D).
Both peptide R5, and R14 bind with 2- fold more affinity to HEK-AMY3 cells based on intrinsic fluorescence measurement compared to HEK-WT cells, which confirmed their specificity for the amylin receptor and thus they were chosen for further investigation.
Source:
University of Alberta
Media Contacts:
Ryan O’Byrne – University of Alberta
Image Source:
The image is in the public domain.
Original Research: Open access
“Short amylin receptor antagonist peptides improve memory deficits in Alzheimer’s disease mouse model”. Rania Soudy, Ryoichi Kimura, Aarti Patel, Wen Fu, Kamaljit Kaur, David Westaway, Jing Yang & Jack Jhamandas.
Scientific Reports doi:10.1038/s41598-019-47255-9.
