By David N. Leff
It's common knowledge — or anyway, received dogma — that amyloid beta peptide is a prime ingredient of senile neuritic plaques, which are the etiological hallmark of Alzheimer's disease (AD).
That smoking gun is triggered by the enormous amyloid fibrils embedded in those toxic plaques.
Maybe, but it's crisply reopened by a paper in the current Proceedings of the National Academy of Sciences (PNAS), dated May 26, 1998. Its title: "Diffusible, nonfibrillar ligands derived from amyloid-beta (1-42) are potent central nervous system neurotoxins." Among its three leading authors is bio-organic chemist Grant Krafft, of Northwestern University Medical School, in Evanston, Ill.
Note that word "nonfibrillar." It challenges the primacy of amyloid-beta fibrils as the molecular gunmen in AD. Instead, the PNAS report arraigns as prime perpetrator a novel protein — ADDL — short for "amyloid-beta-derived diffusible ligands."
"When people have said that amyloid-beta fibrils are toxic," Krafft told BioWorld Today, "what they didn't realize — because they weren't using a technology that could see the ADDL structures — was that they actually had ADDL in their samples, and didn't know it."
Krafft cited another PNAS paper, dated Dec. 6, 1994, by a different group, bearing the headline: "Beta-amyloid neurotoxicity requires fibrils to be toxic."
"In that article," he recalled, "there was an electron micrograph of amyloid-beta fibrils. But in the background, looking like specks or dirt, were these little structures — ADDLs. We think," he added, "that fibrils themselves are not good things. But compared with ADDLs, they might actually be preferred."
ADDLs are tiny globular-shaped clumps of amyloid beta protein, five nanometers in diameter, only a fraction the size of a fibril.
Krafft and his collaborators were able to discern them by putting their samples under the atomic force microscope — an instrument of hyper-high-resolution developed to screen computer chips. "We came to realize," he recounted, "that the aggregated amyloid preparations that were the most toxic were littered with these tiny globular ADDL structures. The specimens that contained only fibrillar material — where we couldn't see any globules — were toxic only at very high concentrations. So the fibrils themselves seemed to be, in our hands, largely inert.
"Most interestingly," he continued, "when we saw what kinds of signals these ADDLs were activating in nerve cells, we got the notion that maybe we should look at long-term potentiation (LTP) in either brain slices or whole mice, to see if these globules could directly interfere with the information storage of learning and memory, as in AD.
"For that storage process to occur," he explained, "one neuron has to receive a signal, which then must interact with and reinforce the interaction of a neighboring nerve cell.
"In our experiment," Krafft narrated, "when we applied a mild stimulus to neuron A in a slice of murine hippocampal tissue, it responded by talking mildly to cell B. But if instead of one pulse of modest voltage, we very rapidly applied 10 pulses to that first cell, the next time we come in with a single jolt, instead of cell A talking gently to cell B, there was quite a significant interaction between them.
ADDLs Addle Memory Potentiation
Krafft pointed out, "In these experiments, it's the repetitive 10 pulses that we deliver that's the learning part. When we come in with the eleventh jolt sometime later, the intensity of the signal that's sent is much greater, and persists for hours. The neurons remember that for a long time. So I can give a nerve cell 10 pulses, then 90 minutes later come in with one small jolt and get a very substantially enhanced response."
However, when Krafft's team treated the brain slices with ADDLs, "within a relatively short time, that ability to remember those 10 pulses went away. So, essentially, these cells had not stored the memory of having received that initial information."
Krafft observed that the "LTP phenomena that most people consider central to learning and memory were directly compromised by ADDLs, and [that] may set off progression of AD."
Moving from in vitro to in vivo, instead of placing their LTP electrode on neurons in a brain-slice preparation, the co-authors implanted them in hippocampal spatial brain areas of living mice and controls. (The hippocampus plays a leading role in processing memory.)
The test animals got shots of ADDL; the controls of media only. Results were comparable to the brain-slice data.
ADDLs bind to receptors on the surface of their target neurons. When the Northwestern group "shaved" these proteins off that surface with the protease trypsin, subsequent incubation with ADDLs had no toxic effect.
"But when we took the culture medium containing those receptor shavings and concentrated it ten-fold, those concentrated proteins were able not only to block the ADDLs from binding to the cell surfaces that still had their receptors, but also to block the toxicity in those brain slice assays."
Researchers Seeking Pharmaceutical Partner
"That tells us," Krafft pointed out, 'that somewhere in the shavings there's an intact extracellular portion of protein that's capable of binding the ADDLs. Obviously," he added, "we're working hard to figure out what the identity of that protein is. But meanwhile, at least, we've been able to use the binding phenomenon to establish assays to start looking for compounds that can block the interaction. That's where we are now."
Where he and his collaborators will be a month or so from now depends on whether they strike a drug discovery deal, now pending, with a major pharmaceutical company. "We've been in fairly late-stage discussions with several large pharmaceutical firms, and are pretty close to a collaborative arrangement," Krafft observed.
In late 1996, he and his two principal co-authors — neurobiologist William Klein at Northwestern (the PNAS paper's senior author) and neurogerontologist Caleb Finch, from University of California at Los Angeles — formed a company, Acumen Pharmaceuticals Inc., based in Glen View, Ill. Krafft is its president and chief scientific officer. He also figures as lead inventor on the pending ADDL patents, and is principal investigator of the National Institute of Health project grant that has funded his research so far.
"Assuming things go well in the next month or so," Krafft concluded, "this first collaborative arrangement with industry will provide funding to take Acumen from a virtual company to a real company." *