The death of synapses, and the neurons that need them, marks the inevitable endgame of Alzheimer’s disease. Despite this eventual synaptic mayhem, a new work, led by Thomas Südhof and Marius Wernig at Stanford University, supports the idea that in its monomeric form, Aβ, the peptide behind amyloid plaques, spurs the growth of synapses. Writing in the October 19 Science Translational Medicine, the authors report that in cultured human neurons harboring the Aβ-boosting, Swedish mutation in amyloid precursor protein (APP), synapse numbers climbed by 20 percent, and synaptic transmission rose accordingly. In agreement with previous studies, neurons harboring this familial AD mutation also had swollen endosomes. Lowering Aβ with a BACE inhibitor, or by nixing APP expression, produced the opposite effect—lowering synapse numbers and shrinking endosomes. The findings support a physiological role for Aβ at the synapse, and the authors suggest this explains the cognitive impairment observed in BACE inhibitor trials.
- In human stem-cell-derived neurons, the Swedish mutation boosted synapses.
- BACE inhibition, or APP deletion, reduced synapse numbers.
- Aβ monomer peptides fueled the synaptic boost.
“The results should not confuse or distract from the accelerating approach to neutralize and remove synaptotoxic Aβ aggregates from the brains of AD patients, which has just received a strong boost through the positive top-line results of the Phase 3 clinical trial of lecanemab,” commented Dennis Selkoe of Brigham and Women’s Hospital in Boston.
While the literature on the synaptic effects of various forms of Aβ is complex, evidence indicates that at low, physiological concentrations, Aβ monomers can promote synaptic signaling, while at high concentrations, or in aggregated forms, the peptide is neurotoxic and extinguishes long-term potentiation, a critical component of functional neuronal circuitry (Puzzo et al., 2008; Nov 2009 news; Apr 2002 news). Previously, scientists found that in transgenic mice expressing the APP Swedish mutation synapses withered. Because the Swedish mutation accelerates Aβ production, this suggested that too much of the peptide is bad, and yet in hippocampal slice cultures and in human stem cell-derived neurons, other Aβ-boosting mutations stoked synaptic transmission (Fitzjohn et al., 2001; Feb 1999 news; Ghatak et al., 2019).
To study the effects of Aβ at the synapse in human neurons, first author Bo Zhou and colleagues used a novel approach to generate stringently isogenic lines of human neurons expressing either wild-type APP or APP bearing the APP-Swe mutation. Previous studies have derived stem cells from carriers of this mutation, and then corrected the mutation using CRISPR to create isogenic controls. However, the authors noted that this approach requires selection and propagation of mutation-corrected clones, a process which promotes genetic changes. To avoid this, the researchers started with a single line of embryonic stem (ES) cells and, in a parallel approach, a line of induced pluripotent stem (tips) cells. Using homologous recombination, they inserted into the endogenous APP gene back-to-back copies of exon 16—one encoding the wild-type APP sequence and the other the Swedish mutation. These different versions of APP could be switched on using different recombinases, thus allowing the researchers to generate lines that expressed either normal or mutant forms of APP. As the approach only affected one copy of APP, the other, normal copy of APP was left intact, yielding ES and iPS lines that were either homozygous for normal APP, or heterozygous for APP-Swe. In neurons differentiated from these lines, the researchers detected more Aβ40 and Aβ42 in cells expressing the Swedish-mutant APP than in those expressing only normal APP. In keeping with previous work, endosomes swelled by 15 to 20 percent in the mutant APP neurons as well (Aug 2019 news).
Next came a finding the scientists had not expected. Relative to neurons expressing normal APP, those expressing a copy of the Swedish mutant boasted 20 percent more synapses, and they expressed substantially more synaptic proteins. Electrophysiological recordings suggested that the synaptic windfall stoked a proportional boost in synaptic transmission. The findings suggested that the Swedish mutation bolstered the growth and function of synapses.
Aβ: Fuel for Synapses? Compared to neurons expressing only normal APP (left), those expressing a copy of APP with the Swedish mutation (right) boasted 20 percent more synapses (green). [Courtesy of Zhou et al., Science, 2022.]
The Swedish mutation promotes Aβ production by facilitating the cleavage of APP by BACE1. Therefore, the researchers reasoned that blocking BACE1 should reverse the synaptic boost. Indeed, treatment with the BACE inhibitor LY2886721 completely prevented the synaptic enhancement. It also decreased synaptic numbers and transmission in wild-type neurons. In agreement with these findings, the researchers detected a similar decrease in synapse numbers and transmission in neurons when they deleted APP. “Thus, the deletion and Swedish mutation of APP induce mirror-image phenotypes: The APP deletion suppresses synapse numbers and synaptic function, but the APP-Swedish mutation enhances them,” the authors wrote.
What is responsible for the synaptogenic effect? Besides producing a cadre of Aβ peptides, the cleavage of APP by β- and γ-secretases yields several other proteolytic fragments. Through a series of experiments where they expressed different APP fragments in neurons and/or added them exogenously, the researchers zeroed in on the synaptic booster—none other than Aβ. They did not distinguish between Aβ40 and Aβ42 in this regard, but their findings suggest that Aβ monomers—as opposed to oligomers or fibrils—promoted synapses.
“This is excellent work, and the experiments are very well controlled,” wrote Bart De Strooper of Dementia Research Institute, UK. “It shows in a convincing way that the APP-Swe mutation promotes synaptogenesis and that the increase in Aβ peptide is responsible for this effect.”
De Strooper also agreed with the authors that the observations help to interpret the BACE1 inhibitor side effects. However, he noted that, as familial AD mutations go, APP-Swe is unique. While other ADAD mutations in APP and the presenilins skew Aβ production to larger, more aggregation-prone forms, APP-Swe is the lone familial AD mutation that boosts overall production of Aβ. Therefore, De Strooper believes that the synaptogenic effect of the mutation is unlikely a disease-promoting effect. Rather, the findings suggest that loss of Aβ, due to its aggregation, could be pathologically relevant.
“The big question that remains is, what is the best therapeutic target?” asked De Strooper. “The relative loss of Aβ peptide might reduce synaptic function but the gain of toxic effects, due to amyloid peptide aggregates and amyloid plaques causing glial pathology, might also drive the neurons also into dysfunction and cell death,” he added.
“This is a convincing and exciting study providing intriguing new evidence for Aβ-dependent effects on synapses and endosomes,” commented Gunnar Gouras of Lund University in Sweden. “Nevertheless, how does this enhancement in synapse number and endosome size from the APP Swedish mutation relate to what occurs in AD where synapses are lost?” Gouras asked. He noted that human stem-cell-derived neurons cannot account for the effects of aging that occur in the human brain, making it difficult to know if Aβ might influence synapses differently with age (see full comment below).
How to explain the findings in light of other studies that have deemed Aβ toxic to synapses? For one, the authors noted that many animal and cell culture studies were performed with high concentrations of Aβ. Furthermore, they acknowledged decades of work indicating oligomeric forms of Aβ are neurotoxic, while monomeric forms are beneficial. They agreed that oligomeric Aβ may only cause harm after it accumulates over decades.
Of course, the latter statement is a key tenant of the amyloid cascade hypothesis formulated by Selkoe and others. Selkoe noted that Zhou’s findings are quite consistent with previous studies from his lab and others, in which soluble Aβ dimers/oligomers isolated from the AD brain unleashed a bevy of pathogenic events in cultured neurons, whereas Aβ monomers isolated in the same experiments had no deleterious effects (Jin et al., 2011). “The new data from Zhou et al. rigorously extend this concept in a physiologically relevant system,” Selkoe wrote.
In an email to Alzforum, Südhof was reticent to speculate about what the findings imply about AD pathogenesis. “Ours is a basic cell biology study that unbiasedly examines the effect of an APP mutation and Aβ on human synapses at physiological levels, but does not intend to construe any disease mechanisms,” he wrote. “Of course, observing such dramatic effects of a familial Alzheimer's disease mutation on synapses is relevant for Alzheimer's disease, but understanding what the results mean for the disease will require additional work.”—Jessica Shugart
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