The peptide Aβ42 builds up, misfolds, and aggregates in Alzheimer’s disease, but scientists still don’t understand its normal role in the cell. Super-resolution microscopy offers a new clue. Packets of the peptide turn up at the presynapses of both mature and immature healthy hippocampal neurons, according to results published June 14 in Life Science Alliance. Since the machinery needed to make Aβ42 also lurks in the presynapse, the results imply that the peptide may help develop and maintain neuronal connections, said authors led by Sophia Schedin-Weiss and Lars Tjernberg, Karolinska Institutet, Huddinge, Sweden.

  • Super-resolution microscopy exposes pockets of Aβ42 in neurons.
  • Aβ42 vesicles sit in presynapses of hippocampal neurons.
  • Does this mean Aβ42 helps develop and maintain synapses?

Scientists had previously localized Aβ42 to the synapse (Takahashi et al., 2002). However, even the best confocal microscopy, with a resolution of 200 nm, cannot resolve the tiny synaptic cleft, so researchers couldn’t tell on which side the peptide was sitting. With super-resolution microscopy techniques, researchers can breach the 200 nm diffraction limit of confocal microscopy and resolve structures as small as 20 nm—the size of the synaptic cleft (Jan 2009 news). 

“This interesting study uses incredible resolution to assess the localization of Aβ,” wrote John Cirrito, Washington University in St. Louis, to Alzforum (full comment below). “It supports data from our group and others that a lot of Aβ is made presynaptically.”

Choosing Sides.

At left, Aβ42 (red) comingles with presynaptic marker synaptophysin (green); at right, the peptide keeps more distance from postsynaptic protein PSD95 (cyan). Axons and dendrites are labeled in white. [Courtesy of Yu et al., 2018.]

First author Yang Yu and colleagues used two super-resolution microscopic methods—stimulated emission depletion (STED) microscopy and stochastic optical reconstruction microscopy (STORM)—to image cultured and fixed hippocampal neurons from embryonic mice. They labeled the presynapse, postsynapse, and neurites with fluorescent labels for synaptophysin, PSD95, and actin filaments, respectively. They then labeled Aβ42 with an antibody specific for the C-terminal end of the peptide, which bound neither Aβ40 nor APP. 

What emerged in these young mouse neurons was a picture of Aβ42 in two main spots—an abundance of small vesicles at the presynapse, and sparser, larger vesicles in neurites. The smaller ones appeared among vesicles containing the presynaptic marker synaptophysin, and they were clearly separate from postsynaptic marker PSD95 (see images above). Curiously, at the presynapse, Aβ42 and synaptophysin overlapped in some but not all of the vesicles.

Less prevalent were larger Aβ42-containing vesicles in axons and dendrites located farther away from synapses (see image below). Similar to the multivesicular bodies seen via electron microscopy in a previous study by Gunnar Gouras of Lund University, Sweden, Aβ42 seemed to be concentrated at the outer edges of these structures (Takahashi et al., 2002). 

See the Orbs?

Large vesicles containing Aβ42 (yellow arrow) appear in neurites away from the synapses, with higher concentrations toward the rim. [Courtesy of Yu et al., 2018.]

In a prior study, Schedin-Weiss and colleagues found that γ-secretase also hides out at the presynapse (Schedin-Weiss et al., 2016). To these researchers, this suggests that Aβ42 is playing a functional role at the synapse. “We think it’s unlikely that the machinery for producing and secreting Aβ42 are in the same place by coincidence,” Tjernberg told Alzforum. “The peptide may have a function there.”

Synaptophysin marks vesicles that release neurotransmitters upon activation. Since synaptophysin was absent from some but not all Aβ42-containing vesicles, it’s possible that Aβ42 comes in two different types of vesicle, some released upon neuronal activity, and some not, the authors suggest. That could explain why studies find both activity-dependent and -independent release of Aβ42 (Dec 2005 news; Lundgren et al., 2014). 

Cirrito was more cautious about this point. Synaptophysin may simply have gone undetected in some Aβ42 vesicles of the presynapse. “That there could be two types of Aβ-containing vesicles is intriguing, but I believe still not conclusive,” he said. Similarly, some Aβ could exist postsynaptically, as has been suggested in other studies (Wu et al., 2011). 

Gouras, who was not involved in the study, found the results convincing. “Whether Aβ42 is found at the pre- or postsynapse has been a big question in Alzheimer’s disease research,” he said, adding, “It is exciting and new that they clearly see endogenous Aβ in the presynapse.” Gouras predicts that the vesicles at the presynapse will turn out to be endosomes, which would align with his prior electron microscopy results. He thinks it’s plausible that Aβ42 may function at the synapse, but cautions that more data is needed.

The authors note that the neurons they observed were from young mice, not from humans, much less from old or Alzheimer’s brains. The cultured neurons also lacked glial cells and other components of a normal neuronal environment. Nevertheless, observing neurons and their synapses gives some insight about where Aβ42 appears in healthy cells and what it might be doing, Tjernberg said.

Next, the researchers want to film living cells with super-resolution microscopy to get a moving picture of Aβ42 trafficking. The peptide might turn up in other places, Tjernberg said. He also plans to repeat these studies with even more powerful microscopes to better characterize the Aβ42-containing vesicles at the presynapse. —Gwyneth Dickey Zakaib

Comments

  1. This is an interesting study using incredible resolution to assess localization of Aβ. It supports data from our group and others that a lot of Aβ is made presynaptically. Our in vivo data suggest that 70 percent of Aβ (40 and 42) found in the interstitial fluid is made via presynaptic mechanisms.

    The imaging methods used here are very good at demonstrating where Aβ42 is located, but I think it is tough to make conclusions on the negative data regarding location. That some vesicles lack synaptophysin does not necessarily mean it isn’t present; that there could be two types of Aβ-containing vesicles is intriguing, but I believe still not conclusive.

    Similarly, there are other conclusive studies that demonstrate that some Aβ is made postsynaptically. For instance, Paul Worley has a nice paper looking at the role of arc postsynaptically in Aβ generation (Wu et al., 2011).

    My interpretation here is that Aβ42 is enriched presynaptically in these vesicles, but we cannot rule out it is present in other locations that may be below the level of detection.

    References:

    . Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent β-amyloid generation. Cell. 2011 Oct 28;147(3):615-28. PubMed.

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References

News Citations

  1. Method of the Year—Microscopy Advances Take Biology by STORM
  2. Paper Alert: Synaptic Activity Increases Aβ Release

Paper Citations

  1. . Intraneuronal Alzheimer abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology. Am J Pathol. 2002 Nov;161(5):1869-79. PubMed.
  2. . Super-resolution microscopy reveals γ-secretase at both sides of the neuronal synapse. Acta Neuropathol Commun. 2016 Mar 31;4:29. PubMed.
  3. . Activity-independent release of the amyloid β-peptide from rat brain nerve terminals. Neurosci Lett. 2014 Apr 30;566:125-30. Epub 2014 Mar 3 PubMed.
  4. . Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent β-amyloid generation. Cell. 2011 Oct 28;147(3):615-28. PubMed.

Further Reading

Papers

  1. . Analysis of microdissected neurons by (18) O mass spectrometry reveals altered protein expression in alzheimer's disease. J Cell Mol Med. 2011 Aug 30; PubMed.
  2. . ADAM10 and BACE1 are localized to synaptic vesicles. J Neurochem. 2015 Nov;135(3):606-15. Epub 2015 Sep 17 PubMed.

Primary Papers

  1. . Neuronal Aβ42 is enriched in small vesicles at the presynaptic side of synapses. Life Science Alliance, June 14, 2018