26 Nov 2014

For more than a decade, scientists have used microdialysis to estimate how the concentration of Aβ in the brain’s interstitial fluid changes in response to synaptic activity. Unfortunately, the technique allows sampling every half-hour at most, but neurons fire much more frequently. “We were orders of magnitude off the timescale for Aβ generation and release,” said John Cirrito, Washington University in St. Louis. Cirrito spoke to an audience at the Society for Neuroscience annual meeting, held in Washington, D.C., November 15 to 19. To close that gap, Cirrito’s group has developed a carbon fiber electrode that measures the concentration of Aβ every 30 seconds. This micro-immunoelectrode specifically binds to Aβ and oxidizes the peptide’s lone tyrosine, detecting the electrons released as current. Using this new technique, scientists will be able to observe up-to-the-minute Aβ fluctuations.

In regular microdialysis, researchers insert a 2-mm long, 300-μm diameter probe into the brain, where it collects Aβ that crosses its semi-permeable membrane. An ELISA then measures the concentration of Aβ every 30 to 60 minutes (see Cirrito et al., 2003). When it debuted, this technique gave scientists their first opportunity to measure the concentration of Aβ over time in the same mouse. When working at David Holtzman's lab at WashU, Cirrito and colleagues found that, on average, Aβ production rose when synapses were excited, and fell when they were quieter (see Dec 2005 news story). 

A few years later, Cirrito caught a presentation by Chenzhong Li, Florida International University, Miami, about an antibody-tipped microelectrode that could rapidly detect vascular endothelial growth factor coming from tumor cells (see Prabhulkar et al., 2009). After visiting Li’s lab, Cirrito realized that the same technique could speed up detection of Aβ.

Together, Li and Cirrito developed a 5-μm diameter micro-immunoelectrode that varies between 20 and 50 μm in length (see image above). It is five orders of magnitude smaller than the probe used in microdialysis, about half the size of a neuron. A previous publication described its first use in vitro (see Prabhulkar et al., 2012). Carla Yuede, who works in Cirrito's lab, helped adapt it for use in vivo. Inserted into the hippocampus of an anesthetized APP/PS1 transgenic mouse, the microelectrode, charged to 0.65 volts, oxidized the tyrosine residue on the human Aβ, capturing the electrons released. “The amount of current generated is directly proportional to the number of tyrosines that bind the electrode,” said Cirrito. Because rodent Aβ lacks this tyrosine, the micro-immunoelectrode picks up only human Aβ, he told Alzforum. 

To ensure that the electrode detects Aβ specifically, the tip is coated with an anti-Aβ antibody, and any remaining spaces are filled in with bovine serum albumin (see image below). The researchers can substitute different antibodies, depending on whether they want to detect Aβ40 or Aβ42. While these antibodies should be highly specific, they also need to have a reasonably low affinity for Aβ, so as not to bind the peptide too tightly and saturate the electrode immediately. Even so, a given electrode only lasts about 90 minutes. Cirrito said he is still working to figure out why they eventually give out.

So far, Cirrito’s group has conducted initial experiments that refine previous estimates about Aβ production and synaptic activity. For instance, when the researchers injected anesthetized APP/PS1 mice with picrotoxin to block inhibitory GABAergic signaling, Aβ levels rose gradually between three and 15 minutes later, and then stabilized out to 90 minutes, the lifetime of the electrode. Before, the researchers only knew that levels rose, on average, within an hour. In another experiment, Cirrito and colleagues blocked Aβ production with a γ-secretase inhibitor. They calculated that half the Aβ in the interstitial fluid cleared in 40 minutes, slightly faster than the 54-minute half-life estimated from microdialysis.

On her SfN poster, Yuede further broke down the clearance measurements. She found that when mice were injected with a γ-secretase inhibitor, Aβ dropped in two phases: for the first 15 minutes, the peptide cleared rapidly, but for the next hour, disappeared at a slower rate. The researchers predict that these two phases correspond to different mechanisms of clearance from the brain. For instance, when Yuede blocked p-glycoprotein, a protein thought to pump Aβ through the blood-brain barrier, the first phase stayed the same, but the second was slower, suggesting that p-glycoprotein handles the slower clearance mechanism. “We do not know yet what is responsible for the faster clearance,” Cirrito told Alzforum. Since research suggests that people with Alzheimer’s disease clear Aβ more slowly from the brain (see Jul 2010 news story), clearance mechanisms may be important to target for therapeutics. “If you want to enhance clearance to help AD, you may want to prioritize one pathway over another,” said Cirrito.

This technique won’t replace microdialysis, said Cirrito, but it will give researchers another option if they want to measure rapid Aβ fluctuations. For example, previous research by Holtzman’s lab suggests that Aβ production wanes during sleep and ramps up again during wakefulness (see Kang et al., 2009). This technique will allow researchers to see how quickly those changes take place, and whether a lag phase follows sleeping or waking, Cirrito said.

Researchers praised this as a “fantastic” new methodology. “This will revolutionize the analysis of interstitial fluid levels of Aβ,” Gary Landreth, Case Western Reserve University, Cleveland, wrote to Alzforum. The real-time measurements it allows represent a vast improvement over the more cumbersome microdialysis. “One of the most compelling aspects of this work is that it should be applicable to other analytes which have an appropriately placed tyrosine and for which there is a good antibody,” wrote Landreth. Cirrito's group has already developed a prototype electrode that uses an anti-α-synuclein antibody, and researchers have expressed interest in developing it for tau. The release of both peptides may depend on synaptic activity (see Fortin et al., 2005; Yamada et al., 2014).

At Cirrito’s presentation, Miranda Reed, West Virginia University, asked whether different types of anesthesia could influence Aβ measurements from the micro-immunoelectrode. Cirrito responded that he had no comparative data to present yet, but expected it might. Sleep promotes glymphatic clearance of solutes, including Aβ, from the brain (see May 2014 news story). In the future, Cirrito hopes to perform measurements in awake mice and avoid problems with anesthesia altogether. In answer to another audience question, Cirrito explained that the 30-second time window for the electrode was necessary to enable enough Aβ to bind and give a readable signal, but not so long that the all the available antibodies would become laden with unoxidized Aβ.—Gwyneth Dickey Zakaib

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References

News Citations

1. Paper Alert: Synaptic Activity Increases Aβ Release 22 Dec 2005
2. Honolulu: Wake-Up Call—Aβ Clearance, Not Production, Awry in AD 26 Jul 2010
3. Glymphatic Flow, Sleep, microRNA Are Frontiers in Alzheimer’s Research 6 May 2014

Research Models Citations

1. APPPS1

Paper Citations

1. Cirrito JR, May PC, O'Dell MA, Taylor JW, Parsadanian M, Cramer JW, Audia JE, Nissen JS, Bales KR, Paul SM, Demattos RB, Holtzman DM. In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life. J Neurosci. 2003 Oct 1;23(26):8844-53. PubMed.
2. Prabhulkar S, Alwarappan S, Liu G, Li CZ. Amperometric micro-immunosensor for the detection of tumor biomarker. Biosens Bioelectron. 2009 Aug 15;24(12):3524-30. Epub 2009 May 13 PubMed.
3. Prabhulkar S, Piatyszek R, Cirrito JR, Wu ZZ, Li CZ. Microbiosensor for Alzheimer's disease diagnostics: detection of amyloid beta biomarkers. J Neurochem. 2012 Feb 28; PubMed.
4. Kang JE, Lim MM, Bateman RJ, Lee JJ, Smyth LP, Cirrito JR, Fujiki N, Nishino S, Holtzman DM. Amyloid-beta dynamics are regulated by orexin and the sleep-wake cycle. Science. 2009 Nov 13;326(5955):1005-7. PubMed.
5. Fortin DL, Nemani VM, Voglmaier SM, Anthony MD, Ryan TA, Edwards RH. Neural activity controls the synaptic accumulation of alpha-synuclein. J Neurosci. 2005 Nov 23;25(47):10913-21. PubMed.
6. Yamada K, Holth JK, Liao F, Stewart FR, Mahan TE, Jiang H, Cirrito JR, Patel TK, Hochgräfe K, Mandelkow EM, Holtzman DM. Neuronal activity regulates extracellular tau in vivo. J Exp Med. 2014 Mar 10;211(3):387-93. Epub 2014 Feb 17 PubMed.

Further Reading

Papers

1. Bero AW, Yan P, Roh JH, Cirrito JR, Stewart FR, Raichle ME, Lee JM, Holtzman DM. Neuronal activity regulates the regional vulnerability to amyloid-β deposition. Nat Neurosci. 2011 Jun;14(6):750-6. Epub 2011 May 1 PubMed.
2. Kang JE, Cirrito JR, Dong H, Csernansky JG, Holtzman DM. Acute stress increases interstitial fluid amyloid-beta via corticotropin-releasing factor and neuronal activity. Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10673-8. PubMed.

News

1. Link Between Synaptic Activity, Aβ Processing Revealed 15 Apr 2008
2. Brain Microdialysis Reveals Tau, Synuclein Outside of Cells 23 Sep 2011
3. Soluble Aβ—Bane or Boon? Real-time Data in Humans Yield New Insight 2 Sep 2008
4. Brain Activity and Aβ—The Interstitial Plot Thickens 10 Aug 2011
5. Sleep Deprivation Taxes Neurons, Racks Up Brain Aβ? 25 Sep 2009