See Q&A below with David Holtzman
23 September 2011. In many neurodegenerative diseases, pathologic proteins present tough therapeutic targets because they are intracellular—or at least scientists have assumed they were. New evidence from in-vivo microdialysis studies now suggests that at least some of these cytosolic proteins routinely get out of cells in healthy brains. Researchers led by David Holtzman at Washington University in St. Louis, Missouri, developed a microdialysis technique for measuring soluble tau levels in the interstitial fluid (ISF) of awake, active mice. In the September 14 Journal of Neuroscience, the scientists report that the ISF of both wild-type and tau transgenic mice contains soluble tau. Tau levels were much higher in ISF than in cerebrospinal fluid (CSF); surprisingly, the two were uncoupled. High CSF tau levels are widely used as a biomarker of Alzheimer’s disease (see, e.g., ARF related news story). Although it is not yet clear how tau gets out of cells, or what effect extracellular tau has on the brain, one encouraging implication of the finding is that tau might be more accessible to therapies than scientists have thought.
Tau is not the only cytosolic protein to escape from cells into the ISF. Researchers led by Kostas Vekrellis at the Academy of Athens, Greece, used a similar in-vivo microdialysis technique to measure extracellular concentrations of α-synuclein, the major component of the Lewy bodies found in Parkinson’s disease and some dementias. The results, published online July 14 in PLoS One, showed α-synuclein in the ISF of wild-type and transgenic mice. Importantly, Vekrellis and colleagues also found the protein in ISF collected from people with brain injuries. Although such experiments are in early days yet, the tau and synuclein results hint that cytosolic proteins leaking into the ISF could be a common phenomenon.
Scientists have just begun to tap the potential of in-vivo microdialysis, with but a handful of such studies to date. Holtzman’s group used the technique to examine the dynamics of soluble and insoluble Aβ in AD mice (see ARF related news story). With colleagues elsewhere, they have also measured ISF Aβ in injured human brains, finding that Aβ levels rise as brains recover, and suggesting its secretion is part of normal physiology (see ARF related news story on Brody et al., 2008). A prior study has looked at tau: Researchers at Uppsala University Hospital, Gothenburg, Sweden, detected high levels of both interstitial tau and Aβ in people receiving microdialysis after brain injury (see Marklund et al., 2009). Because this was done in injured brains, as was the α-synuclein study, it could not address the question of whether extracellular tau is present in healthy brains.
To get at this question, Holtzman and colleagues used a mouse model. First author Kaoru Yamada adapted the Aβ microdialysis technique for use with tau. As monomeric tau is larger than Aβ, about 50 kDa instead of four kDa, Yamada used a membrane with a pore size of 100 kDa. With such large pores, fluid diffuses from the probe and builds up in the animal’s brain, a hurdle the scientists overcame by using a push-pull pump and a high concentration of albumin protein in the probe to increase the osmotic pressure. After perfecting the method in vitro, the authors slid the tiny probe, 0.5 to 1 millimeter long, through a guide cannula into the mouse hippocampus. Animals remained awake and freely moving around the cage while fluid flowed through the tubing and sampled ISF proteins over the course of two days.
Yamada and colleagues measured tau levels in the collected fluid by enzyme-linked immunosorbent assay (ELISA). The authors found 45 ng/ml soluble tau in the ISF of three-month-old wild-type mice. They found about five times that in young transgenic tau P301S mice, consistent with the five times higher expression level of the protein in these mice. This showed that extracellular tau is present in healthy mouse brain and implied that the assay accurately reflects total soluble tau levels. One limitation of the microdialysis technique is that it captures only monomeric tau, not larger aggregates. In wild-type animals, ISF tau levels were around 10-fold higher than CSF levels.
Next, the authors checked what happens with age. In wild-type mice, tau ISF levels stayed the same, but in the P301S animals, levels began to drop around six months of age, when tau starts aggregating. Conversely, CSF tau levels increased with age in the transgenics. Surprisingly, the ISF and CSF changes did not correlate. The reason is not clear, but it suggests ISF and CSF pools are independently regulated, the authors note. This parallels findings for Aβ, where mouse ISF and CSF levels have also been shown to be unlinked (see Cirrito et al., 2003). The finding that monomeric ISF tau drops when aggregates emerge implies that the two forms might be in equilibrium. Indeed, when the authors injected tau aggregates into the brains of young transgenic mice, soluble tau dropped by 30 percent, suggesting it was getting sequestered onto aggregates.
Microdialysis has several potential applications, Holtzman told ARF. The technique could help to screen the efficacy of tau-lowering treatments. Because researchers could follow change over time, “The pharmacodynamic assessment is potentially much richer” than current methods, Holtzman told ARF. Microdialysis could also provide insight into the normal behavior of tau in the brain, Holtzman suggested. For example, researchers could study how tau gets out of cells by using reverse microdialysis to infuse a drug that inhibits exocytosis and see if that changes tau levels.
Henrik Zetterberg at Sahlgrenska University Hospital in Molndal, near Gothenburg, Sweden, a coauthor on the earlier human tau microdialysis study, suggested that physiological release of tau might be a consequence of synaptic remodeling, noting that tau levels are very high in newborn CSF. “Personally, I believe that extracellular tau in the absence of pathological processes may reflect neuroaxonal plasticity,” Zetterberg wrote to ARF (see full comment below).
One of the crucial questions is what extracellular tau does in the brain. Work from several groups, including Marc Diamond at Washington University and researchers led by Michel Goedert at MRC Laboratory of Molecular Biology, Cambridge, U.K., has shown that misfolded tau can propagate from cell to cell, helping disease spread across the brain (see, e.g., ARF related conference story; ARF related news story; and ARF news story). Extracellular tau could be a culprit in this process. To investigate this, Holtzman’s group is working on adapting microdialysis to detect molecules bigger than 100 kDa and collaborating with Diamond to look for tau aggregates in ISF. If extracellular tau spreads pathology, the good news is that it may also provide an accessible target for tau-lowering therapies. This might help explain preliminary reports that passive and active immunization strategies lower tau pathology in mice (see ARF related conference story and ARF conference story).
The tau findings jibe with those of other proteins implicated in neurodegenerative disease that also seem to spread between cells. For example, deposits of α-synuclein can migrate from diseased tissue into healthy grafts (see ARF related news story; ARF related conference story; and ARF news story). In addition, Aβ has been shown to spread through the brain (see ARF related news story and ARF news story), as does Huntingtin protein (see ARF news story).
In their paper, Vekrellis and colleagues report detecting α-synuclein in the ISF of healthy mouse brains. Vekrellis initially presented this work at the AD/PD 2011 conference in Barcelona, Spain, this past March. First author Evangelia Emmanouilidou used a 100 kDa microdialysis pore size to capture the protein. Although the synuclein monomer is 14 kDa, recent studies suggest the protein may have a native tetrameric form (see ARF related news story), which would be around 56 kDa. After developing an ELISA sensitive between 0.01 ng/ml to 25 ng/ml of α-synuclein, the Greek scientists found about 0.15 ng/ml of ISF α-synuclein in wild-type mice, and about three times that in A53T mutant α-synuclein transgenic mice, matching their threefold overexpression. Extending the study to people, the scientists examined ISF samples from eight patients with severe brain injuries who received microdialysis as part of standard medical monitoring. They found α-synuclein concentrations varying from 0.5 to 8 ng/ml, showing the protein also gets out in human brain. None of the patients had a diagnosis of Parkinson’s or dementia, and the probe sampled healthy brain tissue, not sites of injury. Even so, synuclein levels could have been affected by overall poor brain health in these patients. Although the effect of extracellular α-synuclein in vivo is unknown, in previous cell culture experiments, the authors showed that secreted α-synuclein lowers neuronal survival (see Emmanouilidou et al., 2010).—Madolyn Bowman Rogers.
Yamada K, Cirrito JR, Stewart FR, Jiang H, Finn MB, Holmes BB, Binder LI, Mandelkow EM, Diamond MI, Lee VM, Holtzman DM. In vivo microdialysis reveals age-dependent decrease of brain interstitial fluid tau levels in P301S human tau transgenic mice. J Neurosci. 2011 Sep 14;31(37):13110-7. Abstract
Emmanouilidou E, Elenis D, Papasilekas T, Stranjalis G, Gerozissis K, Ioannou PC, Vekrellis K. Assessment of α-synuclein secretion in mouse and human brain parenchyma. PLoS One. 2011;6(7):e22225. Abstract
Q&A With David Holtzman
ARF: Why measure interstitial fluid (ISF) tau in mice?
Holtzman: To understand the normal or disease function of a protein, I think it is really important to understand its metabolism. What controls its level? This technique should allow us to make measurements we were never able to make before in the brain of a living animal. The field has not previously been able to make in-vivo brain tau measurements dynamically. Tau is critically important in several diseases, and previous work on Aβ microdialysis led us to believe we can do this for tau, too.
ARF: How did you make the technique work for tau?
Holtzman: We knew the Aβ method would not work for tau. Because tau is a much bigger protein than the microdialysis probe cutoff size, there was no way it would go through the membrane. We obtained probes that have larger cutoff sizes to see if it was feasible. It took a while to get it to work even just in a test tube. We (Kaoru Yamada, post-doctoral fellow) also had to modify the constituents of what was flowing through the microdialysis probe itself. We used albumin to make sure tau did not stick to the plastic. We realized with tau, if you increase the concentration of albumin, you get much better recovery of the protein. That was the biggest trick other than the size. When we went in vivo, the other big adaptation was that we realized we had to use a different pump. With a normal pump you just push fluid through the probe and collect what drips out at whatever rate you set the machine at. If the molecular cutoff is too big, you get a positive flow through the brain, but if you use a push-pull pump, you do not get pressure building up in the brain. The brain, because of the skull, cannot tolerate any increase in pressure.
ARF: What did you find?
Holtzman: The concentration of tau in the extracellular space was a lot higher than in human cerebrospinal fluid (CSF). That was the first big surprise. In human CSF, tau is around 250 pg/ml in a normal person. In wild-type mice, ISF tau is 50 ng/ml, 200 times higher. We thought, “This cannot be right.” But it is true; we have now done many measurements in many mice.
I think what is likely happening is that when proteins get out of the cell, they are getting retained in the brain. There are lots of sticky things in the extracellular space of the brain. Proteins are sticking to membranes, to extracellular matrix, and few reach the CSF. That is a guess.
In tau transgenic mice, tau is higher. Yoshiyama et al. had shown in their paper that tau is overexpressed fivefold in the P301S mice (line PS19), and that is exactly what we found in ISF. That told us the method was likely working reasonably well.
ARF: Does tau change with age?
Holtzman: One of the main points is that when we looked at wild-type up to a year of age, there was no change in tau levels, but in transgenic mice, there was a big drop from 250 ng/ml down to 80 ng/ml. No change in tau expression. We think as tau is aggregating, there is a sink of aggregated material, so a lot of the tau does not float freely around because it is in equilibrium with aggregates.
We do not know if tau is in equilibrium with intracellular aggregates or if there are extracellular aggregates. If aggregates spread from cell to cell, there is likely a phase where they are in the extracellular space before they get into the next cell. We would like to assess if there are extracellular aggregates. If you inject aggregates into the brain, it causes an immediate drop in ISF tau.
ARF: What forms does microdialysis measure?
Holtzman: Not aggregates, probably. I do not know how it could measure even a dimer. In Western blots, it looks like we measure a monomer of full-length tau. The fact that tau level goes down with age in transgenic mice implies there is a change in the aggregation state, and we keep detecting the monomer.
I was very surprised to see that tau went down with age. I thought that, as the mice develop neurodegeneration as they get older, tau would go up as the cells were being damaged. But remember, the transgenic mice are a model for frontotemporal dementia. In human frontotemporal dementia, tau does not go up, at least in CSF. In retrospect, maybe it was not surprising that we saw nothing going on.
ARF: Why does CSF tau go up in AD but not in frontotemporal dementia?
Holtzman: I have no answer yet. I have thought about that a lot. One would think if the elevation of CSF tau in AD was due to the presence of neurofibrillary tangles or tau aggregates, then tau should be elevated also in frontotemporal dementias. But that is not the case. While the amount of CSF tau in AD correlates with the amount of neurofibrillary pathology, it also correlates with amyloid deposition. I wonder if the reason tau is elevated higher in AD than in frontotemporal dementia has to do with the amount of neuritic degeneration. I wonder whether it is due to smoldering injury, that tau release from synaptic regions is higher. There is a fair amount of that kind of injury that is occurring in an ongoing fashion in AD. But that is just speculation.
ARF: Tau, unlike Aβ, is a cytoplasmic protein. It is not released.
Holtzman: Yes, but with cytoplasmic proteins, if you look carefully in CSF you can find them. Most of them have been described to be in CSF. These are abundant cytoplasmic proteins in normal people like GFAP, SOD. To me it is unlikely it has to do with a normal function of the proteins. But they do get out. Maybe our cells are normally leakier than we thought.
The concentrations of these proteins inside cells are much higher. It is probably orders of magnitude higher inside, both for tau and α-synuclein.
ARF: Are you doing ISF microdialysis in humans?
Holtzman: Not right now. We are just interested in further understanding the biology of proteins like tau. What regulates it normally?
ARF: What are the therapeutic implications?
Holtzman: It is exciting: Some people want to lower tau as a potential treatment. This would be a nice method to screen treatments, just like we have done for Aβ. For example, if you knock down tau expression with short interfering RNA, do you see ISF tau levels go down, and how fast does that happen? Days? Hours?