24 Mar 2004

As part of the tangled web of pathology that leads to Alzheimer’s disease, inflammation is associated with local immune responses. For example, recent data indicate that both AD patients and elderly healthy people have circulating Aβ-reactive T cells, despite an age-related decrease in T cell priming (Monsonego et al., 2003). These and other unusual immunologic responses beg further investigation, and two papers by German scientists in the current issue of the Journal of Neuroscience are tying to do just that. One study combines laser microdissection with quantitative PCR to explore the possible role of brain-derived neurotrophic factor (BDNF) in the inflammatory reactions surrounding amyloid deposits. The other uses two-photon microscopy to observe as marauding T cells wreak havoc upon the brain.

First, the BNDF paper. Why study this cytokine? Widely expressed in the brain, this neurotrophic protein is generally thought of as beneficial, and as decreasing in AD brain (see ARF Live Discussion). However, it has recently been connected to immune processes, and some papers describe its association with amyloid plaques, suggesting it might be detrimental. In an attempt to clarify contradictory data about BDNF’s role in inflammation, Guido Burbach, Thomas Deller, and colleagues at J. W. Goethe University in Frankfurt analyzed BDNF mRNA and protein levels in and immediately around plaque-burdened brain tissue from APP23 transgenic mice , and compared them with levels at a distance from plaques and in control animals. In brief, they found an upregulation of BDNF near plaques and suggest that BDNF may modulate inflammatory processes.

In examining cortical sections from aged APP23 mice, both immunostaining and in-situ hybridization signaled a BDNF mRNA expression gradient that radiated out from amyloid plaques into surrounding plaque-free tissue. To verify these results using a different technique called laser microdissection, the researchers dissected individual plaques and subjected them to quantitative PCR (qPCR). They observed a sixfold and threefold higher BDNF mRNA expression within amyloid plaques and in the plaques’ vicinity, respectively. Immunostaining for glial markers and BDNF revealed BDNF-positive glial cells associated with plaques, and BDNF-negative glia in plaque-free areas, again indicating a gradient of BDNF expression. Furthermore, other BDNF-positive balloon-like structures resembled dystrophic axonal boutons. The authors point to glial cells as the main source of this plaque-associated BDNF gradient.

Burbach and colleagues next turned to investigating BDNF protein concentrations in the frontal cortices of APP23 mice. They studied mice at three different ages: at five months (just prior to amyloid deposition), 10.5 months (intermediate amyloid load), and 20 months (severe amyloid load). Both ELISA and Western blot analyses confirmed a 17-fold increase in BDNF levels over control mice in 20-month-old APP23 mice, while five-month-old and 10-month-old mice only exhibited slightly elevated BDNF levels. Moreover, these increased BDNF levels correlated with increases in Aβ40 and Aβ42 concentrations, suggesting to the authors that amyloid deposition leads to increases in BDNF protein concentration. In addition, CSF of APP23 mice lacked detectable BDNF levels, indicating that these observed changes in BDNF are local rather than global.

The biological relevance of plaque-associated increases in BDNF remains elusive. The authors argue that the late-stage and general decrease in BDNF levels reported by other groups may be masking plaque-associated and local increases of this cytokine. Furthermore, they suggest that glial BDNF expression links BDNF to the inflammatory response and speculate that it possesses an immunomodulatory function, since it is known to inhibit major histocompatibility complex (MHC) molecules, which present antigen to T lymphocytes.

A Lethal Touch
A key pathogenic event in neuroinflammatory disease is the trafficking of T cells to their site of action. The second study summarized here examines T cells that infiltrate the brain of a mouse model for multiple sclerosis. In the same issue of the Journal of Neuroscience, Robert Nitsch, Frauke Zipp, and colleagues at the Charité Hospital of Humboldt University in Berlin report that not only do T cells damage myelin and oligodendrocytes during neuroinflammation (as is well-known), but they also inflict neuronal damage during their march to these sites. Specifically, the scientists found that T cells that make contact with neurons trigger a lethal rise in intracellular calcium in them.

Play movie [.avi]

Hippocampal neurons in cultured brain slices contain physiological levels of calcium (green). Addition of glutamate induces a transient increase in intracellular calcium, as indicated by the sudden rise in fluorescence.

Play movie [.avi]

In this MHC mismatch experiment, PLP-specific T cells (red) obtained from SJL/J (H-2s) mice make contact with neurons (green) as they move through hippocampal slices from B10.PL (H-2u) mice. This kiss of death induces high, oscillating levels in intracellular calcium, to which the neuron eventually succumbs.

Using brain slices from SJL/J mice (which suffer from experimental autoimmune encephalomyelitis, or EAE), the researchers used a two-photon confocal microscope to track T cells as they invaded the tissue. They generated in the mice T cells specific to proteolipid protein (PLP) and T cells specific to non-murine protein ovalbumin (OVA), and labeled both populations with the red fluorescent tracer 4-chloromethyl benzoyl amino tetramethyl rhodamine (CMTMR). Next, Nitsch and colleagues monitored the invasion of the T cells into the brain tissue and noted that they mimicked an antigen search strategy characteristically exhibited in the lymph nodes.

How, then, does this army of T cells affect the neurons it encounters during this search? To answer this question, Nitsch and colleagues incubated the brain slices in Fluo-4, a calcium- sensitive dye, again unleashed labeled T cells onto the slices, and watched the T cells make contact with neurons in the brain parenchyma. Sixty percent of all sustained T cell contacts with neuronal dendrites resulted in an irreversible rise in neuronal intracellular calcium, in some cases, up to 80 percent above controls. To confirm that this rush of calcium was lethal to the neurons, the researchers visualized dead neurons located adjacent to T cells used with propidium iodide.

The scientists were able to inhibit the T cells’ lethal power by blocking perforin (a membrane-puncturing protein found in the cytoplasmic granules that activated T cells release) and with glutamate receptor antagonists, but not by MHC mismatch. This suggested to the authors that both PLP and OVA T cells can damage bystanding neurons as they invade tissue, independent of MHC. It also suggests that glutamatergic network activity is involved in T cell-induced calcium overload and eventual death. What’s more, since activated T cells are thought to release glutamate themselves, the authors propose that this released glutamate acts through the glutamatergic network of the neurons the T cells encounter.

Overall, the authors draw attention to the collateral damage caused by T cells, and point to a need for new therapies that protect neurons from the lymphocyte infiltration that is common in neuroinflammatory diseases. In other words, the journey itself might be as important as the final destination.—Erene Mina.

Play movie [.avi]

T cells (red) on the move elongate their ovoid bodies in an hourglass manner. They often stop abruptly and travel in the opposite direction at a velocity dependent on the temperature of the hippocampal slice.

 

Comments

1. • Jacob Mack
     bio-chemistry-psychology/neuroscience graduate.
   • Posted: 04 Feb 2005
   • Paper: Increased T cell reactivity to amyloid beta protein in older humans and patients with Alzheimer disease.

   Although it is true that immune response, and the resulting higher levels of selective immunoglobulins, are present in AD, this can only (and at best) imply immune system components. We cannot jump the gun on proposing an autoimmune response. It may be wiser to hypothesize that inflammation is a result of (not genesis of) Alzheimer's disease.

   Furthermore, I did not see enough relevant research on ligand binding as an underlying cause of overaccumulation of amyloid beta protein or the link between genectics and amyloid precursor protein.

   View all comments by Jacob Mack
2. • Vijendra Singh
     
   • Posted: 08 Feb 2005
   • Paper: Increased T cell reactivity to amyloid beta protein in older humans and patients with Alzheimer disease.

   This is a very good paper for increased T cell reactivity in Alzheimer disease, but the authors failed to refer to previous research, including our own report of abnormal lymphocyte proliferation by amyloid protein-β in AD patients (Singh, 1994). I wish people would read up on the previous literature on faulty immune regulation and autoimmunity in AD (Singh, 1997).

   Dr. Jacob Mack's point might be well taken, but the most important thing is the "immune activation" that precedes inflammation and/or autoimmunity. Both are directly relevant to a cell-mediated autoimmune mechanism of pathogenesis in AD, which we proposed previously (see reference below). And Dr. Howard Weiner's group is now finding experimental evidence in favor of this hypothesis. Unfortunately, because nobody was giving us funding, we had to stop working on the immunopathogenesis of AD. So, I convey my heartfelt good wishes to those who are continuing immunology-autoimmunity research in AD, especially since nobody knows what causes AD.

   To that end, thinking outside the box would help us find some new clues.

   References:

   Singh VK. Studies of neuroimmune markers in Alzheimer's disease. Mol Neurobiol. 1994 Aug-Dec;9(1-3):73-81. PubMed.

   Singh VK. Immune-activation model in Alzheimer disease. Mol Chem Neuropathol. 1996 May-Aug;28(1-3):105-11. PubMed.

   Singh VK. Neuroautoimmunity: pathogenic implications for Alzheimer's disease. Gerontology. 1997;43(1-2):79-94. PubMed.

   View all comments by Vijendra Singh

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References

Paper Citations

1. Monsonego A, Zota V, Karni A, Krieger JI, Bar-Or A, Bitan G, Budson AE, Sperling R, Selkoe DJ, Weiner HL. Increased T cell reactivity to amyloid beta protein in older humans and patients with Alzheimer disease. J Clin Invest. 2003 Aug;112(3):415-22. PubMed.

Other Citations

1. ARF Live Discussion

Further Reading

No Available Further Reading