I wanted to thank Serge Rivest, Mathias Jucker, Tony Wyss-Coray, Joseph El Khoury, and Pritam Das for their helpful and thought-provoking comments, and to address some of their questions. I find it terribly interesting that the recent report by Richard, Rivest, and colleagues showed spontaneously increased TGF-β expression in immune cells near plaques of Tg APP/TLR2-/- mice. I agree that these striking findings are in line with the interpretation that increased TGF-β1 levels in AD patient brains, as shown by Wyss-Coray, Masliah, Mucke, and colleagues, likely serve the maladaptive role of maintaining an “immune privileged” brain milieu in AD patients and in these transgenic mouse models of the disease. We believe that overcoming this non-productive immune state will likely be key in targeting beneficial immune-mediated clearance of cerebral amyloid—and what better immune cell to target than the blood-borne macrophage (Greek etymology—“big eater”)? We also agree with Joseph El Khoury that a key aspect of this therapeutic modality will be promoting the Aβ...  Read more

I wanted to thank Serge Rivest, Mathias Jucker, Tony Wyss-Coray, Joseph El Khoury, and Pritam Das for their helpful and thought-provoking comments, and to address some of their questions. I find it terribly interesting that the recent report by Richard, Rivest, and colleagues showed spontaneously increased TGF-β expression in immune cells near plaques of Tg APP/TLR2-/- mice. I agree that these striking findings are in line with the interpretation that increased TGF-β1 levels in AD patient brains, as shown by Wyss-Coray, Masliah, Mucke, and colleagues, likely serve the maladaptive role of maintaining an “immune privileged” brain milieu in AD patients and in these transgenic mouse models of the disease. We believe that overcoming this non-productive immune state will likely be key in targeting beneficial immune-mediated clearance of cerebral amyloid—and what better immune cell to target than the blood-borne macrophage (Greek etymology—“big eater”)? We also agree with Joseph El Khoury that a key aspect of this therapeutic modality will be promoting the Aβ phagocytosis response while opposing the proinflammatory response, both of which likely exist as a continuum of innate immune cell activation profiles (Town et al., 2005). But, if we can accomplish this, will amyloid-reducing therapies ultimately be successful AD therapeutics? As stated by Dave Morgan and others on this forum, the first test of the amyloid cascade hypothesis of AD in humans will likely be the Aβ vaccine. We anxiously await whether the hypothesis holds up and delivers an efficacious AD therapy. If it does, then the floodgates will open for a whole host of amyloid-targeted AD therapeutics—both immune and non-immune.

About the issue raised by Mathias Jucker and Tony Wyss-Coray of CD11c as a marker for blood-borne innate immune cells/macrophages versus microglia, I should mention that we initially thought that CD11c would be a microglial marker in the context of AD. However, after examining numerous brain sections from various ages of wild-type versus Tg2576 or mutant APP/PS1 doubly transgenic mice for CD11c expression, we concluded that while microglia in the parenchyma around Aβ deposits were CD11b, CD45, MHC II, F4/80 Ag, and CD68 positive, they were negative for CD11c. However, we did observe a small number of round, non-process bearing CD11c positive cells within the lumen of blood vessels in both Tg2576 and APP/PS1 mice, consistent with Stalder and colleagues’ report of invading hematopoietic cells in brains of aged Tg2576 mice. At the time that we were checking for CD11c expression in AD mice, Alon Monsonego and Harold Weiner published a review in Science where they mentioned (as data not shown) that plaque-associated microglia were CD11c positive. I called Alon and asked him about the methodological details. However, after trying various tissue handling techniques, antibodies, and confocal settings, I was unable to reproduce this despite getting microglia in day 20 MOG-EAE brain sections to light up like a Christmas tree with CD11c. I came away thinking that it is possible to acutely activate microglia with the necessary vigor to promote CD11c expression, for example, in the context of EAE. However, I believe that this form of activation does not occur in AD mice, where the profile more closely resembles a chronic, persistent, low-level inflammation.

I have recently read the paper by Bulloch and coworkers with great interest, which shows the presence of CD11c/EYFP “dendritic-like” mouse microglia in multiple stages of life. However, because the authors did not quantify their observations, it is unclear how prevalent these cells are in the brain, and/or whether these cells arose from the blood or were long-term CNS residents. Further, the authors had difficulty in co-staining these cells with CD11c antisera in tissue sections, raising a possibility that those who work with transgenics are all too aware of: expression of transgenes is often more promiscuous than expected. In our study, we demonstrated a seven- to eightfold increase in CD45+CD11b+CD11c+CD68+Ly-6C- cells (presumed “anti-inflammatory” macrophages initially immunophenotyped by Littman’s group in Geissmann et al., 2003) in our crossed mice, and immunohistochemical approaches revealed prominent vascular cuffing, where these cells appeared to be entering the brain via cerebrovessels. Regarding the questions from Joseph El Khoury and Pritam Das about the origin of these brain macrophages, we agree that the “acid test” of whether the macrophage-like cells that we see in and around cerebral vessels and β amyloid plaques arise from the periphery or from within the CNS would either be a chimeric approach or parabiosis. We moved away from the chimeric approach following recent reports in Nature Neuroscience (Ajami et al., 2007; Mildner et al., 2007) showing that the act of irradiating the mice leads to brain infiltration of monocytes/macrophages—the very dependent variable that we are interested in testing. However, we believe that 1) parabiosis of AD mice with GFP+CD11c-DNR mice or 2) chemical methods of ablating hematopoietic cells in AD mice followed by reconstitution with GFP+CD11c-DNR bone marrow containing or depleted of macrophages represent possible strategies that we are currently pursuing.

Finally, Pritam Das raises the interesting questions of the long-term consequences of inhibiting TGF-β signaling on peripheral macrophages and the effects on T cells. We did not observe increased peripheral numbers of innate immune cells (including macrophages and dendritic cells), CD4+ or CD8+ T cells, or B cells in CD11c-DNR mice alone or in Tg2576xCD11c-DNR crossed mice, suggesting that an autoimmune state was not generated and that the increased abundance of macrophages in the brains of our crossed mice was β amyloid-directed. We also quantified T cells in brains of our crossed mice versus singly transgenic animals, and detected that about 4-5 percent of brain hematopoietic cells were TcRαβ positive (presumed T cells), and they were divided about equally between CD4+ and CD8+ subsets—however, these numbers were similar amongst wild-type, CD11c-DNR, APP/PS1, and APP/PS1xCD11c-DNR mice, suggesting that neither the CD11c-DNR nor the APP/PS1 transgenes were able to modify brain entry of T cells. Finally, regarding the issue of assessing neurodegeneration, we are currently pursuing this line of investigation by quantitative synaptophysin immunohistochemistry and hope to answer this question in the near future.

References:
Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FM. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat Neurosci. 2007 Dec;10(12):1538-43. Abstract

Bulloch K, Miller MM, Gal-Toth J, Milner TA, Gottfried-Blackmore A, Waters EM, Kaunzner UW, Liu K, Lindquist R, Nussenzweig MC, Steinman RM, McEwen BS. CD11c/EYFP transgene illuminates a discrete network of dendritic cells within the embryonic, neonatal, adult, and injured mouse brain. J Comp Neurol. 2008 Jun 10;508(5):687-710. Abstract

Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003 Jul;19(1):71-82. Abstract

Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch UK, Mack M, Heikenwalder M, Brück W, Priller J, Prinz M. Microglia in the adult brain arise from Ly-6C(hi)CCR2(+) monocytes only under defined host conditions. Nat Neurosci. 2007 Dec 1;10(12):1544-53. Abstract

Monsonego A, Weiner HL. Immunotherapeutic approaches to Alzheimer's disease. Science. 2003 Oct 31;302(5646):834-8. Abstract

Richard KL, Filali M, Préfontaine P, Rivest S. Toll-like receptor 2 acts as a natural innate immune receptor to clear amyloid beta 1-42 and delay the cognitive decline in a mouse model of Alzheimer's disease. J Neurosci. 2008 May 28;28(22):5784-93. Abstract

Stalder AK, Ermini F, Bondolfi L, Krenger W, Burbach GJ, Deller T, Coomaraswamy J, Staufenbiel M, Landmann R, Jucker M. Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice. J Neurosci. 2005 Nov 30;25(48):11125-32. Abstract

Town T, Nikolic V, Tan J. The microglial "activation" continuum: from innate to adaptive responses. J Neuroinflammation. 2005 Oct 31;2:24. Abstract

Wyss-Coray T, Masliah E, Mallory M, McConlogue L, Johnson-Wood K, Lin C, Mucke L. Amyloidogenic role of cytokine TGF-1 in transgenic mice and in Alzheimer's disease. Nature. 1997 Oct 9;389(6651):603-6. Abstract

View all comments by Terrence Town

I am glad that the researchers studying transgenic models are finally confirming our results published in 2002 (Fiala et al., 2002), which showed transmigration of macrophages across the brain vessel wall and clearance of plaques by these large macrophages.

The migrating macrophages broke through ZO-1 tight junction barrier and aggregated around brain vessels similarly as in HIV encephalitis. This has been followed by a recent publication in PNAS (Fiala et al., 2007). The animal studies cannot resolve the crucial question: are macrophages of patients with AD different from those of control subjects? The answers for interested readers are available in our PNAS article and more current work presented at ICAD. Not only macrophages penetrate across the blood-brain barrier but also clear oligomeric amyloid-β from neurons.

References:
Fiala M, Liu QN, Sayre J, Pop V, Brahmandam V, Graves MC, Vinters HV. Cyclooxygenase-2-positive macrophages infiltrate the Alzheimer's disease brain and damage the blood-brain barrier. Eur J Clin Invest. 2002 May;32(5):360-71. Abstract

Fiala M, Liu PT, Espinosa-Jeffrey A, Rosenthal MJ, Bernard G, Ringman JM, Sayre J, Zhang L, Zaghi J, Dejbakhsh S, Chiang B, Hui J, Mahanian M, Baghaee A, Hong P, Cashman J. Innate immunity and transcription of MGAT-III and Toll-like receptors in Alzheimer's disease patients are improved by bisdemethoxycurcumin. Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12849-54. Abstract

View all comments by Milan Fiala

The manuscript by Rademakers and colleagues provides evidence that increased binding of miR-659 to the 3’UTR of the GRN gene could underlie an important risk for TDP-43-positive frontotemporal dementia (FTLD-U). These data bring strong clinical support for the role of microRNAs in neurodegenerative disorders in humans. These results are consistent with a loss of function of the GRN gene in the disease, further linking gene dosage effects in neurodegenerative disorders (as seen, e.g., with APP in Alzheimer disease and SNCA in Parkinson disease).

I think Amber Dance did a fantastic job reviewing the highlights of this paper. I would like to discuss additional issues with regard to certain technical and mechanistic aspects of these findings, which could be taken into account when interpreting the data.

First, miR-659, located on chromosome 22 in humans, seems to be relatively very weakly expressed in adult brain (with cycle threshold [Ct] values of approximately 32 as measured by qRT-PCR). Therefore, whether endogenous miR-659 levels are sufficient to regulate GRN levels...  Read more

The manuscript by Rademakers and colleagues provides evidence that increased binding of miR-659 to the 3’UTR of the GRN gene could underlie an important risk for TDP-43-positive frontotemporal dementia (FTLD-U). These data bring strong clinical support for the role of microRNAs in neurodegenerative disorders in humans. These results are consistent with a loss of function of the GRN gene in the disease, further linking gene dosage effects in neurodegenerative disorders (as seen, e.g., with APP in Alzheimer disease and SNCA in Parkinson disease).

I think Amber Dance did a fantastic job reviewing the highlights of this paper. I would like to discuss additional issues with regard to certain technical and mechanistic aspects of these findings, which could be taken into account when interpreting the data.

First, miR-659, located on chromosome 22 in humans, seems to be relatively very weakly expressed in adult brain (with cycle threshold [Ct] values of approximately 32 as measured by qRT-PCR). Therefore, whether endogenous miR-659 levels are sufficient to regulate GRN levels in vivo remains speculative. Mechanistically, one must envisage that regulation of GRN mRNA by miR-659 occurs in a cell-autonomous fashion. One possibility, not shown here, is that miR-659 is expressed in specific cell types, such as the granular cell layer of the cerebellum where GRN protein is decreased (it should be noted that the qRT-PCR for miR-659 was performed on whole tissues). In my opinion, this would strongly strengthen the biological significance of the proposed mode of regulation.

Here, the authors use basic, but widely accepted in vitro systems to validate their hypothesis. First, artificial overexpression of miR-659 (at a concentration of 12 nM) in human M17 neuroblastoma cells leads to decreased expression of endogenous GRN protein levels (note that inverse experiments using antisense oligonucleotides to block endogenous miR-659 was not performed, possibly due to the extremely low levels of this microRNA in these cells). Whether GRN mRNA levels are affected in these conditions is not shown. Then, additional studies were conducted in mouse Neuro2A cells using luciferase-based constructs containing the GRN 3’UTR. In these latter experiments, functional effects on GRN expression are seen with the mutant TT construct at concentrations starting at 5 pM of exogenous miR-659. Again from a mechanistic point of view, it would be interesting to see whether the “increased” binding (i.e., increased sequence complementarity) of miR-659 to the mutant TT allele causes an siRNA effect (thus degradation of mRNA). It should be noted, however, that, in affected patients, GRN mRNA (from total tissue sections) is not affected.

Interestingly, the predicted target site (more particularly the “seed” sequence) for miR-659 in the GRN 3’UTR is only conserved in humans, and is not found in other mammals including mouse and dog (e.g., see www.targetscan.org). Similarly, miR-659 is, at least for now, only found in humans. Interestingly, the GRN 3’UTR is quite short (approximately 300 bp in length). In comparison, the BACE1 and APP 3’UTRs, which equally have functional microRNA target sites, are approximately 4,000 bp and 2,000 bp in length, respectively.

Overall, these findings provide novel and important clues into the development of FTLD-U. In addition, this study contributes to the potential role of microRNA pathways in the development of neurodegenerative disorders in human. I agree that relatively few patients were analyzed here to make definitive conclusions with regard to the biological relevance of these findings.

View all comments by Sebastien S. Hebert

This work illustrates two frequently under-emphasized points about animal models of disease. First, although mice have proven fantastically useful and easy to manipulate experimentally, they rarely provide perfect models of any human disease. Second, genetic manipulations in mice often produce complex phenotypes that are more closely related to the function of the transgene than to the human disease that they are aiming to model. Our high rate of failure in getting therapeutically useful compounds from preclinical mouse models to the target human population is certainly related to both of these points; more work on complementary models (canines, primates, etc.) is essential.

View all comments by M. Paul Murphy

I think that the explanation of microhemorrhages in the brain of vaccinated transgenic mice by the "washing out" of vascular or perivascular amyloid, and the recommendation of early treatment, "before amyloid deposition," is lacking rationale. Lumping all forms of vascular amyloid deposits into "CAA" does not take into account the difference between so-called "congophilic angiopathy," with amyloid inside the wall of medium-sized vessels, and "dysoric angiopathy," so named because amyloid seems to leak out of capillaries (in fact, the converse is probably true).

The first one is contemporary to the initiation of AD; I have seen it (Foncin, 1974; Foncin et al., 1985) in a cortical biopsy of a 42-year-old woman who died demented aged 51; she was the index case of FAD4 (Sherrington et al., 1995); congophilic angiopathy is seen prominently in AD with lobar hemorrhages. On the opposite, dysoric angiopathy is probably secondary.

My conclusion is what is called AD really is the result of the lumping together of various conditions with various pathogenies, and inferences for AD...  Read more

I think that the explanation of microhemorrhages in the brain of vaccinated transgenic mice by the "washing out" of vascular or perivascular amyloid, and the recommendation of early treatment, "before amyloid deposition," is lacking rationale. Lumping all forms of vascular amyloid deposits into "CAA" does not take into account the difference between so-called "congophilic angiopathy," with amyloid inside the wall of medium-sized vessels, and "dysoric angiopathy," so named because amyloid seems to leak out of capillaries (in fact, the converse is probably true).

The first one is contemporary to the initiation of AD; I have seen it (Foncin, 1974; Foncin et al., 1985) in a cortical biopsy of a 42-year-old woman who died demented aged 51; she was the index case of FAD4 (Sherrington et al., 1995); congophilic angiopathy is seen prominently in AD with lobar hemorrhages. On the opposite, dysoric angiopathy is probably secondary.

My conclusion is what is called AD really is the result of the lumping together of various conditions with various pathogenies, and inferences for AD therapy in general drawn from any particular mouse model are hazardous at best.

References:
FONCIN J.-F. (1974): Angiopathie amyloïde et maladie d'Alzheimer familiale. In "Biologie et pathologie des parois artérielles et artériolo-capillaires" Lyon, ACEML, pp. 49-50.

Foncin JF, Salmon D, Supino-Viterbo V, Feldman RG, Macchi G, Mariotti P, Scoppetta C, Caruso G, Bruni AC. Démence présénile d'Alzheimer transmise dans une famille étendue. Rev Neurol (Paris). 1985;141(3):194-202. Abstract

Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H, Lin C, Li G, Holman K. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature. 1995 Jun 29;375(6534):754-60. Abstract

View all comments by Jean-François FONCIN

Deacetylation is a wide and complex epigenetic mechanism, which could involve undesired targets. The use of specific compounds to obtain epigenetic silencing of genes in AD treatment is much more preferable and safe. We published several papers in which we show the involvement of gene methylation in AD pathology.

References:
Fuso A, Nicolia V, Cavallaro RA, Ricceri L, D'Anselmi F, Coluccia P, Calamandrei G, Scarpa S. B-vitamin deprivation induces hyperhomocysteinemia and brain S-adenosylhomocysteine, depletes brain S-adenosylmethionine, and enhances PS1 and BACE expression and amyloid-beta deposition in mice. Mol Cell Neurosci. 2008 Apr;37(4):731-46. Abstract

Cavallaro RA, Fuso A, D'Anselmi F, Seminara L, Scarpa S. The effect of S-adenosylmethionine on CNS gene expression studied by cDNA microarray analysis. J Alzheimers Dis. 2006 Aug;9(4):415-9. Abstract

View all comments by Sigfrido Scarpa

My wife participated in an LY-450139 Phase 3 trial. She had to drop out when her legs would no longer support her. Physicians admitted her to hospital as a cardiac patient. She has DHF [diastolic heart failure].

Her Alzheimer's appeared to be stable during the months she took the trial medication. It appears to have deteriorated markedly during the few weeks since she stopped the medication.

Has anyone observed a developing weakness possibly related to LY-450139?

View all comments by Paul Murray

The comment that the cleavage of neuropeptide Y to generate a biologically active fragment by neprilysin (Neutral EndoPeptidase-24.11) is the first such example for the enzyme is incorrect. At least one example has previously been reported in the metabolism of calcitonin gene-related peptide (CGRP) (Davies et al., 1992).

References:
Davies D, Medeiros MS, Keen J, Turner AJ, Haynes LW. Endopeptidase-24.11 cleaves a chemotactic factor from alpha-calcitonin gene-related peptide. Biochem Pharmacol. 1992 Apr 15;43(8):1753-6. Abstract

View all comments by Tony Turner