Marutle A, Ohmitsu M, Nilbratt M, Greig NH, Nordberg A, Sugaya K.
Modulation of human neural stem cell differentiation in Alzheimer (APP23) transgenic mice by phenserine.
Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12506-11.
PubMed.
In response to the comments by Frautschy and others, the objectives of our paper are first to explain the immune mechanisms of amyloidosis in Alzheimer disease patients and second to find out what can be done about clearance of amyloidosis from the patient’s brain. The emerging answers are that amyloidosis is contributed by insufficient clearance by the Alzheimer patients’ innate immune system and that modulation of the innate immune system has positive effects on amyloid-β clearance.
There is no problem in distinguishing FITC-amyloid-β by fluorescence microscopy from curcuminoids, which (at 0.1 microM) are not visible by fluorescence microscopy. Amyloid-β is also revealed by immunostaining with amyloid-β antibody or by electron microscopy. This can be seen in the pictures of FITC-Aβ in Figs. 2, 3, 5 in the current PNAS publication (1) or the Figs. 2 and 3 (using anti-Aβ immunofluorescence or electron microscopy) in our previous publication (2). The responses of individual patients and clinical data correlations were examined in a previous publication (3).
Our work relates to human tissues and blood cells from patients with Alzheimer disease, which makes a direct comparison with transgenic animals (which do not have a specific immune defect) difficult. We performed the studies in macrophages and monocytes from Alzheimer patients over a 6-year period. Logistically, it is difficult to ask that the blood specimens of over 140 patients collected over a 6-year period would be analyzed by the same techniques that were developed later during the course of this study. However, the immune defects in phagocytosis have been observed by fluorescence microscopy in a majority of patients and the biochemical defects in a small number of patients (MGAT3 in over 20 patients, TLR defects in four patients) but with a remarkable consistency. Without doubt, many factors might affect the immune system, including drugs, hormones, stress, infection, etc. However, the patients in Phase 1 – preclinical study (where we are now) have to be examined, as they present themselves. Phase 2 and 3 studies will be possible at a later stage of investigation.
Bisdemethoxycurcumin showed greatest effect on phagocytosis when compared to unfractionated curcuminoids or other fractions. In order to obtain the most reproducible results, we chose to work with a pure chemical, bisdemethoxycurcumin, not the unfractionated material.
Regarding MGAT3 and TLR results, the data, such as Fig. 4, speak for themselves since they are consistent (patients vs. controls). Recent results continue to support the conclusions about transcriptional effects of curcuminoids. The studies of MGAT3 protein levels in the brain are difficult since a good antibody is not available. We agree that more flow cytometric testing of TLR proteins in PBMCs treated with bisdemethoxycurcumin is warranted (the legend had an error and we plan to correct this).
The results of PBMC clearance of Aβ in brain sections in Fig. 6 are striking and deserve closer scrutiny of the legends. Similar results have been obtained in at least five other experiments. The tissues were obtained from the UCLA brain bank. We have not seen the effects on neuritic plaques in these frozen tissues, but further work is ongoing.
Our intention in this study was to identify and characterize the most potent anti-Alzheimer disease agent in mixture of curcuminoids, not to study curcumin SAR. In fact, bisdemethoxycurcumin does possess antioxidant activity (4). In the AAPH-induced linoleic acid antioxidation test or the DPPH-radical scavenging test, bisdemethoxycurcumin does possess significant activity as an antioxidant.
We are aware of the chemical properties of curcumins and related materials, and the compounds are readily handled with proper care. All of the compounds were fully characterized spectrally, and instability was not a problem during our analytical and synthetic studies. We are aware of the metabolic properties of curcumins and in fact “Dynamic Medicinal Chemistry” has been a major effort leading our studies in this and other areas (5). Our intention was not to study bisdemethoxycurcumin in a pharmaceutical sense. For our purposes, of greatest relevance was the apparent effective concentration of the active pharmacological agent at the target site and not the relative percent of material in dietary supplements.
The purpose of the study was to biotrack the most pharmacologically active constituent. That no curcumin was present in the final HPLC in the purification of bisdemethoxycurcumin was not surprising and speaks to the alacrity of our separation approach. Agreeably, the retention time was short, but the solvent polarity gradient was steep and quite effective. Of course, elution profiles are a function of the matrix employed and the history of the matrix. To confirm the activity of bisdemethoxycurcumin, indeed, synthetic bisdemethoxycurcumin was prepared and fully characterized spectrally. That synthetic bisdemethoxycurcumin is also highly active supports the exciting observation that the minor constituent in curcuminoids contains remarkable biological properties.
It was beyond the scope of this study to examine the glucuronidation of bisdemethoxycurcumin, but it is important to point out that 1) glucuronides undergo enterohepatic cycling, and urinary metabolite levels may not reflect metabolic disposition in the blood; 2) bisdemethoxycurcumin may not be “free” in the biological context but in fact associated with proteins, thus confounding apparent observations about solution stability study data; and 3) the effective concentration or accumulation of bisdemethoxycurcumin in the target tissue or cell may be much different than that estimated from plasma levels. The examination of these points is the subject of additional studies.
References:
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.
PubMed.
Fiala M, Lin J, Ringman J, Kermani-Arab V, Tsao G, Patel A, Lossinsky AS, Graves MC, Gustavson A, Sayre J, Sofroni E, Suarez T, Chiappelli F, Bernard G.
Ineffective phagocytosis of amyloid-beta by macrophages of Alzheimer's disease patients.
J Alzheimers Dis. 2005 Jun;7(3):221-32; discussion 255-62.
PubMed.
Zhang L, Fiala M, Cashman J, Sayre J, Espinosa A, Mahanian M, Zaghi J, Badmaev V, Graves MC, Bernard G, Rosenthal M.
Curcuminoids enhance amyloid-beta uptake by macrophages of Alzheimer's disease patients.
J Alzheimers Dis. 2006 Sep;10(1):1-7.
PubMed.
Somparn P, Phisalaphong C, Nakornchai S, Unchern S, Morales NP.
Comparative antioxidant activities of curcumin and its demethoxy and hydrogenated derivatives.
Biol Pharm Bull. 2007 Jan;30(1):74-8.
PubMed.
Cashman JR, MacDougall JM.
Dynamic medicinal chemistry in the elaboration of morphine-6-glucuronide analogs.
Curr Top Med Chem. 2005;5(6):585-94.
PubMed.
Comments
UCLA
Reply to Frautschy, Teter Comment
In response to the comments by Frautschy and others, the objectives of our paper are first to explain the immune mechanisms of amyloidosis in Alzheimer disease patients and second to find out what can be done about clearance of amyloidosis from the patient’s brain. The emerging answers are that amyloidosis is contributed by insufficient clearance by the Alzheimer patients’ innate immune system and that modulation of the innate immune system has positive effects on amyloid-β clearance.
There is no problem in distinguishing FITC-amyloid-β by fluorescence microscopy from curcuminoids, which (at 0.1 microM) are not visible by fluorescence microscopy. Amyloid-β is also revealed by immunostaining with amyloid-β antibody or by electron microscopy. This can be seen in the pictures of FITC-Aβ in Figs. 2, 3, 5 in the current PNAS publication (1) or the Figs. 2 and 3 (using anti-Aβ immunofluorescence or electron microscopy) in our previous publication (2). The responses of individual patients and clinical data correlations were examined in a previous publication (3).
Our work relates to human tissues and blood cells from patients with Alzheimer disease, which makes a direct comparison with transgenic animals (which do not have a specific immune defect) difficult. We performed the studies in macrophages and monocytes from Alzheimer patients over a 6-year period. Logistically, it is difficult to ask that the blood specimens of over 140 patients collected over a 6-year period would be analyzed by the same techniques that were developed later during the course of this study. However, the immune defects in phagocytosis have been observed by fluorescence microscopy in a majority of patients and the biochemical defects in a small number of patients (MGAT3 in over 20 patients, TLR defects in four patients) but with a remarkable consistency. Without doubt, many factors might affect the immune system, including drugs, hormones, stress, infection, etc. However, the patients in Phase 1 – preclinical study (where we are now) have to be examined, as they present themselves. Phase 2 and 3 studies will be possible at a later stage of investigation.
Bisdemethoxycurcumin showed greatest effect on phagocytosis when compared to unfractionated curcuminoids or other fractions. In order to obtain the most reproducible results, we chose to work with a pure chemical, bisdemethoxycurcumin, not the unfractionated material.
Regarding MGAT3 and TLR results, the data, such as Fig. 4, speak for themselves since they are consistent (patients vs. controls). Recent results continue to support the conclusions about transcriptional effects of curcuminoids. The studies of MGAT3 protein levels in the brain are difficult since a good antibody is not available. We agree that more flow cytometric testing of TLR proteins in PBMCs treated with bisdemethoxycurcumin is warranted (the legend had an error and we plan to correct this).
The results of PBMC clearance of Aβ in brain sections in Fig. 6 are striking and deserve closer scrutiny of the legends. Similar results have been obtained in at least five other experiments. The tissues were obtained from the UCLA brain bank. We have not seen the effects on neuritic plaques in these frozen tissues, but further work is ongoing.
Our intention in this study was to identify and characterize the most potent anti-Alzheimer disease agent in mixture of curcuminoids, not to study curcumin SAR. In fact, bisdemethoxycurcumin does possess antioxidant activity (4). In the AAPH-induced linoleic acid antioxidation test or the DPPH-radical scavenging test, bisdemethoxycurcumin does possess significant activity as an antioxidant.
We are aware of the chemical properties of curcumins and related materials, and the compounds are readily handled with proper care. All of the compounds were fully characterized spectrally, and instability was not a problem during our analytical and synthetic studies. We are aware of the metabolic properties of curcumins and in fact “Dynamic Medicinal Chemistry” has been a major effort leading our studies in this and other areas (5). Our intention was not to study bisdemethoxycurcumin in a pharmaceutical sense. For our purposes, of greatest relevance was the apparent effective concentration of the active pharmacological agent at the target site and not the relative percent of material in dietary supplements.
The purpose of the study was to biotrack the most pharmacologically active constituent. That no curcumin was present in the final HPLC in the purification of bisdemethoxycurcumin was not surprising and speaks to the alacrity of our separation approach. Agreeably, the retention time was short, but the solvent polarity gradient was steep and quite effective. Of course, elution profiles are a function of the matrix employed and the history of the matrix. To confirm the activity of bisdemethoxycurcumin, indeed, synthetic bisdemethoxycurcumin was prepared and fully characterized spectrally. That synthetic bisdemethoxycurcumin is also highly active supports the exciting observation that the minor constituent in curcuminoids contains remarkable biological properties.
It was beyond the scope of this study to examine the glucuronidation of bisdemethoxycurcumin, but it is important to point out that 1) glucuronides undergo enterohepatic cycling, and urinary metabolite levels may not reflect metabolic disposition in the blood; 2) bisdemethoxycurcumin may not be “free” in the biological context but in fact associated with proteins, thus confounding apparent observations about solution stability study data; and 3) the effective concentration or accumulation of bisdemethoxycurcumin in the target tissue or cell may be much different than that estimated from plasma levels. The examination of these points is the subject of additional studies.
References:
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. PubMed.
Fiala M, Lin J, Ringman J, Kermani-Arab V, Tsao G, Patel A, Lossinsky AS, Graves MC, Gustavson A, Sayre J, Sofroni E, Suarez T, Chiappelli F, Bernard G. Ineffective phagocytosis of amyloid-beta by macrophages of Alzheimer's disease patients. J Alzheimers Dis. 2005 Jun;7(3):221-32; discussion 255-62. PubMed.
Zhang L, Fiala M, Cashman J, Sayre J, Espinosa A, Mahanian M, Zaghi J, Badmaev V, Graves MC, Bernard G, Rosenthal M. Curcuminoids enhance amyloid-beta uptake by macrophages of Alzheimer's disease patients. J Alzheimers Dis. 2006 Sep;10(1):1-7. PubMed.
Somparn P, Phisalaphong C, Nakornchai S, Unchern S, Morales NP. Comparative antioxidant activities of curcumin and its demethoxy and hydrogenated derivatives. Biol Pharm Bull. 2007 Jan;30(1):74-8. PubMed.
Cashman JR, MacDougall JM. Dynamic medicinal chemistry in the elaboration of morphine-6-glucuronide analogs. Curr Top Med Chem. 2005;5(6):585-94. PubMed.
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