Brain Research, 594 (1992) 273-278 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
273
BRES 25393
Short Communications
Progressive transformation of the cy < skeleton associated with normal aging and Alzhei ner's disease J a m e s C. Vickers a, A n d r 6 D e l a c o u r t e c and J o h n H. M o r r i s o n a,b Departments of a Neurobiology and b Geriatrics and Adult Development, Mount Sinai School of Medtcine, New York, IVY 10029-6574 (USA) and c INSERM U156, Lille (France)
(Accepted 21 July 1992)
Key words: Alzheimer's disease; Aging; Neurofibrillary tangle; Thioflavine; Tau; Neurofilament; Dementia
Transitional and end-stage forms of neurofibrillary tangles associated with normal aging and Alzheimer's disease were identified using thioflavme staining combined with tau and neurofilament protein immunofluorescence. Normal aging was marked by transitional pathology in layer !I of the entorhinal cortex but no neurofibrillary tangles in prefrontal cortex, whereas, in Alzheimer's disease cases, layer I1 entorhinal neuron~ had progressed to end-stage neurofibrillary tangles and the prefrontal cortex contained a high representation of transitional forms of the ueurofibrillaly tangle.
The clinical course of Alzheimer's disease (AD) is generally characterized by initial disturbances in memory followed by the global deterioration of higher cognitive processes. It has been demonstrated that specific neuronal subpopulations are vulnerable in this disease and that their degeneration may underlie the principal clinical fL.atures. For instance, neurofibriilary tangle (NFI') formation and neuronal degeneration is found in those specific neurons that provide the corticocortical projections that interconnect association areas within the neocortex ~'13'14'22'2s'37and those that interconnect the hippocampal formation with the rest of the brain 2't7. With regard to the hippocampal connections, the projection from the entorhinal cortex to the dentate gyrus, often referred to as the perforant path, is particularly vulnerable. The cellular changes that reflect the progression of the disease are essentially unknown although it is possible that cytoskeletal alterations that lead to development of the "neurofibrillary" hallmarks may underlie the neuronal degeneration. We have examined both AD and control cases using a multiple immuno- and histo-fluorescent labelling paradigm to determine the alterations in cytoskeletal
proteins within the vulnerable subset of neurons that form NFTs. Seven control cases (ages 16, 43, 47, 49, 66, 73 and 85 years) and seven AD cases (ages 66, 72, 73, 82, 84, 85 and 87 years) were utilized for this study (3-5 h post-mortem interval). Coronal blocks were immersionfixed in 4% paraformaldehydc (0.1 M phosphate buffer, pH 7.6) for 24-48 h followed by washes in a series of graded sucrose solutions (12, 16 and 18%) or graded glycerol solutions (10 and 20% glycerol including 2% dimethyl sulphoxide). Sections (40 ~m) of the prefrontal cortex (area 9) and hippocampal formation were processed for either immunoperoxidase single labeling (ABC Vectastain kit, Vector Lab., Burlingame, CA), or for multiple fluorescent labeling using an autofluorescence quenching procedure 3° (0.25% potassium permanganate for 20 min followed by destaining in a 1% potassium metabisulfite/l% oxalic acid solution) and, for some sections, a solution of 0.0125% thioflavine S (Sigma, St. Louis, MO) '~°'33. Primary antibodies consisted of mouse monoclonal antibodies to either non-phosphorylated (SMI32) !s'2°'27'3'~ or phosphorylated (SMI310) 39 epitopes on the middle and
Correspondence: J.H. Morrison, Fishberg Research Center for Neurobiology, Box 1065, Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY 10029-6574, USA. Fax (1) (212) 996-9785.
274 htavy molecular weight subunits of the mammalian neurofilament (NF) triplet (Sternberger Monoclonals Inc., Baltimore, MD; diluted 1:5000) and a rabbit polyclonal antibody raised against human tau protein 7. The NF antibodies do not cross-react with tau on immunoblots ~8'2°'27"3'~and the tau antibody is known to label abnormally phosphorylated tau in paired helical filament preparations 7a°. In order to expose possible hidden epitopes within NFTs, some sections were pre-
treated with 20% formic acid for 20 min prior to the application of the primary antibodies 6. For doubk, immunolabeling preparations, mouse monoclonals were visualized with a horse anti-mouse IgG antibody conjugated to fluorescein isothiocyanate (Vector Laboratories; diluted 1:200) and the rabbit polyclonal to tau was visualized with a goat anti-rabbit IgG antibody conjugated to biotin (Vector Laboratories; diluted 1:200) f(4iowed by streptavidin-Texas Red
Fig. I. Photomicrographsshowing multiple labeling for NF proteins, tau and thioflavine S in layer II of the entorhinal cortex in the older control cases. A, B, C show an example of layer II cells labeled for non-phosphorylated NF protein (SM132, A), tau (B) and thioflavine S (C). Thioflavine staining presents as yellow fluorescence with the filter block (filter set 487916, Zeiss) used to visualize FITC (green) in A. Tau and thioflavine positive NFTs (e.g. arrows) were present within the neurons labeled with SM132. C and D show an example of a NIT double labeled for both SM132 (D) and tau (E). Note that $M132 labels bundles of filaments within the tau immunoreactive tangle. E and F show double labeling for phosphorylated NF protein epitopes (SMI310) (F) and tau (G) in a formic acid pretreated tissue section. SMI310, in addition to labeling nerve fibres in this region, also labels the NFTs immunoreactive for tau (arrows). Bars = 30 p,m (A,B,C,F,G), 20 p.m (D,E).
275 ( A m e r s h a m , Arlington Heights, IL; diluted 1:200). Secondary antibodies were tested for possible cross reactivity. As previously described m3'26, n e u r o n s in layer II of the entorhinal cortex and pyramidal cells in o t h e r layers of the entorhinal cortex and the prefrontal cortex of all control cases were selectively labeled with SMI32. In contrast, SMI310 preferentially labeled axons in these control cases.
In the five older control cases (47-85 years of age), multiple fluorescent labeling d e m o n s t r a t e d that many layer I1 cells in the entorhinal cortex were preferentially involved in the process o f / o r m i n g NFTs. In these cases, most thioflavine-positive NF Ts occurred inside intact SM132-1abeled neurons and the N F T structure itself was intensely immunoreactive for tau and often labeled, in part, with SM132 (Fig. 1 A - E ) . All taulabeled NFT-like structures and most neuropil threads
ii ii!iiii!iii!iilii!ii!lii!i!fiiiii!i!i!!ii!i!li!ii!i !
ili !iii!i
Fig. 2. Photomicrographs showing multiple labeling in layer II of the entorhinal cortex and prefrontal cortex of AD cases. A, B and C show an example of labeling for non-phosphorylated NF proteins (SM132) (A), tau (B) and thioflavine S in layer II of the entorhinal cortex. Whereas NFTs in this region are thioflavine positive (C and yellow fluorescence in A, arrows), these NFTs are not contained within SM132 labeled neurons and show little or no immunoreactivity for tau (e.g. arrows). In layers IH (D,E,F) and V (G,H,I) of the prefrontal cortex (area 9) of these AD cases, many neurons demonstrate NFT-related alterations in SM132 (D,G) and tau (E,H) labelling. F and I are double exposure microphotographs showing the relationship between alterations in these cytoskeletal proteins with overlapping immunoreactivity appearing as yellow. Many of the SM132-1abeled neurons in D, E and F contain tau-immunoreactive NFT material which is in part labeled with SMl32 (arrows). The NFT in G, H, I is labeled in part with SM132 (arrow). Bars -- 20 ,¢m (A,B,C), 30 gm (D,E,F,G,H,I).
276 were also thioflavine-positive. Many of these NFT were labeled with SMI310. Formic acid pretreatment of tissue sections resulted in the immunolabeling of more NFT in layer 11 of the entorhinal cortex with SMI310, but did not alter the number of NFT labeled with SM132 or the antibody to tau. Formic acid pretreatmerit also eliminated thioflavine staining which suggests that this process may degrade some of the conformational features of the substructure of the tangle to expose the phosphorylation-dependent NF protein epitopes. Double labeling demonstrated that all tau-immunoreactive NFTs in these cases were also labeled with SMI310 following formic acid pretreatment (Fig. IF, G). A few NFTs were present.in the hippocampal CA1 field and in deeper entorhinal layers of the 66and 85-year-old control cases. NFTs were not detected with the above markers in the prefrontal cortex of the control cases, in the AD cases, thioflavine-positive NFT corresponding to end-stage extracellular NFT were abundant in layer li of the entorhinai cortex. Virtually all of these NFT were unlabeled with SMI32, SMI310 or the antibody to tau (Fig. 2A-C). In contrast, many neurons in the prefrontal cortex were in various transitional states that reflec,:d the formation of a NFT. In a similar fashion to layer I1 cells of the entorhinal cortex of the older control cases, many of the neocortical NITs were labeled with thioflavine and the tau antibody and were present within morphologically intact SM132.1abeled pyramidal cells (Fig. 2D-F). In addition, many of the NITs in this cortical region were immunoreactiv¢ for tau and labeled, in part, with SM!32 (Fig. 2D-l). Some end-stage NFT, stained with thioflavine but not labeled for tau or SM132, were also present in the prefrontal cortex. All tau immunoreactire NFT-like structures and most neuropil threads in these AD cases were also thioflavine-positive. Only a few NFTs in the prefrontal cortex were labeled with SM1310. Formic acid pretreatment of tissue sections dramatically increased the number of NFTs labeled with SMI310 in the neocortex and in layer.~ I!! to V! of the entorhinal cortex but did not increase the number of NFTs labeled in layer !1 of the entorhinal cortex. Formic acid pretreatment did not increase the number of NFT labeled with SMI32 or tau in the entorhinal cortex or neocortex of the AD cases. On the basis of their cytoskeletal protein immunoreactivity, NFTs can therefore be subdivided into transitional forms that are labeled with thioflavine and contain NF and tau protein epitopes or end-stage NFTs that are stained with thioflavine but are non-immunoreactive for particular tau epitopes and NF proteins. These results support the proposal that NFT
formation follows progressive alterations in the normal cytoskeleton 24'25. In addition, the cellular correlate of the differentiation between normal aging and dementia appears to be the point at which cytoskeletal changes progress from tr,~nsitional to end-stage in layer II of the entorhinal cortex, and begin to involve a large enough contingent of neocortical pyramidal cells to affect cort~cocortical integration. The localization of NFT in the entorhinal cortex using conventional methods has been noted in a significant number of control cases from other studies 5.23,29,32 whereas, in AD, severity of dementia has been correlated with increasing numbers of NFTs in the neocortex I's'29'37. Therefore, cytoskeletal pathology manifested as NFT-like changes in entorhinal cortex may not be uncommon in neurologically normal individuals in their fourth and fifth decades while, in AD, this process is facilitated by unknown factors to result in the progression of NFTs to their end-stage in most entorhinal layer II cells and the appearance of NFT-as. sociated cytoskeletal alterations in neocortical areas. Indeed, the specific pathological changes observed in the entorhinal cortex during normal aging may form the basis of subtle memory impairments known as "benign senescent forgetfulness". Whereas immunohistochemical and biochemical analysis has confirmed that some region of the tau polypeptide forms part of the core of PHFs that comprise NIT 7's,l°'t2't~,zt,'~s, the role of the neurofilament proteins in NIT formation is controversial, partly due to the cross-reactivity of some of the anti-NF antibod. ies that recognize NFT with tau proteins ta.27. The results of this study further support the hypothesis that the neurons that selectively contain the NF triplet are also the cells that are particularly vulnerable to NFT formation tx~4':6'~6. Cortical neurons that lack these NF proteins, such as GABAergic interneurons, are not as prone to degeneration in the disease 4s.~s,'6`2z,2s. The current results present evidence that NF protein antibodies that do not cross-react with tau can label particular forms of NFTs. NF polypeptides possibly become incorporated into NFTs initially in a non-phosphory. lated form and subsequently become abnormally phosphorylated, as has been shown for the tau component of NFTs 10,19,21. Phosphorylation of similar sequences present in both tau and the NF proteins, possibly by an abnormally functioning kinase, may underlie the alterations and association of these cytoskeletal proteins within NFTs. Further conformational changes associated with the formation of end-stage NFTs may mask NF and tau epitopes, or, alternatively, these regions may be cleaved from the essential NFT subcomponents. Particularly, it
277
has been noted that antibodies that recognize the repeat region in the tau carboxyterminal domain recognize NFTs that correspond to endstage as presented in this report 3':'4. The issue of the incorporation of regions of NF proteins into NFTs "~slimited by difficulties in the detection of further particular NF epitopes (e.g. the 68 kDa NF subunit, peripherin, alpha-internexin) in aldehyde-fixed tissues 34-36 and the unknown identity of the insoluble fractions of current paired helical filament preparations. Regardless, the progressive sequestering of microtubule-associated proteins and some region of the NF proteins into NFT may be the factor that leads to the destabilization and dissolution of the normal neuronal cytoskeleton. Hence, even the early stages of NFT formation in morphologically intact neurons would have profound effects on normal neuronal function. While the precise causative factors leading to NFT formation are unclear, the progressive alteration of cytoskeletal proteins may provide the basis for the neuronal dysfunction and degeneration underlying the development of dementia. The present data are, therefore, most compelling when placed in the context of a continuum that begins with aberrant intraneuronal biochemical processing that is manifested as differential cellular vulnerability and ends with dementia. The importance of the pathological involvement of subsets of neocortical neurons in AD is exemplified by the role of the neocortex as the putative substrate for long-term declarative memory 3~ and as the region underlying higher cognitive processes ~, Further, it has been proposed that memory functions that are initially dependent on hippocampal circuits may become largely independent once stored in neocortex 3~, which may in turn explain the relatively benign disruption of neurological function in the controls with significant hippocampai damage but no neocortical pathology. Agents that stabilize the neuronal cytoskeleton may prove useful in ameliorating this progressive degeneration, and, if used in conjunction with sensitive neuropsychological tests that distinguish between age-related and AD-related cognitive dysfunction, the cytoskeletal pathology might be halted prior to development of the devastating aspects of dementia that result from neocortical involvement. We would like to thank our colleagues at the Fishberg Research Center for Neurobiology as well as Dr. John Trojanowski aad Dr. Neil Kowall for their valued comments on the manuscript. We also thank William Janssen for technical support and Bob Woolley for photographic assistance, in addition, we would like to acknowledge the Institute of Biogerontology Research Tissue Donation Program (Sun City, Arizona) for the provision of many of the cases in the present study. This work was funded by AG05138 and AG06647 from the National Institute for Aging and AHAF. James Vickers is a
recipient of a CJ. Martin Postdoctoral Fellowship from the Australian National Health and Medical Research Council. I Arriagada, P.V., Growdon, ,I.H., Hedley-Whyte, E.T. and Hyman, B.T., Neurofibrillary tangles but not sende plaques parallel duration and severity of dementia, Neurology, 42 (1992) 631-639. 2 Ball, M.,I., Hachinski, V., Fox, A., Kirshen, A.J., Fisman, M., Blume, W., Kral, V.A., Fox, H. and Merskey, H., A new definition of Alzheimer's disease: a hippocampal dementia, Lancet, I (1985) 14-16. 3 Bondareff, W., Wischik, C.M., Novak, M., Amos, W.B., Klug, A and Roth, M., Molecular analysis of neurofibrillary degeneration in Alzheimer's disease, Am. J. Pathol., 137 (1990) 711-723. 4 Braak, H. and Braak, E., Ratio of pyramidal cells versus nonpyramidal cells in the human frontal isocortex and changes in ratio with ageing and Alzheimer's disease. In D.F. Swaab, E. Fliers, M. Mirmiran, W.A. van Gool and F. van Haaren, F. (Eds.), The Brain in Aging and Alzheimer's Disease, Progress m Bra#~ Research, Vol. 70, Elsevier, Amsterdam, 1986, pp. 185-212. 5 Braak, If. and Braak, E., Neuropathological stageing of Alzheimer-related changes, Acta NeuropathoL, 82 (1991) 239-259. 6 Cammarata, S., Mancardi, G. and Tabaton, M., Formic acid treatment exposes hidden neurofilament and tau epitopes in abnormal cytoskeletal filaments from patients with progressive supranuclear palsy and AIzheimer's disease, Neurosct. Lett., 115 (1990) 351-355. 7 D6fossez, A., Beauvillain, .I.C., Delacourte, A. and Mazzuca, M., Alzheimer's disease: a new evidence for common epitopes between microtubule associated protein tau and paired helical filaments (PHF), Virchows Arch., 413 (1988) 141-145. 8 Delacourte, A. and D6fossez, A., AIzheimer's disease: tau proteins, the promoting factors of microtubule assembly, are major components of paired helical filaments, J. NeuroL Sci., 76 (1986) 173-186. 9 Esiri, M.M., Pearson, R.C.A., Steele, ,I.E. and Powell, T.P.S., A quantitative study of the neurofibrillary tangles and the choline acetyltransferase activity in the cerebral cortex and the amygdala in AIzheimer's disease, J. Neurol. Neurosurg. P~'ychiatry, 53 (199{)) 161-165. 10 Flament, S., Delacourtc, A., H~mon, B. and D~fossez, A., Characterization of two pathological tau variants in Alzheimer brain cortices, J. Neurol. Sci., 92 (1989) 133-141. 11 Ooldman.Rakic, P.S., Topography of cognition: parallel distributed networks in primate association cortex, Atom. Rev. Neu. roscL, 11 (1988) 137-156. 12 Grundke-lqbal, I., Vorbrodt, A.W., iqbal, K,, Tung, Y,-C., Wang, G.P. and Wisniewski, H.M., Microtubule-associated polypeptides are altered in AIzheimer paired helical filaments, MoL Brain Res., 4 (1988) 43-52. 13 Hof, P.R., Cox, K. and Morrison, J.H., Quantitative analysis of a vulnerable subset of pyramidal neurons in AIzheimer's disease: l. Superior frontal and inferior temporal cortex, J. Comp. NeuroL, 301 (1990) 44-54. 14 Hof, P.R. and Molrison, J.H., Quantitative analysis of a vulnerable subset of pyramidal neurons in Alzheimer's disease: !1. Primary and secondary visual cortex, J. Comp. NeuroL, 301 (1990) 55-64. 15 Hof, P.R., Cox, K., Young, W., Celio, M.R., Rogers, J. and Morrison, `I.H., Parvalbumin-immunoreactive neurons in the neecortex are resistant to degeneration in Alzheimer's disease, J. Neuropathol. Exp. Neurol., 50 (1991) 451-462. 16 Hof, P.R. and Morrison, J.H., Neocortical neuronal subpopulations labeled by a monoclonal aatibody to calbindin exhibit differential vulnerability in Alzheimer's disease, Exp. NeuroL, 111 (1991) 293-301. 17 Hyman, B.T., Van Hoesen, G.W., Damasio, A.R. and Barnes, C.L., AIzheimer's disease: cell specific pathology isolates the hippocampal formation, Science, 225 (1984) 1168-1170. 18 Ksiezak-Reding, H., Dickson, D.W., Davies, P. and Yen, S.-H., Recognition of tau epitopes by anti-neurofilament antibodies that bind to Alzheimer neurofibrillary tangles, Prec. NatL Acad. Scl. USA, 84 (1987) 3410-3414.
278 19 Ksiezak-Reding, H. and Yen, S.-H., Structural stability of paired helical filaments requires microtubule-binding domains of tau: a model for self-association, Neuron° 6 (1991) 717-728, 20 Lee, V.M.-Y., Otvos, L., Carden, M.J., Hollosi, M., Dietzschold, B. and Lazzarini, R.A., Identification of the major multiphosphorylation site in mammalian neurofilaments, Prec. Natl. Acad. ScL USA, 85 (1988) 1998-2002. 21 Lee, V.M.-Y., Balin, B.J., Owos, L. and Trojanowski, J.O.. A68: a major subunit of paired helical filaments and derivatized forms of normal tau, Scwnce, 251 (1991) 675-678. 22 Lewis, D.A., Campbell, M.J., Ten3', R.D. and Morrison, J.H., Laminar and regional distributions of neurofibrillary tangles and neuritic plaques in AIzheimer's disease: a quantitative study of visual and auditory cortices, J. Neuroso., 7 (1987) 1799-1808. 23 McKee, A.C., Kosik, K.S. and Kowail, N.W., Neuritic pathology and dementia in AIzheimer's disease, Ann. Neurol., 30 (1991) 156-165. 24 Mena, R., Wischik, C.M., Novak, M., Milstein, C. and Cuello, A.C., A progressive deposition of paired helical, filaments (PHF) in the brain characterizes the evolution of dementia in AIzheimer's disease, J. Neuropathol. Exp. NeuroL, 50 (1991) 474490. 25 Metuzah, J., Robitaille, Y., Houghton, S., Gauthier, S., Kang, C.Y. an,. Leblanc, R., Neuronal transformations in Alzheimer's disease, Cell Ttssue Res., 252 (1988) 239-248. 26 Morriso,i, J.H.. Lewis, D.A., Campbell, M.J., Huntley, G.W., Benson, D.L. and Bouras, C., A monoclonal antibody to nonphosphorylated neurofilament protein marks the vulnerable cortical neurons in AIzheimer's disease, Bn~in Res., 416 (1987) 331336. 27 Nukina, N.. Kosik. K.S. and Selkoe, D.J., Recognition of Alzheimer paired helical filaments by monoclonal neurofilament antibodies is due to crossreaction with tau protein. Prec. Natl. Acad. S¢i. USA, 84 (1987) 3415-3419. 28 Pearson, R.C.A., Esiri, M.M., Hiorns, R.W., Wilcock, G.K. and Powell, T.P.S., Anatomical correlates of the distribution of the pathological changes in the neocortex in Alzheimer disease, Prec. Natl. A ad. Sci. USA, 82 (1985) 4531-4534. 29 Price, J.L, Davis, P,B., Morris, J.C. and White, D.L., The distribution o" tangles, plaques and related immunohislochemical
markers in healthy aging and Alzheimcr's disease, Neurobiol. Aging, 12 (1991) 295-312. 30 Styren, G., Civin, W.H. and Rogers, J., Quenching lipofus,-in autofluorescence in aged and AIzheimer's brain: a double-label fluorescence method with thiofiavin counterstain, Soc. Neurosci. Abstr., 17 (1991) 694. 31 Squire, L.R. and Zola-Morgan, S., The medial temporal lobe memory system, Science, 253 (1991) 1380-1386. 32 Ulrich, J., Alzheimer changes in nondemented patients younger than sixty-five: possible early stages of AIzheimer's disease and senile dementia of Alzheimer type, Ann. Neurol., 17 (1985) 273-277. 33 Vallet, P.G., Guntern, R., Hof, P.R., Golaz, J., Delacourte, A., Robakis, N.K. and Bouras, C., A comparative study of histological and immunohistochemical methods for neurofibrillary tangles and senile plaques in Alzheimer's disease, Acta. Neuropathol., 83 (1992) 170-178. 34 Vickers, J.C., Costa, M., Vitadello, M., Dahl, D. and Marotta, C.A., Neurofilament protein-triplet immunoreactivity in distinct subpopulations of peptide-containing neurons in the guinea-pig coeliac ganglion, Neuroscience, 39 (1990) 743-759. 35 Vickers, J.C., Vitadello, M., Parysek, L.M. and Costa, M., ComplemeW.ary immunohistochemical distribution of the neurofilament triplet and novel intermediate filament proteins in the autonomic and sensory nervous system of the guinea-pig, J. Chem. Neuroanat., 4 (1991) 259-270. 36 Vickers, J.C. and Costa, M., The neurofilament triplet is present in distinct subpopulations of neurons in the central nervous system of the guinea-pig, Neuroscience, 49 (1992) 73-100. 37 Wilcock, G.K. and Esiri, M.M., Plaques, tangles and dementia, 3,. Neurol. Sci., 56 (1982) 343-356. 38 Wischik, C.M., Novak, M., Thogerson, H.C., Edwards, P.C., Runswick, MJ., ,lakes, R., Walker, J.E., Milstein, C., Roth, M. and Klug, A., Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease, Proc. Natl. Acad. Sci. USA, 85 (1988) 4506-4510. 39 Zhang, H., Sternberger, N,H., Rubinstein, LJ., Herman, M.M., Binder, L.I. and Sternberger, LA,, Abnormal processing of multiple proteins in Alzheimer disease, Prec, Natl, Acad, Sci, USA, 8fi (I 989) 8045-8049,
273
BRES 25393
Short Communications
Progressive transformation of the cy < skeleton associated with normal aging and Alzhei ner's disease J a m e s C. Vickers a, A n d r 6 D e l a c o u r t e c and J o h n H. M o r r i s o n a,b Departments of a Neurobiology and b Geriatrics and Adult Development, Mount Sinai School of Medtcine, New York, IVY 10029-6574 (USA) and c INSERM U156, Lille (France)
(Accepted 21 July 1992)
Key words: Alzheimer's disease; Aging; Neurofibrillary tangle; Thioflavine; Tau; Neurofilament; Dementia
Transitional and end-stage forms of neurofibrillary tangles associated with normal aging and Alzheimer's disease were identified using thioflavme staining combined with tau and neurofilament protein immunofluorescence. Normal aging was marked by transitional pathology in layer !I of the entorhinal cortex but no neurofibrillary tangles in prefrontal cortex, whereas, in Alzheimer's disease cases, layer I1 entorhinal neuron~ had progressed to end-stage neurofibrillary tangles and the prefrontal cortex contained a high representation of transitional forms of the ueurofibrillaly tangle.
The clinical course of Alzheimer's disease (AD) is generally characterized by initial disturbances in memory followed by the global deterioration of higher cognitive processes. It has been demonstrated that specific neuronal subpopulations are vulnerable in this disease and that their degeneration may underlie the principal clinical fL.atures. For instance, neurofibriilary tangle (NFI') formation and neuronal degeneration is found in those specific neurons that provide the corticocortical projections that interconnect association areas within the neocortex ~'13'14'22'2s'37and those that interconnect the hippocampal formation with the rest of the brain 2't7. With regard to the hippocampal connections, the projection from the entorhinal cortex to the dentate gyrus, often referred to as the perforant path, is particularly vulnerable. The cellular changes that reflect the progression of the disease are essentially unknown although it is possible that cytoskeletal alterations that lead to development of the "neurofibrillary" hallmarks may underlie the neuronal degeneration. We have examined both AD and control cases using a multiple immuno- and histo-fluorescent labelling paradigm to determine the alterations in cytoskeletal
proteins within the vulnerable subset of neurons that form NFTs. Seven control cases (ages 16, 43, 47, 49, 66, 73 and 85 years) and seven AD cases (ages 66, 72, 73, 82, 84, 85 and 87 years) were utilized for this study (3-5 h post-mortem interval). Coronal blocks were immersionfixed in 4% paraformaldehydc (0.1 M phosphate buffer, pH 7.6) for 24-48 h followed by washes in a series of graded sucrose solutions (12, 16 and 18%) or graded glycerol solutions (10 and 20% glycerol including 2% dimethyl sulphoxide). Sections (40 ~m) of the prefrontal cortex (area 9) and hippocampal formation were processed for either immunoperoxidase single labeling (ABC Vectastain kit, Vector Lab., Burlingame, CA), or for multiple fluorescent labeling using an autofluorescence quenching procedure 3° (0.25% potassium permanganate for 20 min followed by destaining in a 1% potassium metabisulfite/l% oxalic acid solution) and, for some sections, a solution of 0.0125% thioflavine S (Sigma, St. Louis, MO) '~°'33. Primary antibodies consisted of mouse monoclonal antibodies to either non-phosphorylated (SMI32) !s'2°'27'3'~ or phosphorylated (SMI310) 39 epitopes on the middle and
Correspondence: J.H. Morrison, Fishberg Research Center for Neurobiology, Box 1065, Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY 10029-6574, USA. Fax (1) (212) 996-9785.
274 htavy molecular weight subunits of the mammalian neurofilament (NF) triplet (Sternberger Monoclonals Inc., Baltimore, MD; diluted 1:5000) and a rabbit polyclonal antibody raised against human tau protein 7. The NF antibodies do not cross-react with tau on immunoblots ~8'2°'27"3'~and the tau antibody is known to label abnormally phosphorylated tau in paired helical filament preparations 7a°. In order to expose possible hidden epitopes within NFTs, some sections were pre-
treated with 20% formic acid for 20 min prior to the application of the primary antibodies 6. For doubk, immunolabeling preparations, mouse monoclonals were visualized with a horse anti-mouse IgG antibody conjugated to fluorescein isothiocyanate (Vector Laboratories; diluted 1:200) and the rabbit polyclonal to tau was visualized with a goat anti-rabbit IgG antibody conjugated to biotin (Vector Laboratories; diluted 1:200) f(4iowed by streptavidin-Texas Red
Fig. I. Photomicrographsshowing multiple labeling for NF proteins, tau and thioflavine S in layer II of the entorhinal cortex in the older control cases. A, B, C show an example of layer II cells labeled for non-phosphorylated NF protein (SM132, A), tau (B) and thioflavine S (C). Thioflavine staining presents as yellow fluorescence with the filter block (filter set 487916, Zeiss) used to visualize FITC (green) in A. Tau and thioflavine positive NFTs (e.g. arrows) were present within the neurons labeled with SM132. C and D show an example of a NIT double labeled for both SM132 (D) and tau (E). Note that $M132 labels bundles of filaments within the tau immunoreactive tangle. E and F show double labeling for phosphorylated NF protein epitopes (SMI310) (F) and tau (G) in a formic acid pretreated tissue section. SMI310, in addition to labeling nerve fibres in this region, also labels the NFTs immunoreactive for tau (arrows). Bars = 30 p,m (A,B,C,F,G), 20 p.m (D,E).
275 ( A m e r s h a m , Arlington Heights, IL; diluted 1:200). Secondary antibodies were tested for possible cross reactivity. As previously described m3'26, n e u r o n s in layer II of the entorhinal cortex and pyramidal cells in o t h e r layers of the entorhinal cortex and the prefrontal cortex of all control cases were selectively labeled with SMI32. In contrast, SMI310 preferentially labeled axons in these control cases.
In the five older control cases (47-85 years of age), multiple fluorescent labeling d e m o n s t r a t e d that many layer I1 cells in the entorhinal cortex were preferentially involved in the process o f / o r m i n g NFTs. In these cases, most thioflavine-positive NF Ts occurred inside intact SM132-1abeled neurons and the N F T structure itself was intensely immunoreactive for tau and often labeled, in part, with SM132 (Fig. 1 A - E ) . All taulabeled NFT-like structures and most neuropil threads
ii ii!iiii!iii!iilii!ii!lii!i!fiiiii!i!i!!ii!i!li!ii!i !
ili !iii!i
Fig. 2. Photomicrographs showing multiple labeling in layer II of the entorhinal cortex and prefrontal cortex of AD cases. A, B and C show an example of labeling for non-phosphorylated NF proteins (SM132) (A), tau (B) and thioflavine S in layer II of the entorhinal cortex. Whereas NFTs in this region are thioflavine positive (C and yellow fluorescence in A, arrows), these NFTs are not contained within SM132 labeled neurons and show little or no immunoreactivity for tau (e.g. arrows). In layers IH (D,E,F) and V (G,H,I) of the prefrontal cortex (area 9) of these AD cases, many neurons demonstrate NFT-related alterations in SM132 (D,G) and tau (E,H) labelling. F and I are double exposure microphotographs showing the relationship between alterations in these cytoskeletal proteins with overlapping immunoreactivity appearing as yellow. Many of the SM132-1abeled neurons in D, E and F contain tau-immunoreactive NFT material which is in part labeled with SMl32 (arrows). The NFT in G, H, I is labeled in part with SM132 (arrow). Bars -- 20 ,¢m (A,B,C), 30 gm (D,E,F,G,H,I).
276 were also thioflavine-positive. Many of these NFT were labeled with SMI310. Formic acid pretreatment of tissue sections resulted in the immunolabeling of more NFT in layer 11 of the entorhinal cortex with SMI310, but did not alter the number of NFT labeled with SM132 or the antibody to tau. Formic acid pretreatmerit also eliminated thioflavine staining which suggests that this process may degrade some of the conformational features of the substructure of the tangle to expose the phosphorylation-dependent NF protein epitopes. Double labeling demonstrated that all tau-immunoreactive NFTs in these cases were also labeled with SMI310 following formic acid pretreatment (Fig. IF, G). A few NFTs were present.in the hippocampal CA1 field and in deeper entorhinal layers of the 66and 85-year-old control cases. NFTs were not detected with the above markers in the prefrontal cortex of the control cases, in the AD cases, thioflavine-positive NFT corresponding to end-stage extracellular NFT were abundant in layer li of the entorhinai cortex. Virtually all of these NFT were unlabeled with SMI32, SMI310 or the antibody to tau (Fig. 2A-C). In contrast, many neurons in the prefrontal cortex were in various transitional states that reflec,:d the formation of a NFT. In a similar fashion to layer I1 cells of the entorhinal cortex of the older control cases, many of the neocortical NITs were labeled with thioflavine and the tau antibody and were present within morphologically intact SM132.1abeled pyramidal cells (Fig. 2D-F). In addition, many of the NITs in this cortical region were immunoreactiv¢ for tau and labeled, in part, with SM!32 (Fig. 2D-l). Some end-stage NFT, stained with thioflavine but not labeled for tau or SM132, were also present in the prefrontal cortex. All tau immunoreactire NFT-like structures and most neuropil threads in these AD cases were also thioflavine-positive. Only a few NFTs in the prefrontal cortex were labeled with SM1310. Formic acid pretreatment of tissue sections dramatically increased the number of NFTs labeled with SMI310 in the neocortex and in layer.~ I!! to V! of the entorhinal cortex but did not increase the number of NFTs labeled in layer !1 of the entorhinal cortex. Formic acid pretreatment did not increase the number of NFT labeled with SMI32 or tau in the entorhinal cortex or neocortex of the AD cases. On the basis of their cytoskeletal protein immunoreactivity, NFTs can therefore be subdivided into transitional forms that are labeled with thioflavine and contain NF and tau protein epitopes or end-stage NFTs that are stained with thioflavine but are non-immunoreactive for particular tau epitopes and NF proteins. These results support the proposal that NFT
formation follows progressive alterations in the normal cytoskeleton 24'25. In addition, the cellular correlate of the differentiation between normal aging and dementia appears to be the point at which cytoskeletal changes progress from tr,~nsitional to end-stage in layer II of the entorhinal cortex, and begin to involve a large enough contingent of neocortical pyramidal cells to affect cort~cocortical integration. The localization of NFT in the entorhinal cortex using conventional methods has been noted in a significant number of control cases from other studies 5.23,29,32 whereas, in AD, severity of dementia has been correlated with increasing numbers of NFTs in the neocortex I's'29'37. Therefore, cytoskeletal pathology manifested as NFT-like changes in entorhinal cortex may not be uncommon in neurologically normal individuals in their fourth and fifth decades while, in AD, this process is facilitated by unknown factors to result in the progression of NFTs to their end-stage in most entorhinal layer II cells and the appearance of NFT-as. sociated cytoskeletal alterations in neocortical areas. Indeed, the specific pathological changes observed in the entorhinal cortex during normal aging may form the basis of subtle memory impairments known as "benign senescent forgetfulness". Whereas immunohistochemical and biochemical analysis has confirmed that some region of the tau polypeptide forms part of the core of PHFs that comprise NIT 7's,l°'t2't~,zt,'~s, the role of the neurofilament proteins in NIT formation is controversial, partly due to the cross-reactivity of some of the anti-NF antibod. ies that recognize NFT with tau proteins ta.27. The results of this study further support the hypothesis that the neurons that selectively contain the NF triplet are also the cells that are particularly vulnerable to NFT formation tx~4':6'~6. Cortical neurons that lack these NF proteins, such as GABAergic interneurons, are not as prone to degeneration in the disease 4s.~s,'6`2z,2s. The current results present evidence that NF protein antibodies that do not cross-react with tau can label particular forms of NFTs. NF polypeptides possibly become incorporated into NFTs initially in a non-phosphory. lated form and subsequently become abnormally phosphorylated, as has been shown for the tau component of NFTs 10,19,21. Phosphorylation of similar sequences present in both tau and the NF proteins, possibly by an abnormally functioning kinase, may underlie the alterations and association of these cytoskeletal proteins within NFTs. Further conformational changes associated with the formation of end-stage NFTs may mask NF and tau epitopes, or, alternatively, these regions may be cleaved from the essential NFT subcomponents. Particularly, it
277
has been noted that antibodies that recognize the repeat region in the tau carboxyterminal domain recognize NFTs that correspond to endstage as presented in this report 3':'4. The issue of the incorporation of regions of NF proteins into NFTs "~slimited by difficulties in the detection of further particular NF epitopes (e.g. the 68 kDa NF subunit, peripherin, alpha-internexin) in aldehyde-fixed tissues 34-36 and the unknown identity of the insoluble fractions of current paired helical filament preparations. Regardless, the progressive sequestering of microtubule-associated proteins and some region of the NF proteins into NFT may be the factor that leads to the destabilization and dissolution of the normal neuronal cytoskeleton. Hence, even the early stages of NFT formation in morphologically intact neurons would have profound effects on normal neuronal function. While the precise causative factors leading to NFT formation are unclear, the progressive alteration of cytoskeletal proteins may provide the basis for the neuronal dysfunction and degeneration underlying the development of dementia. The present data are, therefore, most compelling when placed in the context of a continuum that begins with aberrant intraneuronal biochemical processing that is manifested as differential cellular vulnerability and ends with dementia. The importance of the pathological involvement of subsets of neocortical neurons in AD is exemplified by the role of the neocortex as the putative substrate for long-term declarative memory 3~ and as the region underlying higher cognitive processes ~, Further, it has been proposed that memory functions that are initially dependent on hippocampal circuits may become largely independent once stored in neocortex 3~, which may in turn explain the relatively benign disruption of neurological function in the controls with significant hippocampai damage but no neocortical pathology. Agents that stabilize the neuronal cytoskeleton may prove useful in ameliorating this progressive degeneration, and, if used in conjunction with sensitive neuropsychological tests that distinguish between age-related and AD-related cognitive dysfunction, the cytoskeletal pathology might be halted prior to development of the devastating aspects of dementia that result from neocortical involvement. We would like to thank our colleagues at the Fishberg Research Center for Neurobiology as well as Dr. John Trojanowski aad Dr. Neil Kowall for their valued comments on the manuscript. We also thank William Janssen for technical support and Bob Woolley for photographic assistance, in addition, we would like to acknowledge the Institute of Biogerontology Research Tissue Donation Program (Sun City, Arizona) for the provision of many of the cases in the present study. This work was funded by AG05138 and AG06647 from the National Institute for Aging and AHAF. James Vickers is a
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