J. pswhiaf. Res ., vol . 24, No . I, pp 37-50, 1990 . Printed in Great Britain .
0022-3956/90 53 .00 + .00 G' 1990 Pergamon Press plc
ALZHEIMER CORTICAL NEURONS CONTAINING ABUNDANT AMYLOID mRNA . RELATIONSHIP TO AMYLOID DEPOSITION AND SENILE PLAQUES WEN-GANG CHOU, * SAYEEDA B . ZAIN,* RONALD
E.
S.
REFm4AN, • BARBARA TATE-OSTROFF,ttlI
MASOCHA,tt§II FRANCINE M . BENESttII
and
CHARLES A . MAROTTAt$§II
-Cancer Center and Department of Biochemistry, University of Rochester Medical School, Rochester, NY 14642, U .S .A . ; tDepartment of Psychiatry and Neuroscience Program ; tMailman Research Center, McLean Hospital, Belmont MA 02178, U .S .A . ; §Massachusetts General Hospital, Boston, MA 02114, U .S .A. ; and [Harvard Medical School, Boston, MA 02115, U .S .A .
(Received 6 July 1989 ; revised 29 September 1989) Summary-Since the detailed molecular events leading to the formation of amyloid-containing senile plaques of the Alzheimer's disease (AD) brain are incompletely understood, the present studies were undertaken to address this issue using a combination of molecular and cytochemical approaches . Amyloid precursor protein riboprobes containing the A4 (5-amyloid) domain were applied to cortex using the in situ hybridization method to examine the distribution of neuronal amyloid mRNA in relation to the laminar pattern of amyloid deposition and the localization of plaques . The derived data indicated that high levels of amyloid mRNA can be synthesized by AD cortical neurons that appeared to be morphologically intact . The distribution of these cells was not coincident with the cortical laminar pattern that is typical of amyloid deposits observed after immunostaining with anti-A4 monoclonal antibodies . Further, there was no obvious relationship between neurons containing abundant amyloid mRNA and the distribution of plaques identified by thiofavin S staining . While the neuronal synthesis of amyloid may be a significant factor at some point during plaque formation, it may not be the exclusive determinant . The possibility is raised that processes affecting secretion, diffusion, and/or transport of amyloid away from neuronal or non-neuronal cells of origin to sites of deposition may be meaningful aspects of the molecular pathology of Alzheimer's disease . INTRODUCTION
senile plaques are found in higher density in Alzheimer's disease (AD) than in normal aging and are characteristic pathologic markers that have been linked to intellectural decline (BLESSED, TOMLINSON, & ROTH, 1968) . Thus, understanding genetic and cellular mechanisms that affect amyloid accumulation may provide insight into diseaserelated degenerative changes of the AD brain as well as the normal aging process . Using in situ hybridization methods a number of studies have addressed the localization of amyloid A4 (/3-peptide) mRNA with regard to areas of the AD brain that are commonly affected (BAHMANYAR, HIGGINS, GOLDABER, LEWIS, MORRISION, WILSON, SHANKAR, & GAJDUSEK, 1987 ; GOEDERT, 1987 ; HIGGINS, LEWIS, BAHMANYAR, GOLDABER, GAIDUSEK, YOUNG, MORRISON, & WILSON, 1988 ; LEWIS, CAMPBELL, TERRY, & MORRISION, 1988 ; COHEN, GOLDE, USL4K, YOUNKIN, & YOUNKIN, 1988 ; PALMERT, GOLDE, COHEN, KOVACS, TANzi, AMYLOm-CONTAINING
Address for correspondence : Dr . Charles A . Marotta, Neurobiology Laboratory, Bullfinch 4, Massachusetts General Hospital, Boston, MA 02114 U .S .A ., Tel . (617) 729-5211 37
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Wen-GANG CBOU et a!.
GUSELLA, USIAK, YOUNKIN, & YOUNKIN, 1988) . It is generally agreed that neurons contain appreciable amounts of amyloid mRNA . There is uncertainty, however, concerning the significance of amyloid mRNA levels with regard to cortical and subcortical sites that are typically affected by neuritic plaques and neurofibrillary tangles . Indeed, both relatively high and low levels of neuronal amyloid mRNA have been observed in cortex, hippocampus, and other brain regions in AD (BAHMANYAR et al., 1987 ; CHOU, MAJOCHA, BENES, ZAIN, & MAROTTA, 1987 ; GOEDERT, 1987 ; HIGGINS et al., 1988 ; LEwis et al., 1988 ; COHEN et
al., 1988 ; PALMERT et al ., 1988) . Consequently, the detailed mechanism by which amyloid mRNA regulation may influence the development of the characteristic neuropathological changes is an unsettled issue . The frequency of AD cases with high neuronal amyloid precursor protein mRNA levels in cortical and subcortical regions in not clear . However, the occurrence of such cases, once one is identified, permits questions to be addressed concerning the relationship, if any, between abundant amyloid mRNA production and the molecular pathogenesis of AD . Thus, in the present studies we asked whether or not there is a correlation between high levels of amyloid mRNA per neuron, as measured by quantitative in situ hybridization methods, with altered neuronal morphology and the distribution of senile plaques in various cortical layers . The latter was of particular interest since in an earlier report we observed the non-random deposition of A4 amyloid in the lamellae of the AD cortex using highly specific monoclonal antibodies (MAJOCHA, BENES, RODENRYS, & MAROTTA, 1988) . In the present study we compared plaque-containing areas of an AD brain to unaffected sites of the same cortex ; thus, we avoided the inherent variability that results from comparison of different postmortem cases . A portion of the present data were reported previously in preliminary form (CHOU et al., 1988a) . MATERIALS AND METHODS Tissues Normal aged and AD cases were obtained from the Massachusetts Alzheimer's Disease Research Center (Neuropathology core at the Massachusetts General Hospital) and the McLean Hospital Brain Tissue Resource Center .
Riboprobe preparation A cloned amyloid precursor protein cDNA, referred to as amy37, was prepared from AD mRNA and was previously characterized (ZAIN, SALIM, CHOU, SAJDEL-SULKOW5IA, MAJOCHA, & MAROTTA, 1988) . The cDNA insert was cut with Eco RI at the beginning of the A4 site to generate a 1 .1 Kb fragment spanning nucleotides 1795-2851 (KANG, LEMAIRE, UNTERBECK, SALBAUM, MASTERS, CRZESCHIK, MULTHAUP, BEYREUTHER, & MULLER-HILL, 1987 ; ZAIN et al., 1988) . The 1 .1 Kb fragment was purified from an agarose gel (SALIM, REHMAN, SAJDEL-SULKOWSKA, CHOU, MAJOCHA, MAROTTA, & ZAIN, 1988) and subcloned into the Eco RI site of plasmid pGEM I (Promega) in both orientations . The recombinant plasmids were digested with BamHI to linearize them . Single-stranded RNA probes (riboprobes) were generated in the sense and antisense orientation using T7 RNA polymerase and ( 3 H)UTP and ('H)CTP to a specific activity of 3 .3x107cpm/ug . The riboprobes were partially hydrolyzed to approximately 300 bases (average) by mild alkaline treatment (Fig . I) .
ALZHEIMER Amnom
MESSENGER
RNA
39
In situ hybridization Formalin fixed, paraffin embedded sections of post-mortem human control or AD frontal cortex area 9 were used for in situ hybridization and cytochemical studies . The general procedure of ANGERER, Cox, and ANGERER (1985) was applied . Five µm-thick sections were mounted on clean glass slides pre-treated with a 2% solution of 3 aminopropyltriethoxysilane (Aldrich) in acetone . The slides were dipped in xylene and rehydrated in decreasing concentrations of ethanol in water . The sections were then treated for 30 min with a I µg/ml solution of Proteinase K (Sigma) in 50 mM EDTA, 100 mM Tris pH 8 .0 at 37°C . Sections were then washed in water and dipped in a solution of 0 .25% acetic anhydride (freshly made, immediately before use) in 0 .1 M triethanolamine pH 8 .0 for 10 min and washed with 2x SSC (1 x SSC = 0 .15 M NaCl, 0 .015 M sodium citrate) . Sections were dehydrated with increasing concentrations of ethanol to 100% . Tissue was prehybridized in hybridization buffer (see below) for 30 min at 37°C . Radiolabeled probes were diluted to 2 .7-5 .4 x 10 4 cpm/µl in 37 µl hybridization buffer : 0 .3 M NaCl, 10 mM Tris pH 8 .0, 1 mM EDTA, 10% dextran sulfate, 0 .25% BSA, 0 .25% Ficoll 400, 0 .25% polyvinylpyrrolidone 360, 50% deionized formamide (Baker) and 500 µg/ml t-RNA . Annealing was carried out at 45°C for 16 h . Excess probe was removed by rinsing in 4 x SSC four times (5 min each) . Sections were treated for 30 min at 37°C with a solution of RNase A (20 µg/ml) and RNase TI (1 unit/ml) (Sigma) in 0 .5 M NaCl, 10 mM Tris pH 8 .0, then immersed in 2 x SSC for 30 min at room temperature . High stringency washing was done in 0 .1 x SSC at 65°C for 15 min and transferred to another 0 .1 x SSC solution for 30 min at room temperature . The sections were dehydrated in a series of increasing ethanol solutions with 0 .3 M ammonium acetate and finally in 100% ethanol . They were dipped in KodakT" NTB-2 emulsion diluted 1 :1 with H2O and ammonium acetate at a 0 .3 M final concentration . After drying, all slides were stored desiccated at 4°C for up to three weeks . They were developed in Kodak D-19 for 5 min, washed and put into Kodak rapid fixer for 5 min, then rinsed . Sections were counterstained with hematoxylin and eosin and coverslipped with permount . A D and control cases were processed simultaneously . Immunocytology and tissue staining
The procedure for immunostaining cortical sections with anti-A4 monoclonal antibodies and for quantitating amyloid deposits followed the previously described protocols (MA ocHA et a!., 1988 ; MAJOCHA, MAROTTA, & BENES, 1985) except that the average of five fields from a single case were examined in the present study . Staining with thioflavin S was described elsewhere (TATS-OSTROFF, MAJOCHA, & MAROTTA, 1989) . Tissue used for immunostaining was obtained from the same block as tissue used for in situ hybridization . Sections of 40 µm were cut on a cryostat from tissue infiltrated with sucrose . Grain counting in cortical layers
Specimens processed autoradiographically to visualize hybridization probes at the light microscopic level were subjected to quantitative analyses using a Bioquant Advanced System IV (R & M Biometrics) that permits automated image processing and grain counting . The specimens were visualized with a Leitz Laborlux 12 microscope . The system was calibrated to detect particles of a size and density of the autoradiographic grains visualized with a
40
WEN-GANG CHOU et al.
100x oil immersion objective . The data are expressed as grains per microscopic field . Grain counting in relation to neuronal size
In the case of probes that were localized to neuronal cell bodies, the images of individual cells were processed through the analysis system so that the corresponding pixels could be quantitated . Highlighted pixels of each neuron were computed automatically and the numbers of grains present were expressed either per cell or as a function of cellular area (µm 2) using a bivariate regression analysis . Data for normal control and AD cases were compared using a two-tailed Student's t-test . Simple linear regression analysis was used to examine the relationship between grain counts and cell size expressed in µm 2 by generating a correlation coefficient (r) . Grain counting in relation to senile plaques Tissue sections subjected to both in situ hybridization and thioflavin S staining were
visualized and photographed using the 40 x objective under dark field and fluorescence optics of a Zeiss microscope . All photomicrographs, with a final magnification of 570 x , were taken with a Zeiss MC63, photographic system . Structures stained by thioflavin S were visualized with an epifluorescent illuminator equipped with a fluorescein excitation and barrier filter set . Using the same AD case (A67), forty-seven fields with an area equal to 940µm 2 were obtained and the number of plaques and cells within each region were counted . The mean number of grains per cell plus surrounding neuropil, in regions with or without plaques, was then determined . RESULTS
Selection of riboprobes and tissues
Previously we cloned and characterized amyloid precursor protein cDNA that had been obtained from mRNA of an AD brain (ZAIN et al., 1988) . The region encompassing the A4 domain was excised by digestion with EcoRI and isolated (MAROTTA, CHOU, MAJOCHA, WATKINS & ZAIN, 1989) . The resulting 1 .1 Kb fragment was used to prepare tritiated ripoprobes in the sense and antisense orientation (Fig . 1, lanes a and c) . After a limited alkaline digestion to yield polynucleotides of smaller sizes (Fig . 1, lanes b and d) the probes were applied to formalin fixed cortical sections for in situ hybridization, as described in the Materials and Methods section . Four AD and 4 normal control cortices were screened with the riboprobes . Based on this preliminary data, we selected for detailed study a 67 yr old case (referred to as A67, post-mortem interval 11 h) that had the highest density of antisense tritated grains in the series (see below) . For comparison we examined a matched control (69 yr old, referred to as C69, post-mortem interval 12 h) . Both tissues were from Brodman area 9 of Caucasian females . Immunostaining with anti-A4 Mobs
A combination of three Mabs to A4 amyloid, previously characterized (MAJOCHA el al ., 1988) were applied to the AD and control cases . In A67, extensive amyloid deposition was observed throughout the layers of the cortex (Fig . 2A) . The normal control was unreactive with the Mab mixture (Fig . 2B) .
ALZHEIMER AMYLOID MESSENGER RNA
41
1 .4-
0.8-
0 .2-
a b c d
FIG . 1 . Characterization of riboprobes . The riboprobes amy12 (sense, lane a) and amyl5 (anti-sense, lane c) were synthesized using T7 polynterase and tritiated UTF and CTP . Shown are the products before (lanes a and c) and after (lanes h and d) mild alkaline treatment . The samples were electrophoresed on a 1 .4% agarose gel containing 6% formaldehyde . The gel was treated with enhancer (NEN) and dried for autolluorography . The size of markers, in Kb, is indicated .
42
WEN-GANG Cnou et a!.
FIG . 2 . 1 in munostaining of AD and control cortices with monoclonal antibodies to A4 polypeptide (see Materials
and Methods). (A) A67 frontal cortex, layer V . (R) C69 frontal cortex, layer V . In both cases the bar = 50 µm .
ALZHEIMER AMYLOID MESSENGER
RNA
43
Ftc . 3 . In situ hybridization of AD and control frontal cortices (layer V except where indicated) . (A) Antisense probe applied to A67 (dark field) . (B) Antisense probe applied to A67 and shown at higher magnification to demonstrate hybridization to neurons (bright field) . Arrows indicate two neurons labeled with grains . (C) Antisense probe applied to A67 layer VI . (D) Anrisense probe applied to C69 at low magnification (dark field) . (E) Same as D, at higher magnification (bright field) . (F) Sense probe applied to A67 . In all cases the bar = 20 µm .
44
WFN-GANG CH0r et at.
Fur . 4 . 'I - hioflavin stain of tissue section processed for in situ hybridization to demonstrate the co-localization of riboprobe and senile plaques . AD cortex was processed for in situ hybridization and then stained with thioflavin S as described in the Material and Methods . Shown are grains and plaques (Fluorescent stain) of cortical layer III . Bar - 20 µm .
ALZHEIMER AMVLOIO MESSENGER RNA
45
In situ hybridization Both control and AD brains were processed for in situ hybridization using the antisense riboprobe and viewed with both dark and bright field illumination (Fig . 3) . Large and small neurons of grey matter were labeled in AD case A67 . Although tritiated grains were present to an extent in all AD cortical layers (see below for quantitation, Table 2), layer III and V neurons exhibited the most intense signal . Layer V is shown in Fig . 3A . The indicated neurons (arrows, Fig . 3B) were typical of those in A67 cortical layers III and V and frequently appeared to have normal morphology . However, some of the smallest apparently normal neurons, including those at the junction of gray and white matter, also contained tritiated grains (Fig . 3C) . The AD case under scrutiny had high signal levels when compared to 3 other AD cases and when compared to four neurologically normal controls ; the matched case, C69, is shown for comparison (Fig . 3D, E) . The sense strand riboprobe applied to both control and AD cortices did not appear to exhibit consistent clustering of grains over perikarya (Fig . 3F) . Quantitation of amyloid mRNA in cortical cells
The occurrence of an AD case with the qualitative appearance of high levels of amyloid mRNA relative to a matched control was verified by quantitating the grain distribution . The mean number of tritiated grains per large pyramidal neuron (of approximately 100 µm 2) was determined (Table 1) . For both layers III and V of the AD brain, antisense and TABLE 1 . MEAN NUMBER OF GRAINS PER CELL
f
Layer III A67
SEW
A4 antisense
A4 sense
probe
probe
7 .37
f
0 .88
1 .82 t 0 .14
n=75 C69
2 .56 t 0 .17
n=51 2 .46 i 0 .22
n=77
n=56
Layer V A67 C69
12 .55
3
1 .02
n=44
3 .15
4 .33 i 0 .43 n=49
3
0 .38
n=33 1 .69 t 0 .18 n=64
*The mean number of grains per cell were counted in the large pyramidal neurons of layers IIl and V of Alzheimer case A69 by the methods previously described .
sense strands of the riboprobe showed significant differences in the mean number of grains per cell (p < 0 .01) . There was also a significant increase (p < 0 .01) in the mean number of grains of the antisense probe in the AD case relative to the same probe in the control for both layers III and V (Table 1) . The results of the regression analysis showed the correlation coefficient for cellular area and number of grains was high for the antisense probe in AD layer III and layer V neurons (r = 0 .861 and 0 .690, respectively) . Distribution of amyloid mRNA in cortical layers
AD brain A67 was used to assess the laminar distribution of neurons with high levels
46
WEN-(TANG CHOH et at.
of amyloid mRNA in relation to the deposition of immunologically detectable A4 amyloid . The average number of immunodetectable amyloid deposits of all morphologic types, per unit area, were! 68 .6 f 4 .5 ; 36 .2 t 7 .7 ; 41 .2 t 7 .7 ; 15 .0 t 4 .6 ; and 8 .4 t 1 .2 for layers 1, 11, III, V and VI, respectively . The distribution was in general agreement with previous observations made on four other AD cases (MAJOCHA et al ., 1988) . Table 2 shows the mean number of grains of the antisense and sense probes per field TABLE 2 . MEAN NUMBER OF GRAINS PER FIELD t SEM PER ALZHEIMER CORTICAL LAYER' Cortical layer
n
A4 antisense probe
Layer 1 Layer II Layer 111 Layer V Layer VI
10 10 10 10 10
27 .90 56 .40 83 .70 71 .30 54 .30
f 3 + t L
1 .63 5 .01 6 .17 6 .88 5 .42
A4 sense probe 32 .00 28 .50 33 .40 32 .00 28 .00
t 3 t : t
4 .71 3 .24 4 .30 3 .74 3 .47
'Field size, Bioquant screen at x 100 magnification .
in each cortical layer of case A67 . Layers II-VI had the highest hybridization signals and of these, layers III and V were the most highly labeled . Grain counts for the sense probe were nearly the same in all layers (Table 2) . Unlike the distribution of the A4 amyloid peptide, which showed decreasing numbers of deposits with cortical depth, the distribution of the antisense hybridization probe appeared to reflect the pattern of neuronal size and numbers characteristic of the cortical layers . Neuronal amyloid mRNA and senile plaques AD case A67 examined by in situ hybridization, was stained with thioflavin S to demonstrate neuritic plaques . The photomicrograph of layer III, shown in Fig . 4, is a typical result . Rarely did we observe high levels of tritiated grains clustered around thioflavinpositive material . For quantitative studies, grain counts were carried out in the vicinity of plaques (neuropil and cellular areas) and compared with similar determinations of grains present over cells or in areas containing neither cells nor plaques (area = 940 µm 2) . The data revealed that the mean number of grains (25 .9 t 1 .3) was highest in regions containing cells but unassociated with plaques (N= 12 areas examined) ; the average value was more than twice that in neuropil (12 .0 t 1 .5 grains ; N = 15 areas examined) . The lowest values were obtained for regions containing plaques (7 .6 t 1 .4 grains ; N= 20 areas examined ; p < 0 .001 when compared to cellular areas) . In the latter study, only 3 cells were found in close proximity to plaques after analysis of twenty 940 µm 2 tissue areas . However, when cells were present, their mean density of grains (13 .7 grains/cell) was equivalent to cells unassociated with plaques (13 .4 grains per cell, and see Table 1) . DISCUSSION
Application of the in situ hybridization procedure to detect mRNA at the cellular level has been a useful tool for over a decade and has been validated for neuron-specific markers in brain (e .g ., Urn. & SASEK, 1986) . Using different procedural approaches, several previous
47
ALZHEIMER AMrioID MESSENGER RNA
reports focussed on amyloid mRNA in control and AD brains with attention to RNA levels, differential expression in various cell types, and relationship to pathologic markers characteristic of AD (BAHMANYAR et al., 1987 ; CHOU et al ., 1988b ; GOEDERT, 1987 ; HIGGINS et al ., 1988 ; LEWIS et al ., 1988 ; COHEN et al., 1988 ; PALMERT et al., 1988) . In the most widely applied technique, and the one adopted in the present study, the 1 .1 Kb segment of the amyloid precursor protein mRNA which contains the A4 region was lebeled and used for in situ hybridization on fixed post-mortem tissues . However, unlike probes labeled with tritium, as described here, earlier studies relied on 35S or 3zP radiolabels which resulted in grain clusters that tended to obscure cellular morphology . Nevertheless, it has been generally concluded that cortical and subcortical neurons have appreciable levels of amyloid mRNA (BAHMANYAR et al.,
1987 ; CHou et al., 1988b ;
GOUDERT, 1987 ; HIGGINS et al., 1988 ; LEWIS et al., 1988) . In certain instances in situ hybridization studies led to the conclusion that the amyloid mRNA was often decreased in AD relative to controls when frontal cortex was examined, and this result was consistent with Northern blot analyses (BAHMANYAR et al., 1987 ; GOEDERT, 1987) . However, other studies have led to the opposite conclusion concerning cortical and/or subcortical areas (HIGGINS et al., 1988 ; LEWIS et al., 1988 ; COHEN et al., 1988 ; PALMERT et al., 1988) . At the very least, earlier studies have observed that certain neurons of the AD brain may contain increased levels of amyloid mRNA at some point during the course of the disease. With this view as a starting point, we screened a series of cortices to identify those with notably high levels of amyloid mRNA within neurons . Having identified a suitable case, we asked whether or not conclusions could be drawn concerning sites of abundant amyloid mRNA and plaque localization . Further, we utilized a double labeling method which allowed us to assess mRNA abundance in the vicinity of thioflavin S positive plaques . An AD frontal cortex with dense deposits of immunologically detectable amyloid contained neurons with abundant amyloid mRNA . With regard to this neuronal population, overproduction of amyloid mRNA did not appear to be associated with an alteration in gross cellular morphology of fixed brain tissue as assessed at the light microscope level . These data do not bear on possible biochemical changes and fine structural alterations that may have occurred and which were not evaluated in the present investigation . Further, no conclusions can be drawn concerning the long-term consequence of high levels of amyloid synthesis since post-mortem studies provide solely a static picture during one phase of the disease . It is possible that certain neocortical and hippocampal neurons that produce abundant amyloid for prolonged periods may undergo degeneration . When compared to a control cortex, the antisense probe gave a much higher signal than the sense probe in all AD cortical layers except the first, where the values were nearly the same . This may be related to the paucity of neurons in layer 1, although it has been shown that glia contain the amyloid precursor protein (TATE-OSTROFF et al., 1989) . In terms of relative abundance, the distribution of amyloid mRNA in the AD cortex was not coincident with the distribution of amyloid deposits identified by anti-A4 Mabs which show a gradient of density with highest levels occurring in Layer I and lowest in Layer VI (MASOCHA et
a!., 1988 and see Results and Table 2) . Further, in double labeling studies utilizing cortical sections subjected to both in situ hybridization and thioflavin S staining, we did not observe a preponderance of riboprobe in the immediate vicinity of senile plaques in twenty separate
48
WEN-GANG CHOU
et al.
determinations . These data and the previous observations suggest that the amyloid component of senile plaques may be derived from non-adjacent sources . For example, while synthesis of amyloid precursor protein occurs in cell bodies, the release and deposition of amyloid may occur distally at axon terminations . Thus, amyloid deposits corresponding to mRNA localized in a neuron somata may be present in another region of the cortex to which the neuron projects . Other factors, however, bear on this result . The possibility exists that extensive cell loss in the vicinity of plaques is a major contributing factor to the decreased hybridization signal . Since the progression of disease is not amenable to direct observation by post-mortem studies, we cannot rule out the possibility that numerous cells with abundant amyloid mRNA were present at earlier stages . It was interesting to note that surviving neurons adjacent to senile plaques contained as much amyloid mRNA as cells in non-plaque areas . While evidence has accumulated to implicate neuronal amyloid as a contributing factor to plaque formation (MASTERS, MULTHAUP, SIMMS, POTTGIESSER, MARTINS, & BEYREUTHER, 1985), additional mechanisms may be influential determinants . A similar conclusion was reached by LEWIS et al. (1988) . Previously we suggested that the distribution and morphology of prefrontal cortical amyloid deposits, as revealed by immunocytochemistry, may be dependent upon underlying laminar-specific structures of the neocortex (MAJOCHA et al., 1988) . These may include dendrites and/or cortically projecting axons from within the cortex or subcortical structures (LEWIS et al ., 1988 ; ISHII, 1966 ; MAJOCHA et al., 1988 ; MASTERS el al., 1985 ; SuzuKI & TERRY, 1967) . Further, we considered that diffusional processes, possibly secondary to
secretion, may be involved in extracellular amyloid accumulation
(BENES, REtFEL, MAJocHA,
This possibility was supported by the recent finding that an extracytoplasmic domain of the APP is found at extracellular sites in the AD brain and can occur within senile plaques (TATE-OSTROFF et al., 1989) . Since the cellular source of amyloid that contributes to senile plaques has not been definitively identified, the present and previous studies do not exclude a vascular origin for plaque amyloid (GLENNER, 1979) or the possible contribution of non-neuronal cells & MAROTTA, 1989 ; MAJOCHA et al ., 1988 ; TATE-OSTROFF et al., 1989) .
(WISNIEWSKI & MERZ, 1983) .
The combined use of in situ hybridization and selective staining methods applied to large numbers of brains may help to define the temporal relationship between vascular or cellular events that influence senile plaque formation . With regard to cellular functioning, we can hypothesize that amyloid mRNA is normally present at basal levels . In AD, an event, as yet undefined, may cause amyloid to be overproduced, possibly transiently . Disregulation at the transcriptional or translational level may be involved . At the cytoplasmic level, the effects of proteolytic processing must also be taken into account . Subsequently, amyloid or cleavage products may be transported from the cell by secretion and diffusion away from the site of synthesis and contribute to amyloid plaques ; and/or there may be transport of amyloid along the axon to the terminus with subsequent deposition . Both mechanisms would result in amyloid-containing plaques occurring at sites that are non-adjacent to immediately surrounding neurons . Acknowledgements-The authors thank Jennifer Reifet for excellent technical assistance . This research was supported by the following grants : AG 02126, CA 11198, CA 36432 from the National Institutes of Health ; and
ALZHEIMER AMYLOID MESSENGER RNA
49
by the Axelrod Family Fund, the Katherine Smith Bolt Fund, the American Health Assistance Foundation and the Sandoz Foundation for Gerontological Research . Brain tissues were supplied by the Massachusetts Alzheimer's Disease Research Center, supported by National Institutes of Health grant AGO5134 ; and the McLean Hospital Brain Tissue Resource Center, supported by grant MH/NS 31862 . REFERENCES ANGERER, K ., Cox, K ., & ANGERER, L. (1985) . In situ hybridization to cellular RNAs . Genetic Engineering . Principles and Methods 7, 43-65 . BAHMANYAR, S ., HIGGws, G ., GOLDGABER, D ., LEwts, D . A ., MORRISON, J . H ., WILSON, M. C ., SHANKAR, S . K ., & GAJDUSEK, D . C . (1987) . Localization of amyloid p protein messenger RNA in brains from patients with Alzheimer's disease . Science 237, 77-80. BENES, F . M ., RErnEL, J ., MAJOCIrA, R . E ., & MAROTTA, C . A . (1989). Evidence for diffusion of amyloid during plaque formation in Alzheimer brain . Neuroscience 33, 483-488 . BLESSED, G ., TOMLINSON, B ., & ROTH, M . (1968) . The association between quantitative measures of dementia and of senile changes in the cerebral grey matter of elderly subjects . British Journal of Psychiatry 114, 797-811 . COHEN, M . L ., GOLDE, T . E ., UsLAK, M . F ., YOUNKIN, L . H ., & YOUNKrN, S . C . (1988) . In situ hybridization of nucleus basalis neurons shows increased d-amyloid mRNA in Alzheimer disease . Proceedings of the National Academy of Science, U.S .A . 85, 1227-1231 . CHOU, W . G ., MAJOCHA, R . E ., BENES, F . M ., ZAIN, S . B ., & MAROTTA, C . A . (1988a) . Amyloid messenger RNA in the Alzheimer frontal cortex . Intraneuronal levels, relationship to cell size and laminar distribution . Society for Neuroscience 14, 638 . CHOU, W . G ., MAJocHA, R . E ., SAIDEL-SULKOWSKA, E . M ., BENES, F . M ., SALIM, M ., STOLER, M ., FULWILER, C . E, VENTOSA-MICHELMAN, M ., RODENRYS, A . M ., MOORE, K . E ., WEBB, T ., ZAIN, S . B ., & MAROTTA, C . A . (1988b) . Immunologic and molecular genetic studies on amyloid deposition in the Alzheimer's disease brain. Brain Dysfunction 1, 133-145 . GLENNER, G . G . (1979) . Congophilic microangiopathy in the pathogenesis of Alzheimer's syndrome (presenite dementia) . Medical Hypotheses 5, 1231-1236 . GOEDERT, M . (1987) . Neuronal localization of amyloid beta protein precursor mRNA in normal human brain and in Alzheimer's disease . EMBO Journal 6, 3627-3632 . HIGGINS, G . A ., LEwis, D . A ., BAHMANYAR, S ., GOLDGABER, D ., GAJUUSEK, D . C ., YOUNG, W . G ., MORRISON, J . H ., & Wnso&, M . C . (1988) . Differential regulation of amyloid-13-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease. Proceedings of the National Academy of Science, U.S.A . 85, 1297-1301 . ISHII, T . (1966) . Distribution of Alzheimer's neurofibrillary changes in the brain stem and hypothalmus of senile dementia . Acta Neuropathology 6, 181-187 . KANG, J ., LEMAIRE, J . G ., UNTERBECK, A ., SALBAUM, J . M ., MASTERS, C . L ., CRZESccLK, K . H ., MULTHAUP, G ., BEYREUTHER, K ., & MULLER-HILL, B . (1987) . The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor . Nature 325, 733-736 . LEwis, D . .A ., CAMPBELL, M, J ., TERRY, R . D ., & MoaxrsoN, J . H . (1987) . Laminar and regional distributions of neurofibrillary tangles and neuritic plaques in Alzheimer's disease : a quantitative study of visual and auditory cortices . Journal of Neuroscience 7, 1799-1808 . MAJOCHA, R . E ., BENES, F . M ., REIFEL, J . L ., RODENRYS, A . M ., & MAROTTA, C. A . (1988) . Laminar-specific distribution and infrastructural derail of amyloid in the Alzheimer cortex visualized by computer-enhanced imaging of unique epitopes . Proceedings of the National Academy of Science, U.S.A . 85, 6182-6186 . MAJOCHA, R . E ., MAROTTA, C . A ., & BENES, F . (1985) . Immunostaining of neurofilament protein in human postmortem cortex : a sensitive and specific approach to the pattern analysis of human cortical cytoarchitecture . Canadian Journal of Biochemistry and Cell Biology 63, 577-584 . MAROTTA, C . A ., CHOU, W . G ., MAJCJCHA, R . E ., WATKINS, R ., & ZAIN, S . B . (1989) . Overexpression of amyloid precursor protein A4 (p-amyloid) immunoreactivity in genetically transformed cells . Implications for a cellular model of Alzheimer amyloidosis. Proceedings of the National Academy of Science, U.S .A . 86, 337-341 . MASTERS, C ., MULTHAUP, G ., SIMMS, G ., POTTGIESSER, J ., MARTINS, R ., & BEYREUTHER, K . (1985) . Neuronat origin of a cerebral amyloid : neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels . EMBO Journal 4, 2757-2763 . MASTERS, C ., SIMMS, G ., W EINMAN, N . A ., MULTHAUT, G ., MCDONALD, B ., & BEYREUTHER, K . (1985) . Amylold plaque core protein in Alzheimer's disease and Down Syndrome . Proceedings of the National Academy of Science, U.S.A . 82, 4245-4249 . PALMERT, M . R ., GOLDE, T . E ., COHEN, M . L ., KOVACS, D . M ., TANZI, R . E ., GUSELLA, J . G ., USIAK, M . F ., YOUNKtN, L . H ., & YOUNKN, S . G . (1988) . Amylold protein precursor messenger RNAs : differential expression in Alzheimer's disease . Science 241, 1080-1084 .
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SALIM, M ., REHMAN, S ., SAJDEL-SULKOWSKA, E . M ., CHOU, W . G ., MAJocHA, R . E ., MAROTTA, C. A ., & ZAIN, S . B . (1988) . Preparation of a recombinant eDNA library from poly(A+) RNA of the Alzheimer brain . Identification and characterization of a eDNA copy encoding a glial-specific protein . Neurobiology of Aging 9, 163-171 . SUZOKI, K ., & TERRY, R . D . (1967) . Fine structural localization of acid phosphatase in senile plaques in Alzheimer's presenile dementia . Aria Neuropathologica (Berlin) 8, 276-284 . TATE-OSTROFF, B ., MAJOCHA, R . E ., & MARO'rTA, C . A . (1989) . Identification of cellular and extracetlular sites of amyloid precursor protein extracytoplasmic domain in normal and Alzheimer's brains . Proceedings of the National Academy of Science, U.S .A . 86, 745-749 . Urn., G . R ., & SASEK, C . A . (1986) . Somatostatin mRNA : Regional variation in hybridization densities in individual neurons . Journal of Neuroscience 6, 3258-3264 . WlsNtewsKr, H ., & MERZ, G . S . (1983) . Neuritic and amyloid plaques in senile dementia of the Alzheimer type . In R . KATZMAN (Ed .), Banbury report 15: Biological aspects of Alzheimer's disease (pp . 145-151) . New York : Cold Spring Harbor Laboratory . ZAIN, S . B ., SALIM, M ., CHOU, W . G ., SAJDEL-SULKOWSKA, E . Nl ., MAJOCHA, R . E ., & MAROTTA, C . A . (1988) . Molecular cloning of amyloid eDNA derived from mRNA of the Alzheimer brain . Coding and non-coding regions of the fetal precursor mRNA are expressed in the Alzheimer cortex . Proceedings of the National Academy of Science, U .S .A . 85, 929-933 .
0022-3956/90 53 .00 + .00 G' 1990 Pergamon Press plc
ALZHEIMER CORTICAL NEURONS CONTAINING ABUNDANT AMYLOID mRNA . RELATIONSHIP TO AMYLOID DEPOSITION AND SENILE PLAQUES WEN-GANG CHOU, * SAYEEDA B . ZAIN,* RONALD
E.
S.
REFm4AN, • BARBARA TATE-OSTROFF,ttlI
MASOCHA,tt§II FRANCINE M . BENESttII
and
CHARLES A . MAROTTAt$§II
-Cancer Center and Department of Biochemistry, University of Rochester Medical School, Rochester, NY 14642, U .S .A . ; tDepartment of Psychiatry and Neuroscience Program ; tMailman Research Center, McLean Hospital, Belmont MA 02178, U .S .A . ; §Massachusetts General Hospital, Boston, MA 02114, U .S .A. ; and [Harvard Medical School, Boston, MA 02115, U .S .A .
(Received 6 July 1989 ; revised 29 September 1989) Summary-Since the detailed molecular events leading to the formation of amyloid-containing senile plaques of the Alzheimer's disease (AD) brain are incompletely understood, the present studies were undertaken to address this issue using a combination of molecular and cytochemical approaches . Amyloid precursor protein riboprobes containing the A4 (5-amyloid) domain were applied to cortex using the in situ hybridization method to examine the distribution of neuronal amyloid mRNA in relation to the laminar pattern of amyloid deposition and the localization of plaques . The derived data indicated that high levels of amyloid mRNA can be synthesized by AD cortical neurons that appeared to be morphologically intact . The distribution of these cells was not coincident with the cortical laminar pattern that is typical of amyloid deposits observed after immunostaining with anti-A4 monoclonal antibodies . Further, there was no obvious relationship between neurons containing abundant amyloid mRNA and the distribution of plaques identified by thiofavin S staining . While the neuronal synthesis of amyloid may be a significant factor at some point during plaque formation, it may not be the exclusive determinant . The possibility is raised that processes affecting secretion, diffusion, and/or transport of amyloid away from neuronal or non-neuronal cells of origin to sites of deposition may be meaningful aspects of the molecular pathology of Alzheimer's disease . INTRODUCTION
senile plaques are found in higher density in Alzheimer's disease (AD) than in normal aging and are characteristic pathologic markers that have been linked to intellectural decline (BLESSED, TOMLINSON, & ROTH, 1968) . Thus, understanding genetic and cellular mechanisms that affect amyloid accumulation may provide insight into diseaserelated degenerative changes of the AD brain as well as the normal aging process . Using in situ hybridization methods a number of studies have addressed the localization of amyloid A4 (/3-peptide) mRNA with regard to areas of the AD brain that are commonly affected (BAHMANYAR, HIGGINS, GOLDABER, LEWIS, MORRISION, WILSON, SHANKAR, & GAJDUSEK, 1987 ; GOEDERT, 1987 ; HIGGINS, LEWIS, BAHMANYAR, GOLDABER, GAIDUSEK, YOUNG, MORRISON, & WILSON, 1988 ; LEWIS, CAMPBELL, TERRY, & MORRISION, 1988 ; COHEN, GOLDE, USL4K, YOUNKIN, & YOUNKIN, 1988 ; PALMERT, GOLDE, COHEN, KOVACS, TANzi, AMYLOm-CONTAINING
Address for correspondence : Dr . Charles A . Marotta, Neurobiology Laboratory, Bullfinch 4, Massachusetts General Hospital, Boston, MA 02114 U .S .A ., Tel . (617) 729-5211 37
38
Wen-GANG CBOU et a!.
GUSELLA, USIAK, YOUNKIN, & YOUNKIN, 1988) . It is generally agreed that neurons contain appreciable amounts of amyloid mRNA . There is uncertainty, however, concerning the significance of amyloid mRNA levels with regard to cortical and subcortical sites that are typically affected by neuritic plaques and neurofibrillary tangles . Indeed, both relatively high and low levels of neuronal amyloid mRNA have been observed in cortex, hippocampus, and other brain regions in AD (BAHMANYAR et al., 1987 ; CHOU, MAJOCHA, BENES, ZAIN, & MAROTTA, 1987 ; GOEDERT, 1987 ; HIGGINS et al., 1988 ; LEwis et al., 1988 ; COHEN et
al., 1988 ; PALMERT et al ., 1988) . Consequently, the detailed mechanism by which amyloid mRNA regulation may influence the development of the characteristic neuropathological changes is an unsettled issue . The frequency of AD cases with high neuronal amyloid precursor protein mRNA levels in cortical and subcortical regions in not clear . However, the occurrence of such cases, once one is identified, permits questions to be addressed concerning the relationship, if any, between abundant amyloid mRNA production and the molecular pathogenesis of AD . Thus, in the present studies we asked whether or not there is a correlation between high levels of amyloid mRNA per neuron, as measured by quantitative in situ hybridization methods, with altered neuronal morphology and the distribution of senile plaques in various cortical layers . The latter was of particular interest since in an earlier report we observed the non-random deposition of A4 amyloid in the lamellae of the AD cortex using highly specific monoclonal antibodies (MAJOCHA, BENES, RODENRYS, & MAROTTA, 1988) . In the present study we compared plaque-containing areas of an AD brain to unaffected sites of the same cortex ; thus, we avoided the inherent variability that results from comparison of different postmortem cases . A portion of the present data were reported previously in preliminary form (CHOU et al., 1988a) . MATERIALS AND METHODS Tissues Normal aged and AD cases were obtained from the Massachusetts Alzheimer's Disease Research Center (Neuropathology core at the Massachusetts General Hospital) and the McLean Hospital Brain Tissue Resource Center .
Riboprobe preparation A cloned amyloid precursor protein cDNA, referred to as amy37, was prepared from AD mRNA and was previously characterized (ZAIN, SALIM, CHOU, SAJDEL-SULKOW5IA, MAJOCHA, & MAROTTA, 1988) . The cDNA insert was cut with Eco RI at the beginning of the A4 site to generate a 1 .1 Kb fragment spanning nucleotides 1795-2851 (KANG, LEMAIRE, UNTERBECK, SALBAUM, MASTERS, CRZESCHIK, MULTHAUP, BEYREUTHER, & MULLER-HILL, 1987 ; ZAIN et al., 1988) . The 1 .1 Kb fragment was purified from an agarose gel (SALIM, REHMAN, SAJDEL-SULKOWSKA, CHOU, MAJOCHA, MAROTTA, & ZAIN, 1988) and subcloned into the Eco RI site of plasmid pGEM I (Promega) in both orientations . The recombinant plasmids were digested with BamHI to linearize them . Single-stranded RNA probes (riboprobes) were generated in the sense and antisense orientation using T7 RNA polymerase and ( 3 H)UTP and ('H)CTP to a specific activity of 3 .3x107cpm/ug . The riboprobes were partially hydrolyzed to approximately 300 bases (average) by mild alkaline treatment (Fig . I) .
ALZHEIMER Amnom
MESSENGER
RNA
39
In situ hybridization Formalin fixed, paraffin embedded sections of post-mortem human control or AD frontal cortex area 9 were used for in situ hybridization and cytochemical studies . The general procedure of ANGERER, Cox, and ANGERER (1985) was applied . Five µm-thick sections were mounted on clean glass slides pre-treated with a 2% solution of 3 aminopropyltriethoxysilane (Aldrich) in acetone . The slides were dipped in xylene and rehydrated in decreasing concentrations of ethanol in water . The sections were then treated for 30 min with a I µg/ml solution of Proteinase K (Sigma) in 50 mM EDTA, 100 mM Tris pH 8 .0 at 37°C . Sections were then washed in water and dipped in a solution of 0 .25% acetic anhydride (freshly made, immediately before use) in 0 .1 M triethanolamine pH 8 .0 for 10 min and washed with 2x SSC (1 x SSC = 0 .15 M NaCl, 0 .015 M sodium citrate) . Sections were dehydrated with increasing concentrations of ethanol to 100% . Tissue was prehybridized in hybridization buffer (see below) for 30 min at 37°C . Radiolabeled probes were diluted to 2 .7-5 .4 x 10 4 cpm/µl in 37 µl hybridization buffer : 0 .3 M NaCl, 10 mM Tris pH 8 .0, 1 mM EDTA, 10% dextran sulfate, 0 .25% BSA, 0 .25% Ficoll 400, 0 .25% polyvinylpyrrolidone 360, 50% deionized formamide (Baker) and 500 µg/ml t-RNA . Annealing was carried out at 45°C for 16 h . Excess probe was removed by rinsing in 4 x SSC four times (5 min each) . Sections were treated for 30 min at 37°C with a solution of RNase A (20 µg/ml) and RNase TI (1 unit/ml) (Sigma) in 0 .5 M NaCl, 10 mM Tris pH 8 .0, then immersed in 2 x SSC for 30 min at room temperature . High stringency washing was done in 0 .1 x SSC at 65°C for 15 min and transferred to another 0 .1 x SSC solution for 30 min at room temperature . The sections were dehydrated in a series of increasing ethanol solutions with 0 .3 M ammonium acetate and finally in 100% ethanol . They were dipped in KodakT" NTB-2 emulsion diluted 1 :1 with H2O and ammonium acetate at a 0 .3 M final concentration . After drying, all slides were stored desiccated at 4°C for up to three weeks . They were developed in Kodak D-19 for 5 min, washed and put into Kodak rapid fixer for 5 min, then rinsed . Sections were counterstained with hematoxylin and eosin and coverslipped with permount . A D and control cases were processed simultaneously . Immunocytology and tissue staining
The procedure for immunostaining cortical sections with anti-A4 monoclonal antibodies and for quantitating amyloid deposits followed the previously described protocols (MA ocHA et a!., 1988 ; MAJOCHA, MAROTTA, & BENES, 1985) except that the average of five fields from a single case were examined in the present study . Staining with thioflavin S was described elsewhere (TATS-OSTROFF, MAJOCHA, & MAROTTA, 1989) . Tissue used for immunostaining was obtained from the same block as tissue used for in situ hybridization . Sections of 40 µm were cut on a cryostat from tissue infiltrated with sucrose . Grain counting in cortical layers
Specimens processed autoradiographically to visualize hybridization probes at the light microscopic level were subjected to quantitative analyses using a Bioquant Advanced System IV (R & M Biometrics) that permits automated image processing and grain counting . The specimens were visualized with a Leitz Laborlux 12 microscope . The system was calibrated to detect particles of a size and density of the autoradiographic grains visualized with a
40
WEN-GANG CHOU et al.
100x oil immersion objective . The data are expressed as grains per microscopic field . Grain counting in relation to neuronal size
In the case of probes that were localized to neuronal cell bodies, the images of individual cells were processed through the analysis system so that the corresponding pixels could be quantitated . Highlighted pixels of each neuron were computed automatically and the numbers of grains present were expressed either per cell or as a function of cellular area (µm 2) using a bivariate regression analysis . Data for normal control and AD cases were compared using a two-tailed Student's t-test . Simple linear regression analysis was used to examine the relationship between grain counts and cell size expressed in µm 2 by generating a correlation coefficient (r) . Grain counting in relation to senile plaques Tissue sections subjected to both in situ hybridization and thioflavin S staining were
visualized and photographed using the 40 x objective under dark field and fluorescence optics of a Zeiss microscope . All photomicrographs, with a final magnification of 570 x , were taken with a Zeiss MC63, photographic system . Structures stained by thioflavin S were visualized with an epifluorescent illuminator equipped with a fluorescein excitation and barrier filter set . Using the same AD case (A67), forty-seven fields with an area equal to 940µm 2 were obtained and the number of plaques and cells within each region were counted . The mean number of grains per cell plus surrounding neuropil, in regions with or without plaques, was then determined . RESULTS
Selection of riboprobes and tissues
Previously we cloned and characterized amyloid precursor protein cDNA that had been obtained from mRNA of an AD brain (ZAIN et al., 1988) . The region encompassing the A4 domain was excised by digestion with EcoRI and isolated (MAROTTA, CHOU, MAJOCHA, WATKINS & ZAIN, 1989) . The resulting 1 .1 Kb fragment was used to prepare tritiated ripoprobes in the sense and antisense orientation (Fig . 1, lanes a and c) . After a limited alkaline digestion to yield polynucleotides of smaller sizes (Fig . 1, lanes b and d) the probes were applied to formalin fixed cortical sections for in situ hybridization, as described in the Materials and Methods section . Four AD and 4 normal control cortices were screened with the riboprobes . Based on this preliminary data, we selected for detailed study a 67 yr old case (referred to as A67, post-mortem interval 11 h) that had the highest density of antisense tritated grains in the series (see below) . For comparison we examined a matched control (69 yr old, referred to as C69, post-mortem interval 12 h) . Both tissues were from Brodman area 9 of Caucasian females . Immunostaining with anti-A4 Mobs
A combination of three Mabs to A4 amyloid, previously characterized (MAJOCHA el al ., 1988) were applied to the AD and control cases . In A67, extensive amyloid deposition was observed throughout the layers of the cortex (Fig . 2A) . The normal control was unreactive with the Mab mixture (Fig . 2B) .
ALZHEIMER AMYLOID MESSENGER RNA
41
1 .4-
0.8-
0 .2-
a b c d
FIG . 1 . Characterization of riboprobes . The riboprobes amy12 (sense, lane a) and amyl5 (anti-sense, lane c) were synthesized using T7 polynterase and tritiated UTF and CTP . Shown are the products before (lanes a and c) and after (lanes h and d) mild alkaline treatment . The samples were electrophoresed on a 1 .4% agarose gel containing 6% formaldehyde . The gel was treated with enhancer (NEN) and dried for autolluorography . The size of markers, in Kb, is indicated .
42
WEN-GANG Cnou et a!.
FIG . 2 . 1 in munostaining of AD and control cortices with monoclonal antibodies to A4 polypeptide (see Materials
and Methods). (A) A67 frontal cortex, layer V . (R) C69 frontal cortex, layer V . In both cases the bar = 50 µm .
ALZHEIMER AMYLOID MESSENGER
RNA
43
Ftc . 3 . In situ hybridization of AD and control frontal cortices (layer V except where indicated) . (A) Antisense probe applied to A67 (dark field) . (B) Antisense probe applied to A67 and shown at higher magnification to demonstrate hybridization to neurons (bright field) . Arrows indicate two neurons labeled with grains . (C) Antisense probe applied to A67 layer VI . (D) Anrisense probe applied to C69 at low magnification (dark field) . (E) Same as D, at higher magnification (bright field) . (F) Sense probe applied to A67 . In all cases the bar = 20 µm .
44
WFN-GANG CH0r et at.
Fur . 4 . 'I - hioflavin stain of tissue section processed for in situ hybridization to demonstrate the co-localization of riboprobe and senile plaques . AD cortex was processed for in situ hybridization and then stained with thioflavin S as described in the Material and Methods . Shown are grains and plaques (Fluorescent stain) of cortical layer III . Bar - 20 µm .
ALZHEIMER AMVLOIO MESSENGER RNA
45
In situ hybridization Both control and AD brains were processed for in situ hybridization using the antisense riboprobe and viewed with both dark and bright field illumination (Fig . 3) . Large and small neurons of grey matter were labeled in AD case A67 . Although tritiated grains were present to an extent in all AD cortical layers (see below for quantitation, Table 2), layer III and V neurons exhibited the most intense signal . Layer V is shown in Fig . 3A . The indicated neurons (arrows, Fig . 3B) were typical of those in A67 cortical layers III and V and frequently appeared to have normal morphology . However, some of the smallest apparently normal neurons, including those at the junction of gray and white matter, also contained tritiated grains (Fig . 3C) . The AD case under scrutiny had high signal levels when compared to 3 other AD cases and when compared to four neurologically normal controls ; the matched case, C69, is shown for comparison (Fig . 3D, E) . The sense strand riboprobe applied to both control and AD cortices did not appear to exhibit consistent clustering of grains over perikarya (Fig . 3F) . Quantitation of amyloid mRNA in cortical cells
The occurrence of an AD case with the qualitative appearance of high levels of amyloid mRNA relative to a matched control was verified by quantitating the grain distribution . The mean number of tritiated grains per large pyramidal neuron (of approximately 100 µm 2) was determined (Table 1) . For both layers III and V of the AD brain, antisense and TABLE 1 . MEAN NUMBER OF GRAINS PER CELL
f
Layer III A67
SEW
A4 antisense
A4 sense
probe
probe
7 .37
f
0 .88
1 .82 t 0 .14
n=75 C69
2 .56 t 0 .17
n=51 2 .46 i 0 .22
n=77
n=56
Layer V A67 C69
12 .55
3
1 .02
n=44
3 .15
4 .33 i 0 .43 n=49
3
0 .38
n=33 1 .69 t 0 .18 n=64
*The mean number of grains per cell were counted in the large pyramidal neurons of layers IIl and V of Alzheimer case A69 by the methods previously described .
sense strands of the riboprobe showed significant differences in the mean number of grains per cell (p < 0 .01) . There was also a significant increase (p < 0 .01) in the mean number of grains of the antisense probe in the AD case relative to the same probe in the control for both layers III and V (Table 1) . The results of the regression analysis showed the correlation coefficient for cellular area and number of grains was high for the antisense probe in AD layer III and layer V neurons (r = 0 .861 and 0 .690, respectively) . Distribution of amyloid mRNA in cortical layers
AD brain A67 was used to assess the laminar distribution of neurons with high levels
46
WEN-(TANG CHOH et at.
of amyloid mRNA in relation to the deposition of immunologically detectable A4 amyloid . The average number of immunodetectable amyloid deposits of all morphologic types, per unit area, were! 68 .6 f 4 .5 ; 36 .2 t 7 .7 ; 41 .2 t 7 .7 ; 15 .0 t 4 .6 ; and 8 .4 t 1 .2 for layers 1, 11, III, V and VI, respectively . The distribution was in general agreement with previous observations made on four other AD cases (MAJOCHA et al ., 1988) . Table 2 shows the mean number of grains of the antisense and sense probes per field TABLE 2 . MEAN NUMBER OF GRAINS PER FIELD t SEM PER ALZHEIMER CORTICAL LAYER' Cortical layer
n
A4 antisense probe
Layer 1 Layer II Layer 111 Layer V Layer VI
10 10 10 10 10
27 .90 56 .40 83 .70 71 .30 54 .30
f 3 + t L
1 .63 5 .01 6 .17 6 .88 5 .42
A4 sense probe 32 .00 28 .50 33 .40 32 .00 28 .00
t 3 t : t
4 .71 3 .24 4 .30 3 .74 3 .47
'Field size, Bioquant screen at x 100 magnification .
in each cortical layer of case A67 . Layers II-VI had the highest hybridization signals and of these, layers III and V were the most highly labeled . Grain counts for the sense probe were nearly the same in all layers (Table 2) . Unlike the distribution of the A4 amyloid peptide, which showed decreasing numbers of deposits with cortical depth, the distribution of the antisense hybridization probe appeared to reflect the pattern of neuronal size and numbers characteristic of the cortical layers . Neuronal amyloid mRNA and senile plaques AD case A67 examined by in situ hybridization, was stained with thioflavin S to demonstrate neuritic plaques . The photomicrograph of layer III, shown in Fig . 4, is a typical result . Rarely did we observe high levels of tritiated grains clustered around thioflavinpositive material . For quantitative studies, grain counts were carried out in the vicinity of plaques (neuropil and cellular areas) and compared with similar determinations of grains present over cells or in areas containing neither cells nor plaques (area = 940 µm 2) . The data revealed that the mean number of grains (25 .9 t 1 .3) was highest in regions containing cells but unassociated with plaques (N= 12 areas examined) ; the average value was more than twice that in neuropil (12 .0 t 1 .5 grains ; N = 15 areas examined) . The lowest values were obtained for regions containing plaques (7 .6 t 1 .4 grains ; N= 20 areas examined ; p < 0 .001 when compared to cellular areas) . In the latter study, only 3 cells were found in close proximity to plaques after analysis of twenty 940 µm 2 tissue areas . However, when cells were present, their mean density of grains (13 .7 grains/cell) was equivalent to cells unassociated with plaques (13 .4 grains per cell, and see Table 1) . DISCUSSION
Application of the in situ hybridization procedure to detect mRNA at the cellular level has been a useful tool for over a decade and has been validated for neuron-specific markers in brain (e .g ., Urn. & SASEK, 1986) . Using different procedural approaches, several previous
47
ALZHEIMER AMrioID MESSENGER RNA
reports focussed on amyloid mRNA in control and AD brains with attention to RNA levels, differential expression in various cell types, and relationship to pathologic markers characteristic of AD (BAHMANYAR et al., 1987 ; CHOU et al ., 1988b ; GOEDERT, 1987 ; HIGGINS et al ., 1988 ; LEWIS et al ., 1988 ; COHEN et al., 1988 ; PALMERT et al., 1988) . In the most widely applied technique, and the one adopted in the present study, the 1 .1 Kb segment of the amyloid precursor protein mRNA which contains the A4 region was lebeled and used for in situ hybridization on fixed post-mortem tissues . However, unlike probes labeled with tritium, as described here, earlier studies relied on 35S or 3zP radiolabels which resulted in grain clusters that tended to obscure cellular morphology . Nevertheless, it has been generally concluded that cortical and subcortical neurons have appreciable levels of amyloid mRNA (BAHMANYAR et al.,
1987 ; CHou et al., 1988b ;
GOUDERT, 1987 ; HIGGINS et al., 1988 ; LEWIS et al., 1988) . In certain instances in situ hybridization studies led to the conclusion that the amyloid mRNA was often decreased in AD relative to controls when frontal cortex was examined, and this result was consistent with Northern blot analyses (BAHMANYAR et al., 1987 ; GOEDERT, 1987) . However, other studies have led to the opposite conclusion concerning cortical and/or subcortical areas (HIGGINS et al., 1988 ; LEWIS et al., 1988 ; COHEN et al., 1988 ; PALMERT et al., 1988) . At the very least, earlier studies have observed that certain neurons of the AD brain may contain increased levels of amyloid mRNA at some point during the course of the disease. With this view as a starting point, we screened a series of cortices to identify those with notably high levels of amyloid mRNA within neurons . Having identified a suitable case, we asked whether or not conclusions could be drawn concerning sites of abundant amyloid mRNA and plaque localization . Further, we utilized a double labeling method which allowed us to assess mRNA abundance in the vicinity of thioflavin S positive plaques . An AD frontal cortex with dense deposits of immunologically detectable amyloid contained neurons with abundant amyloid mRNA . With regard to this neuronal population, overproduction of amyloid mRNA did not appear to be associated with an alteration in gross cellular morphology of fixed brain tissue as assessed at the light microscope level . These data do not bear on possible biochemical changes and fine structural alterations that may have occurred and which were not evaluated in the present investigation . Further, no conclusions can be drawn concerning the long-term consequence of high levels of amyloid synthesis since post-mortem studies provide solely a static picture during one phase of the disease . It is possible that certain neocortical and hippocampal neurons that produce abundant amyloid for prolonged periods may undergo degeneration . When compared to a control cortex, the antisense probe gave a much higher signal than the sense probe in all AD cortical layers except the first, where the values were nearly the same . This may be related to the paucity of neurons in layer 1, although it has been shown that glia contain the amyloid precursor protein (TATE-OSTROFF et al., 1989) . In terms of relative abundance, the distribution of amyloid mRNA in the AD cortex was not coincident with the distribution of amyloid deposits identified by anti-A4 Mabs which show a gradient of density with highest levels occurring in Layer I and lowest in Layer VI (MASOCHA et
a!., 1988 and see Results and Table 2) . Further, in double labeling studies utilizing cortical sections subjected to both in situ hybridization and thioflavin S staining, we did not observe a preponderance of riboprobe in the immediate vicinity of senile plaques in twenty separate
48
WEN-GANG CHOU
et al.
determinations . These data and the previous observations suggest that the amyloid component of senile plaques may be derived from non-adjacent sources . For example, while synthesis of amyloid precursor protein occurs in cell bodies, the release and deposition of amyloid may occur distally at axon terminations . Thus, amyloid deposits corresponding to mRNA localized in a neuron somata may be present in another region of the cortex to which the neuron projects . Other factors, however, bear on this result . The possibility exists that extensive cell loss in the vicinity of plaques is a major contributing factor to the decreased hybridization signal . Since the progression of disease is not amenable to direct observation by post-mortem studies, we cannot rule out the possibility that numerous cells with abundant amyloid mRNA were present at earlier stages . It was interesting to note that surviving neurons adjacent to senile plaques contained as much amyloid mRNA as cells in non-plaque areas . While evidence has accumulated to implicate neuronal amyloid as a contributing factor to plaque formation (MASTERS, MULTHAUP, SIMMS, POTTGIESSER, MARTINS, & BEYREUTHER, 1985), additional mechanisms may be influential determinants . A similar conclusion was reached by LEWIS et al. (1988) . Previously we suggested that the distribution and morphology of prefrontal cortical amyloid deposits, as revealed by immunocytochemistry, may be dependent upon underlying laminar-specific structures of the neocortex (MAJOCHA et al., 1988) . These may include dendrites and/or cortically projecting axons from within the cortex or subcortical structures (LEWIS et al ., 1988 ; ISHII, 1966 ; MAJOCHA et al., 1988 ; MASTERS el al., 1985 ; SuzuKI & TERRY, 1967) . Further, we considered that diffusional processes, possibly secondary to
secretion, may be involved in extracellular amyloid accumulation
(BENES, REtFEL, MAJocHA,
This possibility was supported by the recent finding that an extracytoplasmic domain of the APP is found at extracellular sites in the AD brain and can occur within senile plaques (TATE-OSTROFF et al., 1989) . Since the cellular source of amyloid that contributes to senile plaques has not been definitively identified, the present and previous studies do not exclude a vascular origin for plaque amyloid (GLENNER, 1979) or the possible contribution of non-neuronal cells & MAROTTA, 1989 ; MAJOCHA et al ., 1988 ; TATE-OSTROFF et al., 1989) .
(WISNIEWSKI & MERZ, 1983) .
The combined use of in situ hybridization and selective staining methods applied to large numbers of brains may help to define the temporal relationship between vascular or cellular events that influence senile plaque formation . With regard to cellular functioning, we can hypothesize that amyloid mRNA is normally present at basal levels . In AD, an event, as yet undefined, may cause amyloid to be overproduced, possibly transiently . Disregulation at the transcriptional or translational level may be involved . At the cytoplasmic level, the effects of proteolytic processing must also be taken into account . Subsequently, amyloid or cleavage products may be transported from the cell by secretion and diffusion away from the site of synthesis and contribute to amyloid plaques ; and/or there may be transport of amyloid along the axon to the terminus with subsequent deposition . Both mechanisms would result in amyloid-containing plaques occurring at sites that are non-adjacent to immediately surrounding neurons . Acknowledgements-The authors thank Jennifer Reifet for excellent technical assistance . This research was supported by the following grants : AG 02126, CA 11198, CA 36432 from the National Institutes of Health ; and
ALZHEIMER AMYLOID MESSENGER RNA
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by the Axelrod Family Fund, the Katherine Smith Bolt Fund, the American Health Assistance Foundation and the Sandoz Foundation for Gerontological Research . Brain tissues were supplied by the Massachusetts Alzheimer's Disease Research Center, supported by National Institutes of Health grant AGO5134 ; and the McLean Hospital Brain Tissue Resource Center, supported by grant MH/NS 31862 . REFERENCES ANGERER, K ., Cox, K ., & ANGERER, L. (1985) . In situ hybridization to cellular RNAs . Genetic Engineering . Principles and Methods 7, 43-65 . BAHMANYAR, S ., HIGGws, G ., GOLDGABER, D ., LEwts, D . A ., MORRISON, J . H ., WILSON, M. C ., SHANKAR, S . K ., & GAJDUSEK, D . C . (1987) . Localization of amyloid p protein messenger RNA in brains from patients with Alzheimer's disease . Science 237, 77-80. BENES, F . M ., RErnEL, J ., MAJOCIrA, R . E ., & MAROTTA, C . A . (1989). Evidence for diffusion of amyloid during plaque formation in Alzheimer brain . Neuroscience 33, 483-488 . 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