Alterations in Alzheimer's Disease\p=m-\AssociatedProtein in Alzheimer's Disease Frontal and Temporal Cortex Garth Bissette, PhD; Wayne H. Smith; Kenneth C. Dole; Barbara Crain, MD, PhD; Barney Miller, PhD; Charles B. Nemeroff, MD, PhD \s=b\ Alzheimer's disease (AD)\p=m-\associatedprotein is present in brain and cerebrospinal fluid of patients with AD but not in adult, nondemented, normal controls. This protein may represent an abnormal epitope of the "tau" microtubuleassociated protein and has been detected before the appearance of senile plaques and neurofibrillary tangles. The amount of AD\p=m-\associatedprotein in the frontal and temporal cortices in 93 cases of neuropathologically confirmed AD was compared with the amount that was present in 20 cases without AD. The amount of AD\p=m-\associatedprotein was significantly increased in the cases of AD for both brain regions compared with that in the cases without AD. The presence of high levels of this protein is a useful adjunct, postmortem marker of the presence of AD and may eventually lead to tests that allow early detection of individuals at risk for this disease. (Arch Gen Psychiatry. 1991;48:1009-1012) Wolozin and colleagues1 reported the presence In of1986, novel antigen in brain with Alzheimer's disease that monoclonal detected a (AD) (ALZ-50) by raising a antibody homogenates of ventral forebrain from four patients with AD. Partial purification and Western blot techniques showed that this antigen was a single protein with a molecular weight of 68 000 d, and it was subsequently named A68.2 The A68 protein was recently reported to be one of three ALZ-50 immunoreactive ele¬ ments of a larger protein termed Alzheimer's diseaseassociated protein (ADAP),3 with a total aggregate weight of 200000 d as measured by size-exclusion column elution. The presence of ALZ-50 immunoreactivity in brain tis¬ sue from normal human infants aged younger than 2 years,4 its subsequent disappearance in normal adults, and its apparent reexpression in AD have generated con¬ siderable interest in this protein as the basis of a potential was to the Accepted for publication February 14, 1991. From the Departments of Psychiatry (Drs Bissette and Nemeroff and Messrs Smith and Dole), Pharmacology (Dr Nemeroff), and Pathology (Dr Crain), and the Joseph and Kathleen Bryan Alzheimer's Disease Research Center (Drs Crain and Bissette), Duke University Medical Center, Durham, NC, and the Abbott Diagnostics Divisions, Abbott Laboratories, Abbott Park, III (Drs Ghanbari and Miller). Reprint requests to Duke University Medical Center, 3859, Durham, NC 27710 (Dr Bissette). PO Box Hossein Ghanbari, PhD; diagnostic marker for AD. While there have been several confirmatory reports of ALZ-50 immunoreactivity in AD,5,6 neurofibrillary tangles in non-AD dementia and Pick bodies also react with the ALZ-50 antibody.7,8 In ad¬ dition, microtubule-associated "tau" protein may be rec¬ ognized by ALZ-50,9,10 even though tauprotein is different from the A68 protein in that tau-associated ALZ-50 immunoreactivity is not destroyed by trypsin. Thus, a more specific means of detecting ADAP was necessary before such measurements could be developed into a useful clinical test. For this purpose, we have constructed two-antibody sandwich, enzyme-linked immunoassay was designed to avoid cross-reactivity with such nonspecific epitopes as tau protein. This goal has been successfully reached with the development of the ALZ-EIA, which was used in this study, and it has been reported in detail elsewhere.11 In the present study, we evaluated the potential diagnostic utility of this labo¬ ratory test by measuring ADAP immunoreactivity in temporal and frontal cortical punch samples from post¬ mortem brains with and without neuropathological diag¬ a (ALZ-EIA) that noses of AD. SUBJECTS AND METHODS Brain tissue was obtained from the Kathleen Price Bryan Brain Bank of the Joseph and Kathleen Bryan Alzheimer's Disease Re¬ search Center at Duke Medical Center, Durham, NC. Tissues that were sampled represented 93 patients with a confirmed neuropathological diagnosis of AD1213 and 20 patients without AD. The patients in the group with AD included 11 cases with additional diagnoses (six with Parkinson's disease, four with diffuse Lewy body disease, and one with progressive supranuclear palsy). Diagnoses were reviewed by a single neuropathol¬ ogist (B.C.) who provided counts of senile plaques and neu¬ rofibrillary tangles on representative silver-stained14 sections of solution-fixed temporal and frontal cortex. Counts were made on the most severely affected area in each section. The control group without AD included four neurologically normal, nondemented controls, three nondemented pa¬ tients with neurological disease (one each with amyotrophic lat¬ eral sclerosis, idiopathic Parkinson's disease, and Huntington's disease), and 13 patients with non-AD dementias. The neuro¬ pathological diagnoses that were assigned to these demented patients without AD included two with Binswanger's disease, two with unclassified neurodegenerative disease, two with Par¬ kinson's disease, two with Pick's disease, and one each with chronic subdural hematoma, chronic meningoencephalitis, formaldehyde Downloaded From: https://jamanetwork.com/ by a Texas Medical Center Library User on 12/25/2022 Mean ± SEM No. of Patients Group Nondemented Normal Neurological disease Demented Without Alzheimer's disease With Alzheimer's disease Sex 4 3 3 2 13 93 F, 1 M F, 1 M F, 9 M 50 F, 43 M 4 multi-infarct dementia, anoxie encephalopathy, and no discern¬ ible anatomical cause for dementia. Micropunch (2-mm diameter) plugs of the middle temporal gyrus (middle of Brodmann's area 21) and the superior frontal gyrus (middle of Brodmann's area 11) that contained from 15 to 20 mg of brain protein were taken from the frozen brain hemi¬ sphere of each case and placed in 1.5-mL plastic microcentrifuge tubes. All samples were coded, and personnel were blind to the diagnosis of individual samples. Tissues were homogenized with a motor-driven Teflon pestle in 4 µ of buffer (0.1 mmol of TRIS, 150 mL of sodium chloride, and 0.5% ethylene glycol bis, , ', '-tetra acetic acid [EGTA], gentamicin sulfate, and so¬ dium alkyl paraben [Nipaset, Nipa Labs Ine, Wilmington, Del; pH 7.5]) per milligram of wet tissue weight. This homogenate was centrifuged in a fluorescence polarization immunoassay (TDx, Abbott Laboratories, Abbott Park, 111) centrifuge for 1 minute, and duplicate samples that contained 50-µ aliquote of the su¬ pernatant were placed in the assay wells. As described in detail elsewhere,11 the ALZ-EIA uses a rabbit poly clonai antiserum to ADAP (a complex with three ALZ-50 immunoreactive subunits, one of which is the A68 protein) that was developed by Abbott Laboratories. The IgG fraction of this poly clonai antiserum is bound to polystyrene beads and binds to and separates effec¬ tively the ADAP epitope from the other nonspecific, supernatant ALZ-50 immunoreactive species. This bead-antigen complex is next reacted with ALZ-50 antibody, followed by incubation with a horseradish peroxidase-conjugated goat anti-mouse IgM. Af¬ ter incubation with an o-phenylenediamine hydrochloric acid solution, the resulting yellow liquor is read at a 492-nm wave¬ length with the use of an (Quantum) analyzer (Abbott Labo¬ ratories) on 60-well plastic plates. This photometric assay measures the amount of ALZ-50 bound to the bead. Absorbance is calculated as units per milligram of protein. Protein was measured by a modification of the Folin-phenol method of Lowry et al.15 The ADAP-positive standards were manufactured from two di¬ lutions of brain extract that were obtained from Abbott Laborato¬ ries and one dilution of brain extract that was obtained from the Duke University Medical Center; these extracts were derived from tissues that previously were determined to have high levels of ADAP. All samples were measured in duplicate, and infra-assay and interassay variation was 9% and 15%, respectively. Data were analyzed by analysis of variance and Student's f test. Correlations among regional concentrations of ADAP, numbers of senile plaques and neurofibrillary tangles, age, sex, postmortem delay, freezer storage time, and length of disease were sought with the Spearman Rank Correlation Coefficient test. RESULTS The group means and SEMs (mean ± SEM) for age, postmortem delay time, and frozen tissue storage time are shown in the Table for the group with AD (n 93), the demented controls without AD (n 13), and the nondemented controls (n 7, including three nondemented controls with neurologic disease and four nondemented, neurologically normal controls). For the group with AD, the average length of illness of 70 patients was = = = (Range) Postmortem Interval, h Age, y Freezer Storage Time, mo 6.3 ±4.6 (0.8-20) 2.0 ±1.1 (1.0-3.2) 20.5 ±10.4 (4-49) 61.7 ±8.7 (52-79) 72.2 ± 3.4 (47-90) 6.5 ±2.5 (0.7-26.0) 7.1 ±1.0 (0.3-36) 23.6 ±4.2 66.2 ±5.5 (51-78) 77.0 ±1.0 (63-92) 42.0 ±3.1 (40-48) (2-45) 24.5 ±1.5 (2-63) 111.3±9.5 months (mean±SEM), while for 12 of the demented patients without AD, the average length of illness was 65.8± 14.5 months (mean±SEM). One of the neurologically normal, nonde¬ mented controls exhibited moderate plaque density in the frontal and temporal cortices, as did three of the 13 demented controls without AD. However, only one of these 13 demented patients without AD had a single neurofibrillary tangle per 20 x field in these cortical regions. In contrast, all 93 of the patients with AD had demonstrable plaques, and only 21 did not exhibit neu¬ rofibrillary tangles in either the frontal or temporal cortex. As illustrated in the Figure, the patients with AD exhibited markedly higher ADAP concentrations in both the frontal and temporal cortices (0.398+0.029 and 0.594±0.43 absorbance U/mg of protein, respectively) compared with that in the group without AD (0.175±0.028 and 0.281 ±0.054 absorbance U/mg of protein, respec¬ tively). The difference was significant to the P<.001 level for both brain regions. None of the four neurologically normal, nonde¬ mented controls had ADAP concentrations above 0.200 absorbance U/mg of protein in either the temporal or frontal cortex. However, two of the three nondemented patients with neurological disease (Huntington's disease and Parkinson's disease) had ADAP con¬ centrations well above 0.200 absorbance U/mg of protein in the temporal but not the frontal cortex. Only two of the 13 demented controls without AD had ADAP levels above 0.200 absorbance U/mg of protein in the frontal cortex, while four of the 13 demented patients without AD had ADAP concentrations greater than 0.200 absorbance U/mg of protein in the temporal cortex. In contrast, only 10 of the 93 patients with AD had ADAP concentrations below 0.200 absorbance U/mg of protein in both the frontal and temporal cortices. The samples of patients without AD but with substantial concentrations of ADAP were examined by Western blot analysis and did indeed have authentic ADAP present, while all the sam¬ ples with AD but without ADAP, as measured by the ALZ-EIA, were also negative by Western blot techniques (data not shown). of freezer Postmortem delay, length of disease, and storage time were not strongly correlated to any of the variables that were examined. The ADAP concentrations in the frontal and temporal cortices were positively correlated (P<.001) for both groups with and without AD. Temporal cortex concentrations of ADAP were significantly correlated with senile plaque numbers in both the temporal and frontal cortices of the patients with AD, but the concentration of frontal cortex ADAP was not similarly correlated. In the group with AD, ADAP concentrations in the frontal cortex were negatively correlated (P<.001) with patient age; temporal cortical ADAP was not. In addition, and not sur¬ prisingly, a significant positive correlation between numbers of senile plaques in the frontal cortex and either numbers of tem¬ poral cortex plaques (P<.001) or numbers of frontal (P<.007) or temporal (P<.04) cortex neurofibrillary tangles was present in the group with AD. iength COMMENT goal identifying definitive premortem or post¬ mortem diagnostic markers in AD has not yet been real¬ ized. Clinicians remain rather poor at diagnosing AD,16 The of Downloaded From: https://jamanetwork.com/ by a Texas Medical Center Library User on 12/25/2022 2.0 v*0 1.5 0 2 2 '5 o- S-I 3 y u > . g <-8 Q < O J3 advanced Down's < 0.5 syndrome,4,21 and the dense ALZ-50 immunoreactivity that is found in plaques and tangles of AD tissue all mandate further investigation of the phys¬ iological and pathological roles of the ALZ-50 antigen. n 0.0L stained for ALZ-50 may eventually have developed tan¬ gles. Similarly, Mattiace et al21 have reported preliminary findings of ALZ-50 immunoreactivity in Down's syn¬ drome that precedes development of the characteristic changes of AD that are seen in patients with Down's syn¬ drome aged older than 30 years, such as neuritic plaque amyloid and paired helical filament immunoreactivity. The presence of the A68 protein in developing normal human brain until about the age of 2 years,4 the reported presence of ALZ-50 immunoreactivity in senescent mon¬ keys,22 the presence of ALZ-50 immunoreactivity in Temporal Cortex Frontal Cortex Alzheimer's disease-associated protein (ADAP) concentration in absorbance units per milligram of protein for temporal and frontal cortex samples from 93 patients with AD (diamonds) and20 controls without AD (open circles), including four nondemented, neuro¬ logically normal patients (X symbols), three nondemented patients with neurological disease (N symbols), and 13 demented patients without AD (O symbols). Bars represent the group mean with SEM brackets. The concentration of ADAP in the group with AD was sig¬ nificantly (?<. 01) increased in both the frontal and temporal cortex compared with the demented controls without AD alone, the non¬ demented controls alone, or both control groups together. postmortem diagnosis still requires quantitative or semiquantitative evaluation of neurofibrillary tangles and senile plaques in stained tissue. This histological diagno¬ sis is time consuming and relatively subjective. Because precise diagnoses, both premortem and postmortem, is a necessary prerequisite for the elucidation of the etiology and pathogenesis of AD, investigators have sought neuand rochemical alterations in AD on which to base novel di¬ agnostic tests. The discovery of the A68 protein raises the question of whether measurement of this protein will provide the basis for a diagnostic test for AD in postmor¬ tem tissue or a premortem test in brain biopsy samples or cerebrospinal fluid. The data presented here confirm findings from previous reports17,18 of increased brain ADAP concentrations in patients with a neuropathologically confirmed diagnosis of AD. The demented controls without AD but with high ADAP concentrations were found to have true ADAP immunoreactivity as shown by Western blot analysis and may represent patients at risk for AD who contracted another dementing disease that led to death before classical neuropathological changes associated with the diagnosis of AD could develop. The regional distribution of ADAP within the brain of patients with AD is similar to the regional distribution of other neuropathological and neurochemical alterations that are seen in AD. The affected brain regions with increased levels of ADAP in AD include the hippocam¬ pus, subiculum, amygdala, temporal, frontal, and pari¬ etal cortices, and nucleus basalis of Meynert.17 However, a recent report19 of ALZ-50 immunoreactivity in the somatosensory cortex in a neuropathologically confirmed case of AD with only moderate cholinergic deficiency and normal somatostatin immunoreactivity emphasized the wide variability among individual cases of AD. Hyman et al20 have reported ALZ-50 staining of both normalappearing and tangle-bearing neurons in AD, and they have postulated that those neurons without tangles that While much remains unknown, at present, the A68 to be one of three ALZ-50 immunoreac¬ tive subunits of a complex termed ADAP.3 The ALZ-50 antibody has also been shown to recognize a nonphosphatase-sensitive epitope of the microtubuleassociated protein known as tau.9-23 The A68 protein im¬ munoreactivity is distinct from tau-1, however, as the ALZ-50 immunoreactive epitope on the A68 protein is destroyed by trypsin and the ALZ-50 immunoreactive component of tau is not.9 Tau protein has also recently been reported to be decreased, not increased as ADAP is, in AD.10 The ALZ-50 immunoreactivity has been shown to be present in neurons of the neonatal rat and to decrease with ensuing development.24 This rodent ALZ-50 immunoreactivity codistributes with microtubule-associated protein 2. The ALZ-50 immunoreactiv¬ ity has also been reported to be present in neocortical subplate neurons that are marked for naturally occurring cell death.25 Recent evidence has shown that ALZ-50 im¬ munoreactivity is degraded by the cyclic adenosine monophosphate-mediated action of ubiquitin.26 Until the abnormal epitope represented in ADAP can be purified and sequenced and the gene cloned, the func¬ tional relationship between the presence of this antigen and the dementia and neuropathological degeneration associated with AD will likely remain obscure. The pres¬ ence of the ADAP antigen in an accessible physiological compartment, such as cerebrospinal fluid, might allow early identification of patients at risk for AD. Intensive study of such individuals is required for development of rational and novel pharmacotherapies for this common dementing illness. protein appears This study was supported by grant MH-40524 from the National Institute of Mental Health and by grant AG 05128 from the National Institute on Aging, Bethesda, Md. We are grateful to Sharon Rhoden for preparation of the manu¬ script. References 1. Wolozin Bl, Pruchnicki A, Dickson DW, Davies P. A neuronal antigen in the brains of AlzheimersScience. 1986;232:648\x=req-\ 650. 2. Wolozin Bl, Davies P. Alzheimer related neuronal protein A68: specificity and distribution. Ann Neurol. 1987;22:521-526. 3. Miller BE, Simon AB, Ghanbari HA. Chromatographic analysis by HPLC of ADAPs. Soc Neurosci. 1989;15:1038. Abstract 414.3. 4. Wolozin Bl, Scicutella A, Davies P. Re-expression of a deand velopmentally regulated antigen in Down's syndrome Alzheimer's disease. Proc Natl Acad Sci U S A. 1988;85:6202\x=req-\ 6206. Downloaded From: https://jamanetwork.com/ by a Texas Medical Center Library User on 12/25/2022 5. Hyman BT, VanHoesen GW, Wolozin Bl, Davies P, Kromer LJ, Damasio AT. Alz-50 antibody recognizes Alzheimers related neronal changes. Ann Neurol. 1988;23:371-379. 6. Doebler JA, Markesbery WR, Anthony A, Davies P, Scheff SW, Rhoads RE. Neuronal RNA in relation to Alz-50 immuno- reactivity in Alzheimers 1988;23:20-24. related neronal changes. Ann Neurol. S, Saitoh T, Quijada S, Cole GM, Terry RD. ALZ-50, and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies. J Neuropathol Exp Neurol. 7. Love ubiquitin 1988;47:393-405. 8. Love S, Burrola P, Terry RD, Wiley CA. Immunoelectron microscopy of Alzheimer and Pick brain tissue labelled with the monoclonal antibody Alz-50. Neuropathol Appl Neurobiol. 1989;15:223-231. 9. Ksiezak-Reding H, Davies P, Yen S-H. ALZ-50, a monoclonal antibody to Alzheimer's disease antigen, cross reacts with T proteins from bovine and normal human brain. J Biol Chem. 1988;263:7943-7947. 10. Ksiezak-Reding H, Binder LI, Yen S-H. Immunochemical and biochemical characterization of T proteins in normal and Alzheimer disease brains with Alz 50 and tau-1. J Biol Chem. 1988;263:7948-7953. 11. Ghanbari HA, Kosak T, Miller BE, Riesing S. A sandwich enzyme immunoassay for detecting and measuring Alzheimer disease\p=m-\associatedproteins in human brain tissue. J Clin Lab Anal. 1990;4:189-192. 12. Khachaturian ZS. Diagnosis of Alzheimer's disease. Arch Neurol. 1985;42:1097-1105. 13. Mirra SS, Brownlee LM, Sumi SM, Hughes JP, Crain BJ, Heyman A. The CERAD neuropathology protocol: observations on cases clinically diagnosed as probable Alzheimer's disease and controls. J Neuropathol Exp Neurol. 1989;48:334. 14. Lloyd B, Brinn N, Burger PC. Silver-staining of senile plaques and neurofibrillary change in paraffin-embedded tissues. J Histotechnol. 1985;8:155-156. 15. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-phenol reagent. J Biol Chem. 1951;193:265-275. 16. Risse SC, Raskind MA, Nochlin D, Sumi SM, Lampe TH, Bird TD, Cubberley L, Peskind ER. Neuropathological findings in patients with clinical diagnoses of probable Alzheimer's dis- Am J Psychiatry. 1990;147:168-172. 17. Ghanbari HA, Miller BE, Chong JK, Heigler HF, Whetsell WO Jr. Distribution of Alzheimer's disease associated proteins in human brain tissue: detection, measurement, specificity and distribution. In: lqbal K, McLachan D, Winblad B, Wisniewski H, eds. Alzheimer's Disease: Basic Mechanisms, Diagnosis and Therapeutic Strategies. West Sussex, England: Wiley; 1991:569\x=req-\ 576. 18. Ghanbari HA, Miller BE, Haigler HJ, Arato M, Bissette G, Davies P, Nemeroff CB, Perry EK, Perry R, Ravid R, Swaab DF, Whetsell WO, Zemlan FP. Biochemical assay of Alzheimer's disease\p=m-\associatedproteins in human brain tissue: a clinical ease. study. JAMA. 1990;263:2907-2910. 19. Jagust WJ, Davies P, Tiller-Borich JK, Reed BR. Focal Alzheimer's disease. Neurology. 1990;40:14-19. 20. Hyman BT, VanHoesen GW, Wolozin Bl, Davies P, Damasio AR. ALZ-50: recognition of Alzheimer related neuronal changes before neurofibrillary tangle formation. Neurology. 1987;37:728. 21. Mattiace LA, Dickson DW, Kress YS, Davies P. Alz 50 im- munoreactivity preceeds PHF formation and neuritic change in Down's syndrome. Soc Neurosci. 1989;15:1038. Abstract 414.2. 22. Cork LC, Walker LC, Davies P, Price DL. A68 immunoreactivity in brains of nonhuman primates. Soc Neurosci. 1988;14:1085. Abstract 437.11. 23. Nukina N, Kosik KS, Selkoe DJ. The monoclonal antibody, ALZ-50 recognizes tau proteins in Alzheimer's disease brain. Neurosci Lett. 1988;87:240-246. 24. Hamre KM, Hyman BY, Goodlett CR, West JR, Van Hoesen GW. ALZ-50 immunoreactivity in the neonatal rat: changes in development and co-distribution with MAP-2 immunoreactivity. Neurosci Lett. 1989;98:264-271. 25. Al-Ghoul W, Miller MW. Alz-50 as a marker for naturally occurring neuronal death in the neocortical subplate of the rat. Soc Neurosci. 1988;14:1118. Abstract. 26. Vincent I, Davies P. ATP dependent loss of Alz 50 immunoreactivity with A68. Soc Neurosci. 1989;15:1038. Abstract 414.2. Downloaded From: https://jamanetwork.com/ by a Texas Medical Center Library User on 12/25/2022