Preservation of Brain Nerve Growth Factor in Mild Cognitive Impairment and Alzheimer Disease

ORIGINAL CONTRIBUTION Preservation of Brain Nerve Growth Factor in Mild Cognitive Impairment and Alzheimer Disease

Elliott J. Mufson, PhD; Milos D. Ikonomovic, MD; Scot D. Styren, PhD; Scott E. Counts, PhD; Joanne Wuu, MA; Sue Leurgans, PhD; David A. Bennett, MD; Elizabeth. J. Cochran, MD; Steven T. DeKosky, MD

Background: The status of nerve growth factor (NGF) Setting and Patients: Subjects were from the Rush levels during the prodromal phase of Alzheimer disease Religious Orders Study and the University of Pittsburgh (AD), characterized by mild cognitive impairment (MCI), Alzheimer’s Disease Research Center (Pittsburgh, Pa). remains unknown. Results: We found no changes in cortical or hippocam- Objective: To investigate whether cortical and/or hip- pal NGF levels across groups; in MCI, levels did not cor- pocampal NGF levels are altered in subjects with MCI relate with an increase in choline acetyltransferase ac- or different levels of AD severity. tivity in these regions.

Design and Main Outcome Measures: An NGF Conclusion: Brain NGF levels appear sufficient to sup- enzyme-linked immunosorbent assay determined pro- port the cholinergic plasticity changes seen in MCI and tein levels in the hippocampus and 5 cortical areas in remain stable throughout the disease course. people clinically diagnosed as having no cognitive impairment, MCI, mild AD, or severe AD. Arch Neurol. 2003;60:1143-1148

T HAS BEEN hypothesized that cho- METHODS linergic basal forebrain (CBF) cortical and hippocampal pro- We evaluated 54 individuals (Table 1) who jection neurons degenerate in participated in a longitudinal study of aging and Alzheimer disease (AD) because AD among the Catholic clergy, the Religious Iof the loss of neurotrophic support from Orders Study (ROS).15,17-20 Subjects were cat- their target sites, which produce nerve egorized as having NCI, MCI, or mild AD. growth factor (NGF).1 This protein has Twelve subjects with severe AD were selected well-known survival effects on CBF from the University of Pittsburgh Alzheimer’s neurons.2,3 Studies have reported un- Disease Research Center (Pittsburgh, Pa). At changed,4-7 decreased,8 or increased8-13 their last evaluation (performed within 12 NGF levels in the cortex and hippocam- months of death), they had a mean Mini- pus in patients with end-stage AD. De- Mental State Examination score of 8.3 (significantly lower than all 3 ROS groups; PϽ.001). spite these inconsistencies and the initia- These subjects were similar to the 3 ROS groups From the Department of tion of a clinical trial to test the effect of in age at death, sex, level of education, APOE Neurological Sciences and Rush NGF gene therapy as a treatment for mild ⑀4 status, and postmortem delay (Table 1). The 14 Alzheimer’s Disease Center, AD, there are scant data concerning al- human investigations committees of Rush Pres- Rush Presbyterian–St Luke’s terations in NGF levels in the cortex and byterian–St Luke’s Medical Center (Chicago, Medical Center, Chicago, Ill hippocampus throughout the course of Ill) and the University of Pittsburgh approved (Drs Mufson, Counts, Leurgans, AD. Interestingly, choline acetyltransfer- this study. Bennett, and Cochran and ase (ChAT) activity is increased in the fron- Ms Wuu); Departments of tal cortex and hippocampus in people with CLINICAL EVALUATION Neurology and Psychiatry and mild cognitive impairment (MCI).15,16 To the Alzheimer’s Disease determine whether NGF levels exhibit a Details of the clinical evaluation in the ROS and Research Center, University of University of Pittsburgh cases have been pub- Pittsburgh School of Medicine, similar plasticity response, we evaluated lished elsewhere.15,17-21 Our MCI population was Pittsburgh, Pa (Drs Ikonomovic these levels in the hippocampus and 5 cor- defined as subjects rated as impaired on neu- and DeKosky); and Aventis tical areas in people clinically diagnosed ropsychological testing but not having demen- Pharmaceuticals, Bridgewater, as having no cognitive impairment (NCI), tia.17-20 The University of Pittsburgh patients NJ (Dr Styren). MCI, mild AD, or severe AD. were diagnosed as having AD following a stan-

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ROS Cases

NCI MCI Mild AD Total Severe AD Cases* (n = 24) (n = 18) (n = 12) (N = 54) (n = 12) P Value† Age at death, y Mean ± SD 81.6 ± 7.3 84.5 ± 5.7 86.0 ± 7.1 83.6 ± 6.9 79.6 ± 8.0 .15‡ Range 66-92 75-97 70-95 66-97 66-90 M, % 58 44 67 56 25 .47§ Level of education, y Mean ± SD 18.3 ± 4.3 17.7 ± 3.4 15.1 ± 4.2 17.4 ± 4.0 NA .09‡ Range 8-25 8-22 6-19 6-25 APOE ⑀4 allele, % 20 28 42 26 92 .43§ Postmortem interval, h Mean ± SD 8.1 ± 6.8 6.9 ± 5.5 6.2 ± 3.2 7.3 ± 5.7 5.9 ± 3.3 .63‡ Range 2.8-29.0 0.2-23.0 3-12 0.2-29.0 2.5-12.0 MMSE score Mean ± SD 27.9 ± 1.6 26.1 ± 2.5 18.2 ± 6.1 25.1 ± 5.1 8.1 ± 5.3࿣ Ͻ0.001‡ Range 25-30 20-30 5-25 5-30 3-17 ROS ADϽMCI or NCI¶

Abbreviations: AD, Alzheimer disease; MCI, mild cognitive impairment; MMSE, Mini-Mental State Examination; NA, not applicable; NCI, no cognitive impairment; ROS, Religious Orders Study. *Subjects with severe AD were from the University of Pittsburgh Alzheimer’s Disease Research Center (Pittsburgh, Pa). †Comparison among the 3 ROS groups. ‡One-way analysis of variance. §Fisher exact test. ࿣Four subjects had missing MMSE scores. The MMSE scores of the other 8 cases were significantly lower than for each of the 3 ROS groups (PϽ.001). ¶Tukey test for multiple comparisons.

dardized Alzheimer’s Disease Research Center evaluation at a hibitors. An aliquot was then removed for total protein deter- consensus conference using criteria from the National Insti- mination (BCA Protein Assay Kit; Pierce Biotechnology, Inc, tute of Neurological and Communicative Disorders and Stroke– Rockford, Ill). The remaining tissue lysate was centrifuged at Alzheimer’s Disease and Related Disorders Association 17000g for 60 minutes, and NGF was measured in the resulting (NINCDS-ADRDA)22 and Diagnostic and Statistical Manual of supernatant using a sandwich enzyme immunoassay. All tissue Mental Disorders, Revised Third Edition23 (now the Diagnostic samples were run in triplicate. The capture antibody was mono- and Statistical Manual of Mental Disorders, Fourth Edition, Text clonal antibody anti-␤ (2.5 Svedberg flotation units [Sf]; 7 Sf) Revision). NGF, whereas the detection antibody was monoclonal antibody anti-␤ (2.5 Sf; 7 Sf) NGF-␤-Gal, a ␤-galactosidase– TISSUE PREPARATION AND conjugated form of the capture antibody (both from Roche Di- NEUROPATHOLOGIC DESIGNATION agnostics, Mannheim, Germany). Detailed assay description and the sensitivity and specificity of human NGF antibodies have been Brain tissue collection and processing were virtually identical published previously.8,28,29 The amount of NGF present in tis- for ROS and University of Pittsburgh subjects.15,17-19,21 Cases were sue samples was determined by monitoring the color change of excluded from these studies if brain abnormalities were sub- a substrate solution (chlorophenol red galactopyranoside; Sigma stantial. Tissue was snap frozen in liquid nitrogen and frozen Chemical, St Louis, Mo) at 570 nm using a plate reader (Dyna- at −80°C until analysis. All cases were assigned a neuropatho- tech, Vienna, Va) and comparing the results with human NGF logic diagnosis based on Braak and Braak staging scores24 and standards (Roche Diagnostics) run in parallel with the same plate. National Institute on Aging (NIA)–Reagan criteria25 and geno- To control for any nonspecific binding of NGF in the tissue ly- typed for apolipoprotein E (APOE) using restriction isotyping sates, tissue samples were spiked with a known amount of NGF of genomic DNA isolated from plasma, performed by polymer- standard, and the percentage of NGF recovered corrected the read- ase chain reaction amplification.18,21,26 Designations of normal ing value. Samples from all 4 diagnostic groups were run in par- (with respect to AD or other dementia processes), possible or allel. To ensure reproducibility of the assay, randomly chosen probable AD, and definite AD were based on criteria from the samples were run using multiple plates, yielding comparable val- Consortium to Establish a Registry for Alzheimer’s Disease27 ues for the same sample. The NGF measurements were ex- and NINCDS-ADRDA.22 pressed as picograms of NGF per milligrams of protein. Semiquantitative counts of diffuse and neuritic plaques were performed on Bielschowsky silver-stained paraffin-embedded STATISTICAL ANALYSES cortical sections and scanned at original magnification ϫ10 to determine the area of greatest plaque density. A grid reticule Demographic and clinical characteristics of diagnostic groups was placed over the area; at original magnification ϫ20, only were compared using analysis of variance for continuous vari- plaques identified within the grid boundaries were counted. The ables and the Fisher exact test for categorical variables; pair- grid was moved across and up and down the field using fidu- wise comparisons were performed using the Tukey test. To as- ciary landmarks to prevent counting objects twice. sess NGF levels across diagnostic groups, we used the nonparametric Kruskal-Wallis test. Distribution of neuropatho- NGF ASSAY logic lesions according to NIA-Reagan criteria and Braak and Braak staging was compared using the Fisher exact test. Cor- Homogenate samples were prepared using brain tissue sonica- relations were performed using the Spearman rank correlation in phosphate buffer saline (pH, 7.2) containing protease in- tion. Statistical significance was set at PϽ.05 (2-tailed test).

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40 80 25

30 20 60

15 20 40 10 NGF, pg/mg of Protein NGF,

20 10 5

0 0 0 NCI MCI Mild AD NCI MCI Mild AD NCI MCI Mild AD

Superior Temporal Middle Temporal Hippocampus

40 60 80

50 30 60 40

20 30 40

NGF, pg/mg of Protein NGF, 20 10 20

0 NCI MCI Mild AD NCI MCI Mild AD Severe AD NCI MCI Mild AD Severe AD

Figure 1. Box plots of nerve growth factor (NGF) levels by diagnostic group (no cognitive impairment [NCI], mild cognitive impairment [MCI], and Alzheimer disease [AD]) in 5 cortical regions and the hippocampus. No statistically significant differences were found among the 3 clinical groups for any region. Black dots indicate outliers.

RESULTS comparing severe AD cases with any of the ROS groups examined. DEMOGRAPHICS NGF LEVELS, ChAT ACTIVITY, AND NUCLEUS Individuals with NCI, MCI, or mild AD were similar in BASALIS OF MEYNERT NEURON COUNTS age, education level, sex representation, and postmortem interval (Table 1). Pairwise comparisons showed that the We found no correlations between cortical or hippocam- difference in Mini-Mental State Examination score among pal NGF levels and ChAT activity (Figure 2). We also the 3 ROS groups (PϽ.001) was primarily due to lower found decreases in nucleus basalis of Meynert neurons scores in the AD group (P=.04). Of the mild AD cases, 42% containing ChAT, tyrosine receptor kinase A, or low- contained at least 1 APOE ⑀4 allele compared with 28% affinity neurotrophin receptor, as derived from previ- for the MCI group and 20% for the NCI group (Table 1); ous studies.15,17,18,21 We have not performed cell counts the differences were not statistically significant. Of the pa- of cholinergic septal diagonal band–hippocampal pro- tients with end-stage AD, 90% had at least 1 ⑀4 allele. jection neurons, so we were unable to determine the correlations with hippocampal NGF levels. BRAIN NGF LEVELS NGF LEVELS VS COGNITIVE SCORES There were no differences in NGF levels across clinical groups for any of the cortical regions examined (Figure 1 For each brain region examined, we evaluated the asso- and Table 2). The hippocampus of the subjects with mild ciations between NGF levels and scores on cognitive tests AD contained the highest NGF levels (Table 2); levels referable to these regions. Scores for these analyses were were not statistically different among the 3 clinically di- obtained from the last annual evaluation, performed agnostic groups in the ROS. Mean±SD NGF levels were within 1 year of death. Hippocampal (Spearman rank cor- 13.4±5.5 for the middle temporal cortex and 25.9±13.9 relation; r=−0.31; P=.04) and middle temporal cortex for the hippocampus in patients with end-stage AD. No (Spearman rank correlation; r=−0.35; P=.01) NGF lev- significant differences in NGF levels were found when els were negatively correlated with Word List Recogni-

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ROS Cases

NCI MCI Mild AD Total P Value* Anterior cingulate No. of subjects 21 18 12 51 Mean ± SD 15.4 ± 12.4 14.5 ± 11.8 23.4 ± 29.9 17.0 ± 17.8 .99 Range 1.9-35.9 2.3-37.8 1.3-97.7 1.3-97.7 Superior frontal cortex No. of subjects 23 16 12 51 Mean ± SD 17.0 ± 11.9 16.9 ± 13.4 10.5 ± 12.4 15.4 ± 12.6 .14 Range 1.0-41.6 0.6-42.2 0.3-44.7 0.3-44.5 Inferior parietal cortex No. of subjects 22 16 12 50 Mean ± SD 6.6 ± 5.7 9.3 ± 8.1 7.2 ± 4.3 7.6 ± 6.3 .54 Range 1.0-25.4 1.7-30.5 1.9-13.3 1.0-30.5 Superior temporal cortex No. of subjects 23 17 12 52 Mean ± SD 21.2 ± 9.6 18.9 ± 11.6 20.2 ± 10.5 20.2 ± 10.4 .82 Range 5.9-41.6 5.2-35.5 2.4-34.8 2.4-41.6 Middle temporal cortex No. of subjects 23 17 12 52 Mean ± SD 12.2 ± 7.5 18.8 ± 15.8 14.6 ± 10.4 14.9 ± 11.6 .16 Range 5.2-35.3 5.2-61.9 5.2-41.0 5.2-61.9 Hippocampus No. of subjects 18 15 12 45 Mean ± SD 23.6 ± 15.2 22.7 ± 15.2 31.3 ± 26.8 25.4 ± 18.9 .79 Range 10.7-64.3 4.8-60.8 5.5-84.2 4.8-84.2

Abbreviations: AD, Alzheimer disease; MCI, mild cognitive impairment; NCI, no cognitive impairment; ROS, Religious Orders Study. *Comparison among the 3 ROS groups was performed using the Kruskal-Wallis test.

Hippocampus Superior Frontal NCI MCI 12 Mild AD 5

4 8

3 h per Gram of Protein

4 2 mol/ µ

1 ChAT, ChAT,

0 0

10 30 50 70 90 0 10 20 30 40 NGF, pg/mg of Protein NGF, pg/mg of Protein

Figure 2. Correlation of hippocampal and superior frontal cortex nerve growth factor (NGF) levels and choline acetyltransferase (ChAT) activity in individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), or Alzheimer disease (AD).

tion Test1 scores; superior frontal cortex levels were posi- els was found. Across the clinical spectrum of severity, tively correlated with these scores (Spearman rank there were no differences based on Braak and Braak stag- correlation; r=0.29; P=.04). The NGF levels and cogni- ing or NIA-Reagan criteria (Figure 3). tive functions in other brain regions had no statistically significant correlations (data not shown). COMMENT NGF LEVELS VS REGIONAL QUANTITATIVE The NGF levels in the cortex and hippocampus of people PATHOLOGIC FEATURES with MCI, mild AD, or severe AD are stable, consistent with most other studies showing stability of NGF in end- For 3 cortical regions (superior frontal, superior tempo- stage AD.4,7 In this study, clinical information and diag- ral, and inferior parietal), neuropathologic plaque counts nosis were based on a standard, uniform clinical evalu- were determined; no association between mean NGF lev- ation prior to death.20 Therefore, this approach was not

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 confounded by selection bias resulting from case catego- rization and selection of subjects using neuropathologic A NCI 8 80 MCI criteria. In contrast, the earlier study clinically defined Mild AD

its subjects by a retrospective evaluation of case notes and 60 postmortem interviews of relatives and medical staff as well as the display of ␤-amyloid 4 cortical plaques, none 40

of which may provide an accurate representation of a pa- Cases, % tient’s clinical status. These methodological differences 20

make it difficult to compare observations between these 0 studies. Not AD Low Intermediate High Prior studies indicating an increase in NGF mes- NIA-Reagan Diagnosis: Likelihood of AD senger RNA and protein were performed in cases of end- B stage AD.6,8-13 Scott et al11 reported a wide range of NGF- like activity in a population of patients who had early- 60 to late-onset AD. In their cohort some of the highest and lowest levels of NGF were seen in end-stage AD, suggest- 40 ing that within a given disease cohort, NGF levels can be differentially affected by age at disease onset. Despite dif- Cases, % 20 ferences in age at onset, we and others11 found no correlation between ChAT activity and NGF levels, nor be- 0 0 I-II III-IV V-VI tween the reduction in the number of neurons containing Braak and Braak Stage ChAT,17 tyrosine receptor kinase A,18 or low-affinity neurotrophin receptor and the NGF levels in MCI and mild Figure 3. Bar graph showing percentages of no cognitive impairment (NCI), AD. Moreover, the lack of correlation between any APOE mild cognitive impairment (MCI), or Alzheimer disease (AD) cases represented ⑀4 genotype and NGF levels across disease stages is inter- within each of the 4 diagnostic groups according to National Institute on Aging (NIA)–Reagan criteria (A) and Braak and Braak staging (B). esting because APOE ⑀3 and ⑀4 alleles are associated with a greater reduction in cholinergic markers in end-stage AD.30 The similarity in the degree of pathologic charac- movic, Bennett, Cochran, and DeKosky); analysis and in- teristics between the MCI and AD groups indicates that terpretation of data (Drs Mufson, Ikonomovic, and De- global AD-like pathologic features generally do not in- Kosky); drafting of the manuscript (Drs Mufson); critical duce changes in NGF regulation within the septohippo- revision of the manuscript for important intellectual con- campal or basocortical projection systems early in the dis- tent (Drs Ikonomovic, Styren, Counts, Leurgans, Ben- ease state. These findings are consistent with other nett, Cochran, and DeKosky and Ms Wuu); statistical ex- investigations demonstrating that the underlying patho- pertise (Dr Leurgans and Ms Wuu); obtained funding (Drs logic characteristics of MCI are similar to those seen in Mufson, Bennett, and DeKosky); administrative, techni- AD.15,16 This suggests that AD-like abnormalities do not cal, and material support (Drs Mufson, Ikonomovic, Ben- produce alterations in the cholinotrophic projection sys- nett, Cochran, and DeKosky); study supervision (Drs Muf- tems during the prodromal stage of the disease process. son and Bennett). Taken together, these observations do not support the hy- This study was supported by grants AG05133, pothesis that alterations in the central cholinergic corti- AG14449, AG10161, AG10688, AG09466, and AG00257 cal projection system affect NGF levels during the dis- from the National Institute on Aging, Bethesda, Md, and the ease progression. Illinois Department of Public Health, Springfield. We previously demonstrated an up-regulation of We gratefully acknowledge the altruism and support ChAT activity in the hippocampus and superior frontal of the nuns, priests, and brothers from the following groups cortex in MCI,15,16 but there was no concomitant in- in the ROS: Archdiocesan priests, Chicago, Ill, Dubuque, crease in NGF concentration in these or other regions Iowa, and Milwaukee, Wis; Benedictine Monks, Lisle, Ill, examined in the present study. Elevation of hippocam- and Collegeville, Minn; Benedictine Sisters of Erie, Erie Pa; pal ChAT activity in MCI may be due to a cholinergic Benedictine Sisters of the Sacred Heart, Lisle; Capuchins, plasticity response related to the degeneration of ento- Appleton, Wis; Christian Brothers, Chicago, and Mem- rhinal stellate neurons early in the disease,19,31 which in- phis, Tenn; Diocesan priests, Gary, Ind; Dominicans, River duces sprouting of septal cholinotrophic projections into Forest, Ill; Felician Sisters, Chicago; Franciscan Hand- the denervated hippocampus.32,33 maids of Mary, New York, NY; Franciscans; Chicago; Holy Finally, because NGF levels in CBF cortical target sites Spirit Missionary Sisters, Techny, Ill; Maryknolls, Los Al- are unchanged across all stages of AD, it is unlikely that a tos, Calif, and Maryknoll, NY; Norbertines, DePere, Wis; simple defect in NGF production underlies the reduction Oblate Sisters of Providence, Baltimore, Md; Passionists, Chi- of this protein seen in CBF neurons in end-stage AD.34 The cago; Presentation Sisters, Dubuque; Servites, Chicago; Sin- stability of brain NGF levels appears sufficient to support sinawa Dominican Sisters, Chicago, and Sinsinawa, Wis; the cholinergic plasticity response seen in MCI.15,16 Sisters of Charity, Blessed Virgin Mary, Chicago and Dubu- que; Sisters of the Holy Family, New Orleans, La; Sisters of Accepted for publication April 1, 2003. the Holy Family of Nazareth, Des Plaines, Ill; Sisters of Mercy Author contributions: Study concept and design (Drs of the Americas, Chicago, Aurora, Ill, and Erie; Sisters of Mufson and DeKosky); acquisition of data (Drs Ikono- St Benedict, Minn, St Cloud, Minn, and St Joseph, Minn; Sis-

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 ters of St Casimir, Chicago; Sisters of St Francis of Mary 15. DeKosky ST, Ikonomovic MD, Styren S, et al. Upregulation of choline acetyl- Immaculate, Joliet, Ill; Sisters of St Joseph of LaGrange, transferase activity in hippocampus and frontal cortex of elderly subjects with mild cognitive impairment. Ann Neurol. 2002;51:145-155. LaGrange Park, Ill; Society of Divine Word, Techny; Trap- 16. Ikonomovic MD, Mufson EJ, Woo J, Cochran EJ, Bennett DA, DeKosky ST. pists, Gethsemane, Ky, and Peosta, Iowa; Wheaton Fran- Cholinergic plasticity in hippocampus of individuals with mild cognitive ciscan Sisters, Wheaton, Ill. We are also indebted to the dedi- impairment: correlation with Alzheimer’s neuropathology. J Alzheimers Dis. 2003; cation and hard work of J. Bach, MSW, ROS coordinator; 5:39-48. 17. Gilmor ML, Erickson JD, Varoqui H, et al. Preservation of nucleus basalis neu- B. Howard, W. Longman, and S. Shafaq of the Rush Brain rons containing choline acetyltransferase and the vesicular acetylcholine trans- Bank, Chicago; and G. Klein and W. Fan for data re- porter in the elderly with mild cognitive impairment and early Alzheimer’s dis- trieval. We thank William R. Paljug, Michael Paulin, and ease. J Comp Neurol. 1999;411:693-704. Mohamed Nadeem for technical support. 18. Mufson EJ, Ma SJ, Cochran EJ, et al. Loss of nucleus basalis neurons contain- Corresponding author and reprints: Elliott J. Mufson, ing trkA immunoreactivity in individuals with mild cognitive impairment and early Alzheimer’s disease. J Comp Neurol. 2000;427:19-30. PhD, Department of Neurological Sciences, Rush Presby- 19. Kordower JH, Chu Y, Stebbins GT, et al. Loss and atrophy of layer II entorhinal terian–St Luke’s Medical Center, 2242 W Harrison St, Chi- cortex neurons in elderly people with mild cognitive impairment. 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