Neurond Loss Correlates with but Exceeds Newofibriiary Tangles in Alzheimer's Disease

Teresa G6mez-Isla, MD,* Richard Hollister, BA,* Howard West, MD,* Stina Mui, BA,* John H. Growdon, MD,* Ronald C. Petersen, MD, PhD,? Joseph E. Parisi, MD,? and

Bradley T. Hyman, MD, PhD*

To assess the relationship between dementia, neuronal loss, and neuropathological findings in Alzheimer's disease (AD), we counted the number of neurons, senile plaques, and neurofibrillary tangles in a high-order association cortex. We studied the superior temporal sulcus of 34 individuals with AD and 17 nondemented control subjects, using statistically unbiased, stereological counting techniques. The number of superior temporal sulcus neurons in nondemented control subjects was stable across the sixth to ninth decades. In AD, more than 50% of the neurons were lost. Both neuronal loss and neurofibrillary tangles increased in parallel with the duration and severity of illness, but the amount of neuronal loss exceeded by manyfold the amount of neurofibrillary tangles accumulated. In contrast to the correlation between neurofibrillary tangles and neuronal loss, the number of senile plaques and the percentage of the superior temporal sulcus that was covered by AP (amyloid burden) were not related to neuronal loss, number of neurofibrillary tangles, or duration of disease. Neither the amount nor the rate of neuronal loss in the superior temporal sulcus in AD correlated with apolipoprotein E genotype. These data suggest that neuronal loss in association areas such as the superior temporal sulcus contributes directly to cognitive impairment in AD.

Gomez-Isla T, Hollister R, West H, Mui S, Growdon JH, Petersen RC, Parisi JE, Hyman BT. Neuronal loss correlates with but exceeds neurofibrillary tangles in Alzheimer's disease. Ann Neurol 1997;4 1 : 17-24

Neuropathological study of the Alzheimer's disease (AD) brain highlights the appearance of neurofibrillary tangles (NFTs) and senile plaques (SPs), as well as the loss of neurons and synapses. NFTs occur primarily in projection neurons of limbic and association cortices and it is believed that their presence disrupts the flow of information along axons linking association cortices and the limbic cortical areas in the medial temporal lobe [I-71. NFTs specifically affect neurons in layers 11, 111, and V in association cortex, corresponding to feedforward and feedback projections [3, 8, 31. SPs have a broader and more varied distribution than NFTs. Although they affect association cortices, SPs may be present in primary motor and sensory cortices that remain clinically silent, even at the very late stages of the disease [3, 51. Previous studies suggested that the number of SPs in the cerebral cortex tends to re- main stable independently of duration of illness, while the number of NFTs correlates more closely with the severity of dementia [5 , 7, 10-121. From this perspec- tive, the presence of NFTs qualitatively and quantita- tively matches the clinical deficits of the disease better than the number or distribution of SPs.

By contrast to NFTs and SPs, neuronal loss, as a negative phenomenon, cannot be easily quantitated but

only calculated from normative data on age-matched control populations. Moreover, the cause of neuronal loss in neurodegenerative processes is unknown, and there are no biochemical markers to examine neurons at risk for death. As a consequence, no topographic map has been established to evaluate whether or not there are consistent patterns of neuronal loss. There- fore, our first goal was to determine whether neuronal loss correlates with NFTs or SPs.

Recent studies suggested that inheritance of the apolipoprotein E (APOE) ~4 gene is a risk factor for AD [13, 141. Therefore, our second goal was to test the hypothesis that APOE4 is associated with faster or a greater extent of neuronal loss.

The recent application of stereological counting techniques for counting neurons has provided a power- ful tool to address these questions, overcoming the problems of correcting for cortical atrophy and ad- justing appropriately for cell shrinkage and different sizes of cells that lead to potential errors in traditional two-dimensional counting schemes [ 15, 161.

We chose to study the region that configures the superior temporal sulcus (STS) for several reasons. First, it is a high-order association cortex that receives input from multiple modalities. It is one of only three

From the *Neurology Service, Massachusetts General Hospital, Bos- ton, MA, and ?Departments of Neurology and Pathology, Mayo Clinic, Rochester, MN.

Received Apr 12, 1996, and in revised form Jul 5. Accepted for publication Jul 10, 1996.

Address correspondence to Dr Hyman, Neurology Service, 149 13th Street, Room 6405, Massachusetts General Hospital, Charles- town, MA 02123.

Copyright 0 1997 by the American Neurological Association 17

association areas i n the nonhuman primate brain that receives input from each sensory modality [ G , 171. Sec- ond, it is well demarcated anatomically a n d shows little brain-to-brain variability, making the task of defining a region of interest in different brains easier. Third, it is known to be consistently affected by both NFTs and SPs in AD [3, 5, 12, 181.

Materials and Methods Patient Selection The 34 cases of AD selected for study met the following inclusion and exclusion criteria: The patients had been exam- ined and followed in thc clinical units of the Massachusetts General Hospital Alzheimer’s Disease Research Center or the Mayo Clinic Alzheimer’s Disease Center, had clear clinical histories of AD dementia with well-documented duration of disease, and had a confirmed neuropathological diagnosis of AD [13] without evidence of vascular insults, cortical Lewy bodies, or other lesions. O f the 34 cases, 33 came from the Massachusetts General Hospital. All of them had undergone biannual serial evaluations that included general medical and neurological examinations as well as neuropsychological as- sessments over an average of 39.1 ? 25.9 months (mean ? standard deviation [SD]). T o test whether neuropathological changes and neuronal loss were correlated with severity of dementia, we charted measures of cognitive impairment de- rived from the information, memory, and concentration (ICM) subtest of the Blessed Dementia Scale (BDS) [20]. BDS scores within 18 months before death were available for 9 patients. There were 17 control subjects with an age range (sixth decade to the tenth decade) chat matched that of the AD group. Of the 17 control brains, 14 were from the Massachusetts Alzheimer’s Disease Research Center brain bank. These control subjects were believed to be cognitively normal based on retrospective clinical assessment using previ- ously published criteria 1211. Three control brains and 1 AD brain were obtained from the Mayo Clinic Alzheimer’s Disease Center. All 4 of these subjects had participated in several com- munity-based studies on cognitive function [22-251 and all had been examined clinically within 1 year of death. None of the control brains met neuropathological criteria for AD [ 191.

Tissue Processing All brains were fixed in 10% buffered formalin or 4% para- formaldehyde, within 24 hours after death. Two to 7 days later, blocks were placed in a cryoprotection solution con- taining 4% buffered formalin, 10%) glycerol, and 10% su- crose. We alternated between using right and left hemi- spheres of the AD brains, freezing the other half for biochemical assays. Fifty-micrometer-thick sections were ob- tained from blocks containing the STS region by using a freezing sledge microtome. Sections were stained using the Nissl procedure to determine neuronal counts, monoclonal antibodies against paired helical filaments (PHF- 1, courtesy of Dr Peter Davies, Bronx, New York) to count NFTs, and P-amyloid (10D5, courtesy of Dr Dale Schenk, Athena Neu- rosciences, South Sail Francisco, CA) to count SPs. Quanti- tation was previously described [ l o , 121. Sections were pre- treated with 70% formic acid for 10 minutes for AP

Fig 1. Photomicrogruph o f a Nissl-stained cross section through the superior temporal sulcus (STS) region in a 95- year-old contpot subject. The box represents a 700-pm-wide counting chamber. SF = sylvian fissure; LV = laterul ven- tricle.

Quantitation of Neurons, Neurojbrillary Tangles, and Senile Plaques Counts were performed following the stereological optical dissector procedure described by West [16]. Data were re- corded by a Bioquant Image Analysis System (Nashville, TN). The region counted was located in the inferior bank of the STS, approximarely 1 cm medial to the crown of the gyrus (Fig 1). Volume density was assessed in an area 700 p m wide by the full width of the cortex. The entire width of the cortex in each STS region was sampled to avoid the possibility of a relative overrepresentation of the more densely packed lamina, layers I1 and IV, in AD brains. The total number of STS neurons per section was calculated for each brain by multiplying the volume density obtained from the optical dissector counts by volume of STS measured on each cross section (“reference volume”). Preliminary experi- ments demonstrated that this estimate was equivalent to counting five 150-pm-wide strips spaced equally throughout the inferior lip, depth, and superior lip of the STS. This estimate of neuronal number accounts for shrinkage in the width of the cortex but might underestimate loss in AD be- cause atrophy in the anterior-posterior direction is not ac- counted for. Ideally, one would measure the anterior-poste- rior direction length of the STS in each brain and derive an estimate of total number. Unfortunately, both the anterior and posterior boundaries of the STS are somewhat variable in humans, making this approach untenable. Therefore, we chose the conservative approach of counting neurons at a single well-described level. Approximately 1,000 neurons in each brain were counted, and the percent of neuronal loss in AD brains was calculated by subtracting the number of neurons in an individual AD brain from the average number obtained in the control group. immunostaining.

18 Annals of Neurology Vol 41 No 1 January 1997

Table I . Neuronal, Neurofibrillary Tangle (NFT), and Senile Plaque (SP) Counts in the Superior Temporal Sulcus (STS) Region in A D and Control Groups"

Control Group AD Group (n = 17) (n = 34)

(yr) Gender

M F

No. of neurons per section (X lo4)

Reference volume (mm?

Neuronal density X (neurons/ w 3 )

N ~ . of^^^^ (x 104) Amyloid burden (Yo) No. of SPs/mm2

70.9 2 16.9 80.2 2 8.7

8 17 9 17 9.70 2 0.87 4.57 2 1.77h

6.8 2 0.1 4.3 2 0.1'

1.45 t 0.18 1.05 t 0.29d

0.68 2 0.42 <1Yo 7.20 2 2.75 <6 174 2 83.4

'In the AD group, neuronal loss in the STS represented a 53% loss compared to the control group ( p < 0.001). Neuronal loss was accompanied by a volume reduction of the STS (38%) ( p < 0.01). Pdeuronal density in the STS was 28% less than that in control brains. The values represent the average 5 standard error.

' b < 0.01. h p < 0.001.

< p < 0.05.

N F T counts were obtained using the same stereological methods and following identical sampling schemes, but using I'HF-1 immunostaining [26] in adjacent sections to identify both intraneuronal and extraneuronal NFTs. The percentage of cortex covered by SPs (burden amyloid) were assessed in the same region by using an anti-AP monoclonal antibody, 10D5 [27]. The video images were captured and an optical density threshold able to discriminate the immunostaining was obtained. In each field manual editing eliminated arti- ficts and the staining associated with blood vessels. The number of SPs and the percentage of cortex covered by plaques were calculated.

APOE genotypes were carried out as previously described [14]. APOE genotype, N F T number, and amyloid burden for many of these cases have been reported elsewhere [I21 and are included here for comparison to neuronal counts.

Statistical Analysis A linear regression analysis was used to compare neuron number in the STS between AD and control brains and to correlate neuron number with duration of illness and num- ber of NFTs and SPs in AD brains. The total number of neurons, neuronal density, and volume of STS between the AD and the control group were compared by the t test at p < 0.05 level.

Results The average values for reference volumes, total number of neurons and NFTs, and amyloid burden per 50- lm-thick section estimated in the STS of AD and con- trol groups are shown in Table 1.

Number of Neurons per 5n pm-thick section

60000 j_l 1 20000

Fig 2. No statistically signiJicant differences in the number o f neurons in the superior temporal sulcus volume assessed were observed in the control brains (n = 17) according to age (r = 0.001, not signiJicant [NS]).

'zoooo 1 I 7

100000

60000

Number of Neurons per 50 pm-thick section

60000

40000

20000

53% *

T

A D c

Fig 3. The average total number of neurons in the superior temporal sulcus volume assessed was reduced by 53% in the A D group (n = 34) when compared to the control (C) group (n = 17) (p < 0.001).

The number of neurons in the STS volume assessed in the control brains remained unchanged over the four decades sampled. The absolute number of neurons per cross section in this group ranged from 8.02 X lo4 to 11.44 X lo4, with an average value of 9.70 * 0.87 X lo4 (Fig 2). No significant differences were observed even between the subjects at the extremes of the ages analyzed. Among the oldest subjects were the 3 from the Mayo Clinic Patient Registry (ages 80, 80, and 84) who had had normal findings on neurological and neuropsychological evaluations prior to death.

The total number of STS neurons was reduced by 53% in AD brains when compared to control brains (4.57 +- 1.77 X l o 4 vs 9.70 +- 0.87 X lo4, respec- tively; p < 0.001) (Fig 3). The amount of neuronal loss in AD brains was correlared strongly with the du- ration of illness ( Y = 0.74, p < 0.001), ranging from

G6mez-Isla et al: Neuronal Loss in the Superior Temporal Sulcus in Alzheimer's Disease 19

luuouu 1 80000

18n0u 1 A A -AA

Number of Neurons per SO pm-thick section

0 4 ' 8 " ' 3 ' '

n 5 10 1 5 2 0

Duration (years)

A

Number of NPTs per 50 pm-thick section

Nenrannl Las, 40u0u

3uuuu 1- 200m/-/ , , , , , , , , , ,

per 50 pm-Ihiek section

10u00

U n s 1 0 i s 2 0 2s 30 3s 4 0

Last HDS xore

B

r d . 8 4 p <O.UUl

A

----- U 5 1 0 1 5 20

Duration (years)

A

Fig 4. (A) The amount of neuronal loss in the superior tem- poral sulcus (STS) in the AD group (n = 34) correlated with duration o f illness (r = 0.74, p < 0.001). (B) The amount of neuronal loss in the STS in the A D group corre- lated with the clinical severity o f dementia (r = 0.73, p < 0.02). The graph represents 9 A D patients fiom whom a Blessed Dementia Scale (BDS) score was obtained within 18 months fiom death. In the remaining 25 A D patients this information was not available.

no measurable loss in the patients with clinical symp- toms for the shortest duration (1 year) to more than 75% reduction of STS neurons in several with more advanced AD (Fig 4A). In the 9 AD patients with a BDS score obtained within 18 months before death, neuronal loss had in addition a strong correlation with severity of cognitive impairment ( r = 0.73, p < 0.02) (Fig 4B) .

The loss of neurons in the AD group was accompa- nied by a reduction of the volume of the region, which was estimated at 38% compared to the non-AD brains (6.8 vs 4.3 mrn3). The degree of atrophy increased with duration of dementia ( r = 0.44, p = 0.01).

NFT number in the STS of AD brains progressed in parallel with duration and severity of illness ( r = 0.84, p < 0.001) (Fig 5A) and the amount of neuronal

i 8OOUU

7UUUU f A

6UUU0

Number of NFTs per 50 pm-thick section

6000 9 7

'".-

A

20000 4OUUU 6UUUU 8OU0U

Neuronal Loss

B

Fig 5. (A) The neurojbrillary tangle (NFT) number in the superior temporal sulcus (STS) o f AD brains (n = 34) pro- gressed in parallel with duration of illness (r = 0.84, p < 0.001). (B) The N F T number in the STS of A D brains (n = 34) correlated with the amount of neuronal loss (r = 0.66 p < 0.01).

loss ( r = 0.66, p < 0.01) (see Fig 5B), with absolute numbers per section ranging from 155 to 16,687. 'The number of neurons lost (calculated by subtracting the number of neurons in each AD brain from the average number of neurons estimated in the control group), however, dramatically exceeded NFT number by more than sevenfold (Fig 6).

Assessment of SPs showed no correlation between the percentage of STS covered by AD (amyloid burden) or number of SPs and severity of neuronal depopula- tion ( r = 0.16, p = 0.34, not significant [NS]) (Fig 7), the amount of NFTs ( r = 0.03, p = 0.85, NS), or duration or severity of illness ( r = 0.04, p = 0.80, NS). A few scattered SPs in the STS were observed in two of the control brains. The amyloid burden in these latter brains was less than 1%.

Finally, we evaluated the possibility that the rapidity or amount of neuronal loss in AD might be influenced by APOE genotype. Comparison of the amount of neuronal loss calculated per year among APOE geno-

20 Annals of Neurology Vol 41 No 1 January 1997

80000 1 /

70000 I A

A Number Of NFTs 40000

30000

20000

ioono

0 0 5 10 1 5 20

Duration (years)

Fig 6 The number of neurons lost in the superior temporal sulcus o f AD brains (n = 34) correlated but dramatically exceeded the neurofibrillary tangle (NFT) number by ouer seuenfi ld.

151 I a

Amyloid Burden (a) as e%i ~

5

I .I .

0 zoono 40000 mnno m o o

Eleuronal LO?,

Fig Z The percentage o f superior temporal sulcus covered by Ap (amyloid burden) in AD brains (n = 34) did not corre- late with neuronal loss (r = 0.16 p = 0.34, not signijcant [NSI).

Gb1e 2. Comparison o f Rate of Neuronal Loss in the Superior Temporal Sulcus among APOE Genotypesa ~ ~

APOE Genotype N Age (yr) at Onsetb Duration (yr) Neuronal Loss (X 104)b Neuronal Loss (X 1O7)/yrb

313 314 4J4

10 72.25 f 16.06 9.5 f 5.3 5.65 f 1.85 7.71 2 3.92 12 74.33 2 7.15 7.6 t 4.4 4.64 f 1.93 7.89 f 6.14 7 65.14 t 11.20 10 2 4.0 6.03 f 0.66 6.76 +- 2.35

‘The comparison of rate of neuronal loss in AD brains among different genotypes showed no statistically significant differences. The infrequent cases with genotypes 2/3 (n = 1) and 2 / 4 (n = 1) were excluded in these analyses. hData are means +- standard deviations.

type subgroups (313, n = 10; 314, n = 12; 414, n = 7) showed no statistically significant differences (Table

Discussion Clarifying the relationship among loss of neurons, de- velopment of NFTs and SPs, and measures of dementia is a central goal in defining the pathophysiological basis of AD. The results of this study support four main conclusions: ( I ) In nondemented individuals, the num- ber of neurons in the STS region remains stable be- tween the sixth and tenth decades of life. (2) In AD, neuronal loss is dramatic in the STS and parallels the chronological evolution of dementia. (3 ) NFT forma- tion correlates significantly with duration of illness and with number of neurons lost in the STS, but the amount of neuronal loss in the STS is severalfold greater than the number of NFTs. (4) Amyloid deposi- tion does not correlate with neuronal loss, NFT forma- tion, or severity of illness. Each of these points is dis- cussed in turn.

Studies on neuronal loss in different neurodegenera- tive diseases and the aging process first began a hun-

dred years ago [28, 291. More recently, studies have been directed at assessing the occurrence, extent, topo- graphical distribution, and role of neuronal loss in AD and normal aging [30-351. Unfortunately, data from these studies are frequently contradictory and difficult to compare, because the populations selected and the sampling schemes and counting techniques used varied widely [35]. There is overall consensus, however, that neuronal loss occurs selectively in vulnerable areas of AD brains when compared to age-matched normal specimens 1341. There is more controversy than con- sensus regarding the issue of neuronal depletion as a common concomitant of aging not associated with cog- nitive decline 130, 31, 35-37]. Our findings clearly address this question in the STS: Across the four de- cades of life assessed in this study there was no measur- able neuronal loss in the STS region. The total number of neurons estimated per cross section was stable across age in nondemented subjects. This finding coincides with our previous report of neuronal stability in the entorhinal cortex of nondemented individuals of about the same age range [38], and supports the conclusion that neuronal loss in these regions is not inevitable in

G6mez-Isla er a]: Neuronal Loss in the Superior Temporal Sulcus in Alzheimer’s Disease 21

normal aging. Even though the complexity of the hu- man brain does not allow us to extrapolate these find- ings to every brain region, we believe that these data support the notion that a dramatic loss of cortical neu- rons does not occur in normal healthy elderly subjects.

Assuming that individuals who ultimately develop AD start with about the same number of neurons as those who do not, out analysis of neuronal counts in the STS of AD brains reveals that more than half of the original STS neuronal population is lost during the course of the disease. This concurs with other reports of major neuronal losses in specific cell populations of the limbic and association cortices in AD [12, 16, 391. A single report [40] failed to find neuronal loss in AD, but did not directly examine limbic and association regions that are known to be selectively affected by AD.

Neuronal loss in the STS region is not apparent early in the disease process, despite the presence of many SPs and occasional NFTs. This result contrasts with our observation that there is marked (>50%) loss of layer I1 entorhinal cortex neurons in AD individuals wit$ very mild cognitive impairment [38]. These ob- servations support the hypothesis that there is a topo- graphical hierarchy of neuronal loss that closely paral- lels that described for the development of NFTs. These data are consistent with the idea that memory loss (usually impairment of delayed recall), which is often the earliest symptom in AD, stems from neuronal de- population in the entorhinal cortex and other medial temporal lobe structures. During this early stage of de- mentia, association cortices are relatively preserved in terms of neuronal numbers. As the clinical illness evolves from barely detectable memory loss to wide- spread cognitive failure with language impairment, there is a progressive loss of STS neurons. The strong correlation between neuronal loss in the STS and sever- ity of cognitive impairment assessed by the BDS (see Fig 4B) supports this observation.

From a theoretical standpoint, loss of neurons and the anatomical connections that make the STS a high- order association cortex would contribute to AD de- mentia. There is substantial evidence from experimen- tal studies that lesions in the STS would be expected to impair complex cognitive processes. Based on ana- tomical studies, the polysensory nature of the STS region is well known [41-431. The STS receives con- nections from auditory, somatosensory, and visual asso- ciation areas [41, 441. Along these lines, single cell re- cordings in macaque brain have shown that a population of cells located in the STS is involved in visual processing concerned with faces, with separate functional subsystems sensitive to face and head orien- tation, eye gaze direction, facial expression, and facial identity [45, 461. The dramatic neuronal loss found here in the STS of AD brains could underly similar

damage in visual and auditory processing systems dur- ing the course of the disease.

The contribution of NFT and AP deposits to neu- ronal loss and to the clinical evolution of AD remains controversial. Early studies emphasized a prime role of amyloid accumulation in the progression and severity of dementia [20, 47-49]. Other studies, including our own, found a closer correlation between NFT accumu- lation and clinical evolution of the disease than with amyloid [5 , 7, 10, 11, 50, 511. Based on our previous observations, we had anticipated that the correlation between the magnitude of neuronal loss and the degree of neuropathological changes in AD may be better for NFTs than SPs. The data confirmed our prediction: The number of NFTs in the STS of AD brains in- creased in close relation with the number of neurons lost and with the progression of dementia. However, when absolute numbers of neurons and NFTs ate com- pared, neuronal loss in the STS exceeded by far the number of NFTs. Taking into account the fact that the majority of NFTs in the STS (>90%) are intracel- lular, only about 1 to 2% of neuronal loss in the STS can be attributed to neurons for which an extracellular “tombstone” remains. This observation suggests one of two possibilities: (1) NFTs might be formed, be in- volved in the death of neurons, and then be cleared leaving no trace; or (2) the majority of neuronal loss in advanced AD occurs through a non-NFT mecha- nism. The first possibility cannot be excluded, but the relatively insoluble nature of NFTs qualifies them more as permanent markers of neuronal loss than as indices of a transient stage in cell death. The second possibility suggests that stereological techniques demonstrate an underappreciated alteration in AD brain than numeri- cally is of substantially greater impact than NFTs. The observations of a laminar disparity between NFT distri- bution and losses of large projection neurons in visual cortices of AD brains [9], and earlier observations by Terry and colleagues [52] support this view.

By contrast with the parallel between neuronal loss and NFT development, AP deposition does not corre- late with neuronal loss, number of NFTs, or duration or severity of illness. These findings are consistent with the suggestion that the number of AP deposits remains stable despite progression of dementia [5, 10-121. Our data do not rule out the possibility that there is a rela- tionship between neuronal loss and specific subsets of SPs (e.g., neuritic plaques, cored plaques, or SPs con- taining specific forms of A@), but argue against a local direct toxic effect of AD on neurons.

APOE genotype influences the clinical and neuro- pathological phenotype of AD [12, 14, 531. APOE4 is associated with increased AP deposition [14] but has no effect on rate of NFT accumulation [la]. Recent data suggest that APOES (more than APOE4) might have a trophic effect on neurons by supporting neurite

22 Annals of Neurology Vol 41 No 1 January 1997

outgrowth [54, 551. In this context, APOE genotype - - . _ was correlated with the magnitude of neuronal loss, but no differences in rates of neuronal loss among genotype groups were found. This finding is internally consistent with previous studies [12, 561 showing that APOE ge- notype does not influence rates of cognitive decline in AD.

This work was supported by National Institutes of Health grants AG08031, AG06786, AG05134, and AG08487.

We thank Dr Mark West for his consultations and advice regarding stereological techniques.

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