Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer's disease

Abstract

The entorhinal cortex (EC) plays a crucial role as a gateway connecting the neocortex and the hippocampal formation. Layer II of the EC gives rise to the perforant pathway, the major source of the excitatory input to the hippocampus, and layer IV receives a major hippocampal efferent projection. The EC is affected severely in Alzheimer disease (AD), likely contributing to memory impairment. We applied stereological principles of neuron counting to determine whether neuronal loss occurs in the EC in the very early stages of AD. We studied 20 individuals who at death had a Clinical Dementia Rating (CDR) score of 0 (cognitively normal), 0.5 (very mild), 1 (mild), or 3 (severe cognitive impairment). Lamina-specific neuronal counts were carried out on sections representing the entire EC. In the cognitively normal (CDR = 0) individuals, there were approximately 650,000 neurons in layer II, 1 million neurons in layer IV, and 7 million neurons in the entire EC. The number of neurons remained constant between 60 and 90 years of age. The group with the mildest clinically detectable dementia (CDR = 0.5), all of whom had sufficient neurofibrillary tangles (NFTs) and senile plaques for the neuropathological diagnosis of AD, had 32% fewer EC neurons than controls. Decreases in individual lamina were even more dramatic, with the number of neurons in layer II decreasing by 60% and in layer IV by 40% compared with controls. In the severe dementia cases (CDR = 3), the number of neurons in layer II decreased by approximately 90%, and the number of neurons in layer IV decreased by approximately 70% compared with controls. Neuronal number in AD was inversely proportional to NFT formation and neuritic plaques, but was not related significantly to diffuse plaques or to total plaques. These results support the conclusion that a marked decrement of layer II neurons distinguishes even very mild AD from nondemented aging.

Fig. 1.

No statistically significant differences in the total number of neurons in the EC were observed in the nondemented group (CDR = 0; n = 10) according to age (r = 0.0001, p = 0.98, NS).

Fig. 2.

No statistically significant differences in the number of neurons per layer in the EC were observed in the nondemented group (CDR = 0; n = 10) according to age (layer II, r = 0.001, p = 0.91; layer III,r = 0.04, p = 0.57; layer IV,r = 0.05, p = 0.54; layers V, VI,r = 0.03, p = 0.60, NS).

Fig. 3.

The average total number of neurons in the EC was reduced by 48% in the AD group (n = 10) when compared with the nondemented group (n = 10) (p < 0.001).

Fig. 4.

The volume of the EC was reduced by 40% in the AD group (n = 10) when compared with the nondemented group (n = 10) (p < 0.001).

Fig. 5.

Significant decreases in neuronal number were present in all layers of EC in the AD brains (n = 10) when compared with the nondemented group (n = 10). Decrease in cell number in the AD group was highest in layer II compared with other layers (layer II, 72%, p < 0.001; layer III, 41%, p < 0.01; layer IV, 55%,p < 0.001; layers V, VI, 40%, p < 0.01).

Fig. 6.

The number of neurons in the EC in the AD group (n = 10) compared with CDR = 0 controls (n = 10), correlated with the clinical severity of dementia. The difference increased from 32% in the CDR = 0.5 subgroup (n = 4) to 69% in the CDR = 3 subgroup (n = 5).

Fig. 7.

The number of neurons in the EC of AD brains (n = 10) compared with CDR = 0 controls (n = 10), negatively correlated with clinical severity of dementia in all layers. Layers II and IV, in particular, showed the highest and earliest changes. In layer II, a difference of 57% was estimated in the CDR = 0.5 subgroup (n = 4) and 87% in the CDR = 3 subgroup (n = 5). In layer IV, differences of 41 and 69%, respectively, were estimated in the same CDR subgroups.

Fig. 8.

The topographical distribution of NFTs assessed by PHF-1 immunostaining (Price et al., 1991) in the EC and CA1 zone of the hippocampus is illustrated in each CDR category (CDR = 0, 0.5, and 3).

Fig. 9.

The rank order of cases for NFT density in the EC was negatively correlated with the number of neurons (Spearman rank correlation test n = 17, Z = 3.40, p < 0.001). We also calculated the relationship between the rank order of NFT densities and the number of neurons in the AD group alone (n = 9, Z = −2.24,p = 0.02). The graph represents data from nine AD brains. In one case, the PHF-1 immunostaining for NFTs was not available.

Fig. 10.

a, Total SP density (neuritic and cored plus diffuse plaques), as assessed from a Bielschowsky-stained section in the EC, did not correlate with number of neurons in AD brains (n = 10) (Spearman rank correlation test;Z = −0.17, p = 0.86, NS).b, The density of neuritic and cored plaques in the EC of AD brains (n = 10), as assessed from a Bielschowsky-stained section, negatively correlated with the number of neurons (Spearman rank correlation test; Z = −2.44,p = 0.01). c, The density of diffuse plaques in the EC of AD brains (n = 10), as assessed from a Bielschowsky-stained section, did not correlate with the number of neurons (Spearman rank correlation test; Z = 1.72, p = 0.09, NS).