Neuropathological correlates of vulnerability and resilience in the cerebellum in Alzheimer's disease

Abstract

Introduction: We investigated whether the cerebellum develops neuropathology that correlates with well-accepted Alzheimer's disease (AD) neuropathological markers and cognitive status.

Methods: We studied cerebellar cytoarchitecture in a cohort (N = 30) of brain donors. In a larger cohort (N = 605), we queried whether the weight of the contents of the posterior fossa (PF), which contains primarily cerebellum, correlated with dementia status.

Results: Although there was no granular layer (GL) cell loss, GL area was lower in AD cases, particularly in the lateral cerebellum. Lower numbers of mossy fiber synaptic terminals in the cerebellar GL of AD cases correlated with Braak stages IV-VI. PF content weight correlated with dementia independently of age, neuropathology, and education. In addition, we found that a measure of the relative size of the PF content weight to total brain weight correlated with less dementia.

Discussion: These results confirm that the cerebellum is not spared neuropathological damage in AD.

Highlights: Novel evidence of cerebellar atrophy in the granule cell layer of the lateral cerebellar cortex (or 'cognitive cerebellum'), and loss of a specific cerebellar synapse type in this region, the cerebellar glomerulus. Both correlated with dementia status and Braak stages IV through VI, in a cohort with complete neuropathological characterization. Although there have been recent brain imaging studies suggesting a role for cerebellum in Alzheimer's disease, we believe our study constitutes some of the most concrete neuropathological evidence to date of anatomic and synaptic substrates that are disrupted in AD. These changes in this cerebellar region may even play a role in the etiology of cognitive symptoms. Novel evidence that individuals with lower postmortem cerebellar weights showed more cognitive decline, independent of classical neuropathology markers such as Braak stage, Thal phase, or Corsortium to Establish a Registry for Alzheimer's Disease (CERAD) score, suggesting a role for this brain region in dementia, using advanced statistical analysis of a large unbiased population cohort (n = 605), the Adult Changes in Thought (ACT) study. Conversely, a measure of how intact the cerebellum was correlated with less dementia, independent of classical neuropathology markers and cerebral cortical weight, again, in the ACT cohort of 605 brain donors. We believe that this novel finding has relevance and implications for the identification of resilience factors, which may protect against the development of dementia.

Keywords: cerebellar glomerulus; cerebellar reserve; cognition; granule cells; posterior fossa contents; synapse loss.

FIGURE 1

Lower granular layer areas are associated with dementia status and AD neuropathology. (A) Sample labeling with H&E‐ and LFB‐stained slides (5 µm thickness) of granular and molecular cell layers, white matter, and dentate nucleus using Visiopharm deep learning applications. Scale bar = 6 mm. The average ratio of ML:GL for each participant plotted as a function of their (B) dementia status. Means compared with an unpaired t‐test with Welch's correction. *p < 0.05. (C) Braak stage: one‐way ANOVA: F _{(3, 26)} = 3.11, p < 0.05. On post hoc Dunnett's multiple comparison's test, comparison between Braak stages 0–3 and Braak stages 4 and 5 were not significant; comparison between Braak stages 0–3 and Braak stage 6 was significant (*p < 0.04), and (D) Thal phase: one‐way ANOVA: F _{(3, 26)} = 3.99, p = 0.018. On post hoc Dunnett's multiple comparison's test, Thal phase 5 was greater than Thal phase 0–2, p = 0.017. (E) Comparison of the average ratio of ML:GL from the vermis and lateral hemisphere for each participant plotted as a function of their dementia status. For dementia status, two factors were significant: cerebellar cortical region (F(_1,54) = 15.15, ***p < 0.001), and dementia status (F_(1,54) = 10.6, **p < 0.01) but not their interaction (F(_1,54) = 0.005, p < 0.95) on two‐way ANOVA. The average ratio of ML:GL area in the Dementia vs No Dementia comparison was significant only in the lateral cerebellar cortex (*p < 0.05), using Šídák's multiple comparisons test. (F, G) ML:GL ratio was graphed as a function of age in the histology cohort. The ratio of the ML:GL was anticorrelated with age in sections from the lateral cerebellar cortex when analyzed with a simple linear regression. The slope was significantly non‐zero in the lateral cerebellar cortex (F) F _(1,28) = 6.423, p = 0.017, F _(1,26) = 6.53, p = 0.017 with presenilin cases removed; but not the vermis (G) F _(1,28) = 2.76 p = 0.108. In B, C, D, E, and F, Presenilin cases are in teal. AD, Alzheimer's Disease; ANOVA, Analysis of Variance; H&E, Hematoxylin and eosin; LFB, Luxol fast blue; µm, micron; mm, millimeter; ML:GL, molecular layer to granular layer ratio.

FIGURE 2

VGlut1+ glomeruli are significantly reduced in the cerebellar granular layer with dementia and high Braak stage, and VGlut1+ staining is increased in the molecular layer of subjects with dementia. (A) Representative staining of VGlut1+ staining (red) in the granular layer (GL) of cerebellar cortex in a control case. Scale bar = 100 µm. Inset is zoomed to show two distinct morphological structures stained by VGlut1 co‐localized with DAPI (gray) in the GL: glomeruli (arrows) and small punctate synapses (arrowheads). ML = molecular layer. (B) Same image as in (A), but with co‐staining for VGlut1 (red), mGluR2/3 (green), and GFAP (blue). Inset is zoomed to show distinct VGlut1+, mGluR2/3+, and GFAP‐positive glomeruli and small VGlut1+ puncta. (C) Representative image of VGlut1+ (red) staining in the molecular layer. Scale bar = 100 µm. (D) Same image as in (C), but with co‐staining for VGlut1 (red), mGluR2/3 (green), and GFAP (blue). (E) The average density of small VGlut1+ puncta within the granular layer, (F) the average density of large VGlut1+ glomeruli within the granular layer, and (G) the average VGlut1+ staining intensity within the molecular layer for each participant plotted as a function of their dementia status. Means compared with an unpaired t‐test with Welch's correction in (E), (F), and (G) *** p < 0.001, * p < 0.05. (H) VGlut1+ glomerular density in GL correlates with Braak staging. Braak stage: one‐way ANOVA: F _{(3, 26)} = 6.5, p < 0.02. Comparison between Braak stages 0–3 and Braak stage 4: **p < 0.018; Comparison between Braak stages 0–3 and Braak stage 5: p < 0.101 (not significant); comparison between Braak stages 0–3 and Braak stage 6: *p < 0.016. (I) Average VGlut1+ stain intensity in ML does not correlate with Braak staging. Section thickness is 5 µm. In A–D, the dotted red line indicates the area for Visiopharm app to quantify staining per area. In E through I, Presenilin cases are in teal. DAPI, 4',6‐diamidino‐2‐phenylindole; GFAP, Glial fibrillary acidic protein; mGluR2/3, metabotropic glutamate receptor 2/3; Vglut1+, Vesicular glutamate transporter 1 positive.

FIGURE 3

PF Content weight quintile predicts the risk of dementia independent of known AD risk factors. Forest plot of relative risk (RR) from the final linear model for dementia score as a function of Braak stage, CERAD score, age at death, sex, education, and posterior fossa contents weight quintile from Table 4. CERAD, Corsortium to Establish a Registry for Alzheimer's Disease; CI, Confidence Interval; PFW, Posterior fossa content weight quintile; RR, Relative Risk.