Histopathology of the cerebellar cortex in essential tremor and other neurodegenerative motor disorders: comparative analysis of 320 brains

Abstract

In recent years, numerous morphologic changes have been identified in the essential tremor (ET) cerebellar cortex, distinguishing ET from control brains. These findings have not been fully contextualized within a broader degenerative disease spectrum, thus limiting their interpretability. Building off our prior study and now doubling the sample size, we conducted comparative analyses in a postmortem series of 320 brains on the severity and patterning of cerebellar cortex degenerative changes in ET (n = 100), other neurodegenerative disorders of the cerebellum [spinocerebellar ataxias (SCAs, n = 47, including 13 SCA3 and 34 SCA1, 2, 6, 7, 8, 14); Friedreich's ataxia (FA, n = 13); multiple system atrophy (MSA), n = 29], and other disorders that may involve the cerebellum [Parkinson's disease (PD), n = 62; dystonia, n = 19] versus controls (n = 50). We generated data on 37 quantitative morphologic metrics, grouped into 8 broad categories: Purkinje cell (PC) loss, heterotopic PCs, PC dendritic changes, PC axonal changes (torpedoes), PC axonal changes (other than torpedoes), PC axonal changes (torpedo-associated), basket cell axonal hypertrophy, and climbing fiber-PC synaptic changes. Principal component analysis of z scored raw data across all diagnoses (11,651 data items) revealed that diagnostic groups were not uniform with respect to pathology. Dystonia and PD each differed from controls in only 4/37 and 5/37 metrics, respectively, whereas ET differed in 21, FA in 10, SCA3 in 10, MSA in 21, and SCA1/2/6/7/8/14 in 27. Pathological changes were generally on the milder end of the degenerative spectrum in ET, FA and SCA3, and on the more severe end of that spectrum in SCA1/2/6/7/8/14. Comparative analyses across morphologic categories demonstrated differences in relative expression, defining distinctive patterns of changes in these groups. In summary, we present a robust and reproducible method that identifies somewhat distinctive signatures of degenerative changes in the cerebellar cortex that mark each of these disorders.

Keywords: Cerebellum; Dystonia; Essential tremor; Histopathology; Neurodegenerative; Spinocerebellar ataxia.

Fig. 1

Principal component analyses of cerebellar morphologic metrics across diagnoses. Principal component analysis of the z-scored raw data across all eight diagnostic categories (a) or across select diagnoses d, f shows how individual patients distribute across the two major axes of variation in the data, identifying distinct disease groupings. Each dot represents data from an individual patient. Panel b is an enlargement of the boxed area in panel (a). Color coded vertical lines d, f denote the median change along the x-axis in principal component 1 for the corresponding diagnostic category. The loading graphs c, e portray the strength and directionality of the correlation between each metric with each principal component, with metrics that are a greater distance from the center (0.0, 0.0) being most correlated with the principal components. Metrics are color coded based on the eight categories of morphologic metrics, as indicated in the legend including Purkinje cell loss (purple), Purkinje cell heterotopia (gray), Purkinje cell dendrites (yellow), Purkinje cell torpedoes (green), Purkinje cell axons, other (orange), Purkinje cell axons, torpedo associated (blue), Basket cell plexus (red) and climbing fibers (light blue). 1c includes all diagnoses and 1e is limited to controls, ET, PD and dystonia. CB = calbindin, GAD glutamic acid decarboxylase, LH&E Luxol fast blue/hematoxylin and eosin, PC Purkinje cell, PC1 principal component 1, PC2 principal component 2, VGlut2 vesicular glutamate transporter type 2

Fig. 2

Differences in fold-change for each metric across diseases and key pathologic drivers. a Metrics (rows) and diagnoses (columns) are shown. Each cell in the heatmap is the average log₂-fold-change (disease vs. control) for each metric. The scale ranges from dark purple (high relative to control) to dark green (low relative to control). Elements with a black asterisk indicate a statistically significant difference vs. controls (Mann–Whitney test, p values corrected for false discovery by Benjamini–Hochberg method; * = FDR < 0.01). b Hierarchical clustering of the correlation coefficients between all pairwise combinations of metrics, with colored scale of red (positive correlation), white (no correlation) and blue (negative correlation). Many of the prominent positively correlated (red and orange asterisks) or negatively correlated (blue asterisk) variables are the same variables that differ across disease categories (see black asterisks in 2a), and are designated here as “Key Drivers” in (a). c Three scores were computed across disease categories by combining the average fold-change (disease/control) for selected metrics and plotted on a log₂-transformed scale, including a “Severity Score” (large block of 25 positively correlated red and orange metrics in panel (b)), a “Purkinje Cell Loss Score” (inverse Purkinje cell body and percent empty baskets), and a score reflecting climbing fibers (CFs) in the outer 20% of the molecular layer. Within each violin, the dashed line shows the median value and the dotted lines indicate outer quartiles in the data distribution. Data for Friedreich’s ataxia was derived from 4 cases in whom metrics from calbindinD_28k staining was performed and 13 cases for all other metrics performed in paraffin sections

Fig. 3

CalbindinD_28k immunohistochemistry on 100-μm cerebellar neocortex sections across diagnoses. Several categories of morphologic changes in Purkinje cells are identified, including cell body loss, heterotopia (white arrow, b, c, f, g, n, p), dendrite swelling (white caret, b–f, h, p), and axonal changes including torpedoes (large black arrow, b–h, –p), thickened axons (arrowhead, b, c [inset], d–h, n, o) and recurrent collaterals on torpedo bearing axons (black caret, h, j–o). a Normal appearance of Purkinje cell dendrites and cell bodies and thin axon profiles in the granule cell layer of a control. b, c SCA3 and Friedreich’s ataxia (FA) with intermediate axonal changes including torpedoes, thickened axons and recurrent collaterals. Heterotopic Purkinje cells and dendrite swellings are present. In FA, a focus with multiple torpedoes associated with recurrent collaterals is shown (inset, c). d Axonal changes and Purkinje cell loss are more prominent in essential tremor (ET). A dendrite swelling is present. e–i Spectrum of changes in SCA1/2/6/7/8 with moderate to severe Purkinje cell body and dendrite loss, abundant torpedoes, thickened axons and torpedo associated recurrent collaterals. J–m Foci in ET (j), SCA1 (k), SCA2 (l) and SCA7 (m) where there are clusters of torpedo bearing axons with recurrent collaterals. n–o Spectrum of change in MSA, being mild in cases with predominant striatonigral degeneration (MSA-SND, n) and severe in cases with predominant olivopontocerebellar atrophy (MSA-OPCA, p). In MSA with mixed striatonigral and olivopontocerebellar atrophy (MSA-SNA-OPCA, o), there are numerous torpedoes that predominantly have recurrent collaterals. SCA spinocerebellar ataxia, MSA multiple system atrophy. Scale bar, 100 μm in m, p, inset in c

Fig. 4

Dendritic and spine abnormalities in highly degenerate Purkinje cells. CalbindinD_28k immunohistochemistry on 100-μm cerebellar neocortex sections in essential tremor (ET, a, b), Friedreich’s ataxia (FA, c), SCA1 (d), SCA2 (e), SCA6 (f, g), SCA8 (h) and MSA (i) identifies Purkinje cells with a halo-like arrangement of multiple thin dendrites from the Purkinje cell soma and somatic spines. Dendrite swellings are associated with residual dendrites from these cells in the molecular layer (white caret, a, b, d, e). SCA spinocerebellar ataxia, MSA multiple system atrophy. Scale bars (in a, i), 50 μm

Fig. 5

Skyline Plot—Patterns of degenerative changes across disorders. The median value for each of our eight core metrics are graphed in essential tremor (ET) cases in comparison to primary disorders of cerebellar degeneration (i.e., SCAs), including the following: basket cell axonal hypertrophy rating scale (red bar, Bielschowsky stain), heterotopic Purkinje cells per Purkinje cell (gray bar, LH&E stain), PC terminal sprout rating (orange bar, calbindinD_28k immunostain), torpedoes per Purkinje cell (green bar, LH&E stain), torpedoes with recurrent collaterals/mm (blue bar, calbindinD_28k immunostain), Purkinje cell dendritic swellings per Purkinje cell (yellow bar, Bielschowsky stain), inverse (−1) Purkinje cell body/mm (purple bar, LH&E stain), Climbing fibers in outer 20% of ML (light blue bar, VGlut2 immunostain). ET essential tremor, ML molecular layer, SCA spinocerebellar ataxia