Essential new insights into human cerebellar Purkinje cell biology from studies of essential tremor

The groundbreaking work by Widner et al. (1) marks a significant leap in our understanding of human Purkinje cell biology and cerebellar disease, definitively establishing, for the first time, that human Purkinje cells exhibit spatially defined selective vulnerability to disease reflected in their structure and likely underlying molecular biology.

Cerebellar Purkinje cells have long fascinated neurobiologists. These massive neurons with intricately branched and flat dendritic trees were first described in 19th century by Jan Evangelista Purkyně. Camillo Golgi’s silver nitrate staining technique enabled him to describe their very large cell bodies, followed by Santiago Ramón y Cajal who refined Golgi’s staining protocol and revealed their elaborate dendritic arbors (2) (Fig. 1). Purkinje cells are central to the function of the cerebellum, which is essential for normal motor coordination in addition to cognition, language, social, and emotional processes (3). Purkinje cells are the sole output neuron of the cerebellar cortex. In humans, their large dendritic trees are estimated to receive ~1 million synaptic inputs, largely from hundreds of thousands of synapses from hundreds of thousands of parallel fibers of granule cell axons traversing through the dendrites at right angles, making Purkinje cells capable of extraordinarily complex computational integration (4).

Fig. 1.

Human Purkinje cell as illustrated by Santiago Ramón y Cajal. Illustration reproduced with permission from Legado Cajal-Consejo Superior de Investigaciones Científicas.

Although the Purkinje cell circuit is mostly uniform across the cerebellum, a multitude of studies have clearly defined a high degree of functional and regional specialization across the cerebellum, with distinct areas dedicated to different motor and cognitive functions. This regional patterning, also known as zonal organization, has been well characterized in mice (5). It is reflected in the molecular heterogeneity of developing and adult Purkinje cell transcriptomes, local circuit electrophysiology, Purkinje cell morphology, and the patterned input of climbing fibers from the inferior olivary nucleus of the medulla. Patterned Purkinje cell axons synapse on neurons of cerebellar nuclei that are located deep within the center of the cerebellum. These nuclei comprise the major output of the entire cerebellum as multisynaptic cerebellar to neocortical loops which underlie a wide spectrum of behaviors (6).

Studies of Purkinje cell function have a strong historical foundation in the analysis of several spontaneous mouse mutants where motor deficits, an easily recognizable phenotype, were found to be associated with Purkinje cell degeneration. Quantitative histology demonstrated temporally coincident or progressive loss of Purkinje neurons strictly paralleling the onset and severity of cerebellar motor deficits. Further, it was noted that Purkinje cell loss was not uniform, but rather, there was regional sparing and loss of Purkinje cells. More and more sophisticated mapping in many degeneration models across mice, rats, cats, and dogs show consistent alternating sagittal bands of surviving or depleted Purkinje cells (“stripes” or “zones”). These often, but not always, correspond to molecular markers (zebrin II/aldolase C, EAAT4, HSP25), which in turn, often correlate to distinct electrophysiological features (7). It remains unclear however why Purkinje cells have differing sensitivities to loss; however, it is postulated that glutamate clearance via EAAT4, differential stress/protection pathways, and metabolic adaptability are likely key determinants, though most studies remain correlative rather than directly causal (8).

The groundbreaking work by Widner et al. (1) marks a significant leap in our understanding of human Purkinje cell biology and cerebellar disease, definitively establishing, for the first time, that human Purkinje cells exhibit spatially defined selective vulnerability to disease reflected in their structure and likely underlying molecular biology.

A longstanding question to both basic biologists and clinicians has been whether selective Purkinje cell vulnerability to disease in mice translates to cerebellar disease in humans. Notably, almost all mouse models of human cerebellar degenerative disorders also show clear, reproducible regional (nonuniform) Purkinje cell degeneration and Purkinje cell loss. These include multiple well-validated mouse models of human cerebellar degenerative disorders—such as Niemann–Pick type C (Npc1^–/–), spinocerebellar ataxia (SCA) type 1 (ATXN1[82Q]), ARSACS (Sacs^–/–), and CACNA1A channelopathies (“tottering,” “leaner”), with Purkinje cell loss in aldolase C negative stripes in anterior lobules and sparing in both posterior lobules and aldolase C-positive zones (7). Determining whether humans also show similar patterns of loss has been difficult. For example, MRI studies consistently detect disproportionate atrophy in anterior and select posterior lobules, inferring (but not proving) that these findings reflect Purkinje cell loss in SCA patients (9). However, these studies are limited in resolution at the lobule level, with no molecular correlates of Purkinje cell regional identity or function. In human autopsy samples, histological assessments have reported patterned Purkinje cell loss in SCAs, alcoholism, and aging with limited regional mapping. Both bulk and single-nuclei RNA sequencing of SCA1 and SCA7 human cerebellar tissue suggest disruptions in aldolase C-related cell signatures. However, regional information is lost for sample preparation with these sequencing studies and further, the altered signatures were attributed to another cerebellar cell type, molecular layer interneurons, rather than Purkinje cells, since Purkinje cells represent a very small fraction of all cells found in the adult cerebellum and are not well represented in the data (10, 11). To date, rigorous human subregional histological-based mapping studies have not been designed to demonstrate precise relationships among anatomical regions and either Purkinje cell molecular or functional identity.

Widner et al. present the first demonstration of spatially defined, molecularly distinct Purkinje cell subtype populations in human cerebella (1). Through rigorous analyses, they also show that this molecular heterogeneity has distinct regional distribution and differential disease vulnerability between primarily motor vs. nonmotor regions of the cerebellum in essential tremor (ET) patients. Why ET? ET is a very common, progressive movement disorder affecting at least 2.2% of the USA population (12). In addition to tremors, mild-to-moderate gait ataxia and other signs of cerebellar dysfunction may occur (i.e., subtle saccadic eye movement abnormalities and abnormalities of motor timing) as well as cognitive features, some of which may be due to cerebellar dysfunction. Neuroimaging studies have repeatedly demonstrated the presence of functional, metabolic, and structural abnormalities in the cerebellum in individuals with ET (13, 14) The genetics of ET are complex and can be polygenic involving common variants (15). Nevertheless, despite likely considerable genetic heterogeneity, rigorous tissue-based studies across multiple affected individuals have identified a series of structural changes in the ET cerebellum. Most of these pathological changes are centered on the Purkinje cell and connected neuronal populations, which can result in partial loss of Purkinje cells and circuitry reorganizations that would disturb cerebellar function (14).

Widner et al. (1) first set out to find markers for human Purkinje cell subpopulations, searching for differential expression of neurofilament heavy chain (NEFH) and differential sizes of axon caliber in six control samples. In mice, these are known markers of Purkinje cell heterogeneity which also correlate with aldolase C expression (16, 17). To rigorously measure axon caliber, the authors used fluorescent immunohistochemistry to label samples with a pan-Purkinje cell marker, PCP2, and devised a method to generate hundreds of 3 dimensional reconstructions of individual axon segments from confocal images. They specifically restricted their search to Purkinje cell axons segments in cerebellar cortical folial white matter and in regions with known predominant motor or cognition functions: 1) hemisphere lobule V (HV), a motor-related cerebellar region that processes upper limb and hand movement-related information vs. 2) the nearby hemispheric lobule Crus I lobule with significant function in cognition. Higher NEFH expression and larger caliber axons were found in the HV motor region, similar to distributions of these Purkinje cell subtypes in the mouse cerebellum.

Having established a reliable means to define human regional Purkinje cell heterogeneity, the authors next assessed axon caliber in 10 ET samples and six age-matched controls to specifically address the question of regionally selective vulnerability of Purkinje cells. Impressively, hundreds of Purkinje cell axon segments were compared showing that PCP2 expression was unchanged by disease. Next the authors showed that in the nonmotor Crus I region, control axons were of smaller caliber than in the motor HV region. Additionally, in nonmotor Crus I, ET axons were structurally indistinguishable from those in controls. In striking contrast however, ET axons in HV, the motor predominant region, showed a highly statistically significant, specific reduction of axon cross-sectional area. Not only were these white matter axons much thinner, they also exhibited a decreased intra-axon variable range and dysregulation of this range across axons compared to controls. Further, this variability, but not mean size, correlated with clinical tremor score and duration in this initial cohort of ET cases. Weaker associations with other pathological features in ET were also noted but require further data and analyses. Prior studies of Purkinje cell axons in ET were performed in the granule cell layer, above the cortical folial white matter, and show distinctive phenotypes including axonal thickenings and swellings (torpedoes) (13). Thus, this now more distal, white matter Purkinje cell axonal thinning phenotype adds another distinctive metric to the repertoire of described degenerative changes in ET.

This paper presents a tour de force analysis and sets the standard for detailed human pathological studies of cerebellar disease going forward. While the current findings certainly have importance for the understanding of ET pathology specifically, they are also foundational for both our understanding of normal human Purkinje cell biology and human cerebellar disease more generally. Yes, there are clearly many more things we would like to know about human Purkinje cell heterogeneity. How extensive is the heterogeneity? Do the zones of heterogeneity organize in sagittal bands as in other mammals? Are the Purkinje cell vulnerabilities in ET shared in other human cerebellar diseases? For all those who have studied model organisms, where tissue availability is unlimited and can be studied in extremely fine temporal, spatial and molecular detail, I remind you that this current study was accomplished in human postmortem tissue. The seemingly straightforward analyses presented belie their technical complexity and underline why it is difficult to quickly answer all of the many outstanding questions. Human pathological analyses are only possible because of extensive prior work by dedicated teams and donor families to ensure availability of appropriate tissue resources. Variation in postmortem interval and tissue integrity is challenging and can compromise study outcome if not properly controlled. Complete slices of human cerebellum banked to retain spatial orientation are not commonly available. Banked cerebellar samples of ET patients or any disease which are also associated with significant relevant clinical data involves additional layers of complexity, extensive expertise, and interdisciplinary collaboration. Yet, these precious resources are essential for progress. This current foundational study was only possible because of prior substantial long-term, broad scope, multinational commitments to research funding. Over many decades, these investments first enabled the discovery of basic science principles of mouse neurological mutant phenotypes, cerebellar organization, and physiology and now are yielding unique insights into the physiology normal human cerebellum function and the disease pathogenesis of a very common human neurological disease. It is essential that our collective society remains committed to long-term science investment if we are ever to achieve the very real promise of improved diagnostics and therapeutics for important clinical disorders. Sharing science stories, such as this current study, with the additional context of their long historical roots and important current basic and clinical impact represents a powerful way to continue to advocate for broad and sustained science funding.