Climbing fiber-Purkinje cell synaptic pathology in tremor and cerebellar degenerative diseases

Abstract

Changes in climbing fiber-Purkinje cell (CF-PC) synaptic connections have been found in the essential tremor (ET) cerebellum, and these changes are correlated with tremor severity. Whether these postmortem changes are specific to ET remains to be investigated. We assessed CF-PC synaptic pathology in the postmortem cerebellum across a range of degenerative movement disorders [10 Parkinson's disease (PD) cases, 10 multiple system atrophy (MSA) cases, 10 spinocerebellar ataxia type 1 (SCA1) cases, and 20 ET cases] and 25 controls. We observed differences in terms of CF pathological features across these disorders. Specifically, PD cases and ET cases both had more CFs extending into the parallel fiber (PF) territory, but ET cases had more complex branching and increased length of CFs in the PF territory along with decreased CF synaptic density compared to PD cases. MSA cases and SCA1 cases had the most severely reduced CF synaptic density and a marked paucity of CFs extending into the PF territory. Furthermore, CFs in a subset of MSA cases formed collateral branches parallel to the PC layer, a feature not seen in other diagnostic groups. Using unsupervised cluster analysis, the cases and controls could all be categorized into four clusters based on the CF pathology and features of PC pathology, including counts of PCs and their axonal torpedoes. ET cases and PD cases co-segregated into two clusters, whereas SCA1 cases and MSA cases formed another cluster, separate from the control cluster. Interestingly, the presence of resting tremor seemed to be the clinical feature that separated the cases into the two ET-PD clusters. In conclusion, our study demonstrates that these degenerative movement disorders seem to differ with respect to the pattern of CF synaptic pathology they exhibit. It remains to be determined how these differences contribute to the clinical presentations of these diseases.

Keywords: Climbing fiber; Essential tremor; Multiple system atrophy; Parkinson’s disease; Purkinje cell; Spinocerebellar ataxia.

Fig. 1

Decreased CF synaptic density on PC dendritic shafts in the molecular layer of tremor and cerebellar degenerative disorders. Representative cerebellar cortical sections labeled with anti-VGlut2 antibody of a control (a), a PD case (b), an ET case (c), an MSA case (d), and an SCA1 case (e) are shown. The rectangular areas (a–e) are shown below at higher magnification and demonstrate the decreased CF linear synaptic density in the ET case, the MSA case, and the SCA1 case as compared to the control and the PD case. The quantifications of the CF synaptic density across different diagnostic groups are shown (f). Ctrl control, PD Parkinson’s disease, ET essential tremor, MSA multiple system atrophy, SCA1 spinocerebellar ataxia type 1, CF climbing fiber. NS non-significant, *p < 0.05, ***p < 0.005, ****p < 0.001

Fig. 2

CFs in the outer portion of the molecular layer in the cerebellar cortex of tremor and cerebellar degenerative disorders. The dotted line indicates the border between the outer 20 % and inner 80 % of the molecular layer (a–e). Representative cerebellar sections of a control (a), a PD case (b), an ET case (c), an MSA case (d), and an SCA1 case (e) labeled with anti-VGlut2 antibodies are shown. The square areas are shown at higher magnification, to the right of each panel (a–c), and demonstrate that an ET case and a PD case have an increased number of CFs passing the dotted line as compared to a control. Representative cerebellar sections of an MSA case (d) and an SCA case (e) demonstrate that the CFs regressed from the outer portion of the molecular layers. Abnormal CFs parallel to the PC layer (d, an arrow) and a dystrophic CF (d, asterisk) can be observed in the MSA case. Torpedoes are seen in the SCA1 case (e, arrowheads). When compared to controls, ET cases and PD cases have a significantly increased percentage of total CF profiles extending into the outer 20 % of the molecular layer, whereas this pathology is markedly decreased in MSA cases and SCA1 cases. Ctrl control, PD Parkinson’s disease, ET essential tremor, MSA multiple system atrophy, SCA1 spinocerebellar ataxia type 1, CF climbing fiber. NS non-significant, ****p < 0.001

Fig. 3

CF morphology in the outer 20 % of the molecular layer and CF synapses on the thin PC dendritic branchlets. The dotted line indicates the border between the outer 20 % and inner 80 % of the molecular layer (a). Representative cerebellar sections labeled with anti-VGlut2 antibody of a control, a PD case, an ET case, an MSA case, and an SCA1 case demonstrate more complex CF synaptic pathology in the ET case (a). In ET cases, there are increased CF length (b), increased CF synapses (c), and increased number of CF branches (d) in the outer 20 % of the molecular layer when compared to other diagnostic groups. Representative images of dual immunofluorescence with anti-VGlut2 (Alexa 488, green) and anti-calbindin_D28k antibody (Alexa 594, red) of cerebellar sections of a control, an ET case and a PD case (e) and CF synapses on the thin PC dendritic branchlet <1 μm in diameter are shown (arrowheads in e). ET cases have increased percentage of CF synapses on the thin PC dendritic branchlets <1 μm in diameter when compared to PD cases and controls (f), and this feature is also increased, but to a lesser extent, in PD compared to controls. Ctrl control, PD Parkinson’s disease, ET essential tremor, MSA multiple system atrophy, SCA1 spinocerebellar ataxia type 1, CF climbing fiber. ML molecular layer, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 4

Abnormal CFs in MSA cases. The cerebellar cortical sections labeled with anti-VGlut2 antibody of MSA cases demonstrated abnormal CFs oriented parallel to the PC layer (a). Thickened CFs extended from the granule cell layer (arrows) to the proximal part of PC dendrites (arrowheads) before transitioning into normal appearing CFs (b). Dual immunofluorescence with anti-calbindin_D28k antibody (Alexa 594, red) (c, f, i) and anti-VGlut2 (Alexa 488, green) (d, g, j) of cerebellar sections of MSA cases show that abnormal CFs (d, e, arrows) run perpendicular to fragmented PC dendrites (c, e, arrowheads). In addition, some abnormal CFs (g, h, j, k) form synapses with the very proximal part of the dystrophic PC dendrites (f, h) or fragmented PC dendritic dendrites (i, k) in the cerebellar cortex. The abnormal CF synapses (arrows, m, n) that are parallel to PC layers partially colocalize with neurofilament, supporting that these fibers are CF axons (arrows, l, n). ML molecular layer. PCL Purkinje cell layer

Fig. 5

Unsupervised cluster analysis in degenerative movement disorders. We performed unsupervised cluster analysis to assess whether the clinical diagnosis would segregate based on the four pathological features in the cerebellum: [1] CF synaptic density; [2] the percentage of the CFs extending into the outer 20 % of the molecular layer; [3] PC counts; and [4] torpedo counts. Four clusters were identified. The blue cluster consists mainly of MSA cases and SCA1 cases. The gray cluster is composed primarily of controls. ET cases and PD cases segregated both in the yellow cluster and in the orange cluster. Ctrl control, PD Parkinson’s disease, ET essential tremor, MSA multiple system atrophy, SCA1 spinocerebellar ataxia type 1, CF climbing fiber, ML molecular layer