Research ArticleESSENTIAL TREMOR

Cerebellar oscillations driven by synaptic pruning deficits of cerebellar climbing fibers contribute to tremor pathophysiology

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Science Translational Medicine  15 Jan 2020:
Vol. 12, Issue 526, eaay1769
DOI: 10.1126/scitranslmed.aay1769
  • Fig. 1 PC synaptic pathology in ET correlates with reduced GluRδ2 expression.

    (A) Representative images of CF synapses in the postmortem human cerebellar cortex of a patient with ET and a control (Ctrl). Patients had more CF synapses, as visualized by VGlut2 puncta (green), on the thin, spiny branchlet of Purkinje cell (PC) dendrites (arrows). (B) Quantified VGlut2 puncta counts (15 ET and 19 controls, Student’s t test). (C and D) Representative cerebellar cortical sections (C) and quantification (D) of VGlut2 immunohistochemistry for CF-PC synapses. The dotted lines indicated the border between the outer 20% and the inner 80% of the molecular layer. (E to H) Images and quantification of Western blot analysis of GluRδ2, mGluR1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in frozen cerebellar cortex [(F) 15 ET and 15 controls, Student’s t test; (G and H) available in 11 ET and 8 controls, Spearman’s correlation]. CF, climbing fiber; ET, essential tremor; VGlut2, vesicular glutamate transporter type 2; PC, Purkinje cell; ML, molecular layer.

  • Fig. 2 GluRδ2 protein insufficiency in mice recapitulates ET-like CF synaptic pathology.

    (A) RT-PCR of the cerebellar cortex of Grid2 cDNA from hotfoot17J mice. PCR fragments that include exon 3 showed an increase-sized fragment. kbp, kilo base pair. (B) Diagrams of WT and hotfoot17J Grid2 cDNA allele. bp, base pair. (C) Quantitative PCR of genomic DNA of exon 3 and exon 6 of Grid2 gene from WT or homozygous hotfoot17J (Grid2dupE3) mice (n = 3 in each group, Kruskal-Wallis, one-way ANOVA). (D) Western blots of the cerebellar cortex of WT and homozygous hotfoot17J (Grid2dupE3) mice [(D) n = 5 in each group, Student’s t test). (E) Representative images of GluRδ2 immunohistochemistry. (F) Representative images of dual immunofluorescence of calbindin to visualize PCs (green) and GluRδ2 (magenta). (G) Representative images of dual immunofluorescence of GRP78 to visualize endoplasmic reticulum (magenta) and of GluRδ2 (green). (H) Western blots of cerebellar slices incubated with either proteasomal (MG-132) or lysosomal (NH4Cl and leupeptin) inhibition in a WT mouse or a hotfoot17J (Grid2dupE3) mouse and (I) the quantification of GluRδ2 expression [(I) n = 8 in each group, Kruskal-Wallis one-way ANOVA). DMSO, dimethyl sulfoxide. (J) Representative images of dual immunofluorescence of calbindin and VGlut2 in a WT mouse or a Grid2dupE3 mouse. The dotted lines indicate the border between the outer 20% and the inner 80% of the molecular layer. (K) Quantification of the percentage CF synapses in the outer 20% of the molecular layer in WT and Grid2dupE3 mice (n = 8 in each group, Student’s t test). Error bars denote SEM. *P < 0.05, **P < 0.01, and ****P < 0.001. dupE3, Grid2dupE3; WT, wild type.

  • Fig. 3 Reduced expression of GluRδ2 protein in mice causes progressive kinetic tremor.

    (A) Scheme showing tremor recording with simultaneous motion monitoring in a freely moving mouse. (B and C) Representative time-frequency plots (B) and normalized power spectral density (PSD) diagram [(C) n = 10 mice in each group] of tremor in 3-month-old mice (see Materials and Methods). FFT, fast Fourier transformation. (D to F) Kinetic tremor in Grid2dupE3 mice. A time-frequency plot and the corresponding PSD diagram, coregistered by video-based motion detection, showed kinetic predominant tremor [(F) n = 9 mice]. (G to I) Tremor progression with age. Representative time-frequency plots (G) and corresponding PSD diagrams (H) in Grid2dupE3 mice and group analysis of peak PSDs (I) from WT or Grid2dupE3 mice in different ages (n = 5 mice in each group). (J) Construct design for Sindbis virus (SINV) expressing GluRδ2 for the rescue experiment. (K) Schematic demonstrating SINV-mediated GluRδ2 expression in the cerebellar cortex. DAPI, 4′,6-diamidino-2-phenylindole. (L) GFP expression in a Grid2dupE3 mouse motor cerebellum injected with SINV-GluRδ2WT-GFP. (M) GluRδ2 protein expression in Grid2dupE3 mouse cerebellum transfected with either SINV-GluRδ2WT-GFP or control SINV-GFP at postinjection day 5. (N) Representative time-frequency plots of tremor in a Grid2dupE3 mouse before SINV-GluRδ2WT-GFP injection in the cerebellum (day 0) and at postinjection day 5 and day 14. (O) Representative PSD diagram of tremor in a Grid2dupE3 mouse injected with SINV-GluRδ2WT-GFP at different time points. (P) Group analysis of tremor PSD peaks by SINV-GluRδ2WT-GFP intervention (n = 7 mice). (Q) Quantification of peak tremor PSD of Grid2dupE3 mice transfected with SINV-GluRδ2WT-GFP (n = 7 mice) when compared with control SINV-GFP at different time points (n = 6 mice). n.s., not significant. *P < 0.05, **P < 0.01 by Wilcoxon signed-rank tests. See also fig. S4.

  • Fig. 4 CF-to-PC pathway contributes to tremor generation.

    (A) Scheme showing the IO-CF-PC-DCN axis of the cerebellar pathway. (B to E) Cryoinjury of cerebellar cortex. Thirty seconds of dry ice exposure (B) created loss of anatomical architecture of lobules IV to VI of the cerebellar cortex (C). A representative time-frequency plot of a Grid2dupE3 mouse before and after cryoinjury to the cerebellum (D). Group data of PSD diagrams (left) and quantification of corresponding peak PSD (right) in Grid2dupE3 mice before and after cryoinjury to the cerebellum [(E) n = 5 mice]. (F to J) Optogenetic inhibition of PC outputs. We virally expressed NpHR 3.0 in PCs and suppressed PC outputs by optic inhibition of PC axonal terminals in DCN (F). NpHR expression was colocalized with calbindin, a specific marker of PCs, in both cerebellar cortex and DCN [(G) right and left panels, respectively]. Green light suppressed tremor instantaneously, and the light removal caused immediate reappearance of tremor (H and I). Quantitative spectrum analysis demonstrated significant and reversible effects of PC outputs in tremor generation [(J) n = 3 mice, each had two runs]. (K to O) IO silencing by in situ lidocaine microinfusion. Lidocaine suppressed IO firings in representative multiunit traces (L, right) and quantitative single-unit analysis (L, left, n = 15 units in two mice). Behaviorally, lidocaine effects on tremor followed the time course of IO activity changes shown in representative plots (M to N) and group analysis [(O) n = 7 mice]. *P < 0.05 and ***P < 0.001 by Wilcoxon signed-rank tests. Error bars denote SEM. DCN, deep cerebellar nuclei; IO, inferior olive.

  • Fig. 5 Optogenetic silencing of CF synapses suppresses tremor.

    (A) Schematic illustration of SYP-miniSOG functions. Illumination of miniSOG with blue light causes synaptic protein damage and, thus, inhibits synaptic vesicle release. (B and C) Expression of SYP-miniSOG in the CF synaptic terminals. Viral injection of SYP-miniSOG-citrine in the IO (B) and the expression of citrine in the CF synaptic terminals across the cerebellar cortex, confirmed by the colocalization with VGlut2 on PCs, visualized by calbindin (C). (D to H) Blue light scanning of lobules V and VI of the cerebellar surface and tremor in Grid2dupE3 mice. We scanned the cerebellar surface with blue light through a cranial window in the sequence indicated by arrows [(D) see Supplementary Materials and Methods for details] and measured mouse tremor in different time points (E to G). Effective blue light and controlled green light illumination showed different effects on tremor [(H) n = 4 mice in two runs]. (I to N) Effect of slicing synaptic terminals from IO-to-DCN collaterals. Virus was injected into IO and optic fiber was placed in DCN (I), confirmed by fluorescence imaging showing citrine (+) synaptic terminals in DCN (J). Representative time-frequency plot (K) and corresponding PSD (L and M) of mouse tremor are shown in different time points. PSD peaks were compared in between baseline and postillumination hour 1 in a group of Grid2dupE3 mice [(N) n = 3 mice in two runs]. *P < 0.05 by Wilcoxon signed-rank tests. Error bars denote SEM. SYP, synaptophysin.

  • Fig. 6 Cerebellar oscillations correlate to tremor generation.

    (A) Scheme showing simultaneous recordings of cerebellar LFPs of lobules V and VI and tremor in a freely moving mouse. (B and C) Representative time-frequency plots and corresponding PSD diagrams in a WT mouse and a Grid2dupE3 mouse. (D) Group analysis of PSD peaks (n = 7 mice in each group, *P < 0.05 by Mann-Whitney tests). (E) Scheme demonstrating AAV-mediated ChR2 expression in the PCs, and an optic fiber was implanted in the DCN to activate PC axonal terminals. (F) AAV-CaMKIIa-eChR2-mCherry expression in lobules IV to VI. In the cerebellar cortex, CaMKIIa is a promoter specific for PCs. (G and H) Representative time-frequency plots and PSD diagrams of cerebellar oscillations, tremor, and coherence for optogenetic PC stimulation in a WT mouse. (I and J) Quantitative data of cerebellar LFP and tremor before, during, and after blue light stimulation (I) and also nonactivating green light control (J) (n = 4 mice in each group; **P < 0.01 by Wilcoxon signed-rank tests). Error bars denote SEM. CB, cerebellum. LFP, local field potential.

  • Fig. 7 Patients with ET develop excessive cerebellar oscillations.

    (A and B) Human cerebellar electroencephalogram (EEG) recorded in the different cerebellar regions in both patients and controls. (C and D) Representative time-frequency plots (C), and corresponding PSD diagrams and group analysis [(D) n = 10 patients and 10 age-matched controls; ***P < 0.001 by Mann-Whitney test]. (E to H) Source localization and bipolar comparison of cerebellar oscillations in patients with ET. EEG was recorded in awake patients under eyes-open condition to suppress occipital alpha rhythm. Location of EEG leads (E) and representative PSD diagrams (F) with color-coded EEG intensity at human tremor frequency (4 to 12 Hz). The highest intensity (red) is located in the cerebellar region. Direct comparison between cerebellar leads (Cb2-Cb1) and occipital leads (O2-O1) in a representative patient (G) and group analysis [(H) n = 5 patients; *P < 0.05 by Wilcoxon signed-rank test]. (I) Cerebellar oscillatory index (COI) and its correlation with tremor severity in patients in an extended cohort (n = 20 patients, r = 0.64, P = 0.002 by Pearson’s correlation coefficient). Error bars denote SEM.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/526/eaay1769/DC1

    Supplementary Materials and Methods

    Fig. S1. mGluR1 expression in the postmortem ET cerebellum.

    Fig. S2. The amino acid alignment of the protein products of WT and hotfoot17J GluRδ2 cDNA.

    Fig. S3. Quantification of cellular and subcellular expression of GluRδ2 in WT and Grid2dupE3 cerebellum.

    Fig. S4. Suppression of mouse tremor by ET medications.

    Fig. S5. Regression of CF synapses after rescue of GluRδ2 expression.

    Fig. S6. Tremor in Grid2dupE3 mice receiving intracerebellar injection of control SINV.

    Fig. S7. Tremor in hotfoot4J mice.

    Fig. S8. Cryoinjury of mouse cerebellum by dry ice.

    Fig. S9. Frequency profiles of tremor in Grid2dupE3 mice by IO silencing.

    Fig. S10. Spike-phase coupling between IO spikes and tremor phases.

    Fig. S11. Spike-phase coupling between IO spikes and cerebellar LFPs.

    Fig. S12. Suppression of cerebellar oscillations and tremor by IO silencing.

    Fig. S13. Optogenetic stimulation of PC outputs at 10 Hz.

    Fig. S14. Optogenetic stimulation of PC outputs by nonactivating green light.

    Fig. S15. Signals and characteristics of cerebellar EEG leads and nearby muscle leads.

    Fig. S16. Signal comparison between cerebellar EEG leads and nearby muscle leads in patients with ET.

    Fig. S17. Occipital alpha rhythm and cerebellar oscillations in a patient with ET.

    Fig. S18. CF synaptic pruning deficits, cerebellar oscillations, and tremor.

    Table S1. Clinical and pathological features of postmortem cerebellum in patients with ET and control subjects.

    Table S2. Demographic data of patients with ET and control subjects for the EEG study.

    Table S3. Demographic data of patients with ET and control subjects for cerebellar oscillation index.

    Movie S1. Tremor characteristics in WT versus Grid2dupE3 mice.

    Movie S2. Tremor modulation by dry ice–mediated cerebellar lesioning.

    Movie S3. Tremor modulation by optogenetic inhibition of cerebellar outputs.

    Movie S4. Tremor modulation by synaptic inhibition of CFs.

    Movie S5. Tremor induction by PC stimulation in a WT mouse.

    Data file S1. Raw data (provided as separate excel file).

    References (6467)

  • The PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. mGluR1 expression in the postmortem ET cerebellum.
    • Fig. S2. The amino acid alignment of the protein products of WT and hotfoot17J GluRδ2 cDNA.
    • Fig. S3. Quantification of cellular and subcellular expression of GluRδ2 in WT and Grid2dupE3 cerebellum.
    • Fig. S4. Suppression of mouse tremor by ET medications.
    • Fig. S5. Regression of CF synapses after rescue of GluRδ2 expression.
    • Fig. S6. Tremor in Grid2dupE3 mice receiving intracerebellar injection of control SINV.
    • Fig. S7. Tremor in hotfoot4J mice.
    • Fig. S8. Cryoinjury of mouse cerebellum by dry ice.
    • Fig. S9. Frequency profiles of tremor in Grid2dupE3 mice by IO silencing.
    • Fig. S10. Spike-phase coupling between IO spikes and tremor phases.
    • Fig. S11. Spike-phase coupling between IO spikes and cerebellar LFPs.
    • Fig. S12. Suppression of cerebellar oscillations and tremor by IO silencing.
    • Fig. S13. Optogenetic stimulation of PC outputs at 10 Hz.
    • Fig. S14. Optogenetic stimulation of PC outputs by nonactivating green light.
    • Fig. S15. Signals and characteristics of cerebellar EEG leads and nearby muscle leads.
    • Fig. S16. Signal comparison between cerebellar EEG leads and nearby muscle leads in patients with ET.
    • Fig. S17. Occipital alpha rhythm and cerebellar oscillations in a patient with ET.
    • Fig. S18. CF synaptic pruning deficits, cerebellar oscillations, and tremor.
    • Table S1. Clinical and pathological features of postmortem cerebellum in patients with ET and control subjects.
    • Table S2. Demographic data of patients with ET and control subjects for the EEG study.
    • Table S3. Demographic data of patients with ET and control subjects for cerebellar oscillation index.
    • Legends for movies S1 to S5
    • Legend for data file S1
    • References (6467)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Tremor characteristics in WT versus Grid2dupE3 mice.
    • Movie S2 (.mp4 format). Tremor modulation by dry ice–mediated cerebellar lesioning.
    • Movie S3 (.mp4 format). Tremor modulation by optogenetic inhibition of cerebellar outputs.
    • Movie S4 (.mp4 format). Tremor modulation by synaptic inhibition of CFs.
    • Movie S5 (.mp4 format). Tremor induction by PC stimulation in a WT mouse.
    • Data file S1. Raw data (provided as separate excel file).

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