Fig 2: Yo and rCDR antibody internalization depletes calbindin D28k in rat Purkinje neurons. Significant depletion of calbindin-immunoreactive cells was observed after (A) Yo antibody and (B) rCDR antibody incorporation, whereas control sera (non-hCDR1–4,100p) but IgG controls showed no impact on the Calb+ cell count. (B) The incubation with antibodies against Purkinje neurone specific protein 2 (PCP2) or ataxia-related GAD65 (1000 ng, 6 days) revealed no reduction in the Calb+ cell count. (C) Calbindin depleted Purkinje neurons were positive for the neurodegeneration marker Fluoro-Jade-C (*, green) and showed high incorporation of Yo antibodies (#, magenta) (C1). The cell death marker propidium iodine (#) (C2, magenta) was not found either in Calb+ (yellow) or Jade-C+ (green) Purkinje neurons after 6 days of Yo antibody incorporation. Scale bar 25 µm.

Fig 3: Yo antibody binding to CDR2 impacts cannabinoid 1 receptor activity. (A–C) Calb+ cells per mm3 in cerebellar organotypic slice culture in response to treatment with anti-Yo/rCDR and co-treatment with AM281 (n = 3) and cannabidiol (CBD, n = 6). The antagonistic effects on anti-Yo-induced pathology are represented in panels A, C, and for anti-rCDRs-induced pathology in panels B, C. Panels A and B are showing the data collected when the antagonists were directly applied at paraneoplastic cerebellar degeneration (PCD) onset and panels C when applied at day 4 after PCD pathology was established. The neuroprotective capacity of the tested compounds was evaluated after 6 days for both treatment strategies. AM281 and CBD minimized the Yo-induced depletion of calbindin-immunoreactive cells but caused depletion of calbindin-immunoreactive cells in naive, IgG and serum controls, respectively. Data are collected at treatment day 6 (onset-PCD) or 4 and 10 (post-PCD) and expressed as mean ± SEM [*P < 0.05; **P < 0.01; ***P < 0.001; nonparametric two-tailed paired Mann–Whitney's U-test; asterisk (*): significance to naive control; hash (#): significance to anti-Yo/rCDR]. (D) z-stack confocal micrographs collected at day 10 of treatment showed that CBD co-applied for 6 days from day 4 of Yo antibody incorporation (post-PCD) was not affecting the uptake of anti-Yo (green) itself (top row) but reduced depletion of calbindin-immunoreactive cells (magenta, middle row) in comparison to Yo incorporation alone. Yo+ cells (green) negative for calbindin with shrunken Purkinje neurone somata as well as Yo+ cells (green) positive for calbindin (white) without Purkinje neurone somata shrinkage were observed under CBD treatment (bottom row). Scale bar 25 µm.

Fig 4: Postnatal Lgr5+ cells are exclusively cerebellar granule neurons. a) Sections from Lgr5EGFP-CreERT2 P14 mouse brains were stained for EGFP to mark Lgr5+ cells, Gabra6 to identify cerebellar granule neuron, NeuN to identify neurons and DAPI to mark cell nuclei. Lgr5-EGFP+ cells are restricted to the internal granule layer (IGL) and all Lgr5+ cells are also positive for GABRA6 and NeuN. Lgr5-EGFP+ processes, presumably from Lgr5+ CGNs in the IGL can be seen in the molecular layer (ML). Top left panel, scale bar, 100 microns. Remaining 5 panels taken from marked area of top left panel. Scale bar, 25 microns. b,c,d) Representative images of Lgr5EGFP-CreERT2 P10 and P14 mouse brains stained for Lgr5-EGFP and b) BLBP to identify Bergmann glia, c) Calb1 to identify Purkinje neurons and d) Olig2 to stain cells of the oligodendrocyte lineage. Top panels, scale bars, 100 microns. Bottom two panels taken from marked area of left panel. Scale bar, 50 microns. e) Cells that stained positive for one of the analyzed markers were counted and examined for Lgr5 expression. Numbers presented are aggregated from all sections analyzed (=4 per marker). There was no overlap of Lgr5 with Calb1, BLBP or Olig2 in any sections analyzed. EGL –external granule layer; ML – molecular layer; PL – Purkinje layer; IGL – internal granule layer; OC – occipital cortex; WM – white matter tract.

Fig 5: Dark type of PN apoptosis.(A) Cerebellar cortices show caspase-3 (Casp3) activation (green) in P20 pcd PNs. Calb (red) PNs, DAPI (blue) cell nuclei. Scale bar: 50 µm. (B) Cerebellar cell cultures show that Nna1 shRNA moderately suppresses deglutamylation and induces Casp3 (green) activation in cultured PNs. Scale bar: 75 µm. (C) Cerebellar cortices show dark apoptotic PNs (arrowheads), nuclear condensation, and cellular shrinkage in P20 pcds. Scale bar: 20 µm. (D and E) Increased PN TUNEL stain (red) in cell cultures given Nna1 shRNA and DAPI (blue) cell nuclei. Dot plots with mean ± SD, n = 6; 2-tailed Student’s t test, compared with control, **P < 0.01. Scale bar: 50 µm. PNs, Purkinje neurons; pcd, Purkinje cell degeneration; Nna1, neuronal nuclear protein induced by axotomy.