The ethical principles set forth in the EU Directive 2010/63/EU concerning the utilization and welfare of laboratory animals were strictly adhered to throughout the execution of our experiments. Prior approval was granted by both the veterinary office of the canton of Basel and the relevant Swiss authorities. For this study, we utilized SCA14 conditional transgenic mice with an FVB background, which has been previously described in detail [12]. The transgenic experiments were performed in mice with an FVB genetic background at the Transgenic Animal Facility of the Biozentrum, University of Basel, utilizing the pronuclear microinjection technique. To identify founder animals, genotyping was carried out using genomic DNA extracted from biopsy samples via PCR analysis. In the initial genotyping step, forward primer 1 (5^′-GACCCCTCCAGACCGCCTAGTCCTG-3^′) and reverse primer 1 (5^′-GCCTATGGAAAAACGCCAGCAACGC-3^′) were employed, while in the second round, a 585 bp fragment was detected using forward primer 2 (5^′-GAGACTTGATGTACCACATTCAACAG-3^′) and reverse primer 2 (5^′-GGCGGGGTCTGAAAGGAGGCGGG-3^′). Subsequently, the presence of the transgenic human PRKCG gene was confirmed through DNA sequencing, with the DNA fragment for sequencing amplified by PCR using genomic DNA samples and a different primer pair: forward primer (5^′-GTCGAGTTTACTCCCTATCAGTGATAG-3^′) and reverse primer (5^′-TAGTCCTGTCGGGTTTCGCCACCTC-3^′). After confirming the transgenic founder animals, they were bred with FVB-Tg (Pcp2-tTA) 3 Horr/J transgenic mice obtained from the Jackson Laboratory in Sacramento, CA, USA, to generate Pcp2-tTA/TRE-PKC$\gamma$ double transgenic mice, with genotyping of the Pcp2-tTA transgene involving detection of a 472 bp band using specific primers: forward primer (5^′-GCGCTGTGGGGCATTTTACTTTAGG-3^′) and reverse primer (5^′-CAACATGTCCAGATCGAAATCGTC-3^′). These mice were specifically engineered to express the human PKC$\gamma$ (G118D) mutation, associated with SCA14, exclusively in Purkinje cells.

Organotypic slice cultures were generated according to a previously established protocol detailed by Kapfhammer and Gugger (2012) [13]. On postnatal day 8, mouse pups were euthanized via decapitation, and their brains were aseptically extracted and dissected. In a chilled minimal essential medium (MEM) (Gibco, cat. no. 11090-081, Thermo Fisher Scientific, Reinach, Switzerland) supplemented with 1% glutamax (Gibco, cat. no. 35050-061, Invitrogen, Thermo Fisher Scientific), the cerebellum was isolated, and sagittal slices of 350 micrometers in thickness were precisely cut using a McIlwain tissue chopper (model McIlwain TC752, Mickle Laboratory Engineering Co. Ltd., Guildford, Surrey, UK; distributed by Thermo Fisher Scientific) under sterile conditions. The cerebellar slices were then meticulously separated and placed onto a permeable membrane (Millicell-CM, cat. no. PICM03050, Merck Millipore, Merck AG, Buchs, Switzerland). These slices were incubated in either incubation medium, composed of 50% MEM (Gibco, cat. no. 11090-081), 25% Basal Medium Eagle (BME; Gibco, cat. no. 21010-046), 25% horse serum (HS; Gibco, cat. no. 26050-088), 1% glutamax (Gibco, cat. no. 35050-061), and 0.65% glucose (Sigma-Aldrich, cat. no. G7021, Merck AG), or in Neurobasal medium, consisting of 97% Neurobasal medium (Gibco, cat. no. 21103-049), 2% B27 (Gibco, cat. no. 17504-044), and 1% glutamax (Gibco, cat. no. 35050-061). Both incubations were carried out under 5% CO₂ at 37 °C, with the medium being renewed every 2 to 3 days to ensure optimal culture conditions.

Primary Purkinje cell cultures were generated from neonatal transgenic mice based on established methods [12]. At postnatal day 0, cerebella were harvested from the mice and processed for dissociation. Subsequently, cells were seeded onto Poly-D-lysine-treated glass chambers to promote adhesion. Cultures were maintained in medium containing 90% DFM (DMEM/F-12, Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12, cat. no. 21331020, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with essential supplements: 1% N₂, 1% glutamax, and 10% fetal bovine serum (cat. no. 10270106, Life Technologies, Zug, Switzerland). Within the initial 2–4 hours after plating, each well received 500 µL of supplemental DFM containing 1% N₂ and 1% glutamax. To preserve favorable growth conditions, 50% medium was refreshed every 4 days. Following a 14-day culture period, cells were fixed for downstream assessment.

For immunocytochemical analysis, primary cerebellar Purkinje cells derived from PKC$\gamma$ knockout mice were fixed in 4% paraformaldehyde for 30 minutes at room temperature. Throughout the staining process, all reagents were diluted in a 100 mM phosphate buffer (PB) adjusted to pH 7.3. After fixation, the cells were incubated with primary antibodies diluted in a blocking solution comprising PB, 3% non-immune goat serum, and 0.5% Triton X-100 for 1 hour at room temperature. The primary antibodies used in this study included rabbit anti-Calbindin D-28K (1:500, CB38, Swant, Marly, Switzerland), mouse anti-Calbindin D-28K (1:500, 300, Swant), Guinea pig anti-Calbindin (1:4000, 214 005, SYSY, Göttingen, Germany), rabbit anti-GFP (1:2000, NB600-308, Novus Biologicals, Zug, Switzerland), and chicken anti-GFP (1:2000, ab13970, Abcam, Cambridge, UK). Following rinsing with PB, the cells were incubated with fluorescence-tagged secondary antibodies specific to mouse (Alexa 568, A-11004, Molecular Probes, Eugene, OR, USA), guinea pig (Alexa 568, A-11075), chicken (Alexa Fluor Plus 488, A-32931), and rabbit (Alexa 488, A-11008), all diluted 1:2000 and sourced from Molecular Probes (Eugene, OR , USA). The secondary antibodies were suspended in PB containing 0.1% Triton X-100 and incubated for 2 hours at room temperature. Once stained, the cells were mounted using Mowiol (Sigma-Aldrich) and imaged using an Olympus AX-70 fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a Spot Insight digital camera (Spot Imaging, Sterling Heights, MI, USA). This immunocytochemical approach allowed for the visualization and identification of specific cellular markers within the primary cerebellar Purkinje cell cultures.

Purified protein extracts were obtained by homogenizing cerebellar slices in radioimmunoprecipitation assay (RIPA) lysis buffer (containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (cat. no. 11836170001, Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (cat. no. 4906845001, PhosSTOP, Roche, Basel, Switzerland). Protein lysates were then electrophoretically separated via SDS-PAGE and trans-blotted onto nitrocellulose membranes for immunodetection. Preceding primary antibody exposure, membrane blocking was conducted with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) (1 hr) to reduce non-specific interactions. Membranes were probed with the following primary antibodies: sheep anti-Rgs8 (cat. no. AF6880, R&D Systems, Minneapolis, MN, USA; 1:1000) and rabbit anti-α-Tubulin (cat. no. 11224-1-AP, Proteintech, Rosemont, IL, USA; 1:1000). After TBS-T washes to clear unbound antibodies, detection was performed using horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-sheep HRP conjugate antibody (cat. no. HAF016, R&D Systems, 1:1000), anti-mouse HRP conjugate antibody (cat. no. W402B, Promega, Madison, WI, USA; 1:10,000), and anti-rabbit HRP conjugate antibody (cat. no. W401B, Promega, 1:10,000). Alternatively, membranes were incubated with IRDye Secondary Antibodies, specifically IRDye 680LT-conjugated goat anti-rabbit IgG (cat. no. 926-68021, LI-COR Biosciences, Lincoln, NE, USA; 1:10,000) and IRDye 800CW-conjugated goat anti-mouse IgG (cat. no. 926-32210, LI-COR Biosciences, 1:10,000), for quantitative analysis. Protein visualization was achieved using the ECL method provided by Pierce, a brand of Thermo Fisher Scientific. For quantitative assessment of protein expression, Band quantification was performed utilizing C-Digit Blot software (Image Studio version 5.2.5, LI-COR Biosciences, Bad Homburg, Germany) for precise densitometric analysis.

To measure the dendritic area of Purkinje cells, we employed a validated image analysis program as previously described [7]. This protocol designated the average dendritic area of control Purkinje cells as baseline (normalized to 1). For controls, we selected green fluorescent protein (GFP)-negative Purkinje cells adjacent to GFP-positive counterparts, detected through specific protein markers. This strategy guaranteed comparable growth environments for both cell types, enabling direct comparisons. Using ImageJ software (Version 1.53t, National Institutes of Health, Bethesda, MD, USA), we scrupulously outlined both control and experimental Purkinje cells to calculate total soma and dendritic areas. These measurements underwent additional analysis in GraphPad Prism (Version 9.0.0, GraphPad Software, San Diego, CA, USA). ImageJ computed mean somatic fluorescence intensity, while raw images were applied to intensity analysis. Images underwent linear brightness/contrast adjustments. Sholl analysis centered on mean/max intersection counts assessed dendritic complexity and measured soma areas in primary cultures. Statistical variations were examined via unpaired t-tests (95% confidence interval (CI)), with significance ascertained at p 0.05 cutoff.

The PRKCG gene harbors more than 40 mutations, one of which is the G118D mutation, identified in patients with SCA14 (Fig. 1A). This particular mutation is located in the regulatory domain of the PKC$\gamma$ protein. Notably, our earlier investigation revealed no abnormalities in the development of Purkinje cells within a transgenic G118D mouse model, referred to as a non-manifesting SCA14 line for organotypic slice culture [12]. For organotypic slice culture, brains were dissected from P8 mouse pups. The cerebellum was isolated from each pup’s brain, and cerebellar tissue was sectioned into slices. Cultured slices were transferred onto the inserts for extended maintenance. After a one-week culture period, tissues were fixed for staining or protein extraction (Fig. 1B). Slices from the control or non-manifesting SCA14 mice were immunostained with anti-Rgs8 antibody (Fig. 1C). The fluorescence intensity reflecting Rgs8 expression in Purkinje cell somas was quantified. Outcomes indicated elevated Rgs8 expression levels in Purkinje cells from transgenic mice (Fig. 1D). To further validate that increased Rgs8 expression changes occur in the tissues of slice cultures from these non-manifesting SCA14 mouse lines, we extracted proteins from both control and SCA14(G118D) mouse line slice cultures. Western blot analysis demonstrated a significant upregulation of Rgs8 protein levels in extracts from the SCA14(G118D) mouse line (Fig. 1E,F). These findings indicate that Rgs8, a Purkinje cell-specific protein, sensitively reflects signaling pathway perturbations and may provide insights into early pathological processes in SCAs.

Fig. 1.

Increased Rgs8 expression in cerebellar slice cultures of SCA14(G118D) mouse line. (A) Mutation overview in protein kinase C gamma (PKC$\gamma$). A schematic representation highlights the presence of more than 40 mutations in the PKC$\gamma$, all identified in patients with SCA14; locating the G118D mutation (shown in red) in the regulatory C1 domain. (B) The procedure begins with P8 mouse pups from which brains are dissected. The cerebellum is isolated from each brain specimen, and the cerebellar tissue is sectioned into culture slices. These cultured slices are transferred onto the inserts for extended culture maintenance. Following a one-week culture period, the tissue is fixed for staining or protein extraction. (C) Immunohistochemical analysis of Purkinje cells and Rgs8 expression. Purkinje cells were immunostained with an antibody against Calbindin (green). Concurrently, Rgs8 expression (red) was visualized using an anti-Rgs8 antibody. The merged image reveals the coexistence of Calbindin and Rgs8 within the Purkinje cells. (D) Rgs8 signal intensity was measured via ImageJ software. Control cells displayed mean Rgs8 expression of 1.0 $\pm$ 0.17 (n = 12). In transgenic SCA14(G118D) cells, Rgs8 exhibited a 1.5 $\pm$ 0.47-fold elevation versus controls (n = 12; from three independent experiments). (E) Western blot analysis of Rgs8 expression. Protein extracts from organotypic cerebellar slice cultures, maintained for one week in vitro, were subjected to Western blot analysis. Both control and G118D samples exhibited clear Rgs8 expression. $\alpha$-Tubulin served as a loading control. (F) Band intensities were digitally analyzed (n = 4 mice/group). G118D samples showed Rgs8 abundance of 1.5 $\pm$ 0.27 relative to control (normalized = 1.0). Intergroup variance reached significance (**p 0.01; unpaired t-test). Data are presented as mean $\pm$ SD. Scale bar: 20 µm. SCA14, Spinocerebellar ataxia type 14; G118D, Gly118Asp.

To further assess potential Rgs8 expression changes, we established primary cerebellar cell culture methods, which allowed systematic characterization of individual Purkinje cell phenotypes. Cerebellar tissue from P0 pups was dissociated into single-cell suspensions, cultured for two weeks, and then fixed for immunohistochemical analysis (Fig. 2A). The SCA14(G118D) transgenic mouse model attains cell-specific expression of the transgene along with GFP via the Tet-Off system driven by the Purkinje Cell Protein 2 / L7 (PCP2/L7) Promote. This approach allowed us to isolate the effects of the mutation, excluding other potential factors involved in SCA pathogenesis. Within the mixed dissociated cultures that were obtained from both G118D-transgenic and control mouse pups, we identified transgenic Purkinje cells through their endogenous GFP expression (Fig. 2B). A quantitation of immunoreactivity was then conducted between GFP-positive Purkinje cells from G118D-transgenic mice and GFP-negative Purkinje cells from control mice within the same culture well. Our findings revealed that, although there were no significant morphological differences between Purkinje cells from G118D-transgenic mice and control mice (Fig. 2C), there was a notable increase in Rgs8 immunoreactivity. Specifically, Rgs8 expression was 1.2 $\pm$ 0.22 fold higher in Purkinje cells from G118D-transgenic mice compared to their GFP-negative counterparts from control mice (Fig. 2D,E). We further carried out two sets of measurements on primary cultures of Purkinje cells, measuring the soma area (Fig. 2F) first and assessing dendritic complexity using Sholl analysis with a focus on the mean and maximum intersection counts (Fig. 2G,H), and the results showed no significant differences. These results are consistent with our previous observations from organotypic slice cultures, indicating that Rgs8 serves as a sensitive indicator of early disruptions in intracellular pathways, even before morphological abnormalities appear in Purkinje cells.

Fig. 2.

Increased Rgs8 expression in Purkinje cells of SCA14(G118D) transgenic mice suggests early disruption of intracellular signaling pathway. (A) The illustrated workflow begins with the dissection of postnatal day 0 (P0) mouse pups. Cerebellar tissue is isolated and dissociated into a single-cell suspension, which is transferred into a conical tube. The suspension is then cultured in a dish containing the medium. After a two-week incubation period, the cultured cells are fixed and stained. (B) A depiction of the SCA14(G118D) mouse line is shown, illustrating Purkinje cells that either express the G118D mutant (and are thus GFP-positive) or do not express it (and are GFP-negative). This expression is facilitated by the L7 promoter-based Tet-Off system. Cerebellar dissociated cultures were employed, in which Purkinje cells from both transgenic mice and their control littermates were cultured under identical conditions. Within the culture dish, GFP-positive cells (appearing green) were observed, indicating the expression of the PKC$\gamma$-G118D mutant. (C) Increased Rgs8 expression in Purkinje cells of transgenic mice. In mixed dissociated cultures containing Purkinje cells from both transgenic and control mice, an elevation in Rgs8 immunoreactivity (red) was observed in Purkinje cells from SCA14(G118D) transgenic mice. Purkinje cells were identified through anti-calbindin staining (blue), while transgenic cells were distinguished through anti-GFP fluorescent staining (green). (D) Dendritic area measurements were conducted on Purkinje cells sourced from three independent experiments. Control cells consisted of GFP-non-expressing Purkinje cells originating from identical culture wells. The average dendritic area for control cells was 1.0 $\pm$ 0.44 (n = 27), while for PKC$\gamma$-G118D expressing, GFP-positive Purkinje cells, it was 0.8 $\pm$ 0.39 (n = 26). (E) Quantification of the fluorescence intensity from anti-Rgs8 staining was carried out via ImageJ software. The arithmetic mean of Rgs8 expression in control cells was established at 1.0 $\pm$ 0.25 (n = 27). In contrast, the average Rgs8 expression magnitude in SCA14(G118D) transgenic Purkinje cells was increased by 1.2 $\pm$ 0.22-fold compared to control cells (n = 26). This difference was statistically significant (**p 0.01) as established via an unpaired t-test. (F) Quantitative analysis of the soma area of Purkinje cells. Relative soma area: Control: 1.0 $\pm$ 0.25; G118D: 1.0 $\pm$ 0.19; p = 0.94; n = 12. (G) Quantitative measurement of the dendritic complexity of Purkinje cells. Sholl mean intersection counts: Control: 7.9 $\pm$ 3.40; G118D: 7.3 $\pm$ 2.64; p = 0.62; n = 12. (H) Sholl max intersection counts: Control: 12.0 $\pm$ 5.37; G118D: 11.5 $\pm$ 4.53; p = 0.81; n = 12. No significant difference (ns) exists between the two groups, as established by an unpaired t-test. Data are reported in terms of mean $\pm$ SD, and a scale bar is provided to mark a distance of 20 µm. GFP, green fluorescent protein; TRE, tetracycline responsive element promoter.