Heat-inactivated fetal bovine serum (FBS), antibiotic solution (10,000 U/mL penicillin, 10,000 µg/mL streptomycin), 0.25% trypsin/EDTA and Hank’s Balanced Salt Solution with calcium and magnesium (HBSS) were obtained from PAN-Biotech (Aidenbach, Germany). PKmito RED probe was supplied by Spirochrome (Stein am Rhein, Switzerland). All other reagents used in this study were obtained from Merck (Darmstadt, Germany), unless otherwise specified.

(S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)−1-(4-fluorobenzyl)−1H-indazole-3-carboxamide (ADB-FUBINACA) was provided by TicTac Communications Ltd, UK. ADB-FUBINACA replicates the pharmacological effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) with up to 85 and 140 times more potency, presenting a half-maximal effective concentration (EC₅₀) of 1.2 nM and 3.5 nM at CB1 and CB2, respectively [29]. Stock solutions of ADB-FUBINACA (5 mM) were prepared in dimethyl sulfoxide (DMSO). Prior to cell exposure, these stock solutions were serially diluted in HBSS to achieve a final DMSO concentration below the threshold known to induce NG108-15 cell differentiation under low serum conditions (i.e., below 0.1%) [30].

The mouse neuroblastoma × rat glioma NG108-15 cell line, sourced from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), was cultured according to established protocols [21]. Cells were grown in 75 cm² flasks containing Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% (v/v) heat-inactivated FBS and an antibiotic solution comprising 100 U/mL penicillin and 100 µg/mL streptomycin. Cell cultures were maintained at 37° C in a humidified atmosphere with 5% CO₂. Upon reaching 80–90% confluence, cell detachment for subculturing or seeding was carried out using a 0.25% trypsin–EDTA solution. Differentiation of NG108-15 cells into cholinergic neurons followed a well-established protocol [21], comprising the replacement of the complete cell culture medium (hereafter referred to as maintenance medium) with DMEM supplemented with 1% FBS, 10 µM retinoic acid, and 30 µM forskolin (hereafter denoted as differentiation medium). ADB-FUBINACA exposure was performed at biologically-relevant concentrations (ranging from 1 pM to 1 µM) immediately after the replacement of the maintenance medium by differentiation medium. Noteworthy, within a span of 72 h, cells undergo morphological changes, transitioning from polygonal flattened bodies to oval shapes, while developing cholinergic traits and forming a distinct neurite network (Supplementary Information, Figure SI-1), serving as a reliable model for studying neuritogenesis and synaptogenesis, crucial processes in neuronal development [31]. Importantly, NG108-15 cells inherently express the CB1 receptor, providing an added advantage for studying the neurotoxic effects of cannabinoids.

Quantitative analysis of mitochondrial morphology and mobility was conducted using fluorescence confocal microscopy and 2D image processing [32]. NG108-15 cells were seeded at a density of 1.0 × 10⁴ cells per well in 8-well chamber slides (Ibidi, Germany). After an overnight incubation period, neurodifferentiation was initiated, and the cells were exposed in parallel to ADB-FUBINACA (1 pM – 1 μM) or 0.1% DMSO (vehicle control) for 24 or 72 h. At each timepoint, the cells were incubated (37 °C, 5% CO₂) with differentiation medium containing PKmito RED (1:3000) for 30 min. Notably, PKMito Red relies on membrane potential for accumulation, which restricts labelling of depolarized mitochondria. Nevertheless, although not covalently bound, PKMito Red remains strongly retained within mitochondria, enabling extended imaging [33]. The medium containing the probe was then aspirated and replaced with fresh medium. A condition in which cells were incubated for 15 min with 10 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) before probe loading was also included (positive control). CCCP, a potent mitochondrial uncoupler, is known to induce significant morphological changes in mitochondria, such as fragmentation of the mitochondrial network [34, 35]. Neuronal cell live-imaging was performed using a fluorescence confocal microscope (Nikon Crest X-Light V3) equipped with a 60 × objective, in combination with DeepSIM X-Light super-resolution system.

During imaging, the cells were maintained at 37° C and 5% CO₂ on the microscope stage. To analyse mitochondrial morphology, images were processed using the Mitochondria Analyzer plugin from ImageJ/Fiji software[36] (Supplementary Information, Figure SI-2). Various parameters were assessed, including the number of mitochondria/networks, mitochondrial area, perimeter, aspect ratio, form factor, number of branches, branch length, and branch junctions. Time-lapse series of image stacks were captured every 1.7 s over a span of 10 min to evaluate the mitochondria transport along neurites [37]. Maximum intensity Z-projections were generated for each time point. To ensure accurate tracking, 100 μm neurite segments starting at least 20 μm from the soma were selected. The MTrackJ plugin was used to track individual mitochondrion in the image sequences and perform data statistics. Mitochondria exhibiting movement of at least 20 μm from their initial location over 10 min were considered motile.

Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA). The normality of each distribution was assessed through the Anderson–Darling, D’Agostino–Pearson, and Shapiro–Wilk normality tests, taking into account the acceptability of skewness and kurtosis values. The tests employed, the number of independent experiments and the number of replicates (if applicable) are specified in the figure legends.

Cells were exposed to ADB-FUBINACA for 24 and 72 h to assess its impact on mitochondrial network morphology and connectivity, followed by PKMito Red staining and live-cell imaging. Separate analyses were performed for mitochondria in the soma and those in the neurites to thoroughly evaluate how ADB-FUBINACA affects mitochondrial dynamics in different cellular compartments.

As shown in Fig. 1A, mitochondria in the soma from control neurons, both at 24 and 72 h, were observed as either separate and distinctly shaped small organelles or as part of a complex and interconnected network with extensive branching. Notably, no changes in this network were observed between these time points. Nonetheless, exposure to CCCP for 15 min resulted in severe mitochondrial fragmentation, producing smaller, spherical mitochondrial units, consistent with previous reports [34, 35, 38–40]. This fragmentation was evident for all analysed endpoints, such as mitochondrial area, perimeter, shape, and network branching, highlighting CCCP's profound impact on mitochondrial structure and function, thereby validating our method.Fig. 1

Assessment of mitochondrial morphology in the soma of NG108-15 cells, following 24 and 72 h treatment with 1 µM ADB-FUBINACA. (A) The mitochondrial network images stained with the PKMito RED probe are presented in their raw format as well as following thresholding and skeletonization processing. Images were quantified and analyzed with the Mitochondria Analyzer Plugin from Fiji (ImageJ), enabling the assessment of various parameters such as (B) mean mitochondrial area, (C) perimeter, (D) aspect ratio, (E) form factor, (F) number of branches per mitochondrion, (G) mean branch length, and (H) number of branch junctions per mitochondrion. Results are presented as mean ± SEM, based on at least seven independent experiments. Between 7 and 14 cells were analysed per condition in each independent experiment, resulting in a minimum of 70 cells evaluated per condition across all experiments. *p < 0.05, **p < 0.01, compared to the respective vehicle control (72 h), using Nested one-way ANOVA, followed by Dunnett’s post-test

Notably, 1 µM ADB-FUBINACA disrupted mitochondrial morphology and connectivity (i.e. network branching) in the neuronal soma after 72 h exposure, with no noticeable effects observed at 24 h. In particular, ADB-FUBINACA reduced the mean mitochondrial area per mitochondrion (0.91 ± 0.05 µm² vs. 1.05 ± 0.06 µm² in control, Fig. 1B), suggesting a more condensed structure or possible content loss at 72 h. The mean perimeter also decreased in the presence of the SC (4.88 ± 0.24 µm vs. 5.48 ± 0.19 µm in controls, Fig. 1C), reflecting the formation of smaller, more compact mitochondrial structures [41]. Additionally, exposure to ADB-FUBINACA also altered mitochondrial shape, as shown by the reduction in aspect ratio (a measure of mitochondrial elongation) and form factor (quantification of how closely the shape of mitochondria gets of a perfect circle) (Figs. 1D and 1E, respectively). The aspect ratio decreased from 2.20 ± 0.04 in control to 2.09 ± 0.04 in ADB-FUBINACA-exposed cells, while the form factor decreased from 2.36 ± 0.07 in the control to 2.15 ± 0.09 in SC-treated cells. Together, these changes indicate a shift from elongated to more spherical and compacted forms over the differentiation period.

We further analysed mitochondrial branching due to its critical role in establishing an interconnected network that supports efficient energy production and the proper distribution of mitochondrial contents. As represented in Fig. 1F, the number of branches per mitochondrion remained unchanged after 72 h exposure to ADB-FUBINACA (1.62 ± 0.05 vs 1.76 ± 0.07 in control). However, the average branch length was significantly reduced in ADB-exposed cells (1.69 ± 0.13 µm) compared to the control (1.98 ± 0.10 µm), as depicted in Fig. 1G. Additionally, the number of branch junctions decreased (0.31 ± 0.03 vs. 0.40 ± 0.08 in control, Fig. 1H), indicating a loss of mitochondrial connectivity.

Given the high energy demands of neurite outgrowth, we also ascertained whether ADB-FUBINACA interfered with mitochondrial dynamics within neurites. This assessment was carried out at 72 h, when neurite extensions were sufficiently developed for a comprehensive analysis. Under control conditions, mitochondria typically display tubular or ovoid structures, distributed along the entire length of the neurites (Fig. 2A, top image). As expected, treatment with CCCP resulted in mitochondrial fragmentation, reducing their overall area and number within the neurite, as observed in Fig. 2A, center image. Notably, 1 µM ADB-FUBINACA increased both the number and area of mitochondria within neurites, compared to the vehicle control (Figs. 2B and 2 C, respectively). Specifically, the mitochondrial count per 100 µm-segment increased from 29.55 ± 1.42 in control cells to 36.44 ± 1.29 in ADB-FUBINACA-treated cells, whereas total mitochondrial area increased from 31.11 ± 1.22 µm² in control cells to 45.02 ± 2.73 µm² in ADB-FUBINACA-treated cells, indicating a substantial enhancement of mitochondrial retention within neurites. Notably, the average perimeter per mitochondrion increased from 5.69 ± 0.42 µm in control cells to 6.29 ± 0.61 µm in ADB-FUBINACA-treated cells (Fig. 2D), while the area per mitochondrion increased from 1.04 ± 0.07 µm² in control cells to 1.27 ± 0.12 µm² (Fig. 2E), suggesting an overall enlargement of the mitochondria. Despite this, no alterations in mitochondrial shape were detected following exposure to the SC, as reflected by the unchanged aspect ratio (3.76 ± 0.31 vs. 3.92 ± 0.24 in control, Fig. 2F) and form factor (2.61 ± 0.22 vs. 2.56 ± 0.16 in the control, Fig. 2G).Fig. 2

Evaluation of mitochondria in neurites after 72 h exposure to 1 µM ADB-FUBINACA. (A) Representative images depicting the mitochondrial network in neurons stained with the PKMito RED probe, displayed in their raw form and after thresholding. (B) Quantification of Mitochondrial Number and (C) Total Mitochondrial Area per 100 µm length of neurite. (D) Measurement of Mitochondrial Perimeter and (E) Area per mitochondrion. Assessment of mitochondrial shape profile, including (F) Aspect Ratio and (G) Form Factor. Results are presented as mean ± SEM from seven independent experiments, with a minimum of 61 neurites assessed per condition (ranging from 5 to 18 neurites per experiment), totaling 339 neurites analyzed. ****p < 0.0001 compared to the respective vehicle control, using Nested one-way ANOVA, followed by Dunnett’s post-test

We also analyzed whether ADB-FUBINACA altered the levels of different markers of mitochondrial fusion and fission in differentiating NG108-15 cells, given that the balance between these processes is key to regulate mitochondrial shape and size, and to maintain mitochondrial structure. In particular, we targeted three key dynamin family GTPases fusion-related proteins: Optic atrophy-1 (Opa1), which is crucial for inner membrane fusion, and Mitofusins 1 (Mfn1) and 2 (Mfn2), which regulate outer membrane fusion [42].

We observed a gradual increase in the levels of all these markers throughout NG108-15 differentiation, with highest levels being attained at 72 h (differentiated cells), as noted in Fig. 3. Specifically, Opa1, Mfn1, and Mfn2 levels in the control increased by around 2.5, 1.6, and 1.7-fold, respectively, compared to pre-differentiation levels, underscoring an enhancement of the mitochondrial fusion machinery. However, exposure to ADB-FUBINACA resulted in the cells’ failure to sustain the increase in Opa1 and Mfn2 levels observed in control cells. Specifically, at 72 h, Opa1 levels were about 60–65% lower following 1 nM and 1 µM ADB-FUBINACA exposure, compared to the control (Fig. 3A), while Mfn2 levels decreased by 57% at 1 µM (Fig. 3C) compared to the respective control. The SC did not seem to change any of these mitochondrial fusion markers 24 h after exposure. Notably, Mfn1 levels remained unchanged by ADB-FUBINACA at both 24 and 72 h.Fig. 3

Impact of ADB-FUBINACA on Mitochondrial Fusion during neurodifferentiation. Analysis of (A) Opa1 (80–90 kDa), (B) Mfn1 (60, 85, and 90 kDa), and (C) Mfn2 (86 kDa) levels during 72 h of NG108-15 cell differentiation and the impact of ADB-FUBINACA after 24 and 72 h, using Western blot. Protein bands were normalized by the amount of β-actin in the respective lane. Data are presented as mean ± SEM from at least six independent experiments. ^ap < 0.05, ^aap < 0.01, compared to vehicle-control at 0 h. ^bp < 0.05, compared to vehicle-control at 24 h, using one-way ANOVA followed by Tukey’s post-hoc test. ***p < 0.001, ****p < 0.0001 compared to vehicle-control at 24 h. ^#p < 0.05, ^##p < 0.01, compared to vehicle-control at 72 h, using two-way ANOVA followed by Dunnett’s post-hoc test

Noteworthy, OPA1 was detected as five major isoforms ranging from 80 to 90 kDa. This aligns with reports of Opa1 undergoing alternative splicing, and generating eight mRNAs that produce five major protein isoforms within the referred molecular weight range (80–90 kDa) [43]. MFN1 appeared as three distinct bands at approximately 85 kDa, 90 kDa, and 60 kDa, which can also be attributed to this protein’s different isoforms and that have been previously detected by others [44].

Analysis of the impact of ADB-FUBINACA on mitochondrial fission mainly focused on four key proteins: Dynamin-related protein 1 (Drp1) and its phosphorylated form (p-Drp1) at Ser616, the primary regulator of mitochondrial fission, which assembles at construction sites to facilitate the division of mitochondria; Fis1 (Mitochondrial Fission 1), a tail-anchored protein in the mitochondrial outer membrane crucial for recruiting Drp1; and MiD51 (or SMCR7L), an adaptor protein that facilitates Drp1 assembly [42].

The levels of mitochondrial fission markers increased during NG108-15 differentiation in the control. As illustrated in Fig. 4A (left), Drp1 levels showed a significant increase, reaching a maximum at 48 h that persisted until 72 h. Fis1 and MiD51 levels displayed a gradual increase over time, reaching a twofold increase at 72 h compared to pre-differentiated cells (Fig. 4C and 4D). However, the phosphorylated form of Drp1, which represents the fission-competent state, decreased over the same period (Fig. 4B). This indicates that although Drp1 abundance was higher, the protein was not being converted into its active form. Exposure to 1 µM ADB-FUBINACA increased the levels of both fission proteins at 72 h, with no notable changes observed at 24 h. Specifically, Drp1 levels increased by 1.5-fold, while Fis1 levels rose by approximately 1.3-fold in the presence of 1 µM ADB-FUBINACA, compared to their respective controls, as shown in Figs. 4A and 4B (right). Meanwhile, MiD51 and p-Drp1 levels were not affected by ADB-FUBINACA exposure (Figs. 4B and 4D).Fig. 4

Evaluation of mitochondrial fission markers levels following exposure to ADB-FUBINACA. Quantification of (A) Drp1 (80 kDa), (B) p-Drp1 (74 kDa), (C) Fis1 (17 kDa) and (D) MiD51 (51 kDa) was performed by Western blot over a 72-h period to evaluate their expression profiles during NG108-15 cells differentiation and the effects of ADB-FUBINACA evaluated following 24 and 72 h of exposure. The density of bands was normalized by the amount of β-actin per lane. Each bar represents the mean ± SEM, for at least five independent experiments. ^aap < 0.01, ^aaaap < 0.0001 compared to vehicle-control at 0 h. ^bp < 0.05, ^bbbbp < 0.0001 compared to vehicle-control at 24 h, using one-way ANOVA followed by Tukey’s post-hoc test. ****p < 0.0001, compared to vehicle-control at 24 h. ^#p < 0.05 compared to vehicle-control at 72 h, using two-way ANOVA followed by Dunnett’s post-hoc test

The increased number of mitochondria within neurites may suggest changes in mitochondrial mobility and trafficking. As such, we evaluated the movement of individual mitochondria in neurites at 72 h, when neurite extensions were sufficiently developed to allow the comprehensive tracking of mitochondrial movement across extensive lengths. Exposure of NG108-15 cells to 1 µM ADB-FUBINACA reduced mitochondrial movement, with the percentage of motile mitochondria dropping to 50.88 ± 7.08%, compared to 61.82 ± 5.10% in the control (Fig. 5A), as observed in the supplementary video. Notably, ADB-FUBINACA reduced overall mitochondrial motility without altering parameters such as total run length per mobile mitochondrion (51.13 ± 2.55 μm vs. 53.92 ± 6.07 μm in the control, Fig. 5B) and average speed (0.26 ± 0.05 µm/s vs. 0.27 ± 0.04 µm/s in the control, Fig. 5C). No significant differences were observed in the proportion of mitochondria exhibiting anterograde or retrograde movement (Supplementary Information, Figure SI-3).Fig. 5

Assessment of mitochondrial trafficking in NG108-15 cell neurites, labelled with PKmito RED. Monitoring and tracking of mitochondrial movement along 100 μm neurite segments over 10 min, using MTrackJ plugin. (A) Percentage of mobile mitochondria, defined as those showing movement of 20 μm or more. (B) Mean path length for each mobile mitochondrion. (C) Average speed of mobile mitochondria. Each bar represents the mean ± SEM, for at least six independent experiments. *p < 0.05, compared to vehicle-control, using Nested one-way ANOVA, followed by Dunnett’s post-test

Considering that ADB-FUBINACA alters mitochondrial mobility within neurites, we also ascertained its potential effects on proteins involved in mitochondrial transport. The mitochondrial transport machinery comprises motor proteins that move mitochondria along microtubules, and adaptor proteins, such as Mitochondrial Rho GTPase 1 (Miro1), which tether mitochondria to these motor proteins [45]. We observed that Miro1 levels increased by approximately 2.5-fold at 72 h after the neurodifferentiation of NG108-15 cells was initiated, compared to undifferentiated cells (Fig. 6B).Fig. 6

Assessment of mitochondria transport machinery expression levels. (A) Representative western blot images. Quantification of (B) Miro1 (∼71 kDa), (C) Tau (35–55 kDa), (D) p-Tau (45, 58 and 60 kDa) and (E) p-Tau/Tau ratio over a 72-h period to evaluate their expression profiles during NG108-15 cells differentiation. Evaluation of 1 µM ADB-FUBINACA effects on (E) Miro1 and (F) p-Tau/Tau levels following 24 and 72 h of exposure. The density of bands was normalized by the amount of β-actin per lane. Each bar represents the mean ± SEM, for at least five independent experiments. ^aap < 0.01, ^aaaap < 0.0001, compared to vehicle-control at 0 h. ^bbp < 0.01, ^bbbbp < 0.0001 compared to vehicle-control at 24 h. ^ccp < 0.01, compared to vehicle-control at 48 h, using one-way ANOVA followed by Tukey’s post-hoc test

Moreover, we measured the levels of the microtubule-associated protein Tau and its phosphorylated form (p-Tau), which is known to primarily regulate the assembly and stabilization of microtubules, a process that is crucial for neurite outgrowth and neuronal differentiation. However, excessive phosphorylation of Tau can destabilize microtubules, impairing axonal transport [46]. The six bands detected for Tau likely correspond to the six Tau isoforms (35–56 kDa) that are known to be generated by alternative splicing of the Tau-encoding gene mapt, all of which are subject to extensive post-translational modifications, most notably phosphorylation [47].

As represented in Fig. 6C and 6D, both Tau and p-Tau levels significantly increased during the differentiation process, with Tau levels increasing by 2.7-fold and p-Tau by 3.1-fold at 72 h, consistent with the known role of this protein in axonal transport [46]. Despite these increases, the Tau/p-Tau ratio remained consistent throughout the differentiation period (Fig. 6E), indicating that the balance between Tau and its phosphorylated form was maintained during this process. Notably, analysis of these key transport markers showed no changes in cells exposed to 1 µM ADB-FUBINACA (Fig. 6F-6G), indicating that this SC did not impact the adaptor protein levels or microtubule stability.