Patients

This study was approved by the local Institutional Review Board of the Friedrich-Alexander-Universität Erlangen-Nürnberg (No. 17-259-B, No. 17-347-B, No. 21-498-D, No. 484_20 B), and all individuals provided written informed consent. Brain sampling followed the ethical rules of each country.

Postmortem tissue

“SPG11 (UKER)” postmortem tissue was donated by a female SPG11–HSP patient (SPG11 donor SPG11-3 in the iMGL experiments, Online Resource Table 3) with a disease onset at the age of 31, progressive gait disorder, muscle cramps, and loss of dexterity. Genetic testing identified a heterozygous nonsense variant at c.267G > A/p.Trp89X in exon 2 and the splice site variant c.1457-2A > G in intron 6 of SPG11. During disease progression, the patient developed a spastic paraparesis, muscle cramps, distal muscle wasting, and pseudobulbar dysarthria. At age 46, she had lost ambulation and exhibited progressive dementia and dysphagia. The patient died at the age of 51 of aspiration, without clinical evidence of an inflammatory disorder, and without antibiotic or immunomodulatory therapy. Postmortem brain tissue derived from a female without neurological disease deceased at age 42 from arterial lung embolism was used as non-inflammatory “control”.

The “SPG11 (BG2)” case was previously described in Denora et al. 2016 [25]. Briefly, this postmortem tissue is derived from a female SPG11–HSP patient with disease onset at age 10, rapidly progressing spastic cerebellar ataxic gait and mild intellectual disability. The patient died at the age of 46 years, and familial genetic testing revealed a homozygous truncating variant c.6739_6742del (p.Glu2247Leufs14) in exon 36 of SPG11. Corresponding control tissue derived from a female deceased at age 59 from the same center was used.

The “SPG11 (UWA)” postmortem tissue was donated by a male SPG11–HSP patient from the Genetic Medicine Clinic at University Washington Medical Center, USA, who died at the age of 42. This patient had delayed language acquisition as a child and required special education through high school. He presented at age 20 with gait and balance symptoms that had begun a few years earlier. Examination showed nystagmus, distal upper extremity amyotrophy and spasticity. He was diagnosed with optic atrophy at age 34. By the time of death, he was mute and markedly bradykinetic. Genetic testing in this case identified a homozygous 4 base-pair deletion variant c.6439_6442del leading to a frameshift. A detailed clinical workup of this case will be published elsewhere (Scherpelz et al., in review). Corresponding control tissues derived from two males deceased without neurological diseases at age 38 (control UWA1) and at age 42 (control UWA2) from the same center were used.

Each SPG11 postmortem case was compared only with corresponding control tissue subjected to comparable processing and staining procedures at the same facility.

Blood samples

Peripheral blood samples were collected from SPG11–HSP patients and healthy controls (Online Resource Tables 1 and 2). All individuals were asked to avoid excessive physical activity within the preceding week, and blood samples were withdrawn in the morning (between 8 and 11 am) after a fasting period of at least 8 h, including abstinence from caffeine and sweeteners. Serum samples were cooled immediately after blood drawing, and supernatants were prepared within 4 h and subsequently stored at  – 80 °C [81]. Individuals receiving immunomodulatory medications were not included in the study. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with non-neurodegenerative or non-inflammatory diseases (controls, n = 38) and SPG11 patients (n = 8). The PBMC control cohort consisted of non-inflammatory and non-degenerative neurological diseases, i.e., meningioma (n = 1), idiopathic intracranial hypertension (n = 8), dysesthesia (n = 7), pain syndrome (n = 2), primary headache (migraine, tension headaches; n = 6), normal pressure hydrocephalus (n = 2), somatization disorder (n = 2), polyneuropathy (n = 1), fatigue (n = 1), phobic vertigo (n = 1), non-specific white matter lesions (n = 4) and without a diagnosis (n = 3). PBMCs underwent processing using a Ficoll gradient method and were subsequently preserved in liquid nitrogen (Ficoll–Paque PLUS density gradient media, Cytiva) [84]. Viability rates exceeded 80%, and routine quality assessments showed unchanged phenotypes of frozen PBMCs compared to the analysis of fresh PBMCs [12, 84].

iPSC lines

IMGL were differentiated from iPSCs derived from three Caucasian females with compound heterozygous pathogenic variants in the SPG11 gene, including the “SPG11 (UKER)” postmortem case, and three age- and sex-matched healthy controls (Online Resource Table 3). Their precise clinical phenotype and generation of iPSCs have been described previously [5, 39, 72, 90].

Animals

Generation and characterization of Spg11–KO mice was described previously [46, 99]. All animal experiments were approved by the “Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz” (TLLV) in Germany (Approval number: 02–039-14). Mice were housed in a 12 h light/dark cycle and fed on a regular diet ad libitum. Five Spg11+/+ and five Spg11−/− mice were analyzed at the age of 16 months.

MSD® multi-spot assay system

Multiplex electrochemiluminescence (ECL) was performed using the Meso Scale Discovery® system (MSD®; Rockville, MD, USA) according to the manufacturer’s instructions, using the U-PLEX Custom Biomarker Group 1 (human) Assay (Cat.: K15067L-1), including U-PLEX 10-Assay, 96-Well SECTOR Plate (Cat.: N05235A-1), U-PLEX Human Antibody Sets for IFNγ (Cat.: B21TT-2), IL-1β (Cat.: B21TU-2), IL-6 (Cat.: B21TX-2), IL-8 (Cat.: B21TY-2), IL-10 (Cat.: B21TZ-2), TNFα (Cat.: B21UC-2), IP-10 (Cat.: B21UF-2), MCP-1 (Cat.: B21UG-2), IL-1α (Cat.: B21UN-2) and IL-18 (Cat.: B21VJ-2). In addition, calibrator 1 (Cat.: C0060-2), calibrator 2 (Cat.: C0061-2) and calibrator 3 (Cat.: C0062-2) were applied together with the respective buffers, Diluent 43 (Cat.: R50AG) and Diluent 3 (Cat.: R50AP). Patient serum and cell culture supernatants were diluted 1:1 in Diluent 43. Provided plates were coated with Linker-coupled antibodies 1 day before the assay was performed. The biotinylated antibodies were combined with the assigned Linker (Linker 1 for IFN-γ, Linker 2 for IL-1β, Linker 3 for IL-6, Linker 4 for IL-8, Linker 5 for IL-10, Linker 6 for TNF-α, Linker 7 for IP-10, Linker 8 for MCP-1, Linker 9 for IL-1α and Linker 10 for IL-18) and adjusted to 6 ml with the Stop Solution (Cat.: R50AO-1). 50 µl of the coating solution was added to each well and the plates were subsequently incubated for 1 h at room temperature, washed trice with phosphate-buffered saline (PBS)/ 0.05% Tween-20. The plates were stored at 4 °C overnight. The calibrators were diluted in a fourfold serial dilution using Diluent 43 to generate eight standards. 25 µl of Diluent 43 was added to each well, followed by 25 µl of prepared calibrator standards or sample dilutions. All standards and samples were measured in duplicates. The plates were incubated at RT with shaking for 1 h. After the plates were washed three times as described above, 50 µl of detection antibody solution was added to each well. The 100X stock solution of the detection antibodies was diluted in Diluent 3. After an incubation for 1 h, the plates were washed again twice and 150 µl of MSD GOLD Read Buffer B was added to each well. The measurement was performed on MESO® QuickPlex® SQ 120MM (Cat.: Al1AA) and subsequent analysis were carried out using MSD® Discovery Workbench® Version 4.0. Protein concentrations within the iMGL supernatants were further normalized to total protein amount, which was measured using the Pierce BCA protein assay Kit (Thermo Fisher) according to the manufacturer’s protocol.

Immunohistochemistry (IHC) of human postmortem tissue

Formalin-fixed paraffin-embedded tissue blocks of cortical areas and basal ganglia sectioned at 5 µm underwent standard analyses, including Hematoxylin and Eosin (H&E) and luxol fast blue (LFB) staining. Chromogenic (DAB) immunohistochemistry was performed for GFAP, CD8, CD4, CD68, STAT1 and IBA1. For immunofluorescence (IF) stainings, sections were deparaffinized at 60 °C for 30 min and incubated in Neo-Clear (Sigma-Aldrich) for 10 min. After rehydration, antigen retrieval was performed by incubating sections in 100X Tris Buffer (pH 10.0; abcam) using a pressure cooker (2100 Antigen Retriever, Aptum Biologics). Sections were permeabilized for 10 min with 0.2% Triton-X-100 (Sigma-Aldrich) and 2% bovine serum albumin (BSA) in PBS followed by blocking of non-specific antibody binding by PBS with 2% BSA for 1 h at RT. Primary antibody incubation in PBS 2% BSA overnight at 4 °C (Online Resource Table 4). On the next day, sections were first blocked with 2% donkey serum (Pan Biotech) and 0.2% Triton-X-100 in PBS and afterward incubated in respective secondary Alexa Fluor coupled donkey antibody (1:500, Thermo Fischer, Online Resource Table 4) in PBS 2% BSA for 1 h at RT. Next, sections were washed in PBS with 0.2% Triton-X-100 for 5 min followed by PBS washes. In addition, sections were then stained with 0.5 mg/ml 49,6-diamidino-2-phenylindole (DAPI) and autofluorescence was quenched with TrueBlack (20X in 70% Ethanol, Biotium). Sections were subsequently washed and mounted with Prolong Gold anti-fade with DAPI Mounting Media (Invitrogen). Microscopic analysis was performed on an Observer.Z1 microscope (ZEISS) and ZEN 2.6 blue software (ZEISS). For the quantification of the IF images, ten randomly selected fields of view were counted manually, blinded for genotype for each brain region.

IHC of murine brain tissue

Animals were euthanized with a fivefold overdose of Ketamin (500 mg/kg body weight) and Xylazin (80 mg/kg body weight) and perfused transcardially with 4% PFA in 1 × PBS. Brains were removed and post-fixed in 4% PFA overnight at 4 °C.

IHC was performed on 40 µm cryosections that were first washed three times with TBS buffer (100 mM TRIS, 1.5 M NaCl, pH 8 in H2O) for 5 min. Afterward, antigen retrieval was performed. Sections were incubated in Dako TRS, Citrate pH 6 (10x; Aglilent Technologies) for 30 min at 80 °C followed by 30 min at RT. After washing with TBS, sections were incubated in blocking buffer (3% donkey serum, 0.1% Triton-X-100 in TBS) for 30 min. Incubation of the primary antibody (Online Resource Table 4) diluted in blocking buffer was performed at 4 °C overnight. On the next day, sections were first washed with TBS and afterward incubated in respective secondary Alexa Fluor coupled donkey antibody (Online Resource Table 4) for 1 h at RT. Sections were subsequently stained with 0.5 mg/ml DAPI and washed with TBS. Mounting was performed using Prolong Gold anti-fade Mounting Media (Invitrogen). Microscopic analysis was performed as described above. For quantification of the IF images, five randomly selected fields of view per mouse were counted manually, blinded for genotype.

Electron microscopy

Postmortem brain tissue fixed in 4% PFA underwent two different routes for further processing. For ultrastructural analysis, tissue was post-fixed in Itho-buffer containing 3% glutaraldehyde (GA). Embedding in resin and sectioning was performed as described before [1]. For immuno-gold labeling, tissue samples were rehydrated, frozen and cryo-sectioned to 50 µm. These 50 µm sections were then incubated with an anti-IBA1 antibody (Wako, Online Resource Table 4) and a gold-labeled secondary antibody. After washing the section to remove unbound secondary antibodies, silver enhancement was performed to increase signal intensity in transmission electron microscopic (TEM) analysis. Sections were then fixed in Itho-buffer (with 3% GA), embedded and sectioned as described above. All ultrathin sections were transferred into a 1400Plus TEM (JEOL) operating at 120 kV.

iPSC derivation and culture

Fibroblasts from patients and controls had been reprogrammed as previously described [72, 76]. Briefly, fibroblasts obtained from dermal punch biopsies were reprogrammed with viral transduction [34, 72] of the four Yamanaka factors (KLF4, c-Myc, Oct4 and Sox2). All obtained iPSC lines were tested for pluripotency (Tra1-60 expression by Flow cytometry) and stable karyotype using the G-banding chromosomal analysis and analysis of copy number variations > 100 kb [76]. All iPSC lines were described in our previous studies which focused on the neural lineage [34, 51, 60, 71, 72, 77]. Two established iPSC lines were analyzed per individual.

iPSCs were maintained in mTeSR Plus (STEMCELL Technologies) on Geltrex™-coated plates (500 µg for 57 cm2, Thermo Fisher Scientific). Cells were passaged as clumps using Gentle Cell Dissociation reagent (STEMCELL Technologies).

iMGL generation

iPSCs were differentiated into iMGL via hematopoietic progenitor cells (HPCs) as previously described [52]. For HPC generation, STEMDiff hematopoietic kit (STEMCELL Technologies) was used according to the manufacturer’s protocol. Briefly, iPSCs were seeded as small clumps and cultivated in the media provided by the STEMDiff hematopoietic kit. HPCs were collected on days 12, 14, and 16 of differentiation and combined in RPMI1640 with 10% FCS (Gibco), 50 U/ml Penicillin/streptomycin (Gibco), and 10 ng/ml GM–CSF (Peprotech). On day 16 of differentiation, HPCs were either frozen or further seeded in maturation media additionally containing 50 ng/ml IL-34 (Peprotech) on glass slides containing plates to generate iMGL. HPCs were differentiated for 2 weeks into iMGL with the addition of media every 2–3 days and one 50% media change after 1 week. All analyses were performed on differentiated iMGL, i.e., after 2 weeks on day 28, including these stimuli: IFNγ (10 ng/ml for 24 h, Peprotech), LPS (100 ng/ml for 24 h, SIGMA), Oleic Acid (200 µm for 24 h; SIGMA), serum depletion for 24 h with BafilomycinA1 (BAF) exposure (100 nM for 6 h; Thermo Fisher) and Ruxolitinib (5–100 µM for 24 h before IFNγ stimulation, in DMSO, Selleckchem). For immunocytochemical analysis, either 100,000 HPCs per well were differentiated in a 24-well plate or differentiated iMGLs were reseeded with TrypLE Express (Invitrogen) on a 96-well staining plate (Ibidi; 10,000 cells/ well).

To generate microglia-conditioned media (MCM), control and SPG11 iMGL (5 clones each) were re-seeded on day 25 of differentiation on 24-well plates with 200,000 cells per well. On day 27, Ruxolutinib treatment (50 µM, Selleckchem) was performed, 24 h prior to IFNγ (10 ng/ml, Peprotech). After 24h of IFNγ stimulation, MCM was collected and centrifuged at 1000 × g for 10 min. The supernatant was diluted 1:1 in neuronal media NMM and applied to neurons.

Generation of iPSC-derived neurons

Differentiation of iPSC into cortical neurons was based on the published protocol by Shi et al. [86]. First, iPSCs were seeded at a density of 300,000 cells/cm2 on Geltrex™-coated plates. At 100% confluency, medium was changed to neural induction medium which is composed of neural maintenance medium (NMM; 50% Neurobasal (Life Technologies), 50% DMEM/F12 + GlutaMAX (Life Technologies), B27 + VitA (50x; Life Technologies), N2 (200x; Life Technologies), MEM Non-essential amino acids solution (100x, Life Technologies), 2-Mercaptoethanol (50 µM; Life Technologies), Penicillin/streptomycin (50 U*ml−1; Life Technologies)) supplemented with LDN (100 nM; Tocris) and SB431542 (10µM; Tocris). Medium was changed every day until day 12 when the cells were split 1:3 in aggregates using Collagenase IV (Life Technologies) and medium was changed to NMM with subsequent media changes every other day. As soon as neural rosettes appeared, NMM was supplemented with FGF2 (20 ng/ml; Peprotech) for 2 days. After withdrawal of FGF2, approximately 16–20 days after neural induction, cells were split again 1:2 using Collagenase IV. On day 27 of differentiation, neural progenitor cells (NPCs) were detached by Accutase (Life Technologies) and cryopreserved. For further differentiation into neurons, NPCs were seeded at a density of 50,000 cells/cm2 on Geltrex™-coated plates and cultured in NMM with half media changes twice weekly. For conditioned media experiments, neurons were split after 2 weeks of differentiation on 48-well plates with 300,000 cells per well. After 48 h of incubation with MCM, neurons were fixed for 10 min in 4% PFA in PBS, followed by three washes with PBS. As a positive control, neurons were incubated with sodium arsenite (10 nM for 24 h) to induce cell death.

Phagocytosis assay

To confirm and quantify the phagocytic activity of iMGL, pHrodo™ Red S. aureus BioParticles™ (Thermo Fisher) were used according to the manufacturer’s protocol, as described previously [52]. Shortly, iMGL were dissociated on day 26 of differentiation using TrypLE Express and 20,000 cells/ 96-well were seeded in a fluorescence reader compatible 96-well plate (Corning). After 24 h, iMGL were exposed to IFNγ (10 ng/µl for 24 h) or left untreated and on the next day, the phagocytosis assay was performed. iMGL of each line were additionally treated with CytochalasinD (10 µM; Life Technologies) for 30 min at 37 °C as a negative control, before pHrodo particles were added (200 ng/ml in maturation media). All iMGL were incubated with bacterial particles for 2 h at 37 °C before cell nuclei were counterstained with NucBlue (Thermo Fisher) for 20 min according to the manufacturer’s protocol. Fluorescence intensity was measured in triplicates at 2 h, 4 h, 6 h and 8 h after the addition of pHrodo particles on a CLARIOstar Plus fluorescent plate reading device (BMG LABTECH).

Immunocytochemistry

Differentiated iMGL and MCM-exposed neurons were fixed for 10 min in 4% PFA in PBS, followed by three washes with PBS. To permeabilize the cells and block unspecific antibody binding, cells were first incubated for 1h in PBS with 3% donkey serum and 0.1% Triton-X-100 at RT. Primary antibodies diluted in PBS with 3% donkey serum were applied (Online Resource Table 5) and incubated overnight at 4 °C or 1 h at RT. Afterward, cells were washed three times with PBS and incubated for 1 h at RT with the respective Alexa Fluor coupled secondary antibody diluted in PBS with 3% donkey serum (Online Resource Table 4).

To visualize lysosomal proteins in iMGL, cells were first incubated in 50 mM NH4Cl in PBS for 10 min to reduce the autofluorescence. After washing one time with PBS, cells were permeabilized with 0.1% saponin in PBS for 10 min followed by blocking of non-specific antibody binding with 5% donkey serum and 0.05% saponin in PBS for 1 h. Primary antibodies were diluted in 3% donkey serum and 0.05% saponin in PBS and incubated overnight at 4 °C or 1 h at RT. Next, cells were washed three times with PBS followed by 1 h incubation at RT with secondary antibodies which were diluted in PBS with 3% donkey serum and 0.05% saponin.

After staining with secondary antibodies, nuclei were counterstained with DAPI (0.5 mg/ml) for 5 min followed by three PBS washes. Cells stained on coverslips were mounted using Aqua-Poly/Mount (Polyscience). If staining was performed in a 96-well plate (ibidi), cells were mounted with Mowiol 4–88 (Sigma-Aldrich). Before microscopic analysis, mounted coverslips and plates were dried overnight at RT.

For visualization of apoptotic cells, the One-step TUNEL In Situ Apoptosis Kit (Red, Elab Fluor® 647; Elabscience) was used before antibody staining according to the manufacturer’s instructions.

Flow cytometry (FC)

For FC analysis of PBMCs, frozen PBMCs were thawed and allowed to rest in a medium at a temperature of 37 °C. Subsequently, centrifugation was carried out at 300 g for 10 min. The PBMCs were then suspended in FACS buffer, which consisted of PBS (PAA Laboratories GmbH) supplemented with 2% fetal calf serum (FCS; Invitrogen). Blocking was performed using human Fc-block (BD Bioscience). PBMCs were stained for 30 min at 4 °C, washed, and finally analyzed using a Cytek Northern Lights instrument (Cytek Biosciences, Fremont, CA, USA). The following antibodies were used for staining: LIVE/DEAD Zombie NIR Fixable Viability Kit (Biolegend), CD3 cFluor® V420 (Cytek, CloneSK7), HLA-DR PerCP (BD, Clone L243), CD14 PerCP eFluor 710-A (Invitrogen, Clone 61D3), CD19 cFluorBYG710-A (Cytek, Clone HIB19), CD16 cFluor R668-A (Cytek, Clone 3G8), CD56 cFluor R720-A (Cytek Clone 5.1H11). A detailed gating strategy is provided in Online Resource Fig. 3.

FC analysis of IBA1 expression in iMGL was performed as described previously [52]. Briefly, iMGL were dissociated and collected in FC buffer (2% FCS and 0.01% sodium azide in PBS) on day 28 of differentiation. After centrifugation, 100,000 cells per staining were transferred in a V-bottom 96-well plate. Cells were fixed by 10 min incubation in 50 µl BD Cytofix per well (BD Bioscience) followed by permeabilization for 5 min in BD Perm/Wash (BD Bioscience). Afterward, cells were incubated for 30 min at RT with primary antibody anti-IBA1 (Wako; Online Resource Table 4) diluted 1:50 in BD Perm/Wash. Cells were washed and secondary antibody incubation for 30 min was performed (Thermo Fisher; Online Resource Table 4) followed by additional washing steps. Finally, cells were resuspended in FC buffer for analysis on a Beckman Coulter Cytoflex S FACS platform. As negative controls, iMGL stained with respective rabbit isotype control (Invitrogen) and iPSC stained for IBA1 were included. Data were analyzed using CytExpert software (version 2.4.0.28).

Western blot

For protein isolation, cells were pelletized in cold PBS. Pellets were resuspended in 100–150 µl Radio-immunoprecipitation buffer (RIPA; 50 mM Tris/HCl pH 7.4, 0.5% Deoxycholic acid sodium salt, 1% NP-40, 1% Sodium deoxycholate, 0.1% Sodium dodecyl Sulfate (SDS), 150 mM Sodium chloride, 2 mM Ethylenediaminetetraacetic acid (EDTA), 50 mM Sodium fluoride) freshly supplemented with EDTA free complete mini protease inhibitor cocktail (Roche) and PhosphoSTOP (Roche). Cells were incubated for 30 min on ice followed by sonication with Diagenode Bioruptor Pica (setting: 30 s ON, 30 s OFF, 5 cycles, high frequency). Protein concentration was measured using the Pierce BCA protein assay Kit (Thermo Fisher) according to the manufacturer’s protocol. Color change was measured at 564 nm using SpectraMax 190 plate reader (Molecular Devices) and the SpectraMax software (SoftMax Pro 7.1). Protein samples were prepared with 5X laemmli-buffer (300 mM Tris–HCl pH 6.8, 10% SDS, 50% glycerol, 5% β-Mercaptoethanol) and boiled at 95 °C for 10 min. Equal amounts of protein were used to run SDS–PAGE using the NuPAGE™ system (Thermo Fisher). All immunoblots were run on 4–12% Bis–Tris gels with NuPAGE™ MOPS SDS running buffer (20x). SDS–PAGE was performed according to manufacturer’s protocol. Gels containing separated proteins were blotted onto methanol pre-activated PVDF membranes (0.2 µm) at 10 V for 14 h at 4 °C in NuPAGE™ transfer puffer (20x) with 10% Methanol. Afterward, membranes were blocked for 1 h at RT with 5% milk in Tris-buffered saline supplemented with Tween (TBS-T; TBS supplemented with 0.1% Tween). For antibodies against phosphorylated proteins, membranes were blocked with 5% BSA in TBS-T. Subsequently, the membranes were incubated with primary antibodies diluted in TBS-T 5% milk/BSA overnight: anti-HA, anti-LAMP1, anti-LC3, anti-p62, anti-STAT1, anti-P-STAT1 and anti-β-actin (Online Resource Table 4). On the next day, the membranes were washed three times for 5 min with TBS-T, followed by incubation with secondary antibody (diluted in TBS-T 5% milk/BSA) for 1 h at RT (Online Resource Table 4). Blots were developed using ECL blotting solution (Amersham), and chemiluminescence was detected on an automated detection system (ChemiDoc MP, Biorad). To visualize total protein, membranes were incubated in Direct Blue (DB) 71 (Sigma-Aldrich) stain solution (0.008% DB71 in 40% ethanol, 10% acetic acid) for 5 min. Blot signals were quantified densitometrically using ImageJ (version 1.54d). The signal was normalized to the corresponding signal of β-actin.

RNA isolation and quantitative real-time PCR (qPCR)

For gene expression analysis, RNA was isolated from iMGL at day 28 of differentiation. Cells were lysed in 0.5–1 ml QIAzol Lysis Reagent (Qiagen) and incubated for 5 min at RT. 100–200 μl chloroform (Carl Roth) was added and the suspension was mixed vigorously by shaking manually for 15 s. The suspension was incubated for 3 min at RT and centrifuged for 15 min at 12,000 g and 4 °C. The upper aqueous phase containing RNA was transferred into a new 1.5 ml tube and an equal volume of 70% ethanol was added. RNA extraction was continued using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. gDNA digestion by DNase I treatment was performed for bulk RNA sequencing. The purity and final concentration of the isolated RNA were measured using a Nanophotometer NP80™ (Implem). Isolated RNA was stored at  – 80 °C. For cDNA synthetization, QuantiTect Reverse Transcription Kit (Qiagen) was used according to manufacturer’s instructions. PCR reactions were set up with respective primers (Online Resource Table 5) using SYBR Green PCR Master Mix (Thermo Fisher) according to manufacturer’s instructions. For each PCR reaction, 2.5 ng of cDNA were used. Signal was analyzed on a Roche LightCycler 480 real-time PCR platform. Ct values were normalized to two housekeeping genes (GAPDH and HRPT) and expression values relative to untreated samples were calculated using the ddCt method [55].

RNA sequencing

Isolated RNA from three SPG11 and four control iMGL, each untreated and IFNγ treated, underwent paired-end RNA sequencing with Poly-A selection using an Illumina NovaSeq, 2 × 150 bp configuration (Genewiz Germany). Samples were sequenced to a depth of 40,000,000 reads. Fastq files were first trimmed using Trimmomatic (v0.39) [10] and aligned to the human genome (GRCh38) using STAR (v2.7.9a) [26]. The feature Counts module within the Subread package (version 27) was utilized to assign reads to genes in the gencode annotation (version 26). In every sample, > 80% of reads are mapped uniquely to the human genome. Reads Per Kilobase of transcript, per Million mapped reads (RPKM) were calculated from the obtained counts to normalize for gene expression. Only genes with a mean RPKM value of 1 across the data set were considered for further analysis. PCA analysis was performed on RPKM values in Python 3 (v3.9.7). DESeq2 was used to determine differentially expressed genes (v1.34.0) [58]. DESeq2 output was filtered for adjusted p < 0.05 and |log2(fold change)|> 1. All downstream analyses were performed in Python 3 (v3.9.7) and graphs were generated using seaborn (v0.11.2).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9 Software (GraphPad Software Inc.). Normal distribution was examined using the Shapiro–Wilk test. When data were normally distributed, a two-sided unpaired t test was used to compare two groups, and a one-way ANOVA test to compare more than two groups, followed by Bonferroni’s post-hoc test. For determining differences between data that were not normally distributed, Mann–Whitney U test was conducted. For grouped analyses (e.g., NT vs. IFNγ in Ctrl vs. SPG11), two-way ANOVA was performed. P values < 0.05 were considered significant (*P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).