Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. SSADHD leads to impaired GABA metabolism and results in accumulation of GABA and γ-hydroxybutyrate (GHB), which alter neurotransmission and are thought to lead to neurobehavioral symptoms. However, why increased inhibitory neurotransmitters lead to seizures remains unclear. We used induced pluripotent stem cells from SSADHD patients (one female and two male) and differentiated them into GABAergic and glutamatergic neurons. SSADHD iGABA neurons show altered GABA metabolism and concomitant changes in expression of genes associated with inhibitory neurotransmission. In contrast, glutamatergic neurons display increased spontaneous activity and upregulation of mitochondrial genes. CRISPR correction of the pathogenic variants or SSADHD mRNA expression rescue various metabolic and functional abnormalities in human neurons. Our findings uncover a previously unknown role for SSADHD in excitatory human neurons and provide unique insights into the cellular and molecular basis of SSADHD and potential therapeutic interventions.
Biallelic loss-of-function variants in the subunits of the adaptor protein complex 4 lead to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1), and SPG52 (AP4S1). Here, we describe the generation of induced pluripotent stem cells (iPSCs) from three AP-4-HSP patients with biallelic, loss-of-function variants in AP4M1 and their sex-matched parents (asymptomatic, heterozygous carriers). Following reprogramming using non-integrating Sendai virus, iPSCs were characterized following standard protocols including karyotyping, embryoid body formation, pluripotency marker expression and STR profiling. These first iPSC lines for SPG50 provide a valuable resource for studying this rare disease and related forms of hereditary spastic paraplegia.
Abstract CAPN1 ‐associated hereditary spastic paraplegia (SPG76) is a rare and clinically heterogenous syndrome due to loss of calpain‐1 function. Here we illustrate a translational approach to the case of an 18‐year‐old patient who first presented with psychiatric symptoms followed by spastic gait, intention tremor, and neurogenic bladder dysfunction, consistent with a complex form of HSP. Exome sequencing showed compound‐heterozygous missense variants in CAPN1 (NM_001198868.2: c.1712A>G (p.Asn571Ser)/c.1991C>T (p.Ser664Leu)) and a previously reported heterozygous stop‐gain variant in RCL1 . In silico analyses of the CAPN1 variants predicted a deleterious effect and in vitro functional studies confirmed reduced calpain‐1 activity and dysregulated downstream signaling. These findings support a diagnosis of SPG76 and highlight that the psychiatric symptoms can precede the motor symptoms in HSP. Our results also suggest that multiple genes can potentially contribute to complex neuropsychiatric diseases.
Background/Purpose: Adaptor protein complex 4-associated hereditary spastic paraplegia (AP-4-HSP) is caused by biallelic loss-of-function variants in AP4B1, AP4M1, AP4E1, or AP4S1 which constitute the four subunits of this obligate complex. While the diagnosis of AP-4-HSP relies on molecular testing, the interpretation of novel missense variants remains challenging. Here we address this diagnostic gap by using patient-derived fibroblasts to establish a functional assay that measures the subcellular localization of ATG9A, a transmembrane protein that is sorted by AP-4.
Imaging data from the article "AP-4-mediated axonal transport controls endocannabinoid production in neurons", published in Nature Communications by Davies et al., from Fig. 5 and S4. Super resolution structured illumination microscopy (SR-SIM) was used to image HeLa cells expressing endogenously-tagged SERINC1-Clover or SERINC3-Clover, treated with siRNA to knock down AP-4 or non-targeting control siRNA, and labelled with anti-GFP and either anti-ATG9A or anti-DAGLB. SR-SIM was performed on a Zeiss Elyra PS.1 microscope equipped with a 63x/1.46 oil objective (alpha Plan-Apochromat 63x/1.46 Oil Korr M27) and a PCO pco.edge 4.2 sCMOS Camera, and controlled with Zeiss ZEN 2 software with the SR-SIM module. Imaging was performed sequentially, with SERINC1 or SERINC3 (labelled with Alexa Fluor Plus 488) imaged first and DAGLB or ATG9A (labelled with Alexa Fluor 568) imaged second, using 3 rotations of the grid pattern and 5 phases for each rotation. Experiments were performed in biological duplicate, from separate dishes of cells, and with immunofluorescence and microscopy performed independently for each replicate. 20 cells were imaged per condition per replicate, and cell selection and manual focus were performed on the first acquired channel only, without viewing the second channel. For channel alignment, slides with multi-coloured fluorescent beads were imaged before and after each experiment using the same acquisition settings. The data were processed using the SIM module in Zen 2 software in manual mode, using default settings and a theoretical PSF model, except for the following settings: Noise filter -1; SR frequency weighting 2; Max.Isotrop on; Baseline shifted on; Raw scale on. Channel alignment fit parameters were calculated in Zen 2 software using a multi-coloured bead image as input, in affine mode. The resulting parameters were tested on further bead images and then applied to the experimental images. The channel-aligned images were used for colocalisation analysis (see methods of article for details). This dataset includes the raw unprocessed data and the processed and channel aligned input data used for the colocalisation analysis. See article for methods and further detail.
Supplementary data of the manuscript: High-Content Small Molecule Screen Identifies a Novel Compound That Restores AP-4-Dependent Protein Trafficking in Neuronal Models of AP-4-Associated Hereditary Spastic Paraplegia
Imaging data associated with Fig. 6c,e,f, 7e-h, S5a,b, S6b-d and S7 from the article "AP-4-mediated axonal transport controls endocannabinoid production in neurons", published in Nature Communications by Davies et al. DAGLB_Axon_iPSC_derived_neurons: High-throughput confocal imaging was used to assay the density of DAGLB puncta in axons of iPSC neurons from a patient with AP-4 deficiency syndrome (patient 1) and their matched control, labelled with anti-DAGLB, the axonal marker antibody cocktail SMI312 and DAPI. DAGLB_Soma_iPSC_derived_neurons: High-throughput confocal imaging was used to assay the distribution of DAGLB in iPSC-derived neurons from two patients with AP-4 deficiency syndrome (LoF/LoF) and their matched unaffected controls (WT/LoF). Neurons in 96-well plates were labelled with antibodies against DAGLB, GOLGA1 (a TGN marker) and TUJ1, and DAPI. The ratio between the area of high intensity (HI; overlaps with TGN) and low intensity (LI) DAGLB labelling was quantified from three differentiations per cell line. iPSC_derived_neurons_neurite_outgrowth: Neurite outgrowth was assayed in iPSC-derived cortical neurons from two patients with AP4B1-associated AP-4 deficiency syndrome (SPG47) and their unaffected same sex heterozygous parents (control), using automated live cell imaging. Neurons were cultured in the presence of DMSO (vehicle control) or the MGLL inhibitor ABX-1431 at 10, 50, 100 or 500 nM (the highest two doses were administered only to the patient neurons). Neurons were monitored from 4 h post-plating, with images captured every 3 h until 25 h post-plating. See article for methods and further detail.
Imaging data from the article "AP-4-mediated axonal transport controls endocannabinoid production in neurons", published in Nature Communications by Davies et al., from Fig. 2, 3, 4, 6 and S2. Widefield images were captured on a Leica DMi8 inverted microscope equipped with an iTK LMT200 motorised stage, a 63x/1.47 oil objective (HC PL APO 63x/1.47 OIL) and a Leica DFC9000 GTC Camera, and controlled with LAS X (Leica Application Software X). HeLa_HADAGL_RUSC2_Fig_4: Widefield imaging of wild-type HeLa and HeLa cells stably expressing GFP-RUSC2, transiently expressing HA-DAGLB or HA-DAGLA, and labelled with anti-HA (Alexa Fluor 680), anti-ATG9A (Alexa Fluor 568) and DAPI. HeLa_KD_DAGLB_TGN_Fig_S2a,b: Widefield imaging of immunofluorescence double labelling of DAGLB (Alexa Fluor 568) and TGN46 (Alexa Fluor 680) in HeLa cells transfected with a non-targeting siRNA (control) or with siRNA to knock down AP-4. DAPI labelling of the nucleus is also shown. Note, the antibody used to label AP4E1 (Alexa Fluor 488) in replicate 1 had high non-specific background and so was not used in the analysis. HeLa_KO_DAGLB_TGN_Fig_2a,b: Widefield imaging of immunofluorescence double labelling of DAGLB (Alexa Fluor 568) and TGN46 (Alexa Fluor 680) in wild-type, AP4B1 knockout, and AP4B1 knockout HeLa cells stably expressing AP4B1 (functional rescue). DAPI labelling of the nucleus is also shown. HeLa_RUSC2_DAGLB_Fig_3: Widefield imaging of DAGLB (Alexa Fluor 568) labelling in wild-type and AP-4 knockout (AP4B1 KO or AP4E1 KO) HeLa cells, stably expressing GFP-tagged RUSC2. AP4B1 knockout HeLa expressing RUSC2-GFP were also transiently transfected with AP4B1 (rescue) or mock transfected without DNA as a negative control. DAPI labelling of the nucleus is also shown. iPSC_Neurons_DAGLB_TGN_Fig_6a,b: Widefield imaging of immunofluorescence triple labelling of DAGLB (Alexa Fluor 568), TGN46 (Alexa Fluor 680) and TUJ1 (Alexa Fluor 488; a marker to distinguish neurons from co-cultured astrocytes) in iPSC neurons from a patient with AP-4 deficiency syndrome (patient 1) and their matched control. SHSY5Y_DAGLB_TGN_Fig_2c,d_Fig_S2c,d: Widefield imaging of immunofluorescence double labelling of DAGLB (Alexa Fluor 568) and TGN46 (Alexa Fluor 680) in control (parental Cas9-expressing), AP4B1-depleted (BKO) and AP4E1-depleted (EKO) undifferentiated and neuronally-differentiated SH-SY5Y cells. DAPI labelling of the nucleus is also shown. See article for methods and further detail.
Abstract Unbiased phenotypic screens in patient-relevant disease models offer the potential to detect therapeutic targets for rare diseases. In this study, we developed a high-throughput screening assay to identify molecules that correct aberrant protein trafficking in adapter protein complex 4 (AP-4) deficiency, a rare but prototypical form of childhood-onset hereditary spastic paraplegia characterized by mislocalization of the autophagy protein ATG9A. Using high-content microscopy and an automated image analysis pipeline, we screened a diversity library of 28,864 small molecules and identified a lead compound, BCH-HSP-C01, that restored ATG9A pathology in multiple disease models, including patient-derived fibroblasts and induced pluripotent stem cell-derived neurons. We used multiparametric orthogonal strategies and integrated transcriptomic and proteomic approaches to delineate potential mechanisms of action of BCH-HSP-C01. Our results define molecular regulators of intracellular ATG9A trafficking and characterize a lead compound for the treatment of AP-4 deficiency, providing important proof-of-concept data for future studies.
