High-content screening identifies a small molecule that restores AP-4-dependent protein trafficking in neuronal models of AP-4-associated hereditary spastic paraplegia.

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.

ALDH5A1-deficient iPSC-derived excitatory and inhibitory neurons display cell type specific alterations.

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.

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.

Unbiased phenotypic screens in patient-relevant disease models offer the potential to detect novel therapeutic targets for rare diseases. In this study, we developed a high-throughput screening assay to identify molecules that correct aberrant protein trafficking in adaptor 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, C-01, 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 putative molecular targets of C-01 and potential mechanisms of action. 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 Investigational New Drug (IND)-enabling studies.

Functional validation of novel variants in B4GALNT1 associated with early-onset complex hereditary spastic paraplegia with impaired ganglioside synthesis.

Childhood-onset forms of hereditary spastic paraplegia are ultra-rare diseases and often present with complex features. Next-generation-sequencing allows for an accurate diagnosis in many cases but the interpretation of novel variants remains challenging, particularly for missense mutations. Where sufficient knowledge of the protein function and/or downstream pathways exists, functional studies in patient-derived cells can aid the interpretation of molecular findings. We here illustrate the case of a 13-year-old female who presented with global developmental delay and later mild intellectual disability, progressive spastic diplegia, spastic-ataxic gait, dysarthria, urinary urgency, and loss of deep tendon reflexes of the lower extremities. Exome sequencing showed a novel splice-site variant in trans with a novel missense variant in B4GALNT1 [NM_001478.5: c.532-1G>C/c.1556G>C (p.Arg519Pro)]. Functional studies in patient-derived fibroblasts and cell models of GM2 synthase deficiency confirmed a loss of B4GALNT1 function with no synthesis of GM2 and other downstream gangliosides. Collectively these results established the diagnosis of B4GALNT1-associated HSP (SPG26). Our approach illustrates the importance of careful phenotyping and functional characterization of novel gene variants, particularly in the setting of ultra-rare diseases, and expands the clinical and molecular spectrum of SPG26, a disorder of complex ganglioside biosynthesis.

Novel CAPN1 missense variants in complex hereditary spastic paraplegia with early-onset psychosis.

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.

AP-4-mediated axonal transport controls endocannabinoid production in neurons.

The adaptor protein complex AP-4 mediates anterograde axonal transport and is essential for axon health. AP-4-deficient patients suffer from a severe neurodevelopmental and neurodegenerative disorder. Here we identify DAGLB (diacylglycerol lipase-beta), a key enzyme for generation of the endocannabinoid 2-AG (2-arachidonoylglycerol), as a cargo of AP-4 vesicles. During normal development, DAGLB is targeted to the axon, where 2-AG signalling drives axonal growth. We show that DAGLB accumulates at the trans-Golgi network of AP-4-deficient cells, that axonal DAGLB levels are reduced in neurons from a patient with AP-4 deficiency, and that 2-AG levels are reduced in the brains of AP-4 knockout mice. Importantly, we demonstrate that neurite growth defects of AP-4-deficient neurons are rescued by inhibition of MGLL (monoacylglycerol lipase), the enzyme responsible for 2-AG hydrolysis. Our study supports a new model for AP-4 deficiency syndrome in which axon growth defects arise through spatial dysregulation of endocannabinoid signalling.

High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia.

Adaptor protein complex 4-associated hereditary spastic paraplegia 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 adaptor protein complex 4-associated hereditary spastic paraplegia 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 adaptor protein complex 4. Using automated high-throughput microscopy, we determine the ratio of the ATG9A fluorescence in the trans-Golgi-network versus cytoplasm and ascertain that this metric meets standards for screening assays (Z'-factor robust >0.3, strictly standardized mean difference >3). The 'ATG9A ratio' is increased in fibroblasts of 18 well-characterized adaptor protein complex 4-associated hereditary spastic paraplegia patients [mean: 1.54 ± 0.13 versus 1.21 ± 0.05 (standard deviation) in controls] and receiver-operating characteristic analysis demonstrates robust diagnostic power (area under the curve: 0.85, 95% confidence interval: 0.849-0.852). Using fibroblasts from two individuals with atypical clinical features and novel biallelic missense variants of unknown significance in AP4B1, we show that our assay can reliably detect adaptor protein complex 4 function. Our findings establish the 'ATG9A ratio' as a diagnostic marker of adaptor protein complex 4-associated hereditary spastic paraplegia.

Generation and characterization of six human induced pluripotent stem cell lines (iPSC) from three families with AP4M1-associated hereditary spastic paraplegia (SPG50).

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.