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.
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by epilepsy, intellectual disability, and benign tumors of the brain, heart, skin, and kidney. Animal models have ...contributed to our understanding of normal and abnormal human brain development, but the construction of models that accurately recapitulate a human pathology remains challenging. Recent advances in stem cell biology with the derivation of human-induced pluripotent stem cells (hiPSCs) from somatic cells from patients have opened new avenues to the study of TSC. This approach combined with gene-editing tools such as CRISPR/Cas9 offers the advantage of preserving patient-specific genetic background and the ability to generate isogenic controls by correcting a specific mutation. The patient cell line and the isogenic control can be differentiated into the cell type of interest to model various aspects of TSC. In this review, we discuss the remarkable capacity of these cells to be used as a model for TSC in two- and three-dimensional cultures, the potential variability in iPSC models, and highlight differences between findings reported to date.
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and their differentiation into neural lineages is a revolutionary experimental system for studying neurological disorders, ...including intellectual and developmental disabilities (IDDs). However, issues related to variability and reproducibility have hindered translating preclinical findings into drug discovery. Here, we identify areas for improvement by conducting a comprehensive review of 58 research articles that utilized iPSC-derived neural cells to investigate genetically defined IDDs. Based upon these findings, we propose recommendations for best practices that can be adopted by research scientists as well as journal editors.
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In this article, Anderson et al. review 58 research articles to assess the state of intellectual and developmental disabilities (IDDs) research utilizing iPSC-derived neural cells. Major sources of biological and technical variability are identified, and potential areas of improvement are proposed. To define reproducible cellular phenotypes linked to IDDs, the stem cell research community needs to develop shared standards, approaches, and benchmarks.
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.
We introduce a novel all-optical assay for functional studies of biological neural networks
. We created a novel optogenetic construct named OptoCaMP which is a combination of a channelrhodopsin ...variant (CheRiff) and a red genetically encoded calcium indicator (GECI) (jRCaMP1b). It enables simultaneous optical stimulation and recording from large population of neurons with single-cell readout. Additionally, we have developed a spatio-temporal all-optical assay to simultaneously stimulate a sub-section of a neural network and record evoked calcium activity, in both stimulated and non-stimulated neurons, thus allowing the investigation of the spread of excitation through an interconnected network. Finally, we demonstrate the sensitivity of this assay to the change of neural network connectivity.
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.
Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is an ultra-rare autosomal recessive neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. Here, we report ...the generation and characterization of human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of three unrelated SSADHD patients – one female and two males with the CRISPR-corrected isogenic controls. These individuals are clinically diagnosed and are being followed in a longitudinal clinical study.
Succinic semialdehyde dehydrogenase deficiency (SSADHD) represents a model neurometabolic disease at the fulcrum of translational research within the Boston Children's Hospital Intellectual and ...Developmental Disabilities Research Centers (IDDRC), including the NIH-sponsored natural history study of clinical, neurophysiological, neuroimaging, and molecular markers, patient-derived induced pluripotent stem cells (iPSC) characterization, and development of a murine model for tightly regulated, cell-specific gene therapy.
SSADHD subjects underwent clinical evaluations, neuropsychological assessments, biochemical quantification of γ-aminobutyrate (GABA) and related metabolites, electroencephalography (standard and high density), magnetoencephalography, transcranial magnetic stimulation, magnetic resonance imaging and spectroscopy, and genetic tests. This was parallel to laboratory molecular investigations of in vitro GABAergic neurons derived from induced human pluripotent stem cells (hiPSCs) of SSADHD subjects and biochemical analyses performed on a versatile murine model that uses an inducible and reversible rescue strategy allowing on-demand and cell-specific gene therapy.
The 62 SSADHD subjects 53% females, median (IQR) age of 9.6 (5.4-14.5) years included in the study had a reported symptom onset at ∼ 6 months and were diagnosed at a median age of 4 years. Language developmental delays were more prominent than motor. Autism, epilepsy, movement disorders, sleep disturbances, and various psychiatric behaviors constituted the core of the disorder's clinical phenotype. Lower clinical severity scores, indicating worst severity, coincided with older age (R= -0.302, p = 0.03), as well as age-adjusted lower values of plasma γ-aminobutyrate (GABA) (R = 0.337, p = 0.02) and γ-hydroxybutyrate (GHB) (R = 0.360, p = 0.05). While epilepsy and psychiatric behaviors increase in severity with age, communication abilities and motor function tend to improve. iPSCs, which were differentiated into GABAergic neurons, represent the first in vitro neuronal model of SSADHD and express the neuronal marker microtubule-associated protein 2 (MAP2), as well as GABA. GABA-metabolism in induced GABAergic neurons could be reversed using CRISPR correction of the pathogenic variants or mRNA transfection and SSADHD iPSCs were associated with excessive glutamatergic activity and related synaptic excitation.
Findings from the SSADHD Natural History Study converge with iPSC and animal model work focused on a common disorder within our IDDRC, deepening our knowledge of the pathophysiology and longitudinal clinical course of a complex neurodevelopmental disorder. This further enables the identification of biomarkers and changes throughout development that will be essential for upcoming targeted trials of enzyme replacement and gene therapy.
Reprogramming somatic cells from one cell fate to another can generate specific neurons suitable for disease modeling. To maximize the utility of patient-derived neurons, they must model not only ...disease-relevant cell classes, but also the diversity of neuronal subtypes found in vivo and the pathophysiological changes that underlie specific clinical diseases. We identified five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus-detecting (nociceptor) neurons. These recapitulated the expression of quintessential nociceptor-specific functional receptors and channels found in adult mouse nociceptor neurons, as well as native subtype diversity. Moreover, the derived nociceptor neurons exhibited TrpV1 sensitization to the inflammatory mediator prostaglandin E2 and the chemotherapeutic drug oxaliplatin, modeling the inherent mechanisms underlying inflammatory pain hypersensitivity and painful chemotherapy-induced neuropathy. Using fibroblasts from patients with familial dysautonomia (hereditary sensory and autonomic neuropathy type III), we found that the technique was able to reveal previously unknown aspects of human disease phenotypes in vitro.
Optical approaches to fluorescent, spectroscopic, and morphological imaging have made exceptional advances in the last decade. Super-resolution imaging and wide-field multiphoton imaging are now ...underpinning major advances across the biomedical sciences. While the advances have been startling, the key unmet challenge to date in all forms of optical imaging is to penetrate deeper. A number of schemes implement aberration correction or the use of complex photonics to address this need. In contrast, we approach this challenge by implementing a scheme that requires no a priori information about the medium nor its properties. Exploiting temporal focusing and single-pixel detection in our innovative scheme, we obtain wide-field two-photon images through various turbid media including a scattering phantom and tissue reaching a depth of up to seven scattering mean free path lengths. Our results show that it competes favorably with standard point-scanning two-photon imaging, with up to a fivefold improvement in signal-to-background ratio while showing significantly lower photobleaching.
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