Molecular pathology, developmental changes and synaptic dysfunction in (pre-) symptomatic human C9ORF72-ALS/FTD cerebral organoids.

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD, even before symptom onset. Because these early disease phenotypes remain incompletely understood, we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients, presymptomatic C9ORF72-HRE (C9-HRE) carriers, and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition, single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids, including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further, molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly, organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology, whereas the extent to which more downstream cellular defects, as found in C9-ALS/FTD models, were detected varied for the different presymptomatic C9-HRE cases. Together, these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies.

ATAXIN-2 intermediate-length polyglutamine expansions elicit ALS-associated metabolic and immune phenotypes.

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules, neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43Tg-ALS mice, ATXN2-Q33 causes reduced motor function, NMJ alterations, neuron degeneration and altered in vitro stress granule dynamics. Furthermore, gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together, these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy.

Disturbed retinoid metabolism upon loss of rlbp1a impairs cone function and leads to subretinal lipid deposits and photoreceptor degeneration in the zebrafish retina.

The RLBP1 gene encodes the 36 kDa cellular retinaldehyde-binding protein, CRALBP, a soluble retinoid carrier, in the visual cycle of the eyes. Mutations in RLBP1 are associated with recessively inherited clinical phenotypes, including Bothnia dystrophy, retinitis pigmentosa, retinitis punctata albescens, fundus albipunctatus, and Newfoundland rod-cone dystrophy. However, the etiology of these retinal disorders is not well understood. Here, we generated homologous zebrafish models to bridge this knowledge gap. Duplication of the rlbp1 gene in zebrafish and cell-specific expression of the paralogs rlbp1a in the retinal pigment epithelium and rlbp1b in Müller glial cells allowed us to create intrinsically cell type-specific knockout fish lines. Using rlbp1a and rlbp1b single and double mutants, we investigated the pathological effects on visual function. Our analyses revealed that rlbp1a was essential for cone photoreceptor function and chromophore metabolism in the fish eyes. rlbp1a-mutant fish displayed reduced chromophore levels and attenuated cone photoreceptor responses to light stimuli. They accumulated 11-cis and all-trans-retinyl esters which displayed as enlarged lipid droplets in the RPE reminiscent of the subretinal yellow-white lesions in patients with RLBP1 mutations. During aging, these fish developed retinal thinning and cone and rod photoreceptor dystrophy. In contrast, rlbp1b mutants did not display impaired vision. The double mutant essentially replicated the phenotype of the rlbp1a single mutant. Together, our study showed that the rlbp1a zebrafish mutant recapitulated many features of human blinding diseases caused by RLBP1 mutations and provided novel insights into the pathways for chromophore regeneration of cone photoreceptors.

A New Zebrafish Model for CACNA2D4-Dysfunction.

Purpose: Mutations in CACNA2D4, encoding the α2δ4 subunit of retinal voltage-gated calcium channels (Cav), cause a rare type of retinal dysfunction in human, mainly affecting cone vision. Here, we investigate the role of CACNA2D4 in targeting of Cav, its influence on cone-mediated signal transmission, and the cellular and subcellular changes upon loss of α2δ4 by exploiting the advantages of the cone-dominant zebrafish as model system. Methods: We identified two zebrafish CACNA2D4 paralogs (cacna2d4a and cacna2d4b), analyzed their expression by RNA in situ hybridization and introduced truncating frameshift mutations through CRISPR/Cas9-mediated mutagenesis. We analyzed retinal function and morphology of the single and double mutant lines by electroretinography, immunohistochemistry, light- and electron microscopy. Results: Knockout of cacna2d4b reduces the expression of Cacna1fa, the pore-forming subunit of retinal Cav1.4, whereas loss of cacna2d4a did not. Only knockout of both paralogs impaired cone-mediated ERG b-wave amplitude. The number of "floating" ribbons is increased in double-KO, while retinal morphology and expression of postsynaptic mGluR6b remain largely unaffected. Both Cacna1fa and Ribeyeb show ectopic punctate expression in cacna2d4b-KO and double-KO photoreceptors. Conclusions: We find that increasing the expression of Cav at the synaptic membrane is an evolutionarily conserved function of Cacna2d4b. Yet, since both paralogs participate in cone synaptic transmission, we propose partial subfunctionalization in zebrafish. Similar to human patients, our double KO zebrafish model shows mild cone dysfunction, which was not associated with signs of retinal degeneration. Therefore, cacna2d4-KO zebrafish is a suitable model to study the pathophysiological mechanisms underlying CACNA2D4 dysfunction in human.