Modeling native and seeded Synuclein aggregation and related cellular dysfunctions in dopaminergic neurons derived by a new set of isogenic iPSC lines with SNCA multiplications.
Angelo IannielliMirko LuoniSerena Gea GiannelliRosangela FereseGabriele OrdazzoMatteo FossatiAndrea RaimondiFelipe OpazoOlga CortiJochen H M PrehnStefano GambardellaRonald MelkiVania BroccoliPublished in: Cell death & disease (2022)
Triplication of the SNCA gene, encoding the protein alpha-Synuclein (αSyn), is a rare cause of aggressive and early-onset parkinsonism. Herein, we generated iPSCs from two siblings with a recently described compact SNCA gene triplication and suffering from severe motor impairments, psychiatric symptoms, and cognitive deterioration. Using CRISPR/Cas9 gene editing, each SNCA copy was inactivated by targeted indel mutations generating a panel of isogenic iPSCs with a decremental number from 4 down to none of functional SNCA gene alleles. We differentiated these iPSC lines in midbrain dopaminergic (DA) neuronal cultures to characterize αSyn aggregation in native and seeded conditions and evaluate its associated cellular dysfunctions. Utilizing a new nanobody-based biosensor combined with super-resolved imaging, we were able to visualize and measure αSyn aggregates in early DA neurons in unstimulated conditions. Calcium dysregulation and mitochondrial alterations were the first pathological signs detectable in early differentiated DA neuronal cultures. Accelerated αSyn aggregation was induced by exposing neurons to structurally well-characterized synthetic αSyn fibrils. 4xSNCA DA neurons showed the highest vulnerability, which was associated with high levels of oxidized DA and amplified by TAX1BP1 gene disruption. Seeded DA neurons developed large αSyn deposits whose morphology and internal constituents resembled Lewy bodies commonly observed in Parkinson's disease (PD) patient brain tissues. These findings provide strong evidence that this isogenic panel of iPSCs with SNCA multiplications offers a remarkable cellular platform to investigate mechanisms of PD and validate candidate inhibitors of native and seeded αSyn aggregation.
Keyphrases
- early onset
- spinal cord
- genome wide
- copy number
- crispr cas
- induced pluripotent stem cells
- genome wide identification
- late onset
- gene expression
- cerebral ischemia
- climate change
- genome editing
- physical activity
- drug induced
- gold nanoparticles
- high throughput
- depressive symptoms
- small molecule
- protein protein
- brain injury
- amino acid
- cancer therapy
- quantum dots
- transcription factor
- mass spectrometry
- blood brain barrier
- drug delivery
- resting state
- binding protein