Pietro De Camilli

This protocol describes the genetic modification of induced pluripotent cells (iPSCs) using CRISPR-Cas9, including synthesis of gRNA plasmids, transfection, selection of clones, and sequencing of genomic DNA to confirm knockout generation. The steps described in this protocol is based on Skarnes et al.(2019) and Fernandopulle et al.(2018).

Synaptojanin-1 (SJ1) is a major neuronal-enriched PI(4, 5)P2 4- and 5-phosphatase implicated in the shedding of endocytic factors during endocytosis. A mutation (R258Q) that impairs selectively its 4-phosphatase activity causes Parkinsonism in humans and neurological defects in mice (SJ1RQKI mice). Studies of these mice showed, besides an abnormal assembly state of endocytic factors at synapses, the presence of dystrophic nerve terminals selectively in a subset of nigro-striatal dopamine (DA)-ergic axons, suggesting a special lability of DA neurons to the impairment of SJ1 function. Here we have further investigated the impact of SJ1 on DA neurons using iPSC-derived SJ1 KO and SJ1RQKI DA neurons and their isogenic controls. In addition to the expected enhanced clustering of endocytic factors in nerve terminals, we observed in both SJ1 mutant neuronal lines increased cilia length. Further analysis of …

From the early stages of my career in Milano (Italy), I had been fascinated by synaptic vesicles, the homogeneously sized small vesicles that store and secrete neurotransmitters at synapses. As a postdoctoral researcher with Paul Greengard at Yale University, I worked at their molecular characterization. In my lab, I became interested in the mechanisms underlying their regeneration by endocytic recycling. Electron microscopy studies had suggested a role for clathrin-mediated budding in this process and genetic studies in Drosophila had implicated the GTPase dynamin in the fission of endocytic buds from the plasma membrane. Building on these studies, we found that dynamin assembles into a ring at the neck of clathrin-coated buds, where, as we and others proved later, it functions as a pinchase. It remained unclear how the function of clathrin coats and dynamin are coupled.Insight into this coupling, with …

This protocol describes the preparation of cell lysate from and iPSC-derived neurons (I 3 Neurons, dopaminergic) and the immunoblotting procedure.

The endoplasmic reticulum (ER) is a continuous cellular endomembrane network that displays focal specializations. Most notable examples of such specializations include the spine apparatus of neuronal dendrites, and the cisternal organelle of axonal initial segments. Both organelles exhibit stacks of smooth ER sheets with a narrow lumen and interconnected by a dense protein matrix. The actin-binding protein synaptopodin is required for their formation. Here, we report that expression in non-neuronal cells of a synaptopodin construct targeted to the ER is sufficient to generate stacked ER cisterns resembling the spine apparatus with molecular properties distinct from the surrounding ER. Cisterns within these stacks are connected to each other by an actin-based matrix that contains proteins also found at the spine apparatus of neuronal spines. These findings reveal a critical role of a synaptopodin-dependent actin matrix in generating cisternal stacks. These ectopically generated structures provide insight into spine apparatus morphogenesis.

This protocol describes the differentiation of iPSCs (WTC11 and KOLF2. 1) to dopaminergic neurons according to Bressan et al 2021.

This protocol describes the maintenance, transfection, immunocytochemistry, and imaging of RPE1 and also transfection, immunocytochemistry, and imaging of iPSCs, i 3 Neurons and DA neurons.

This protocol describes the co-culturing of iPSC-derived dopaminergic (DA) neurons and iPSC-derived medium spiny neurons (MSNs) in a microfluidic compartmentalization device.