ATG9 vesicles comprise the seed membrane of mammalian autophagosomes
By savannah onThe ratios of ATG9 and LC3-II at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the expanding autophagosome membrane.
Structure and activation of the human autophagy-initiating ULK1C:PI3KC3-C1 supercomplex
By savannah onThe presence of PI3KC3-C1 induces a rearrangement of ULK1C from a FIP200:ATG13:ULK1 2:1:1 to a 2:2:2 stoichiometry by dislocating an ATG13 loop from an inhibitory site on the dimeric FIP200 scaffold. This suggests a mechanism for the initiation of autophagy through PI3KC3-C1-induced dimerization of ULK1 as bound to FIP200, followed by an activating trans-autophosphorylation of ULK1.
Basal ganglia neurons in healthy and Parkinsonian primates generate recurring sequences of spikes
By savannah onThe authors conclude that basal ganglia neurons fire in recognizable sequences of ISIs, whose incidence is influenced by the induction of parkinsonism.
Single-cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, aging, and disease
By savannah onPublished: The authors used two approaches to ask which genes are expressed during aging in dopaminergic neurons; the cell type affected in Parkinson's disease. View original preprint.
Early deficits in an in vitro striatal microcircuit model carrying the Parkinson’s GBA-N370S mutation
By savannah onPublished: The results highlight the unique utility of modeling striatal neurons in a modular and highly physiological circuit, which is essential to reveal mechanistic insights of the loss of electrical functional integrity in the striata of GBA1 PD patients. View original preprint.
Differentiation of human cortical neurons (CNs) from induced pluripotent stem cells (iPSCs) and their coculture with rat astrocytes
By savannah onThis protocol described the production of human cortical neurons from induced pluripotent stem cells in cocultured with commercially available rat astrocytes.
Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and Arf6 GTPase disrupts the axonal transport of autophagosomes
By savannah onThese findings support a model where a regulatory imbalance between LRRK2-hyperphosphorylated RABs and ARF6 induces an unproductive “tug-of-war” between dynein and kinesin, disrupting processive autophagosome transport. This disruption may contribute to PD pathogenesis by impairing the essential homeostatic functions of axonal autophagy.
Datasets and key resources used in Do, Quyen B. et al. (2023) – Early striatal hyperexcitability in an in vitro human striatal microcircuit model carrying the Parkinson’s GBA-N370S mutation
By savannah onThe results highlight the unique utility of modelling striatal neurons in a modular and highly physiological circuit which is essential to reveal mechanistic insights of the loss of electrical functional integrity in the striata of GBA1 PD patients.
Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
By savannah onThe authors adapted a previously described method (Pantazis et al., 2022) for employing Piggybac transfection to stably express doxycycline-inducible NGN2 in human iPSCs. After stable integration of NGN2, proceed to differentiate iPSCs using protocol “iNeuron differentiation from human iPSCs.”
Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
By savannah onThe authors describe procedure and equipment used for live-imaging of axonal cargoes. This was performed both using primary mouse cortical neurons and human iPSC-derived excitatory glutamatergic neurons. Equipment and software used varied based on laboratory site and scheduled upgrades to microscopy equipment during the course of this study.
Cell lysis and immunoblotting for protein and phospho-protein quantification
By savannah onThe authors describe the procedure by which human iPSC-derived neurons or mouse embryonic fibroblasts (MEFs) were lysed and probed for levels of proteins of interest using Western blot.
CRISPR/Cas9 generation of knock-out iPSCs
By savannah onThis protocol describes RNP-based CRISPR/Cas9 gene-editing to generate knock-out iPSCs. iPSCs are transfected with sgRNA, recombinant Cas9, and GFP using Lipofectamine Stem. After FACS sorting to enrich for successfully transfected cells, individual clones are picked and expanded for further analysis.
Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes
By savannah onThe authors plate, culture, and transfect human iPSC-derived excitatory glutamatergic neurons for the purpose of observing transport of axonal cargoes under spinning disk confocal microscopy.
Immunostaining of iPSC-derived neurons
By savannah onThe authors fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal and axonal abundance of proteins of interest. For preceding culture of neurons, see “Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes.”
iNeuron differentiation from human iPSCs
By savannah onThe authors adapted a previously-described method (Boecker et al., 2020, 2021; Fernandopulle et al., 2018) for differentiating iPSCs stably expressing mNGN2 at a safe-harbor locus into human excitatory glutamatergic neurons. Pre-i 3Neuron iPSCs (human iPSCs with an integrated doxycycline-inducible mNGN2 transgene in the AAVS1 safe-harbor locus) were a gift from M. Ward (National Institutes of Health, Maryland).
Differentiation of human medium spiny neurons (MSNs) from induced pluripotent stem cells (iPSCs)
By savannah onThis protocol generates human medium spiny neurons (MSNs) from induced human pluripotent stem cells (iPSCs).
GFP immunoprecipitation and sample preparation for Tandem Mass Tag (TMT) mass spectrometry analysis
By savannah onA method to identify potential interactors of any Green Fluorescent Protein (GFP) tagged protein expressed in mammalian cells by GFP immunoprecipitation coupled to Tandem Mass Tag (TMT) mass spectrometry analysis. The authors used a GFP-tagged phosphoRab interactor protein (RILPL1-GFP), and its non-binding mutant (RILPL1 -GFP, which cannot interact with phosphorylated Rab proteins) as a control.
Organelle isolation from mouse embryonic fibroblasts (MEFs) stably expressing organelle tags for subsequent immunoblotting or proteomic analysis
By savannah onThis protocol can be adapted to isolate organelles from commonly cultured cells, such as HEK293 and A549 cells, that express an organelle tag.
