Preparing plasmids for nucleofection of hPSCs
By onThis protocol describes the standard procedure for the preparation of plasmids to be delivered into human pluripotent stem cells (hPSCs) using nucleofection.
A possible role for VPS13-family proteins in bulk lipid transfer, membrane expansion and organelle biogenesis
By onAt organelle–organelle contact sites, proteins have long been known to facilitate the rapid movement of lipids. Classically, this lipid transport involves the extraction of single lipids into a hydrophobic pocket on a lipid transport protein. Recently, a new class of lipid transporter has been described with physical characteristics that suggest these proteins are likely to function differently. They possess long hydrophobic tracts that can bind many lipids at once and physically span the entire gulf between membranes at contact sites, suggesting that they may act as bridges to facilitate bulk lipid flow. Here, we review what has been learned regarding the structure and function of this class of lipid transporters, whose best characterized members are VPS13 and ATG2 proteins, and their apparent coordination with other lipid-mobilizing proteins on organelle membranes. We also discuss the prevailing hypothesis in the field, that this type of lipid transport may facilitate membrane expansion through the bulk delivery of lipids, as well as other emerging hypotheses and questions surrounding these novel lipid transport proteins.
Optogenetic experiments with iLID system
By onThis protocol details experiments with the iLID optogenetic system as performed to acutely recruit Miro to mitochondria.
Quantifying the LAMP1 positive puncta
By onThis protocol describes quantifying the LAMP1 positive puncta.
Endosomal escape of RNA therapeutics: How do we solve this rate-limiting problem?
By onWith over 15 FDA approved drugs on the market and numerous ongoing clinical trials, RNA therapeutics, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), have shown great potential to treat human disease. Their mechanism of action is based entirely on the sequence of validated disease-causing genes without the prerequisite knowledge of protein structure, activity or cellular location. In contrast to small molecule therapeutics that passively diffuse across the cell membrane's lipid bilayer, RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and instead are taken up into cells by endocytosis. However, endosomes are also composed of a lipid bilayer barrier that results in endosomal capture and retention of 99% of RNA therapeutics with 1% or less entering the cytoplasm. Although this very low level of endosomal escape has proven sufficient for liver and some CNS disorders, it is insufficient for the vast majority of extra-hepatic diseases. Unfortunately, there are currently no acceptable solutions to the endosomal escape problem. Consequently, before RNA therapeutics can be used to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a nontoxic manner.
Peripheral neuronal activation shapes the microbiome and alters gut physiology
By onThe authors specifically activate ChAT- or TH-expressing gut-associated neurons in mice and perform multi-omics, finding that subsets of peripherally-activated neurons differentially regulate the gut microbiome and host GI physiology.
Neurite_FISH_Quant–Python code for quantification of FISH puncta in neurites
By onThis repository contains Jupyter Notebooks with Python 3 code for quantification of FISH puncta within neuronal dendrites or axons. However, prior identification of FISH puncta in the images (and optional neurite segmentation) is required. We use ImageJ plugins for this purpose, as specified below. The workflow assumes Z-stack, multi-channel fluorescence images, with one channel used for identification of neurites and other channels for FISH.
Nuclear Isolation of Post-Mortem Brain Tissue for snRNAseq
By onThis protocol details how to isolate nuclei from frozen brain tissue for single nuclear RNA sequencing using 10x Genomics GEM isolation using the Chromium accessory and Single Cell 3ʹ Reagent Kits.
Plasmid: V5-KANSL1 pLVX-EF1α-IRES-Puro
By onPlasmid: Human KANSL1 cDNA with N-terminal V5 tag cloned into the pLVX-EF1α-IRES-Puro plasmid (EcoRI and SpeI restriction sites) from Clontech, Takara Bio.
pCAG-OSF-ATG13 (2-197)-K15D
By onPlasmid for expression of human ATG13 HORMA K15D mutant in mammalian cells.
Determination of NM Concentration
By onThis is the protocol for determining neuromelanin concentration and data.
cDNA clones for expression of LysoTag (TMEM192) in mammalian cells
By onPlasmids for expression of TMEM192 (LysoTag) in human and mouse cells.
Immunohistochemistry for Carbon Fiber Thread Electrodes
By onMethods for immunofluorescent staining of brain tissue with indwelling electrodes.
A step forward for LRRK2 inhibitors in Parkinson’s disease
By onIn common with the majority of neurodegenerative diseases, there is an urgent and pressing need for novel disease modifying therapies for Parkinson’s disease (PD). Reporting the results of the first human trial for kinase inhibitors of Leucine Rich Repeat Kinase 2 (LRRK2), Jennings and co-workers presented an important advance along the drug development pathway for a target that has long been a priority for the Parkinson’s research community. The focus discusses several topics including: functional characterisation of LRRK2 and the impact of mutations and a key role for altered kinase function in disease; the human genetics of the LRRK2 locus; the outcome of a first-in-human clinical trial for LRRK2 kinase inhibitors by Denali therapeutics, LRRK2 kinase inhibition as a therapeutic strategy in humans; the strategy of using antisense oligonucleotide knockdown approach and the challenges faced by clinical trials – measuring outcomes in chronic, slowly progressing disorders with variable rates of progression.
pCAG- WIPI2dC93E-cs- TEV-STREP
By onPlasmid: Mammalian expression of human WIPI2d C93E with C-terminal Strep.
Immunofluorescent staining for neuronal marker MAP2
By onThis is the protocol for immunofluorescent staining for neuronal marker MAP2.
