Bioorthogonal labeling of live dissociated and organotypic slice culture neurons

Over the past couple decades, the explosion in the development of high-resolution and super-resolution microscopy techniques has led to the need for the development of new protein labeling techniques. Click-labeling via genetic code expansion (GCE) has received particular attention given its potential has the ultimately small labelling probe for proteins. Click-labeling via GCE offers a reliable and sterically minimally demanding capacity to label proteins, but its application in non dividing cells such as neurons remains poorly exploited due to its low efficiency. Here, we describe a simple, efficient and reproducible protocol that allows to fluorescently label transmembrane proteins in live neurons using click-labeling via GCE, both in dissociated culture and organotypic brain slices.

Tau assemblies enter the cytosol of neurons in a cholesterol sensitive manner

The microtubule associated protein tau forms filamentous assemblies in the cytosol of neurons in Alzheimer's disease and other neurodegenerative diseases. Assemblies of tau have been proposed to transit between cells of the brain in a 'prion-like' manner, resulting in templated aggregation of native tau in recipient neurons. Interactions between tau assemblies, surface receptor LRP1 and heparan sulphate proteoglycans promote the uptake of tau assemblies to membrane-bound vesicles. A subsequent escape from these vesicles is postulated for assemblies to enter the cytosol and contact cytosolic tau pools. However, the process by which tau assemblies enter the cytosol is poorly defined. Here we establish assays that permit the study of tau entry in real time and at physiological concentrations. Modulation of entry by genetic or pharmacologic means alters levels of seeded aggregation, confirming the role of cytosolic entry as the rate-limiting, upstream step in seeded aggregation. Entry to HEK293, a commonly used reporter cell line, depended on clathrin-mediated pathways with late endosomal Rab7 GTPase involvement. In contrast, entry to primary neurons was via a clathrin- and dynamin-independent route that was sensitive to cholesterol levels. Extraction of cholesterol from neurons increased tau entry to the cytosol, consistent with cholesterol's established role in maintaining vesicle stability. Importantly, reducing cholesterol levels increased seeded aggregation in both primary neurons and organotypic slice culture models of tau pathology. Finally, we find no evidence that tau assemblies mediate their own entry to the cytosol by membrane rupture. Our results establish cytosolic entry as a distinct event from uptake and is upstream and essential to seeded aggregation. They further describe a cholesterol-sensitive, clathrin-independent pathway of tau entry to the cytosol of neurons.

Neurogenic and Neuroprotective Potential of Stem/Stromal Cells Derived from Adipose Tissue

Currently, the number of stem-cell based experimental therapies in neurological injuries and neurodegenerative disorders has been massively increasing. Despite the fact that we still have not obtained strong evidence of mesenchymal stem/stromal cells’ neurogenic effectiveness in vivo, research may need to focus on more appropriate sources that result in more therapeutically promising cell populations. In this study, we used dedifferentiated fat cells (DFAT) that are proven to demonstrate more pluripotent abilities in comparison with standard adipose stromal cells (ASCs). We used the ceiling culture method to establish DFAT cells and to optimize culture conditions with the use of a physioxic environment (5% O2). We also performed neural differentiation tests and assessed the neurogenic and neuroprotective capability of both DFAT cells and ASCs. Our results show that DFAT cells may have a better ability to differentiate into oligodendrocytes, astrocytes, and neuron-like cells, both in culture supplemented with N21 and in co-culture with oxygen–glucose-deprived (OGD) hippocampal organotypic slice culture (OHC) in comparison with ASCs. Results also show that DFAT cells have a different secretory profile than ASCs after contact with injured tissue. In conclusion, DFAT cells constitute a distinct subpopulation and may be an alternative source in cell therapy for the treatment of nervous system disorders.

TMOD-26. ESTABLISHING A PATIENT-DERIVED, IN-VITRO ORGANOTYPIC SLICE CULTURE MODEL OF GBM

Glioblastoma Multiforme (GBM) is the most common primary malignant brain tumour. This tumour is universally fatal with a median survival of 15 months. Driving this pathology is an extremely heterogeneic tumour and complex tumour microenvironment. GBM research is primarily conducted using immortalized or primary cell lines due to their practicality and reproducibility. However, these cell lines do not effectively recapitulate the tumour microenvironment. Mouse models address these shortcomings but are laborious and expensive. We propose to utilize a patient derived organotypic culture model of GBM as an intermediary. We have utilized this model to test genetic manipulation via lentiviral transduction and the feasibility of utilizing this model to understand patient derived extracellular vesicles (EVs). We have sectioned and cultured patient derived organotypic models for 14 days without loss of viability. To determine if these organotypic cultures are amenable to lentiviral manipulation, tissue sections were transduced with far-red fluorescent lentivirus and efficiency determined by confocal laser scanning microscopy (CLSM) and flow cytometry (FC). To determine feasibility as a model for EVs, media obtained from patient-derived organotypic cultures was analyzed by western blot, nanoparticle tracking analysis (NTA), and nanoFlow Cytometry (nFC). In the future these EVs will be compared to those found in patient serum. The model of GBM has been lentivirally transduced to express a far-red fluorescent vector in approximately 15% of the slice, quantified by CLSM and FC. EV-sized particles positive for canonical EV markers have been identified in the media by NTA, nFC and western blot. Using lentiviral-mediated genetic engineering and emerging EV science, this organotypic slice culture models yields exciting utility in GBM research. The established organotypic slice culture model will likely be a valuable tool in the study of GBM biology and EV dynamics.

POSCAbilities: The Application of the Prion Organotypic Slice Culture Assay to Neurodegenerative Disease Research

Prion diseases are fatal, transmissible neurodegenerative disorders whose pathogenesis is driven by the misfolding, self-templating and cell-to-cell spread of the prion protein. Other neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and Huntington’s disease, share some of these prion-like features, with different aggregation-prone proteins. Consequently, researchers have begun to apply prion-specific techniques, like the prion organotypic slice culture assay (POSCA), to these disorders. In this review we explore the ways in which the prion phenomenon has been used in organotypic cultures to study neurodegenerative diseases from the perspective of protein aggregation and spreading, strain propagation, the role of glia in pathogenesis, and efficacy of drug treatments. We also present an overview of the advantages and disadvantages of this culture system compared to in vivo and in vitro models and provide suggestions for new directions.