Rapid astroglial stress response and elevation of endogenous BiP levels promote neuronal survival in hippocampal slice cultures

A hallmark of neurodegenerative diseases is the accumulation of protein aggregates, the formation of which is prevented by chaperone proteins. BiP is the central chaperone in the endoplasmic reticulum. In this study we investigated the pattern of BiP in tunicamycin-stressed murine organotypic hippocampal slice cultures (OHCs). In stressed OHCs highest apoptotic rates occur in neurons of the CA1 regions and the dentate gyrus, in which we found BiP levels to be lowest. Highest BiP protein levels were found in astrocytes. Cell culture experiments indicated that the stress response of glial cells is faster and stronger than in neuronal cells. We hypothesize that the rapid and pronounced BiP expression in astrocytes helps to maintain the fine-balanced micromilieu necessary for survival of neurons. SubAB is a toxin, which cleaves and inactivates BiP. Low dosages of SubAB did not elicit a specific glial response and apoptosis was not induced in a specific hippocampal subfield. Mild prestressing with SubAB promoted neuronal viability in tunicamycin-treated OHCs. We conclude that preconditioning of hippocampal tissue with stressors that elevate endogenous chaperone levels exert a protective effect thereby promoting neuronal survival. These experiments strengthen the thesis that preconditoning with mild stressors positively affects the survival of neuronal cells.

Development of P301S tau seeded organotypic hippocampal slice cultures to study potential therapeutics

Intracellular tau inclusions are a pathological hallmark of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and other sporadic neurodegenerative tauopathies. Recent in vitro and in vivo studies have shown that tau aggregates may spread to neighbouring cells and functionally connected brain regions, where they can seed further tau aggregation. This process is referred to as tau propagation. Here we describe an ex vivo system using organotypic hippocampal slice cultures (OHCs) which recapitulates aspects of this phenomenon. OHCs are explants of hippocampal tissue which may be maintained in culture for months. They maintain their synaptic connections and multicellular 3D architecture whilst also permitting direct control of the environment and direct access for various analysis types. We inoculated OHCs prepared from P301S mouse pups with brain homogenate from terminally ill P301S mice and then examined the slices for viability and the production and localization of insoluble phosphorylated tau. We show that following seeding, phosphorylated insoluble tau accumulate in a time and concentration dependent manner within OHCs. Furthermore, we show the ability of the conformation dependent anti-tau antibody, MC1, to compromise tau accrual in OHCs, thus showcasing the potential of this therapeutic approach and the utility of OHCs as an ex vivo model system for assessing such therapeutics.

Tau assemblies do not behave like independently acting prion-like particles in mouse neural tissue

A fundamental property of infectious agents is their particulate nature: infectivity arises from independently-acting particles rather than as a result of collective action. Assemblies of the protein tau can exhibit seeding behaviour, potentially underlying the apparent spread of tau aggregation in many neurodegenerative diseases. Here we ask whether tau assemblies share with classical pathogens the characteristic of particulate behaviour. We used organotypic hippocampal slice cultures from P301S tau transgenic mice in order to precisely control the concentration of extracellular tau assemblies in neural tissue. Whilst untreated slices displayed no overt signs of pathology, exposure to recombinant tau assemblies could result in the formation of intraneuronal, hyperphosphorylated tau structures. However, seeding ability of tau assemblies did not titrate in a one-hit manner in neural tissue. The results suggest that seeding behaviour of tau arises at high concentrations, with implications for the interpretation of high-dose intracranial challenge experiments and the possible contribution of seeded aggregation to human disease.

Tau assemblies do not behave like independently acting prion-like particles in mouse neural tissue

A fundamental property of infectious agents is their particulate nature: infectivity arises from independently-acting particles rather than as a result of collective action. Assemblies of the protein tau can exhibit seeding behaviour, potentially underlying the apparent spread of tau aggregation in many neurodegenerative diseases. Here we ask whether tau assemblies share with classical pathogens the characteristic of particulate behaviour. We used organotypic hippocampal slice cultures from P301S tau transgenic mice in order to precisely control the concentration of extracellular tau assemblies. Whilst untreated slices displayed no overt signs of pathology, exposure to tau assemblies could result in the formation of intraneuronal, hyperphosphorylated tau structures. However, seeding ability of tau assemblies did not titrate in a one-hit manner in neural tissue. The results suggest that seeding behaviour of tau only arises at supra-physiological concentrations, with implications for the interpretation of high-dose intracranial challenge experiments and the possible contribution of seeded aggregation to human disease.

Protective Effect of N-Arachidonoyl Glycine-GPR18 Signaling after Excitotoxical Lesion in Murine Organotypic Hippocampal Slice Cultures

N-arachidonoyl glycine (NAGly) is an endocannabinoid involved in the regulation of different immune cells. It was shown to activate the GPR18 receptor, which was postulated to switch macrophages from cytotoxic to reparative. To study GPR18 expression and neuroprotection after NAGly treatment we used excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). The effect of NAGly was also tested in isolated microglia and astrocytes as these cells play a crucial role during neuronal injury. In the present study, the GPR18 receptor was found in OHSC at mRNA level and was downregulated after N-Methyl-D-aspartate (NMDA) treatment at a single time point. Furthermore, treatment with NAGly reduced neuronal damage and this effect was abolished by GPR18 and cannabinoid receptor (CB)2 receptor antagonists. The activation but not motility of primary microglia and astrocytes was influenced when incubated with NAGly. However, NAGly alone reduced the phosphorylation of Akt but no changes in activation of the p44/42 and p38 MAPK and CREB pathways in BV2 cells could be observed. Given NAGly mediated actions we speculate that GPR18 and its ligand NAGly are modulators of glial and neuronal cells during neuronal damage.