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Summary: Bioorthogonal labeling and click chemistry techniques allow the detailed examination of cellular physiology through tagging and visualizing newly synthesized proteins. Here, we describe three methods applying bioorthogonal non-canonical amino acid tagging and fluorescent non-canonical amino acid tagging to quantify protein synthesis in microglia. We describe steps for cell seeding and labeling. We then detail microscopy, flow cytometry, and Western blotting techniques. These methods can be easily adapted for other cell types to explore cellular physiology in health and disease.For complete details on the use and execution of this protocol, please refer to Evans et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Microglia and astrocytes are implicated in aging and age-related diseases. Here, we present a protocol to isolate and culture these glia cells from the murine brain. The protocol consists of two parts: magnetic sorting of adult microglia and mechanical/magnetic sorting of adult microglia and astrocytes. We then describe the characterization of these glial cells by flow cytometry and immunohistochemistry. Microglia isolated from aged mice maintain age-related phenotype during culture. These purified glia cells can be applied in ex vivo studies.

Summary: Neurons are highly dependent on mitochondria, but little is known about how they react to a local mitochondrial oxidative insult. We therefore developed a protocol in primary hippocampal cultures that combines the photosensitizer mito-KillerRed with fluorescent biosensors and photoactivatable GFP. We found in both the soma and dendrites that neurons restrict the local increase in mitochondria-derived reactive oxygen species and the decrease in ATP production to the damaged compartment, by quarantining mitochondria. Although the cytosol of both the soma and dendrites became oxidized after mito-KillerRed activation, dendrites were more sensitive to the oxidative insult. Importantly, the impaired mitochondria exhibited decreased motility and fusion, thereby avoiding the spread of oxidation throughout the neuron. These results establish how neurons manage oxidative damage and increase our understanding about the somatodendritic regulation of mitochondrial functions after a local oxidative insult. : Physiology; Neuroscience; Cell Biology Subject Areas: Physiology, Neuroscience, Cell Biology

Histopathological studies on the brains of tauopathy cases including cases with Alzheimer's disease (AD) demonstrate that neurons with hyperphosphorylated protein tau display granulovacuolar degeneration (GVD), as evidenced by vacuolar lesions harboring a central granule, together with markers of the activated unfolded protein response (UPR). In order to examine whether this hallmark is reproduced in animal models we studied the presence of GVD and the activated UPR in two complementary mouse models, pR5 mice with a tau pathology and APPSLxPS1mut mice with an amyloid plaque pathology. Neither GVD nor a significant activation of the UPR was found in both APPSLxPS1mut mice and in those regions in the pR5 brain where only neurons with an early stage of tau hyperphosphorylation were present. In contrast, those neurons that displayed a tau phospho-epitope signature that only appeared in old pR5 mice and also correlated with Gallyas-positive tangle staining harbored granulovacuolar lesions that were labeled with the GVD markers casein kinases 1δ and 1ε. Granulovacuolar lesions in pR5 mice were also labeled with the UPR markers phosphorylated PKR-like endoplasmic reticulum kinase, phosphorylated inositol-requiring enzyme 1α and phosphorylated eukaryotic initiation factor 2α. However, GVD was rarely observed in neurons bearing mature neurofibrillary tangles as evidenced by Congo red staining. Our results suggest that NFT-formation activates the UPR in pR5 mice and that it is the early stages of neurofibrillary tangle formation that are accompanied by GVD, in line with observations from studies on human autopsy cases.

Amyloid β-peptide (Aβ) containing plaques and neurofibrillary tangles (NFT) are the two major histopathological hallmarks of Alzheimer's disease (AD). According to the amyloid cascade hypothesis, deposition of Aβ is an initial and essential step in the pathogenesis of AD, and formation of NFT has been proposed to be caused by increased Aβ levels. Several previous studies revealed that Aβ plaque formation can be reduced or even prevented by active immunization with Aβ preparations or by administration of Aβ-specific antibodies. To assess the role of fibrillar preparations of Aβ42 in NFT formation, we previously performed intracerebral (i.c.) injections of Aβ42 into brains of NFT-forming P301L tau transgenic mice which caused significant increases in NFT numbers. To determine whether these increases in NFT can be blocked or reduced by active immunization, P301L tau mice were immunized with intraperitoneal injections of preaggregated Aβ42. Aβ42-specific titers were monitored and the mice injected i.c. with Aβ42. We found that i.c. injection of Aβ42 caused significant increases in NFT formation. However, this induction was not affected by active immunization despite high serum anti-Aβ42 titer levels and binding of anti-Aβ42 antibodies to the injected Aβ42 aggregates. We conclude that active immunization is not sufficient to prevent the effect of Aβ42 on tau aggregation in our model system. Further studies are required to determine whether modifications of our protocol could affect the Aβ42-mediated induction of NFT formation.

Neurofibrillary tangles, insoluble protein deposits composed of filamentous tau aggregates, are neuropathological hallmarks of Alzheimer's disease and familial frontotemporal dementia (FTDP-17). Transgenic mice expressing the FTDP-17 mutation P301L of tau recapitulate key features of the human pathology, that is, tau proteins aggregate and neurofibrillary tangles begin to appear in the amygdala at 6 months of age. To detect early signs of tau aggregate-associated changes, we investigated behavioral alterations and cognitive deficits in such mice using an amygdala-specific test battery for anxiety-related and cognitive behavior. P301L mice had anxiety levels not different from wild-types, but their exploratory behavior was significantly increased. Acquisition of a fear response to tone and context as well as taste aversion was comparable to wild-types. However, extinction of a conditioned taste aversion was significantly accelerated. We conclude that already aggregation of tau proteins not yet accompanied by massive formation of neurofibrillary tangles causes selective behavioral deficits.