The unfolded protein response (UPR) is commonly associated with a range of neurodegenerative diseases, and targeting UPR components has been suggested as a therapeutic strategy. The UPR surveys protein folding within the endoplasmic reticulum. However, many of the misfolded proteins that accumulate in neurodegeneration are localized so that they do not directly cause endoplasmic reticulum triggers that activate this pathway. Here, using a transgenic mouse model and primary cell cultures along with quantitative PCR, immunoblotting, and immunohistochemistry, we tested whether the UPR is induced in in vivo and in vitro murine models of tauopathy that are based on expression of mutant tauP301L. We found no evidence for the UPR in the rTg4510 mouse model, in which mutant tau is transgenically expressed under the control of tetracycline-controlled transactivator protein. This observation was supported by results from acute experiments in which neuronal cultures expressed mutant tau and accumulated misfolded cytoplasmic tau aggregates but exhibited no UPR activation. These results suggest that the UPR is not induced as a response to tau misfolding and aggregation despite clear evidence for progressive cellular dysfunction and degeneration. We propose that caution is needed when evaluating the implied significance of the UPR as a critical determinant across major neurodegenerative diseases.

We report a technologically novel microscopy system for bioimaging based on a 100 fs titanium:sapphire (Ti:Sa) laser pumped coherent continuum from a tailored, 9-cm long, all normal dispersion (ANDi) fiber, enabling concurrent image contrast with (a) spectral focusing coherent anti-Stokes Raman scattering (SF-CARS) (spanning 900-3200 cm −1) and (b) sum frequency generation (SFG). Both modalities were efficiently excited with power levels at the microscope focus compatible with biological samples. Moreover, using the continuum, images were recorded in the back-scattering (epi-detection) geometry, without the necessity for an expensive, computer-controlled, spatial light modulator (SLM), clearly demonstrating the strong signal levels achieved. Image contrast from the multiple modalities provided greater chemical and structural insights than imaging with any single technique in isolation. Numerical simulations supported these developments in regard to both the optimum fiber length for SC generation and the achievement of high spectral resolution in SF-CARS via careful group delay dispersion matching across the pump and Stokes pulses using just an inexpensive sequence of short glass blocks inserted into the Stokes beam. We show bio-images of mouse tissue recorded concurrently via label/stain-free contrast from multiple modalities: CARS, two-photon auto-fluorescence (TPaF) and second harmonic/sum frequency generation (SHG/ SFG). Overall, our approach delivers optimum performance in back-scattered (epi-) detection configuration, suited for thick samples, at reduced complexity and cost. The addition of this simple fiber add-on to lasers already widely used for TPF microscopy can thus extend the capabilities of a significant number of existing microscopy laboratories.

Glutamate receptors of the N-methyl-D-aspartate (NMDA) family are coincident detectors of pre- and postsynaptic activity, allowing Ca 2+ influx into neurons. These properties are central to neurological disease mechanisms and are proposed to be the basis of associative learning and memory. In addition to the well-characterised canonical GluN2A NMDAR isoform, large-scale open reading frames in human tissues had suggested the expression of a primate-specific short GluN2A isoform referred to as GluN2A-S. Here, we confirm the expression of both GluN2A transcripts in human and primate but not rodent brain tissue, and show that they are translated to two corresponding GluN2A proteins present in human brain. Furthermore, we demonstrate that recombinant GluN2A-S co-assembles with the obligatory NMDAR subunit GluN1 to form functional NMDA receptors. These findings suggest a more complex NMDAR repertoire in human brain than previously thought.

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Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl‐prolyl‐tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here, we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild‐type cells, cells lacking GCN2 and those incapable of phosphorylating eIF2α. Proline supplementation rescues the observed HF‐induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2‐to‐eIF2α pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic.