The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance and splicing alterations during the course of disease in prion-inoculated mice. Prion infection induced PrP-dependent transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical signs, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, could be the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed signs in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets.

2The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance, splicing and editing alterations during the course of disease in prion-inoculated mice. Prion infection induced transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical symptoms, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, represents the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed symptoms in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets. MainAfter the onset of clinical signs, prion diseases typically progress very rapidly to a terminal stage, which is characterized by micro-and astrogliosis, vacuolation and neuronal loss. Sporadic Creutzfeldt-Jakob disease (CJD), the most common human prion disease, can lead to death within months of symptom onset 1 .Conversely, prion infections are often characterized by very long incubation times: iatrogenic cases of CJD after administration of prion-contaminated growth hormone display incubation times of >20 years 2 and Kuru, an acquired form of prion disease, was reported to arise decades after consumption of contaminated materials 3 . The extraordinary duration of the latency phase has led to the presumption that CJD may be caused by a "slow virus". Although this hypothesis was dismissed 4 , prion pathogenesis is initially insidious and only acquires a rapid rate of progression in the late stages of disease.The seemingly incongruous combination of a very long latency and a rapidly progressing clinical disease can be reproduced in mouse models of prion infection 5 . After inoculation of prion-containing brain homogenate, wild-type mice experience an incubation period of several months followed by rapidly progressive neurological dysfunction. It was suggested that prion replication occurs without causing any neuronal damage until a plateau level of infectious particles is reached, whereas neurotoxicity arises because of a toxic form of PrP named "PrP L " 6-8 . However, no physical evidence for the existence of "PrP L " has ever come forth, and these experimental observations can be explained by alternative models. For example, small numbers of prion seeds may cause early molecular alterations that elicit late-onset clinical signs. This question may be addressed by File 3). Consistent with the hierarchical clustering and the PCA, 813 genes changed at 8 wpi and the number of DEGs gradually increased during the later timepoin...

While the initial pathology of Parkinson's disease and other αsynucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of αsynuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analysed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α-synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α-synuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion, and the seeded induction of αsynuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α-synucleinopathies is not universally valid.

Prions consist of pathological assemblies of normal cellular prion protein and cause infectious neurodegenerative diseases, a phenomenon mirrored in many other prion-like neurodegenerative diseases. However, despite their key importance in disease, the individual processes governing this formation of pathogenic aggregates, as well as their rates, have remained challenging to elucidate in vivo. Here we bring together a mathematical framework with kinetics of the accumulation of prions in mice and microfluidic mea-

Background The persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). Methods We investigated HIV-1 promoter activity after integration into specific sites in BACH2 in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean–/mCherry+) HIV-1 promoters were characterised. Results Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Conclusion Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity.