Bradley, D. P. | bioRxiv Science

bioRxiv2024

Development of a novel, non-invasive and whole brain biomarker of demyelination in a mouse model of multiple sclerosis

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), affecting 2.8 million people worldwide, that presents multiple features, one of which is demyelination. Although treatments exist to manage the condition, no cure has been found to stop the progression of neurodegeneration. To develop new treatments and investigate the multiple systems impacted by MS, new imaging technologies are needed at the preclinical stage. Functional ultrasound imaging (fUS) has recently been demonstrated to robustly measure brain cerebral blood volume (CBV) dynamics as an indirect measure of neural activity. This study aimed at proposing a new biomarker of de- and/or re-myelination in a mouse model of MS induced by cuprizone. We demonstrate first that extended demyelination induces an increased hemodynamic response in the primary sensory cortex both spatially and temporally, which is consistent with fMRI data collected on MS patients. Second, using descriptors of the evoked hemodynamic response, we show that 3 of these descriptors allows the prediction of the level of myelin in the primary sensory cortex (p=5. 10-5) and the thalamus (p=6. 10-6). The development of such a non-invasive biomarker is crucial in the MS field as is provides an extremely useful tool for both disease follow-up and drug development. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSMultiple sclerosis (MS) is an autoimmune neurodegenerative disorder of the central nervous system. It is the most common cause of neurological disability in young adults, affecting approximately 2.8 million worldwide. While the field of studies in MS has been very active at identifying the neurobiological cellular and molecular mechanisms underlying MS progression, the number of new treatments has been very limited so far, due to several factors, such as the lack of robust and non-invasive biomarkers of myelin loss in longitudinal studies (measurements during the development of the disease). Unfortunately, quantification of myelin loss, (one of the key neurobiological markers of MS progression) is classically performed post-mortem on fixed tissues, preventing longitudinal studies. Longitudinal follow up of an indirect measure of myelin loss is possible, using magnetic resonance imaging. However, the small size of rodent brains poses a challenge for conventional imaging techniques, requiring the use of high field magnet to achieve the necessary sensitivity and resolution. Added value of this studyIn this study, using a sensitive neuroimaging technique, we developed a simple, non-invasive, predictive biomarker able to quantify the individual amount of myelin content consistently and accurately in brain structures in mice. Implications of all the available evidenceThe development of such a biomarker is extremely important for the MS field as it will accelerate the pre-clinical tests for drug efficacy. The benefits provided by our biomarker encompass: 1) Enhanced sensitivity in individually quantifying myelin content, providing a more comprehensive assessment across diverse brain regions 2) Speeding up the process of the discovery, by reducing the number of animals required per group and 3) It will also likely lead to new scientific outcomes, as many more structures will be studied (most teams and drug compagnies only study the demyelination at the level of the corpus callosum). Finally, from a clinical perspective, given the brain alterations observed in this animal model closely mirror those observed in early stages in patients with MS, we anticipate our biomarker, with minimal additional refinements, to be readily applicable in clinical settings.

neuroscience↗

bioRxiv2022

Predicted structure of the hepatitis B virus polymerase reveals an ancient conserved protein fold

Hepatitis B virus (HBV) replicates by protein-primed reverse transcription. It chronically infects >250 million people, and the dominant anti-HBV drugs are nucleos(t)ide analogs targeting the viral polymerase (P). P has four domains, the terminal protein (TP) that primes DNA synthesis, a spacer, the reverse transcriptase (RT), and the ribonuclease H (RNaseH). Despite being a major drug target and catalyzing a reverse transcription pathway very different from the retroviral pathway, HBV P has resisted structural analysis for decades. Here, we exploited advances in protein structure prediction to model the structure of P. The predicted HBV RT and RNaseH domains aligned to the HIV RT-RNaseH at 3.75 [A] RMSD, had a nucleic acid binding groove spanning the two active sites, had DNA polymerase active site motifs in their expected positions, and accommodated two Mg++ ions in both active sites. Surprisingly, the TP domain wrapped around the RT domain, with the priming tyrosine poised over the RT active site. This model was validated using published mutational analyses, and by docking RT and RNaseH inhibitors, RNA:DNA heteroduplexes, and the HBV {varepsilon} RNA stem-loop that templates DNA priming into the model. The HBV P fold, including the orientation of the TP domain, was conserved among hepadnaviruses from rodents to fish and in P from a fish nackednavirus, but not in other non-retroviral RTs. Therefore, this protein fold has persisted since the hepadnaviruses diverged from nackednaviruses >400 million years ago. This model will guide drug development and mechanistic studies into Ps function. SignificanceThe hepatitis B virus (HBV) polymerase (P) catalyzes an unusual reverse transcription pathway and is a major drug target. However, Ps insolubility and instability have long prevented its structural analyses. This work predicts the structure of P proteins from human to fish viruses, revealing an unanticipated conserved protein fold that is at least 400 million years old. The HBV P model was validated by mechanistically interpreting mutations with strong phenotypes and by computationally docking nucleic acids to P and inhibitors into both of its active sites. The model will advance mechanistic studies into the function of P and enable drug discovery against targets on P other than the reverse transcriptase and ribonuclease H active sites.

microbiology↗