Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue

Hijacked NAD+ metabolism during axon degeneration in Drosophila

Axon loss is the earliest shared and detectable feature of nervous systems being challenged in degenerative disorders, by chemotherapy or mechanical forces. Yet intrinsic molecular mechanisms that execute axon degeneration remain largely unknown, making the development of therapeutics to attenuate axon degeneration challenging. Injury-induced axon degeneration (Wallerian degeneration) is a simple and well-established system to study how axons execute their destruction (axon death). After injury, in the axon separated from the soma, a temporal rise of the metabolite NMN occurs, followed by rapid depletion of NAD+, a metabolite crucial for axonal survival. To date, four essential and evolutionary conserved axon death genes dictate levels of NMN and NAD+ in Drosophila: Highwire regulating levels of the NMN-consuming and NAD+-synthesising dNmnat upstream, the NAD+ hydrolase dSarm in the centre, and Axed downstream in this signalling cascade. My Ph.D. thesis aims to gain further insights into i) how NAD+ metabolites modulate dSarm activity and ii) the precise function of Axed during axon death signalling in Drosophila. I could demonstrate in the fly that lowering NMN levels through the expression of a newly generated prokaryotic NMN-Deamidase (NMN-D) preserves severed axons for months and keeps them circuit-integrated for weeks. In contrast, elevated NMN levels, through the expression of mouse nicotinamide phosphoribosyltransferase (mNAMPT), lead to faster axon degeneration after injury. I also show that dSarm mediates axon degeneration through NMN- induced activation, which is regulated by the NMN/NAD+ ratio in vivo. Finally, NM-D delays neurodegeneration caused by the loss of the sole NMN-consuming and NAD+-synthesising enzyme dNmnat. Axed is the most downstream candidate of the signalling pathway. I performed in vivo pulldowns using endogenously tagged Axed (AxedeGFP::3xFLAG) to identify Axed-interacting proteins. They were classified by DAVID analyses in GO terms for biological processes, cellular compartments, and molecular function. The predicted 3D structure of Axed was modelled by implementing several described structural homologues. It will help to decipher the precise mechanistic role of Axed or at least identify additional proteins required for the axon death signalling cascade. Axon death signalling is also activated in the absence of injury, such as in diseased and challenged nervous systems. Thus, understanding the mechanisms underlying axon death signalling could help define therapeutic targets to block axon loss. RESUMÉ La perte d'axones est la première caractéristique partagée et détectable des systèmes nerveux mis à l'épreuve dans les troubles dégénératifs, par la chimiothérapie ou les forces mécaniques. Pourtant, les mécanismes moléculaires intrinsèques qui exécutent la dégénérescence des axones restent largement inconnus, ce qui rend difficile le développement de thérapies pour atténuer la dégénérescence des axones. La dégénérescence des axones induite par une blessure (Wallerian degeneration) est un système simple et bien établi pour étudier comment les axones exécutent leur destruction (mort des axones). Après blessure, dans l'axone séparé du soma, une élévation temporelle du métabolite NMN se produit, suivie d'une déplétion rapide du NAD+, un métabolite crucial pour la survie axonale. À ce jour, quatre gènes essentiels et évolutifs conservés de la mort des axones dictent les niveaux de NMN et de NAD+ chez la Drosophila : les niveaux de régulation élevés du dNmnat consommant du NMN et synthétisant du NAD+ en amont, le dSarm hydrolase de NAD+ au centre et Axed en aval dans cette cascade de signalisation. Ma thèse de doctorat vise à mieux comprendre i) comment les métabolites NAD+ modulent l'activité de dSarm et ii) la fonction précise d'Axed lors de la signalisation de la mort des axones chez la drosophile. J'ai pu démontrer chez la mouche que l'abaissement des niveaux de NMN par l'expression d'une NMN-Deamidase procaryote nouvellement générée (NMN-D) préserve les axones coupés pendant des mois et les maintient intégrés au circuit pendant des semaines. En revanche, des niveaux élevés de NMN, grâce à l'expression de la nicotinamide phosphoribosyltransférase de souris (mNAMPT), conduisent à une dégénérescence axonale plus rapide après une blessure. Je montre également que dSarm intervient dans la dégénérescence des axones par l'activation induite par le NMN, qui est régulée par le rapport NMN/NAD+ in vivo. Enfin, NMN-D retarde la neurodégénérescence causée par la perte de la seule enzyme dNmnat consommatrice de NMN et synthétisant NAD+. Axed est le candidat le plus en aval de la voie de signalisation. J'ai effectué des immunoprécipitations in vivo à l'aide d'Axed marqué de manière endogène (AxedeGFP::3xFLAG) pour identifier les protéines interagissant avec Axed. Ils ont été classés par des analyses DAVID en termes GO pour les processus biologiques, les compartiments cellulaires et la fonction moléculaire. La structure 3D prédite d'Axed a été modélisée en mettant en œuvre plusieurs homologues structurels décrits. Cela aidera à déchiffrer le rôle mécaniste précis d'Axed ou au moins à identifier les protéines supplémentaires nécessaires à la cascade de signalisation de la mort des axones. La signalisation de mort axonale est également activée en l'absence de blessure, comme dans les systèmes nerveux malades et en difficulté. Ainsi, comprendre les mécanismes sous-jacents à la signalisation de la mort des axones pourrait aider à définir des cibles thérapeutiques pour bloquer la perte d'axones

Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue.Team Carlas SmithTeam Michel VerhaegenBN/Kristin Grussmayer LabImPhys/Computational Imagin

The NAD+ precursor NMN activates dSarm to trigger axon degeneration in Drosophila

Axon degeneration contributes to the disruption of neuronal circuit function in diseased and injured nervous systems. Severed axons degenerate following the activation of an evolutionarily conserved signaling pathway, which culminates in the activation of SARM1 in mammals to execute the pathological depletion of the metabolite NAD+. SARM1 NADase activity is activated by the NAD+ precursor nicotinamide mononucleotide (NMN). In mammals, keeping NMN levels low potently preserves axons after injury. However, it remains unclear whether NMN is also a key mediator of axon degeneration and dSarm activation in flies. Here, we demonstrate that lowering NMN levels in Drosophila through the expression of a newly generated prokaryotic NMN-Deamidase (NMN-D) preserves severed axons for months and keeps them circuit-integrated for weeks. NMN-D alters the NAD+ metabolic flux by lowering NMN, while NAD+ remains unchanged in vivo. Increased NMN synthesis, by the expression of mouse nicotinamide phosphoribosyltransferase (mNAMPT), leads to faster axon degeneration after injury. We also show that NMN-induced activation of dSarm mediates axon degeneration in vivo. Finally, NMN-D delays neurodegeneration caused by loss of the sole NMN-consuming and NAD+-synthesizing enzyme dNmnat. Our results reveal a critical role for NMN in neurodegeneration in the fly, which extends beyond axonal injury. The potent neuroprotection by reducing NMN levels is similar to the interference with other essential mediators of axon degeneration in Drosophila

The NAD+ precursor NMN activates dSarm to trigger axon degeneration in Drosophila.

Funder: John and Lucille Van Geest FoundationAxon degeneration contributes to the disruption of neuronal circuit function in diseased and injured nervous systems. Severed axons degenerate following the activation of an evolutionarily conserved signaling pathway, which culminates in the activation of SARM1 in mammals to execute the pathological depletion of the metabolite NAD+. SARM1 NADase activity is activated by the NAD+ precursor nicotinamide mononucleotide (NMN). In mammals, keeping NMN levels low potently preserves axons after injury. However, it remains unclear whether NMN is also a key mediator of axon degeneration and dSarm activation in flies. Here, we demonstrate that lowering NMN levels in Drosophila through the expression of a newly generated prokaryotic NMN-Deamidase (NMN-D) preserves severed axons for months and keeps them circuit-integrated for weeks. NMN-D alters the NAD+ metabolic flux by lowering NMN, while NAD+ remains unchanged in vivo. Increased NMN synthesis by the expression of mouse nicotinamide phosphoribosyltransferase (mNAMPT) leads to faster axon degeneration after injury. We also show that NMN-induced activation of dSarm mediates axon degeneration in vivo. Finally, NMN-D delays neurodegeneration caused by loss of the sole NMN-consuming and NAD+-synthesizing enzyme dNmnat. Our results reveal a critical role for NMN in neurodegeneration in the fly, which extends beyond axonal injury. The potent neuroprotection by reducing NMN levels is similar to the interference with other essential mediators of axon degeneration in Drosophila

Data_Sheet_1_Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains.pdf

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue.</p