On the relevance of precision autophagy flux control in vivo – Points of departure for clinical translation

Figures & data

Table 1. Transgenic mouse model systems for in vivo assessment of autophagy activity.

Probe/Model System	Benefit	Notes	Reference
Transgenic GFP-LC3- RFP-LC3∆G mouse model	ATG4-facilitated cleavage generates equimolar GFP-LC3 and RFP-LC3∆G, with RFP-LC3∆G serving as internal control. Ratiometric GFP/RFP signal enables direct flux readout. Flux readout can be ranked, standardization achievable.	Identification of region-specific flux distribution achieved. Kinetic monitoring allows identification and monitoring of steady state flux and dynamic flux changes.	[11]
Neuron-specific murine mRFP-eGFP-LC3 model	Allowing autophagosome and autolysosome assessment.	Visualizing altered neuronal autophagosome/lysosome dynamics. With lysosome marker (here TRGL6), all three pathway intermediate pool sizes quantifiable.	[45]
Cardiac-specific mRFP-GFP-LC3 transgenic mouse	Allowing quantification of myocyte autophagosomal and autolysosomal pool size.	Dissection of neonatal, adult and whole heart autophagy response to ischemia or oxidative stress.	[47]
Intraventricular delivery of monomeric tandem mCherry-GFP-LC3 through adeno- associated viruses, mouse model.	Allowing quantification of autophagosome and autolysosome pool in central and peripheral nervous system.	Spinal chord, cerebellum and peripheral nerve autophagy flux assessment.	[44]
CAG-RFP-eGFP-LC3 transgenic mouse	Autophagosome and autolysosome assessment in primary renal epithelial cells, glomeruli and tubules.	Counterstain with LAMP1 allows detection of lysosome pool.	[46]
Systemic murine LC3- GFP expression	Specific marker for autophagic membranes. Enables monitoring of autophagy activity and autophagic response, discerning differential autophagy induction	Weakly labeling autolysosomes. Could underestimate autophagy activity in high clearing tissue. Direct flux readout achievable with lysosomal inhibition and counterstain.	[37]
Mt-Keima transgenic mouse model	The ratio of dually excited Keima reflects level of mitophagy activity due to Keima inherent protease-resistance.	Detection of in vivo mitophagy flux. Addition of lysosensor probe provides lysosome pool size context.	[48]

Figure 1. In vivo autophagosome flux imaging. In vivo imaging and acquisition of fluorescent signal using light sheet microscopy enables autophagic flux visualization in the living whole organism, identifying regions of high, intermediate and low flux. Flux may be indicated as ratiometric signal, or, if resolution is sufficient, as autophagy pathway intermediates, upon the complete inhibition of autophagosome lysosome fusion using bafilomycin A₁ (Baf).

Figure 2. Revealing the presence of a steady state autophagy flux and kinetics of flux changes in vivo. Autophagosome flux measures can be used to quantify and visualize the organ-specific transition time, i.e. the time required for an organ to clear its complete autophagosomal pool (nA). Note, the change in autophagosome number (indicated by the slope) describes the autophagosome flux (J) in autophagosomes/cell/h.

Table 2. Desirable conditions for measuring autophagy flux in vivo and using light sheet microscopy specifically [52–55].

Desirable Conditions For Autophagy Flux Measurement in vivo	Desirable Outcomes Inherent To Light Sheet Fluorescence Microscopy
Enabling prolonged (several days) time lapse acquisitions	Image acquisition along multiple directions
Enabling large field of view for whole organ or organism acquisitions	Deeper tissue penetration due to low NA objectives
Achieving rapid acquisition of whole volume in living tissue or organism	Illumination-based optical sectioning (decoupling of excitation and emission light pathway)
Achieving single cell resolution, allowing autophagy pathway intermediates (A, AL, L) to be quantified	Gentle acquisition, fluorophore only once exposed to illumination light per volume data set
Enabling dynamic assessment, allowing kinetics of pathway intermediates or ratiometric signal to be monitored	Reduced fluorophore bleaching and phototoxicity
Limiting phototoxicity, avoiding inherent autophagy induction/stress response, even after prolonged monitoring	Improved health and viability of organ/organism
Limiting photobleaching, allowing enhanced accuracy in quantitative analysis of ratio signal or vesicle abundance, even after prolonged monitoring	Enabling 3-dimensional orientation and sample control, use of fiducial markers
Enabling a physiological microenvironment (temperature-, gas and metabolite control)	Benefit of custom design for multimodal imaging, if light path is set up for simultaneous use of two different objectives
Enabling autophagy manipulation by allowing drug/treatment intervention, access to sample chamber	Benefit of geometric flexibility and optical design adaptations for different mounts, cleared tissue, i.e. large brain tissue and alike
Achieving acquisition with limited background noise, enhancing accuracy in vesicle or ratiometric readout analysis	Magnitude of pixels that are recorded in parallel
	Enormous compatibility with fluorescent probes and proteins
	Achieving a high signal to noise ratio

Figure 3. Measuring autophagy induction. (A) How high is high enough? Many autophagy inducers exist and may be drug-, diet- or lifestyle-derived, incrementally (J_induced1 and J_induced2) inducing autophagy above baseline levels (J_basal) upon repeated exposure. Plotting this sequential enhancement of autophagosome flux may indicate the maximal achievable autophagosome flux for the cellular system and the flux that is most favorable for a given therapeutic intervention. (B) How good is the inducer? A most sensitive assessment of autophagy inducers is required that allows concentration-dependent titration of autophagosome flux and selection of the most suitable concentration to achieve a desirable flux induction above reduced or pathological autophagy activity. (C) Matching drug concentration with flux response. What drug concentration is required to achieve, for example, a 10% increase in autophagosome flux? The cartoon is depicting 3 distinct autophagy inducers and the concentration that leads to a 25, 50 and 75% increase in autophagosome flux.

Figure 4. Assessing the steady state. Knowing whether the autophagy system is at steady state (A), reaches a new steady state (B), becomes unstable or in transition (C) or maintains a heightened flux for a given time (D) can be achieved by plotting the autophagosome pool under defined interventions. Likewise, a cellular system may be non-responsive to an autophagy induction, despite having a basal steady state flux or may be responding robustly to an autophagy inducer without establishing a new steady state and steady state flux. Note, the change in autophagosome number (indicated by the slope) describes the autophagosome flux (J) in autophagosomes/cell/h.

Figure 5. Autophagy response dynamics. (A) Tuning autophagosome flux with minimal side effects. A physiological autophagic activity exists (green) that homeostatically tunes cellular metabolism with autophagic activity. However, autophagic flux can be drug-induced or inherently pathologically altered, leading to the manifestation of side effects (e.g. thrombocytopenia in chloroquine treatment). The percent autophagosome flux plotted against the clinical side effect of interest allows the tuning of autophagic flux with desired parameters of health. (B) Dosing and scheduling. Autophagosome flux is tissue specific. Plotting autophagosome flux in PBMCs together with the flux of the tissue of interest (e.g. tumor biopsies) as well as the plasma concentration of the autophagy-modulating drug present at that time, may reveal the desirable autophagy response that is required in the target tissue of interest. PBMCs may serve as a suitable autophagic flux reference, due to their relative accessibility. (C) Dosing and scheduling. Assessing autophagy response dynamics: The change of the total autophagosome pool in the target organ reveals the time when steady state is reached (t₁) and lost, and whether flux is sufficiently (green shaded area) lifted above dysfunctional (red shaded area) levels. Shown here is a single application of 4 different autophagy inducers.

Figure 6. Tissue-specific autophagic activity distribution imagined: a systemic human autophagy flux distribution profile, describing regions of high (red), intermediate (yellow) and low (blue) autophagy flux.

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