In vivo brain imaging of mitochondrial Ca2+ in neurodegenerative diseases with multiphoton microscopy

Abstract

Mitochondria are involved in a large number of essential roles related to neuronal function. Ca²⁺ handling by mitochondria is critical for many of these functions, including energy production and cellular fate. Conversely, mitochondrial Ca²⁺ mishandling has been related to a variety of neurodegenerative diseases. Investigating mitochondrial Ca²⁺ dynamics is essential for advancing our understanding of the role of intracellular mitochondrial Ca²⁺ signals in physiology and pathology. Improved Ca²⁺ indicators, and the ability to target them to different cells and compartments, have emerged as useful tools for analysis of Ca²⁺ signals in living organisms. Combined with state-of-the-art techniques such as multiphoton microscopy, they allow for the study of mitochondrial Ca²⁺ dynamics in vivo in mouse models of the disease. Here, we provide an overview of the Ca²⁺ transporters/ion channels in mitochondrial membranes, and the involvement of mitochondrial Ca²⁺ in neurodegenerative diseases followed by a summary of the main tools available to evaluate mitochondrial Ca²⁺ dynamics in vivo using the aforementioned technique.

Keywords: Alzheimer's disease; Calcium; Fluorescent proteins; GECIs; Mitochondria; Multiphoton microscopy.

Fig. 1.

Main components of the mitochondrial Ca²⁺ homeostasis in neurons The main components of Ca²⁺ regulation in the cell are part of the plasma membrane (VGCCs and ROCs among other channels and pumps), endoplasmic reticulum, and mitochondria. In the mitochondria, the MCU complex is the part of the main Ca²⁺ influx pathway. VDAC contributes to this movement by allowing the flow of ions through the OMM. Ca²⁺ efflux is predominantly regulated by the NCLX and the H⁺/Ca²⁺ exchangers. In addition to this, the mPTP can also vent Ca²⁺ ions out of the cell. Mitochondria-ER communication is moderated through the MAMs (comprised of the MCU complex, VDAC, and IP₃R among other proteins and tethers), and allow the flow of Ca²⁺ ions into the mitochondria from the ER for further regulation. In the ER, Ca²⁺ release is regulated through the RyR and IP₃R pathways, while SERCA is involved in its extrusion from the cytosol. Abbreviations: ΔΨm (mitochondrial membrane potential), ER (endoplasmic reticulum), ETC (electron transport chain), GRP75 (glucose-regulated protein 75), H⁺/Ca²⁺ (H⁺/Ca²⁺ exchanger), IMM (inner mitochondrial membrane), IP₃R (inositol trisphosphate receptor), MAM (mitochondrial associated membranes), MCU (mitochondrial Ca²⁺ uniporter), MICU (mitochondrial Ca²⁺ uptake proteins), (mtDNA (mitochondrial DNA), NCLX (Na⁺/Ca²⁺ Li⁺ permeable exchanger), mPTP (mitochondrial permeability transition pore), ROCs (receptor-operated Ca²⁺ channels), RyR (ryanodine receptor), SERCA (sarco/endoplasmic reticulum Ca²⁺-ATPase), TCA (tricarboxylic acid), VDAC (voltage dependent anion channel), VGCCs (voltage gated Ca²⁺ channels).

Figure 2.. Schematic of the study of mitochondrial function in vivo

The use of fluorophores in conjunction with in vivo imaging to evaluate intracellular function can be summarize as such: (1) a fluorescent indicator is injected into the cortex of the mouse model, (2) and after 3 weeks viral expression should be sufficient for imaging. (3) At this point a cranial widow is surgically implanted and (4) in vivo imaging with multiphoton microscopy may commence. Left image represents a cranial window in the mouse skull. Field of view shows the reporter in neuronal mitochondria (green) and the blood vessels labeled with fluorescent Dextran Texas Red (red). Pseudocolor images represent the color coded (according to the lower bar) mitochondrial Ca²⁺ concentrations for the corresponding neurites. Warm colors represent high Ca²⁺, whereas blue colors represent low Ca²⁺ concentration. Scale bar represents 15 μm.

Figure 3.. In vivo multiphoton microscopy images of mitochondrial Ca²⁺ in neurons in Wt and APP/PS1 mice.

APP/PS1 transgenic (Tg) mice and wild-type (Wt) mice were injected with the mitochondrial Ca²⁺ reporter AAV.hSyn.2mtYC3.6 and a cranial window was implanted. Mitochondrial Ca²⁺ was assessed with multiphoton microscopy. Pictures represent Wt (top) and APP/PS1 Tg mice (bottom). Field of view shows the reporter in neuronal mitochondria (green), the blood vessels labeled with fluorescent Dextran Texas Red (red) and amyloid plaques labeled with the dye HS169 (red, [198]). Pseudocolor images represent the color coded (according to the lower bar) mitochondrial Ca²⁺ concentrations for the corresponding neurons. Warm colors represent high Ca²⁺, whereas blue colors represent low Ca²⁺ concentration. Scale bar represents 15 μm.