Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity.

Mitochondrial dysfunction is implicated in the pathogenesis of multiple neurological diseases, but elucidation of underlying mechanisms is limited experimentally by the inability to damage specific mitochondria in defined neuronal groups. We developed a precision chemoptogenetic approach to target neuronal mitochondria in the intact nervous system in vivo. MG2I, a chemical fluorogen, produces singlet oxygen when bound to the fluorogen-activating protein dL5** and exposed to far-red light. Transgenic zebrafish expressing dL5** within neuronal mitochondria showed dramatic MG2I- and light-dependent neurobehavioral deficits, caused by neuronal bioenergetic crisis and acute neuronal depolarization. These abnormalities resulted from loss of neuronal respiration, associated with mitochondrial fragmentation, swelling and elimination of cristae. Remaining cellular ultrastructure was preserved initially, but cellular pathology downstream of mitochondrial damage eventually culminated in neuronal death. Our work provides powerful new chemoptogenetic tools for investigating mitochondrial homeostasis and pathophysiology and shows a direct relationship between mitochondrial function, neuronal biogenetics and whole-animal behavior.

Most life processes require the energy produced by small cellular compartments called mitochondria. Many internal and external factors can harm these miniature powerhouses, potentially leading to cell death. For instance, in patients with Parkinson's or Alzheimer's disease, dying neurons often show mitochondrial damage. However, it is unclear exactly how injured mitochondria trigger the demise of these cells. Gaining a better understanding of this process requires studying the impact of mitochondrial damage in live neurons, something that is still difficult to do. As a response to this challenge, Xie, Jiao, Bai, Ilin et al. designed a new tool that can specifically injure mitochondria in the neurons of live zebrafish larvae at will, and fine-tune the amount of damage inflicted. The zebrafish are genetically engineered so that the mitochondria in their neurons carry a protein which can bind to a chemical compound called MG2I. When attached to each other, MG2I and the protein respond to far-red light by locally creating highly damaging chemicals. This means that whenever far-red light is shone onto the larvae, mitochondria in their neurons are harmed ­ the brighter the light, the stronger the damage. Zebrafish larvae exposed to these conditions immediately stopped swimming: mitochondria in their neurons could not produce enough energy and these cells could therefore no longer communicate properly. The neurons then started to die about 24 hours after exposure to the light, suggesting that the mitochondrial damage triggered other downstream processes that culminated in cell death. This new light-controlled tool could help to understand the consequences of mitochondrial damage, potentially revealing new ways to rescue impaired neurons in patients with Parkinson's or Alzheimer's disease. In the future, the method could be adapted to work in any type of cell and deactivate other cell compartments, so that it can be used to study many types of diseases.

Chemoptogenetic damage to mitochondria causes rapid telomere dysfunction.

Reactive oxygen species (ROS) play important roles in aging, inflammation, and cancer. Mitochondria are an important source of ROS; however, the spatiotemporal ROS events underlying oxidative cellular damage from dysfunctional mitochondria remain unresolved. To this end, we have developed and validated a chemoptogenetic approach that uses a mitochondrially targeted fluorogen-activating peptide (Mito-FAP) to deliver a photosensitizer MG-2I dye exclusively to this organelle. Light-mediated activation (660 nm) of the Mito-FAP-MG-2I complex led to a rapid loss of mitochondrial respiration, decreased electron transport chain complex activity, and mitochondrial fragmentation. Importantly, one round of singlet oxygen produced a persistent secondary wave of mitochondrial superoxide and hydrogen peroxide lasting for over 48 h after the initial insult. By following ROS intermediates, we were able to detect hydrogen peroxide in the nucleus through ratiometric analysis of the oxidation of nuclear cysteine residues. Despite mitochondrial DNA (mtDNA) damage and nuclear oxidative stress induced by dysfunctional mitochondria, there was a lack of gross nuclear DNA strand breaks and apoptosis. Targeted telomere analysis revealed fragile telomeres and telomere loss as well as 53BP1-positive telomere dysfunction-induced foci (TIFs), indicating that DNA double-strand breaks occurred exclusively in telomeres as a direct consequence of mitochondrial dysfunction. These telomere defects activated ataxia-telangiectasia mutated (ATM)-mediated DNA damage repair signaling. Furthermore, ATM inhibition exacerbated the Mito-FAP-induced mitochondrial dysfunction and sensitized cells to apoptotic cell death. This profound sensitivity of telomeres through hydrogen peroxide induced by dysregulated mitochondria reveals a crucial mechanism of telomere-mitochondria communication underlying the pathophysiological role of mitochondrial ROS in human diseases.

Antibody-Linked Fluorogen-Activating Proteins for Antigen Detection and Cell Ablation.

We demonstrate selective labeling of cell surface proteins using fluorogen-activating proteins (FAPs) conjugated to standard immunoglobulins (IgGs). Conjugation was achieved with a polypeptide reagent comprised of an N-terminal photoactivatable Fc-binding domain and a C-terminal FAP domain. The resulting FAP-antibody conjugates were effective agents for protein detection and cell ablation in cultured mammalian cells and for visualizing cell-cell contacts using a tethered fluorogen assay. Because our approach allows FAP-antibody conjugates to be generated for most currently available IgGs, it should have broad utility for experimental and therapeutic applications.