Brain-derived exosomal hemoglobin transfer contributes to neuronal mitochondrial homeostasis under hypoxia

Reviewer #1 (Public Review):

Summary:

This study investigates the hypoxia rescue mechanisms of neurons by non-neuronal cells in the brain from the perspective of exosomal communication between brain cells. Through multi-omics combined analysis, the authors revealed this phenomenon and logically validated this intercellular rescue mechanism under hypoxic conditions through experiments. The study proposed a novel finding that hemoglobin maintains mitochondrial function, expanding the conventional understanding of hemoglobin. This research is highly innovative, providing new insights for the treatment of hypoxic encephalopathy.

Overall, the manuscript is well organized and written, however, there are some minor/major points that need to be revised before this manuscript is accepted.

Major points:

(1) Hypoxia can induce endothelial cells to release exosomes carrying hemoglobin, however, how neurons are able to actively take up these exosomes? It is possible for other cells to take up these exosomes also? This point needs to be clarified in this study.

(2) The expression of hemoglobin in neurons is important for mitochondrial homeostasis, but its relationship with mitochondrial homeostasis needs to be further elucidated in the study.

Reviewer #2 (Public Review):

Summary:

This is an interesting study with a lot of data. Some of these ideas are intriguing. But a few major points require further consideration.

Major points:

(1) What disease is this model of whole animal hypoxia supposed to mimic? If one is focused on the brain, can one just use a model of focal or global cerebral ischemia?

(2) If this model subjects the entire animal to hypoxia, then other organs will also be hypoxic. Should one also detect endothelial upregulation and release of extracellular vesicles containing hemoglobin mRNA in non-CNS organs? Where do these vesicles go? Into blood?

(3) What other mRNA are contained in the vesicles released from brain endothelial cells?

(4) Where do the endothelial vesicles go? Only to neurons? Or to other cells as well?

(5) Neurons can express endogenous hemoglobin. Is it useful to subject neurons to hypoxia and then see how much the endogenous mRNA goes up? How large is the magnitude of endogenous hemoglobin gene upregulation compared to the hypothesized exogenous mRNA that is supposed to be donated from endothelial vesicles?

(6) Finally, it may be useful to provide more information and data to explain how the expression of this exogenous endothelial-derived hemoglobin binds to neuronal mitochondria to alter function.