Mitochondrial fission and fusion impact numerous cellular functions and neurons are particularly sensitive to perturbations in mitochondrial dynamics. Here we describe that male mice lacking the mitochondrial A-kinase anchoring protein 1 (AKAP1) exhibit increased sensitivity in the transient middle cerebral artery occlusion model of focal ischemia. At the ultrastructural level, AKAP1^−/−mice have smaller mitochondria and increased contacts between mitochondria and the endoplasmic reticulum in the brain. Mechanistically, deletion of AKAP1 dysregulates complex II of the electron transport chain, increases superoxide production, and impairs Ca²⁺homeostasis in neurons subjected to excitotoxic glutamate. Ca²⁺deregulation in neurons lacking AKAP1 can be attributed to loss of inhibitory phosphorylation of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) at the protein kinase A (PKA) site Ser637. Our results indicate that inhibition of Drp1-dependent mitochondrial fission by the outer mitochondrial AKAP1/PKA complex protects neurons from ischemic stroke by maintaining respiratory chain activity, inhibiting superoxide production, and delaying Ca²⁺deregulation. They also provide the first genetic evidence that Drp1 inhibition may be of therapeutic relevance for the treatment of stroke and neurodegeneration.SIGNIFICANCE STATEMENTPrevious work suggests that activation of dynamin-related protein 1 (Drp1) and mitochondrial fission contribute to ischemic injury in the brain. However, the specificity and efficacy of the pharmacological Drp1 inhibitor mdivi-1 that was used has now been discredited by several high-profile studies. Our report is timely and highly impactful because it provides the first evidence that genetic disinhibition of Drp1 via knock-out of the mitochondrial protein kinase A (PKA) scaffold AKAP1 exacerbates stroke injury in mice. Mechanistically, we show that electron transport deficiency, increased superoxide production, and Ca²⁺overload result from genetic disinhibition of Drp1. In summary, our work settles current controversies regarding the role of mitochondrial fission in neuronal injury, provides mechanisms, and suggests that fission inhibitors hold promise as future therapeutic agents.

Obesity genes working together: The biological causes of obesity are not well understood. Sobreira et al. examined the chromatin interactions between key genes in a locus known for its associations with obesity in human patients. In addition to directly interrogating the connections between these genes and examining the mechanisms that regulate their activity, the authors used mouse models to study the target genes' effects on both adipose tissue and brain cells that play a role in regulating dietary preferences. Science , abf1008, this issue p. 1085

Mitochondrial fission catalyzed by dynamin-related protein 1 (Drp1) is necessary for mitochondrial biogenesis and maintenance of healthy mitochondria. However, excessive fission has been associated with multiple neurodegenerative disorders, and we recently reported that mice with smaller mitochondria are sensitized to ischemic stroke injury. Although pharmacological Drp1 inhibition has been put forward as neuroprotective, the specificity and mechanism of the inhibitor used is controversial. Here, we provide genetic evidence that Drp1 inhibition is neuroprotective. Drp1 is activated by dephosphorylation of an inhibitory phosphorylation site, Ser637. We identify Bβ2, a mitochondria-localized protein phosphatase 2A (PP2A) regulatory subunit, as a neuron-specific Drp1 activatorin vivo. Bβ2 KO mice of both sexes display elongated mitochondria in neurons and are protected from cerebral ischemic injury. Functionally, deletion of Bβ2 and maintained Drp1 Ser637 phosphorylation improved mitochondrial respiratory capacity, Ca²⁺homeostasis, and attenuated superoxide production in response to ischemia and excitotoxicityin vitroandex vivo. Last, deletion of Bβ2 rescued excessive stroke damage associated with dephosphorylation of Drp1 S637 and mitochondrial fission. These results indicate that the state of mitochondrial connectivity and PP2A/Bβ2-mediated dephosphorylation of Drp1 play a critical role in determining the severity of cerebral ischemic injury. Therefore, Bβ2 may represent a target for prophylactic neuroprotective therapy in populations at high risk of stroke.SIGNIFICANCE STATEMENTWith recent advances in clinical practice including mechanical thrombectomy up to 24 h after the ischemic event, there is resurgent interest in neuroprotective stroke therapies. In this study, we demonstrate reduced stroke damage in the brain of mice lacking the Bβ2 regulatory subunit of protein phosphatase 2A, which we have shown previously acts as a positive regulator of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). Importantly, we provide evidence that deletion of Bβ2 can rescue excessive ischemic damage in mice lacking the mitochondrial PKA scaffold AKAP1, apparently via opposing effects on Drp1 S637 phosphorylation. These results highlight reversible phosphorylation in bidirectional regulation of Drp1 activity and identify Bβ2 as a potential pharmacological target to protect the brain from stroke injury.

Uncoupling protein 1 (UCP1) is a mitochondrial protein critical for adaptive thermogenesis in adipose tissues, and it is typically believed to be restricted to thermogenic adipose tissues. UCP1-Cre transgenic mice are utilized in numerous studies to provide “brown adipose-specific” conditional gene targeting. Here, we examined the distribution of Cre and UCP1 throughout the body in UCP1-Cre reporter mice. UCP1-Cre mice crossed to Ai14-tdTomato and Ai9-tdTomato reporter mice were used to explore the tissue distribution of Cre recombinase and Ucp1 mRNA in various tissues. UCP1-Cre mice were independently infected with either a Cre-dependent PHP.eB-tdTomato virus or a Cre-dependent AAV-tdTomato virus to determine whether and where UCP1 is actively expressed in the adult central nervous system. In situ analysis of the deposited single cell RNA sequencing data was used to evaluate Ucp1 expression in the hypothalamus. As expected, Ucp1 expression was detected in both brown and inguinal adipose tissues. Ucp1 expression was also detected in the kidney, adrenal glands, thymus, and hypothalamus. Consistent with detectable Ucp1 expression, tdTomato expression was also observed in brown adipose tissue, inguinal white adipose tissue, kidney, adrenal glands, and hypothalamus of both male and female UCP1-Cre; Ai14-tdTomato and UCP1-Cre; Ai9-tdTomato mice by fluorescent imaging and qPCR. Critically, expression of tdTomato, and thus UCP1, within the central nervous system was observed in regions of the brain critical for the regulation of energy homeostasis, including the ventromedial hypothalamus (VMH). TdTomato expression in UCP1-Cre; tdTomato mice is not restricted to thermogenic adipose tissues. TdTomato was also expressed in the kidneys, adrenal glands, and throughout the brain, including brain regions and cell types that are critical for multiple aspects of central regulation of energy homeostasis. Collectively, these data have important implications for the utility of UCP1-Cre mice as genetic tools to investigate gene function specifically in brown adipose tissue.