A dominant negative mitofusin causes mitochondrial perinuclear clusters because of aberrant tethering.

In vertebrates, mitochondrial outer membrane fusion is mediated by two mitofusin paralogs, Mfn1 and Mfn2, conserved dynamin superfamily proteins. Here, we characterize a variant of mitofusin reported in patients with CMT2A where a serine is replaced with a proline (Mfn2-S350P and the equivalent in Mfn1, S329P). This serine is in a hinge domain (Hinge 2) that connects the globular GTPase domain to the adjacent extended helical bundle. We find that expression of this variant results in prolific and stable mitochondrial tethering that also blocks mitochondrial fusion by endogenous wild-type mitofusin. The formation of mitochondrial perinuclear clusters by this CMT2A variant requires normal GTPase domain function and formation of a mitofusin complex across two membranes. We propose that conformational dynamics mediated by Hinge 2 and regulated by GTP hydrolysis are disrupted by the substitution of proline at S329/S350 and this prevents progression from tethering to membrane fusion. Thus, our data are consistent with a model for mitofusin-mediated membrane fusion where Hinge 2 supports a power stroke to progress from the tethering complex to membrane fusion.

Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye.

Here we report multiple lines of evidence for a comprehensive model of energy metabolism in the vertebrate eye. Metabolic flux, locations of key enzymes, and our finding that glucose enters mouse and zebrafish retinas mostly through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. We used human retinal epithelial cells to show that lactate can suppress consumption of glucose by the retinal pigment epithelium. Suppression of glucose consumption in the retinal pigment epithelium can increase the amount of glucose that reaches the retina. This framework for understanding metabolic relationships in the vertebrate retina provides new insights into the underlying causes of retinal disease and age-related vision loss.

A tale of two circuits: CCKNTS neuron stimulation controls appetite and induces opposing motivational states by projections to distinct brain regions.

Cholecystokinin (CCK)-expressing neurons within the nucleus of the solitary tract (CCKNTS) of the mouse are responsive to satiety signals and their chemogenetic activation suppresses appetite. Optogenetic activation of CCKNTS axon terminals within either the parabrachial nucleus (PBN) or the paraventricular nucleus of the hypothalamus (PVH) is sufficient to suppress feeding. An interesting dichotomy has been revealed when assessing the motivational valence of these two circuits. Activating CCKNTS cell bodies is aversive as demonstrated by conditioned taste aversion and place-preference assays. Activation of the CCKNTS→PBN pathway is also aversive; however, stimulating the CCKNTS→PVH pathway is appetitive when assayed using a real-time, place-preference task. Thus, these two projections from CCKNTS neurons reduce food intake through opposite motivational states; one pathway signals positive valence (CCKNTS→PVH) and the other signals negative valence (CCKNTS→PBN).

Mitochondria Maintain Distinct Ca2+ Pools in Cone Photoreceptors.

Ca2+ ions have distinct roles in the outer segment, cell body, and synaptic terminal of photoreceptors. We tested the hypothesis that distinct Ca2+ domains are maintained by Ca2+ uptake into mitochondria. Serial block face scanning electron microscopy of zebrafish cones revealed that nearly 100 mitochondria cluster at the apical side of the inner segment, directly below the outer segment. The endoplasmic reticulum surrounds the basal and lateral surfaces of this cluster, but does not reach the apical surface or penetrate into the cluster. Using genetically encoded Ca2+ sensors, we found that mitochondria take up Ca2+ when it accumulates either in the cone cell body or outer segment. Blocking mitochondrial Ca2+ uniporter activity compromises the ability of mitochondria to maintain distinct Ca2+ domains. Together, our findings indicate that mitochondria can modulate subcellular functional specialization in photoreceptors.SIGNIFICANCE STATEMENT Ca2+ homeostasis is essential for the survival and function of retinal photoreceptors. Separate pools of Ca2+ regulate phototransduction in the outer segment, metabolism in the cell body, and neurotransmitter release at the synaptic terminal. We investigated the role of mitochondria in compartmentalization of Ca2+ We found that mitochondria form a dense cluster that acts as a diffusion barrier between the outer segment and cell body. The cluster is surprisingly only partially surrounded by the endoplasmic reticulum, a key mediator of mitochondrial Ca2+ uptake. Blocking the uptake of Ca2+ by mitochondria causes redistribution of Ca2+ throughout the cell. Our results show that mitochondrial Ca2+ uptake in photoreceptors is complex and plays an essential role in normal function.

Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina.

Symbiotic relationships between neurons and glia must adapt to structures, functions, and metabolic roles of the tissues they are in. We show here that Müller glia in retinas have specific enzyme deficiencies that can enhance their ability to synthesize Gln. The metabolic cost of these deficiencies is that they impair the Müller cell's ability to metabolize Glc. We show here that the cells can compensate for this deficiency by using metabolites produced by neurons. Müller glia are deficient for pyruvate kinase (PK) and for aspartate/glutamate carrier 1 (AGC1), a key component of the malate-aspartate shuttle. In contrast, photoreceptor neurons express AGC1 and the M2 isoform of pyruvate kinase, which is commonly associated with aerobic glycolysis in tumors, proliferating cells, and some other cell types. Our findings reveal a previously unidentified type of metabolic relationship between neurons and glia. Müller glia compensate for their unique metabolic adaptations by using lactate and aspartate from neurons as surrogates for their missing PK and AGC1.