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During brain development, synapses are initially formed in excess and are later eliminated in an activity-dependent manner. Weak synapses are preferentially removed, but the mechanism linking neuronal activity to synapse removal is unclear. Here, we show that, in the developing mouse visual pathway, inhibiting synaptic transmission induces postsynaptic activation of caspase-3. Caspase-3 deficiency results in defects in synapse elimination driven by both spontaneous and experience-dependent neural activity. Notably, caspase-3 deficiency blocks activity-dependent synapse elimination, as evidenced by reduced engulfment of inactive synapses by microglia. Furthermore, in a mouse model of Alzheimer’s disease, caspase-3 deficiency protects against synapse loss induced by amyloid-β deposition. Our results reveal caspase-3 activation as a key step in activity-dependent synapse elimination during development and synapse loss in neurodegeneration.

Photosynthesis occurs in thylakoids, a highly specialized membrane system. In the cyanobacterium Synechocystis sp PCC 6803 (hereafter Synechocystis 6803), the thylakoids are arranged parallel to the plasma membrane and occasionally converge toward it to form biogenesis centers. The initial steps in PSII assembly are thought to take place in these regions, which contain a membrane subcompartment harboring the early assembly factor PratA and are referred to as PratA-defined membranes (PDMs). Loss of CurT, the Synechocystis 6803 homolog of Arabidopsis thaliana grana-shaping proteins of the CURVATURE THYLAKOID1 family, results in disrupted thylakoid organization and the absence of biogenesis centers. As a consequence, PSII is less efficiently assembled and accumulates to only 50% of wild-type levels. CurT induces membrane curvature in vitro and is distributed all over the thylakoids, with local concentrations at biogenesis centers. There it forms a sophisticated tubular network at the cell periphery, as revealed by live-cell imaging. CurT is part of several high molecular mass complexes, and Blue Native/SDS-PAGE and isoelectric focusing demonstrated that different isoforms associate with PDMs and thylakoids. Moreover, CurT deficiency enhances sensitivity to osmotic stress, adding a level of complexity to CurT function. We propose that CurT is crucial for the differentiation of membrane architecture, including the formation of PSII-related biogenesis centers, in Synechocystis 6803.

The marine cyanobacterium Synechococcus is the second most abundant phytoplanktonic organism in the world's oceans. The ubiquity of this genus is in large part due to its use of a diverse set of photosynthetic light-harvesting pigments called phycobiliproteins, which allow it to efficiently exploit a wide range of light colors. Here we uncover a pivotal molecular mechanism underpinning a widespread response among marine Synechococcus cells known as “type IV chromatic acclimation” (CA4). During this process, the pigmentation of the two main phycobiliproteins of this organism, phycoerythrins I and II, is reversibly modified to match changes in the ambient light color so as to maximize photon capture for photosynthesis. CA4 involves the replacement of three molecules of the green light-absorbing chromophore phycoerythrobilin with an equivalent number of the blue light-absorbing chromophore phycourobilin when cells are shifted from green to blue light, and the reverse after a shift from blue to green light. We have identified and characterized MpeZ, an enzyme critical for CA4 in marine Synechococcus . MpeZ attaches phycoerythrobilin to cysteine-83 of the α-subunit of phycoerythrin II and isomerizes it to phycourobilin. mpeZ RNA is six times more abundant in blue light, suggesting that its proper regulation is critical for CA4. Furthermore, mpeZ mutants fail to normally acclimate in blue light. These findings provide insights into the molecular mechanisms controlling an ecologically important photosynthetic process and identify a unique class of phycoerythrin lyase/isomerases, which will further expand the already widespread use of phycoerythrin in biotechnology and cell biology applications.

Light-harvesting antennae are critical for collecting energy from sunlight and providing it to photosynthetic reaction centers. Their abundance and composition are tightly regulated to maintain efficient photosynthesis in changing light conditions. Many cyanobacteria alter their light-harvesting antennae in response to changes in ambient light-color conditions through the process of chromatic acclimation. The control of green light induction (Cgi) pathway is a light-color-sensing system that controls the expression of photosynthetic genes during chromatic acclimation, and while some evidence suggests that it operates via transcription attenuation, the components of this pathway have not been identified. We provide evidence that translation initiation factor 3 (IF3), an essential component of the prokaryotic translation initiation machinery that binds the 30S subunit and blocks premature association with the 50S subunit, is part of the control of green light induction pathway. Light regulation of gene expression has not been previously described for any translation initiation factor. Surprisingly, deletion of the IF3-encoding gene infCa was not lethal in the filamentous cyanobacterium Fremyella diplosiphon , and its genome was found to contain a second, redundant, highly divergent infC gene which, when deleted, had no effect on photosynthetic gene expression. Either gene could complement an Escherichia coli infC mutant and thus both encode bona fide IF3s. Analysis of prokaryotic and eukaryotic genome databases established that multiple infC genes are present in the genomes of diverse groups of bacteria and land plants, most of which do not undergo chromatic acclimation. This suggests that IF3 may have repeatedly evolved important roles in the regulation of gene expression in both prokaryotes and eukaryotes.

During brain development, synapses are initially formed in excess and are later eliminated in an activity-dependent manner. Weak synapses are preferentially removed, but the mechanism linking neuronal activity to synapse removal is unclear. Here, we show that, in the developing mouse visual pathway, inhibiting synaptic transmission induces postsynaptic activation of caspase-3. Caspase-3 deficiency results in defects in synapse elimination driven by both spontaneous and experience-dependent neural activity. Notably, caspase-3 deficiency blocks activity-dependent synapse elimination, as evidenced by reduced engulfment of inactive synapses by microglia. Furthermore, in a mouse model of Alzheimer’s disease, caspase-3 deficiency protects against synapse loss induced by amyloid-β deposition. Our results reveal caspase-3 activation as a key step in activity-dependent synapse elimination during development and synapse loss in neurodegeneration.

My dissertation research addresses the signal transduction mechanisms through which the cyanobacterium Fremyella diplosiphon regulates its photosynthetic pigments called phycobiliproteins in response to light quality and nutrient levels. Red light induces production of phycocyanin while green light induces phycoerythrin. This light color induced differential accumulation of phycobiliproteins allows the cells to maximize photosynthesis and growth rate. In red light the induction of phycocyanin genes is controlled by the phytochrome-based Rca system, which also partially represses the phycoerythrin genes. Further repression of phycoerythrin genes in red light is performed by a second pathway, called the Cgi system. To identify components of this pathway, I performed transposon mutagenesis and found several genes required for proper regulation of phycoerythrin. One encodes a translation initiation factor 3 (IF3). F. diplosiphon has two IF3 encoding genes and both appear to be dispensable. This raises the possibility that in cyanobacteria, core components of the protein synthesis process were duplicated and recruited for regulatory purposes. The phycobiliproteins are also regulated by the availability of nutrients in their environments. When F. diplosiphon experiences low sulfate levels, it drastically down-regulates two phycocyanins known as PC1 and PC2 and expresses another form of phycocyanin (PC3), which has almost no sulfur containing amino acids. Using gene knockouts and gene expression assays I found that a non-coding portion of the RNA encoding PC3 destabilizes the RNA produced by the PC1 and PC2. This is a novel form of regulation through which the cells use the non-coding portion of a structural RNA to repress the expression of functionally related genes.

During brain development, synapses are initially formed in excess and are later eliminated in an activity-dependent manner. Weak synapses are preferentially removed, but the mechanism linking neuronal activity to synapse removal is unclear. Here we show that, in the developing mouse visual pathway, inhibiting synaptic transmission induces postsynaptic activation of caspase-3. Caspase-3 deficiency results in defects in synapse elimination driven by both spontaneous and experience-dependent neural activity. Notably, caspase-3 deficiency blocks activity-dependent synapse elimination, as evidenced by reduced engulfment of inactive synapses by microglia. Furthermore, in a mouse model of Alzheimer's disease, caspase-3 deficiency protects against synapse loss induced by amyloid-beta deposition. Our results reveal caspase-3 activation as a key step in activity-dependent synapse elimination during development and synapse loss in neurodegeneration.Competing Interest StatementThe authors have declared no competing interest.Footnotes* We have revised our abstract and title to highlight the causal link between synapse weakening/inactivation and caspase-3 activation. We have also modified our language about specificity of elimination by microglia to reflect that engulfment measurements are only a readout of synapse elimination. Finally, we have added two more supporting datasets to our figures: mEPSC measurements (Figure 4), and dLGN neuron density in wild type and caspase-3 KO animals (Figure S7).

Vipp1 is highly conserved and essential for photosynthesis, but its function is unclear as it does not participate directly in light-dependent reactions. We analyzed Vipp1 localization in live cyanobacterial cells and show that Vipp1 is highly dynamic, continuously exchanging between a diffuse fraction that is uniformly distributed throughout the cell and a punctate fraction that is concentrated at high curvature regions of the thylakoid located at the cell periphery. Experimentally perturbing the spatial distribution of Vipp1 by relocalizing it to the nucleoid causes a severe growth defect during the transition from non-photosynthetic (dark) to photosynthetic (light) growth. However, the same perturbation of Vipp1 in dark alone or light alone growth conditions causes no growth or thylakoid morphology defects. We propose that the punctuated dynamics of Vipp1 at the cell periphery in regions of high thylakoid curvature enable acquisition of photosynthetic competency, perhaps by facilitating biogenesis of photosynthetic complexes involved in light-dependent reactions of photosynthesis.