Targeting genes to specific neuronal or glial cell types is valuable both for understanding and for repairing brain circuits. Adeno-associated viral vectors (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is a challenge. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that ~11% of these AAVs specifically target expression to neuronal and glial cell types in the mouse retina, mouse brain, non-human primate retina in vivo, and in the human retina in vitro. We demonstrate applications for recording, stimulation, and molecular characterization, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast, and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.Despite the central importance for both basic and translational research, most current technologies available for cell-type-targeting rely on transgenic animals, which limits their applicability. Either the genetic tool that senses or modulates brain function, or the enzyme, such as Cre recombinase, that allows the genetic tool to be conditionally expressed, is expressed from the animal's genome. The inclusion of a transgenic component in the cell-type-targeting strategy excludes its use in therapy for humans, limits its range of application in pre-clinical, non-human primate research, and complicates its use in model organisms such as mice. The development of transgenic non-human primates and mice is costly and slow, especially since cell-type targeting is often applied in the context of other genetic manipulations, such as double or triple gene knockouts, or when targeting different cell types with different tools.Viral vectors for cell-type-targeting may overcome such limitations. AAVs are the most frequently used vectors in both basic research and gene therapy, as they are safe for use in all tested species, including humans and non-human primates, and their production is simple, cheap, and fast (Planul and Dalkara, 2017). They have three important components: the capsid for cell entry, the promoter that drives transgene expression, and the gene of interest to be expressed in the transduced cells, and they drive expression episomally (Duan et al., 1998; Penaud-Budloo et al., 2008). Futhermore, many genetic tools are small enough to fit into AAVs, different AAVs can be injected together, and synthetic AAV capsids allow brain-wide delivery (Deverman et al., 2016).Cell-type-targeting by AAVs could be achieved by engineering the capsid and/or by using specific promoters. Capsid protein mutations can be used to tune the efficacy of
Dendrite pruning is critical for sculpting the final connectivity of neural circuits as it removes inappropriate projections, yet how neurons can selectively eliminate unnecessary dendritic branches remains elusive. Here, we show that calcium transients that are compartmentalized in specific dendritic branches act as temporal and spatial cues to trigger pruning in Drosophila sensory neurons. Calcium transients occurred in local dendrites at ~3 hours before branch elimination. In dendritic branches, intrinsic excitability increased locally to activate calcium influx via the voltage-gated calcium channels (VGCCs), and blockade of the VGCC activities impaired pruning. Further genetic analyses suggest that the calcium-activated protease calpain functions downstream of the calcium transients. Our findings reveal the importance of the compartmentalized subdendritic calcium signaling in spatiotemporally selective elimination of dendritic branches.
Enabling near-infrared light sensitivity in a blind human retina may supplement or restore visual function in patients with regional retinal degeneration. We induced near-infrared light sensitivity using gold nanorods bound to temperature-sensitive engineered transient receptor potential (TRP) channels. We expressed mammalian or snake TRP channels in light-insensitive retinal cones in a mouse model of retinal degeneration. Near-infrared stimulation increased activity in cones, ganglion cell layer neurons, and cortical neurons, and enabled mice to perform a learned light-driven behavior. We tuned responses to different wavelengths, by using nanorods of different lengths, and to different radiant powers, by using engineered channels with different temperature thresholds. We targeted TRP channels to human retinas, which allowed the postmortem activation of different cell types by near-infrared light.
We have identified a novel phospholipase A 1 , named mPA-PLA 1 , which is specifically expressed in human testis and characterized it biochemically together with previously identified mPA-PLA 1 ␣. The sequence of mPA-PLA 1  encodes a 460-amino acid protein containing a lipase domain with significant homology to the previously identified phosphatidic acid (PA)-selective PLA 1 , mPA-PLA 1 ␣. mPA-PLA 1  contains a short lid and deleted 9 loop, which are characteristics of PLA 1 molecules in the lipase family, and is a member of a subfamily in the lipase family that includes mPA-PLA 1 ␣ and phosphatidylserine-specific PLA 1 . Both mPA-PLA 1  and mPA-PLA 1 ␣ recombinant proteins exhibited PA-specific PLA 1 activity and were vanadate-sensitive. When mPA-PLA 1 -expressing cells were treated with bacterial phospholipase D, the cells produced lysophosphatidic acid (LPA). In both mPA-PLA 1 ␣ and -expressing cells, most of the PA generated by the phospholipase D (PLD) treatment was converted to LPA, whereas in control cells it was converted to diacylglycerol. When expressed in HeLa cells most mPA-PLA 1 ␣ protein was recovered from the cell supernatant. By contrast, mPA-PLA 1  was recovered almost exclusively from cells. Consistent with this observation, we found that mPA-PLA 1  has higher affinity to heparin than mPA-PLA 1 ␣. We also found that the membrane-associated mPA-PLA 1 s were insoluble in solubilization by 1%
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