A custom-made AAV1 variant (AAV1-T593K) enables efficient transduction of Japanese quail neurons in vitro and in vivo

Figures

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  Fig. 1. Development of primary cultures from embryonic quail brains. a Micrographs of cultures produced from 7, 8, or 9-day-old embryos (days in OVO, DIO) (rows), grown for 7, 14, and 21 days in vitro (DIV) (column). b Cultures remained viable for up to 30 days (arrowheads represent examples of viable cells). c Seven DIV cultures (produced from 9 DIO embryos) co-stained with DAPI (yellow) and NeuroTrace (pink) suggest that the majority (~90%) of cells (inset: pink arrowheads) represent neurons, with a smaller fraction (10%) of non-neuronal cells, summarized in the right most panel. d MAP2-staining of cultures suggests that ~18% of cultured cells are neurons (summarized in the pie chart).

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  Fig. 2. Electrophysiological description of primary cultured quail neurons. a Representative current-clamp (top) and voltage-clamp (bottom) recordings from cultured neurons (left inset—micrograph of the patched cell). The current-clamp recording shows the resting membrane potential of the neuron (−80 mV), excitatory postsynaptic potential (EPSPs), and action potential firing (e.g., dashed rectangle and inset). During voltage-clamp, the cell was clamped at −70 mV, during which barrages of excitatory postsynaptic currents (EPSCs) could be distinguished. b Summary of electrical properties of quail neurons in vitro (n = 12). c Assessment of intrinsic neuronal excitability. Representative voltage traces (top) from a single neuron in response to current injection (bottom protocol) from which we could deduce the maximal firing rates, summarized in d. Data are presented as mean ± SEM. Color-coded traces correspond to colored data points on the plot (For the complete dataset, see Supplementary Fig. 3c).

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  Fig. 3. Single-cell mRNA sequencing of cultured cells reveals a large number (11) of cellular populations. a UMAP plot of 9561 cells, taken from cultures produced from an embryo’s entire hemisphere, classified based on the top ten most differentially expressed genes (DEGs), as shown in the heatmap in (b). The number of cells within each cluster are noted at the left corner of UMAP and clusters’ colors correspond to column colors in b. Red annotated genes (in b) were manually curated (i.e., identity was not automatically found in reference genomes). c Cell type identification dotplot shows mean expression levels (blue to red) and percent of cells (size of data point) in each cluster and for select markers (bottom axis). Red annotated genes were manually curated. d Feature plot showing expression of SFRP1. Mean expression (Log2 fold change) and percent of cells for each cluster are depicted below the plot.

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  Fig. 4. Common viral tools currently used in neuroscience poorly transduce primary quail cultures. Various AAV serotypes, lentivirus, and baculovirus show very limited transduction efficacy of quail cultures. Infection efficacy was dually assessed on cultured cells (HEK293T cells, top lines) and quail cultures (bottom lines). AAVs (including A3V) express CAG-eYFP, Baculovirus expresses CAG-jGCaMP7f, and the lentivirus expresses CMV-eGFP. The strong expression of fluorescent markers within HEK293T cells (except for A3V, which showed no infection of HEK293T cells83) demonstrates the viability of the viruses used. Despite the latter, very few infected cells (mostly neurons) were detected in quail cultures. Note the cytotoxicity of the baculovirus.

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  Fig. 5. The AAVR is present in quail cells, but diverges in sequence from the mammalian receptor. a Feature plot of KIAA0319L, the quail AAV-receptor (qAAVR). Mean expression (log2 fold change) is noted at the left of the plot and the percent of cells per each cluster are depicted below the plot. b (left) Cartoon illustration of the binding of the AAVR (gray transmembrane domain and PKD domains in lavender) by an AAV (magenta icosahedron). The PKD2 domain is highlighted (dark lavender) because of its essential role in the binding the AAV’s (AAV1 and AAV2) capsid86,89. (right) Atomic structure (by cryo-EM) of AAV1’s capsid (magenta) bound to PKD2 (lavender) (pdb: 6JCQ87). Key residues in PKD2 that interact with the capsid monomer are highlighted in yellow. The K464 residue (green) in PKD2 interacts with residue T593 (pink) found in the capsid of a second AAV1 protomer. Inset: binding of the receptor (three yellow highlights) by multiple AAV capsid monomers (magenta) shows the very close proximity between K464 (green) and T593 (pink). c PKD2’s protein sequence alignment between different species shows high conservation (lavender). Residues in the AAVR that directly interact with the AAV1…

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  Fig. 6. The new AAV1 variant (AAV1*) exhibits enhanced transduction efficiency of quail cultures and enables calcium imaging. a Micrographs of quail cultures infected by AAV1* (left) or by AAV1 (right) (both of identical titer—3.3 × 1012). The amount of YFP-positive cells (infected) and fluorescence intensity are summarized in (b). b AAV1* infected significantly more cells compared to AAV1 by the same titer –AAV1* (Mean ± SEM: 396.5 ± 9.5, AAV1 – 27.25 ± 5.7, T-test, p < 0.0001). Fluorescence intensity is significantly higher in cells infected with AAV1* compared to AAV1 (AAV1* - 48.7 ± 1.66, AAV1 – 24.06 ± 1.02, T-test, p < 0.0001). c, d Electrophysiological recordings of AAV1*-infected cells (i.e., expressing YFP). c- Recording from a mature neuron (inset) reveals excitatory postsynaptic currents (top trace) and action potential firing and plateaus (bottom trace, n = 5), whereas amorphic cells (d, inset) do not fire (non-firing cells) and exhibit very health and hyperpolarized resting membrane potentials (n = 4). Small membrane activity could be easily seen, reminiscent of glial activities. e Micrograph showing cultures infected by AAV1*-CAG-GCaMP6m and two representative cells showing…

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  Fig. 7. AAV1* outperforms AAV1 in vivo in young quails. a Coronal brain slices of young quails (4 weeks old), produced after 7 weeks from animals injected, side-by-side, with 1 μl of AAV1*.or AAV1 (b); expressing CAG-eYFP. c AAV1* provides a higher density of infected cells (mostly neurons, top)- AAV1* (Mean ± SEM: 509.2 ± 107.04, AAV1—101.9 ± 18.4, T-test, p < 0.001). AAV1*-infected neurons also show a tendency for higher expression within each cells (bottom)- AAV1* - 207.8 ± 5.9, AAV1—173.4 ± 12.04. Each data point represents one animal (n = 5). The complete data set is provided in Supplementary Fig. 9. Note that the highest density observed in animals injected with AAV1 is equivalent to the lowest density obtained by AAV1* (c, top). Data were presented as mean ± SEM, p values are indicated, and noted by *p < 0.05; **p < 0.01; ***p < 0.001, n.s. non-significant.