Mice
Mouse breeding and husbandry procedures were conducted in strict accordance with the guidelines and approval of UCLA's Animal Care and Use Committee at the University of California, Los Angeles and were supervised by Josh Trachtenberg. Mice were provided with food and water ad libitum and were maintained under a 12-hour day/night cycle, with a maximum of four adult animals per cage. The PcdhγC3 knockout (γC3 KO) mice were generated and previously characterized by the laboratories of Joshua Weiner and Robert W. Burgess (The Jackson Laboratory) [21]. The Pcdhγfcon3/fcon3 transgenic mice have been described previously 43. Additionally, the ROSA26-CAG::lox-Stop-lox-PcdhγA1-mCherry and ROSA26-CAG::lox-Stop-lox-PcdhγC3-mCherry mice were originally generated by Julie Lefebvre and Joshua Sanes at Harvard University [1]. For all experiments, a minimum of three animals were analyzed per genotype.
Tamoxifen Induction for Cre Mice
Tamoxifen (Sigma) was freshly prepared at a concentration of 20 mg/ml in corn oil and allowed to dissolve overnight at 37°C with continuous agitation. To induce gene expression, mice were injected with 5mg/kg of tamoxifen (Sigma T5648) dissolved in corn oil on postnatal day 1 or 2 for three consecutive days. This treatment aimed to eliminate Pcdhγ cluster genes and replace endogenous Pcdhγ cluster genes with either the γC3 or γA1 single isoform in astrocytes. Aldh1l1-Cre/ERT2 x Pcdhγfcon3/fcon3, Aldh1l1-Cre/ERT2 x Pcdhγfcon3/fcon3 x ROSA26-CAG::lox-Stop-lox-PcdhγC3-mCherry, and Aldh1l1-Cre/ERT2 x Pcdhγfcon3/fcon3 x ROSA26-CAG::lox-Stop-lox-PcdhγA1-mCherry mice were used for tamoxifen injections.
Whole-Mount Tissue Optical Clearing
To achieve optical clearing of brain tissues, we employed the FOCM reagent. The FOCM reagent was prepared as a solution containing 30% (wt/vol) urea, 20% (wt/vol) D-sorbitol, and 5% glycerol dissolved in DMSO (D8418). Mice designated for histological analysis were anesthetized with isoflurane and then transcardially perfused with ice-cold 1x PBS, followed by 4% paraformaldehyde in 1x PBS buffer. Brains were subsequently removed, postfixed in 4% paraformaldehyde overnight, and transferred to a 30% sucrose 1XPBS solution for a minimum of 48 hours at 4°C. Sections, 200 μm in thickness, were cryosectioned using a Leica cryostat microtome. These sections were washed three times for 1 hour in 1X PBS, followed by permeabilization and blocking for 4 hours in a solution containing 0.3% Triton X-100 and 10% NGS, 1% BSA, and 0.5% Triton X-100 in 1X PBS. Subsequently, slides were incubated with primary antibodies diluted in 3% NGS, 1% BSA, and 0.5% Triton X-100 in 1X PBS for 72 hours at 37°C. Primary antibodies were used at the following concentrations: Rabbit anti-V5 (Bethyl, 1:500), Chicken anti-V5 (Bethyl, 1:500), anti-c-myc rat antibody (Biorad, 1:500), Anti-GFP antibody (abcam, 1:1000). After six washes of 6 hours with 1X PBS, tissues were incubated with Alexa Fluor 488, 568, 647 secondary antibodies (Invitrogen, 1:500) or anti-RFP nanobody (FluoTag®-X4 anti-RFP, 1:200) for mCherry immunostaining. When necessary, tissues were treated with a nuclear counterstain, either DAPI (Invitrogen, 1:1000) or NeuroTrace Green (Invitrogen, 1:500), for 48 hours at 37°C. Finally, tissues were washed six times for 4 hours with 1X PBS and mounted on glass slides using a holder. The holder was filled with FOCM reagent, and the brain section was incubated for 15 minutes. Once the tissues became optically cleared, a glass coverslip was placed on the holder and sealed with nail polish for imaging.
Plasmids and AAVs
To generate GfaABC1D.Lck-smV5 and Lck-smMyc plasmids, cytosolic smFP-V5 and smFP-Myc DNA sequences were amplified by PCR from the pCAG-smFP-V5 (Addgene plasmid # 59758) and pCAG-smFP-Myc plasmid (Addgene plasmid # 59757). The pCAG-smFP-V5 and pCAG-smFP-Myc plasmids were generously provided by Loren Looger. The GfaABC1D.Lck-GFP plasmid (Addgene ID #61099) was digested with restriction enzyme using XhoI and SalI. PCR amplified smFP-V5 and smFP-Myc genes were annealed into the cleavage sites of the plasmid backbone using T4 ligase to generate GfaABC1D.Lck-smV5 and Lck-smMyc plasmids, respectively. The cDNAs of the full-length γC3 fused with C-terminal 3xV5 epitopes was synthesized by Genewiz Priority Gene (Azenta Life Science). Next, the gene fragments were cut with restriction enzymes to facilitate cloning into the XhoI and XbaI digested GfaABC1D.Lck-GFP plasmid backbone to generate AAV.GfaABC1D -γC3FL construct. To generate the heterophilic binding deficient AAV chimera constructs, the EC1-EC4 domains of the WT full-length γC3 was replaced with the EC1-EC4 domains of the modified γC3 heterophilic binding chimeras (M1, M3, M6, and M8). To enable in vivo detection of the expressed constructs, 3xV5 tags were added to the C-terminus of the M1 and M3 constructs while 3xHA tags were fused to the C-terminus of the M6 and M8 constructs. The cDNAs for the M1, M3, M6, and M8 constructs were synthesized by Genewiz Priority Gene (Azenta Life Science). The synthesized gene fragments were restriction digested and cloned into the AAV.GfaABC1D.Lck-GFP plasmid backbone to generate AAV.GfaABC1D.M1, AAV.GfaABC1D.M3, AAV.GfaABC1D.M6, and AAV.GfaABC1D.M8 constructs.
Generation of γC3 Single-Point Mutants
Two γC3 single-point mutants, γC3-L87E and γC3-L342E, were designed by introducing an unsatisfied negative charge at the EC1::EC4 interface of the γC3 homodimer (Fig. 4C). The biophysical characteristics of these homophilic binding deficient mutants were then assessed in the context of the γC3 EC1-EC4 construct (Table S1). QuickChange site-directed mutagenesis kit from Strategene was used to introduce point mutations on the full-length γC3 cDNA with fused C-terminal 3xV5 tags following the mutagenesis PCR protocol. The resulting AAV.GfaABC1D. γC3-L87E and AAV.GfaABC1D. γC3-L342E constructs were verified by sequencing.
AAV Production and administration
Neonatal mice were gently removed from their holding cages and positioned on their side. Sterile saline and a cotton-tipped applicator were used to clean the injection site. A 33-gauge needle attached to the Hamilton microinjection syringe was inserted at a 45° angle into the left retro-bulbar sinus of the neonatal mice. A total injection volume of 10 µl virus diluted in 1X PBS was delivered into the retro-orbital bulbar sinus. After viral injection, mild pressure was applied at the injection site to minimize backflow of the injected viruses. AAVs were administered at an approximate viral titer of 1x10^11 vg per mouse. All injections were performed on postnatal day 1-2. Mice were sacrificed at designated time points in accordance with approved animal protocols and procedures.
To assess the effects of homophilic binding deficient mutants, separate litters of WT and γC3 KO mice each received AAV.GfaABC1D.γC3FL, AAV.GfaABC1D.γC3-L87E, or AAV.GfaABC1D.γC3-L342E. All groups were also injected with AAV.GfaABC1D.Lck-smMyc to label astrocyte morphology. In the heterophilic binding chimera experiment, the litter of γC3KO mice was equally divided to evaluate the effect of each heterophilic binding pair. To evaluate the phenotypic effect of the M1+M6 heterophilic binding chimera, the litters were equally divided to receive either AAV.GfaABC1D.M1, AAV.GfaABC1D.M6, or AAV.GfaABC1D.M1+ AAV.GfaABC1D.M6 constructs. Following a similar experimental design, other γC3 KO litters were equally divided to receive AAV.GfaABC1D.M3, AAV.GfaABC1D.M8, and AAV.GfaABC1D.M3+M8 treatments to evaluate the effect of M3 and M8 heterophilic binding pairs on astrocyte morphology. Of note, the same WT and γC3 KO control groups were shown in both Fig. 4E and Fig 5D. All AAV viruses were produced by the Janelia Viral Vector Core. Viral titers were measured using reverse transcription PCR (RT-PCR) to quantify the viral particle concentration in the final virus preparations.
Confocal Imaging and Morphology Analysis
Confocal images were acquired by a Zeiss LSM 880 confocal microscope equipped with Zen digital imaging software. Images were acquired at several microscope magnifications including 4x, 20x, 40x with each image frame of 1024 pixels by 1024 pixels and acquired at 4x, 20x, or 40x magnifications. Z stacks images were obtained to capture the entirely of single astrocyte volume based on fluorescence signals. The acquired astrocyte z-stack confocal images were first analyzed to compute the functional surface, which was then used to extract a range of morphometric parameters for individual astrocytes. The functional surface was by creating a region of interest (ROI) that encapsulated the entirety of each individual astrocyte based on fluorescence signal. The background signal was eliminated by adjusting the threshold level. The fluorescence intensities of the GFP or smFPs used to image astrocyte morphology were equivalent across all experimental groups.For 2D morphological analysis, various morphology parameters such as Feret max, Feret min, aspect ratio, territory size, roundness, and circularity, were performed in accordance with established protocols 23,44. This assessment was carried out using maximum intensity projections, and each morphological parameter was quantified using ImageJ software.
Building trans dimer models
We used a crystal structure of γC4 (PDBID: 7JGZ) 35 as a template to build models of trans dimers for γC3 wild-type (WT), γC5 WT, the single point mutants to explain their effects on binding (γC3 L87E, γC3 L342E, γC4 E78A) and the chimeras (Mi, i =1, 2, 3, 6, 8) designed for exclusive heterophilic binding. Sequences of all modelled EC1-EC4 fragments (residues 1-421, numbering as in PDBID: 7JGZ) are juxtaposed with the template in a supplementary Fig. S10. Multiple sequence alignment shown in Fig. S10 was generated with Clustal Omega 45 and visualized via https://espript.ibcp.fr/ESPript/ESPript/ 46.
The variants were modelled by mutating γC4 amino acids of the template structure into amino acids of the target proteins using BuildModel utility in FoldX 47. This method ensures the backbone of the modeled proteins mirrors the template (γC4), gauging their propensity to form a γC4-like trans dimer. For instance, to build models of γC3 and γC5 wild-type trans dimers, we made 215 and 194 mutations to each γC4 protomer within a γC4::γC4 trans dimer, yielding DDG values of 27.2 and 5.9 kcal/mol, respectively. Here, DDG denotes the interaction energy of the trans dimer, derived from its total folding energy using AnalyseComplex utility in FoldX. The chimeras exhibited destabilization in a γC4-dictated trans dimer conformation by 16-18 kcal/mol, similar to γC3. These modelled trans dimers and their binding energies relative to γC4 are shown in Fig. S9A. Of note, the figure shows the precursor chimera models corresponding to merging EC1EC2 (residues 1-205) and EC3EC4 (residues of 206-421) fragments of γC3 (blue) with either γC4 (green) or γC5 (red). Only M1 and M3 match the precursor chimera in sequence. Mi chimeras (where i=2,6,8) carry additional point mutations atop the precursor chimeras, introduced via FoldX to bolster trans dimerization (detailed further in 'Design of chimeras').
Design of chimeras
Utilizing FoldX, we first modelled heterophilic protocadherin chimeras combining sequences of γC3 with either γC4 or γC5 (refer to ‘Building trans dimer models’). Subsequent mutations were introduced to correct the non-complementary surfaces. Largely positive relative FoldX energies of the initial chimera models (Fig. S9A) suggested interface issues that might hinder gC4-like heterophilic chimera binding. Particularly, the issues were likely from the γC3 fragments (as γC3::γC3 was most destabilized compared to γC4::γC4, Fig. S9A).
By examining all model structures, we identified problematic chemical interactions, especially those between polar/charged and hydrophobic residues. A comparison of these destabilizing contacts with the template interactions revealed specific issues. For instance, Fig. S9B shows a ribbon representation of a γC4 trans dimer (template, green) and a model of a γC3 trans dimer (forced to have a backbone identical to γC4, blue). The amino acids at the EC2-EC3 boundary of γC4 are mostly hydrophobic (see labelled residues shown as sticks in a diamond-shaped area, shaded in grey) whereas γC3 has polar/charged residues (N299 and E301, underlined in cyan) that are incompatible with the neighboring hydrophobic residues (L204, A118, L117). Furthermore, a hydrophobic contact in a γC4 dimer formed by two valine residues (V206) was missing in γC3 (as it has Ala in place of Val). Hence, A206, N299, and E301 residues of γC3 would likely destabilize binding in a γC4-like orientation due to polar-hydrophobic mismatches at the EC2::EC3 interface. This logic extends to chimeras containing γC3 fragments. Of note, γC5 interface is similar in chemical composition to that of γC4 featuring V206 and PAM (299-301) (Fig. S9C and Fig.S10).
To validate our observations, we employed computational mutagenesis via FoldX. We mutated residues of the precursor chimera models to make the trans dimer interface at the EC2-EC3 boundary more hydrophobic (see details on computational mutagenesis procedure in 48). Predictions confirmed that A206V, N299P and E301M mutations would stabilize the interface (Table S2).
The E78A mutation enhances homophilic binding in γC4 35. The biophysical effect of this mutation is explained in Fig.S7. The E78A mutation was introduced to M2 and M6 chimera featuring fragments of γC4. The N299P and E301M mutations were introduced to M6 and M8 chimeras together with a predicted neutral (Table S2) P300A mutation (as a triple NPE®PAM mutation) to minimize the number of cloning events in the chimera generation. The A206V mutation was effectively introduced by a shift in the EC2-EC3 domain boundary (i.e. we merged the domains to preserve the Val-Val contact by keeping Val206 of γC4 or γC5 and cutting out Ala206 of γC3, see Fig. S7 for the chosen domain boundary between EC1EC2 and EC3EC4 fragments in the Mi chimeras). The NPE®PAM mutation differentiates M6 (with) from M2 (without) and M8 (with) from M4 (without), and it is crucial for restoring hydrophobic complementarity at the EC2-EC3 boundary in the heterophilic chimeric trans dimers (Fig. S9C) as M1::M6 and M3::M8 both formed heterodimers in AUC whereas M1::M2 and M2::M3 did not (Table S1).
Sedimentation equilibrium measurements
Experiments were performed in a Beckman XL-A/I analytical ultracentrifuge (Beckman-Coulter, Palo Alto CA, USA), utilizing six-cell centerpieces with straight walls, 12 mm path length and sapphire windows. All proteins were dialyzed over-night and then diluted in TRIS 10 mM, NaCl 150 mM, CaCl2 3 mM pH 8.0, 250 mM imidazole. Samples were diluted to 1.1, 0.73 and 0.37 mg/mL in channels A, B and C, respectively. Dilution buffer were used as blank. All samples were run at four speeds, 11000, 14000, 17000 and 20000 rpm, at 25oC. The lowest speed was held for 20 h after which four measuring scans were taken with 1 h interval, the second lowest speed held for 10 h, followed by four scans as above, the third lowest and the highest speed performed identically as the second lowest speed. All measurements were done at 25oC, and detection was by interference at 675 nm. Solvent density and protein v-bar were determined using the program SednTerp. (Alliance Protein Laboratories, Corte Cancion, Thousand Oaks, CA, USA) For calculation of dimeric KD and apparent molecular weight, all useful data were used in a global fit, using the program SedPhat, obtained from National Institute of Health (www.nibib.nih.gov). All measurements are summarized in Table S1.
Protein production
cDNA for mouse Pcdh ectodomain fragments, excluding the predicted signal sequence, were cloned into a pαSHP- H mammalian expression vector (from Daniel J. Leahy, John Hopkins University), modified with the BiP signal peptide (BiP: MKLSLVAAMLLLLSAARA) and a C-terminal octahistidine tag. The signal sequence was predicted using the SignalP 4.0 server. Point mutations were introduced into the constructs using the KOD hot start polymerase (Novagen) following the standard Quikchange protocol (Stratagene). The constructs were expressed in suspension-adapted HEK293 Freestyle cells (Invitrogen) in serum-free media using polyethyleneimine as a transfectant (Polysciences). Media was supplemented with 10 mM CaCl2 4hr after transfection. Conditioned media was harvested 5 days after transfection, and the secreted proteins were purified by nickel affinity chromatography followed by size exclusion chromatography in 10 mM Tris, pH 8.0, 150 mM sodium chloride, 3 mM calcium chloride, and 250 mM imidazole, pH 8.0. The purified proteins were concentrated to > 3 mg/mL before sedimentation equilibrium AUC experiments.
Statistical Analyses
Data were analyzed using a two-tailed Mann–Whitney test for the comparisons between two groups and one-way ANOVA analysis, followed by Tukey’s post hoc analyses, to assess for significant differences between more than two groups. All statistical analyses were performed using GraphPad Prism version 5.0c. Data are expressed as mean ± SEM. Asterisks in the figures denote the following significance levels: *p < 0.05; **p < 0.01; ***p < 0.001. In all cases, p < 0.05 was considered significant.