Fig 2: Gamma-aminobutyric acid transporter 3 is found on nTS astrocytes. (A) Overview of immunoreactivity of GAT-3 (red), astrocyte S100B (green) and GABAergic GAD67 terminals (blue). Dashed box is shown in (B,C). Scale, 25 µm. (B) Super-resolution image of S100B and GAT-3, and their overlay, demonstrating GAT-3 aligns with S100B. Arrowheads denote GAT-3 on the membrane of S100B-labeled astrocyte somas. Scale, 5 µm. (C) GAT-3 surrounds GAD67-labeled GABA terminals (arrow). Scale, 5 µm. Shown are single optical sections.

Fig 3: Nerve Morphometry and Muscle Reinnervation at 1 Month Following Delayed Nerve Repair. (A) Representative confocal images of nerve cross-sections 5 mm distal to the repair site were labeled for Schwann cells (S100), host axons (SMI35), and myelin (myelin basic protein; MBP). (B) No differences in SMI35 expression were detected distal to the repair site, suggesting a comparable number of host axons regenerated into the distal sheath. (C) The mean area of SMI35+ regions found distal to the repair was greater in the TE-NMI cohort, indicating the host axons in the distal nerve were larger than the controls. (D) Greater number of myelinated axons were found distal to the repair in the TE-NMI cohort. (E) Increased S100β expression, a common marker of Schwann cells, was observed in the TE-NMI group. (F) Representative confocal images of the tibialis anterior (TA) muscle cross-section stained for acetylcholine receptors (bungarotoxin) to identify the neuromuscular junctions (NMJs) and synaptophysin, a presynaptic marker (gray-scaled). Sections were counterstained with phalloidin to visualize muscle fibers. (G) No significant difference in the total number of AchR counts between groups. (H) Greater muscle reinnervation, as indicated by the percent of mature NMJ co-labeled for AchR and synaptophysin, was found in animals that previously received a TE-NMI transplantation. Collectively, these findings suggest that both while both groups are exhibiting ongoing regeneration, TE-NMIs may enable earlier axon maturation and muscle reinnervation following delayed nerve repair. Fractional area was calculated by measuring the percent area of positive fluorescent expression per ROI averaged over three ROIs. Mean values compared using two-tailed unpaired Student's t-tests. Error bars represent standard error. *p < 0.05; **p < 0.01.

Fig 4: Freshly-Cut TE-NMIs Axons in the Otherwise Denervated Distal Nerve Enable an Immediate Evoked Response Following Nerve Fusion. (A) Schematic illustrating the surgical model, delayed nerve fusion paradigm, and outcome measure. At 20 weeks post transplantation and host chronic nerve axotomy, the TE-NMI was removed leaving behind freshly transected axons in the distal nerve. To enable axon fusion, the graft was excised in hypotonic saline containing a calcium chelating agent, similar to previous protocols. (B) Intraoperative image at 20 weeks post transplantation showing the proximal common peroneal nerve secured to a nearby muscle, the TE-NMI secured to the distal nerve, and the uninjured tibial nerve coursing above it. (C) Intraoperative image immediately after delayed nerve repair showing the previously uninjured tibial nerve sutured to the distal portion of the common peroneal nerve following TE-NMI excision. The blue staining is due to methylene blue application during the fusion protocol. (D) Compound nerve action potentials recorded immediately after delayed nerve fusion were obtained in all animals that had received a TE-NMI. Greater nerve conductivity was found in the TE-NMI group compared to acellular controls. (E) Compound muscle action potentials were recoded after eliciting an evoked muscle response by stimulating proximal to the repair site. Greater evoked muscle response was observed in the TE-NMI group compared to acellular controls. (F) At 20 weeks post repair, the surgical site was re-exposed and the TE-NMI transplant was harvested for histological analyses. Representative longitudinal images are shown labeling neurons and dendrites with MAP2 (far red) and sensory and motor TE-NMI neurons and axons with endogenous expression of GFP and tdTomato, respectively. Robust TE-NMI neuron survival was found within the lumen at 20 weeks post transplantation. (G) At high magnification, healthy neurons were readily visualized within the micro-column co-labeling with MAP2. (H) Representative longitudinal nerve sections and (I) axial nerve cross-sections immediately distal to the excised transplant are shown labeled for Schwann cells (S100). Robust TE-NMI sensory outgrowth (GFP, green) was visualized. TE-NMI outgrowth was found (TD-Tomato, red), but the expression was weaker. Error bars represent standard error. Mean values compared using two-tailed unpaired Student's t-tests. *p < 0.05; **p < 0.01. Scale bars: (F) 25 μm.

Fig 5: Two weeks MLi-2 treatment does not alter ciliogenesis in R1441C LRRK2 striatal interneurons or astrocytes.Mice (8 weeks old) were fed MLi-2 LRRK2 inhibitor-containing chow or control chow for 2 consecutive weeks prior to perfusion and staining. (A) Confocal images of sections of the dorsal striatum from 8 week R1441C LRRK2 KI mice; ChAT (green, white outline); Adenylate cyclase 3 (AC3) (magenta), and DAPI (blue). (B) Quantitation of the percentage of ChAT+ and ChAT- neurons containing a cilium. (C) Percentage of ChAT+ neurons containing a cilium± MLI-2. (D) Quantitation of ChAT+ neuron ciliary length. (E) Confocal images of astrocytes identified by antibodies to GFAP (magenta, white outline), Arl13B (green), and DAPI (blue), ± MLi-2. (F, G) Quantitation of the percentage of total astrocytes (F) or GFAP/S100B+ astrocytes (G) containing a cilium. Scale bars, 10 µm. Significance was determined by t-test. B, *, p = 0.0417.