Mitochondrial transfer from glia to neurons protects against peripheral neuropathy

Abstract

Primary sensory neurons in dorsal root ganglia (DRG) have long axons and a high demand for mitochondria, and mitochondrial dysfunction has been implicated in peripheral neuropathy after diabetes and chemotherapy^1,2. However, the mechanisms by which primary sensory neurons maintain their mitochondrial supply remain unclear. Satellite glial cells (SGCs) in DRG encircle sensory neurons and regulate neuronal activity and pain³. Here we show that SGCs are capable of transferring mitochondria to DRG sensory neurons in vitro, ex vivo and in vivo by the formation of tunnelling nanotubes with SGC-derived myosin 10 (MYO10). Scanning and transmission electron microscopy revealed tunnelling nanotube-like ultrastructures between SGCs and sensory neurons in mouse and human DRG. Blockade of mitochondrial transfer in naive mice leads to nerve degeneration and neuropathic pain. Single-nucleus RNA sequencing and in situ hybridization revealed that MYO10 is highly expressed in human SGCs. Furthermore, SGCs from DRG of people with diabetes exhibit reduced MYO10 expression and mitochondrial transfer to neurons. Adoptive transfer of human SGCs into the mouse DRG provides MYO10-dependent protection against peripheral neuropathy. This study uncovers a previously unrecognized role of peripheral glia and provides insights into small fibre neuropathy in diabetes, offering new therapeutic strategies for the management of neuropathic pain.

Fig. 1. Mitochondrial transfer from SGCs to neurons in co-culture and TNT-like structures in the mouse DRG.

a, Schematic of SGC–neuron co-cultures from mouse DRG. SGCs are labelled with MitoTracker dye and DRG neurons are from Trpv1:Ai9 mice. b, Left, image from an SGC–neuron co-culture showing a Trpv1⁺ neuron interacting with an SGC (scale bar, 20 μm). Right, enlarged view of the boxed area showing a TNT (white arrow) and a mitochondrion (Mito; red arrow) within the TNT (scale bar, 5 μm). c, Percentage of MitoTracker-positive (Mito⁺) and TNT-positive neurons. A total of 109 neurons from 6 independent experiments were included for quantification. d, Schematic for SEM of whole-mount DRG without sectioning. e, High-magnification SEM images of whole-mount mouse DRG showing a TNT-like structure (TNT-LS) with a bulge. n = 4 biological repeats. Scale bars: 5 μm (left); 1 μm (right). f, Schematic for SEM of sectioned mouse DRG. g, Representative SEM images of sectioned mouse DRG showing TNT-LS with bulges from SGCs to neuron. n = 4 biological repeats. Scale bars: 5 μm (left); 1 μm (right). h, Schematic for TEM of mouse DRG. i, Representative low-magnification TEM imaging showing the DRG neuron and SGCs. n = 4 biological repeats. The green asterisk indicates the nucleus of the neuron. Scale bar, 5 μm. j–l, Representative high-magnification TEM imaging showing a TNT-LS between SGC and neuron. ‘Mito’ in black indicates mitochondria in the cell (j–l); ‘Mito’ in red indicates mitochondria in the TNT-LS (k); ‘Vesicle’ indicates a vesicle in the TNT-LS (k,l); the black asterisk indicates the nucleus of an SGC. l, Enlarged view from k showing mitochondria (red arrows) and a vesicle inside the smooth TNT-LS. ER, endoplasmic reticulum. n = 3 biological repeats. Scale bars: 2 μm (j,k); 800 nm (l). Source Data

Fig. 2. In vivo mitochondrial transfer from SGCs to neurons in the DRG of MitoTag mice.

a, Diagram of Aldh1l1-cre^ERT2:MitoTag mice with timeline of Tam injections and DRG collection. IP, intraperitoneal injection; IT, intrathecal injection. b, Top, MitoTag signal in DRG sections. Bottom, enlarged views showing double staining with FABP7 and Nissl. White arrows, MitoTag⁺ SGCs; red arrows, MitoTag⁺ neurons; blue arrows, MitoTag^– neurons. c, The percentage of MitoTag⁺ neurons in mice treated with Tam for 5 days (n = 4), 10 days (n = 4), 10 days with Tam plus CytoB (n = 4), 10 days with Tam plus Pitstop2 (n = 4), Tam with STZ (5 days, n = 3), STZ (10 days) with Tam (n = 4), Tam with CFA (4 h, n = 4), and Tam with CFA (5 days, n = 4). **P = 0.0034. d, Diagram of SNI surgery in Aldh1l1-cre^ERT2:MitoTag mice. e, MitoTag signal in the contralateral and ipsilateral DRG. f, The percentage of MitoTag⁺ neurons in ipsilateral DRG (n = 4) and contralateral DRG (n = 4). **P = 0.002. g, Diagram of SNI surgery with ipsilateral peri-sciatic bupivacaine film implantation. h, MitoTag signal in DRG. Bupi, bupivacaine. i, The percentage of MitoTag⁺ neurons in ipsilateral (n = 4) and contralateral (n = 4) DRG. j, Schematic of injection of AAV-MaCPNS2-Syn-jRGECO1a into Aldh1l1-MitoTag mice, SNI surgery and ex vivo calcium imaging in DRG. k, DRG calcium imaging of jRGECO1a in Aldh1l1-MitoTag mice. l, Heat map of MitoTag⁺ (green) and jRGECO1a⁺ neurons (red) from k. m, Quantification of jRGECO1a fluorescence. n = 45 cells from 9 DRG of 3 different mice. n, Colocalization of MitoTag⁺ neurons and jRGECO1a⁺ neurons. Data are mean ± s.e.m. One-way ANOVA followed by Tukey’s multiple comparisons test (c); two-sided unpaired t-test (f,i,m). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant (P ≥ 0.05). Source Data

Fig. 3. MYO10 in DRG SGCs regulates TNT formation, mitochondrial transfer, axonal degeneration and pain in mice.

a, Analysis of scRNA-seq data (reproduced with license number 6122760569440) reveals the expression of Myo10 in mouse DRG. b, Immunohistochemistry of MYO10 in mouse DRG. N denotes neurons. Scale bar, 10 μm. c, Schematic of mitochondrial transfer from SGCs to neurons and its blockade by knockdown of Myo10. d, Signals of MYO10 and MitoTracker in SGCs and neuron co-cultures treated with control siRNA (siCtrl) or siMyo10. Scale bars, 10 μm. e, Quantification of integrated MYO10 density. n = 4 per group. *P = 0.0285. f,g, Percentage of TNT⁺ neurons (f) and MitoTracker⁺ neurons (g) in control and Myo10 siRNA groups. h, Diagram of experimental setup for Aldh1l1-cre^ERT2:MitoTag mice treated with Tam and siMyo10. i, MitoTag signal in DRG of siCtrl and siMyo10 groups. Red and blue arrows indicate MitoTag⁺ and MitoTag⁻ neurons, respectively. j, Quantification of the percentage of MitoTag⁺ neurons in control (n = 3) and siMyo10 (n = 4) groups. k, Schematic of wild-type (WT) and Myo10^+/− mice for behavioural study. l, von Frey test in wild-type (n = 6) and Myo10^+/− mice (n = 6). PWT, paw withdrawal threshold. m,n, Representative SEM images of DRG from wild-type (m) and Myo10^+/− (n) mice. Asterisks indicate neurons with visible gaps between surrounding SGCs. N1 and N2 indicate neurons; S1 to S7 indicate SGCs; the white arrow (m) indicates a TNT with bulge; the blue arrow (n) points to an irregular TNT spanning a gap between SGC and neuron. o, The percentage of DRG neurons having gaps. n = 4 (wild type), n = 6 (Myo10^+/−). p, Quantification of TNTs within SGCs. n = 4 (wild type), n = 6 (Myo10^+/−). Data are shown as means ± s.e.m. Two-sided unpaired t-test. cLTMR, C-fibre low-threshold mechanoreceptor; Firbo, fibroblast cells; Immune, immune cells; NF, neurofilament⁺ neurons; NP, non-peptidergic neurons; PEP, peptidergic neurons; Schwann, Schwann cells; SST, somatostatin⁺ neurons; TRPM8, TRPM8⁺ neurons; Vascular, endothelial cells. Source Data

Fig. 4. MYO10 expression, TNT formation and mitochondrial transfer in human DRG SGCs and the impact of diabetes.

a, Schematic of sectioned human DRG processed with SEM and immunostaining using adjacent sections. b, Left, representative three-dimensional SEM images. Middle and right, staining of FABP7 (SGCs), Nissl (neurons) and DAPI (nuclei). Neurons are labelled N1 to N8, and N1 is enlarged in the bottom panels. SGCs are labelled S1 to S18. Asterisks indicate outer SGCs. c, High-magnification SEM images showing TNT-LS between an SGC and a neuron (left) and between two SGCs (right). Arrows point to TNT-LS with bulges. Scale bars, 1 μm. d, Schematic for snRNA-seq. e, Uniform manifold approximation and projection (UMAP) of 11,576 DRG nuclei identifies 17 clusters corresponding to 6 major cell types, including SGCs, neurons, connective tissue cells, endothelial cells, immune cells and Schwann cells. f, Proportions of six major DRG cell types. g, UMAP plot showing MYO10 expression across different clusters. h, Violin plot illustrating distinct MYO10 expression levels in single SGCs and neurons. Two-sided Mann–Whitney test. i, In situ hybridization of MYO10 in human DRG tissue from non-diabetic and diabetic donors. Arrows indicate MYO10 mRNA puncta that co-localize with FABP7. Scale bars, 20 μm. j, Quantification of MYO10 puncta surrounding individual DRG neurons per 3,000 μm² area. Two-sided unpaired t-test. n = 4 donors without diabetes; n = 5 donors with diabetes; 4 images (each represented by a small dot) from each donor. k, Schematic of primary SGC–neuron co-culture from non-diabetic and diabetic DRG. l, Representative images of MitoTracker in SGC–neuron co-cultures. Red arrows point to mitochondria within TNTs. m, Quantification of MitoTracker density as the integrated fluorescence intensity in neurons, normalized to the corresponding intensity in interacting SGCs. Two-sided unpaired t-test. n = 6 neurons from 2 independent experiments (no diabetes); n = 9 neurons from 3 independent experiments (diabetes). Data are mean ± s.e.m. Source Data

Fig. 5. Adoptive transfer of SGCs or SGC-derived mitochondria protects against axonal degeneration and alleviates neuropathic pain.

a, Schematic illustrating human SGC culture treated with siCtrl or siMYO10 and intra-DRG microinjection into db/db diabetic mice. b, Results of von Frey test showing the analgesic effect of transferred SGCs, which is reversed by siMYO10 treatment. 1 day vehicle versus siCtrl SGCs: **P = 0.0099; 2 days vehicle versus siCtrl SGCs: **P = 0.006; 3 days vehicle versus siCtrl SGCs: *P = 0.0241. c, OCR of DRG from mice treated with vehicle, or with SGCs pretreated with siCtrl or siMYO10. n = 6 per group. **P = 0.0048. d, Schematic of SGCs from wild-type and Myo10^+/− mice and intra-DRG microinjection. e, Analgesic effect following intra-DRG injection of wild-type SGCs. 1 day vehicle versus wild-type SGCs: *P = 0.0225; 2 days vehicle versus wild-type SGCs: *P = 0.0251. f, Schematic of human SGC culture, mitochondria isolation and intra-DRG injection. g, Analgesic effect of mitochondrial transfer from non-diabetic human SGCs. 6 h: ***P = 0.0009 (vehicle versus non-diabetic SGCs), *P = 0.0157 (vehicle versus diabetic SGCs), *P = 0.0295 (diabetic versus non-diabetic SGCs). 1 day: ****P < 0.0001 (vehicle versus non-diabetic SGCs). 2 days: **P < 0.0014 (vehicle versus non-diabetic SGCs), *P < 0.0123 (vehicle versus diabetic SGCs), ** P < 0.0054 (diabetic versus non-diabetic SGCs). h, Schematic of mitochondrial isolation and intra-DRG injection. i, Analgesic effect of mitochondrial transfer from a non-diabetic donor. Mitochondria from non-diabetic SGCs, baseline versus 1 day: *P = 0.0417; 2 days diabetic versus non-diabetic SGCs: *P = 0.0442. j, Left, images of PGP9.5 staining. Asterisks indicate IENFs. Scale bar, 50 μm. Right, quantification of IENFs. n = 7 per group. *P = 0.0475. Data are mean ± s.e.m. Two-way ANOVA with Tukey’s multiple comparisons tests (b,e,g,i); one-way ANOVA followed by Tukey’s multiple comparisons (c); two-sided unpaired t-test (j). k, Schematic showing potential therapeutic strategies using mitochondrial transfer for neuropathic pain: (1) adoptive transfer of SGCs; and (2) transfer of isolated SGC mitochondria into the DRG. Source Data

Extended Data Fig. 1. Characterization of mitochondrial transfer in mouse DRG cultures.

(a) Immunostaining FABP7, Kir4.1, AQP4, CX43, and GS in mouse SGC cultures, along with quantification of the percentage of marker-positive cells. (b) Triple staining showing mitochondrial (red arrow) transfer from a FABP7-labeled SGC to a Trpv1+ neuron. n = 3 biological repeats. (c-d) Schematic (c) and representative live-cell imaging (d) of TNT formation between SGCs and neurons. Top panels in d: MitoTracker labeling. Red arrows indicate mitochondria within a TNT, and white arrows indicate TNT breakage. Bottom panels in d: contrast images showing a broken TNT (white arrow). (e-f) Schematic (e) and representative images (f) showing mitochondrial transfer from SGCs to neurons in vehicle- and TTX-treated groups. White arrows indicate TNTs and red arrows indicate mitochondria (Mito). (g) Quantification of MitoTracker signal density in neurons interacting with SGCs. n = 31 neurons (TTX) and n = 32 neurons (vehicle) from 3 independent experiments. **** P < 0.0001. (h) Percentage of TNT-positive neurons. n = 60 neurons (TTX) and n = 66 neurons (vehicle) from 3 independent experiments. (i) Top, schematic of SGC and neuron co-cultures treated with different inhibitors. Bottom, representative MitoTracker images showing inhibitory effects of CytoB, Y-27632, Pitstop 2, and CBX on mitochondrial transfer from SGCs to neurons. Arrow indicates TNT. Scale bars are as indicated. (j) Quantification of MitoTracker signal density in neurons interacting with SGCs in 5 different groups. n = 41 (vehicle), 33 (CytoB), 32 (Y-27532), 30 (Pitstop2), and 37 (CBX) over 4 independent experiments each group. **** P < 0.0001, * P = 0.0187, * P = 0.0377. (k) Percentage of TNT-positive neurons showing inhibitory effects of CytoB, Y-27632, and CBX on TNT formation. Sample sizes are as indicated in j. Data are shown as means ± s.e.m. Unpaired t-test (two-sided, g), one-way ANOVA followed by Tukey’s multiple comparisons test (j). Source Data

Extended Data Fig. 2. Characterization of mitochondrial transfer in mouse whole-mount DRG preparations.

(a) Schematic of the ex vivo co-culture setup: SGCs were labeled with MitoTracker and co-cultured with half-cut whole-mount DRG tissue. (b) Low-magnification image of MitoTracker fluorescence showing strong signals in DRG cells, TNTs, and SGCs. Notably, SGC-derived TNTs extend to the edge of the DRG (TNT-4/5, white arrow) and penetrate deep into the tissue (TNT-1 to −3, white arrows). The DRG boundary is outlined by a dotted line. n = 7 biological repeats. (c) High-magnification image (enlarged from boxed area in b) showing TNTs (TNT-1 to −3, white arrows) between SGCs and neurons (blue arrows) within the DRG. (d) Schematic of co-cultures using primary SGC or macrophage cultures and whole mount DRG from Trpv1:Ai9 reporter mice. (e) Top panels: representative MitoTracker (red) and Ai9 (green) fluorescence images of DRG co-cultures with SGCs without (left) and with CytoB treatment (middle), and with macrophages (right). DRG boundary is marked with a dotted line. Scale bars are as indicated. Bottom panels: high-magnification images (enlarged from boxed area in top panels) showing mitochondrial transfer to Trpv1+ neurons and the inhibitory effect of CytoB (middle). Notably, TNT-like structures were observed between SGCs and neurons (left and middle, indicated by arrows), but not between macrophages and neurons (right). In the left panels, strong red fluorescence obscures TNT visibility. Scale, 50 μm. (f) Quantification of MitoTracker signal density in neurons interacting with SGCs, demonstrating the effect of CytoB. n = 5/group. * P = 0.0379. (g) Comparison of mitochondrial transfer to DRG neurons from SGCs versus macrophages. n = 5/group. P = 0.6181. Data are shown as means ± s.e.m. Mice from both sexes were included. Unpaired t-test (two-sided). Source Data

Extended Data Fig. 3. Scanning electron microscopy (SEM) imaging of mouse DRG and structured illumination microscopy (SIM) imaging of mitochondria in SGC cultures.

(a) Schematic of the trypsin-treated whole mount DRG preparation and SEM imaging. (b) Representative SEM images showing destruction of the tube structures in DRG after trypsin treatment. n = 2 biological repeats. (c) Schematic for the paclitaxel (PTX) model using intraperitoneal injections (2 mg/kg, every other day, four injections). (d) Representative SEM images showing gap between SGCs and neurons in the DRG at 1-, 2-, and 4-weeks post-paclitaxel treatment. “N” indicates sensory neuron; “S” indicates SGC; and arrow points to TNT-like structures; gap between the SGC and the neuron is also indicated. (e) Quantification of the percentage of DRG neurons showing increased gaps with SGCs one-week (n = 4), two-week (n = 4), and four-week (n = 3) post PTX treatment compared to naïve mice (n = 4). *** P = 0.0003, ** P = 0.0044, * P = 0.0495. (f) Quantification of TNT-like structures in the gaps between SGCs and neurons showing a significant increase at 4 weeks compared to 1-week post-PTX. n = 4 (PTX-1w), 4 (PTX-2w), and 3 (PTX-4w). * P = 0.021, P = 0.1513. (g-i) SIM imaging of mitochondria and the effects of PTX in SGC cultures. (g-h) Representative SIM images of mitochondria before (g) and 15 min after treatment with 1 μg/mL PTX (h). Right panels showing enlarged views of the boxed areas from the left panels. Arrows indicate mitochondria with short-length. Scale bars are as indicated. (i) PTX treatment increases the short-length mitochondria in SGCs. n = 6/group. *** P = 0.0007. Data are shown as means ± s.e.m and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (e and f). Unpaired t-test (two-sided, i). Mice from both sexes were included. Source Data

Extended Data Fig. 4. Transmission electron microscopy (TEM) imaging of mouse DRG.

(a) Low magnification TEM image showing DRG neurons and SGCs. n = 4 biological repeats. (b) Enlarged image (from the boxed area in a), showing a TNT-like structure (blue dotted line). (c) Enlarged image (from the boxed area in b), showing a tube containing mitochondria (red arrows) and vesicles (orange arrows), as well as ER from adjacent SGC and neuron. Black and green asterisks (*) indicate the nuclei of SGCs and neurons, respectively; ER: endoplasmic reticulum. Scale bars are as indicated.

Extended Data Fig. 5. Characterization of mitochondrial transfer in DRG of MitoTag mice.

(a) Schematic of the immunocapture of mitochondria in Aldh1l1:MitoTag mouse DRG. (b) Western blot showing the expression of GFP and mitochondrial marker COX4 in crude mitochondrial fraction (CMF), immunocapture (IP), and supernatant (Sup) groups. n = 2 biological repeats. (c) DRG fluorescence image of Aldh1l1:MitoTag mice without tamoxifen injection. n = 2 biological repeats. (d) Colocalization of MitoTag fluorescence signal with mitochondrial marker TOM20 in DRG section. (e) Schematic of experimental design of tamoxifen (Tam) treatment (5 daily injections) with DRG tissue collection on day 10, and additional treatment with Pitstop2 and streptozotocin (STZ). (f) Representative DRG images of MitoTag green fluorescence. Bottom panels: enlarge boxed areas from the top panels. Red and blue arrows indicate MitoTag positive and negative neurons, respectively. n = 4 biological repeats in control, Pitstop2, STZ-5d groups, and n = 3 biological repeats in STZ-15d group. (g) Schematic of experimental design of tamoxifen (Tam) treatment with DRG tissue collection on day 5 and additional treatment with complete Freund’s adjuvant (CFA). (h) Representative DRG images of MitoTag green fluorescence. Bottom panels: enlarge boxed areas from the top panels. Red and blue arrows indicate MitoTag positive and negative neurons, respectively. Scale bars are as indicated. Related to Fig. 2b, c. (i) Schematic of pain behavioral testing before (baseline, BL) and after spared nerve injury (SNI) surgery combined with peri-sciatic nerve implantation of bupivacaine-loaded film in Aldh1l1:MitoTag mice. (j) von Frey testing showing paw withdrawal threshold. Data are shown as means ± s.e.m. and analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test. **** P < 0.0001. Source Data

Extended Data Fig. 6. Characterization of mitochondrial transfer in the spinal cord dorsal horn of Aldh1l1: MitoTag mice and SEM imaging in the dorsal horn.

(a) Left: Timeline of tamoxifen treatment (5 daily injections) followed by spinal cord tissue collection at day 0, day 5, and day 10 from Aldh1l1:MitoTag mice. Right: MitoTag fluorescence imaging showing no signal at day 0 without tamoxifen treatment. (b-c) In the spinal cord dorsal horn, MitoTag signal is only present in astrocytes (GFAP+) but not in neurons (NeuN+) 5 days (b) and 10 days (c) after tamoxifen treatment. Boxed areas are enlarged in the bottom panels showing Aldh1l1:MitoTag fluorescence (bottom left) and triple staining of Mitotag/GFAP/NeuN (bottom right). Notably, no mitochondrial transfer was observed in neurons (NeuN+). n = 3 biological repeats. (d-e) SEM imaging on the L4-L5 segment of mouse spinal cord dorsal horn. (d) Schematic of tissue preparation for SEM imaging. (e) Left: low-magnification SEM image showing dorsal root, dorsal horn (grey matter), and white matter. Dotted curve line indicates the dorsal horn boundary. Middle: enlarged boxed area from the left panel. Right: enlarged boxed area from the middle panel. Arrows indicate the cilia-like structures. n = 4 biological repeats. Scale bars are as indicated.

Extended Data Fig. 7. Characterization of mitochondrial transfer in the DRG of Advillin: MitoTag and Cx3cr1: MitoTag mice.

(a) Schematic of crossing Advillin-cre and MitoTag mice. (b) Images of DRG sections showing Advillin:MitoTag (green) and FABP7 (red) fluorescence signal. Red arrows indicate colocalization of MitoTag and FABP7; blue arrows indicate FABP7 without MitoTag. (c) DRG section images showing Advillin:MitoTag fluorescence signal (green) and Iba1 staining (red). Red arrows indicate colocalization of MitoTag and Iba1, and blue arrows indicate macrophages (Iba1+) without MitoTag. (d) Diagram of crossing Cx3cr1-cre/ERT2 and MitoTag mice, along with the timeline of tamoxifen injection and DRG collection at day 5 and day 10. (e-f) MitoTag fluorescence signal (green), Iba1 signal (red), and Nissl signal (gray) at 5 days (e) and 10 days (f) after tamoxifen injection. Red arrows indicate colocalization of MitoTag and Iba1 in macrophages, and blue arrows indicate neurons without MitoTag. n = 3 biological repeats (b, c, e, and f). Scale bars are as indicated.

Extended Data Fig. 8. Additional characterization of ex vivo calcium imaging in mouse DRG.

(a) Representative images of calcium signal (jRGECO1a⁺, red) and mitochondrial signal (MitoTag⁺, green) in DRG neurons of Aldh1l1-Mito mice with tamoxifen (Tam) treatment for 5 days, followed by tissue collection on day 5. Left, low-magnification image showing both red and green channels. The boxed area was enlarged in the right panels for single red or green channel. G1 and G2 indicate two GECO1a-positive neurons. M1 indicates a mitoTag-labeled neuron. n = 3 biological repeats. (b-c) Heatmap of MitoTag- and jRGECO1a-positive neurons from (a) with MitoTag (green) and jRGECO1a (red) fluorescence signal. (d) Lack of MitoTag and jRGECO1a co-expression in 20 MitoTag⁺ neurons and 81 jGECO1a+ neurons in naïve mice without nerve injury. (e) Cell size frequency distribution of total DRG neurons (n = 12 DRG from 4 mice), MitoTag⁺ neurons (n = 9 DRG from 3 mice), and jRGECO1a⁺ neurons (n = 9 DRG from 3 mice) 5 days after SNI surgery. (f) Representative images of calcium indicator jRGECO1a in DRG of Aldh1l1:MitoTag mice following KCl treatment (60 mM, 1 min). Scale bars are as indicated. (g) Size frequency distribution of the jRGECO1a⁺ neurons in DRG of naïve mice with KCl treatment, demonstrating extensive activation in neurons with various diameters. n = 3. Data are shown as means ± s.e.m. Sample sizes are indicated by individual dots inside columns. Source Data

Extended Data Fig. 9. Behavioral and SEM analyses following Myo10 si-RNA knockdown and Myo10 knockout in mice.

(a) Triple staining (left) of MYO10 (red), FABP7 (green), and DAPI (blue) and double staining (right) showing co-localization of MYO10 and FABP7 in SGCs of mouse DRG. n = 3 biological repeats. (b-c) Altered mechanical pain sensitivity following siRNA knockdown of Myo10 in naïve mice. (b) Schematic of intra-DRG injection of si-RNA (Myo10, 4 μg in 1 μl) and associated pain behavior testing. (c) von Frey test showing decreased paw withdrawal threshold 3 days after Myo10-targeting siRNA treatment. n = 12 mice per group. ** P = 0.004. (d) Schematic of the intra-DRG injection of si-RNA (Myo10) and SEM imaging. (e-f) SEM images of DRG from control mice (e) and mice with si-RNA knockdown of Myo10 (f), showing a loss of TNT-like structures after Myo10 knockdown. Arrow indicates a TNT-like tube. Scale bars are as indicated. (g-i) Behavioral test in WT and Myo10^+/− mice. (g) Schematic of mechanical and thermal pain tests in wild-type (WT) and Myo10^+/− mice. (h) Paw withdrawal frequency to 0.16 g von Frey filament, showing heightened response in Myo10^+/− mice (n = 6) compared to WT mice (n = 6). *** P = 0.0003. (i) Hargreaves test showing the decreased paw withdrawal latency in Myo10^+/− mice (n = 6) compared to WT mice (n = 6). * P = 0.0351. Data are shown as means ± s.e.m. and analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test (c). Unpaired t-test (two-sided, h and i). Source Data

Extended Data Fig. 10. CytoB treatment blocks mitochondrial transfer to mouse neurons in vivo and in vitro.

(a-c) CytoB treatment blocks mitochondrial transfer to mouse neurons in vivo. (a-b) Representative images of vehicle-treated (a) and CytoB-treated (b) mice 1 day following DRG microinjection of MitoTracker dye. (a) DRG images from vehicle-treated mice and the box is enlarged in right panels showing MitoTag, GFAP, Nissl, and merged images. Arrows indicate MitoTracker-positive neurons. (b) DRG images from CytoB-treated mice and the box is enlarged in right panels showing MitoTag, GFAP, Nissl, and merged images. Notably, MitoTag labeling in neurons is blocked by CytoB. (c) Quantification of MitoTracker density (left) and the percentage of Mito+ neurons (right). n = 6/group. *** P = 0.0004; ** P = 0.0024. (d-f) CytoB treatment blocks mitochondrial transfer to healthy mouse neurons in vitro. (d) Left panel: schematic of DRG neuron-SGC co-cultures under 3 different conditions: 1) no treatment, 2) SGCs treated with PTX (1 μg/ml, 1 h), 3) SGCs treated with both PTX and CytoB (3.5 μM, 24 h). Right panels: SGC-neuron co-culture images of bright-field (grey color) and JC-1 aggregate signal (red color) under different conditions as indicated. Scale bar is indicated. (e) Quantification of JC-1 aggregate signal density in co-cultures. n = 12/group. *** P = 0.0002, ** P = 0.0039, * P = 0.0445, P > 0.9999, n.s., no significance. (f) Oxygen consumption rate (OCR) showing an inhibitory effect of CytoB treatment (3.5 μM) in DRG SGC-neuron co-cultures. n = 6/group. ** P = 0.0086. Data are shown as means ± s.e.m. and analyzed by Unpaired t-test (two-sided, c and f) and Two-way ANOVA followed by Sidak’s multiple comparisons test (e). Mice from both sexes were included. Source Data

Extended Data Fig. 11. Mitochondrial transfer from SGCs to neurons prevents chemotherapy-induced neuronal hyperactivation and oxidative stress.

(a) Calcium imaging of DRG neurons co-cultured with SGCs from Advillin:GCamp6f mice before (baseline) and after capsaicin perfusion (100 nM, 2 min), demonstrating the effects of PTX (1 μg/mL) and CytoB (3.5 μM). Scale bar: 100 μm. (b-c) Traces of calcium responses to capsaicin and KCl (b), and Amplitudes of capsaicin-induced calcium responses, showing the effects of paclitaxel and CytoB (c) in dissociated DRG neurons under four indicated conditions. n = 288 neurons (vehicle), 296 neurons (PTX), 110 neurons (PTX + SGCs + vehicle), and 101 neurons (PTX + SGCs + CytoB). **** P < 0.0001, P = 0.1938, * P = 0.022. (d) Reactive oxygen species (ROS) assay in DRG SGC-neuron co-cultures showing the effects of paclitaxel and CytoB. n = 12 cultures/group. ** P = 0.0046, * P = 0.0209, * P = 0.024. (e-f) TRPV1 antagonist capsazepine (CPZ) blocks PTX-induced calcium response in DRG neurons. (e) Traces of calcium responses to capsaicin (100 nM, 2 min) in dissociated DRG neurons with PTX and CPZ (100 μM) treatment in 4 different groups. (f) Amplitudes of calcium responses. n = 162 (vehicle), 157 (PTX), 198 (PTX + SGCs + vehicle), and 200 (PTX + SGCs + CytoB). P = 0.9704, P = 0.5901. Data are shown as means ± s.e.m. and analyzed by One-way ANOVA followed by Tukey’s multiple comparisons test (c, d, and f). n.s. no significance. Mice from both sexes were included. Source Data

Extended Data Fig. 12. Chemotherapy impairs mitochondrial transfer from SGCs to nerve axons and causes nerve degeneration.

(a) Schematic of adoptive transfer of SGCs to DRG. (b) Mitochondrial fluorescence in neurons and SGCs in DRG. White and red arrows indicate MitoTracker⁺ SGCs and neurons, respectively. (c) PTX treatment decreased OCR in cultured SGCs. n = 6 per group. *** P = 0.0004. (d) Mitochondrial fluorescence signal in the spinal nerve after adoptive transfer of SGCs. (e) Quantification of integrated density of MitoTracker fluorescence in vehicle (n = 6) and PTX-treated (n = 5) group. **** P < 0.0001. (f) Left, schematic of neuron-SGC co-cultures. Right, βIII-tubulin immunostaining showing axonal outgrowth of neurons. (g) Quantification of axonal outgrowth in neuron-SGC co-cultures. n = 15 neurons per group from three independent experiments. **** P < 0.0001, *** P = 0.0002, * P = 0.0472. (h) Schematic of microinjection of CytoB and Antimycin A into DRG. (i) Paw withdrawal threshold decreased after CytoB and Antimycin A treatment. n = 6/group. *** P = 0.0008, P = 0.3112, ** P = 0.0012; *** P = 0.0008, P = 0.9875, *** P = 0.0005; ** P = 0.001, P = 0.07, *** P = 0.0005; *** P = 0.0008, ** P = 0.0029, *** P = 0.0006; *** P = 0.0008, ** P = 0.0065, *** P = 0.0005. (j) Representative images of PGP9.5 staining and quantification of IENF density. Asterisks (*) indicate IENFs. n = 6/group. ** P = 0.0015, *** P = 0.0004. (k) Representative images of PGP9.5 staining (k) and quantification of IENF density (l) in naïve and PTX-treated group. n = 5/group. **** P < 0.0001. Data are shown as means ± s.e.m. and were statistically analyzed by unpaired t-test (two-sided, c, e, and l), One-way ANOVA followed by Tukey’s multiple comparisons test (g and j), and Two-way ANOVA followed by Tukey’s multiple comparisons test (i). n.s., not significant. Source Data

Extended Data Fig. 13. SEM images of human DRG from non-diabetic and diabetic donors.

(a) Schematic of non-diabetic human donors for whole mount DRG experiment and SEM imaging. (b) Left, low-magnification SEM image. Middle, enlarged box area from the left. Right, enlarged boxed area from the middle. Arrow points a tube-like structure with a bulge. n = 3 biological repeats. (c) Schematic of non-diabetic human donors for sectioned DRG experiment and SEM imaging. (d) low magnification image of N1 and N2 neurons. (e) Left, enlarged boxed area of d, showing multiple SGCs (S1 to S9). Right, enlarged boxed area of the left, showing TNT-like structure with a bulge (arrows) near S1. n = 3 biological repeats. (f) Schematic of diabetic human donors for sectioned DRG experiment and SEM imaging. (g) Left, SEM image of a neuron (N) and surrounding SGCs (S1 to S7). Right: enlarged boxed area from the left, showing micrometer gap between SGCs and neurons and an unsmooth TNT-LS (indicated by a blue arrow). n = 3 biological repeats. (h) A neuron is surrounded by multi-layers of non-neuronal cells (presumably SGCs) in diabetic DRG (indicated by black circles). n = 3 biological repeats. Scale bars are as indicated.

Extended Data Fig. 14. snRNA-seq transcriptomic analysis reveals six major cell populations in healthy human DRG.

(a-b) UMAP plots showing SGC markers FABP7 and EDNRB (a) and immune cell markers CSF1R and LY86 (b). (c) UMAP plots reveal seven neuronal populations including: 1) neurofilament population (FXYD7+), 2) Aβ-LTMR (HS3ST4 + ), 3) proprioceptors (NXPH1+), 4) peptidergic nociceptors (GAL + ), 5) C-fiber low threshold mechanoreceptor (GFRA2+/ and POU4F2+), 6) somatostatin-positive pruriceptors (SST+), and 7) cold-sensing TRPM8+). (d-f) UMAP plots showing Schwann cell markers MPZ and GLDN (d), connective tissue marker COL1A1 (e), and endothelial cell marker PECAM1 (f).

Extended Data Fig. 15. Comparison of mitochondrial transfer from SGCs to neurons versus neurons to SGCs in human DRG cell cultures.

(a) Schematic of two different mitochondrial transfer approaches between SGCs and neurons: 1) MitoTracker labeled SGCs and co-culture with neurons and 2) MitoTracker labeled neurons and co-culture with SGCs. (b-c) Representative images of MitoTracker labeled mitochondrial transfer from SGC to neuron (b) and from neuron to SGC (c). White arrows point to TNTs, and red arrows point to mitochondria within TNTs. Scale bar: 20 μm. (d) Quantification of mitochondrial transfer efficiency as the receiver/donor ratio of MitoTracker signal intensity in two groups. Data are shown as means ± s.e.m. Unpaired t-test (two-sided). **** P < 0.0001. n = 21 cells (left column) and n = 25 cells (right column) from 2 to 4 independent experiments. (e) Percentage of MitoTracker-positive and TNT-positive neurons of the SGC-neuron co-cultures from human DRG. A total of 54 neurons from 6 independent experiments were included for quantification. Source Data

Extended Data Fig. 16. Effects of adoptive transfer of mouse mitochondria on evoked and spontaneous pain in paclitaxel-injected mice and OCR analysis in human DRG SGCs.

(a-f) Mitochondrial uptake by DRG cells following intra-DRG injection of MitoTracker labeled mitochondria. (a) Schematic of mitochondrial isolation from MitoTracker labeled SGCs and intra-DRG injection. (b-e) Low magnification DRG images showing triple staining of MitoTracker labeled mitochondria (red), FABP7-labeled SGCs (green), and NeuN-labeled neurons (blue) from untreated mice (b-c), CytoB (3.5 μM)-treated mice (d), and Pitstop2 (25 μM)-treated mice (e). (c) Enlarged image from the box in b showing adoptively transferred mitochondria in both NeuN+ neuron (blue arrow) and FABP7+ SGCs (white arrows). Bottom panels in (d-e) are enlarged images from the boxed areas in top panels. Scale bars are as indicated. (f) Mito density quantification shows that MitoTracker+ mitochondrial uptake in DRG neurons and SGCs is blocked by Pitstop2, not by CytoB. n = 4 (Mitochondrial + vehicle), n = 3 (Mitochondrial + CytoB), and n = 3 (Mitochondrial + Pitstop2). ** P = 0.0066, P = 0.3434; n.s., no significance. (g-j) Paclitaxel-induced evoked pain and spontaneous pain and the effects of mitochondrial transfer. (g) Schematic of mitochondrial isolation from mouse SGCs, treatment with the mitochondrial complex III inhibitor myxothiazol (1 mM, 10 min), and intra-DRG injection in the PTX model. (h) Analgesic effects of adoptively transferred mitochondria and its blockade by myxothiazol. n = 10/group. **** P < 0.0001, *** P = 0.0003; **** P < 0.0001, *** P = 0.0003. (i-j) Assessment of spontaneous pain with conditioned place preference test (CPP) scores. * P = 0.0224. n = 10 mice/group. (k) OCR analysis of SGC cultures of human DRG from diabetic and non-diabetic donors, showing mitochondrial dysfunction in diabetes. n = 10 cultures/group. * P = 0.0477. Data are shown as means ± s.e.m. and were statistically analyzed by One-way ANOVA followed by Tukey’s multiple comparisons test (f), Two-way ANOVA followed by Sidak’s multiple comparisons test (h), and unpaired t-tests (two-sided, j and k). Mice from both sexes were included for analysis. Source Data

Extended Data Fig. 17. Schematic illustrations of mitochondrial transfer from SGCs to neurons in DRG in health, disease (CIPN/DPN), and treatment conditions.

Top, SGC-neuron mitochondrial transfer in health conditions in physiological status. Middle, disruption of SGC-neuron mitochondrial transfer in peripheral neuropathy-associated diseases, such as chemotherapy-induced peripheral neuropathy (CIPN) and diabetic peripheral neuropathy (DPN), induces nerve degeneration and neuropathic pain. Bottom, therapeutic approaches targeting mitochondrial transfer via SGC transfer or mitochondrial transfer can enhance nerve regeneration and alleviate neuropathic pain.