
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

39229068
10.1101/2024.08.21.608803
preprint
2
Article
Keratinocyte-Derived Exosomes in Painful Diabetic Neuropathy
http://orcid.org/0000-0003-1173-6331
Coy-Dibley James 1
Jayaraj Nirupa D. 1
Ren Dongjun 2
http://orcid.org/0009-0004-2200-451X
Pacifico Paola 1
Belmadani Abdelhak 12
http://orcid.org/0000-0001-5279-5034
Wang Yi-Zhi 1
Gebis Kamil K. 1
Savas Jeffrey N. 1
Paller Amy S. 3
Miller Richard J. 2
Menichella Daniela M. 12^
1 Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
2 Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
3 Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
^ Corresponding author: Daniela Maria Menichella
03 9 2024
2024.08.21.608803https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
nihpp-2024.08.21.608803.pdf
Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Exosomes are small extracellular vesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived exosomes (KDEs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEs in a well-established high-fat diet (HFD) mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEs through size exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of HFD KDEs compared to KDEs obtained from control mice on a regular diet (RD), including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle (EV) reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEs are retrogradely trafficked into the DRG neuron cell body. Overall, our study presents a potential novel mode of communication between keratinocytes and DRG neurons in the skin, revealing a possible role for KDEs in contributing to the axonal degeneration that underlies neuropathic pain in PDN. Moreover, this study presents potential therapeutic targets in the skin for developing more effective, disease-modifying, and better-tolerated topical interventions for patients suffering from PDN, one of the most common and untreatable peripheral neuropathies.
==== Body
pmcINTRODUCTION

Diabetes mellitus affects 29.3 million adults with pre-diabetes in an additional 115.9 million1. Painful diabetic neuropathy (PDN) is a disabling, intractable, and common syndrome occurring in approximately 25% of diabetics2–5. The associated neuropathic pain significantly impacts the quality of life for patients6. Despite the high prevalence and impact, current therapies for PDN have limited effects in treating pain7–10, fail to remediate the damage to nerves, and have side effects associated with their systemic administration10–12. Therefore, there is an urgent need for better tolerated and more effective therapies for PDN.

PDN is characterized by neuropathic pain associated with dorsal root ganglion (DRG) nociceptor hyperexcitability and the degeneration with loss or retraction of the cutaneous DRG neuron axons that innervate the skin (small fiber neuropathy)13,14. A critical barrier to developing effective treatments for PDN is the lack of understanding of the molecular mechanisms leading to neuropathic pain and small fiber neuropathy.

Keratinocytes are the most abundant epidermal cell type. Recent studies have discovered a new role for keratinocytes in mediating innocuous and noxious touch and thermal sensation in healthy skin15. Keratinocytes detect touch stimuli in the skin and transmit mechanical information related to pressure and brushing16,17. Optogenetic inhibition of keratinocytes in vivo inhibits the responses to noxious mechanical and thermal stimuli17,18. There is also evidence that keratinocytes may contribute to persistent neuropathic pain. A study involving the transplantation of human keratinocytes into rodents with transected nerves showed increased excitability of DRG neurons and chronic pain in vivo19. However, the specific role of keratinocytes in PDN has not been widely investigated.

The skin is a highly complex biological system. Along with nociceptive DRG neurons, various other neuronal subpopulations terminate in the skin, both in the dermis and the epidermis5. Keratinocytes are closely juxtaposed to cutaneous nerve terminals, suggesting that there may be bidirectional communication. Interestingly, in rodents and human skin, cutaneous nerve terminals in the epidermis form synapse-like contacts and also tunnel through keratinocytes, where they form connexin 43-positive gap junctions, enabling direct cellular communication20,21. However, the functional implications of such observations remain unclear. One such ubiquitous mode of intercellular communication recently garnering more appreciation in the skin is mediated by extracellular vesicles (EVs).

Exosomes, which are small EVs composed of lipids, proteins and nucleic acids22–25, were initially posited to be involved in removing cellular waste26. However, a substantial body of research now suggests a wider role in intercellular communication. Exosomes are released from most cell types and have been linked to several neurogenerative diseases27,28 and the progression of different cancers29. Keratinocyte-derived exosomes (KDEs) have demonstrated their ability to modulate melanocyte pigmentation30, regulate dermal fibroblast gene expression31, mediate crosstalk with macrophages in cutaneous wound healing32, and play a crucial role in dermal immune responses in psoriasis33,34. Additionally, EVs derived from mesenchymal stem cells can directly alter the excitability of DRG nociceptors in mice35. Exploration of the role of exosomes in diabetes, however, has primarily focused on adipose tissue and inter-organ communication36–38. Recent studies have unveiled a potential role for exosomes in impaired wound healing associated with diabetes39. Yet, research on the effects of exosomes on diabetic neuropathy is limited and has been conducted using exosomes isolated from mesenchymal40,41 or Schwann cells42. Notably, exosomes isolated from mesenchymal stromal cells have shown promise in ameliorating peripheral neuropathy in a mouse model of diabetes40. Conversely, exosomes derived from high glucose-stimulated Schwann cells have been found to promote the development of diabetic neuropathy in mice42. A rigorous and comprehensive investigation of keratinocyte-derived exosomes and their role in PDN represents an important, yet understudied frontier in pain and peripheral neuropathy research.

Using size exclusion chromatography, we obtained enriched keratinocyte-derived exosomes from mice and performed an unbiased molecular cargo characterization with proteomics and small RNA sequencing. We found significantly altered protein and microRNA content of keratinocyte-derived exosomes from high-fat diet (HFD)-induced PDN compared to regular diet (RD) control mice. Altered pathways were involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional EV reporter mouse line, we demonstrated that epidermal-originating GFP-tagged keratinocyte-derived exosomes are retrogradely trafficked into the DRG neuron cell body. Overall, we present evidence that supports keratinocyte-derived exosomes as a novel interaction pathway between epidermal keratinocytes and DRG neurons and that altered cutaneous EV-trafficking may play a functional role in the development of the small fiber neuropathy observed in PDN.

RESULTS

Keratinocytes release a diverse population of exosomes.

To study keratinocyte-derived exosomes (KDEs), we fractionated cell-conditioned medium (CCM) from cultured adult mouse primary keratinocytes17,18 using a qEV 35-nm size-exclusion chromatography column43 (Figure 1A). We observed an increasing total protein concentration with each successive fraction (Figure 1B; silver stain) and immunoblotted for known exosome markers. Fractions 2 and 3 were enriched with the exosome-associated cargo markers Alix, Tsg101, and Syntenin-1 as well as the transmembrane tetraspanins CD63 and CD8123–25,44. These fractions were devoid of GM130 and Calnexin, which are Golgi-associated proteins45 (Figure 1B). Thus, pooled fractions 2 and 3, representing our highly enriched KDE fractions with minimal free-floating protein contamination, were used for all subsequent analyses46.

To investigate the role of KDEs in painful diabetic neuropathy, we employed the clinically relevant and well-established HFD model of PDN47–50, developed by feeding mice a high fat diet for ten weeks, leading to obesity, glucose intolerance and mechanical allodynia with small-fiber degeneration48–50. Cultured keratinocytes from the RD and HFD mice were grown to 90% confluency in low-calcium, EV-depleted medium for scratch assays. The HFD keratinocytes had impaired wound healing during the 3 days of observation (Supplemental Figure 1A–B), consistent with what has been shown in scratch assays with high glucose exposure51 and what is observed in diabetic patients52. Fraction 2/3 KDEs had sizes within the range of exosomes of 59.2 ± 14.5nm (RD; Figure 1C) and 70 ± 26.3nm (HFD; Supplemental Figure 2B) by dynamic light scattering (DLS) and concentration peaks at 95 ± 6.1nm (RD; Figure 1D) and 86 ± 8.7nm (HFD; Supplemental Figure 2C) by nanoparticle tracking analysis (NTA). Tunable resistive pulse sensing (TRPS) determined that the mean size average for RD KDEs were 97.7 ± 4.5nm with a concentration peak of 72.3 ± 4.5nm (Figure 1E). KDEs from both RD and HFD were visualized via negative staining (Figure 1F) and cryo-electron microscopy (Figure 1G), which revealed the expected crescent morphology and intact vesicular structure.

Keratinocytes release soluble factors that encourage DRG neuron axonal growth.

Given that loss of cutaneous innervation is reported in PDN13,14,49, we next sought to investigate the role of KDEs on DRG axonal growth. We employed a microfluidics co-culture system with primary adult mouse DRG neurons in one chamber and adult mouse keratinocytes in the other (Figure 2A). This setup allowed for cell medium exchange only through the microchannels between the chambers. Interestingly, we found that DRG neuronal axons crossed the microchannels separating the two chambers at a higher rate when grown in a co-culture with keratinocytes compared to when grown alone, indicating that keratinocytes release soluble growth factors that promote axonal growth (Figure 2B–C).

To test whether mouse KDEs are functional, we labeled them with DIR, which is a carbocyanine DiOC18(7) lipophilic fluorescent dye, and added them to primary DRG neuron cultures. We observed a robust uptake of these KDEs by both the cell bodies and neurites of the neurons (Figure 2D). To better model physiological relevance, we then cultured DRG neurons in one chamber of our microfluidic system and allowed their neurites to occupy all the microchannels, thus preventing the free flow of medium between chambers. We then added DIR-labeled KDEs to the empty chamber to test whether they could be transported through the neurites to the DRG neuron cell body. Indeed, DIR-labeled KDEs were readily detected in the DRG neuron cell bodies 16 hours post application, suggesting the retrograde transport of DIR-labeled KDEs through the neurites53 (Figure 2E).

Keratinocyte-derived exosomes alter their protein cargo in painful diabetic neuropathy.

We characterized the KDE protein content in the context of PDN using a proteomic approach54. The analysis was performed on the pooled fractions 2/3 using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for both RD and HFD KDEs (Figure 3A). A gene ontology enrichment analysis on proteins detected in pooled fractions 2/3 for both groups clustered in EV categories, suggesting a robust small EV enrichment for both RD and HFD KDEs (Figure 3B). Importantly, we found similar quantities based on the number of spectral counts of the canonical exosome-associated proteins Alix, Tsg101, and Syntenin-1 in both samples (Figure 3C). Fractions 2/3-enriched proteins also significantly clustered in the GO enrichment term ‘Axon Development,’ (Figure 3C; Insert), suggesting neuron-keratinocyte communication via exosomes. Moreover, we identified 90 differentially expressed exosome-associated proteins (EAPs; FC ≥ 1.5, p < 0.05) between RD and HFD KDEs, with biological replicates clustering by group (Figure 3D–E). The differentially expressed REACTOME pathway with ‘MAPK Family Signaling Cascades’ included both Mapk1 and Mapk3 (Figure 3G left; Adjusted p-value ≤ 0.05), with Mapk1 found in fraction 2/3 (Figure 3F). Furthermore, a gene ontology enrichment analysis on the EAPs revealed ‘Wound Healing’ as differentially expressed in HFD KDEs (3G middle; Adjusted p-value ≤ 0.05), consistent with the known impairment in diabetic patients55 and as shown in our 2D primary HFD keratinocyte scratch assays (Supplemental Figure 1A–B). Notably, several annexins were differentially expressed, with annexin VII in fraction 2/3 of our immunoblots (Figure 3F); annexins play a role in ESCRT-III mediated plasma membrane repair56–59 and wound healing56. Another differentially expressed pathway was ‘Neurotransmitter Secretion’ (Figure 3G right; adjusted p-value ≤ 0.05), consistent with the physical localization of DRG nerve afferents, which tunnel through the keratinocyte cytoplasm to form synapse-like contacts20,21. Taken together, these differentially expressed GO pathways support an alteration in the neuron-keratinocyte communication pathways in diabetes-related PDN.

The microRNA cargo of keratinocyte-derived exosomes is altered in painful diabetic neuropathy.

Exosomes contain gene-modifying RNAs, with microRNAs being the most abundant RNA species43. Several microRNAs have been associated with pain in diabetic neuropathy60, but the KDE microRNA content has not yet been identified in the context of PDN. Hence, we next investigated the microRNA cargo of RD and HFD KDEs using an unbiased small-library RNA sequencing approach.

The top ten most abundant microRNAs identified, including their variants, accounted for 74.8% of the total small RNA sequenced for both groups (Figure 4A). Additionally, the top two hits, the let-7 and miR-23 families, accounted for almost 40% of the total RNA sequenced for both groups. By cross-referencing 3 separate databases (Figure 2B), the predicted protein targets of these small RNAs were used to run a KEGG enrichment analysis for let-7 and miR-23, with both revealing ‘Axon Guidance’ as a predicted target pathway (Figure 4C). Between the RD and HFD KDEs, there were 35 significant differentially expressed microRNAs that clustered by group (Figure 4D) and 33 with a fold change (FC) ≥ 1.5 (Figure 4E). By cross-referencing the same three databases, the predicted target proteins from each microRNA were run through a gene ontology enrichment analysis with three producing significant target pathways (Figure 4F; Supplemental Figure 3F–G). Both miR-684, which has been implicated in multiple sclerosis61, and miR24–3p, which has been studied in the context of cancers62,63 along with its regulation of proliferation related to annexin-6 activity64, revealed predicted target axon-guidance related pathways (Figure 4G), further providing compelling evidence for a neuron-keratinocyte communication pathway via KDEs under both normal physiology and PDN. Given that both the proteomic and small RNA sequencing datasets revealed predicted target pathways involved in axon guidance and that painful diabetic neuropathy is accompanied by peripheral nerve degeneration49, we next designed an in vivo animal model to investigate the interaction between KDEs and DRG neuron afferent fibers.

Epidermal keratinocyte-derived exosomes are fluorescently labeled with CD63-emGFP.

Fraction 2/3 from our keratinocyte CCM consistently immunoblotted for the transmembrane tetraspanins CD63 and CD8124 (Figure 1B), in addition to the luminal exosome markers Alix, Syntenin-1, and Tsg101. We crossed the commercially available CD63-emGFP fl/fl mouse line (JacksonLaboratory Strain#:036865) and crossed it with K14-Cre mice to generate a K14-CD63-emGFP EV reporter mouse model (Figure 5A) to label exosomes originating from basal layer keratinocytes and their progeny. We observed robust GFP expression throughout the keratinocyte progeny, with K14 staining co-localizing in basal layer keratinocytes, confirming the limitation of expression to the epidermis (Figure 5B). Furthermore, we immunoblotted whole epidermis cell lysate and detected both the membrane-bound CD63-GFP fusion protein and soluble GFP (Figure 5C), presumably due to endogenous protein recycling. We next cultured keratinocytes from this EV-reporter mouse. As expected, we observed a strong GFP signal along the outer membrane of these keratinocytes (Figure 5D). We next immunoblotted all ten fractions obtained from CCM of primary keratinocyte cultures for GFP and detected a strong GFP signal, both the fusion and soluble forms, in fractions 2/3, which corresponded to our KDE enriched fractions (Figure 5E). Furthermore, isolated GFP-labeled KDEs were functionally internalized by DRG neurons with GFP detected in both the neuron cell bodies and neurites (Supplemental Figure 4A). Given the gene ontology enrichment pathways highlighted from our proteomic and small RNA sequencing experiments, we next sought to better understand the neuron-keratinocyte communication pathway using our EV reporter mouse line.

Epidermal keratinocyte-derived exosomes are retrogradely transported to the DRG neuron cell bodies in male and female mice.

We further investigated the in vivo conditional EV-reporter mouse, shifting our focus towards the DRGs. Notably, we detected a GFP signal in DRG cross-sections from both RD and HFD EV-reporter mice (Figure 6A; Supplemental Figure 4B) and not in WT DRG controls (Figure 6B). Additionally, the cell lysate of K14-CD63-GFP (KCG) DRG neurons immunoblotted for GFP (Figure 6C), further supporting the evidence for the retrograde transport of GFP-positive epidermal exosomes to the DRG neuron cell bodies. We next cultured primary DRG neurons from the EV-reporter mouse model for both RD and HFD. We observed robust GFP expression in both groups (Figure 6D) with no GFP signal in WT controls (Figure 6E). The GFP observed in these primary DRG neuron cultures presented in the same punctate pattern observed in the DIR-labeled exosomes (Figure 2D) and GFP-labeled exosomes applied to primary DRG cultures46 (Supplemental Figure 4A). Indeed, we detected these puncta in both the neuron cell bodies and along the neurites (Figure 6F). These data strongly suggest that CD63-GFP-positive keratinocyte-derived exosomes are retrogradely transported from the epidermis to DRG neuron cell bodies, representing a novel mode of communication.

DISCUSSION

We isolated and characterized keratinocyte-derived exosomes (KDEs) morphologically and molecularly using an unbiased proteomic and small RNA sequencing approach. Our research revealed that KDEs alter their cargo in a mouse model of painful diabetic neuropathy (PDN), and we identified several gene ontology enrichment pathways that were differentially expressed. In both “omic” datasets, neuron-keratinocyte pathways were enriched both under normal physiology and in PDN. Additionally, we created a K14-CD63-emGFP extracellular vesicle (EV)-reporter mouse line and observed a GFP signal in the DRG neurons and neurites, suggesting that keratinocyte-derived GFP-tagged exosomes are retrogradely trafficked from epidermal keratinocytes into the DRG neurons of mice.

These findings suggest a direct communication pathway between the epidermis and the peripheral nervous system, which is altered in our HFD mouse model of PDN. In our microfluidic paradigm, the presence of keratinocytes enhanced neurite outgrowth, indicating release of a soluble factor by keratinocytes that encourages neurite growth (Figure 2A–C). Proteomic analysis of our KDEs revealed that they contained several catenin and plexin isoforms (3C; Insert). The canonical WNT-signaling pathway plays a significant role in axon development in the central65,66 and peripheral67 nervous systems and has been implicated in Schwann cell-axon communication68. Plexins, the surface receptors for semaphorins, are involved in neuron axonal growth and guidance, as well as a host of other functions69–72. Semaphorin 4C-Plexin-b2 signaling has been reported to be markedly increased in states of persistent pain in mice, and downregulation of this pathway led to the impairment of inflammatory hypersensitivity via RhoA-ROCK-dependent mechanisms73. Additionally, Sema3A and Plexin A were reported to be dysregulated in the spinal cord of a HFD model of PDN74. To our knowledge, this is the first report of these proteins being present as KDE cargo. Additionally, notch1, which was recently reported to facilitate neuron-to-neuron communication through EVs in the hippocampus of mice46, was detected in our KDEs for all three biological replicates of both diet groups (3C; Insert), suggesting that this mechanism of internalization may also apply to keratinocyte-neuron terminal endings in the skin. Notch signaling is known to play a prominent role in developing neurons, including in axon guidance75,76. Pathogenic variants in the Notch ligand Jagged1 were implicated in the development of peripheral neuropathy in two independent families and confirmed in a mouse model77. Thus, Notch signaling may represent an unexplored, novel communication pathway between keratinocytes and DRG neuron terminal nerve endings.

The KDEs from both groups contain differentially expressed synaptotagmin, which is an essential component of the presynaptic vesicle release complex that facilitates vesicle fusion with the plasma membrane78,79, along with synaptophysin and synapsin-2, both of which are involved in regulating vesicle docking to the inner plasma membrane80–82 (3G; Right Panel). These proteins have all been previously reported in keratinocytes83. However, it is still unclear why these proteins are present as KDE cargo and what their function in keratinocyte-to-neuron communication might be. Further experiments are required to validate all these cargo proteins, both in mice and human KDEs, and to investigate their mechanism of action on nerve terminals.

Our small RNA sequencing dataset aligned with the proteomic data. The top two most abundant small RNAs, let-7 and miR-23 (Figure 4A), were predicted to target several prominent signaling pathways that overlapped with the proteomic dataset, including MAPK signaling and the WNT signaling pathways (Figure 4C). A recent study has indicated that partially inhibiting p38-MAPK activation in a diabetic neuropathy rat model led to anti-hyperalgesic effects, suggesting a significant role for MAPKs in nociception modulation84. KDEs from both our groups contained a diverse range of MAP kinases (MAPKs), with mapk1 differentially expressed in our HFD model. Both let-7 and miR-23 have been shown to modulate MAPK activity85,86. Additionally, two differentially expressed microRNAs suggested altered keratinocyte-to-neuron communication (Figure 4F–G). An altered expression of miR-24–3p has been reported in cancer62,63,87,88 and diabetes89. By cross-referencing three separate databases for predicted targets and running a gene ontology enrichment analysis, one predicted pathway for miR-24–3p modulation is plexin-semaphorin activity, and our proteomic dataset suggested plexin-b2 as a cargo protein (Figure 3C). However, the precise mechanism by which these microRNAs regulate axon guidance and potentially contribute to PDN remains unclear. Further studies are necessary to understand how these microRNAs modulate target proteins and pathways, either directly or indirectly.

The DRG transcriptome is substantially altered in PDN90, resulting in hyperexcitability of the nociceptive neurons that drive neuropathic pain49,90 and presenting a possible druggable system for future therapeutics. In our studies, using an EV-reporter mouse line in which KDEs are labelled with CD63-emGFP (Figure 6A), we detected a GFP signal in the DRG neuron cell bodies and neurites (Figure 6F). This indicates that GFP-containing exosomes originating in the epidermis are retrogradely trafficked into the DRG neuron cell body, where presumably they can initiate transcriptional changes due to their cargo. It should be noted that the GFP species detected, at least from our immunoblotting, was the non-fused form of GFP rather than the CD63-GFP fusion form (Figure 6C), but that soluble GFP was also detected in the epidermis (Figure 5C) and KDE fractions (Figure 5E) in high abundance. It may be that the GFP-tagged exosomes are trafficked to DRG neurons, where they release their transcription-altering cargo, and the fusion protein is subsequently degraded into the soluble GFP that we detected in immunoblotting.

This study enhances our understanding of the communication between keratinocytes and DRG neurons and reveals a new role for KDEs in possibly promoting axonal degeneration, which underlies neuropathic pain in PDN. As many genes show differential expression in DRG neurons in PDN mice compared to control mice91, our studies support a novel strategy for treating PDN by focusing on the skin rather than the entire body. One of the challenges with current PDN treatments is their systemic administration and off-target effects10–12. Here, we present evidence for a delivery pathway from epidermal keratinocytes to the peripheral nervous system, which could be utilized to develop and deliver improved topical treatments for PDN and other nervous system diseases. Additionally, our investigation into the role of exosome-mediated communication between keratinocytes and DRG neurons has broader implications. Indeed, exosomes hold great promise as novel disease biomarkers, therapeutic agents, and drug delivery systems. This potential extends beyond PDN, laying the groundwork for exploring new avenues in pain and peripheral neuropathy research and treatment.

METHODS

Animals.

Animals were housed on a 12-hour light/12-hour dark cycle with ad libitum access to food and water. We used the following mouse lines: K14-Cre, homozygous; CD63-emGFP fl, homozygous; K14-Cre::CD63-emGFP fl heterogenous.

HFD.

Mice were fed 42% fat (Envigo TD88137) for 10 weeks as a rodent model of type 2 diabetes. Control mice were fed a regular diet (RD) of 11% fat. After 10 weeks of RD or HFD, a glucose tolerance test was performed as described49. A cutoff of (≥140 mg/dl) at 2 SD above the mean for glucose 120 minutes after glucose challenge in WT littermate mice was used as a ‘diabetic’ classification49.

Behavioral testing.

von Frey behavioral studies were performed as previously described49 with random experimental group assignments and double-blind investigator and endpoint analysis conditions.

Primary keratinocyte cultures.

Glabrous paw skin is dissected from the mouse and incubated in dispase (2.3 mg/ml) overnight. The epidermis is separated from the dermis and incubated in TrypLE Express (10 min, 37C; Gibco 12604–013); the keratinocytes are dislodged using gentle agitation with forceps and then plated on 15cm2 plates with 154CF epidermal medium (M154CF500) supplemented with 170ul of 0.2M CaCl2 and 5ml of HKGS (S-001–5), which was depleted of EVs following 18 hours of ultracentrifugation at 100,000g. Medium change occurs 24 hours after plating and then every 48 hours. Complete cell culture medium and all other reagents have been confirmed to be EV-free prior to use with the keratinocyte cultures.

Wound healing scratch assay.

Primary keratinocyte cultures were grown on 6-well plates (Fisherbrand FB012927) with a 300,000-seeding density and grown to 90% confluency. The tip of an Eppendorf 200ul pipette tip (Fisher 02707409) was used to form a vertical scratch down the center. Cultures were tracked every 24 hours on a Leica 2000 LED microscope and analyzed using ImageJ software to measure the rate of gap closure. One-tail paired t-tests were used on the raw dataset to obtain a p-value between groups for each time point (n=58 RD, n=61 HFD for each time point across three separate biological replicates for each group; significance p ≤ 0.05).

Exosome isolation with size exclusion columns.

Cell-conditioned medium from keratinocyte cultures is collected between culture confluency of 60–90%, is centrifuged at 3000g for 30 minutes to remove cell debris and is then concentrated to 500 μl using centrifuge size filters (Pierce Protein Concentrators PES 10K, 88528). The sample is then run through an IZON SEC qEV 35nm column (IZON, ICO-35) as previously reported43. Fractions 2–3 are used for downstream applications.

Western Blot.

Fractions 1–10 of 400ul each are concentrated to 20ul using Millipore centrifuge filters (Microcon 30kDa, MRCF0R030) and combined with one volume equivalent of BioRad 2x Lammalenni loading buffer (BioRad 1610737) with 5% BME. Each fraction is run through a 3–15% gradient gel (BioRad 45610840) alongside full keratinocyte cell lysate (+) and a 10K pellet (large EVs), running a BCA assay (Thermo Scientific) to load ~3 μg of protein per well. Protein was then transferred to PVDF membranes (Millipore) and blocked (BioRad Everyblot 12010020) for 15 minutes. Primary antibodies are applied overnight at 4 °C with secondary antibody at room temperature for 2 hours with 3 TBST washes between each step. Proteins are visualized with a chemiluminescence detection system (Thermo Scientific 32209). All western blot gels were run at minimum in triplicate. Blots were visualized on a Li-Cor Odyssey Fc.

Western blot antibodies.

The following primary antibodies were used: Alix (Abcam ab88388), Syntenin-1 (Abcam ab19903), Tsg101 (Invitrogen PA531260), CD63 (Invitrogen 2H5I1), CD81 (Abcam ab109201), GM130 (Abcam ab52649), Calnexin (Abcam ab22595), K14 (BioLegend 906004), GFP (Abcam ab13970), β-tubulin (ProteinTech 80713–1-RR100UL), GAPDH (Abcam ab181602). The following secondary antibodies were used: Goat anti-rabbit HRP (Abcam ab97080), Goat anti-chicken HRP (Invitrogen A16054).

Dynamic light scattering.

80ul of samples are pipetted into cuvettes (Malvern Catalog#759200) and run through the zetasizer spectrophotometer (Malvern Zetasizer Nano ZSP) with an EV refractive index of 1.35 in ANTEC through Northwestern University. Malvern analytical software is used to analyze the output using particle counts relative to the signal intensity.

Negative stain EM.

Samples suspended in PBS are prepared using the standard uranyl acetate fixation for 5 mins seeded on EMS grids (TMS Catalog#71150) and imaged on a FEI Spirit 2 electron microscope. Images were processed using ImageJ software.

Cryo-EM.

CryoEM images are obtained through the northwestern BioCryo core facility (NUANCE) with samples prepared as previously described92.

Primary DRG Cultures.

DRG sensory neurons from WT and K14-CD63-GFP mice were dissociated as described93 at 18 weeks of age.

Microfluidics.

Primary DRG cultures were deposited into one compartment chamber connected to the microchannel column in a microfluidic system (XONA Microfluidics SND450) with or without primary keratinocyte cultures in the other compartment chambers. Keratinocytes were cultured for 3 days before depositing DRG cultures unless otherwise specified. The medium was a combination of 50% DRG culture medium as previously described93 and 50% keratinocyte culture medium when both cell types were present in the chambers.

DIR Labelling.

Concentrated CCM was labeled with DIR (Invitrogen D12731; 2ug/ul) at a ratio of 1:100 by volume and then passed through the IZON 35nm columns. Isolated DIR-labeled exosomes from fractions 2–3 were then used for downstream applications with a negative DIR control, which was DIR-added to concentrated EV-depleted unconditioned medium passed through the column with the same fractions collected for experiments.

DIR-Exosomes.

DRG Cultures: DIR-labeled exosomes were directly applied to DIV-1 primary DRG cultures and visualized after 16 hours post-treatment by confocal microscopy. Microfluidics: DIR-exosomes were applied to the empty compartment after DRG neuron neurites occupied all microchannels, preventing medium exchange between the two compartments. The microfluidic chambers were visualized after 16 hours by confocal microscopy.

MS sample preparation

Trichloroacetic acid (TCA, Sigma-Aldrich, Cat# T0699) precipitation was used to clean and precipitate proteins from EV samples. Protein pellets were resuspended in 8 M urea (ThermoFisher Scientific, Cat # 29700) prepared in 100 mM ammonium bicarbonate solution (Fluka, Cat # 09830) and processed with ProteaseMAX (Promega, Cat # V2072) according to the manufacturer’s protocol. The samples were reduced with 5 mM Tris(2-carboxyethyl)phosphine (TCEP, Sigma-Aldrich, Cat # C4706; vortexed for 1 hour at RT), alkylated in the dark with 10 mM iodoacetamide (IAA, Sigma-Aldrich, Cat # I1149; 20 min at RT), diluted with 100 mM ABC, and quenched with 25 mM TCEP. Samples were diluted with 100 mM ammonium bicarbonate solution, and digested with Trypsin (1:50, Promega, Cat # V5280) for overnight incubation at 37°C with intensive agitation. The next day, reaction was quenched by adding 1% trifluoroacetic acid (TFA, Fisher Scientific, O4902–100). The samples were desalted using Peptide Desalting Spin Columns (Thermo Fisher Scientific, Cat # 89882). All samples were vacuum centrifuged to dry.

Tandem Mass spectrometry

Three micrograms of each sample were auto-sampler loaded with a Thermo Vanquish Neo UHPLC system onto a PepMap™ Neo Trap Cartridge (Thermo Fisher Scientific, Cat#: 174500, diameter, 300 μm, length, 5 mm, particle size, 5 μm, pore size, 100 Å, stationary phase, C18) coupled to a nanoViper analytical column (Thermo Fisher Scientific, Cat#: 164570, diameter, 0.075 mm, length, 500 mm, particle size, 3 μm, pore size, 100 Å, stationary phase, C18) with stainless steel emitter tip assembled on the Nanospray Flex Ion Source with a spray voltage of 2000 V. An Orbitrap Ascend (Thermo Fisher Scientific) was used to acquire all the MS spectral data. Buffer A contained 99.9% H2O and 0.1% FA, and buffer B contained 80.0% ACN, 19.9% H2O with 0.1% FA. For each fraction, the chromatographic run was for 2 hours in total with the following profile: 0–8% for 6, 8% for 64, 24% for 20, 36% for 10, 55% for 10, 95% for 10 and again 95% for 6 We used Orbitrap HCD-MS2 method for these experiments. Briefly, ion transfer tube temp = 275 °C, Easy-IC internal mass calibration, default charge state = 2 and cycle time = 3 s. Detector type set to Orbitrap, with 60K resolution, with wide quad isolation, mass range = normal, scan range = 375–1500 m/z, max injection time mode = Auto, AGC target = Standard, microscans = 1, S-lens RF level = 60, without source fragmentation, and datatype = Profile. MIPS was set as on, included charge states = 2–7 (reject unassigned). Dynamic exclusion enabled with n = 1 for 60s exclusion duration at 10 ppm for high and low with Exclude Isotopes. Isolation Mode = Quadrupole, isolation window = 1.6, isolation Offset = Off, active type = HCD, collision energy mode = Fixed, HCD collision energy type = Normalized, HCD collision energy = 25%, detector type = Orbitrap, orbitrap resolution = 15K, mass range = Normal, scan range mode = Auto, max injection time mode = Auto, AGC target = Standard, Microscans = 1, data type = Centroid.mins receptively.

MS data analysis and quantification

Protein identification/quantification and analysis were performed with Integrated Proteomics Pipeline - IP2 (Bruker, Madison, WI. http://www.integratedproteomics.com/) using ProLuCID94,95, DTASelect296,97, Census and Quantitative Analysis. Spectrum raw files were extracted into MS1, MS2 files using RawConverter (http://fields.scripps.edu/downloads.php). The tandem mass spectra (raw files from the same sample were searched together) were searched against UniProt mouse (downloaded on 07–29-2023) protein databases98 and matched to sequences using the ProLuCID/SEQUEST algorithm (ProLuCID version 3.1) with 50 ppm peptide mass tolerance for precursor ions and 600 ppm for fragment ions. The search space included all fully and half-tryptic peptide candidates within the mass tolerance window with no-miscleavage constraint, assembled, and filtered with DTASelect2 through IP2. To estimate protein probabilities and false-discovery rates (FDR) accurately, we used a target/decoy database containing the reversed sequences of all the proteins appended to the target database98 (UniProt, 2015). Each protein identified was required to have a minimum of one peptide of minimal length of six amino acid residues. After the peptide/spectrum matches were filtered, we estimated that the protein FDRs were ≤ 1% for each sample analysis. Resulting protein lists include subset proteins to allow for consideration of all possible protein isoforms implicated by at least three given peptides identified from the complex protein mixtures. Then, we used Census and Quantitative Analysis in IP2 for protein quantification. Static modification: 57.02146 C for carbamidomethylation. Quantification was performed by the built-in module in IP2.

Proteomics Statistics.

The spectral counts for each protein accession ID for the three RD and HFD biological replicates are used to run a one-tailed, paired t-test for statistical significance. The average spectral counts for RD and HFD were used to obtain a HFD/RD fold change. The volcano plot was generated in R using ggplot2. The heat map was generated using the R package pheatmap. Gene ontology enrichment analyses were generated using the R packages clusterprofiler, msigdbr and ggplot2. GO enrichment analyses used proteins present in all biological replicates of each group only.

Small RNA sequencing.

Samples underwent small RNA sequencing through Northwestern’s core facilities. Biological triplicate RD and HFD RNA samples were prepared using the total exosome RNA and protein extraction kit from Invitrogen (Invitrogen Catalog#2743605). RNA samples were quantified by Qubit RNA HS assay and the quality was confirmed by Bioanalyzer RNA pico chip assay. Then, 1ng of RNA was used as input for library preparation with NextFlex small RNA-seq kit v4 according to manufacturer’s protocol. Each sample was barcoded with a unique index and multiplexed libraries were pooled for sequencing on Novaseq X Plus 10B flowcell using single end 50nt mode.

RNA sequencing Statistics.

Data analysis was carried out in R using the standard workflow of DESeq2 paired with the libraries apeglm and ggplot2 for figure generation. Bonferroni post-hoc adjustment was used for the reported adjusted p-values ≤ 0.05 for significance. Gene ontology enrichment analyses were generated using the R packages clusterprofiler, msigdbr and ggplot2 with Bonferroni post-hoc adjusted P values < 0.05 for significance.

Protein target analysis.

The direct or indirect protein targets for each microRNA was predicted using several target prediction programs, including miRDB (http://mirdb.org/)99, TargetScan v7.0 (http://www.targetscan.org/vert_72/) and DIANA-microT v5.0 (https://bio.tools/DIANA-microT). Only the predicted proteins identified by all 3 programs were included in the subsequent enrichment analyses.

Immunohistochemistry.

Glabrous hind paw dermis/epidermis was separated from the paw and whole DRGs (lumber 2–4) were isolated from 18 week old mice and fixed with 4% PFA for 1 hour, 30% sucrose for 1 hour, and then embedded in OCT. Samples were processed as previously described50 and analyzed by confocal microscopy.

Antibodies.

We used the following primary antibodies on DRG sections: GFP (chicken; Abcam ab13970). We used the following antibodies on skin sections: K14 (BioLegend 906004). We used the following antibodies on primary DRG cultures: GFP (Abcam ab13970), β-tubulin (ProteinTech 80713–1-RR100UL). We used the following secondary antibodies: Goat anti-chicken AlexaFluor™-598 (Invitrogen A-11042), goat anti-rabbit AlexaFluor-647 (Invitrogen A32733).

EV-Reporter Mouse.

The commercially available CD63-GFP fl/fl mouse line (Jackson Laboratory Strain#:036865) was crossed with K14-Cre mice to generate the EV-reporter K14-CD63-GFP mouse line.

Study approval.

All methods involving animals were approved by the IACUC of Northwestern University.

Supplementary Material

Supplement 1

ACKNOWLEDGEMENTS

Zetasizer Nano ZSP (DLS), Nanosight (NTA), and IZON Exoid (TRPS) experiments were performed in the Analytical bioNanoTechnology Equipment Core Facility of the Simpson Querrey Institute for BioNanotechnology at Northwestern University. ANTEC receives partial support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-2025633) and Feinberg School of Medicine, Northwestern University. Electron microscopy imaging work was performed at the Northwestern University Center for Advanced Microscopy (RRID: SCR_020996) generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. This work made use of the EPIC facility of Northwestern University’s NUANCE Center, which has received support from the SHyNE Resource (NSF ECCS-2025633), the IIN, and Northwestern’s MRSEC program (NSF DMR-2308691). And this work was supported by the Northwestern University NUSeq Core Facility.

STATEMENT OF DATA AND MATERIALS

We will make the raw MS data publicly available in an accessible database upon acceptance.

Figure 1: Keratinocytes release a diverse population of exosomes.

A) Workflow schematic for keratinocyte-derived exosome (KDE) isolation using size exclusion chromatography (SEC). B) Immunoblotting the ten SEC fractions revealed that exosome-associated proteins ALIX, Syntenin, Tsg101, CD63, and CD81 were enriched in fractions 2 and 3 relative to the total protein concentration (silver stain). C) KDEs analyzed with dynamic light scattering had a size range of 59.2 ± 14.5nm (mean ± StDev). N=7 across biological replicates from both male and female mice. D) KDEs analyzed using nanoparticle tracking analysis produced a concentration peak particle size of 95 ± 6.1 nm (mean ± StDev). N=2 male biological replicates. E) KDEs analyzed using tunable resistive pulse sensing (TRPS) determined that the mean size for RD KDEs was 97.7 ± 4.5nm, with a concentration peak of 72.3 ± 4.5nm (mean ± StDev). N=2 biological replicates. F) Nanovesicles from combined fraction 2/3 were visualized with negative stain EM. N=11 across 5 biological replicates from male and female mice. G) Combined SEC fractions 2/3 were visualized with cryo-EM, demonstrating several diverse populations. N=2 biological replicates, one male and one female.

Figure 2: Isolated exosomes from SEC fraction 2/3 are functionally retrogradely trafficked by DRG neurons in vitro.

A) Representative picture of microfluidics device. B) The presence of keratinocytes co-cultured with DRG neurons in a microfluidic paradigm encouraged neurite outgrowth crossing through the microchannels connecting both chambers. C) We observed a significant increase in the neurite microchannel crossing when co-cultured in the presence of keratinocytes (p ≤ 0.05, one-tailed, paired t-test; n=5 with KCs and n=4 without KCs paired with DRG primary cultures). D) KDEs from SEC Fr2/3 are functionally internalized by primary DRG neurons. N=4 across 2 biological replicates. E) KDEs are retrogradely trafficked into the neuron cell body through the neurites in a microfluidic paradigm. N=4 biological replicates.

Figure 3: Keratinocyte-derived exosomes (KDEs) significantly alter their protein cargo in painful diabetic neuropathy.

A) Workflow schematic depicting exosome proteomic analysis. N=3 biological replicates for each group from male mice. B) The top pathways from a gene ontology enrichment analysis of proteins present in each of the three biological replicates in both experimental groups suggested EV enrichment. Bonferroni adjusted p-value ≤ 0.01. C) Select panel of proteins associated with GO Term ‘EV Biogenesis’ revealed similar spectral counts for exosome markers Alix, Syntenin-1, and Tsg101 between both groups. Insert: GO Term ‘Axon Development’ revealed several unverified notable proteins as exosome cargo. Mean ± SEM. GO Terms Bonferroni adjusted p-value ≤ 0.01. D) There were 90 significant differentially expressed exosome-associated proteins (EAPs; FC ≥ 1.5 and paired t-test, one-way, p ≤ 0.05) between RD and HFD KDEs that clustered by group. FC calculated as average HFD/RD spectral count for each protein. E) Representative volcano plot with 90 EAPs with FC ≥ 1.5 and p ≤ 0.05 (labelled red). There were 11 downregulated and 79 upregulated EAPs. F) Western blot confirms two EAPs, Mapk1 (via ERK1/2 expression) and annexin-7 in SEC Fr2/3. n=3 biological replicates for each group. G) Abundances for EAPS in the differentially expressed GO term ‘Wound Healing,’ ‘Neurotransmitter Secretion,’ and the REACTOME term ‘MAPK Family Signaling Cascades.’ Mean ± SEM. Bonferroni adjusted p-value ≤ 0.01.

Figure 4: Keratinocyte-derived exosomes (KDEs) alter their small RNA cargo in painful diabetic neuropathy. N=3 biological replicates for male mice in each diet group.

A) The top ten small RNAs identified in both RD and HFD KDEs represent 74.8% of the total small RNA sequenced. The top two small RNAs, the let-7 and microRNA-23 families, accounted for almost 40% of the total small RNA sequenced. B) The predicted protein targets for let-7 and miR-23 were obtained by cross-referencing 3 separate databases: MirDB, TargetScan, and DIANA-microT. The protein targets predicted by all three programs were used for downstream analysis. C) The enrichment analysis of the predicted protein targets of let-7 and miR-23 both revealed predicted KEGG pathways related to ‘Axon Guidance,’ suggesting a possible role of KDE small RNAs on keratinocyte-to-neuron communication. KEGG pathways Bonferroni adjusted p-value ≤ 0.01. D) There were 35 differentially expressed microRNAs, which clustered by group. Bonferroni adjusted p-value ≤ 0.05. E) Of the 33 differentially expressed microRNAs (FC ≥ 1.5, adjusted p-value ≤ 0.05) between RD and HFD, 22 were overexpressed in HFD. FC is defined as HFDavg/RDavg microRNA counts for each small RNA. Bonferroni adjusted p-value. F) miR-684 was downregulated in HFD while miR-24–3p was upregulated. Using the same three databases as Figure 4B, predicted target proteins lists were obtained for each. G) The gene ontology enrichment analysis for these predicted proteins presented several interesting GO Terms. Both differentially expressed microRNAs predicted axon-related pathways, further providing evidence for an altered keratinocyte-to-neuron communication via exosome cargo. GO Terms Bonferroni adjusted p-value ≤ 0.01.

Figure 5: Epidermal keratinocyte-derived exosomes (KDEs) are enriched with GFP in an EV-reporter mouse model.

A) We created an EV-reporter mouse line by crossing the commercially available CD63-GFP fl/fl mouse line with K14-Cre to create K14-CD63-GFP (KCG) mice. B) IHC on cryo-sections of glabrous mouse skin demonstrated GFP expression in all layers of the epidermis with no detectable GFP in the dermis. The GFP (green) signal also co-localized with K14 staining (red), representing the basal layer of the epidermis. C) Immunoblotting revealed robust GFP signal in epidermal cell lysate from our EV-reporter mouse line. D) GFP was detected in the primary keratinocyte cultures from our EV-reporter mouse line. E) Cell-conditioned medium from KCG keratinocyte cultures was run through IZON 35nm SEC columns. Fraction 2/3 was enriched with CD63-GFP and soluble GFP, supporting that KDEs from primary cultured keratinocytes from our EV-reporter mouse line are tagged with the CD63-GFP fusion protein.

Figure 6: Epidermal keratinocyte-derived exosomes (KDEs) are retrogradely trafficked from the epidermis to DRG neurons in vivo in male and female mice.

A) We detected GFP signal after immunolabel amplification in cryosections of the DRGs from the EV-reporter mice for both RD and HFD. N=3 male biological replicates for both RD and HFD and n=2 for RD female mice. B) As expected, no false GFP signal was detect in WT DRG cryosections. N=3 biological replicates of WT. C) Immunoblotting the DRG cell lysate of EV-reporter mice revealed GFP expression. N=2 biological replicates for both RD and HFD. D) We detected GFP signal after IHC amplification in primary DRG cell cultures from the EV-reporter mice for both RD and HFD. N=3 biological replicates for both RD and HFD. E) As expected, no false GFP signal was detected in WT primary DRG cultures. N=3 biological replicates of WT. F) The GFP signal was not only detected in the cell body of the primary DRG cultures from EV-reporter mice but also along the neurites, further providing evidence that KDEs are trafficked along the neurites. N=6 biological replicates between RD and HFD.

Conflict of interest statement: The authors have declared that no conflict of interest exists.
==== Refs
REFERENCES

1 Martin S. S. 2024 Heart Disease and Stroke Statistics: A Report of US and Global Data From the American Heart Association. Circulation 149 , e347–e913 (2024). 10.1161/CIR.0000000000001209 38264914
2 American Diabetes A. Diagnosis and classification of diabetes mellitus. Diabetes care 34 Suppl 1 , S62–69 (2011). 10.2337/dc11-S062 21193628
3 Dyck P. J. The Rochester Diabetic Neuropathy Study: design, criteria for types of neuropathy, selection bias, and reproducibility of neuropathic tests. Neurology 41 , 799–807 (1991).2046920
4 Spallone V. , Lacerenza M. , Rossi A. , Sicuteri R. & Marchettini P. Painful diabetic polyneuropathy: approach to diagnosis and management. The Clinical journal of pain 28 , 726–743 (2012). 10.1097/AJP.0b013e318243075c 22209797
5 Abbott C. A. , Malik R. A. , van Ross E. R. , Kulkarni J. & Boulton A. J. Prevalence and characteristics of painful diabetic neuropathy in a large community-based diabetic population in the U.K. Diabetes care 34 , 2220–2224 (2011). 10.2337/dc11-1108 21852677
6 daCosta DiBonaventura M. , Cappelleri J. C. & Joshi A. V. A longitudinal assessment of painful diabetic peripheral neuropathy on health status, productivity, and health care utilization and cost. Pain medicine 12 , 118–126 (2011). 10.1111/j.1526-4637.2010.01012.x 21087406
7 Bril V. Evidence-based guideline: Treatment of painful diabetic neuropathy: report of the American Academy of Neurology, the American Association of Neuromuscular and Electrodiagnostic Medicine, and the American Academy of Physical Medicine and Rehabilitation. Neurology 76 , 1758–1765 (2011). 10.1212/WNL.0b013e3182166ebe 21482920
8 Quilici S. Meta-analysis of duloxetine vs. pregabalin and gabapentin in the treatment of diabetic peripheral neuropathic pain. BMC neurology 9 , 6 (2009). 10.1186/1471-2377-9-6 19208243
9 Callaghan B. C. , Cheng H. T. , Stables C. L. , Smith A. L. & Feldman E. L. Diabetic neuropathy: clinical manifestations and current treatments. Lancet Neurol 11 , 521–534 (2012). 10.1016/S1474-4422(12)70065-0 22608666
10 Finnerup N. B. Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis. Lancet Neurol 14 , 162–173 (2015). 10.1016/S1474-4422(14)70251-0 25575710
11 Attal N. & Bouhassira D. Advances in the treatment of neuropathic pain. Curr Opin Neurol 34 , 631–637 (2021). 10.1097/WCO.0000000000000980 34310363
12 Shinu P. Novel Therapies for the Treatment of Neuropathic Pain: Potential and Pitfalls. J Clin Med 11 (2022). 10.3390/jcm11113002
13 Lauria G. & Devigili G. Skin biopsy as a diagnostic tool in peripheral neuropathy. Nat Clin Pract Neurol 3 , 546–557 (2007). 10.1038/ncpneuro0630 17914343
14 Sommer C. & Lauria G. Skin biopsy in the management of peripheral neuropathy. The Lancet. Neurology 6 , 632–642 (2007). 10.1016/S1474-4422(07)70172-2 17582363
15 Stucky C. L. & Mikesell A. R. Cutaneous pain in disorders affecting peripheral nerves. Neuroscience letters 765 , 136233 (2021). 10.1016/j.neulet.2021.136233 34506882
16 Mikesell A. R. Keratinocyte PIEZO1 modulates cutaneous mechanosensation. Elife 11 (2022). 10.7554/eLife.65987
17 Moehring F. Keratinocytes mediate innocuous and noxious touch via ATP-P2X4 signaling. Elife 7 (2018). 10.7554/eLife.31684
18 Sadler K. E. , Moehring F. & Stucky C. L. Keratinocytes contribute to normal cold and heat sensation. Elife 9 (2020). 10.7554/eLife.58625
19 Radtke C. , Vogt P. M. , Devor M. & Kocsis J. D. Keratinocytes acting on injured afferents induce extreme neuronal hyperexcitability and chronic pain. Pain 148 , 94–102 (2010). 10.1016/j.pain.2009.10.014 19932564
20 Talagas M. Keratinocytes Communicate with Sensory Neurons via Synaptic-like Contacts. Annals of neurology 88 , 1205–1219 (2020). 10.1002/ana.25912 32951274
21 Erbacher C. Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit. Elife 13 (2024). 10.7554/eLife.77761
22 van Niel G. , D’Angelo G. & Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 19 , 213–228 (2018). 10.1038/nrm.2017.125 29339798
23 Mathieu M. , Martin-Jaular L. , Lavieu G. & Thery C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat Cell Biol 21 , 9–17 (2019). 10.1038/s41556-018-0250-9 30602770
24 Gurung S. , Perocheau D. , Touramanidou L. & Baruteau J. The exosome journey: from biogenesis to uptake and intracellular signalling. Cell Commun Signal 19 , 47 (2021). 10.1186/s12964-021-00730-1 33892745
25 Kalluri R. & LeBleu V. S. The biology, function, and biomedical applications of exosomes. Science 367 (2020). 10.1126/science.aau6977
26 Pan B. T. , Teng K. , Wu C. , Adam M. & Johnstone R. M. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. The Journal of cell biology 101 , 942–948 (1985). 10.1083/jcb.101.3.942 2993317
27 Rastogi S. The Evolving Landscape of Exosomes in Neurodegenerative Diseases: Exosomes Characteristics and a Promising Role in Early Diagnosis. Int J Mol Sci 22 (2021). 10.3390/ijms22010440
28 Fan Y. , Chen Z. & Zhang M. Role of exosomes in the pathogenesis, diagnosis, and treatment of central nervous system diseases. J Transl Med 20 , 291 (2022). 10.1186/s12967-022-03493-6 35761337
29 Dai J. Exosomes: key players in cancer and potential therapeutic strategy. Signal Transduct Target Ther 5 , 145 (2020). 10.1038/s41392-020-00261-0 32759948
30 Lo Cicero A. Exosomes released by keratinocytes modulate melanocyte pigmentation. Nature communications 6 , 7506 (2015). 10.1038/ncomms8506
31 Nasiri G. , Azarpira N. , Alizadeh A. , Goshtasbi S. & Tayebi L. Shedding light on the role of keratinocyte-derived extracellular vesicles on skin-homing cells. Stem Cell Res Ther 11 , 421 (2020). 10.1186/s13287-020-01929-8 32993791
32 Zhou X. Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing. ACS Nano 14 , 12732–12748 (2020). 10.1021/acsnano.0c03064 32931251
33 Jiang M. Keratinocyte exosomes activate neutrophils and enhance skin inflammation in psoriasis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 33 , 13241–13253 (2019). 10.1096/fj.201900642R 31539277
34 Kotzerke K. Immunostimulatory activity of murine keratinocyte-derived exosomes. Exp Dermatol 22 , 650–655 (2013). 10.1111/exd.12230 24079734
35 Ai M. Role of Human Mesenchymal Stem Cells and Derived Extracellular Vesicles in Reducing Sensory Neuron Hyperexcitability and Pain Behaviors in Murine Osteoarthritis. Arthritis Rheumatol 75 , 352–363 (2023). 10.1002/art.42353 36122169
36 Ashrafizadeh M. , Kumar A. P. , Aref A. R. , Zarrabi A. & Mostafavi E. Exosomes as Promising Nanostructures in Diabetes Mellitus: From Insulin Sensitivity to Ameliorating Diabetic Complications. Int J Nanomedicine 17 , 1229–1253 (2022). 10.2147/IJN.S350250 35340823
37 Sun Y. Human Mesenchymal Stem Cell Derived Exosomes Alleviate Type 2 Diabetes Mellitus by Reversing Peripheral Insulin Resistance and Relieving beta-Cell Destruction. ACS Nano 12 , 7613–7628 (2018). 10.1021/acsnano.7b07643 30052036
38 He Q. Mesenchymal stem cell-derived exosomes exert ameliorative effects in type 2 diabetes by improving hepatic glucose and lipid metabolism via enhancing autophagy. Stem Cell Res Ther 11 , 223 (2020). 10.1186/s13287-020-01731-6 32513303
39 Wei P. Exosomes derived from human amniotic epithelial cells accelerate diabetic wound healing via PI3K-AKT-mTOR-mediated promotion in angiogenesis and fibroblast function. Burns Trauma 8 , tkaa020 (2020). 10.1093/burnst/tkaa020
40 Fan B. Mesenchymal stromal cell-derived exosomes ameliorate peripheral neuropathy in a mouse model of diabetes. Diabetologia 63 , 431–443 (2020). 10.1007/s00125-019-05043-0 31740984
41 Ahmed L. A. & Al-Massri K. F. Exploring the Role of Mesenchymal Stem Cell-Derived Exosomes in Diabetic and Chemotherapy-Induced Peripheral Neuropathy. Mol Neurobiol (2024). 10.1007/s12035-024-03916-z
42 Jia L. Exosomes derived from high-glucose-stimulated Schwann cells promote development of diabetic peripheral neuropathy. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 32 , fj201800597R (2018). 10.1096/fj.201800597R
43 Böing A. N. Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles 3 (2014). 10.3402/jev.v3.23430
44 Arya S. B. , Collie S. P. & Parent C. A. The ins-and-outs of exosome biogenesis, secretion, and internalization. Trends Cell Biol 34 , 90–108 (2024). 10.1016/j.tcb.2023.06.006 37507251
45 Théry C. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7 , 1535750 (2018). 10.1080/20013078.2018.1535750 30637094
46 Wang Y. Z. Notch receptor-ligand binding facilitates extracellular vesicle-mediated neuron-to-neuron communication. Cell Rep 43 , 113680 (2024). 10.1016/j.celrep.2024.113680 38241148
47 George D. S. Mitochondrial calcium uniporter deletion prevents painful diabetic neuropathy by restoring mitochondrial morphology and dynamics. Pain (2021). 10.1097/j.pain.0000000000002391
48 George D. S. The Mas-related G protein-coupled receptor d (Mrgprd) mediates pain hypersensitivity in painful diabetic neuropathy. Pain (2024). 10.1097/j.pain.0000000000003120
49 Jayaraj N. D. Reducing CXCR4-mediated nociceptor hyperexcitability reverses painful diabetic neuropathy. The Journal of clinical investigation 128 , 2205–2225 (2018). 10.1172/JCI92117 29533926
50 Menichella D. M. CXCR4 chemokine receptor signaling mediates pain in diabetic neuropathy. Molecular pain 10 , 42 (2014). 10.1186/1744-8069-10-42 24961298
51 Dam D. H. M. Ganglioside GM3 Mediates Glucose-Induced Suppression of IGF-1 Receptor-Rac1 Activation to Inhibit Keratinocyte Motility. J Invest Dermatol 137 , 440–448 (2017). 10.1016/j.jid.2016.09.028 27729281
52 Nowak N. C. , Menichella D. M. , Miller R. & Paller A. S. Cutaneous innervation in impaired diabetic wound healing. Transl Res 236 , 87–108 (2021). 10.1016/j.trsl.2021.05.003 34029747
53 Frühbeis C. Oligodendrocytes support axonal transport and maintenance via exosome secretion. PLoS Biol 18 , e3000621 (2020). 10.1371/journal.pbio.3000621 33351792
54 Graykowski D. R. , Wang Y. Z. , Upadhyay A. & Savas J. N. The Dichotomous Role of Extracellular Vesicles in the Central Nervous System. iScience 23 , 101456 (2020). 10.1016/j.isci.2020.101456 32835924
55 Greenhalgh D. G. Wound healing and diabetes mellitus. Clin Plast Surg 30 , 37–45 (2003). 10.1016/s0094-1298(02)00066-4 12636214
56 Häger S. C. & Nylandsted J. Annexins: players of single cell wound healing and regeneration. Commun Integr Biol 12 , 162–165 (2019). 10.1080/19420889.2019.1676139 31666917
57 Creutz C. E. The annexins and exocytosis. Science 258 , 924–931 (1992). 10.1126/science.1439804 1439804
58 Sønder S. L. Annexin A7 is required for ESCRT III-mediated plasma membrane repair. Sci Rep 9 , 6726 (2019). 10.1038/s41598-019-43143-4 31040365
59 Williams J. K. , Ngo J. M. , Lehman I. M. & Schekman R. Annexin A6 mediates calcium-dependent exosome secretion during plasma membrane repair. Elife 12 (2023). 10.7554/eLife.86556
60 Fan B. , Chopp M. , Zhang Z. G. & Liu X. S. Emerging Roles of microRNAs as Biomarkers and Therapeutic Targets for Diabetic Neuropathy. Front Neurol 11 , 558758 (2020). 10.3389/fneur.2020.558758 33192992
61 Ibrahim H. M. , AlZahrani A. , Hanieh H. , Ahmed E. A. & Thirugnanasambantham K. MicroRNA-7188–5p and miR-7235 regulates Multiple sclerosis in an experimental mouse model. Mol Immunol 139 , 157–167 (2021). 10.1016/j.molimm.2021.07.002 34543842
62 Mukherjee S. , Shelar B. & Krishna S. Versatile role of miR-24/24–1*/24–2* expression in cancer and other human diseases. Am J Transl Res 14 , 20–54 (2022).35173828
63 Cui M. Interactive functions of microRNAs in the miR-23a-27a-24–2 cluster and the potential for targeted therapy in cancer. J Cell Physiol 235 , 6–16 (2020). 10.1002/jcp.28958 31192453
64 Lin Z. miR-24–3p Dominates the Proliferation and Differentiation of Chicken Intramuscular Preadipocytes by Blocking ANXA6 Expression. Genes (Basel) 13 (2022). 10.3390/genes13040635
65 Herrera A. Neurogenesis redirects β-catenin from adherens junctions to the nucleus to promote axonal growth. Development 150 (2023). 10.1242/dev.201651
66 Huang Y. L. Inhibition of Wnt/β-catenin signaling attenuates axonal degeneration in models of Parkinson’s disease. Neurochem Int 159 , 105389 (2022). 10.1016/j.neuint.2022.105389 35809720
67 Duraikannu A. , Martinez J. A. , Chandrasekhar A. & Zochodne D. W. Expression and Manipulation of the APC-β-Catenin Pathway During Peripheral Neuron Regeneration. Sci Rep 8 , 13197 (2018). 10.1038/s41598-018-31167-1 30181617
68 Lewallen K. A. Assessing the role of the cadherin/catenin complex at the Schwann cell-axon interface and in the initiation of myelination. The Journal of neuroscience : the official journal of the Society for Neuroscience 31 , 3032–3043 (2011). 10.1523/jneurosci.4345-10.2011 21414924
69 Alto L. T. & Terman J. R. Semaphorins and their Signaling Mechanisms. Methods in molecular biology 1493 , 1–25 (2017). 10.1007/978-1-4939-6448-2_1 27787839
70 Janssen B. J. Structural basis of semaphorin-plexin signalling. Nature 467 , 1118–1122 (2010). 10.1038/nature09468 20877282
71 Takamatsu H. & Kumanogoh A. Diverse roles for semaphorin-plexin signaling in the immune system. Trends in immunology 33 , 127–135 (2012). 10.1016/j.it.2012.01.008 22325954
72 Li Y. Macrophages facilitate peripheral nerve regeneration by organizing regeneration tracks through Plexin-B2. Genes Dev 36 , 133–148 (2022). 10.1101/gad.349063.121 35086862
73 Paldy E. Semaphorin 4C Plexin-B2 signaling in peripheral sensory neurons is pronociceptive in a model of inflammatory pain. Nature communications 8 , 176 (2017). 10.1038/s41467-017-00341-w
74 Li J. , Liu H. Q. , Li X. B. , Yu W. J. & Wang T. Function of Adenosine 2A Receptor in High-Fat Diet-Induced Peripheral Neuropathy. J Diabetes Res 2020 , 7856503 (2020). 10.1155/2020/7856503 32566683
75 Kopan R. & Ilagan M. X. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137 , 216–233 (2009). 10.1016/j.cell.2009.03.045 19379690
76 Zhang Y. , Lowe S. , Ding A. Z. & Li X. Notch-dependent binary fate choice regulates the Netrin pathway to control axon guidance of Drosophila visual projection neurons. Cell Rep 42 , 112143 (2023). 10.1016/j.celrep.2023.112143 36821442
77 Sullivan J. M. Dominant mutations of the Notch ligand Jagged1 cause peripheral neuropathy. The Journal of clinical investigation 130 , 1506–1512 (2020). 10.1172/jci128152 32065591
78 Wolfes A. C. & Dean C. The diversity of synaptotagmin isoforms. Curr Opin Neurobiol 63 , 198–209 (2020). 10.1016/j.conb.2020.04.006 32663762
79 Ullah N. , Maaiden E. E. , Uddin M. S. & Ashraf G. M. Synaptotagmin-1: A Multi-Functional Protein that Mediates Vesicle Docking, Priming, and Fusion. Curr Protein Pept Sci 22 , 470–478 (2021). 10.2174/1389203722666210325110231 33823763
80 Thiel G. Synapsin I, synapsin II, and synaptophysin: marker proteins of synaptic vesicles. Brain pathology 3 , 87–95 (1993). 10.1111/j.1750-3639.1993.tb00729.x 7903586
81 Cesca F. , Baldelli P. , Valtorta F. & Benfenati F. The synapsins: key actors of synapse function and plasticity. Progress in neurobiology 91 , 313–348 (2010). 10.1016/j.pneurobio.2010.04.006 20438797
82 Park D. Cooperative function of synaptophysin and synapsin in the generation of synaptic vesicle-like clusters in non-neuronal cells. Nature communications 12 , 263 (2021). 10.1038/s41467-020-20462-z
83 Talagas M. Anatomical contacts between sensory neurons and epidermal cells: an unrecognized anatomical network for neuro-immuno-cutaneous crosstalk. Br J Dermatol 188 , 176–185 (2023). 10.1093/bjd/ljac066 36763869
84 Vieira W. F. Anti-hyperalgesic effects of photobiomodulation therapy (904 nm) on streptozotocin-induced diabetic neuropathy imply MAPK pathway and calcium dynamics modulation. Sci Rep 12 , 16730 (2022). 10.1038/s41598-022-19947-2 36202956
85 Aghaee-Bakhtiari S. H. MAPK and JAK/STAT pathways targeted by miR-23a and miR-23b in prostate cancer: computational and in vitro approaches. Tumour Biol 36 , 4203–4212 (2015). 10.1007/s13277-015-3057-3 25604141
86 Perdas E. , Stawski R. , Nowak D. & Zubrzycka M. The Role of miRNA in Papillary Thyroid Cancer in the Context of miRNA Let-7 Family. Int J Mol Sci 17 (2016). 10.3390/ijms17060909
87 Wang S. MicroRNA-24 in Cancer: A Double Side Medal With Opposite Properties. Front Oncol 10 , 553714 (2020). 10.3389/fonc.2020.553714 33123467
88 Lin Y. miR-24–3p stimulates migration, invasion and proliferation of prostate cancer cells by targeting suppressor of cytokine signaling 6. Int J Clin Exp Pathol 11 , 1803–1810 (2018).31938287
89 Xiang Y. miR-24 in diabetes. Oncotarget 6 , 16816–16817 (2015). 10.18632/oncotarget.4795 26219469
90 George D. S. A subpopulation of peripheral sensory neurons expressing the Mas-related G Protein-Coupled Receptor d (Mrgprd) generates pain hypersensitivity in painful diabetic neuropathy. bioRxiv, 2022.2010.2027.514066 (2022). 10.1101/2022.10.27.514066
91 George D. S. The Mas-related G protein-coupled receptor d (Mrgprd) mediates pain hypersensitivity in painful diabetic neuropathy. Pain (2023). 10.1097/j.pain.0000000000003120
92 Asadi J. Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals. Micron 99 , 40–48 (2017). 10.1016/j.micron.2017.03.019 28419915
93 Menichella D. M. Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice. Molecular pain 12 (2016). 10.1177/1744806916666284
94 Eng J. K. , McCormack A. L. & Yates J. R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5 , 976–989 (1994). 10.1016/1044-0305(94)80016-2 24226387
95 Xu T. ProLuCID: An improved SEQUEST-like algorithm with enhanced sensitivity and specificity. J Proteomics 129 , 16–24 (2015). 10.1016/j.jprot.2015.07.001 26171723
96 Cociorva D. , D L. T. & Yates J. R. Validation of tandem mass spectrometry database search results using DTASelect. Curr Protoc Bioinformatics Chapter 13, Unit 13.14 (2007). 10.1002/0471250953.bi1304s16
97 Tabb D. L. , McDonald W. H. & Yates J. R. 3rd . DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J Proteome Res 1 , 21–26 (2002). 10.1021/pr015504q 12643522
98 UniProt: a hub for protein information. Nucleic Acids Res 43 , D204–212 (2015). 10.1093/nar/gku989 25348405
99 Chen Y. & Wang X. miRDB: an online database for prediction of functional microRNA targets. Nucleic Acids Res 48 , D127–d131 (2020). 10.1093/nar/gkz757 31504780
