Sex as a biological variable

As Fabry disease is an X-linked genetic disorder predominantly affecting males and our Fabry disease rat model displays more robust sensory phenotypes in males (22), all cellular, histological, and electrophysiological studies with Fabry rats in this study exclusively used males. Experiments using SCM in Sprague-Dawley rats used both male and female rats to account for sex as a biological variable.

Animal model

The X-linked genetic Fabry disease rat model (22) (Rat Genome Database symbol: Glaem2Mcwi) was used for in vivo dorsal root teased single-unit recordings, provided primary sensory neuron and Schwann cell cultures, and was compared with age- and sex-matched WT littermate controls. All rats used for in vivo and in vitro electrophysiology and collection of Fabry Schwann cells were hemizygous males between 20 and 40 weeks old. For evaluating the effects of Schwann cell media or p11 protein treatment on naive sensory neuron function, male Sprague-Dawley outbred rats (Envigo) aged 15–30 weeks were used (CTRL). For animal behavior experiments, both male and female Sprague-Dawley rats from Taconic were used. As no sex differences were observed in behavior studies, sexes were combined for analysis.

Light microscopy and transmission electron microscopy

Peripheral nerves. Nerves from Fabry and WT rats were collected (36) and processed (89, 90) as previously described. The relevant method details are presented in the Supplemental Methods.

DRGs. Tissue from Fabry and WT rats aged 13 weeks was harvested and processed as previously described (22, 91). The relevant method details are presented in the Supplemental Methods.

Cell culture

Sensory neuron soma. DRG neurons were harvested, dissociated, and cultured as previously described (22, 92). Further details are presented in the Supplemental Methods.

Schwann cells. Schwann cells were cultured using a previously published protocol (48) with modifications. Further details are presented in the Supplemental Methods.

Schwann cell culture purity. Schwann cell culture purity was assessed using immunofluorescence of the Schwann cell marker SOX10 (93) and DAPI to mark all cells. Analysis was done using a modified colocalization analysis as we previously described (36). Further details are presented in the Supplemental Methods.

SCM. Schwann cell cultures were grown for 2 days in Schwann cell growth media to 70%–80% confluence. Cells were then washed with PBS and replaced with Schwann cell collection media (high-glucose DMEM [Thermo Fisher Scientific], 10 nM neuregulin [Recombinant Heregulin-β1177–244, PeproTech], and 2 μM forskolin [MilliporeSigma]) and cultured for 1–2 days. Schwann cells have been shown to grow in culture with or without crude serum (94); serum was not included in collection media to reduce confounding variables for all experiments. These media were collected, filtered using a 0.22 μm filter (CELLTREAT) to remove debris or cell fragments, flash-frozen in liquid nitrogen, and stored at –20°C or –80°C for less than 1 month for functional assays. Media samples, represented as either SCM or unconditioned media controls, were then thawed and used for all subsequent experiments. Media underwent a maximum of 2 flash-freeze cycles for all assessments and were not diluted.

p11 treatment. Recombinant rat protein p11 with an N-terminal His Tag (S100A10 Recombinant Protein, Aviva Systems Biology, OPCD06771) was dissolved in distilled water to obtain a final concentration of 100 μg/mL and stored at –80°C for less than 1 month. Protein was further diluted in DRG neuron culture media to obtain the respective concentrations (0.1–1,000 ng/mL) for calcium imaging experiments and whole-cell patch clamp electrophysiology experiments; neurons were incubated with this protein overnight. For intraplantar injection to assess rodent behavior, protein was dissolved in saline to a dose of 0.9, 9, or 90 ng per 30 μL injection; saline was used as vehicle injections.

p11 immunofluorescence. Sensory neuron soma isolated from naive rats were cultured and treated with or without 100 ng/mL recombinant rat protein p11 overnight followed by washout. Cells were fixed and underwent an immunofluorescence protocol as detailed in the Supplemental Methods. A negative control stain was also analyzed (p11 no Ig), which consisted of cultures that underwent exposure to 100 ng/mL p11 and the subsequent immunofluorescence protocol without inclusion of the anti-His tag primary antibody.

p11 ELISA of isolated DRG neurons. Details are presented in the Supplemental Methods.

p11 immunodepletion. Fabry-SCM were collected as described above. Media were split into 2 equal aliquots: one undergoing immunodepletion and the other used as a control batch. Anti-p11 antibody (10 μg) (S100A10 polyclonal antibody, ProteinTech, 11250-1-AP) was incubated with 50 μL Dynabeads Protein A (Invitrogen) for 10 minutes, and supernatant was removed with a DynaMag-2 Magnet tube rack (Invitrogen). Afterward, Fabry-SCM was added the anti-p11 antibody–bound Dynabeads and left to incubate on a rotating platform for 60 minutes at room temperature. Samples were then placed onto the magnetic tube rack and supernatant was removed. Depletion of p11 from the media was verified using Rat S100 Calcium Binding Protein A10 (S100A10) ELISA Kit (Biomatik, EKN48271-96T) as per the included instructions.

Dorsal root teased-fiber single-unit recording

Fabry and WT rats underwent in vivo dorsal root single-unit recordings as we reported previously (97). Briefly, rats were anesthetized with subcutaneous injection of urethane (100 mg/kg) followed by isoflurane that was progressively reduced to 0.2% over 30 minutes. A laminectomy was performed to expose the spinal cord from the T13 to the L3 vertebrae, which was covered with warm mineral oil (36°C). Dura was removed, and rats were mounted on a spinal frame with stabilizing vertebral clamps. The L4 dorsal root was gently released from connective tissues and transected at the rootlets adjacent to the spinal cord. The dorsal root was placed onto a glass platform and teased into fine bundles that were individually placed onto a platinum/iridium recording electrode for observation of spontaneous and evoked activity. A reference electrode was placed in adjacent muscle tissue. Signals were collected with an Axoclamp 900 A microelectrode amplifier (Molecular Devices) with the headstage (HS-9A-x0.1U with feedback resistance of 100 MΩ) serving as a preamplifier with gain setting of 500 or higher, were filtered at 1 kHz, and were sampled at 10 kHz using a digitizer (DigiData 1440 A, Molecular Devices). Action potentials were isolated by setting the threshold above background noise.

For recording spontaneous activity, bundles were observed for a 3-minute observation period and recorded for 3–4 minutes if spontaneous activity was present. For recording evoked activity, the receptor field of a unit was identified by low-intensity mechanical stimulation of the glabrous plantar skin of the hind paw with a small glass probe (with 1 mm round tip). Firing frequency to innocuous mechanical stimulation was then examined with graded von Frey monofilaments and a modified von Frey monofilament with a tungsten tip for noxious stimulation for 10 seconds. Prior to evoked stimulation, fibers were assessed for spontaneous activity for 10 seconds, and the mean firing frequency of this spontaneous activity was subtracted from the observed firing frequency during evoked stimulation. To identify unit types, the sciatic nerve was stimulated and used to calculate conduction velocity by dividing the distance between stimulation and recording sites by the response latency of the electrically evoked action potential. Fiber types were then classified based on conduction velocity; 20 m/s or greater for Aβ, between 3 and 20 m/s for Aδ, and less than 3 m/s for C-type. The genotype was blinded for all analyses.

Whole-cell patch clamp electrophysiology

Neuronal soma were categorized into either small (≤32 μm) or large diameter (>32 μm), as the electrophysiology properties of rodent neurons vary based upon size (95, 96), and the majority of small-diameter neurons are considered nociceptors (97). Neuronal capacitance was fully compensated and continuously monitored to ensure stable recording conditions. Whole-cell recordings were obtained using HEKA EPC10 amplifier, and recordings were obtained using Patchmaster Next software (version 1.2). The genotype and treatment were blinded for all analyses. All reagents for patch clamp analysis were obtained from Thermo Fisher Scientific unless otherwise specified.

Current clamp recordings. Isolated sensory neuron soma were superfused with extracellular buffer (140 NaCl, 2.8 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 8.8 sucrose, pH 7.4 ± 0.02 and 310 ± 3 mOsm, adjusted with sucrose). Borosilicate glass pipettes (2–6 MΩ) filled with internal solution (in mM: 135 KCl, 4.1 MgCl2, 2 EGTA, 0.2 GTP, 2.5 ATP, and 10 HEPES, pH 7.2 ± 0.02 and 290 ± 2 mOsm) were pulled using a Sutter Instruments P87 pipette puller and used to perform patch clamp recordings. Series resistance was maintained at less than 15 MΩ and compensated at 80%.

Spontaneous activity. Whole-cell recordings were established in voltage clamp mode, then switched to current clamp mode to measure RMP. Voltage was recorded at RMP for 2 minutes to observe spontaneous, suprathreshold action potentials (>0 mV) with 0 current injection. Cells firing at least 1 action potential during the 2-minute period were considered spontaneously active.

Current-evoked excitability. Neuron soma were held at –70 mV to prevent spontaneous activity from influencing current-evoked recordings, and intrinsic excitability was recorded using the following protocols (98): (i) Voltage-current (V-I) relations were obtained using 20 sweeps of 500 ms alternating ascending/descending current pulses (5 pA steps from holding current). The plateau voltage deflection was plotted against current amplitude, and input resistance was determined from the slope of a V-I plot. (ii) Action potential (AP) properties were measured using an ascending series of 500 ms depolarizing current pulses. Rheobase was defined as the minimal current to elicit at least a single spike. AP threshold was determined from a derivative function, where dV/dt first exceeded 28 mV/ms. AP amplitude was determined relative to AP threshold, and AP half-width was measured as the width at half of the AP amplitude. (iii) A series of eleven 500 ms depolarizing current steps (range, rheobase to 250 pA above rheobase; 25 pA increments, 20-second intervals) or seven 500 ms depolarizing current steps (range, rheobase to 1,600 pA above rheobase; 200 pA increments, 20-second intervals) was used to determine AP firing frequency.

Voltage clamp recordings. Extracellular buffer (in mM: 70 NaCl, 70 choline-Cl, 3 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 10 HEPES, pH 7.35 ± 0.02 and 310 ± 3 mOsm, adjusted with sucrose) was flowed over isolated sensory neuron soma from control rats. The addition of 20 mM TEA-Cl and 0.1 mM CdCl2 was added to extracellular buffer to block voltage-gated K+ channels and Ca2+ channels, respectively (99). Fire-polished borosilicate glass pipettes (1–4 MΩ) were filled with internal solution (in mM: 140 mM CsF, 10 mM NaCl, 2 mM MgCl2, 0.1 CaCl2, 1.1 EGDA, 10 HEPES, pH 7.3 ± 0.02 and 310 ± 3 mOsm, adjusted with sucrose) and pulled using a Sutter Instruments P87 pipette. Soma were established in voltage clamp at a holding potential of –90 mV. Series resistance was maintained at less than 10 MΩ and compensated on 85%, then held at holding potential for 2–4 minutes. Currents were elicited by incremental depolarizing steps (+5 mV increments, 500 ms duration, 5-second intervals) between –80 mV and +40 mV, with a –100 mV hyperpolarizing pulse given before and after each step for 50 ms. The average of 3 sweeps per neuron was taken for determination of peak current density at each step, which were filtered at 2.9 kHz.

Calcium imaging

Sensory neuron soma. Calcium imaging of dissociated neuronal soma was conducted as we have previously published (92). Soma dissociated from DRGs were incubated overnight in media with or without 0.1-1,000 ng/mL p11. Soma were then washed with extracellular buffer solution (150 mM NaCl, 10 mM HEPES, 8 mM glucose, 5.6 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.40 ± 0.03, and 320 ± 3 mOsm) for 30 minutes, incubated with 2.5 mg/mL Fura-2-AM (Life Technologies) for 45 minutes, and washed for 30 minutes. Fluorescence images were captured at 340 nm and 380 nm with a cooled Andor Zyla-SCMOS camera (Oxford Instruments) to calculate the bound to unbound ratio (340/380). NIS-Elements software (Nikon) was used to detect and analyze intracellular calcium changes. To induce membrane depolarization, we added 20 mM KCl to the extracellular buffer solution, while reducing NaCl concentration to maintain an osmolarity of 320 mOsm. The depolarizing solution was applied to neurons for 10 seconds to determine both number of responding neurons and response magnitude. Soma that exhibited a ≥20% increase in 340/380 ratio within 30 seconds after KCl application compared with baseline ratio were considered positive responders. As a positive control, 50 mM KCl was applied near the end of the recordings; soma were considered viable and subsequently analyzed only if they responded to 50 mM KCl. The genotype and treatment were blinded during all calcium imaging analyses.

Schwann cells. A detailed description of the calcium imaging of Schwann cells is presented in the Supplemental Methods.

Mass spectrometry

Peptides were analyzed by nanoLC-MS/MS, and detailed methods are presented in the Supplemental Methods.

Animal behavior

Rat plantar cutaneous mechanical sensitivity was assessed as previously reported (22). Rats underwent 30 μL intraplantar (footpad) injections of saline or treatment with undiluted Schwann cell collection media or p11 (0.9, 9, and 90 ng) and were acclimated on top of a wire mesh for 1 hour, with the experimenter present for 30 minutes of this period. Testing was performed at approximately the same time each day. Mechanical sensitivity threshold was determined with von Frey filaments (up-down method; ref. 100), and values were analyzed after log transforming (101). Hypersensitivity to noxious force (hyperalgesia), which is selectively associated with aversion (102), was tested by needle prick (103). The treatment was blinded for all experiments. For baseline von Frey withdrawal threshold measurements, animals underwent testing within 7 days of treatment administration.

Statistics

Results were considered statistically significant when P < 0.05. All data were analyzed using GraphPad Prism (Version 9.0.0). No potential outliers were removed during data analysis for this study. Statistical analyses used for each data set are indicated within each figure legend. For dorsal root teased-fiber single-unit recording, data were analyzed using χ2 for spontaneous activity per fiber bundle and an unpaired 2-tailed Student’s t test for spontaneous activity per animal and firing rate. Mechanical sensitivity to graded von Frey stimulation was analyzed using a 2-way repeated measures ANOVA, and sensitivity to needle was analyzed using an unpaired 2-tailed Student’s t test. For analysis of myelin pathology, data were analyzed using unpaired 2-tailed Student’s t test. For current clamp experiments, membrane and AP properties were analyzed using a 1-way ANOVA for testing Schwann cell media effects on neuron function and unpaired 2-tailed Student’s t test for testing the effects of p11 on neuron function; current-evoked firing frequency was analyzed using a 2-way repeated measures ANOVA. Percentage of spontaneously active cells was analyzed using χ2. For calcium imaging experiments, proportion of cells responding was analyzed using χ2, while response magnitude data were analyzed using a 1-way ANOVA. For voltage clamp experiments, current densities were measured using a 2-way repeated measures ANOVA, and maximum current density was analyzed using unpaired 2-tailed Student’s t test. For nanoLC-MS/MS, data were analyzed using a Benjamini-Hochberg corrected 2-tailed t test. For ELISA experiments, data were analyzed using a 2-way ANOVA. For von Frey and noxious needle tests, data were analyzed using a 2-way repeated measures ANOVA. Bonferroni post hoc corrections were used for significant ANOVAs. Fisher’s exact tests were used for significant χ2 tests.

Study approval

All protocols were in accordance with NIH guidelines and were approved by the Institutional Animal Care and Use Committee at the Medical College of Wisconsin.

Data availability

All data are available from the Supporting Data Values XLS file and from the corresponding author.