FDA-approved 5-HT_1F Receptor Agonist Lasmiditan Induces Mitochondrial Biogenesis and Enhances Locomotor and Blood-Spinal Cord Barrier Recovery after Spinal Cord Injury

Abstract

Vascular and mitochondrial dysfunction are well-established consequences of spinal cord injury (SCI). Evidence suggests mitigating these dysfunctions may be an effective approach in treating SCI. The goal of this study was to elucidate if mitochondrial biogenesis (MB) induction with a new, selective and FDA-approved 5-hydroxytryptamine receptor 1F (5-HT_1F) receptor agonist, lasmiditan, can stimulate locomotor recovery and restoration of the blood-spinal cord barrier (BSCB) after SCI. Female C57BL/6J mice were subjected to moderate SCI using a force-controlled impactor-induced contusion model followed by daily administration of lasmiditan (0.1 mg/kg, i.p.) beginning 1h after injury. In the naïve spinal cord, electron microscopy revealed increased mitochondrial density and mitochondrial area, as well as enhanced mitochondrial DNA content.

FCCP-uncoupled oxygen consumption rate (OCR), a functional marker of MB, was also increased in the naïve spinal cord following lasmiditan treatment. We observed disrupted mitochondrial DNA content, PGC-1α levels and FCCP-OCR in the injury site 3d after SCI. Lasmiditan treatment attenuated, and in some cases restored these deficits. Lasmiditan treatment also resulted in increased locomotor capability as early as 7d post-SCI, with treated mice reaching a Basso-Mouse Scale score of 3.3 by 21d, while vehicle-treated mice exhibited a score of 2.0. Integrity of the BSCB was assessed using Evans Blue dye extravasation. While SCI increased dye extravasation at 3d and 7d, dye accumulation in the spinal cord of lasmiditan-treated mice was attenuated 7d post-SCI, suggesting accelerated BSCB recovery. Finally, lasmiditan treatment resulted in decreased lesion volume and spared myelinated tissue 7d post-SCI. Collectively, these data reveal that 5-HT_1F receptor agonist-induced MB using the FDA-approved drug lasmiditan may be an effective therapeutic strategy for the treatment of SCI.

Introduction

Spinal cord injury (SCI) is a traumatic event that can generate an array of impairments ranging from partial loss of function to complete paralysis below the injury site. SCI initiated by a mechanical insult, known as primary injury, results in extensive vascular damage, including vasoconstriction. This damage is quickly followed by a progressive local cascade of events, causing further dysfunction and damage and leading to insufficient oxygen delivery and subsequent mitochondrial dysfunction, all of which encompass secondary injury post-SCI (1–3).

While the canonical role for central nervous system (CNS) mitochondria is the generation of energy, they are also involved in the homeostasis and degeneration of neurons (4, 5). Neuronal cells are easily compromised following ischemic events due to their high reliance on ATP-driven processes, in combination with their inadequate energy reserves and limited capacity to buffer oxidative stress (4, 6). Axons are more susceptible to the damage caused by ionic imbalance due to their high concentration of voltage-gated sodium channels in the nodes of Ranvier (1). Taken together, these disruptions manifest in the failure to generate and maintain adequate energy production, thereby exacerbating the pathology of SCI and resulting in further cell dysfunction and death (3, 4, 7).

Mitochondrial biogenesis (MB) is a dynamic process of generating new, functional mitochondria that involves a complex network of transcriptional pathways governed by the “master regulator,” peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) (3, 8, 9). Reports document an immediate and rapid decrease in PGC-1α expression in rodent models of contusion SCI (10, 11), suggesting impaired MB. Evidence indicates that restoration of mitochondrial homeostasis promptly following injury may promote neuronal survival and enhance functional recovery (3, 12–14), suggestive of the potential benefit of pharmacological induction of MB following injury. Additionally, studies have demonstrated a positive correlation between MB induction and angiogenesis (15–17). As compromised vasculature is a delirious consequence of SCI, particularly blood-spinal cord barrier (BSCB) dysfunction (18), enhanced angiogenesis could serve as an effective treatment strategy. Therefore, therapeutics targeting reestablishment of mitochondrial homeostasis through MB activation may improve several facets of secondary injury progression, functional and vascular recovery, and ultimately neuronal survival following SCI (3).

Neuronal 5-hydroxytryptamine (serotonin, 5-HT) receptors are involved in generating and regulating locomotor activity (19). Following SCI, there is a disruption in descending serotonergic projections in spinal motor areas implicated in locomotor dysfunction. Treatment with exogenous serotonin or a 5-HT analog was shown to accelerate locomotor recovery following SCI (19). However, this approach results in global activation of many classes of 5-HT receptors, so the exact contributors promoting recovery remain unknown. Through our drug discovery program to ascertain inducers of MB, we identified the 5-HT_1F receptor as a mediator of MB (8, 20). This receptor, while not fully characterized, is found in the spinal cord and various brain regions (21, 22). In addition, 5-HT_1F receptors have been detected in rodent and human vasculature (23, 24).

We previously showed that treatment with the specific 5-HT_1F agonist LY344864 increases MB in multiple organ systems, including the CNS (24–26). A highly selective and potent 5-HT_1F receptor agonist lasmiditan was recently FDA-approved for the treatment of migraines (27, 28). Using renal endothelial cells, lasmiditan induced MB and angiogenesis (29); however, its effects on mitochondrial function and recovery following CNS trauma remain unknown. Given these data and the detrimental impact of mitochondrial dysfunction post-SCI, the goal of this study was to assess the therapeutic efficacy of lasmiditan-induced MB on recovery after SCI. Ascertaining the mitochondrial and vascular effects of lasmiditan following SCI will reveal its therapeutic potential to be repurposed as a novel pharmaceutical for SCI. We hypothesize that lasmiditan will restore mitochondrial homeostasis, enhance BSCB integrity and promote locomotor recovery after SCI.

Materials and Methods

Animal handling and care

Female C57bl/6J mice (8–10 weeks of age) were purchased from Jackson Laboratories and used in all experiments. All animals were housed in groups of 3–5 in temperature-controlled conditions under a light/dark photo-cycle with food and water supplied ad libitum.

Lasmiditan was obtained from Sigma Aldrich (St. Louis, MO) and dissolved in saline/0.1% DMSO. To determine the MB effect of lasmiditan in the spinal cord under physiological conditions, naïve mice were treated with 0.1 mg/kg lasmiditan, intraperitoneally (i.p.) once daily for 3 days followed by euthanasia using isoflurane overdose and subsequent isolation of the T10–12 region of the spinal cord.

In the SCI model, mice were randomized into sham and SCI groups. Animals were anesthetized with 10 mg/kg ketamine and 6 mg/kg xylazine via i.p injection and continuously monitored for spontaneous breathing. Mice underwent a complete single-level laminectomy at the thoracic vertebral arch (T10–12) and spinal cord was exposed (6, 10). The vertebral column was clamped and stabilized at the upper thoracic and lumbar levels, and a controlled contusion with a force of 80 kilodynes and 0 second dwell time was administered using the Infinite Horizon IH-0400 impactor (Lexington, KY) with the dura intact. Sham mice received laminectomy only. Manual bladder expression was performed twice daily until functional recovery. Injured mice were further randomized into lasmiditan- or vehicle-treated groups and treated i.p with either 0.01, 0.1, 1.0 mg/kg lasmiditan or vehicle control beginning 1h after injury and continuing daily until euthanasia. Among injured animals, criteria for exclusion included broken dura at time of SCI impact, or failure to demonstrate a BMS score below 1.0 by 24h following SCI.

Groups were euthanized 3d, 7d or 21d post-SCI via isoflurane overdose and spinal cords were isolated for analysis. Animal use details provided in Table 1. All studies were approved by the University of Arizona in accordance with the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Table. 1.

Animal use table. For all studies, C57BL/6J female mice were treated daily (i.p) until day of euthanasia. EB, Evan’s blue; EM, electron microscopy; FCCP-OCR, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone-oxygen consumption rate.

Euthanized Time Point	Animal Numbers	Assessments	Surgical Procedure	Treatment (i.p.)
3d	5	FCCP-OCR, copy#	Naïve	Vehicle
3d	5	FCCP-OCR, copy#	Naïve	0.1mg/kg Lasmiditan
3d	5	EM	Naïve	Vehicle
3d	5	EM	Naïve	0.1mg/kg Lasmiditan
3d	5	Protein, copy#	Sham	Vehicle
3d	5	Protein, copy#	SCI	Vehicle
3d	5	Protein, copy#	SCI	0.1mg/kg Lasmiditan
3d	6	FCCP-OCR	Sham	Vehicle
3d	6	FCCP-OCR	SCI	Vehicle
3d	6	FCCP-OCR	SCI	0.1mg/kg Lasmiditan
3d	6	EB	Sham	Vehicle
3d	6	EB	SCI	Vehicle
3d	6	EB	SCI	0.1mg/kg Lasmiditan
3d	2	EB representative	Sham	Vehicle
3d	2	EB representative	SCI	Vehicle
3d	2	EB representative	SCI	0.1mg/kg Lasmiditan
7d	6	EB	Sham	Vehicle
7d	6	EB	SCI	Vehicle
7d	6	EB	SCI	0.1mg/kg Lasmiditan
7d	1	EB representative	Sham	Vehicle
7d	1	EB representative	SCI	Vehicle
7d	1	EB representative	SCI	0.1mg/kg Lasmiditan
7d	6	Histology	Sham	Vehicle
7d	6	Histology	SCI	Vehicle
7d	6	Histology	SCI	0.1mg/kg Lasmiditan
7d	5	Protein	Sham	Vehicle
7d	5	Protein	SCI	Vehicle
7d	5	Protein	SCI	0.1mg/kg Lasmiditan
21d	8	BMS	Sham	Vehicle
21d	9	BMS	SCI	Vehicle
21d	9	BMS	SCI	1.0mg/kg Lasmiditan
21d	10	BMS	SCI	0.1mg/kg Lasmiditan
21d	9	BMS	SCI	0.01 mg/kg Lasmiditan

DNA Isolation and qPCR

DNA was isolated from naïve spinal cords or injury sites (approximately 5mm segment of spinal cord centered at the epicenter (6)) at 3d post-treatment using the Qiagen DNeasy Blood and Tissue Kit (Valencia, CA) with 5 ng used for qPCR of relative mitochondrial DNA (mtDNA) content. ND1, a mitochondrial gene, was measured and normalized to the nuclear encoded gene β-actin. Melting point curves were evaluated for each primer set to ensure specificity. Reference Table 2 for primer sequences.

Table 2.

Primer sequences table.

Target	Sense	Antisense
ND1	5′-TAG AAC GCA AAA TCT TAG GG −3′	5′-TGC TAG TGT GAG TGA TAG GG −3′
β-Actin	5′-GGGATGTTTGCTCCAACCAA-3′	5′-GCGCTTTTGACTCAGGATTTAA-3′

Protein Isolation and Immunoblot Analysis

Protein was extracted from the injury site at 3d or 7d post-treatment using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.4) with protease inhibitor cocktail (1:100), 1 mM sodium fluoride and 1 mM sodium orthovanadate (Sigma-Aldrich, St. Louis, MO). Samples were agitated for 2 h at 4°C and then centrifuged at 14,000 × g for 15 min and the supernatant collected. Protein was quantified using a bicinchoninic acid assay, and 10–12 μg of protein was separated via electrophoresis using 4–15% SDS-polyacrylamide gels, then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were blocked in 5% milk in TBST and incubated overnight with primary antibodies with constant agitation at 4°C. Membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody and visualized using chemiluminescence (Thermo Scientific, Waltham, MA) on a GE ImageQuant LAS4000 (GE Life Sciences, Pittsburgh, PA). Optical density was determined using Image Studio Lite software. Primary antibodies used were as follows: PGC-1α (1:1000), Occludin (1:1000), Claudin-5 (1:1000), and α-tubulin (1:1000). All primary antibodies were purchased from Abcam (Cambridge, UK).

Transmission Electron Microscopy

Approximately 4 mm² samples of T10–12 spinal cord were fixed overnight in 2.5% glutaraldehyde in 0.1 M PIPES buffer (pH 7.4) then transferred to PBS at which point they were delivered to the University of Arizona Microscopy Alliance Core, where they were placed in 0.1 M PIPES buffer (pH 7.4) overnight at 4 °C. Samples were then transferred to 0.1 M PIPES/Glycine buffer for 30 min at room temperature, then washed in deionized water (diH2O) twice for 5 min each, placed in 1% osmium tetroxide for 1h, washed twice in diH2O for 10 min, then stained with 2% aqueous uranyl acetate for 1 h and washed again in diH2O for 10 min. Samples were then dehydrated with increasing ethanol dilutions (5 min, 50, 70, 90, 100%) and acetone (2 × 5min), infiltrated with 1:1 acetonitrile/Spurr’s resin overnight, then 100% Spurr’s resin for 24h and flat-embedded overnight at 60 °C. Cross-sections of the spinal cord samples (80 nm) were cut on an RMC PTXL ultramicrotome, placed onto uncoated 150-mesh coper grids and counterstained with 2% lead acetate for 2 min. Images were viewed by FEI Tecnai Spirit microscope (FEI, Hillsboro, OR) operated at 100 kV and captured using an AMT 4 Mpixel camera (Advanced Microscopy Techniques, Woburn, MA) at a magnification of 4200X. Images of myelinated axons were captured for analysis; our sampling did not discriminate ascending or descending tracts of the spinal cord. Mitochondrial count and morphology were analyzed using the analyze particles plug-in in ImageJ FIJI. In all cases, 5–6 images were evaluated containing 145–241 mitochondria for each spinal cord sample (n=1) by a trained observer blinded to the treatment groups. Images were analyzed for the average mitochondrial number per field and average mitochondrial area per field, and normalized to axonal area per field. Analysis of mitochondria were restricted to axons.

Mitochondria Isolation

Mitochondria were isolated from the naïve spinal cord or the injury site (approximately 5mm of spinal cord centered at the epicenter (6)) 3d post-treatment using techniques modified as previously described (30). The isolated spinal cord segments were immediately transferred into a Teflon-glass dounce homogenizer, each containing approximately 2ml mitochondrial isolation buffer (215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin (BSA), 20 mM HEPES, 1 mM EGTA; pH adjusted to 7.2 with KOH). The tissues were homogenized manually using 20–30 strokes and were centrifuged at 1,300 g for 3 minutes. The supernatants containing mitochondria were transferred to fresh tubes and spun at 13,000 g for 10 minutes. Mitochondrial pellets were resuspended in approximately 700 μL isolation buffer. The mitochondria trapped in the synaptosomal vesicles were released by pressurized N₂ cell disruption at 1,200 psi for 10 minutes. The resulting total mitochondrial suspensions were centrifuged at 10,000 g for 10 minutes. The pellets were resuspended in 50 μL isolation buffer (without EGTA). Mitochondrial protein concentration was determined using BCA protein assay kit (Pierce, Cat # 23,227) recording the absorbance at 560 nm on Biotek Synergy HT plate reader (Winooski, VT).

Analysis of Oxygen Consumption

Oxygen consumption rate (OCR) of isolated mitochondria was measured from the naïve spinal cord or the injury site after 3d of treatment using the Seahorse Bioscience XF-96 Extracellular Flux Analyzer (30). On the day prior to the assay, the sensor cartridge of Extracellular Flux kit was soaked in XF calibrant solution and kept at 37°C overnight. Before loading, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was diluted appropriately in respiration buffer (RB) (215 mM mannitol, 75 mM sucrose, 0.1% BSA, 20 mM HEPES, 2 mM MgCl₂, and 2.5 mM KH₂PO₄, adjusted pH 7.2 with KOH) pre-warmed at 37°C, containing 5 mM pyruvate and 2.5 mM malate. Mitochondria were diluted in RB and 6 μg mitochondria were loaded per well in a volume of 30 μL. The assay plate was centrifuged at 3000 rpm for 4 minutes at 4°C. Final volume per well was brought to 100 μL with RB. Initial OCR was measured before and after injection of FCCP (6 μM) (Sigma Aldrich, St. Louis, MO) to measure the uncoupled OCR (FCCP-OCR) (20). For each n, samples were run in triplicate and OCR averaged.

BMS Assessment

Locomotor capability was assessed using the ten-point (0–9) Basso Mouse Scale (BMS) (30) by two trained observers blinded to experimental groups. Each mouse was observed for 4 min, with bladder expression taking place prior to assessment. Animals were observed 24h after surgery and every week thereafter until euthanasia. Sham animals maintained full locomotor capabilities with a BMS score of 9 throughout the experiment.

Spontaneous locomotor activity

Spontaneous locomotor activity was assessed in naïve and SCI mice in a Digiscan Animal Activity Monitor system (Omnitech Electronics Model RXYZCM(8) TAO, Columbus, OH, USA), (26, 31). Mice were placed in plexiglass chambers with photocell emitters and receptors spaced equally along the perimeter, creating a grid of infrared beams measuring total distance and vertical activity. Total distance was determined by centimeters travelled. Mice were tested in a darkened environment, and data were collected continuously for 10 min at the same time of day for each group.

Lesion Volume Analysis

Histopathological analysis was performed on injured spinal cords following 7d treatment. Mice were transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde (PFA). A 1cm segment of the spinal cord centered on the injury epicenter was removed and post-fixed in PFA for 2h, then washed in 0.2 M phosphate buffer overnight at 4ºC. Tissues were then cryoprotected in 20% sucrose with 0.1% sodium azide at 4ºC until the spinal cords sank (≥3 days). Spinal cords were trimmed to 6 mm segments centered on the injury sight and frozen in OCT at −80ºC. The entire 6mm was cryosectioned into 10 μm coronal sections and every section collected.

Eriochrome cyanine (EC) staining for myelin was used to distinguish damaged and spared myelinated tissue (32). Slides were warmed for 60 minutes at 37ºC, then hydrated in dH₂O, submerged in acetone for 2 minutes, and rehydrated in dH₂O. Slides were exposed to serial dilutions of decreasing concentrations of ethanol and incubated in EC solution for 30 minutes. Selective myelin staining was obtained by differentiation in 0.3% ammonium hydroxide for 30 seconds. Slides were then exposed to serial dilutions of increasing ethanol concentrations. Analyses were performed in a blinded fashion, with respect to treatment group, using the EVOS M5000 Imaging System microscope (ThermoFisher, Waltham, MA) at 40X magnification and ImageJ software. Lesion and spared tissue areas were quantified across 2mm of spinal cord centered on the epicenter at 100 μm intervals using the Cavalieri method (24, 32), totaling 21 sections per animal.

Immunofluorescence

Slides containing frozen sections were washed in 1% Tris-Triton Buffer (TTB) (50 mM Tris Saline, pH 7.6) for 30 min then blocked in 4% normal goat serum for 60 min. Sections were incubated at 4°C overnight in primary antibody claudin-5 (1:50) (Abcam, Cambridge, UK) in TTB containing 4% normal goat serum. Secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (1:1000) (Abcam, Cambridge, UK) in TBB was applied for 60 min at room temp. Sections were counterstained with DAPI (Life Technologies Corporation, Eugene OR), mounted with gold antifade reagent (Life Technologies Corporation, Eugene OR), cover slipped and imaged using the EVOS M5000 Imaging System microscope (ThermoFisher, Waltham, MA) at a final magnification of 200X. Images were captured from the ventral horn 1mm caudal to the epicenter of injury (± 10μm) as determined by the sample’s respective EC analysis.

Evan’s Blue Extravasation

Integrity of the blood-spinal cord barrier (BSCB) was assessed at 7d post-SCI using Evans Blue (EB) dye extravasation (24, 33). Evans Blue dye (2%, 0.08 mL) was administered via intraocular injection and allowed to circulate for 30 minutes. Mice were then transcardially perfused with saline and a 6mm segment of spinal cord centered at the epicenter was collected. EB dye was extracted in formamide at room temperature for ≥ 3d and optical density values of the prepared samples were measured at 620 nm with a Spark multimode microplate reader (Tecan, Switzerland). EB content was calculated as ng dye/mg tissue using a standard curve.

Statistical Analysis

For all studies, an individual mouse represents one n (n = 1). Behavioral assays were n=8 sham mice and n=9–10 injured mice per group and data were analyzed using Two-way ANOVA with repeated measures followed by Tukey’s post-hoc test. Differences in mtDNA, protein expression, total EB content and OCR between all three groups (Sham, SCI + vehicle, SCI + lasmiditan) were analyzed using One-way ANOVA and with n=5–6. Histological analysis between two injured groups (SCI + vehicle, SCI + lasmiditan) as well as analysis of naïve studies (vehicle, lasmiditan) were performed using two-tailed TTEST and with n=5–6. In all cases, GraphPad Prism software (La Jolla, CA) was used and a p<0.05 was considered indicative of a statistically significant difference between mean values. For expression analyses, different superscripts are indicative of statistically significant differences, while bars with the same superscript are not significantly different. For BMS scores (Figure 1.A), “a” denotes a statistically significant difference of 0.1 mg/kg lasmiditan compared to SCI + Vehicle, “b” denotes a statistically significant difference of 0.01 mg/kg lasmiditan compared to SCI + Vehicle and “c” denotes a statistically significant difference compared with day 1.

Fig. 1. Effect of lasmiditan on locomotor recovery post-SCI and MB in the naïve spinal cord.

Mice were subjected to moderate SCI followed by daily administration with either 0.01, 0.1, 1.0 mg/kg lasmiditan or vehicle beginning 1h after injury and continuing for 21d (A). Data are representative of 8 sham mice and 9–10 mice per SCI group and are expressed as mean ± SEM (a p<0.05 0.1 mg/kg lasmiditan compared to SCI + Vehicle, b p<0.05 0.01 mg/kg lasmiditan compared to SCI + Vehicle and c p<0.05 compared to Day 1 by Two-way ANOVA with repeated measures followed by Tukey’s post-hoc test. Injured mice treated with 1.0 mg/kg lasmiditan were not significant from SCI + Vehicle. Naïve mice were exposed to lasmiditan (0.1 mg/kg i.p) or vehicle daily for 3d. The spinal cord was extracted and analyzed for mtDNA content (B). Scale bar=2μm. Mitochondria were also isolated and uncoupled mitochondrial oxygen consumption rates (FCCP-OCR) were measured for complex I-driven respiration using Seahorse XF96 analyzer (C). An additional cohort of naïve mice were treated with lasmiditan or vehicle daily for 3d. Spinal cord was extracted, fixed and imaged using transmission electron microscopy (D) to obtain the number of mitochondria (E) and mitochondrial area (F) per field. Analysis of mitochondrial number and mitochondrial area per field were also normalized to axonal area per field (G,H). Data represent n=5 and are expressed as mean ± SEM (* p < 0.05 compared to Vehicle by two- tailed student’s t-test).

Results

Dose-dependent effect of lasmiditan on locomotor recovery post-SCI

Injured mice were treated daily with either 0.01, 0.1, 1.0 mg/kg lasmiditan or vehicle beginning 1h after SCI and continuing for 21d (Figure 1.A). Vehicle-treated mice demonstrated a BMS score of approximately 2.0 by 21d, similar to that reported previously with this model (6). Compared to 1d, injured mice treated with 0.1 mg/kg showed the earliest recovery compared to 1d by 7d post-SCI (p=0.0165). Lasmiditan-treated mice reached a final BMS score of 3.3 by 21d compared to 2.0 in untreated mice (p=0.0075). In contrast, BMS scores of 2.8 and 2.5 were observed in 0.01 and 1.0 mg/kg lasmiditan-treated groups, respectively, 21d after injury. Analysis revealed a significant effect of treatment/injury [F(4,40)=452.1, p<0.0001, ANOVA] and days post-injury [F(1.982,79.27)=200.2, p<0.0001, ANOVA], as well as a significant interaction [F(12,120)=13.78, p<0.0001, ANOVA]. Based on these data, 0.1 mg/kg lasmiditan was used for the remainder of studies.

Effect of lasmiditan on MB in the naïve spinal cord

Naïve mice were treated with 0.1 mg/kg lasmiditan or vehicle daily for 3 days. Following treatment, the T9–12 portion of the spinal cord was collected and analyzed for mitochondrial endpoints. Lasmiditan-treated mice displayed a 2.0-fold increase in mtDNA content [T(8)=3.082, p=0.015, TTEST] (Figure 1.B) in the spinal cord compared to vehicle-treated mice. FCCP-uncoupled oxygen consumption rate (FCCP-OCR) is a marker of maximal ETC activity and functional MB (20). FCCP-OCR increased by approximately 18% in spinal cords of lasmiditan-treated animals compared to vehicle-treatment [T(8)=2.804, p=0.0230, TTEST] (Figure 1.C). Transmission electron microscopy (TEM) was used to assess mitochondrial content (Figure 1.D). Myelinated axons in the spinal cord from lasmiditan-treated mice displayed a 1.4-fold increase in mitochondrial number [T(8)=2.670, p=0.0283, TTEST] (Figure 1.E) and a 1.3-fold increase in mitochondrial area [T(8)= 2.366, p=0.0455, TTEST] (Figure 1.F). When normalized to axonal area per field, mitochondrial number (Figure 1.G) and mitochondrial area (Figure 1.H) were also increased, [T(8)=2.710, p=0.0267, TTEST] and [T(8)=2.838, p=0.0219, TTEST] respectively. These data provide evidence that lasmiditan induced MB in the naïve spinal cord.

Effect of lasmiditan on MB in the injured spinal cord

Mice were subjected to SCI followed by daily administration of lasmiditan (0.1 mg/kg) or vehicle beginning 1h after injury [df=2,12, F=5.593, p=0.0192, ANOVA]. By 3d post-SCI, mtDNA content was reduced approximately 45% in the injury site compared to sham controls (p=0.015) (Figure 2.A). Lasmiditan administration attenuated this decrease to 21% compared to sham. Immunoblot analysis revealed decreased protein expression of PGC-1α [df=2,12, F=12.55, p=0.0011, ANOVA], similar to that previously reported in this model (6), which was restored to sham levels in lasmiditan-treated mice (Figure 2.B).

Fig. 2. Effect of lasmiditan on MB in the injured spinal cord.

Mice were subjected to moderate SCI followed by daily administration of vehicle or lasmiditan (0.1 mg/kg, i.p) beginning 1h post-injury and continuing for 3d. The injury site was extracted and analyzed for mtDNA content (A) and mitochondrial protein expression (B). An additional cohort of mice were treated similarly. Prior to euthanasia, the injury site was extracted and mitochondria isolated. Uncoupled mitochondrial oxygen consumption rates (FCCP-OCR) were measured for complex I driven respiration using SeahorseXF96 analyzer (C). Data represent 5–6 mice per group and are expressed as mean ± SEM (*p < 0.05 compared to Sham and #p<0.05 compared SCI + Vehicle by one-way analysis of variance followed by Tukey post hoc test).

Similar to that observed with mtDNA, FCCP-OCR was decreased by ~45% in the injury site of vehicle-treated animals compared to sham controls, further indicating mitochondrial dysfunction [df=2,15, F=4.232, p=0.0349, ANOVA] (Figure 2.C). Lasmiditan treatment partially recovered this decrease, restoring FCCP-OCR levels at rates comparable to that of sham. These data provide evidence that lasmiditan induced MB in the injury site 3d post SCI.

Effect of lasmiditan on histopathology in the injured spinal cord

Spinal cord histopathology was assessed at 7d post-SCI using Eriochrome Cyanine staining for myelin (Figure 3.A). Analysis was performed on 2 mm of spinal cord centered on the epicenter revealed. Total volume of the spinal cord was assessed independent of EC stain, revealing similar volumes of spinal cord regardless of treatment [df=10, T=0.8744, p=0.4024, TTEST] (Figure 3.B). Lasmiditan-treated mice had decreased lesion volume [df=10, T=2.870, p=0.0167, TTEST] (Figure 3.C) and increased percent spared myelinated tissue [df=10, T=3.544, p=0.0053, TTEST] (Figure 3.D) compared to vehicle-treated mice. Scale bar=750μm.

Fig. 3. Effect of lasmiditan on spinal cord histopathology post-SCI.

Mice were subjected to moderate SCI followed by daily administration of vehicle or lasmiditan (0.1 mg/kg, i.p) beginning 1h post-injury and continuing for 7d. Spinal cords were extracted and evenly spaced tissue sections stained with Eriochrome Cyanine (A) and analyzed for total volume (B), lesion volume (C) and percent spared tissue (D) across 2 mm of spinal cord centered on the epicenter. Data are representative of 6 mice per group and are expressed as mean ± SEM (*p<0.05 compared to SCI + Vehicle by two-tailed student’s t-test).

Effect of lasmiditan on BSCB integrity in the injured spinal cord

EB dye accumulation increased in injured mice compared to sham controls 3d post-SCI regardless of treatment [F(2,15)=23.45, p<0.0001, ANOVA]. By day 7, vehicle-treated mice still displayed increased dye accumulation compared to sham controls (p= 0.0406), while that of lasmiditan-treated mice was comparable to sham controls (p=0.522) (Figure 4.A, B). Expression of the intracellular scaffolding protein occludin (p=0.0007) and the tight junction protein claudin-5 (p<0.0001) were decreased by more than 60% compared to sham 7d post-SCI in vehicle-treated mice [F(2,12)=87.42, p<0.0001, ANOVA] and [F(2,12)=13.74, p=0.0008, ANOVA] respectively (Figure 4.C). Treatment with lasmiditan attenuated these deficits.

Fig. 4. Effect of lasmiditan on BSCB integrity post-SCI.

Mice were subjected to moderate SCI followed by daily administration of vehicle or lasmiditan (0.1 mg/kg, i.p) beginning 1h post-injury. Subsets of mice were euthanized 3d and 7d post-SCI. For EB experiments, mice were intraocularly injected with EB and the dye was allowed to circulate for 30 minutes prior to euthanasia. Mice were then perfused with saline and a 6mm segment of spinal cord was extracted and analyzed for dye accumulation (A). A subset of mice were transcardially perfused and fixed with 4% PFA and the spinal cords extracted for representative images (B). The injury site was also collected from a separate group of mice 7d post-SCI and analyzed for protein expression (C). A subset of mice were transcardially perfused and fixed with 4% PFA. The spinal cords were extracted, cryopreserved, sectioned at 10μm thickness and stained for claudin-5 or 4′,6-diamidino-2-phenylindole (DAPI) (D). Images were capture from the ventral horn of the spinal cord, 1mm caudal to the epicenter of injury as determined by EC analysis. Data represent 5–6 mice per group and are expressed as mean ± SEM (*p < 0.05 compared to Sham and #p<0.05 compared to SCI + Vehicle by one-way analysis of variance followed by Tukey post hoc test).

Discussion

Disruption of mitochondrial quality control mechanisms has been extensively implicated in the pathology of SCI (7, 12, 34). As such, therapeutic strategies aimed at maintaining and/or restoring mitochondrial homeostasis and related cellular processes are becoming increasingly popular. While we have shown that the 5-HT_1F receptor is a potent inducer of MB peripherally and centrally (4, 24, 26, 35–37), this receptor remains understudied in the spinal cord. MB is a dynamic process of generating new, functional mitochondria that involves a complex network of transcriptional pathways and is markedly impaired following SCI (10, 11). Evidence indicates that pharmacological-induced restoration of mitochondrial homeostasis promptly following injury may encourage neuronal survival and improve functional recovery (3, 12–14). In the naïve spinal cord, EM analysis revealed increased mitochondrial number within myelinated axons of the spinal cord from lasmiditan-treated animals compared to controls. It is important to note that the density of mitochondria may vary across regions and tracts in the spinal cord (38). As such, the effects of lasmiditan on mitochondrial density may not be consistent across all regions and levels of the spinal cord. We also report increases in mitochondrial content and FCCP-OCR, a measurement of maximal respiratory activity and a phenotypic marker of MB (20). Mitochondrial dysfunction was indicated by decreased mitochondrial DNA content, PGC-1α levels and FCCP-OCR at 3d following SCI. Lasmiditan treatment attenuated, and in some cases restored these deficits.

Vascular disruption is a well-established consequence of SCI (39, 40). The BSCB exists at the capillary level and controls the flux of molecules entering the spinal cord. Previous studies have employed therapeutic strategies aimed at stimulating angiogenesis to produce neuroprotection and functional recovery following SCI in rodent models (41, 42). We previously demonstrated that lasmiditan-induced activation of the 5-HT_1F receptor can stimulate MB and angiogenesis in peripheral endothelial cells in vitro (19, 29). Following SCI, reports reveal that restoring BSCB integrity decreases lesion volume and prevents necrosis and apoptosis of neuronal cells, consequently improving functional endpoints (14, 24, 43). Furthermore, functions of the BSCB rely on an intricate network of tight junction proteins including occludin and claudin-5 (18) and expression of these proteins decrease following rodent models of SCI (44). We report increased BSCB permeability at 3d post-SCI and persisting for 7d post-SCI, as well as decreased expression of both occludin and claudin-5 in the injured spinal cord at 7d post-SCI, indicative of BSCB dysfunction (45–48). Following lasmiditan treatment, we report enhanced locomotor recovery beginning 7d post-SCI, as well as attenuated EB dye accumulation and increased tight junction protein expression, representative of accelerated BSCB recovery. In addition to enhanced locomotor and vascular recovery at 7d post-SCI, injured mice subjected to lasmiditan treatment displayed decreased lesion volume and increased spared myelinated tissue, all of which are correlated with recovered locomotor capability (3).

The observed lasmiditan-induced restoration of tight junction protein expression corroborate previous reports documenting that restored BSCB integrity, including attenuated expression of claudin-5, enhances recovery of motor function following SCI (45, 46). Claudin-5 is the most enriched tight junction protein at blood-CNS barriers (49). Disrupted expression of claudin-5 has been implicated in neuropathy-induced allodynia (44) as well as a host of neurodegenerative diseases including Alzheimer’s disease and multiple sclerosis (49). Given the importance of mitochondrial dysfunction in these and many other CNS pathologies, lasmiditan may have a broad therapeutic potential to address multiple facets of disease and injury progression by promoting MB induction and vascular integrity.

Lasmiditan is lipophilic, permeable to CNS-barriers and is a highly specific agonist for the 5-HT_1F receptor (50). Inhibition of glutamate release has been suggested as a potential mechanism for lasmiditan’s therapeutic effect on migraines (49). Neurotoxicity, particularly glutamatergic excitotoxicity at the synapse, is a well-documented component of secondary injury in SCI, generating an array of impairments including mitochondrial dysfunction, demyelination of axons and neuronal cell death, thereby contributing to functional deficits (1, 4). Inhibition of glutamate receptor activity post-SCI may prevent neuronal apoptosis and inhibit the interaction between excitotoxicity and inflammation, both of which can stimulate functional recovery (1). Therefore, mediation of SCI-induced neurotoxicity may also contribute to these observed effects of lasmiditan treatment post-SCI.

Endogenous serotonin is known to play a stimulatory role in locomotor activity (51). As such, we cannot preclude the possibility that the locomotor effects observed with lasmiditan treatment are in part due to actions on other serotonergic systems. While the 5-HT_1F receptor has yet to be fully characterized, previous data suggest that this receptor does not play a pivotal role in basal locomotor function in this model (24). In addition, previous research demonstrated that 5-HT_1F-induced MB enhances functional recovery in periphery organ systems and is not reliant on the serotonergic system (25, 52). Furthermore, we report lasmiditan did not increase spontaneous locomotor activity in the naïve mouse or following spinal cord injury (Supplemental Figure 1). However, reports suggest activation of the 5-HT_1F receptor can inhibit postsynaptic potentials that trigger spasms after SCI (53), and mitigating this serotonergic disruption may stimulate locomotor recovery (19). Therefore, in addition to the MB-related effects, lasmiditan treatment after SCI may attenuate serotonergic disruption or decrease muscle spasms, further enhancing the increased functional behavior observed.

As described in the methods section, these studies employ ketamine as an anesthetic. Reports document a neuroprotective effect of ketamine on acute SCI in rodent models, reducing serum inflammatory markers and increasing cell survival in the spinal cord (54). Therefore, the severity of our model may be mediated by the use of ketamine at the time of SCI surgery.

Though previous reports demonstrate 5-HT_1F agonism induces MB in peripheral and central organs of both male and female mice (10, 25, 55), we have not investigated the effects of lasmiditan in male mice following SCI. Future studies will explore potential sex-related differences in response to 5-HT_1F receptor agonism following SCI.

Conclusion

This is the first report indicating that lasmiditan stimulates MB and enhances recovery of locomotor function and vascular integrity following SCI. Importantly, the 5-HT_1F receptor agonist lasmiditan, trade name Reyvow, was recently approved by the FDA for the treatment of migraines and could therefore be repurposed for the treatment of SCI.

Supplementary Material

MMC1. Supplemental Fig. 1 Effect of lasmiditan on spontaneous locomotor activity in the naïve and injured mouse.

Naïve (A) or injured (B) mice subjected to moderate SCI followed by daily administration of vehicle or lasmiditan (0.1 mg/kg, i.p.) beginning 1h post-injury. Total horizontal activity was assessed prior to SCI then 1d and 21d later. Data represent 8 naive mice and 9–10 injured per group and are expressed as mean ± SEM.

Highlights:

• Lasmiditan treatment induces mitochondrial biogenesis in the naïve and injured spinal cord

• Lasmiditan treatment reduces lesion volume and enhances blood-spinal cord barrier integrity following spinal cord injury

• Lasmiditan treatment enhances locomotor function in a dose-dependent manner following spinal cord injury