Skip to main content

Main menu

  • HOME
  • CONTENT
    • Early Release
    • Featured
    • Current Issue
    • Issue Archive
    • Blog
    • Collections
    • Podcast
  • TOPICS
    • Cognition and Behavior
    • Development
    • Disorders of the Nervous System
    • History, Teaching and Public Awareness
    • Integrative Systems
    • Neuronal Excitability
    • Novel Tools and Methods
    • Sensory and Motor Systems
  • ALERTS
  • FOR AUTHORS
  • ABOUT
    • Overview
    • Editorial Board
    • For the Media
    • Privacy Policy
    • Contact Us
    • Feedback
  • SUBMIT

User menu

Search

  • Advanced search
eNeuro

eNeuro

Advanced Search

 

  • HOME
  • CONTENT
    • Early Release
    • Featured
    • Current Issue
    • Issue Archive
    • Blog
    • Collections
    • Podcast
  • TOPICS
    • Cognition and Behavior
    • Development
    • Disorders of the Nervous System
    • History, Teaching and Public Awareness
    • Integrative Systems
    • Neuronal Excitability
    • Novel Tools and Methods
    • Sensory and Motor Systems
  • ALERTS
  • FOR AUTHORS
  • ABOUT
    • Overview
    • Editorial Board
    • For the Media
    • Privacy Policy
    • Contact Us
    • Feedback
  • SUBMIT
PreviousNext
Research ArticleNew Research, Disorders of the Nervous System

The Mixed-Lineage Kinase Inhibitor URMC-099 Protects Hippocampal Synapses in Experimental Autoimmune Encephalomyelitis

Matthew J. Bellizzi, Jennetta W. Hammond, Herman Li, Mary A. Gantz Marker, Daniel F. Marker, Robert S. Freeman and Harris A. Gelbard
eNeuro 16 November 2018, 5 (6) ENEURO.0245-18.2018; DOI: https://doi.org/10.1523/ENEURO.0245-18.2018
Matthew J. Bellizzi
1Center for Neurotherapeutics Discovery, University of Rochester Medical Center, Rochester, NY 14642
2Department of Neurology, University of Rochester Medical Center, Rochester, NY 14642
3Department of Neuroscience, University of Rochester Medical Center, Rochester, NY 14642
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Matthew J. Bellizzi
Jennetta W. Hammond
1Center for Neurotherapeutics Discovery, University of Rochester Medical Center, Rochester, NY 14642
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Jennetta W. Hammond
Herman Li
1Center for Neurotherapeutics Discovery, University of Rochester Medical Center, Rochester, NY 14642
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mary A. Gantz Marker
4Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Daniel F. Marker
1Center for Neurotherapeutics Discovery, University of Rochester Medical Center, Rochester, NY 14642
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Robert S. Freeman
4Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Harris A. Gelbard
1Center for Neurotherapeutics Discovery, University of Rochester Medical Center, Rochester, NY 14642
2Department of Neurology, University of Rochester Medical Center, Rochester, NY 14642
3Department of Neuroscience, University of Rochester Medical Center, Rochester, NY 14642
5Departments of Pediatrics and Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF
Loading

Abstract

Treatments to stop gray matter degeneration are needed to prevent progressive disability in multiple sclerosis (MS). We tested whether inhibiting mixed-lineage kinases (MLKs), which can drive inflammatory microglial activation and neuronal degeneration, could protect hippocampal synapses in C57BL/6 mice with experimental autoimmune encephalomyelitis (EAE), a disease model that recapitulates the excitatory synaptic injury that occurs widely within the gray matter in MS. URMC-099, a broad spectrum MLK inhibitor with additional activity against leucine-rich repeat kinase 2 (LRRK2) and other kinases, prevented loss of PSD95-positive postsynaptic structures, shifted activated microglia toward a less inflammatory phenotype, and reversed deficits in hippocampal-dependent contextual fear conditioning in EAE mice when administered after the onset of motor symptoms. A narrow spectrum inhibitor designed to be highly selective for MLK3 failed to protect synapses in EAE hippocampi, and could not rescue cultured neurons from trophic deprivation in an in vitro model of MLK-driven neuronal degeneration. These results suggest that URMC-099 may have potential as a neuroprotective treatment in MS and demonstrate that a broad spectrum of inhibition against a combination of MLK and other kinases is more effective in neuroinflammatory disease than selectively targeting a single kinase.

  • microglia
  • multiple sclerosis
  • neurodegeneration
  • neuroprotection
  • synapse

Significance Statement

Multiple sclerosis (MS) causes progressive neurologic decline that correlates with damage to gray matter in the brain, and is not stopped by current treatments. We used a mouse model of MS, in which gray matter injury is associated with inflammatory molecules produced by activated microglia, to test novel neuroprotective treatments. URMC-099, a mixed-lineage kinase (MLK) inhibitor that has anti-inflammatory and direct neuroprotective properties, prevented loss of hippocampal synapses and reversed deficits in learning and memory in a mouse model of MS. An MLK inhibitor with a narrower range of specificity was not effective, suggesting that combination kinase inhibition with URMC-099 may have potential as a neuroprotective treatment in MS.

Introduction

Preventing gray matter degeneration is an important goal in the treatment of multiple sclerosis (MS). Gray matter injury, measured by atrophy in neuroimaging studies, can begin early in the course of disease and progress in parallel with worsening physical and cognitive disability in MS patients (Dalton et al., 2004; Fisniku et al., 2008).

While demyelinated gray matter lesions have been associated with neuronal damage and loss of synapses (Peterson et al., 2001; Wegner et al., 2006; Dutta et al., 2011), neuronal injury occurs in myelinated “normal-appearing” gray matter (NAGM) as well and mirrors patterns of microglial activation within and outside of cortical lesions (Magliozzi et al., 2010). Injury to excitatory synapses with loss of postsynaptic dendritic spines has been found throughout the cortex at similar rates within demyelinated lesions and NAGM (Jürgens et al., 2016). Occurring independently of local demyelination or axon loss, this synaptic injury may underlie the disability that can progress out of proportion to lesion burden and despite current immunomodulatory treatments (Roosendaal et al., 2011; Hauser et al., 2017).

Similar synaptic injury occurs in the hippocampus of mice with experimental autoimmune encephalomyelitis (EAE), providing a model for studying the pathogenesis and treatment of synaptic injury in MS gray matter. Damage to excitatory postsynaptic structures in EAE is mediated by platelet-activating factor (PAF), a signaling molecule that can be released downstream of microglial activation (Bellizzi et al., 2016). Inflammatory microglia also release tumor-necrosis factor-α (TNFα) and interleukin-1β (IL-1β), which have been found to promote neuronal injury in other models of MS gray matter injury (Centonze et al., 2009; Rossi et al., 2012).

Inflammatory molecules can be difficult to target therapeutically due to the redundancy of multiple mediators activated in parallel and because many of these molecules play critical signaling roles in synaptic physiology as well as in pathology. Blockade of PAF signaling could prevent synapse loss in the EAE hippocampus (Bellizzi et al., 2016) but also disrupts learning and memory (Jerusalinsky et al., 1994), and treatments targeting TNFα had deleterious effects in MS clinical trials (van Oosten et al., 1996).

While microglia can produce molecules with neurotoxic effects, they can also become activated to promote neuroprotection and repair in inflammatory disease (Chen and Trapp, 2016). Shifting activated microglia toward a protective phenotype may provide an effective strategy for gray matter neuroprotection in MS. The phosphodiesterase type 4 inhibitor, ibudilast, which can reduce proinflammatory microglial activation in vitro, has slowed rates of brain atrophy in early-phase clinical trials (Barkhof et al., 2010; Fox et al., 2018).

Inhibition of mixed-lineage kinases (MLKs) has potential to both modulate microglial phenotype and directly protect neurons against degeneration. MLKs are activated in response to inflammation and cell stress, and mediate neuronal degeneration (Harris et al., 2002; Tian et al., 2003; Lotharius et al., 2005) and proinflammatory activation of macrophages and microglia (Ciallella et al., 2005; Sui et al., 2006; Eggert et al., 2010) via c-Jun N-terminal kinase (JNK) and other MAP kinase pathways. MLK3, which is widely expressed and can link glutamatergic stimulation to JNK activation at excitatory synapses (Savinainen et al., 2001), has been a target of interest for treatment of several neurodegenerative diseases (Maroney et al., 2001; Goodfellow et al., 2013).

URMC-099 is a brain-penetrant small molecule designed to inhibit MLK3 that has attenuated proinflammatory microglial activation and protected against neuronal and synaptic damage in models of neuroinflammatory disease in vitro and in vivo (Marker et al., 2013; Dong et al., 2016; Kiyota et al., 2018). Here, we investigated whether URMC-099 treatment could protect synapses and hippocampal function in mice with EAE. Because URMC-099 has potent inhibitory activity against several MLKs as well as other kinases including leucine-rich repeat kinase 2 (LRRK2; Goodfellow et al., 2013), we additionally compared URMC-099 against CLFB1134, a highly-specific MLK3 inhibitor with a different chemical scaffold but also with an excellent CNS pharmacokinetic profile (Goodfellow et al., 2016), to determine whether narrow versus broad specificity could enhance neuroprotection in the EAE hippocampus.

Materials and Methods

Animals

Male and female C57BL/6 mice were purchased at age seven weeks (The Jackson Laboratory, catalog #JAX:000664, RRID:IMSR_JAX:000664) and were vivarium-housed for at least one week before EAE induction. All animal experiments were performed in accordance with the University of Rochester University Committee on Animal Resources according to the Public Health Service Policy on Humane Care and Use of Laboratory Animals.

EAE

We immunized mice of either sex with pre-mixed emulsion (Hooke Labs, catalog #EK-2110) containing myelin oligodendrocyte glycoprotein peptide amino acids 35–55 (MOG35–55), in complete Freund’s adjuvant (CFA) containing heat-inactivated mycobacterium tuberculosis H37RA. 100 µl of emulsion was injected subcutaneously at each of two sites over the upper and lower back. Sham-immunized sex-matched littermate controls were injected with control emulsion with MOG35–55 omitted (Hooke Labs, catalog #CK-21100), and otherwise received identical treatment. Mice were injected intraperitoneally with pertussis toxin (Hooke Labs, 90–400 ng, with the dose adjusted according to potency of each lot per manufacturer recommendations) on 0 and 1 d post-immunization (dpi). Mice were subsequently monitored for signs of clinical disease and scored for motor deficits as follows: 0, no deficit; 0.5, partial tail paralysis; 1, complete tail paralysis; 2, hindlimb weakness; 2.5, paralysis of one hindlimb; 3, paralysis of both hind limbs; 3.5, hindlimb paralysis and forelimb weakness; 4, quadriplegia; 5, moribund. Scores are reported relative to day of disease onset (day 0: first score 0.5 or greater) when drug treatment was begun.

Drug treatment

For in vivo experiments, URMC-099 (Califia Bio, Inc., and Pharmaron) and the highly-selective MLK3 inhibitor CLFB1134 (Califia Bio, Inc.) were dissolved at 2 mg/ml in a sterile solution of 5% DMSO, 40% polyethylene glycol 400, and 55% normal saline and stored at room temperature protected from light. Mice were randomized to drug treatment beginning on the first day of motor deficits (EAE score 0.5 or greater). Randomization was stratified for timing and severity of symptom onset, and sham-immunized mice were assigned to begin treatment at the same time. The initial cohort of mice was randomized to treatment with URMC-099 (10 mg/kg, i.p., twice daily), CLFB1134 (30 mg/kg, i.p., twice daily), or vehicle control (all mice received identical volumes of vehicle per weight). Dose selection of URMC-099 and CLFB1134 was based on affinity for MLK3 and prior studies of CNS pharmacokinetics for URMC-099 and similar compounds (Goodfellow et al., 2013), as well as demonstrated in vivo effects of URMC-099 in other models of neuroinflammation (Marker et al., 2013). After establishing the relative efficacy of URMC-099 versus CLFB1134 for protection of hippocampal synapses, subsequent cohorts were treated with URMC-099 or vehicle only. Experimenters were blinded to treatment assignment throughout each experiment and data analysis.

Cued and contextual fear conditioning

Mice underwent fear conditioning 12–14 d after onset of EAE symptoms and drug treatment, by which time mice were acclimated to human handling after daily EAE scoring and injections. For conditioning, we transferred mice individually to an isolation chamber (Coulbourn Habitest H10-24T) with a 7” × 7” square test cage with metal walls and a grid floor of 16 stainless steel bars that were wired to deliver an electric shock. We inserted a Plexiglas plate to support and facilitate mobility for mice with paretic limbs who could not maintain an ambulatory posture on the bars. After 3 min in the chamber, mice were presented with a 15-s white noise cue coterminating with a 2-s shock (0.55 mAmps delivered through the grid floor via a Coulbourn precision animal shocker). Paired cue and shock were delivered three times, 30 s apart. Mice were removed from the chamber to a holding cage until all of their littermates had completed the conditioning protocol, and then returned to their home cage overnight. The chamber floor was removed, wiped with water, and dried between each trial. Twenty-four hours later, we tested contextual conditioning responses by returning mice to the same chamber (prepared identically as for conditioning) for 5 min without stimulus (conditioned context test). We recorded video via a camera fixed directly above the cage for subsequent motion analysis. After returning mice to their home cage (via a holding cage until littermates had completed testing) for 3 h, we tested cued conditioning responses. We placed mice in a round test cage with smooth plastic walls and floor that was wiped with ethanol before each trial, lined on the floor with clean cage bedding, and illuminated in the isolation chamber with a red light to provide a novel context that differed from the conditioned context in shape, texture, odor and color. We recorded video for freezing analysis for 3 min without stimulus (novel context test) followed by 3 min of continuous presentation of the white noise cue (cue test). For each test, mice were placed in the test chambers in the same order which was randomized with respect to treatment groups, with males run before females.

We measured contextual and cued fear conditioning responses using motion analysis software (Freeze Frame/Freeze View, Actimetrics Software) to detect episodes of freezing behavior, which were defined as ≥0.5-s bouts of immobility except for breathing, in which the software-generated motion index was below a threshold value that was confirmed via video review for each mouse to discriminate freezing from exploratory, grooming, sniffing or rearing behaviors. We quantified freezing responses as the percentage of total time spent freezing during the first 3 min in the conditioned context, 3 min in the novel context, and the first 90 s of the cue test (beyond which responses tended to deteriorate at variable rates). Because EAE mice had increased rates of immobility at baseline and across all conditions, reflecting motor deficits and/or decreased exploratory behavior and non-specific to context or cue, we subtracted rates of freezing during exposure to the novel, unconditioned context from rates during the conditioned context and tone tests to generate a measure (Δ freeze) that reflected increased freezing due to the mouse’s ability to discriminate between the conditioned and novel context, and in response to the cue. These values were normalized to those of the sham-immunized controls for the same testing cohort, to account for variations in the magnitude of the conditioning effect among cohorts. Mice with >60% immobility in the unconditioned novel context (4/22 mice in the EAE-vehicle group and 5/26 EAE-099 mice) were excluded from the Δ freeze analysis as high baseline immobility obscured the ability to measure conditioning effects; none of the remaining mice had mean freezing rates of >31% in the novel context.

Immunohistochemistry

After 12–14 d of treatment, mice were anesthetized with ketamine/xylazine (200 and 1 mg/kg, respectively) and intracardially perfused for 1 min with PBS containing EDTA (1.5 mg/ml) followed by 4% paraformaldehyde (PFA; 100 ml perfused over 30 min). Brains were removed, post-fixed for 24 h in 4% PFA and stored at in PBS at 4°C; 40-µm coronal sections were cut on a vibratome (Leica V1000) and stored in cryoprotectant (30% PEG300, 30% glycerol, 20% 0.1 M phosphate buffer, and 20% H2O) at –20°C. Free-floating sections were washed in PBS (3 × 30 min) followed by glycine (100 mM) to reduce autofluorescence, incubated in citrate antigen unmasking solution (Vector H3300) with 0.05% Tween for 30 min at 37° C, and washed again in PBS. We incubated sections for 2 d at room temperature in 1.5% BSA (Sigma), 3% normal goat serum (Vector Laboratories), 0.5% Triton X-100 (Promega), and 1.8% NaCl for blocking and permeabilization, mixed with the following primary antibodies: mouse anti-PSD95 (NeuroMab, catalog #75-028, RRID:AB_2307331; 1:500), rabbit anti-Iba1 (Wako Biochemicals, catalog #019-19741, RRID:AB_839504; 1:1000), rabbit anti-Tmem119 (Abcam, catalog #ab209064, RRID:AB_2728083, 1:200), chicken anti-Iba1 (Synaptic Systems, catalog #234 006, RRID:AB_2619949; 1:500), rat anti-CD68 (Abd Serotec, catalog #MCA1957, RRID:AB_322219; 1:1000), rat anti-Ly-6B (clone 7/4, Abcam, catalog #ab53457, RRID:AB_881409; 1:1000), or mouse anti-MAP2 (Sigma, catalog #M4403, RRID:AB_477193; 1:500). We washed sections in PBS with 1.8% NaCl (3 × 30 min) and then incubated overnight at room temperature with Alexa Fluor-conjugated secondary antibodies (Invitrogen, 1:500) in the same blocking/permeabilization mixture as above. After washing in PBS (3 × 30 min) we mounted sections on slides with Prolong Gold mounting agent (Life Technologies) and #1.5 cover glass.

Tissue section imaging and analysis

Slides were coded during image collection and analysis to blind the experimenters to treatment condition. We imaged tissue sections by grid pseudo-confocal microscopy, using an Olympus BX-51 microscope equipped with Quioptic Optigrid hardware for optical sectioning and a Hamamatsu ORCA-ER camera, controlled by Volocity 3DM software (PerkinElmer Life and Analytical Science). Differences in z-axis registration of different fluors were corrected by calibration with multicolor fluorescent beads. We imaged the dorsal CA1 hippocampal area, collecting images from 6 sections per animal and using identical light intensity and exposure settings for all animals within each image set. We quantified density of postsynaptic puncta from 60× image stacks (z-step = 0.3 µm) of PSD95 staining in the CA1 stratum radiatum, using a custom-designed automated algorithm in Volocity software to identify puncta based on size and local intensity maxima (identified by both Find Objects and Find Spots algorithms). In the same imaging fields, we quantified microglial Iba1 intensity by the sum of Iba1 pixel values within Iba1-positive objects, and microglial CD68 by the proportional volume of Iba1 objects that were also positive for CD68. We measured the ratio of surface area to volume for Iba1-positive objects to quantify microglial changes from a ramified to more amoeboid morphology. For all measurements, values from image stacks in six tissue sections per mouse were averaged and expressed relative to sham-immunized controls from the same treatment cohort. Image stacks are displayed as two-dimensional extended-focus images collapsed along the z-axis.

Hippocampal Western blottings

Mice at 29 dpi were perfused briefly with PBS and hippocampi were isolated, immediately frozen on dry ice, and stored at –80°C. Individual hippocampi were homogenized in RIPA lysis buffer containing a protease inhibitor cocktail (MilliporeSigma: Calbiochem, SetV). The cell lysate was kept on ice with periodic vortexing for 30 min, then centrifuged at 13,000 rpm for 10 min. The supernatant was centrifuged again at 13,000 rpm for 10 min. The protein concentration of the final supernatant lysate was assayed with a detergent-compatible Bradford assay (Pierce). Equivalent amounts of protein between samples were then mixed with loading dye and run on an 8% or 12% SDS-PAGE gel and transferred to PVDF. Membranes were blocked with 5% milk in tris-buffered saline (TBS) with 0.5% Tween-20 (TBST) for 30 min and probed with one of the following primary antibodies in 5% milk in TBST for 1–16 h: Iba1 (Wako Biochemicals, catalog #019-19741, RRID:AB_839504), TNFα (Cell Signaling Technologies, catalog #11948, RRID:AB_2687962), IL1β (Genzyme, catalog #1886-01), iNOS (Cell Signaling Technologies, catalog #2982S, RRID:AB_1078202), FcγR1/CD64 (R&D Systems, catalog #AF2074, RRID:AB_416550), CD86 (BD PharMingen, catalog #553689, RRID:AB_394991), actin (Santa Cruz, catalog #SC-47778, RRID:AB_626632), IL10 (AbD Serotec, catalog #MCA1302G, RRID:AB_2125093), Arg1 (Cell Signaling Technologies, catalog #93668), C1q [1151 (Huang et al., 1999), gift of Andrea Tenner]. Membranes were washed three times in TBST and then incubated with HRP secondary antibodies for 1 h in 5% milk in TBST. After washing we applied ECL substrate (Pierce) and developed membranes using a digital imager (Azure Biosystems). Membranes were stripped using buffer consisting of 200 mM glycine, 0.1% SDS, and 1% Tween 20 (pH 2.2) and re-probed up to four times. Western blotting band densities were quantitated using ImageJ. Band densities for each protein were first normalized to the corresponding actin band densities for each animal from the same gel then normalized to the control group mean.

Primary neuronal cultures

Sympathetic neuron cultures were prepared from superior cervical ganglia (SCG) of newborn (postnatal day 0–1) male and female C57Bl/6J mice. SCG from individual mice were pooled and incubated at 37°C for 20 min in 2.5 mg/ml collagenase (Worthington Biochemical) followed by a second 20-min incubation in 1 mg/ml trypsin (Worthington Biochemical). The SCG were rinsed twice in culture media consisting of modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 20 µM uridine, 20 µM fluorodeoxyuridine, 2 mM L-glutamine, and 50 ng/ml nerve growth factor (NGF; Harlan Products for Bioscience) and then triturated with a fire-polished Pasteur pipette to obtain a single cell suspension. Cells were diluted in fresh NGF-containing media and plated onto poly-L-lysine and laminin-coated glass slides (cell survival assays) or collagen-coated tissue culture dishes (Western blotting experiments). Cultures were maintained in a 5% CO2 incubator at 37°C for 5 d before initiating treatments. For NGF deprivation, neurons were rinsed twice in culture medium lacking NGF and then incubated in the same NGF-free media supplemented with sheep anti-NGF antiserum (Cedarlane Laboratories) to scavenge residual NGF. For treatment with URMC-099 or CLFB1134, we prepared 100-µm stock solutions of each drug in DMSO that were diluted to 100 or 300 nM final concentrations in culture media and added at the start of NGF deprivation.

Cell survival assays

Just before initiating treatments, the number of healthy neurons in each of four pre-marked fields of view (>100 neurons per field of view) was determined using phase-contrast microscopy at 100× magnification. Neurons were scored as healthy if they exhibited a rounded and refractile cell body, a clearly defined nucleus and nucleolus, and smooth and intact neurites. At the end of the treatment period, neurons were fixed in 3% PFA for 20 min and then stained with the DNA-binding dye Hoechst 33 342 (Invitrogen). The number of healthy neurons in the same four pre-marked fields was once again determined based on the criteria described above plus the presence of diffuse Hoechst-stained chromatin completely filling the nucleus. In contrast, unhealthy neurons displayed phase-dark cell bodies, fragmented and beaded neurites, and condensed Hoechst-stained chromatin. Percentage survival equals the average number of healthy neurons across the four fields of view at the end of the treatment divided by the average number of healthy neurons before treatment multiplied by 100. Results correspond to the averages of four wells per condition from two independent experiments.

Cell culture Western blottings

Following treatment, cells were rinsed with PBS and lysed in Laemmli sample buffer containing 5% β-mercaptoethanol. Proteins were separated on 12.5% SDS-PAGE gels and electroblotted onto supported nitrocellulose membranes. Membranes were blocked in 5% non-fat milk in TBST for 1 h at room temperature. Antibodies were diluted in either 2.5% non-fat milk or 5% bovine serum albumin in TBST and incubated with the membranes at 4°C overnight. The following antibodies and dilutions were used: JUN (1:1000, Cell Signaling, catalog #9165, RRID:AB_2130165), phosphorylated Ser63-JUN (1:1000, Cell Signaling, catalog #9261, RRID:AB_2130159), phosphorylated Ser73-JUN (1:1000, Cell Signaling, catalog #3270, RRID:AB_2129575), and tubulin (1:2000, Sigma, catalog #T5168, RRID:AB_477579). Membranes were washed three times in TBS with 0.05% or 0.01% Tween 20 before incubation with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies (Bio-Rad) for 1 h at room temperature. Membranes were washed three more times in TBST before being processed for chemiluminescence using Supersignal West Dura substrate (Pierce). Densitometry was performed on images obtained from scanned films using NIH ImageJ software.

Experimental design and statistical analyses

Statistical analyses and plots were done using Excel (Microsoft) and Prism 7.3 (GraphPad) software. Experiments testing in vivo effects of URMC-099 and CLFB1134 were initially done with male mice, to reduce potential variability in hippocampal synaptic density due to estrous-related changes in females (Woolley et al., 1990). Subsequent treatment cohorts for behavioral testing included female mice as well, and n for each sex is reported for each experiment in the results. For single-sex in vivo and all in vitro experiments, comparisons between groups were done for normally-distributed continuous data by one-way ANOVA followed in the primary analysis by Sidak post hoc tests for hypothesis-driven pairwise comparisons (in vivo experiments: vehicle-treated EAE vs sham-immunized mice, and URMC-099 or CLFB1134 vs vehicle-treated mice; in vitro experiments: URMC-099- vs vehicle-treated cultures). Where other group comparisons were interesting, secondary analysis used Tukey post hoc tests for all pairwise comparisons. For the behavioral experiments, which included both male and female mice, we used two-way ANOVA with sex and treatment as independent factors; after results showed no significant effect of sex or sex-treatment interaction on behavioral test scores, post hoc pairwise comparisons were done as above on the treatment main effect. Non-parametric EAE scores were compared using the two-tailed Mann–Whitney U test for each day after disease onset. Significance was accepted at p < 0.05; p values (reported out to four decimal places or <0.0001) and sample sizes are reported in the text of the results or figure legends, and superscripts following each test in the results refer to the statistical table which reports mean differences with 95% confidence intervals for group comparisons (Table 1). All data in the results and figures are expressed as mean ± SEM.

View this table:
  • View inline
  • View popup
Table 1.

Statistical table

Results

URMC-099 prevents excitatory synapse loss in EAE hippocampus

To test whether MLK3 inhibition could protect excitatory synapses in EAE hippocampus, we treated mice with URMC-099 or the highly-selective MLK3 inhibitor CLFB1134 beginning after the onset of EAE symptoms. URMC-099 treatment preserved excitatory synapses in hippocampal area CA1 of EAE mice (Fig. 1A,B): while PSD95-positive puncta counts were decreased in vehicle-treated EAE mice (n = 6 male mice) to 0.76 ± 0.04 relative to levels of sham-immunized controls (n = 10 male mice, p = 0.028, Sidak post hoc testa), URMC-099 restored counts to 1.01 ± 0.08 relative to controls (n = 6 male mice, p = 0.021 vs EAE-vehicle, Sidak post hoc testb). In contrast, CLFB1134 was not effective at protecting synapses, with hippocampal PSD95-positive puncta reduced to 0.83 ± 0.09 of control levels (n = 7 male mice), similar to vehicle-treated EAE mice. Neither drug prevented development of motor deficits in EAE, with each one causing modest reductions in EAE score that were not significant in this treatment cohortc (Fig. 1C), and became statistically significant but less pronounced in a larger subsequent cohort treated with URMC-099 or vehicled (as discussed below, URMC-099 preserves hippocampal-dependent fear conditioning in EAE mice).

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

URMC-099 prevents excitatory synapse loss in EAE hippocampus. A, Staining for PSD95-positive postsynaptic puncta in the stratum radiatum of hippocampal area CA1 (inset) was decreased in vehicle-treated EAE mice relative to sham-immunized controls. Treatment with URMC-099 beginning after the development of EAE motor deficits preserved synaptic puncta, while treatment with CLFB1134 did not. Scale bar: 10 µm. B, Density of PSD95-positive puncta, quantified relative to sham-immunized controls; *p < 0.05, one-way ANOVA with Sidak post hoc test. C, Treatment with 099 and 1134 in this cohort resulted in a modest reduction in mean severity of EAE motor deficits that was not significantly different from controls; n = 10 male mice (sham-vehicle), n = 6 male mice (EAE-vehicle and EAE-099), n = 7 male mice (EAE-1134). Data in all figures are expressed as mean ± SEM.

URMC-099 modulates the phenotype of microglial activation in EAE hippocampus

Microglial immunostaining in hippocampal area CA1 showed increased Iba1 expression and a shift from a ramified morphology with thin processes to a more amoeboid morphology with thicker, shorter processes consistent with activation in EAE (Fig. 2A). Coexpression of Tmem119 along processes and/or cell bodies (Fig. 2B) suggests that Iba1-positive cells in the hippocampus represented brain-resident microglia rather than monocyte-derived macrophages (Bennett et al., 2016), and staining for Ly-6B, expressed by neutrophils and some recently-generated macrophages (Rosas et al., 2010) showed that infiltrating myeloid cells were sparse in the hippocampus regardless of treatment condition (1.85 ± 0.19 cells per 10× field in n = 5 male sham control mice, vs 2.44 ± 1.12 in n = 4 male EAE-vehicle mice, and 2.05 ± 0.76 in n = 5 male EAE-099 mice, one-way ANOVA F(2,11) = 0.16, p = 0.86), although sporadic clusters could be found in the pial meningeal layer in EAE (Fig. 2B, insets).

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

URMC-099 modulates the phenotype of activated microglia in EAE hippocampus. A, Staining for Iba1 in hippocampal area CA1 shows ramified microglia in sham-immunized mice, and microglia with shorter, thicker processes in EAE. B, Iba1-positive cells in the hippocampi of both sham and EAE mice coexpress microglia-specific marker Tmem119 along their processes and to a more variable extent in the cell body (upper panels). Iba1-positive, Tmem119-negative cells consistent with monocyte-derived macrophages could be found in the pia during EAE (inset), but not in or around the hippocampus. Staining for Ly-6B, expressed on neutrophils and some recently-generated macrophages, is sparse in sham and EAE hippocampi (lower panels, arrowheads), although clusters occurred sporadically in the pia and other area of some EAE brains (inset). Scale bars: 20 µm. s.o., CA1 stratum oriens; pyr, pyramidal layer; s.r., stratum radiatum. C, Quantification of microglial images shows increased Iba1 intensity and reduction in the ratio of surface area to volume in EAE microglia regardless of treatment with URMC-099 or CLFB1134. Microglial expression of lysosomal marker CD68 is increased in many hippocampi from vehicle-treated EAE mice but not in mice treated with URMC-099 or CLFB1134; n = 10 male mice (sham-vehicle), n = 6 male mice (EAE-vehicle and EAE-099), n = 7 male mice (EAE-1134). D, E, Western blottings and band densitometry for markers of inflammation and microglial phenotype in hippocampal protein extracts. Markers associated with proinflammatory microglial activation tended to increase in vehicle-treated EAE hippocampi and were decreased by URMC-099, with significant changes in iNOS (inducible nitric oxide synthase) and immunoglobulin receptor FCγR1/CD64 (lower single band), and a non-significant trend toward increased pro-IL1β. Proinflammatory marker CD86 does not significantly increase in vehicle-treated EAE but is decreased by URMC-099 treatment. Arg1 and IL-10, canonical markers of anti-inflammatory alternative activation, are not significantly changed in EAE or with URMC-099. Consistent with immunostaining results, Iba1 is up-regulated in vehicle-treated EAE hippocampi and is further increased with URMC-099 treatment. Complement component C1q follows a similar pattern; n = 10 male mice (sham-vehicle), n = 7 male mice (EAE-vehicle), n = 11 male mice (EAE-099); one representative blot with three lanes per condition is depicted; *p < 0.05, **p < 0.01, one-way ANOVA with Sidak (iNOS, FCγR1/CD64, CD86) or Tukey (Iba1, SA/Vol, C1q) post hoc test.

Microglial morphologic changes were reflected by a decrease in the ratio of microglial surface area to volume in vehicle-treated EAE mice (n = 6 male mice) and were not prevented by treatment with URMC-099 (n = 6 male mice) or CLFB1134 (Fig. 2C; n = 7 male mice, p = 0.003 for EAE-vehicle vs sham controle, p < 0.0001 for EAE-099 vs sham controlf, and p = 0.002 for EAE-1134 vs sham controlg, Tukey post hoc test). Increases in microglial Iba1 were highest and reached significance only in URMC-099-treated EAE mice (p = 0.023 vs sham control, Tukey post hoc testh). Microglial expression of CD68, a lysosomal marker associated with phagocytosis during activation, was increased in many hippocampal imaging fields in vehicle-treated EAE mice, although the extent was widely variable and quantification did not reach statistical significancei. URMC-099 and CLFB1134 both prevented any increases in microglial CD68 expression, suggesting that while neither agent restores microglia to a resting morphology in the EAE hippocampus, they may modulate their activation state.

We further evaluated microglial phenotypic markers in hippocampal protein extracts from mice treated with URMC-099 or vehicle (Fig. 2D,E). Markers canonically associated with proinflammatory microglial activation tended to be increased in vehicle-treated EAE (n = 7 male mice) compared to sham-immunized controls (n = 10 male mice) and attenuated by URMC-099 (n = 11 male mice), with statistically significant changes in inducible nitric oxide synthase (iNOS; p = 0.003 EAE-vehicle vs shamj and p = 0.04 EAE-vehicle vs EAE-URMC-099k, one-way ANOVA with Sidak post hoc test) and immunoglobulin receptor FCγR1/CD64 (lower single band; p = 0.004 EAE-vehicle vs sham, Sidak post hoc testl), and a non-significant trend toward increased pro-IL1β (p = 0.06 EAE-vehicle vs sham, Sidak post hoc testm). Interestingly, the costimulatory molecule CD86, expressed by antigen-presenting cells as well as macrophages, increased little in vehicle-treated EAE but was significantly decreased by URMC-099 treatment (p = 0.011 EAE-vehicle vs EAE-URMC-099, Sidak post hoc testn). Arg1 and IL10, markers associated with anti-inflammatory alternative activation, were not significantly changed in EAE or with URMC-099. Consistent with immunostaining results, Iba1 expression was increased in EAE hippocampi during both vehicle and URMC-099 treatment (p = 0.025 EAE-vehicle vs shamo, and p = 0.0006 EAE-URMC-099 vs shamp, one-way ANOVA with Tukey post hoc test). Expression of complement component C1q, which is increased in neurons in MS gray matter and is thought to be associated with synapse elimination, followed a similar pattern (p = 0.01 EAE-vehicle vs shamq and p = 0.0006 EAE-URMC-099 vs shamr, Tukey post hoc test) that did not correlate with URMC-099’s synaptic protection. In aggregate, these results suggest that URMC-099 does not prevent microglia from becoming activated in EAE hippocampi but does seem to shift them toward a less inflammatory phenotype that may contribute to synaptic preservation.

URMC-099 prevents neuronal apoptosis in vitro

Given the efficacy of URMC-099 but not CLFB1134 at preserving hippocampal synapses in the face of microglial activation in EAE, we asked whether the drugs differ in their ability to exert direct protective effects on neurons. As direct versus glia-mediated neuroprotection is difficult to differentiate in vivo, we tested both drugs in a culture system in which MLK signaling can drive neuronal apoptosis and rescue by broad-spectrum MLK inhibitors has been firmly established (Xu et al., 2001; Harris et al., 2002). Sympathetic neurons cultured from murine SCG degenerate after withdrawal of NGF from the culture media, with fragmentation of neurites and condensation of chromatin in pyknotic nuclei (Fig. 3A). Addition of 100 or 300 nM URMC-099 prevented neuronal death and maintained healthy neurites up to 48 h after NGF withdrawal in a dose-dependent manner (Fig. 3A,B; n = 4 cultures per condition, p < 0.0001 for each URMC-099 dose vs vehicle at 24 and 48 h, one-way ANOVA with Sidak post hoc tests–v). In similar cultures, we evaluated expression and phosphorylation of JUN, a target of the JNK pathway that mediates apoptosis during NGF withdrawal and can be activated by MLKs and likely other kinases that mediate proinflammatory signaling such as LRRK2 (Puccini et al., 2015). URMC-099 (300 nM) blocked increases in JUN expression as well as phosphorylation at serine residues 63 and 73 (Fig. 3C,D; n = 3 cultures per condition, p = 0.015 for P Ser 73 JUN in 099- vs vehicle-treated cultures at 8 h and p = 0.003 at 12 h, one-way ANOVA with Sidak post hoc testw,x), consistent with reduced JNK activity downstream of MLK inhibition. Treatment with CLFB1134 failed to provide protection or trophic support to NGF-deprived neurons, which underwent neuritic beading and degeneration of cell bodies to an extent that precluded quantification of pyknotic versus healthy nuclei, suggesting that the neuroprotective effects of broad-spectrum MLK inhibition in vitro, like in vivo, are not reproduced by an inhibitor that is highly selective for MLK3. Thus, subsequent experiments focused on further characterizing the effects of URMC-099 in EAE hippocampus.

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3.

URMC-099 protects neuronal cultures during growth factor deprivation. A, Phase-contrast images (upper) of cultured SCG neurons show disruption of neurite integrity after 48 h of NGF deprivation, and Hoechst DNA staining (lower) shows condensed pyknotic nuclei (arrowheads indicate examples) in the same neurons. Cultures treated with URMC-099 (300 nM) maintained neurite integrity and diffuse nuclear chromatin staining indicative of healthy neurons after 48 h of NGF deprivation. Cultures treated with CLFB1134 had marked fragmentation of neurites, and cellular damage severe enough to preclude Hoechst analysis. Scale bars: 60 µm. B, Quantification of dead SCGs identified by pyknotic nuclei shows a dose-dependent protective effect for URMC-099 following NGF deprivation (n = 4 cultures for each condition). C, Western blottings for JUN, which mediates SCG apoptosis during NGF withdrawal and is activated downstream of MLK signaling via phosphorylation by JNK, show increased JUN expression and phosphorylation at serine residues 63 and 73 in protein isolates from SCG cultures following NGF withdrawal, which is nearly abolished by treatment with URMC-099 (300 nM, n = 3 experiments); *p < 0.05, **p < 0.0001, one-way ANOVA with Sidak post hoc test.

URMC-099 preserves hippocampal-dependent fear conditioning in EAE mice

We tested whether URMC-099 could preserve hippocampal-dependent learning and memory in EAE mice, and chose contextual fear conditioning because it is less dependent on locomotion than maze or exploratory learning paradigms and could be adapted to mice with motor deficits. Furthermore, pairing hippocampal-dependent contextual conditioning with a test of cued conditioning which is independent of the hippocampus (Phillips and LeDoux, 1992) but shares the same output measures provided a positive control to test whether conditioning effects could be detected in the presence of EAE motor deficits. Because EAE mice had greater rates of immobility at baseline and during testing in an unconditioned novel context that was not specific to conditioned stimuli (Fig. 4B), we quantified the increase in freezing behavior due to context or cued conditioning as the difference between the percentage of time spent freezing during the conditioned context test and the novel context test (context Δ freeze), and between the cue presentation and the novel context test (cue Δ freeze). This provided a measure of the mouse’s response to the conditioned context and cue while minimizing confound due to motor deficits or reduced exploratory behavior associated with EAE. Cohorts for behavioral testing included both male and female mice, although two-way ANOVA showed no significant effect of sex or of the interaction between sex and treatment on behavioral scores (F(1,62) = 0.40, p = 0.53 for sex main effect and F(2,62) = 1.10, p = 0.34 for interaction effect on context Δ freeze) and further analysis focused on treatment main effects.

Figure 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4.

URMC-099 preserves contextual fear conditioning in EAE mice. A. Contextual and cued fear conditioning protocol for EAE mice. Mice were conditioned in an isolation chamber where they were presented three times with an auditory cue paired with a foot shock; 24 h later, mice were evaluated for episodes of freezing after being returned to the identical chamber (conditioned context), introduced to a chamber modified in shape, texture and odor (novel context), and presented with the auditory cue (cue). B, Group averages of freezing responses during each of the three tests, expressed as % of time spent freezing in 30-s epochs. Mean rates of freezing (dotted lines) increased in the conditioned context and with cue presentation relative to the unconditioned novel context in all groups, although EAE mice with motor deficits had higher rates of immobility in the novel context compared to sham-immunized controls. Mean freezing rates in the novel context were subtracted from those in the conditioned context and during cue presentation (Δ freeze) to differentiate freezing due to conditioning from non-specific immobility. C, Δ freeze values show that deficits in contextual fear conditioning in vehicle-treated EAE mice were restored to control levels by URMC-099 treatment. EAE mice showed no deficits in auditory cue conditioning, with similar Δ freeze values in all groups; *p < 0.05, two-way ANOVA with Sidak post hoc test of the treatment main effect. D, EAE scores for the mouse cohorts that underwent behavioral testing show that URMC-099 treatment after symptom onset did not affect peak EAE severity and was associated with modest recovery that was statistically different from vehicle-treated mice after 9–13 d of disease; *p < 0.5, Mann–Whitney U test. For all panels, n = 29 mice (sham-vehicle), n = 18 mice (EAE-vehicle), n = 21 mice (EAE-099).

Vehicle-treated EAE mice (n = 15 male and n = 3 female mice) showed a decrement in contextual fear conditioning (Fig. 4C), with context Δ freeze reduced to 0.67 ± 0.09 relative to sham-immunized controls (n = 24 male and n = 5 female mice, p = 0.019, two-way ANOVA with Sidak post hoc test on the treatment main effecty). Freezing responses to the conditioned context were restored to control levels in URMC-099-treated EAE mice (0.99 ± 0.12, n = 18 male and n = 3 female mice), a significant improvement compared to vehicle-treated EAE mice (p = 0.038, Sidak post hoc testz). Both vehicle- and URMC-099-treated EAE mice had similar increases in freezing as control mice during presentation of the auditory cue (two-way ANOVA, treatment main effect, F(2,62) = 0.415, p = 0.66), suggesting that EAE deficits do not limit the sensitivity of the test to detect fear conditioning responses. EAE scores from the mouse cohorts that underwent fear conditioning (Fig. 4D) show that URMC-099- and vehicle-treated EAE mice had similar severity of motor deficits at the peak of disease, while URMC-099-treated mice recovered to slightly less severe scores that were statistically significant from 9 to 13 d after disease onset (p < 0.05, Mann–Whitney U testd).

Discussion

These results demonstrate that the kinase inhibitor URMC-099 can protect hippocampal synapses and maintain hippocampal-dependent learning and memory in an EAE model that recapitulates the excitatory synaptic injury that occurs widely within the gray matter in MS. We have previously described URMC-099 as a broad-spectrum MLK3 inhibitor (Goodfellow et al., 2013) because of its ability to modulate multiple kinase “control hubs” related to restoration of the balance between innate immune cells and end-organ target cells such as neurons (Marker et al., 2013), especially with respect to subcellular synaptic elements. URMC-099 appears to modulate activated microglia in the EAE hippocampus toward a less inflammatory phenotype and may have direct protective effects on neurons as well. URMC-099 has similarly reduced markers of proinflammatory microglial activation in mouse models of Alzheimer’s disease (Kiyota et al., 2018) and HIV-associated neuroinflammation (Marker et al., 2013), suggesting that it may have potential to shift microglia toward neuroprotective phenotypes in a variety of neuroinflammatory conditions.

In contrast to other treatments that have shown neuroprotective effects in EAE gray matter when begun before disease onset (Centonze et al., 2009; Kim et al., 2012; Bellizzi et al., 2016; Di Filippo et al., 2016), URMC-099 protected the hippocampus when administered after the onset of EAE motor deficits. At that point in this model, immune activation and infiltration into the CNS is at or near its peak (Murphy et al., 2010) and deficits in hippocampal-dependent learning and memory are already established (Kim et al., 2012). URMC-099 treatment had no effect on the peak severity of EAE motor deficits, which reflect damage due to lymphocyte-driven inflammation in the spinal cord, and did not prevent microglia in the hippocampus from adopting an activated morphology. This suggests that URMC-099 treatment after disease onset did not prevent inflammation in the acute phase of EAE, but was subsequently able to provide hippocampal neuroprotection in the chronic phase.

Microglial activation is the predominant immunopathology in the hippocampus in the chronic phase of this EAE model; prior studies have found no increase in lymphocytes (Ziehn et al., 2010) and we show no change in the sparse peripheral myeloid cells in the hippocampus of EAE versus sham-immunized mice. Converging evidence from multiple studies have established that proinflammatory microglia can disrupt synaptic structure and function in EAE gray matter via production of inflammatory signaling molecules including IL-1β, TNFα, PAF, reactive oxygen species, and nitric oxide (Centonze et al., 2009; Kim et al., 2012; Rossi et al., 2012; Bellizzi et al., 2016; Di Filippo et al., 2016; Mancini et al., 2018). URMC-099 reduced inducible nitric oxide synthase and other markers of proinflammatory microglial activation in the EAE hippocampus, appearing to shift microglia toward a less toxic and perhaps more protective phenotype. Microglial can adopt a diverse range of activation states that can shift during the course of disease and have potential to be harmful or protective depending on their context (Lewis et al., 2014; Ajami et al., 2018; Ayata et al., 2018), and how URMC-099 may modulate these states for neuroprotection needs further study. We propose that the ability to shift already-activated microglia from toxic to protective states may be particularly important for neuroprotection in MS, in which gray matter injury and cognitive dysfunction can begin at the earliest disease stages, are chronically associated with local microglial activation, and almost inevitably progress despite current immunomodulatory therapies (Amato et al., 2004; Dalton et al., 2004; Magliozzi et al., 2010; Weinstock-Guttman et al., 2012).

URMC-099 was more effective at protecting synapses in vivo than the highly-selective MLK3 inhibitor CLFB1134, suggesting that its broader range of kinase inhibition is necessary for its neuroprotective effects. Furthermore, CLFB1134 failed to match URMC-099’s ability to protect cultured sympathetic neurons during NGF withdrawal, a model system in which signaling by MLKs and possibly other kinases drives apoptosis via downstream JNK activation and in which a variety of approaches to MLK inhibition, including introduction of dominant-negative MLK3 isoforms and other MLK inhibitors with broad specificity, have been well established as protective (Xu et al., 2001; Harris et al., 2002; Goodfellow et al., 2013). These data demonstrate that highly selective MLK3 inhibition is less effective for neuroprotection and potential neurorestoration after the onset of disease than broader inhibition of a combination of kinases. This is not surprising given that inflammation and cell stress can activate multiple MLK family members in the brain as well as other kinases targeted by URMC-099, including LRRK2 that can contribute to proinflammatory microglial activation and production of signaling molecules with neurotoxic potential (Moehle et al., 2012; Puccini et al., 2015). These data suggest that overlap and/or synergies among the multiple targets of URMC-099’s kinase inhibition is critical for its neuroprotective properties.

URMC-099 has been effective and well tolerated in a broad variety of in vivo models of degenerative diseases associated with inflammation including HIV-1 associated neurologic disease, Alzheimer’s disease, as well as inflammatory liver injury in non-alcoholic steatohepatitis (Marker et al., 2013; Dong et al., 2016; Tomita et al., 2017). While activity against multiple kinases does raise concern for increasing risk of adverse effects, we propose that evaluating for such effects directly may be more fruitful for therapeutic development than holding out hope that more selective targeting of a single kinase may be as effective. URMC-099’s exemplary safety profile in the above in vivo models, and the fact that the previous non-selective MLK inhibitor CEP-1347 was well tolerated without significant adverse effects compared to placebo in clinical trials (Investigators PSGP, 2007; Ma et al., 2013), further strengthens the argument for advancing this therapeutic approach toward clinical applications.

The combination of URMC-099’s anti-inflammatory and neuroprotective effects was successful at protecting synaptic structures while maintaining synaptic function in EAE hippocampus. Achieving both is necessary for treatment of a chronic neurodegenerative disease, but can be difficult given that many inflammatory signaling molecules with neurotoxic effects including IL-1β, TNFα, PAF, reactive oxygen species, and nitric oxide also play critical roles in physiologic synaptic plasticity (O'Dell et al., 1991; Kato et al., 1994; Klann, 1998; Schneider et al., 1998; Stellwagen and Malenka, 2006). The therapeutic window for targeting signaling at the synapse can be almost impossibly narrow in neuroinflammatory disease: while blocking pathologic versus physiologic NMDA receptor activation has been considered promising for multiple neurodegenerative diseases (Lipton and Rosenberg, 1994), we have shown that similar activation can promote physiologic plasticity or excitotoxic injury depending on the presence of inflammatory mediators (Bellizzi et al., 2005). Indeed, attempts to treat MS patients with the uncompetitive NMDA receptor blocker memantine have resulted in no improvement or even worsening of neurologic deficits (Villoslada et al., 2009; Lovera et al., 2010). Likewise, PAF receptor antagonists can protect synaptic structures in EAE (Bellizzi et al., 2016) but also inhibit performance on tasks of learning and memory (Jerusalinsky et al., 1994). Thus, treatments like URMC-099 that can shift the inflammatory milieu and provide neuroprotection upstream of synaptic activation may hold more promise for restoration of gray matter function in MS and other neurodegenerative diseases.

Footnotes

  • URMC-099 is the proprietary asset of the University of Rochester Medical Center (US Patents: 8,846,909, 8,877,772, and 9,181,247, and international patents/applications to H.A.G.; References: PMCID: PMC3682381; PMC4032177). CLFB-1134 is the proprietary asset of Califia Bio, Inc. with US Patent 9,370,515 to V. S. Goodfellow and H.A.G. H.A.G. has no commercial interest in URMC-099 or CLFB-1134. All other authors declare no competing financial interests.

  • This work was supported by PO1 MH64570 (to H.A.G.), R44 NS092137 (to H.A.G.), TDFs from URMC (to H.A.G.) and by the National Multiple Sclerosis Society RG-1607-25423 (to M.J.B.).

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

References

  1. ↵
    Ajami B, Samusik N, Wieghofer P, Ho PP, Crotti A, Bjornson Z, Prinz M, Fantl WJ, Nolan GP, Steinman L (2018) Single-cell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models. Nat Neurosci 21:541–551. doi:10.1038/s41593-018-0100-x pmid:29507414
    OpenUrlCrossRefPubMed
  2. ↵
    Amato MP, Bartolozzi ML, Zipoli V, Portaccio E, Mortilla M, Guidi L, Siracusa G, Sorbi S, Federico A, De Stefano N (2004) Neocortical volume decrease in relapsing-remitting MS patients with mild cognitive impairment. Neurology 63:89–93. pmid:15249616
    OpenUrlCrossRefPubMed
  3. ↵
    Ayata P, Badimon A, Strasburger HJ, Duff MK, Montgomery SE, Loh YE, Ebert A, Pimenova AA, Ramirez BR, Chan AT, Sullivan JM, Purushothaman I, Scarpa JR, Goate AM, Busslinger M, Shen L, Losic B, Schaefer A (2018) Epigenetic regulation of brain region-specific microglia clearance activity. Nat Neurosci 21:1049–1060.
    OpenUrl
  4. ↵
    Barkhof F, Hulst HE, Drulovic J, Uitdehaag BM, Matsuda K, Landin R; MN166-001 Investigators (2010) Ibudilast in relapsing-remitting multiple sclerosis: a neuroprotectant? Neurology 74:1033–1040. doi:10.1212/WNL.0b013e3181d7d651
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Bellizzi MJ, Lu SM, Masliah E, Gelbard HA (2005) Synaptic activity becomes excitotoxic in neurons exposed to elevated levels of platelet-activating factor. J Clin Invest 115:3185–3192. doi:10.1172/JCI25444 pmid:16276420
    OpenUrlCrossRefPubMed
  6. ↵
    Bellizzi MJ, Geathers JS, Allan KC, Gelbard HA (2016) Platelet-activating factor receptors mediate excitatory postsynaptic hippocampal injury in experimental autoimmune encephalomyelitis. J Neurosci 36:1336–1346. doi:10.1523/JNEUROSCI.1171-15.2016 pmid:26818520
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Bennett ML, Bennett FC, Liddelow SA, Ajami B, Zamanian JL, Fernhoff NB, Mulinyawe SB, Bohlen CJ, Adil A, Tucker A, Weissman IL, Chang EF, Li G, Grant GA, Hayden Gephart MG, Barres BA (2016) New tools for studying microglia in the mouse and human CNS. Proc Natl Acad Sci USA 113:E1738–E1746. doi:10.1073/pnas.1525528113 pmid:26884166
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Centonze D, Muzio L, Rossi S, Cavasinni F, De Chiara V, Bergami A, Musella A, D'Amelio M, Cavallucci V, Martorana A, Bergamaschi A, Cencioni MT, Diamantini A, Butti E, Comi G, Bernardi G, Cecconi F, Battistini L, Furlan R, Martino G (2009) Inflammation triggers synaptic alteration and degeneration in experimental autoimmune encephalomyelitis. J Neurosci 29:3442–3452. doi:10.1523/JNEUROSCI.5804-08.2009
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Chen Z, Trapp BD (2016) Microglia and neuroprotection. J Neurochem 136[Suppl 1]:10–17. doi:10.1111/jnc.13062 pmid:25693054
    OpenUrlCrossRefPubMed
  10. ↵
    Ciallella JR, Saporito M, Lund S, Leist M, Hasseldam H, McGann N, Smith CS, Bozyczko-Coyne D, Flood DG (2005) CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-alpha release from lipopolysaccharide-treated cells and mice. Eur J Pharmacol 515:179–187. doi:10.1016/j.ejphar.2005.04.016 pmid:15904918
    OpenUrlCrossRefPubMed
  11. ↵
    Dalton CM, Chard DT, Davies GR, Miszkiel KA, Altmann DR, Fernando K, Plant GT, Thompson AJ, Miller DH (2004) Early development of multiple sclerosis is associated with progressive grey matter atrophy in patients presenting with clinically isolated syndromes. Brain 127:1101–1107. doi:10.1093/brain/awh126 pmid:14998914
    OpenUrlCrossRefPubMed
  12. ↵
    Di Filippo M, de Iure A, Giampà C, Chiasserini D, Tozzi A, Orvietani PL, Ghiglieri V, Tantucci M, Durante V, Quiroga-Varela A, Mancini A, Costa C, Sarchielli P, Fusco FR, Calabresi P (2016) Persistent activation of microglia and NADPH drive hippocampal dysfunction in experimental multiple sclerosis. Sci Rep 6:20926. doi:10.1038/srep20926 pmid:26887636
    OpenUrlCrossRefPubMed
  13. ↵
    Dong W, Embury CM, Lu Y, Whitmire SM, Dyavarshetty B, Gelbard HA, Gendelman HE, Kiyota T (2016) The mixed-lineage kinase 3 inhibitor URMC-099 facilitates microglial amyloid-β degradation. J Neuroinflammation 13:184. doi:10.1186/s12974-016-0646-z pmid:27401058
    OpenUrlCrossRefPubMed
  14. ↵
    Dutta R, Chang A, Doud MK, Kidd GJ, Ribaudo MV, Young EA, Fox RJ, Staugaitis SM, Trapp BD (2011) Demyelination causes synaptic alterations in hippocampi from multiple sclerosis patients. Ann Neurol 69:445–454. doi:10.1002/ana.22337 pmid:21446020
    OpenUrlCrossRefPubMed
  15. ↵
    Eggert D, Dash PK, Gorantla S, Dou H, Schifitto G, Maggirwar SB, Dewhurst S, Poluektova L, Gelbard HA, Gendelman HE (2010) Neuroprotective activities of CEP-1347 in models of neuroAIDS. J Immunol 184:746–756. doi:10.4049/Jimmunol.0902962 pmid:19966207
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Fisniku LK, Chard DT, Jackson JS, Anderson VM, Altmann DR, Miszkiel KA, Thompson AJ, Miller DH (2008) Gray matter atrophy is related to long-term disability in multiple sclerosis. Ann Neurol 64:247–254. doi:10.1002/ana.21423 pmid:18570297
    OpenUrlCrossRefPubMed
  17. ↵
    Fox RJ, Coffey CS, Conwit R, Cudkowicz ME, Gleason T, Goodman A, Klawiter EC, Matsuda K, McGovern M, Naismith RT, Ashokkumar A, Barnes J, Ecklund D, Klingner E, Koepp M, Long JD, Natarajan S, Thornell B, Yankey J, Bermel RA, et al. (2018) Phase 2 trial of ibudilast in progressive multiple sclerosis. N Engl J Med 379:846–855. doi:10.1056/NEJMoa1803583 pmid:30157388
    OpenUrlCrossRefPubMed
  18. ↵
    Goodfellow VS, Loweth CJ, Ravula SB, Wiemann T, Nguyen TX, Xu Y, Todd DE, Sheppard DW, Pollack S, Polesskaya O, Marker DF, Dewhurst S, Gelbard HA (2013) Discovery, synthesis and characterization of an orally bioavailable, brain penetrant inhibitor of mixed lineage kinase 3. J Med Chem 56:8032–8048.
    OpenUrl
  19. ↵
    Goodfellow VSN, Nguyen TX, Ravula SB, Gelbard, HA (2016) Mixed lineage kinase inhibitors and method of treatments. U.S. Patent 9370515 B2.
  20. ↵
    Harris CA, Deshmukh M, Tsui-Pierchala B, Maroney AC, Johnson EM Jr (2002) Inhibition of the c-Jun N-terminal kinase signaling pathway by the mixed lineage kinase inhibitor CEP-1347 (KT7515) preserves metabolism and growth of trophic factor-deprived neurons. J Neurosci 22:103–113. doi:10.1523/JNEUROSCI.22-01-00103.2002
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Hauser SL, Bar-Or A, Comi G, Giovannoni G, Hartung HP, Hemmer B, Lublin F, Montalban X, Rammohan KW, Selmaj K, Traboulsee A, Wolinsky JS, Arnold DL, Klingelschmitt G, Masterman D, Fontoura P, Belachew S, Chin P, Mairon N, Garren H, et al. (2017) Ocrelizumab versus interferon beta-1a in relapsing multiple sclerosis. N Engl J Med 376:221–234. doi:10.1056/NEJMoa1601277 pmid:28002679
    OpenUrlCrossRefPubMed
  22. ↵
    Huang J, Kim LJ, Mealey R, Marsh HC Jr., Zhang Y, Tenner AJ, Connolly ES Jr., Pinsky DJ (1999) Neuronal protection in stroke by an sLex-glycosylated complement inhibitory protein. Science 285:595–599. doi:10.1126/science.285.5427.595
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Investigators PSGP (2007) Mixed lineage kinase inhibitor CEP-1347 fails to delay disability in early Parkinson disease. Neurology 69:1480–1490.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Jerusalinsky D, Fin C, Quillfeldt JA, Ferreira MB, Schmitz PK, Da Silva RC, Walz R, Bazan NG, Medina JH, Izquierdo I (1994) Effect of antagonists of platelet-activating factor receptors on memory of inhibitory avoidance in rats. Behav Neural Biol 62:1–3. pmid:7945139
    OpenUrlCrossRefPubMed
  25. ↵
    Jürgens T, Jafari M, Kreutzfeldt M, Bahn E, Brück W, Kerschensteiner M, Merkler D (2016) Reconstruction of single cortical projection neurons reveals primary spine loss in multiple sclerosis. Brain 139:39–46. doi:10.1093/brain/awv353 pmid:26667278
    OpenUrlCrossRefPubMed
  26. ↵
    Kato K, Clark GD, Bazan NG, Zorumski CF (1994) Platelet-activating factor as a potential retrograde messenger in CA1 hippocampal long-term potentiation. Nature 367:175–179. doi:10.1038/367175a0 pmid:8114914
    OpenUrlCrossRefPubMed
  27. ↵
    Kim DY, Hao J, Liu R, Turner G, Shi FD, Rho JM (2012) Inflammation-mediated memory dysfunction and effects of a ketogenic diet in a murine model of multiple sclerosis. PLoS One 7:e35476. doi:10.1371/journal.pone.0035476
    OpenUrlCrossRefPubMed
  28. ↵
    Kiyota T, Machhi J, Lu Y, Dyavarshetty B, Nemati M, Zhang G, Lee Mosley R, Gelbard HA, Gendelman HE (2018) URMC-099 facilitates amyloid-β clearance in a murine model of Alzheimer's disease. J Neuroinflammation 15:137. doi:10.1186/s12974-018-1172-y pmid:29729668
    OpenUrlCrossRefPubMed
  29. ↵
    Klann E (1998) Cell-permeable scavengers of superoxide prevent long-term potentiation in hippocampal area CA1. J Neurophysiol 80:452–457. doi:10.1152/jn.1998.80.1.452 pmid:9658063
    OpenUrlCrossRefPubMed
  30. ↵
    Lewis ND, Hill JD, Juchem KW, Stefanopoulos DE, Modis LK (2014) RNA sequencing of microglia and monocyte-derived macrophages from mice with experimental autoimmune encephalomyelitis illustrates a changing phenotype with disease course. J Neuroimmunol 277:26–38. doi:10.1016/j.jneuroim.2014.09.014 pmid:25270668
    OpenUrlCrossRefPubMed
  31. ↵
    Lipton SA, Rosenberg PA (1994) Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 330:613–622. doi:10.1056/NEJM199403033300907 pmid:7905600
    OpenUrlCrossRefPubMed
  32. ↵
    Lotharius J, Falsig J, van Beek J, Payne S, Dringen R, Brundin P, Leist M (2005) Progressive degeneration of human mesencephalic neuron-derived cells triggered by dopamine-dependent oxidative stress is dependent on the mixed-lineage kinase pathway. J Neurosci 25:6329–6342. doi:10.1523/JNEUROSCI.1746-05.2005 pmid:16000623
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Lovera JF, Frohman E, Brown TR, Bandari D, Nguyen L, Yadav V, Stuve O, Karman J, Bogardus K, Heimburger G, Cua L, Remingon G, Fowler J, Monahan T, Kilcup S, Courtney Y, McAleenan J, Butler K, Wild K, Whitham R, et al. (2010) Memantine for cognitive impairment in multiple sclerosis: a randomized placebo-controlled trial. Mult Scler 16:715–723. doi:10.1177/1352458510367662 pmid:20483885
    OpenUrlCrossRefPubMed
  34. ↵
    Ma Q, Gelbard HA, Maggirwar SB, Dewhurst S, Gendelman HE, Peterson DR, DiFrancesco R, Hochreiter JS, Morse GD, Schifitto G (2013) Pharmacokinetic interactions of CEP-1347 and atazanavir in HIV-infected patients. J Neurovirol 19:254–260. doi:10.1007/s13365-013-0172-z pmid:23737347
    OpenUrlCrossRefPubMed
  35. ↵
    Magliozzi R, Howell OW, Reeves C, Roncaroli F, Nicholas R, Serafini B, Aloisi F, Reynolds R (2010) A gradient of neuronal loss and meningeal inflammation in multiple sclerosis. Ann Neurol 68:477–493. doi:10.1002/ana.22230 pmid:20976767
    OpenUrlCrossRefPubMed
  36. ↵
    Mancini A, Tantucci M, Mazzocchetti P, de Iure A, Durante V, Macchioni L, Giampà C, Alvino A, Gaetani L, Costa C, Tozzi A, Calabresi P, Di Filippo M (2018) Microglial activation and the nitric oxide/cGMP/PKG pathway underlie enhanced neuronal vulnerability to mitochondrial dysfunction in experimental multiple sclerosis. Neurobiol Dis 113:97–108. doi:10.1016/j.nbd.2018.01.002 pmid:29325869
    OpenUrlCrossRefPubMed
  37. ↵
    Marker DF, Tremblay ME, Puccini JM, Barbieri J, Gantz Marker MA, Loweth CJ, Muly EC, Lu SM, Goodfellow VS, Dewhurst S, Gelbard HA (2013) The new small-molecule mixed-lineage kinase 3 inhibitor URMC-099 is neuroprotective and anti-inflammatory in models of human immunodeficiency virus-associated neurocognitive disorders. J Neurosci 33:9998–10010. doi:10.1523/JNEUROSCI.0598-13.2013
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Maroney AC, Finn JP, Connors TJ, Durkin JT, Angeles T, Gessner G, Xu Z, Meyer SL, Savage MJ, Greene LA, Scott RW, Vaught JL (2001) Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family. J Biol Chem 276:25302–25308. doi:10.1074/jbc.M011601200 pmid:11325962
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Moehle MS, Webber PJ, Tse T, Sukar N, Standaert DG, DeSilva TM, Cowell RM, West AB (2012) LRRK2 inhibition attenuates microglial inflammatory responses. J Neurosci 32:1602–1611. doi:10.1523/JNEUROSCI.5601-11.2012 pmid:22302802
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Murphy AC, Lalor SJ, Lynch MA, Mills KH (2010) Infiltration of Th1 and Th17 cells and activation of microglia in the CNS during the course of experimental autoimmune encephalomyelitis. Brain Behav Immun 24:641–651. doi:10.1016/j.bbi.2010.01.014 pmid:20138983
    OpenUrlCrossRefPubMed
  41. ↵
    O'Dell TJ, Hawkins RD, Kandel ER, Arancio O (1991) Tests of the roles of two diffusible substances in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger. Proc Natl Acad Sci USA 88:11285–11289. pmid:1684863
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Peterson JW, Bö L, Mörk S, Chang A, Trapp BD (2001) Transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions. Ann Neurol 50:389–400. pmid:11558796
    OpenUrlCrossRefPubMed
  43. ↵
    Phillips RG, LeDoux JE (1992) Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav Neurosci 106:274–285. pmid:1590953
    OpenUrlCrossRefPubMed
  44. ↵
    Puccini JM, Marker DF, Fitzgerald T, Barbieri J, Kim CS, Miller-Rhodes P, Lu SM, Dewhurst S, Gelbard HA (2015) Leucine-rich repeat kinase 2 modulates neuroinflammation and neurotoxicity in models of human immunodeficiency virus 1-associated neurocognitive disorders. J Neurosci 35:5271–5283. doi:10.1523/JNEUROSCI.0650-14.2015 pmid:25834052
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Roosendaal SD, Bendfeldt K, Vrenken H, Polman CH, Borgwardt S, Radue EW, Kappos L, Pelletier D, Hauser SL, Matthews PM, Barkhof F, Geurts JJ (2011) Grey matter volume in a large cohort of MS patients: relation to MRI parameters and disability. Mult Scler 17:1098–1106. doi:10.1177/1352458511404916
    OpenUrlCrossRefPubMed
  46. ↵
    Rosas M, Thomas B, Stacey M, Gordon S, Taylor PR (2010) The myeloid 7/4-antigen defines recently generated inflammatory macrophages and is synonymous with Ly-6B. J Leukoc Biol 88:169–180. doi:10.1189/jlb.0809548 pmid:20400676
    OpenUrlCrossRefPubMed
  47. ↵
    Rossi S, Furlan R, De Chiara V, Motta C, Studer V, Mori F, Musella A, Bergami A, Muzio L, Bernardi G, Battistini L, Martino G, Centonze D (2012) Interleukin-1β causes synaptic hyperexcitability in multiple sclerosis. Ann Neurol 71:76–83. doi:10.1002/ana.22512 pmid:22275254
    OpenUrlCrossRefPubMed
  48. ↵
    Savinainen A, Garcia EP, Dorow D, Marshall J, Liu YF (2001) Kainate receptor activation induces mixed lineage kinase-mediated cellular signaling cascades via post-synaptic density protein 95. J Biol Chem 276:11382–11386. doi:10.1074/jbc.M100190200 pmid:11152698
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Schneider H, Pitossi F, Balschun D, Wagner A, del Rey A, Besedovsky HO (1998) A neuromodulatory role of interleukin-1beta in the hippocampus. Proc Natl Acad Sci USA 95:7778–7783. pmid:9636227
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Stellwagen D, Malenka RC (2006) Synaptic scaling mediated by glial TNF-alpha. Nature 440:1054–1059. doi:10.1038/nature04671 pmid:16547515
    OpenUrlCrossRefPubMed
  51. ↵
    Sui Z, Fan S, Sniderhan L, Reisinger E, Litzburg A, Schifitto G, Gelbard HA, Dewhurst S, Maggirwar SB (2006) Inhibition of mixed lineage kinase 3 prevents HIV-1 Tat-mediated neurotoxicity and monocyte activation. J Immunol 177:702–711. doi:10.4049/Jimmunol.177.1.702
    OpenUrlAbstract/FREE Full Text
  52. ↵
    Tian H, Zhang Q, Li H, Zhang G (2003) Antioxidant N-acetylcysteine and AMPA/KA receptor antagonist DNQX inhibited mixed lineage kinase-3 activation following cerebral ischemia in rat hippocampus. Neurosci Res 47:47–53. pmid:12941446
    OpenUrlCrossRefPubMed
  53. ↵
    Tomita K, Kohli R, MacLaurin BL, Hirsova P, Guo Q, Sanchez LHG, Gelbard HA, Blaxall BC, Ibrahim SH (2017) Mixed-lineage kinase 3 pharmacological inhibition attenuates murine nonalcoholic steatohepatitis. JCI Insight 2.
  54. ↵
    van Oosten BW, Barkhof F, Truyen L, Boringa JB, Bertelsmann FW, von Blomberg BM, Woody JN, Hartung HP, Polman CH (1996) Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal anti-tumor necrosis factor antibody cA2. Neurology 47:1531–1534. pmid:8960740
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Villoslada P, Arrondo G, Sepulcre J, Alegre M, Artieda J (2009) Memantine induces reversible neurologic impairment in patients with MS. Neurology 72:1630–1633. doi:10.1212/01.wnl.0000342388.73185.80 pmid:19092106
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Wegner C, Esiri MM, Chance SA, Palace J, Matthews PM (2006) Neocortical neuronal, synaptic, and glial loss in multiple sclerosis. Neurology 67:960–967. doi:10.1212/01.wnl.0000237551.26858.39 pmid:17000961
    OpenUrlAbstract/FREE Full Text
  57. ↵
    Weinstock-Guttman B, Galetta SL, Giovannoni G, Havrdova E, Hutchinson M, Kappos L, O'Connor PW, Phillips JT, Polman C, Stuart WH, Lynn F, Hotermans C (2012) Additional efficacy endpoints from pivotal natalizumab trials in relapsing-remitting MS. J Neurol 259:898–905. doi:10.1007/s00415-011-6275-7
    OpenUrlCrossRefPubMed
  58. ↵
    Woolley CS, Gould E, Frankfurt M, McEwen BS (1990) Naturally occurring fluctuation in dendritic spine density on adult hippocampal pyramidal neurons. J Neurosci 10:4035–4039. pmid:2269895
    OpenUrlAbstract/FREE Full Text
  59. ↵
    Xu Z, Maroney AC, Dobrzanski P, Kukekov NV, Greene LA (2001) The MLK family mediates c-Jun N-terminal kinase activation in neuronal apoptosis. Mol Cell Biol 21:4713–4724. doi:10.1128/MCB.21.14.4713-4724.2001 pmid:11416147
    OpenUrlAbstract/FREE Full Text
  60. ↵
    Ziehn MO, Avedisian AA, Tiwari-Woodruff S, Voskuhl RR (2010) Hippocampal CA1 atrophy and synaptic loss during experimental autoimmune encephalomyelitis, EAE. Lab Invest 90:774–786. doi:10.1038/Labinvest.2010.6 pmid:20157291
    OpenUrlCrossRefPubMed

Synthesis

Reviewing Editor: Liisa Galea, University of British Columbia

Decisions are customarily a result of the Reviewing Editor and the peer reviewers coming together and discussing their recommendations until a consensus is reached. When revisions are invited, a fact-based synthesis statement explaining their decision and outlining what is needed to prepare a revision will be listed below. The following reviewer(s) agreed to reveal their identity: David Pleasure, Paolo Calabresi. Note: If this manuscript was transferred from JNeurosci and a decision was made to accept the manuscript without peer review, a brief statement to this effect will instead be what is listed below.

In their manuscript, “The mixed-lineage kinase inhibitor URMC-099 protects hippocampal synapses in experimental autoimmune encephalomyelitis”, Bellizzi and colleagues investigated the possible neuroprotective effects of a mixed lineage kinase (MLK) inhibitor (URMC-099), developed by their research group, in an experimental model of MS. In particular, they found that URMC-099, a broad spectrum MLK inhibitor with additional activity against LRRK2 and other kinases, prevented loss of postsynaptic structures in the hippocampus and reversed deficits in hippocampal-dependent contextual fear conditioning in mice affected by EAE mice when administered after the onset of motor symptoms.

All of the reviewers agree that this was a well written manuscript with important implications suggesting potential neuroprotective strategies for MS.

As suggested in the prior review, it would have improved the manuscript if an in vitro dose-response study could help in establishing the neuroprotective effect of the drug but the reviewers are not willing to make this a requirement of acceptance.

The microglial modulation exerted by URMC-099 and CLFB-1134 is not clear at all. Authors stated that “URMC-099 and CLFB-1134 both prevented any increases in microglial CD68 expression, suggesting that treatment with either agent does not block activation of microglia in the EAE hippocampus but may modulate the biologic response of their activated phenotype”. The potential role of persistent microglial activation in the neuro-inflammatory related neurodegeneration has been largely characterized. The fact that microglial activation has not been significantly altered by URMC-099 and CLFB-1134 administration potentially reduces the impact of the work. Interestingly, authors found that URMC-099 significantly reduced the expression of CD68, iNOS and FCγR1/CD64, with a non-significant reduction of IL-1β. Considering these results, authors could take advantage in discussing some works showing the relationship between microglial activation and the activation of NO-pathway in enhancing neuronal mitochondrial dysfunction during EAE (Mancini et al., Neurobiol Dis, 2018), or the relationship existing between hippocampal microglial CD68 expression and the disruption of hippocampal synaptic plasticity and hippocampal-dependent memory during EAE (Di Filippo et al., Sci Rep, 2016).

In their rebuttal and manuscript, the authors overstate the report by Ziehn et al (2010) that there are no inflammatory cells in hippocampus in this model. In fact, Ziehn et al made no effort to discriminate between activated microglia and monocyte-derived macrophages. Further, they did not exclude an early invasion by neutrophils. Immunohistologiccal reagents are now available that can help with this discrimination (though FACS, of course, would be more definitive).

The authors used both sexes but did not analyze by sex and this is a concern as there are noted sex differences in EAE susceptibility (although apparently not in C57BL6 but this should be noted) but there are sex differences in fear conditioning, MS, and immune signalling (i.e. The importance of studying sex differences in disease: The example of multiple sclerosis. Golden LC, Voskuhl R. J Neurosci Res. 2017 Jan 2;95(1-2):633-643; Sex effects on inflammatory and neurodegenerative processes in multiple sclerosis. Ramien C, Taenzer A, Lupu A, Heckmann N, Engler JB, Patas K, Friese MA, Gold SM. Neurosci Biobehav Rev. 2016 Aug;67:137-46; The influence of sex hormones on cytokines in multiple sclerosis and experimental autoimmune encephalomyelitis: a review. van den Broek HH, Damoiseaux JG, De Baets MH, Hupperts RM. Mult Scler. 2005 Jun;11(3):349-59). The advice here would be to use sex as a factor in the analyses as at the very least a covariate but preferably an independent factor. It is possible that the use of sex as a factor in the analyses will provide some insight into the larger errors on some measures. In addition, the sex ratio per study should be given as it is not clear if an equal number of males and females were used.

Minor

Please remove the word “gender” from line 157 and replace it with “sex” as gender is a psychosocial construct (roles on which a given society deems appropriate for men or women) and not appropriate in animal modelling.

Back to top

In this issue

eneuro: 5 (6)
eNeuro
Vol. 5, Issue 6
November/December 2018
  • Table of Contents
  • Index by author
Email

Thank you for sharing this eNeuro article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
The Mixed-Lineage Kinase Inhibitor URMC-099 Protects Hippocampal Synapses in Experimental Autoimmune Encephalomyelitis
(Your Name) has forwarded a page to you from eNeuro
(Your Name) thought you would be interested in this article in eNeuro.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Print
View Full Page PDF
Citation Tools
The Mixed-Lineage Kinase Inhibitor URMC-099 Protects Hippocampal Synapses in Experimental Autoimmune Encephalomyelitis
Matthew J. Bellizzi, Jennetta W. Hammond, Herman Li, Mary A. Gantz Marker, Daniel F. Marker, Robert S. Freeman, Harris A. Gelbard
eNeuro 16 November 2018, 5 (6) ENEURO.0245-18.2018; DOI: 10.1523/ENEURO.0245-18.2018

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Respond to this article
Share
The Mixed-Lineage Kinase Inhibitor URMC-099 Protects Hippocampal Synapses in Experimental Autoimmune Encephalomyelitis
Matthew J. Bellizzi, Jennetta W. Hammond, Herman Li, Mary A. Gantz Marker, Daniel F. Marker, Robert S. Freeman, Harris A. Gelbard
eNeuro 16 November 2018, 5 (6) ENEURO.0245-18.2018; DOI: 10.1523/ENEURO.0245-18.2018
Reddit logo Twitter logo Facebook logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Significance Statement
    • Introduction
    • Materials and Methods
    • Results
    • Discussion
    • Footnotes
    • References
    • Synthesis
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF

Keywords

  • Microglia
  • Multiple Sclerosis
  • neurodegeneration
  • neuroprotection
  • synapse

Responses to this article

Respond to this article

Jump to comment:

No eLetters have been published for this article.

Related Articles

Cited By...

More in this TOC Section

New Research

  • Heterozygous Dab1 null mutation disrupts neocortical and hippocampal development
  • The nasal solitary chemosensory cell signaling pathway triggers mouse avoidance behavior to inhaled nebulized irritants
  • Different control strategies drive interlimb differences in performance and adaptation during reaching movements in novel dynamics
Show more New Research

Disorders of the Nervous System

  • Characterization of the Tau Interactome in Human Brain Reveals Isoform-Dependent Interaction with 14-3-3 Family Proteins
  • Impaired AMPARs translocation into dendritic spines with motor skill learning in the Fragile X mouse model
  • Glycolytic System in Axons Supplement Decreased ATP Levels after Axotomy of the Peripheral Nerve
Show more Disorders of the Nervous System

Subjects

  • Disorders of the Nervous System

  • Home
  • Alerts
  • Visit Society for Neuroscience on Facebook
  • Follow Society for Neuroscience on Twitter
  • Follow Society for Neuroscience on LinkedIn
  • Visit Society for Neuroscience on Youtube
  • Follow our RSS feeds

Content

  • Early Release
  • Current Issue
  • Latest Articles
  • Issue Archive
  • Blog
  • Browse by Topic

Information

  • For Authors
  • For the Media

About

  • About the Journal
  • Editorial Board
  • Privacy Policy
  • Contact
  • Feedback
(eNeuro logo)
(SfN logo)

Copyright © 2023 by the Society for Neuroscience.
eNeuro eISSN: 2373-2822

The ideas and opinions expressed in eNeuro do not necessarily reflect those of SfN or the eNeuro Editorial Board. Publication of an advertisement or other product mention in eNeuro should not be construed as an endorsement of the manufacturer’s claims. SfN does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of any material contained in eNeuro.