Locus coeruleus injury modulates ventral midbrain neuroinflammation during DSS-induced colitis

Parkinson’s disease (PD) is characterized by a decades-long prodrome, consisting of a collection of non-motor symptoms that emerges prior to the motor manifestation of the disease. Of these non-motor symptoms, gastrointestinal dysfunction and deficits attributed to central norepinephrine (NE) loss, including mood changes and sleep disturbances, are frequent in the PD population and emerge early in the disease. Evidence is mounting that injury and inflammation in the gut and locus coeruleus (LC), respectively, underlie these symptoms, and the injury of these systems is central to the progression of PD. In this study, we generate a novel two-hit mouse model that captures both features, using dextran sulfate sodium (DSS) to induce gut inflammation and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to lesion the LC. We first confirmed the specificity of DSP-4 for central NE using neurochemical methods and fluorescence light-sheet microscopy of cleared tissue, and established that DSS-induced outcomes in the periphery, including weight loss, gross indices of gut injury and systemic inflammation, the loss of tight junction proteins in the colonic epithelium, and markers of colonic inflammation, were unaffected with DSP-4 pre-administration. We then measured alterations in neuroimmune gene expression in the ventral midbrain in response to DSS treatment alone as well as the extent to which prior LC injury modified this response. In this two-hit model we observed that DSS-induced colitis activates the expression of key cytokines and chemokines in the ventral midbrain only in the presence of LC injury and the typical DSS-associated neuroimmune is blunted by pre-LC lesioning with DSP-4. In all, this study supports the growing appreciation for the LC as neuroprotective against inflammation-induced brain injury and draws attention to the potential for NEergic interventions to exert disease-modifying effects under conditions where peripheral inflammation may compromise ventral midbrain dopaminergic neurons and increase the risk for development of PD.


Introduction
Prodromal and early Parkinson's disease (PD) is often marked by gastrointestinal distress and in ammation in the intestine.Constipation and defecatory dysfunction emerge in around 80% 1 and 61% 2,3 , respectively, of people with PD, and it appears that gut dysfunction emerges years before a clinical diagnosis [3][4][5] .Additionally, intestinal in ammation, re ected by greater fecal contents of proin ammatory cytokines 6 and greater numbers of reactive glia in the colon 7 , and a deterioration of the colonic epithelium 8,9 are evident in PD.And, people with in ammatory bowel disease (IBD) are at a roughly 22% greater risk of PD later in life 10 , and this risk is mitigated by the use of anti-TNF biologics 11,12 , further cementing a link between gut and brain in PD and highlighting in ammation as a potentially key mediator.
Studies in pre-clinical models have enabled investigators to directly investigate the extent to which gut in ammation can induce brain injury and in ammation.Multiple studies including several from our group have modeled IBD-like in ammation, using oral administration of dextran sulfate sodium (DSS) to denude the colonic epithelium 13 , and reported neuroimmune activation and immune cell in ltration in mice with colitis [14][15][16][17][18] .Critically, DSS-induced colitis has been reported to induce more robust nigrostriatal degeneration in mice expressing PD-relevant mutations in LRRK2 19,20 and SNCA 21,22 relative to their wildtype counterparts.While it is now widely accepted that PD arises from complex gene-by-environment interactions 23,24 , and mice with variants in PD-associated genes would be expected to display dysregulated responses to environmental insults, these mutations are overall low in frequency 24 .It was of greater interest to us to identify how gut in ammation might synergize with other frequent and early pathophysiological events in PD.
The disruption of norepinephrine (NE) signaling from the locus coeruleus (LC) is a common observation in PD and is believed to precede degeneration of the nigrostriatal pathway 25,26 .Emotional dysregulation, manifesting as clinical depression, anxiety, and/or apathy, and sleep disturbances, such as insomnia and daytime sleepiness, each emerge in 30-60% of people with PD [27][28][29] , and LC NE has been implicated in these non-motor PD symptoms 30,31 .A frank loss of LC NE neurons and decreased NE tone in the rest of the brain has been documented in people with PD with in vivo neuromelanin-sensitive MRI 32,33 , [ 11 C]methyl-reboxetine PET imaging studies 34,35 and post mortem immunohistochemical 36,37 and biochemical 38 studies.Interestingly, a similar pattern of global NE denervation has been observed in people with REM sleep behavior disorder (RBD) [39][40][41] , an established prodromal stage of PD or other synucleinopathies 42,43 , suggesting that LC injury occurs earlier than dopaminergic dysfunction.Indeed, mood dysfunction often emerges early and prior to a PD diagnosis 27,44,45 .
Critically, the death of the LC appears to be a central, disease-modifying process in PD.First, the nonmotor symptoms described above seem to be associated with a worsened disease severity 46,47 and lower quality of life 48,49 .Second, studies in pre-clinical models have repeatedly demonstrated the neuroprotective function of the LC.A pharmacological lesion of the LC using N-(2-chloroethyl)-N-ethyl-2bromobenzylamine (DSP-4) resulted in blood-brain barrier disruption 50 , neuroin ammation via the NF-κB in ammasome 51 , and oxidative stress 52 in regions downstream from the LC.Pharmacological blockade of the β2-adrenergic receptor, simulating the loss of NE that occurs with LC degeneration, recapitulates the activation of neuroin ammation seen with LC neuron loss 53,54 .Treating primary microglial cells or organotypic brain slice cultures with NE or adrenergic receptor agonists further demonstrates the protective role of NE, with adrenergic receptor stimulation resulting in a suppression of microglial NF-κB, cytokine production, and motility 55,56 .
More interestingly, LC injury may interact with secondary disease-relevant insults to exacerbate neuroin ammation and neurodegeneration.A DSP-4 lesion has been shown to worsen proteinopathy and behavioral defects in mouse models over-expressing mutant tau 57 and alpha-synuclein 58 .NE depletion through DSP-4 has also exacerbated nigrostriatal death and motor de cits of neurotoxin models of PD that selectively injury the midbrain dopaminergic neurons [59][60][61] .Of most relevance to the gut-brain axis in PD, DSP-4 has exacerbated neuroin ammation after systemic in ammatory insults, including an acute lipopolysaccharide challenge 62-65 and a high-sugar diet 66 .Clearly, the LC is an important regulator of neuroin ammation and brain health, especially amidst other in ammatory challenges, which would be expected to have important disease-modifying consequences on the development and progression of PD.
However, despite the frequency with which both emerge in PD, the role of LC death in neuroin ammation due to intestinal permeability has not yet been evaluated.Our hypothesis is that the LC protects the midbrain against leaky gut-induced neuroin ammation.Here, we pair the well-established DSS experimental colitis model with DSP-4, an LC-speci c neurotoxin, to model the gut in ammation and LC injury that emerge frequently in PD and examine the combined effects of these insults on midbrain neuroimmune gene expression.Notably, we take care to a rm the speci city of this two-hit procedure, ensuring the NE de cit is restricted to the brain and the induction of colitis with DSS is unaffected.
Speci cally, based on relevant data using the DSP-4 model and other NE-related interventions and on our own data with the DSS-induced colitis model 14 , we expect that DSP-4-induced LC degeneration will exacerbate midbrain neuroimmune gene expression due to DSS-induced colitis, implicating an in uential position for the LC as a protective locus against in ammatory stress along the gut-brain axis in PD.A better understanding of the relationship between gut health and LC permeability with respect to brain in ammation would advance our understanding of PD pathogenesis, emphasizing upstream diseaserelated events as potential diagnostic or therapeutic opportunities.

Animals
C57BL/6J mice were purchased from Jackson Laboratories (strain #000664), bred to congenicity, and housed until 2-3 months of age on a 12:12 light-dark cycle with ad libitum access to food and water.For experiments examining the speci city of DSP-4, mice were anesthetized with iso urane and euthanized with transcardiac perfusion with heparinized PBS after their respective dosing schedules.Brains were rapidly extracted, and the frontal cortex was dissected using a razor blade, removing any olfactory bulb, striatum, or other tissue so cortical monoamines could be measured with high-performance liquid chromatography (HPLC).Colons were also extracted so roughly 5cm of tissue on the cecal end of the colon could be cleaned and analyzed with HPLC.For experiments employing a two-hit schedule with both DSP-4 and DSS, mice were again anesthetized and perfused as above, and colon lengths and spleen weights were measured.To assess colonic in ammation and injury, roughly 5cm of tissue on the cecal end of the colon was cleaned and ash-frozen in liquid nitrogen for biochemical and gene expression analyses.To assess neuroin ammation in this two-hit model, brains were extracted and the ventral midbrain was rapidly removed as described in ref. 14 and ash-frozen for gene expression analyses.All procedures were approved by the University of Florida Institutional Animal Care and Use Committee and followed the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health.

Drug administration
Within cages, mice were randomly assigned to DSP-4 or vehicle treatment groups.Mice were given a single intraperitoneal injection of DSP-4 (50mgkg) or sterile saline vehicle.DSP-4 (Sigma, #C8417) was dissolved in sterile saline to yield a 5mg/mL solution which was injected into mice.Due to the rapid halflife of DSP-4 in solutions with a physiological pH 67 , only enough solution was prepared for 3-5 mice at a time, and mice were injected within 2 minutes of the dissolution of DSP-4.
When combining DSP-4 with experimental colitis, cages were randomly assigned to receive dextran sulfate sodium (DSS) or untreated autoclaved tap water as a vehicle control.40kDa DSS (ThermoFisher, #J14489-22) was dissolved in autoclaved tap water at the concentrations described below and placed in sterile water bottles for administration.To elicit peak intestinal injury, 3% DSS (w/v) was used for 7 days, and tissues were harvested.To elicit peak neuroin ammation, 2.5% DSS (w/v) was used for 7 days followed by 2 days of withdrawal with untreated autoclaved tap water.Our group has identi ed these schedules based on a previous study characterizing the kinetics of intestinal and brain in ammation in the DSS-induced colitis model 14 .During and after DSS administration, animal weights and disease severity were measured daily using the rubric described in Supplemental Table 1.In all two-hit studies, mice began receiving DSS treatment 28 days after a single injection of DSP-4 or vehicle.HPLC Tissue samples were weighed and lysed in 0.1M perchloric acid.Colon samples were lysed in a 10:1 (volume to weight) ratio of 0.1M perchloric acid, while frontal cortex samples were lysed in a 20:1 ratio.Tissue was sonicated in pulses using a probe sonicator until tissue was fully dissociated.Tissue lysates were centrifuged at 16,000g for 15min at 4°C, the supernatants were ltered through a Nanosep 0.2µm mesh (Fisher Scienti c, #50-197-9573) with centrifugation at 5,000g for 5min at 4°C, and samples were stored at -20°C until analysis.Monoamine concentrations were analyzed using high-performance liquid chromatography (HPLC) with electrochemical detection (HTEC, Azuma Inc.).A standard curve consisting of 3-methoxy-4-hydroxyphenylglycol (MHPG), NE, 3,4-dihydroxyphenylacetic acid (DOPAC), DA, 5hydroxyindoleacetic acid (5HIAA), homovanillic acid (HVA), 3-methoxytyramine (3MT), and 5hydroxytryptophan (5HT) were used to enable the measurement of these molecules at the picomolar level.Concentration data are shown as percentages of the control group.
Tissues were dehydrated in graded methanol baths, delipidated in a 2:1 solution of DCM and methanol, washed twice in pure DCM, and optically cleared by direct transfer to dibenzyl ether (DBE).Tissue remained in DBE without agitation until imaging.

Fluorescence light-sheet microscopy
Cleared and immunolabeled intact tissues were imaged using an UltraMicroscope Blaze (Miltenyi Biotec).All images were acquired using right-side illumination, adaptive horizontal focus, 15% overlap between mosaic tiles, and a step-size half the width of the light-sheet for optimal axial resolution.Images were rendered in three dimensions using Imaris v. 9.8 (Oxford Instruments).Illumination settings, including laser power and exposure time, and image rendering settings, including any lookup table adjustments, were kept constant between images within an experiment.

Nucleic acid and protein extraction
Protein and RNA were extracted from brain and colon using a TRIzol-based method.TRIzol (ThermoFisher, #15596018) was added to tissue and lysed using a sterile metal bead with rapid agitation in a TissueLyser II (Qiagen).Lysate was then mixed with UltraPure phenol:chloroform:isoamyl alcohol (ThermoFisher, #15593049) in a 5:1 ratio.Samples were centrifuged to separate protein, RNA, and DNA.The top aqueous layer containing RNA was transferred to a QIAshredder column (Qiagen, #79656), and ow-through as mixed 1:1 with 70% ethanol.This solution was transferred to a RNeasy Mini column (Qiagen, #74104) and centrifuged.The column was washed once in 700µL RW1 buffer and twice in 500µL RPE buffer, following the manufacturer's instructions.RNA was eluted in nuclease-free water and a Denovix DS-11 spectrophotometer was used to measure nucleic acid concentrations.
To isolate protein, the white DNA layer and some of the pink layer was discarded, leaving roughly 300µL.1mL of methanol was added to precipitate the protein.Protein precipitation occurred over 10 minutes at room temperature, and the protein was pelleted and washed in 500µL of methanol.The supernatant was decanted and the pellet was air-dried to remove residual methanol after which the protein was resuspended in 1% SDS (w/v) in deionized water.Protein concentrations were measured using a Pierce BCA Protein Assay Kit (ThermoFisher, #23225) according to manufacturer's instructions.

SDS-PAGE and immunoblotting
Within experiments, protein was diluted to an equivalent concentration for each sample in additional 1% SDS with 4X Laemmli sample buffer (Bio-Rad, #1610747) and 2-mercaptoethanol.15µg of protein was loaded into a pre-cast 4-20% polyacrylamide Criterion TGX gel (Bio-Rad, #5678095).To normalize signal between different gels within an experiment, a constant sample was included on every gel.Electrophoresis consisted of one hour of 60V followed by 125V until nished, about 75 minutes.Electrophoresed samples were transferred to a PVDF membrane (Bio-Rad, #1620175) using a Trans-Blot Turbo Transfer system (Bio-Rad, #1704150) using the preset Mixed Molecular Weight program.Signal normalization was achieved using the Revert 700 Total Protein Stain (Li-Cor, #926-11011).The stain was imaged on a Li-Cor Odyssey Fc imager with a 2-minute exposure time.

cDNA synthesis and qPCR
Complementary DNA (cDNA) was synthesized from RNA using the ImProm-II Reverse Transcription System (Promega, #A3800) according to manufacturer's instructions.RNA was hybridized to oligo-dT primers in a total volume of 4µL for 5 minutes at 70°C.Then, the reverse transcription master mix, containing 1X ImProm-II reaction buffer, 5mM MgCl 2 , 0.6mM deoxyribonucleotides, RNase inhibitor, and reverse transcriptase, was added to the RNA with annealed primers.Samples were equilibrated at 25°C for 5 minutes followed by reverse transcription at 42°C for one hour and reverse transcriptase inactivation at 70°C for 15 minutes.Samples were diluted with nuclease-free water to 5-10µg/µL, depending on the abundance of the target gene.
Gene expression was quanti ed using quantitative polymerase chain reaction (qPCR) with SYBR Green chemistry.5µL of diluted cDNA was mixed with 35µL of qPCR master mix, consisting of 1.25µM forward and reverse primers, SYBR Green (ThermoFisher, #A46112), and nuclease-free water.This reaction mix was plated into 10µL triplicates.A QuantStudio 5 Real-Time PCR System (ThermoFisher) was used to perform the qPCR.Triplicates were pruned based on intra-sample standard deviation and deviation from the median value using a custom R script.Data were analyzed using the ΔΔC T method, with data being expressed as a fold change from the average ΔΔC T of the reference group.Primer sequences can be found in Supplemental Table 2.

NanoString nCounter gene expression analysis
To assess how our two-hit model impacts neuroimmune activation in the midbrain, we leveraged the nCounter direct transcript counting platform from NanoString Technologies using the Mouse Neuroin ammation Panel with an additional 55 genes added in that are known to be responsive to NE, affected by colitis, or involved in the immunological crosstalk between brain and periphery.Prior to running the assay, RNA quality was assessed using a Bioanalyzer 2100 (Agilent) with the RNA 6000 Nano Kit (Agilent, #5067 − 1511) according to manufacturer's instructions.We used the 6 samples from each treatment group from both sexes with the highest RINs (48 samples total).From these samples, 100ng of total RNA was input into the nCounter protocol using the MAX/FLEX workstation, following manufacturer's instructions.
Data quality was examined in R (v. 4.2-4.3)using the NACHO package 70 , which revealed two samples to be discarded ahead of downstream analyses (Fig. S1A-B).Inspecting the expression of individual housekeeping genes revealed the gene Asb10 to be only rarely captured (Fig. S1C), so this gene was removed.Data were then normalized using the NanoTube package 71 with the RUVg method from the RUVSeq package 72 , which uses only high-con dence housekeeper probes and removes unwanted technical variance while preserving biological variance.This strategy has been shown to outperform NanoString's commercial normalization strategy, which uses only centrality measures from the housekeeper probes included in the panel 73 .
After normalization, differential expression testing was then performed with DESeq2 74 .Differentially expressed genes (DEGs) were classi ed as genes that showed | log 2 (fold change) | > 0.25 and Benjamini-Hochberg-adjusted p-value < 0.05 for a given comparison.DEGs relative to the H2O + saline control group were identi ed for each of the other treatment groups, collapsed across sex.Then, the lists of DEGs from the DSS + saline group were treated as co-expression modules for which the eigengenes were calculated for each sample using the WGCNA package 75 .

Statistical analysis
All statistical analysis was performed in R v. 4.2-4.3.Tissue monoamine concentration changes due to DSP-4 were analyzed with a Wilcoxon rank sum test as data were not assumed to come from a normal distribution.Daily weight measurements during DSS were analyzed with an ANOVA that included day as a within-subjects variable and DSP-4 as a between-subjects variable.Sphericity of these data were assessed using the Performance package's , which uses Mauchly's test, and the Geisser-Greenhouse correction was applied when appropriate.Colon length, spleen weight, tissue gene expression, and tissue protein concentrations after our two-hit schedule were analyzed using a two-way ANOVA using DSS and DSP-4 as the two between-subjects factors.A three-way ANOVA was conducted for gene expression analyses in brain that included sex as a third between-subjects factor.Post hoc check s phericity testing was done with Tukey's correction using the Emmeans package, and CLDs were generated using the Multcomp package based on the contrasts run by Emmeans.All data visualizations were created using the ggplot2 syntax.

Results
DSP-4 is selective for NE and its action is reversible in the periphery First, we sought to con rm the speci city of DSP-4 for brain NE, as its effect in the gastrointestinal system has not yet been examined.At 7 days post-injection (dpi), NE was depleted signi cantly by about 60% by DSP-4 in the cortex, while DA and 5HT were unaffected (Fig. 1a).However, colonic NE was also depleted by about 25% at 7dpi, while DA and 5HT were again unaffected in the colon (Fig. 1b), suggesting that the DSP-4 effect was not limited to the brain at 7dpi.Although, it has been suggested that the effects of DSP-4 are reversible in the periphery.Indeed, we found that NE concentrations in the colon returned to baseline levels after 28dpi, while cortical NE remained depleted by over 50% during the same timeframe (Fig. 1c).We also found a generalized brain loss of NET + bers (Fig. 1d) and a visible decrease in DBH + cells in the LC (Fig. 1e) by 3D immunolabeling with iDISCO + and uorescence lightsheet microscopy.Meanwhile, colonic NE innervation, visualized by TH+ (Fig. 1f) and NET+ (Fig. 1g) bers in the proximal colon, appeared unaffected by DSP-4.Thus, DSP-4 was selective for NE, and this effect on NE was persistent in the brain.

DSP-4 does not interfere with the induction of colitis with DSS
To further ensure DSP-4's speci city and verify that the colon's response to injury is unaffected by peripheral DSP-4 administration, we pre-treated mice with DSP-4 or saline vehicle and subsequently subjected them to DSS-induced colitis 28dpi.The outcomes of a DSS-induced colitis schedule designed to induce peak colonic injury and in ammation (Fig. 2a) were con rmed not to be affected by DSP-4, as re ected by the lack of differences in weight loss during colitis and no obvious differences in disease severity with or without DSP-4 (Fig. 2b-c).Gross indices of disease, including increases in spleen weight (Fig. 2d) and colon shortening (Fig. 2e) after DSS were also unaffected by DSP-4.Similarly, a colitis schedule designed to induce peak neuroin ammation (Fig. 2f) was not affected by DSP-4, again as re ected by the lack of differences in weight loss (Fig. 2g), disease severity (Fig. 2h), spleen weight (Fig. 2h), or colon length (Fig. 2i) with or without DSP-4.
Next, we examined the extent to which peripheral DSP-4 administered prior to colitis could modify the DSS-induced colitis phenotype.Using the DSS schedule designed to cause peak gut injury (Fig. 2a), we found that ZO-1 protein levels were not affected in any group as measured by immunoblotting (Fig. 3a-b), but the mRNA encoding for ZO-1 was downregulated by DSS treatment without being modi ed by DSP-4 (Fig. 3c).Similarly, the abundance of the tight junction protein occludin was not affected signi cantly by DSS (Fig. 3d-e) but the transcript was signi cantly depleted by DSS without being modi ed by DSP-4 (Fig. 3f).The tight junction protein claudin-5 was signi cantly affected by DSS but not DSP-4 (Fig. 3g-h).In total, gut barrier disintegration phenotypes induced by DSS were not modi ed by prior administration of peripheral DSP-4.
We also examined the extent to which DSP-4 affected colonic in ammation induced in the DSS model.The NLRP3 in ammasome, the assembly that creates mature IL-1B, was induced by DSS in the colon similarly across DSP-4 and vehicle groups (Fig. 3i-j).We observed a signi cant interaction between DSS and DSP-4 in the induction of Tnf transcript expression in the proximal colon (Fig. 3k), but the expression of Cd68 (Fig. 3l), Il6 (Fig. 3m), Il10 (Fig. 3n), Ifng (Fig. 3o), and Lcn2 (Fig. 3p) were all modulated signi cantly by DSS but not DSP-4.Meanwhile, Il1b expression was not affected in any group (Fig. 3q).In sum, peripheral DSP-4 administration prior to colitis did not modify DSS-induced colonic in ammation in the DSS model.Overall, DSP-4's speci city for the central compartment, namely after 28dpi when the colonic NE depletion is reversed, enables one to con dently combine DSP-4 NE denervation with other peripheral in ammatory insults.In this case, DSS-induced colitis phenotypes remain intact despite the prior peripheral DSP-4 administration, permitting unconfounded interpretations of the combinatorial effects of gut in ammation and brain NE depletion on neuroin ammation.

DSP-4 modulates neuroimmune gene expression in the midbrain during DSS-induced colitis
To determine whether LC injury and increased gut permeability interact to affect neuroin ammation in the midbrain, we rst used qPCR to measure the expression of key cytokines and chemokines after a DSSinduced colitis schedule designed to elicit peak neuroin ammation (Fig. 4a).Interestingly, the induction of Il1b expression in the midbrain was achieved only in mice subjected to both injuries, and this appeared to be a male-speci c effect (Fig. 4b).The induction of Ccl2 expression in the midbrain followed a similar pattern, showing an increase only in males treated with both DSS and DSP-4 (Fig. 4c).In contrast, Tnf, Lcn2, and Nfe2l2 were activated by DSS without any signi cant effect of DSP-4, mostly in male mice (Fig. 4d-f).
To extend these ndings, we employed the Mouse Neuroin ammation panel on the NanoString nCounter direct transcript counting platform with their Mouse Neuroin ammation panel with an additional 55 custom genes.Standard differential expression analyses revealed 53 differentially expressed genes (DEGs) in the midbrains of DSS-only mice and 15 DEGs in the midbrains of mice that received both insults, relative to vehicle-treated mice (Fig. 4f-g).Of the upregulated genes in both conditions, none were unique to the two-hit group; DSS-only mice upregulated all the genes upregulated by the two-hit group, including Cdkn1a, Il1r1, Ly6a, Lnc2, and S100a9, as well as unique genes such as C4a, Fcgr3, Il6ra, and Nrros (Fig. 4h).Examining the rst principal component of the genes upregulated by DSS alone revealed that DSS-treated mice expectedly upregulate this module, while mice pre-treated with DSP-4 show a blunted response in males only (Fig. 4i).DSS had a smaller but similarly positive effect on these genes in female midbrains, and DSP-4 did not in uence this upregulation (Fig. 4i).Similarly, of the downregulated genes in both conditions, none were unique to the two-hit group; mice treated with both DSS and DSP-4 downregulated only Hsd11b1, Kcnj10, and Ugt8a, while DSS-only mice downregulated these genes as well as Adrb1, Bdnf, Fcrls, Lrrc3, Shank3, Tnfrsf17, and others (Fig. 4j).The eigengene of this module was expectedly downregulated by DSS alone but not by DSS and DSP-4 together in males, while female mice downregulated this module in both conditions similarly but to a lesser extent than males (Fig. 4k).Taken together, these data suggest that LC injury modulates the midbrain neuroimmune response to intestinal permeability.LC injury activates the expression of speci c cytokines and chemokines in the midbrain, including Il1b and Ccl2, which are normally absent during colitis, and blunts the differential expression of genes normally associated with colitis.

Discussion
In this study, we established and characterized a novel two-hit mouse model that incorporates several common and early features of PD, including gastrointestinal in ammation and LC degeneration.Speci cally, we provide a reevaluation of the DSP-4 neurotoxin model of LC injury, con rming its speci city for NE as expected based on previous studies [76][77][78][79] .We also show gastrointestinal NE depletion via DSP-4 is reversible after 4 weeks, a rming early reports of transient sympathetic denervation with DSP-4 77,79,80 .Importantly for our study, the peripheral reversibility of DSP-4 meant that the induction of colitis via DSS and the colonic features of DSS-induced colitis were left intact.This feature of our two-hit model is critical in the downstream interpretations of any effects observed on ventral midbrain outcomes.Several previous studies have employed pharmacological or genetic tools, such as anti-TNF therapies, paquinimod, avonoids, and targeted LRRK2 mutations 15,19,20,81 , to modulate the brain effect of the colitis.In the DSS model, our group has previously demonstrated that colitis severity is the best predictor of neuroin ammation 14 , and modifying the severity of the disease modi es the neuroin ammatory phenotype 82 .Those efforts, while still critical for the eld, affect the induction of colitis itself, making it di cult to determine whether neuroin ammation itself is modi ed or whether the systemic insult that triggers this neuroin ammation is modi ed.Here, we show that a brain-restricted phenotype, LC injury, affects the neuroin ammatory response in the midbrain induced by a leaky gut.
We observed that Il1b expression is signi cantly activated in the ventral midbrain of mice that received both LC injury and colitis but not in mice that received either insult alone.Previous studies support an interaction between NE and IL-1β during in ammatory challenges.In vitro studies have demonstrated that lipopolysaccharide (LPS) induces IL-1β production in microglia, which can be blocked with the coapplication of NE 56 .NE may activate suppressors of IL-1β signaling, including IL-1ra and IL-1RII 83 , or suppress the expression of Il1b via the suppression of the nuclear action of NF-κB 84 , which may downregulate interferon regulatory factor 1 (IRF1) 85 , a transcription factor that activates Il1b.IL-1β overproduction is thought to be a driver of PD, as IL-1β induction has been shown to worsen motor dysfunction and alpha-synuclein pathology in animal models of PD-like pathology 86 , and circulating IL-1β has been associated with worsened PD severity 87,88 .As such, the observed activation of Il1b expression in the ventral midbrain of mice treated with DSS only in the presence of an injured LC suggests potentially damaging consequences for the nigrostriatal pathway.
Similarly, we found selective activation of Ccl2 expression in the ventral midbrain of mice that received both DSS and DSP-4.CCL2 is a major chemoattractant implicated in the in ltration of myeloid cells, especially monocytes and neutrophils, into the brain during in ammation 89,90 .A relationship between NE and CCL2 has not been extensively examined in microglia, but a recent report demonstrated that NE reduces CCL2 production in mouse and human macrophages in a β-adrenergic receptor-dependent manner 91 .Additionally, recent work in astrocytes has revealed a complicated relationship between NE and CCL2 that may provide insight for the data in this study and others.At rest, NE induces CCL2 production in astrocytes, likely through β-adrenergic receptors 92,93 , while clonidine, an α2-adrenergic receptor agonist, reduces CCL2 production in these cells 94 .Further, endotoxin-exposed astrocytes downregulated CCL2 with NE 95 , suggesting that the immunoregulatory effect of NE depends on the neuroin ammatory milieu.In all, our data are in line with this notion; the expression of Ccl2 was induced by NE depletion via DSP-4 only during peak colitis.This effect raises the interesting prospect of peripheral immune cell invasion into the brain, which future studies will address.The in ux of CCR2 + peripheral phagocytes has been reported in PD and mouse models of PD 24,96 , suggesting that the induction of their ligand CCL2 is a relevant event in our model, and dysregulation of the crosstalk between brain and peripheral immune system is a potential consequence of NE depletion.
We found that the differential regulation of several genes during DSS-induced colitis was dampened by LC injury.At rst glance, this may seem to suggest that LC degeneration is neuroprotective during colitis, but closer inspection of their identity and their associated pathways reveals that upregulation of several ventral midbrain genes (Ms4a4a, Fcgr2, Myc, Lcn2, Osmr, Nqo1, and Nrros) is likely to represent a protective neuroin ammatory and antioxidant response.For example, the rs1582763 variant in the MS4A locus increases MS4A4A expression 97 , whose mouse ortholog Ms4a4a was upregulated by DSS but not DSS and DSP-4 together.This MS4A variant was recently shown to be associated with reduced risk of Alzheimer's disease 98 by promoting healthy lipid metabolism and reducing chemokine signaling in microglia 97 .FcγRIIB is the only inhibitory Fc receptor, known for controlling many immune processes and regulating a healthy defense against infection 99 , and the murine gene encoding for this Fc receptor Fcgr2 was upregulated by DSS-induced colitis alone but not when the LC was injured in mice treated with both DSS and DSP-4.Similarly, MYC, encoded by Myc which was upregulated by DSS alone in our study, has been shown to control in ammatory activity in myeloid cells 100 .Lipocalin-2, encoded by Lcn2, and oncostatin M signaling through its receptor, encoded by Osmr, which are both upregulated during DSS irrespective of DSP-4 treatment, are thought to be highly neuroprotective during in ammation [101][102][103][104] .Finally, DSS-only mice upregulated Nqo1 and Nrros, genes encoding for critical regulators of antioxidant responses [105][106][107] , while mice given both DSS and DSP-4 did not.Together, we conclude that many of the DSS-associated genes identi ed here are protective against in ammatory and oxidative stress, and the DSP-4-associated ablation of their activation would be expected to be damaging to the nigrostriatal pathway.
At the same time, some genes regulated by DSS treatment may be injurious components of the neuroimmune response to colitis.Genes like Il1r1, Cdkn1a, and S100a9 were upregulated by DSS in both the presence and absence of DSP-4.S100A9 activates NF-κB signaling and has been shown to aggravate neuroin ammation in a model of subarachnoid hemorrhage 108 and Alzheimer's disease 109 and has been implicated as an integral component of in ammation along the gut-brain axis in the DSS model 15 .Cyclindependent kinase inhibitor p21, encoded by Cdkn1a, appears to be an in ammatory response element and may be a marker of DNA damage 110,111 , and an upregulation of the IL-1 receptor type 1, encoded by Il1r1, has been associated with pathogenesis in several neurodegenerative diseases 112 .In general, the colitis-induced neuroimmune response is complex, and further work will be needed to unravel this complexity especially amidst LC injury.
We acknowledge that a notable limitation of our study is our inability to determine whether the detectable regulation of neuroimmune gene expression in the ventral midbrain actually compromise dopaminergic neuron survival.To address this, future studies will employ a chronic dosing scheme of DSS to induce colitis after LC injury, that has been used previously to induce neurodegeneration 19,20,113,114 .Another step in this line of investigation might be to investigate in vitro the regulation of genes identi ed by our in vivo study, using NE or adrenergic receptor drugs amidst LPS treatment of microglia or astrocytes, as done previously 56,94 , and/or by mining the ever-growing body of high-throughput sequencing studies of neurodegenerative diseases and their respective mouse models.However, based on the existing literature demonstrating that an LC lesion worsens other models of neuroin ammation and degeneration 58,60,61,63- 66 , we predict that the dampened ventral midbrain neuroin ammatory response to DSS-induced colitis we report here due to LC injury will have a detrimental outcome for the nigrostriatal pathway, which has been shown by numerous studies to be selectively vulnerable to the effects of in ammatory and oxidative stress 24,[115][116][117] .
Another limitation of our study is that we cannot discern whether the changes in neuroimmune gene expression observed here are due to the depletion of NE or another transmitter from the LC.To elucidate the role of NE, future studies would combine our two-hit model with pharmacological drugs for adrenergic receptors or NE reuptake and examine the effects on neuroin ammation.This strategy has been employed in several mouse studies previously; two NE reuptake inhibitors, such as desipramine and atomoxetine, and β2-adrenergic receptor agonists, such as clenbuterol, have lessened neuroin ammation after immune insults and alpha-synuclein overexpression 118-120 , while β-adrenergic receptor blockers, like metoprolol and propranolol, have exacerbated aging-and proteinopathy-related neuroin ammation and subsequent cognitive dysfunction 54,121 .Based on these data, we expect that the loss of NE mediates most of the in ammatory phenotype we observe in our two-hit model, however the anti-in ammatory effects of brain-derived neurotrophic factor 122 and galanin 123 , some of the LC's major co-transmitters, are also worth further consideration.
With regards to the relevance of our ndings within the context of therapeutic interventions, identifying the molecules mediating the in ammatory predisposition that comes with LC injury may inform therapeutic strategies for PD.NE medication is routinely used in the treatment of PD.For example, propranolol is used to treat tremor 124 and control levodopa-induced dyskinesia in PD 125 , desipramine and other norepinephrine reuptake inhibitors are used to treat depression and anxiety in PD [126][127][128][129] , and the NE reuptake inhibitor atomoxetine is being examined as a therapy for cognitive decline in PD 130,131 .Given the role of NE signaling in brain health and immunity, the effect of these drugs on neuroin ammation must be considered.In this vein, a recent phase II trial examining the e cacy of atomoxetine in protecting cognition in Alzheimer's disease demonstrated that NE reuptake inhibitor treatment reduced phosphorylated tau and CD244, a putative marker for cytotoxic T-cells and natural killer cells, in the cerebrospinal uid 132 .Including readouts of systemic and central in ammation in further work studying these therapeutics in PD may build further evidence that NE signaling is protective against neuroin ammation-associated neurodegeneration.
In summary, we have developed a two-hit mouse model that models common and disease-modifying features of early PD.With this model, we have observed that DSS-induced colitis triggers what is likely a compensatory neuroimmune gene program in the ventral midbrain, while activating very little expression of pro-in ammatory cytokines and chemokines.When the LC is injured, these programs are reversed and potentially protective immune and antioxidant responses are dampened.Overall, this study highlights the LC as a regulator of neuroin ammation during colonic permeability and in ammation.This study, alongside the mounting evidence positing the LC as neuroprotective, may inform future therapeutic strategies involving NE drugs to prevent or delay PD progression and draw focus to LC integrity as a predictor of disease progression. Declarations