United States

Abstract Title: Insights into necessary function of interlukin-1 in regulating neuroinflammatory gene expression following a closed-head traumatic brain injury in mice.

Authors: Jonathan C. Vincent1,2,3, Colleen N. Bodnar1,2, Adam D. Bachstetter1,2

Affiliations: 1Department of Neuroscience, University of Kentucky College of Medicine, Lexington, KY, United States, 2Spinal Cord and Brain Injury Research Center, University of Kentucky College of Medicine, Lexington, KY, United States, and 3MD/PhD Program, University of Kentucky College of Medicine, Lexington, KY, United States

Neuroinflammation is a major mechanism contributing to secondary injury cascades following a traumatic brain injury (TBI). Interleukin-1 (IL-1), often called a master cytokine, is elevated in the brain after a TBI, and likely drives a significant amount of the inflammatory response to the TBI. However, it is often assumed that significant redundancies in the cytokine communication network can overcome the loss of a single cytokine. In contrast, we hypothesize that specificity in the neuroinflammatory responses depends on IL-1 signaling. Using a diffuse mouse model of mild traumatic brain injury (mTBI) caused by a closed-head injury (CHI), we tested our hypothesis using a global genetic knockout of the receptor for IL-1 (IL-1R1). The neuroinflammatory response to a TBI is not stable over time but occurs in activation waves and then resolution. Therefore, we also evaluated IL-1R1s role in regulating the pattern of the inflammatory response. The study used wild-type (WT) male mice (n=56) and IL-1 Receptor 1 (IL-1R1) global knock-out (gKO) male mice (n=51) subjected to either a midline CHI (WT n=29, gKO n=29) or sham procedure (WT n=27, gKO n=22). Mice were sacrificed at 3 hrs, 9 hrs, 24 hrs, and 72 hrs post-injury. RNA was extracted from the neocortex, most proximal to the area of injury. Gene expression changes were measured using the Nanostring neuroinflammatory panel. Loss of IL-1R1 signaling blunted the inflammatory response to injury at the early (3 hrs, 9hrs) post-injury time point compared to the WT mice. However, the temporal pattern of gene expression changes was also altered in the IL-1R1 gKO mice with some genes showing a delayed upregulation compared to the WT mice. Out of 757 genes analyzed, 201 genes were found significant at the 3 hr time-point, 367 genes were found significant at the 9 hr time-point, 195 genes were found significant at the 24 hr time-point, and 114 genes were found significant at the 72 hr time-point. IL-1R1 was found to have specificity in regulating aspects of the neuroinflammatory cascade, including chemokine upregulation, while having limited effects on cytokines, such as TNFalpha. This study provides further insight into the necessary function of IL-1R1 in amplifying the neuroinflammatory cascade. 




2022 AMA Research Challenge

Neurology - Insights into IL-1 effect in regulating neuroinflammatory gene expression following a closed-head traumatic brain injury in mice - Neurology

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Background 

Neurodegenerative diseases are the leading source of disability in the world. Parkinson’s Disease (PD) is a progressive disease with motor and non-motor symptoms. The loss of dopaminergic neurons in the substantia nigra and Lewy Bodies composed mostly of alpha-synuclein (α-Syn) aggregates are hallmarks of PD. Many cellular models are used to study α-Syn pathologies, SH-SY5Y human neuroblastoma cells are one of the most widely used. SH-SY5Y cells can differentiate into neurons and offer the advantage of studying impaired dopamine metabolism and other pathologies. This cell line is extremely sensitive, and proliferation tends to be slow. Typical culture involves DMEM F12 + 10% FBS. NuSerum (NuS) is a low-protein alternative serum for newborn calf and fetal bovine sera. With this study, we hypothesize that culturing cells with NuSerum will aid and promote SH-SY5Y cell proliferation and differentiation when compared to traditional culture methods. This study’s relevance exists in the quality of cell culture and, consequently, the quality of research. 

Methods

SH-SY5Y cells were previously cultured in DMEM F12 +10% FBS or DMEM F12 + 10% NuS media. Next, cells were divided into three groups and cultured with serum-free (SF) DMEM F12 media, 10% FBS-containing DMEM F12 and 10% NuS-containing DMEM F12. Cell counting was evaluated on days 2, 4, and 6 after the cells were plated. Cell proliferation rate between the groups was assessed using WST-1 reagent on days 1 through 4 after the cells were plated. Cells were observed with a brightfield microscope each day and imaged on days 2, 4, and 6. 

Results

Cell counting results consistently revealed higher cell numbers and viability in the NuS-treated group, followed by FBS-treated group, and lastly, the SF-treated group. Cells were seen to develop faster in the NuS-treated group morphologically. 

Conclusion

Our preliminary data points to NuSerum as a suitable serum alternative in SH-SY5Y cell culture. NuSerum offers an advantage in proliferation, viability, and morphology for the cell line. Adjustments to cell culture protocols for this cell line can yield better overall cell health and positively influence the quality of research using this cell line, especially in the neuroscience field.


Neurology - NuSerum: An alternative supplement for SH-SY5Y Cell Culture - Neurology

Background
Much has been alluded to previously about the systemic effects that contribute towards the development of stroke, however little is known about how acute ischemic stroke can elicit a systemic response. Research has been done on the brain-immune system axis post-stroke, demonstrating an increase in post-stroke deaths through immunosuppression. To address the gap in systemic responses to stroke we aim to demonstrate: 1) Acute brain disorders induce systemic responses in peripheral proteomes; 2) Brain disorder-induced peripheral proteomic changes are specific to injury conditions. 

Methods
Transient focal cerebral ischemia was modeled in mice by occlusion of the middle cerebral artery (MCAO) for 30 min MCAO or 60 min MCAO, with Sham-operated animals as controls (n=5, 5, and 4, respectively); which was followed by reperfusion. The heart, liver, kidney, and spleen were collected, processed, and analyzed by quantitative mass spectrometry with triplicate replications. Proteins that were downregulated and upregulated in both the 30 and 60 minutes MCAO were analyzed through bioinformatics in Metacore and DAVID for each respective organ. 

Results
Results of bioinformatic analyses indicated variable regulation of proteins related to the immune system in each organ. Proteins associated with chemotaxis were upregulated for both the kidney and liver but downregulated for the spleen. T cell processes were downregulated for the kidney and liver. Antigen processing was downregulation for both the liver and spleen but upregulated in the heart. For neuro-immune pathways, proteins associated with the LRRK2 receptors were upregulated for all four organs.

Conclusion
Our results demonstrate a net immunosuppressive state in both the liver and the spleen after a 30- or 60-minute MCAO. Variable immunosuppression was seen in the kidney and the heart. The global upregulation of LRRK2 receptors demonstrates an increase in neuroinflammation in the systemic response after a transient focal cerebral ischemic event. 


Neurology - Proteomic Analysis of Immune Response After Transient Focal Cerebral Ischemia in Peripheral Organs - Neurology

Background
In 2015, 3.4 million people in the US had active epilepsy. Despite numerous pharmacologic treatments available, only 44% report seizure control consistent with reported rates of drug-resistance. This highlights the need for better defined pathophysiology and mechanism-targeted therapy. Among the numerous implicated mechanisms, mutations in NMDA receptors have been causally implicated in pathogenesis. NMDA receptors are glutamate-gated Ca2+-permeable ion channels with essential roles in the physiology of central excitatory synapses and their dysfunc-tion cause severe neuropsychiatric symptoms. More than 4000 genetic variants have been identi-fied across the seven genes encoding NMDA receptor subunits, however, functional information about these variants is sparse and their role in pathogenesis insufficiently known. Here we reveal the structural mechanism by which two GluN2A variants may be pathogenic. 
Methods
We use all-atom targeted molecular dynamics simulations to build a structural model of channel opening as no structural data yet exists for these channels in the open conformation. This allowed us to screen and identify residues comprising critical allosteric interfaces that strengthen during activation. We identify a conserved structural element that single-molecule electrophysiology to examine the contribution of GluN2A subunit-residues, P552 and F652, and their pathogenic sub-stitutions, P552R and F652V, affect receptor functions.
Results
We found that P552 and F652 interact during the receptors’ normal activity cycle; the interaction stabilizes receptors in open conformations and is required for a normal electrical response. Engi-neering shorter side-chains at these positions (P552A and/or F652V) caused a loss of interaction energy and produced receptors with severe gating, conductance, and permeability deficits. In contrast, the P552R sidechain resulted in stronger interaction and produced a distinct, yet still drastically abnormal electrical response.
Conclusion
These results identify the dynamic contact between P552 and F652 as a critical step in the NMDA receptor activation, and show that both increased and reduced communication through this interaction cause dysfunction. Results show that subtle differences in NMDA receptor pri-mary structure can generate complex phenotypic alterations whose binary classification is too simplistic to serve as a therapeutic guide.

Neurology - Rethinking NMDA Receptor Disease Variant Classification in Epilepsy - Neurology

Previous studies demonstrated that increasing a protein’s thermostability may increase its resistance to protease digestion. Given recent efforts at evolutionary redesign of lysosomal enzymes to treat lysosomal storage diseases, we sought to identify orthologs of tripeptidyl-peptidase 1 (TPP1) with enhanced thermostability for potential use in improving the existing enzyme replacement therapy for late-infantile neuronal ceroid lipofuscinosis (LINCL), a neurodegenerative disease. TPP1 orthologs from Rattus norvegicus, Heterocephalus glaber, Danio rerio, and Gallus gallus domesticus exhibited increased thermal half-life at 60oC, suggesting that these proteins may confer increased in vivo stability via evolutionary redesign of human TPP1. Structure-function analysis is required to identify residues for mutation in human TPP1 to confer this enhanced stability.

Neurology - Thermostability of TPP1 Animal Orthologs: Potential for Drug Development via Evolutionary Redesign - Neurology

Background: Ovarian Cancer (OC) is the leading cause of death from gynecologic malignancies in the United States largely due to the advanced stage at the time of diagnosis. The identification of metastatic triggers is the significant and unmet need in OC therapy. The epithelial-to-mesenchymal transition (EMT), a biological process where epithelial cells lose their adhesive properties and gain invasive, metastatic, or mesenchymal properties, has been well-described in OC. Discovering the “trigger/s,” its downstream targets, and therapeutic targeting are essential to substantively improve the survival of women with OC. The objective of our study is to evaluate the role of Connective Tissue Growth Factor (CTGF), a member of a family of extracellular matrix (ECM) associated heparin-binding proteins in EMT in OC. 

Methods: R182 and R2615 are well-described epithelial OC cell and MR182 and MR2615 are the mesenchymal counterparts. R182/R2615 CTGF knock outs (KO) were derived utilizing a Cas9/CRISPR-Cas9 lentivirus plasmid vector and verified by indel sequencing. Invasion, anoikis resistance, and chemosensitivity assays were performed in wild-type (WT) and KO cells. RNA sequence analysis was performed and analyzed using iPathway guide. Top five upregulated and downregulated genes involved in ECM organization pathway were validated by quantitative PCR (qPCR). Immunofluorescence was performed for F-actin. 

Results: CTGF was expressed in the epithelial and not in the mesenchymal OC cell lines. Loss of CTGF was associated with anoikis resistance, where KO and WT cells displayed 75% and 10% viability, respectively. KO cells were significantly more invasive than WT cells. Administration of exogeneous CTGF in KO cells decreased invasion in a dose dependent manner. No change was seen in chemosensitivity to Cisplatin in KO cells. RNA seq analysis identified ECM organization as the biologic process most affected by loss of CTGF. Upregulated (FREM2, LAMC2, ITGB4) and downregulated (SPP1, SV2A, RELN, COL6A3, COL4A6) genes were validated by qPCR. Immunofluorescence staining demonstrated increased F-actin expression in KO cells.

Conclusion: Our data suggests that CTGF expression maintains the epithelial phenotype in OC and that loss of CTGF may be one of the early triggers of EMT in OC. Loss of CTGF should be further evaluated as a poor prognostic marker in OC.


Loss of Connective Tissue Growth Factor Expression Promotes Remodeling of the Extracellular Matrix and Epithelial-to-Mesenchymal Transition in Ovarian Cancer

Background: The Renin Angiotensin System has a protective axis involving Angiotensin Converting Enzyme 2 (ACE2) and its product, Ang-1-7. ACE2 has received attention as a host cell receptor for the SARS-CoV-2 virus, but it also has a protective role regulating inflammatory pathways in tissues including the retina. This review summarizes what the current literature determines the role of ACE2 to be in the retina, and how this information can be used to develop therapies for treating retinopathies. 

Methods: Medical Subject Headings (MeSH) inquiry in the Medline/PubMed databases was used to search for papers. The Mesh topics used are “Angiotensin Converting Enzyme 2,” “ACE2 protein,” “angiotensin I (1-7),” “Retina”, “Retinal Pigment Epithelium,” and “Macula.” This review includes animal studies, postmortem studies, and studies using primary human retinal pigment epithelium cell cultures (hRPE) and human induced pluripotent stem-cell derived retinal organoids.

Results: The literature indicated that ACE2 is expressed in retinal ganglion cells extending to photoreceptors and the Retinal Pigment Epithelium (RPE) layer, indicating that it spans the entire retina. It has been shown that ACE2 co-localizes with Müller cells, which also stretch across the thickness of the retina. Additionally, the pathways influenced by ACE2 have been investigated. Animal and human studies found that ACE2 attenuates inflammation by regulating MAPK and NF-kB pathways. Human studies additionally found the STAT3 pathway to be another way ACE2 prevents inflammation. ACE2/Ang-1-7’s protective properties have been tested and it was found that it can reduce retinal damage caused by diabetes. It has been suggested to explore ACE2/Ang-1-7 as possible therapy for various retinopathies. 

Conclusion: ACE2 and its product, Ang-1-7, have been shown to have anti-inflammatory effects on the retina. This can be used in the treatment of pathologies that affect the retina, including diabetic retinopathy. Ang-1-7 has a short half-life which may be why it has not been used as a therapeutic intervention. The challenge is to find a way to deliver Ang-1-7 effectively, or to induce endogenous ACE2 to produce more Ang-1-7. Overall, the ACE2/Ang-1-7 protective axis of RAS is involved in the entire retina, affects various anti-inflammatory pathways, and can be used in therapeutic interventions in managing retinopathies. 


A Review of Angiotensin Converting Enzyme 2 and the Retina

Purpose: Glaucoma is a group of optic neuropathies characterized by retinal ganglion cell (RGC) death and visual field loss. A degenerative mechanism associated with RGC death is disrupted delivery of neurotropic factors due to glaucomatous axonal damage. This transport is critical for protection of long-term neuronal function. Thus, we investigate a potential therapeutic target, human Neuritin 1 (NRN1), which has demonstrated neuroprotective effects in rodent axonal injury models. Further, the advent of induced pluripotent stem cell (iPSC) technology allows iPSC-RGCs to be generated in-vitro from commercial iPSCs and reprogrammed corneal fibroblasts. We hypothesize delivery of NRN1 and transplantation of iPSC-RGCs will sustain survival of RGCs and ultimately slow progression of glaucoma-induced neuronal death in our ocular translaminar autonomous system (TAS) perfusion model system. This will allow us to analyze a potential therapeutic approach for both early-stage glaucoma (NRN1 therapy) to protect dying RGCs, and late-stage (iPSC-RGCs), when most RGCs are lost. 

Methods: Human donor eyes were obtained from eye banks according to Declaration of Helsinki. The iPSC-RGCs transfected with AAV2-GFP were seeded into human posterior cups with NRN1 and cultured in TAS model under pressurized conditions for 2 days. Survival of iPSC-RGCs, gliotic and fibrotic pathways were measured through expression by qRT-PCR and immunohistochemistry. Retinal function post-treatment was measured through electroretinogram analysis. 

Results: We successfully maintained the human posterior eye cups in translaminar differentials for 2 days. In contrast to controls, we observed increased RGC survival and retinal function after combination therapy of NRN1 and iPSC-RGC. Additionally, we found differential gene expression of apoptosis, inflammation, and gliotic markers.

Conclusion: Our study identified that NRN1 in conjunction with iPSC-RGC transplantation treatment promotes RGC survival under glaucomatous conditions. This suggests that NRN1 and iPSC-RGCs could be utilized as a potential combination therapy to save retinal neurons and prevent neurodegeneration in glaucoma patients. 


Combinatorial Therapeutic Strategy of Stem Cell Retinal Organoids and Neurotrophic Factor for Glaucoma

Background
Diabetic retinopathy (DR) is one of the leading complications of diabetes mellitus (DM) and is a progressive neovascular disease that causes pathological changes in neuronal, vascular, and immune systems. Inflammation and inflammasome activation have been associated with multiple neurovascular conditions and are implicated in the pathogenesis of diabetic retinopathy. NLRP3 is a well-studied inflammasome made up of the constituents NLRP3, Asc, and Caspase-1. This inflammasome has been demonstrated to be activated by a multitude of danger signals including elevated glucose and free fatty acids, which are present in diabetes mellitus. However, there remains uncertainty regarding the role of the NLRP3 inflammasome in DR development.

Methods
Diabetes was induced using the streptozotocin (STZ) model in C57BL/6J wild type (WT) male mice (N=40) and knock out (KO) male mice lacking certain NLRP3 inflammasome constituents (Caspase-1 KO (N=7) and ASC KO (N=9)). Vehicle control was injected for control (CTL) mice of each genotype. After one and three months of hyperglycemia, retinal neurodegeneration was evaluated using scotopic electroretinography (ERG) and bilateral eyes were harvested for immunoblotting to evaluate the neuroretina for inflammasome activity. 

Results
Immunoblotting of whole retinal lysates showed minimal increase of inflammasome activation in WT DM mice compared to WT CTL mice. However, on ERG evaluation at three months, there was a 23.1% decrease in a-wave and a 32.3% decrease in b-wave between DM and CTL WT mice. For both strains of KO mice, there was a lesser decrease of 10.8% and 21.8% in a- and b-waves respectively (Caspase-1 KO), and of 5% and 18.3% in a- and b-waves respectively (ASC KO) between DM and CTL mice.

Conclusion
Our results suggest that while NLRP3 inflammasome activation may not be evident on protein analysis, the decrease in readings on ERG evaluation may be indicative of a reduction in neuroretinal function by three months of DR. Additionally, the reduced loss of electrical function in both Caspase-1 KO and ASC KO mice may represent a partial rescue of neuroretinal function when components of the NLRP3 inflammasome are absent. This finding helps to establish a direct connection between NLRP3 inflammasome constituents to the pathological processes of DR. 


NLRP3 inflammasome activation in diabetic retinopathy affects neuroretinal function.

Background
Sig1R is a pluripotent modulator of cell survival, the activation of which by the high-affinity, high-specificity ligand pentazocine ((+)-PTZ), has marked retinal neuroprotective effects in the RhoP23H/+ model of adRP compared to non-treated mutants. These findings were obtained in mice administered (+)-PTZ beginning at postnatal day 14 (P14); whether prenatal administration of (+)-PTZ would afford retinal protection was not known and was investigated in the current study. 

Methods
RhoP23H/+ mice were administered (+)-PTZ 3X/week beginning at either (a) P14 (“post”-RhoP23H/+) or (b) RhoP23H/+ mouse dams received (+)-PTZ during adolescence, throughout pregnancy, and pups were treated beginning at P14 (“pre”-RhoP23H/+). Pups from both groups were administered (+)-PTZ through P180 at which time rod and cone photoreceptor function was evaluated by scotopic and photopic electroretinography (ERG), visual acuity by OptoMotry (OMR), in vivo retinal architecture in dorsal and ventral retina by optical coherence tomography (OCT). Retinal structure was examined post-mortem by light microscopy; the thickness of the outer nuclear layer (ONL) of rod/cone nuclei was quantified. Data were analyzed by 2-way ANOVA.

Results
ERG: Scotopic and photopic highest flash intensity b-wave amplitudes were significantly impaired in pre-RhoP23H/+ mice (123.9±13.7μV; 44.3±5.5μV, respectively) at P180 compared to age-matched post-RhoP23H/+ mice (203.4±12.4μV; 70.9±4.5μV) p <0.0001 and 0.0003, respectively. OMR: Acuity in pre-RhoP23H/+ mice was significantly reduced (0.344±0.013 c/d) compared to post-RhoP23H/+ (0.387±0.003c/d) p <0.0001. OCT: ONL thickness was significantly greater in pre-RhoP23H/+ (15.56±0.29μm) versus post-RhoP23H/+(14.3±0.22μm) along the dorsal retina (p = 0.0155). Microscopy: ONL thickness along the dorsal retina in pre-RhoP23H/+ was significantly less than in post-RhoP23H/+ (24.37±3.19 v. 35.15±1.52μm) (p <0.0001).

Conclusion
We anticipated that prenatal plus postnatal treatment with Sig1R would yield a more beneficial response than postnatal treatment alone. However, in our study design, mice in the prenatal Sig1R activation cohort had diminished ERG responses, reduced visual acuity and decreased ONL thickness by histologic examination, although OCT revealed thicker ONL in the dorsal retina. This pilot study raises compelling questions about pre- versus post-natal Sig1R activation and its effects on retinal development. Sig1R is gaining attention in the field of neurodegenerative diseases requiring careful analysis of optimal administration of its ligands.

Prenatal activation of Sigma 1 Receptor (Sig1R) yields intriguing consequences on photoreceptor (PRC) function in a murine model of autosomal dominant Retinitis Pigmentosa (adRP)

Background
Diabetic retinopathy (DR) is a serious diabetes complication that affects millions worldwide. Current therapies are invasive and only slow late-stage progression, thus the development of less invasive, novel therapies that attenuate progression are paramount. In the diabetic milieu, inducible nitric oxide synthase (iNOS) expression is upregulated in retinal macrophages/microglia, which increases production of inflammatory cytokines and reactive oxygen species, worsening ischemia, blood-retinal-barrier (BRB) compromise, and disease progression. Arginase 1 (Arg1) is a ureohydrolase that competes with iNOS for their common substrate, L-arginine. Recent studies revealed a neuroprotective role of Arg1 in murine models of retinal ischemia/reperfusion injury and ischemic stroke and demonstrated the beneficial effects of Arg1 administration in these models. As DR also involves ischemia and inflammation, we hypothesized that exogenous Arg1 would be similarly beneficial through iNOS substrate depletion, resulting in decreased retinal inflammation and oxidative stress.

Methods
Using the obese, type 2 diabetic db/db mouse model that well-approximates human DR pathophysiology, the visual function, BRB, retinal inflammation, and oxidative stress were characterized in these mice aged 16-24-weeks and compared to lean, littermate controls. Visual function was determined by electroretinography (ERG) responses, visual acuity, and contrast threshold measurements. Retinas were collected for immunofluorescence or western blot analyses for iNOS, inflammatory cytokines (IL-1β, TNF-α), oxidative stress (3-nitrotyrosine, 4-hydroxynonenal), and BRB assessment, determined by both retinal albumin extravasation and co-labeling for ZO-1 (tight junction protein) and CD31 (endothelial cell-specific protein). To assess Arg1 therapeutic potential, recombinant Arg1 conjugated to polyethylene glycol (PEG-Arg1), for improved half-life, or a PEG-5000 vehicle control was administered intraperitoneally (25mg/kg) to 16-24-week-old db/db mice thrice weekly for 2 weeks and the same experiments were performed.

Results
Compared to lean littermate controls, db/db mice exhibited significant visual dysfunction, BRB compromise, and elevated retinal levels of iNOS, IL-1β, TNF-α, 3-nitrotyrosine, and 4-hydroxynonenal. PEG-Arg1 treatment significantly ameliorated visual function impairment and BRB compromise in these mice and markedly reduced iNOS levels, retinal inflammation, and oxidative stress compared to vehicle-treated db/db mice. 

Conclusion
The retinal pathology of db/db mice significantly improved with PEG-Arg1 therapy. Though further investigation is necessary, these results suggest that systemic PEG-Arg1 administration has potential as a novel DR therapeutic.

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Neurology - Insights into IL-1 effect in regulating neuroinflammatory gene expression following a closed-head traumatic brain injury in mice - Neurology

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