United States

Background: 
Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neuromuscular disease with no current cure. Disease biomarkers are similarly lacking despite critical importance to accurately diagnose pre-clinical disease, stratify disease subtypes, and improve clinical trial design. Dysfunction of TDP-43, a DNA/RNA binding protein involved in RNA processing, is a hallmark of ALS pathophysiology. One result of disrupted functioning of TDP-43 in ALS is cryptic exons, alternatively spliced RNA variants that change protein structure and function. In vitro, two genes, POLDIP3 and HP1BP3, have increased levels of alternatively spliced RNA variants when TDP-43 is silenced in non-ALS derived cell lines. In this study, we aimed to characterize alternative splicing of these genes in ALS patient derived lymphoblasts as well as plasma samples to determine if these targets may be viable biomarker candidates. 

Methods: 
Lymphoblast lines derived from ALS patients and neurological controls were cultured and harvested for immunocytochemistry, protein, and RNA analyses. Levels and localization of TDP-43 were evaluated for each line by immunostaining. Western blotting was used to probe for levels and variants of TDP-43 and POLDIP-3. RNA transcript levels of POLDIP-3 and HP1BP3 were assessed via quantitative RT-PCR in these lymphoblast samples and in plasma samples from ALS patients and neurological controls.

Results
Immunostaining, Western blotting, and qRT-PCR analysis revealed slight TDP-43 related effects when comparing ALS cases to controls. Levels of the normal and alternatively spliced POLDIP-3 RNA transcripts and proteins were not significantly different between groups. In human plasma samples, RNA transcript levels were detectable, albeit at low concentrations.

Conclusion
This study demonstrates that TDP-43 mediated exon splicing is detectable in human derived blood cells (lymphoblasts). Overall RNA transcript levels are low in plasma samples suggesting that alternative methods of enriching for cells with TDP-43 dysregulation from patient biosamples may be required. Our study provides proof-of-concept that detecting alternative splicing in lymphoblasts is possible, and we plan to carry out additional studies to evaluate splice variants as potential biomarkers for TDP-43 pathology in ALS using more sensitive methods.



AMA Research Challenge 2022

Neurology - Alternative Splicing as a Potential Biomarker for TDP-43 Pathology in Amyotrophic Lateral Sclerosis - Neurology

This annual event showcases and celebrates the neuroscience research in Delaware.  Posters and talks highlight neuroscience research carried out by graduate students, undergraduate students, and research staff at all of Delaware’s research and education institutions. The Lounge section of the symposium allows real-time interactions and networking among attendees.  New this year is an expo of research core facilities in Delaware.  Check them out in the Research Cores section of our poster session, or see the logos below.

Delaware Neuroscience Symposium 2021

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Welcome to the largest national, multi-specialty research event featuring research from 750 medical students, residents and fellows, and international medical graduates.


Abstract Title:
Effects of Hypoxia on Cognitive Function and Anxiety Levels in Rats
Background
Hypoxia is defined as a deficiency in amount of oxygen reaching the tissues. Effects of hypoxia can be
seen at high altitudes, due to the low barometric pressure. Airmen and Guardians participate in military
operations at high altitudes. If low atmospheric oxygen at these high altitudes leads to a reduction in both
physical and cognitive performances, it could jeopardize operation and safety critical tasks, decreasing the
effectiveness of our military. Therefore, the military would greatly benefit from assessing the effects of
hypoxia on behavior as well as developing targeted therapies that would aim to accelerate adaptation to
hypoxic environments. These techniques can eventually be applied to human trials to focus on improving
learning, memory and attention in Airmen and Guardians at high altitudes.
Methods
Rats will undergo two tests to assess cognitive function when exposed to acute hypoxia conditions:
Passive Avoidance Task (PAT) and Novel Object Recognition (NOR). NOR and PAT habituation will
occur after the acclimation period, followed by Training 24hrs later, and testing 24hrs after that. During
test day, acute hypoxia exposure occurs by the rats being placed in a closed environment set to 8%
oxygen for 4 hours total. Elevated Zero Maze (EZM) is a test used to examine a rat’s level of anxiety or
stress. Rats are placed in an elevated circular platform with closed arms and open arms. More time spent
in the closed arms versus the open arms as well as decreased locomotion is associated with increased
anxiety.
Results
No statistically significant difference is seen on the three behavioral tests. However, the results are all
trending in the way we would expect if hypoxia was causing a cognitive deficit.
Conclusion
Although no statistical significance is seen, the results show trends that we would expect to see with
hypoxia causing a cognitive deficit. Future work would include changing hypoxia parameters in order to
see a significant difference compared to control animals. The trends going the way we expect would lead
to future studies possibly just increasing the amount of time spent in hypoxia exposure.

Neurology - Effects of Hypoxia on Cognitive Function and Anxiety Levels in RatsBackground - Neurology

Traumatic brain injury (TBI) resulting from impact to the head can cause long lasting impairments of cognitive processes that lead to increased risk-taking behavior in clinical populations. Our laboratory has recently shown that female, but not age-matched male, rats increase preference for risky choices after multiple experimentally-induced mild TBI’s. The prefrontal cortex (PFC) plays a prominent role in risk-based decision making. Previous studies have demonstrated that both dopamine (DA) and norepinephrine (NE) transmitter levels are increased in the PFC immediately following TBI, which is then followed by a persistent hypo-catecholaminergic state. These results suggest that an imbalance of catecholamine levels within the PFC may underlie aberrant decision-making behavior following TBI. Here we examined how levels of catecholamine neurotransmitter regulatory proteins responsible for packaging (VMAT2) and degrading (COMT and MAO) are altered to explain chronic decreases following TBI. The closed head-controlled cortical impact (CH-CCI) model was used on age-matched adult male and female Long Evans rats to induce either a single or multiple (3) mild brain injuries over the course of one week. Rats were sacrificed and brain tissue from the sub-regions of the PFC (mPFC, OFC, and ACC) were collected and standard western blotting protocols were used to measure the levels of VMAT2, COMT, and MAO in each brain region. We observed no differences in protein levels across all three sub-regions of the PFC using one-way ANOVAs (p > 0.05). Multiple comparisons revealed injury-induced differences in protein levels within the OFC. Decreased VMAT2 levels were found, indicating less capacity for packaging, storage, and release of catecholamine transmitters in this region, leading to a hypo-catecholaminergic tone as previously reported. Decreased levels of COMT in the OFC were found, which suggests reduced capacity for catecholamine degradation; however, this downregulation may result as a secondary compensatory response to lowered catecholamine levels due to less necessary degradation. Interestingly, MAO-A in the OFC was increased in males, but decreased in females following injury which may underlie the sex-specific differences observed in previous risk/reward decision making behavioral data. Based upon these findings, we conclude that there are changes in the levels of catecholamine neurotransmitter regulatory proteins which may underlie functional catecholamine imbalances in the PFC. Additional studies will use pharmacological agents to further probe how sex-specific changes in catecholamine regulatory proteins impact sex-specific preference for risky choice following head injury.

Neurology - Examining levels of catecholamine neurotransmitter regulatory proteins within the prefrontal cortex of rodents following traumatic brain injury - Neurology

Alzheimer’s disease (AD) is the most common cause of dementia with limited treatment options. AD pathology is identified by the accumulation of Aβ plaques (marked by Aβ42), increased neuroinflammation (marked by Iba1), and apoptosis (marked by caspase-3) in cognitively-relevant regions of the brain such as the hippocampus (HP) and the cortex. 5xFAD mouse model of AD (strain #034840-JAX) is advantageous because it recapitulates Aβ42-induced neurodegeneration. Fingolimod, an FDA-approved treatment for multiple sclerosis, is an agonist of sphingosine-1-phosphate receptor which is ubiquitously expressed throughout the brain. Recently our lab has shown that fingolimod treatment in 5xFAD
mice provides neuroprotection, reduces Aβ42, lowers inflammation, and improves spatial memory (Carreras e al., 2019). Here we investigated whether treatment with fingolimod from 1 to 14 months of age reduces apoptosis in the HP and whether it correlates with the effect of fingolimod on neuroinflammation.

Neurology - Fingolimod Treatment Decreases Apoptosis in the Brain of Alzheimer's Transgenic 5xFAD Mice - Neurology

Background: Despite progressive research, recurrent strokes remain a common occurrence affecting approximately 25% of ischemic stroke patients, thereby putting patients at a significantly higher risk for mortality and physical disability. Although gender differences in primary stroke risk are highly studied, recurrent stroke gender risk factors are not. This study focused on a subset of recurrent stroke patients who also experienced a transient ischemic attack to compare recurrent ischemic stroke risk factors between men and women. 

Methods: A retrospective analysis was conducted on patients who presented with an acute ischemic stroke from January 2010 through January 2016 to Prisma Health Upstate. Binary logistic regression was used to determine risk factors associated with recurrent strokes with and without history of TIA. Our model was tested using the Hosmer-Lemeshow test for the overall correct classification percentage, while interactions and multicollinearity between independent variables were examined using variance inflation factors.

Results: In the adjusted analysis, smoking history (OR=2.763, 95% CI, 1.063-7.185, P=0.037) , dyslipidemia (OR=1.073, 95% CI, 1.039-1.108, P< 0.001), and higher heart rate (OR=1.049, 95% CI, 1.017-1.082, P=0.002) were associated with female patients with history of recurrent stroke and TIA. On the other hand, men were shown to have a higher incidence of coronary artery disease (CAD), elevated diastolic blood pressures and creatinine levels.

Conclusion: Results identified modifiable risk factors in ischemic stroke patients with history of TIA that will encourage continued monitoring of varying comorbidities by gender to prevent and manage recurrent ischemic strokes properly.

Neurology - Gender specific risk factors for Recurrent Ischemic Stroke with previous history of TIA - Neurology

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. 




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

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

Next from AMA Research Challenge 2022

Neurology - Effects of Hypoxia on Cognitive Function and Anxiety Levels in RatsBackground - Neurology

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