United States

Melanoma is the second leading invasive cancer with a greater capacity to invade and metastasize. Increased oxidative stress has been shown to be involved in melanoma growth and spread. Several antioxidants, vitamins, and other phytochemicals have been shown to prevent melanoma cancer growth. However, the chemopreventive efficacy of vialinin-A, an edible mushroom-derived p-terphenyl antioxidant, on cancer growth and invasion is unknown. We, therefore, hypothesized that with its potent anti-oxidative and anti-inflammatory actions, vialinin-A could prevent melanoma cell growth and invasion. B16-F10 melanoma cells were treated without or with various concentrations of vialinin-A. The cell viability was determined by MTT assay and apoptosis by annexin-V staining. Our results suggest that vialinin-A prevents melanoma cell growth in a dose-dependent manner. Vialinin-A also prevents the invasion and migration of melanoma cells. Further, vialinin-A prevents the generation of reactive oxygen species and promotes apoptosis of B16-F10 cells. In addition, vialinin-A increases the activation of caspase-3 and cleavage of PARP protein. Vialinin-A also regulated the expression of various anti-apoptotic, pro-apoptotic, and inflammatory markers in melanoma cells. In conclusion, our results suggest that vialinin-A, through regulating oxidative stress-modulated apoptotic pathways, could prevent melanoma cell growth and invasion and has potential chemopreventive properties. 


AMA Research Challenge 2024 

Edible mushroom-derived antioxidant, vialinin-A, prevents melanoma growth and invasion

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Abstract Title
Harnessing the anti-neoplastic potential of novel mannose-binding lectins on human urothelial and breast cell carcinomas.
Authors and Affiliation: 
Julie Marchioni, BS; Alec Skomo, BS; and Prerna Sharma, PhD*
Geisinger Commonwealth School of Medicine, Scranton PA 18509

Background
Mannose-binding lectins (MBLs) are a class of carbohydrate-binding proteins that are important in modulation of viral infections, inflammation, and cancer progression through interactions with glycosylated regions. High grade cancer cell lines have their membranes decorated with oligomannosidic sugars that can be harnessed to develop anticancer drugs. The model MBL, cyanovirin-N (CVN-N) has shown promise as an antiviral. However, its widespread use is limited due to difficult isolations, poor stability, and limited applicability to cancer targets. This led us to identify novel MBLs and test their anti-neoplastic effects on human cancer cell lines.
Methods
The novel proteins were expressed with the pET23a/BL21(DE3) Escherichia coli expression system and purified using Nickel-NTA chromatography. The purified proteins were assessed for their structure, folding, thermal, and chemical stability using circular dichroism (CD) spectroscopy. Mannose-binding studies using model glycoproteins were performed with electrophoretic mobility shift assay, EMSA. Functional studies to assess the antineoplastic effects were performed on human urinary bladder carcinoma, T24, and breast cell adenocarcinoma, MDA-MB-231, using MTS and WST-1-based assays.
Results
We identified two novel fungal lectins, LC-3 (17 kDa) and LC-4 (13 kDa) with proposed mannose binding ability by data mining using BLAST search. Their predicted secondary structure aligned well with the model structure of CVN-N. The far UV-CD spectra of LC-3 and LC-4 proteins displayed a well folded structure with single negative peaks at 215 and 219 nm respectively. Both the proteins were found to be thermostable up to 90°C. Chemical denaturation studies of LC-3 with guanidinium hydrochloride reveal a Cm of 2.75 M and a ΔG of unfolding to be 7241.7 J/mol. Chemical denaturation studies for LC-4 were significant for a ΔG of unfolding of 1458.4 J/mol and for cooperativity of unfolding of two sub-domains. Positive shifts with Fetuin A, a sialylated ligand, and RNase B, a mannosylated ligand were observed for both proteins in EMSA. Significant inhibition of cell proliferation by both the proteins were observed in low micromolar concentrations (1-5 µM) for the mannose expressing cell lines, T24 and MDA-MB-231, when compared to control groups.

Conclusion
The identified fungal lectins, LC-3 and LC-4 are thermochemically stable and have high specificity for mannosylated ligands. The high yield, thermochemical stability, and significant anti-proliferative effects of LC-3 and LC-4 against urinary bladder and breast cell carcinomas make them a potential candidate for clinical applications.

Harnessing the Anti-Neoplastic Potential of Novel Mannose-Binding Lectins on Human Urothelial and Breast Cell Carcinomas.

Immunosuppressive effects of Transforming Growth Factor Beta (TGFβ) on cytotoxic function of human natural killer cells are reversible

Anjali Patel 1, Giuseppe Maiocco 1, Catherine Mirto 1, Donald S. Torry 1,2,3, and Andrew Wilber 1,3

1 Medical Microbiology, Immunology and Cell Biology, 2 Obstetrics and Gynecology, and 3 Simmons Cancer Institute, SIU School of Medicine, Springfield, IL 62702

Background: Natural killer (NK) cells are innate immune cells that are CD56pos/CD16pos and have anti-tumor function. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg and poorly cytotoxic. These features are analogous to human decidual NK (dNK) cells, which are found at the maternal-fetal interface and support development of the placenta development during embryo implantation. The decidua and kidney tumor environment have high levels of transforming growth factor beta (TGFβ), which is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell cytotoxic function is transient and therefore reversible.

Methods: pNK cells were isolated negative selection from blood of healthy donors (HD) or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess levels of TGFβ receptors (R1, R2, and R3) mRNA and protein, respectively. NK cells were cultured for 4-5 days with the proliferative cytokine IL2 without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). For this, cells were mixed at 1:1, 1:2, and 1:5 target to effector ratio and cultured overnight. Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant.

Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed equivalent levels of all three TGFβ receptors and were potently cytotoxic against K562 target cells. NK cells treated with TGFβ retained only 15% cytotoxic function compared with untreated cells (p≤0.05). Cytotoxic function was completely restored when TGFβ treatment was discontinued and NK cells cultured in standard conditions for 4-5 days.

Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective cancer therapy. TGFβ limits this potential in the tumor environment by converting pNK cells to non-cytotoxic dNK-like cells. Importantly, NK regained complete cytotoxic function when they were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment and have implications for anti-cancer therapies. Efforts to explore this possibility using selective inhibitors or genome editing technology are underway. 



Immunosuppressive effects of Transforming Growth Factor Beta (TGFβ) on cytotoxic
function of human natural killer cells are reversible

Abstract Title
Loss of BCLAF1 impairs AML pathogenesis
Background
Acute myelogenous leukemia (AML) pathologically activates self-renewal programs of normal hematopoietic stem cells (HSCs) to promote proliferation and prevent differentiation. We identified that BCLAF1, a transcriptional repressor, has critical roles regulating HSC self-renewal. Both fetal and adult Bclaf1-deficient HSCs have impaired repopulation activity in competitive transplantation experiments. BCLAF1 is upregulated in pediatric and adult AML, and previous literature demonstrated that BCLAF1 suppression is detrimental to AML. We aim to define the function of BCLAF1 in AML transformation and progression, and we hypothesized that BCLAF1 is essential for AML pathogenesis.
Methods
Wild-type and Bclaf1-deficient hematopoietic progenitors were transduced with a retrovirus expressing MLL-AF9 to generate AML, then transferred into recipients. Hematopoietic progenitor cells from Mx-Cre:Bclaf1fl/fl mice, which inducibly delete Bclaf1 after pIpC treatment, were transduced with the MLL-AF9 construct and transplanted. Additionally, primary haploinsufficient Mx-Cre:Bclaf1fl/- and wild-type Bclaf1fl/fl AMLs were transferred into new recipients and treated with pIpC two weeks later. Haploinsufficient Mx-Cre:Bclaf1fl/- cells were also treated with Tat-Cre
recombinase to delete the remaining floxed allele. Lastly, Bclaf1-depletion was performed in human K562 AML cells.
Results
All mice receiving transduced wild-type cells developed AML, while only 50% of mice that received Bclaf1-deficient cells developed AML. Interestingly, mice that received Bclaf1fl/fl or Mx-Cre:Bclaf1fl/fl transduced cells developed AML at similar incidences; genotyping demonstrated all Mx-Cre:Bclaf1fl/fl AMLs were haploinsufficient for Bclaf1. No Bclaf1-deficient AMLs propagated from Mx-Cre:Bclaf1fl/fl, despite 85% of hematopoietic progenitors losing both floxed alleles after pIpC treatment. AML progressed equivalently in recipients of Bclaf1fl/fl or Mx-Cre:Bclaf1fl/- AML cells, and genotyping showed all Mx-Cre:Bclaf1fl/- AMLs remained haploinsufficient. Tat-Cre treatment of Mx-Cre:Bclaf1fl/- cells generated Bclaf1-/- cells (confirmed via genotyping). These Bclaf1-deficient AML cells had reduced colony formation in semisolid culture compared to haploinsufficient (Mx-Cre:Bclaf1fl/-) AML cells. K562 cells expressing shBclaf1 were selectively depleted in culture compared to cells expressing a non-targeting shRNA. Additionally, using an
inducible CRISPR-Cas9 system, K562 cells expressing a guide RNA against Bclaf1 formed fewer colonies after single-cell subcloning than cells expressing a non-targeting guide RNA. Recovered colonies predominantly expressed BCLAF1 despite loss of BCLAF1 in the bulk CRISPR-generated BCLAF1-depleted K562 cells prior to subcloning.
Conclusion
Collectively, these studies demonstrate that BCLAF1 has critical functions in AML and will define its functions in AML transformation and maintenance. Ongoing studies will define chromatin binding patterns of BCLAF1 and BCLAF1-mediated transcriptional programs in AML.

Loss of BCLAF1 impairs AML pathogenesis

Sickle cell disease (SCD) is a genetic blood disorder affecting approximately 100,000 people in the United States, 90% of whom are African American. This disorder causes abnormal hemoglobin production due to defects in the beta-globin protein. As a result, oxygen transport by erythrocytes (a type of erythroid cell) is hindered, leading to severe symptoms and complications. 

Current treatments for this disorder have limited effectiveness and/or accessibility and often have side effects. Recent studies, however, suggest that fetal hemoglobin (HbF) can compensate for the defective adult hemoglobin in SCD patients. HbF is naturally inhibited throughout adulthood by several transcription factors, including ZBTB7A. While transcription factors have historically been deemed “undruggable,” new advancements in therapeutic modalities, like molecular glue degraders, have spurred interest in targeting them. 

We previously identified and characterized SH6, a novel small molecule degrader, to induce gamma-globin (a component of HbF) through ZBTB7A degradation. It is still unclear, though, if SH6 is as effective as other gamma-globin inducers (e.g., thalidomide, decitabine, and hydroxyurea) or if SH6’s action can be amplified with them.

HUDEP-2 cells were treated with 0.05%-0.2% DMSO, pomalidomide (an analog of thalidomide), decitabine, hydroxyurea, SH6, or SH6 with decitabine or pomalidomide during their 5-day long differentiation phase. qPCR was then conducted to measure gamma-globin mRNA expression. HUDEP-2 cells without a combination treatment were also accessed via flow cytometry for gamma-globin protein production. Singlet cells were selected based on FSC/SSC, while CD235+ bright differentiated erythroid cells were gated.

SH6 had a stronger gamma globin induction effect on both mRNA and protein levels than other non-combination treatments. Synergistic effects were also seen between SH6 and decitabine, with mRNA gamma-globin expression increasing 4-fold compared to SH6 alone and 9-fold compared to decitabine alone.

So, although some gamma-globin inducers are available in the market or are in clinical trials, they are less effective than SH6 in HUDEP-2 cells. However, HUDEP-2 cells differentiate into normal erythrocytes and thus are not representative of erythrocytes found in SCD patients. SH6-mediated gamma-globin induction would need further investigation using SCD patient-derived iPSCs or CD34+ cells to ensure SH6’s efficacy in SCD physiological conditions. Despite the limited realm within which it has been studied, SH6 shows promising pre-clinical results as a potential therapy for sickle cell disease.

SH6 Outperforms Known Gamma-Globin Inducers for Treatment of Sickle Cell Disease in Vitro

Background
Hydrocephalus (HC) is a common pediatric neurosurgical condition characterized by the accumulation of cerebrospinal fluid (CSF) and elevated intracranial pressure. Without treatment, HC causes substantial neurological morbidity and mortality. The gold-standard treatment for HC, CSF shunting, is associated with high rates of shunt infection, obstruction, and malfunction and requires lifelong access to prompt neurosurgical care. Endoscopic third ventriculostomy with choroid plexus cauterization (ETV-CPC) was thus developed as an alternative to CSF shunting in low and middle-income countries (LMICs); however, the impact of this treatment on neurological function or brain development remains unclear.

Methods
The current study details the technical development of an experimental, large animal model of ETV-CPC to examine the neurobiological and neurobehavioral effects of ETV-CPC versus CSF shunting.

Results
Hydrocephalus was induced in juvenile pigs by cisternal injection of 25% kaolin and confirmed with high-resolution MRI after 10-14 days post-induction. For surgical treatment of HC, frameless stereotactic Stealth neuronavigation was registered to the animal's head and an orthogonal trajectory to the foramen of Monro was planned with entry 1 cm lateral to midline. After incision, a burr hole was fashioned, the dura cauterized, and a ventricular catheter was passed into the right lateral ventricle. After the collection of ~1 ml of CSF for later analyses, the catheter was removed and a flexible ventriculoscope was inserted along the track. The endoscope was navigated through the foramen of Monro and into the 3rd ventricle, where a flexible Bugbee wire was used to make an ETV in the lamina terminalis. After completion of the ETV, the endoscope was withdrawn into the right lateral ventricule, and the choroid plexus was cauterized, beginning at the foramen of Monro and moving toward the atrium/glomus, and then into the temporal horn to the temporal tip. In most cases, the septum pellucidum was incompetent, and the same procedure was used to cauterize the entirety of the choroid plexus in the left lateral ventricle. After the ETV-CPC, the endoscope was removed and a ventricular catheter was affixed to a ventricular reservoir, inserted into the right lateral ventricle, and secured in place with pericranial sutures. The wound was irrigated and closed.

Conclusion
This experimental juvenile porcine model of ETV-CPC offers the opportunity to study the neurobiological and neurobehavioral effects of ETV-CPC and CSF shunting in a novel way. Understanding the long-term effects of these treatments is paramount in identifying the optimal treatment for children with HC.

Experimental  Endoscopic Third Ventriculostomy With Choroid Plexus Cauterization: A Novel Model For Physiological Investigation

Background: 
The unfolded protein response (UPR) is activated by stresses in tumor cells, including hypoxia, acidosis, reactive oxygen species, radiation, and chemotherapy. It responds to unfolded/misfolded proteins in the endoplasmic reticulum (ER) by initiating pathways to enhance chaperone expression, boost protein degradation, and reduce protein synthesis, thereby regulating ER stress. If ER homeostasis cannot be restored, the UPR can shift from promoting cell survival to cell death. Targeting the UPR is a novel therapeutic approach for glioblastoma (GBM), especially against GBM stem-like cells (GSCs) that drive tumor regrowth, heterogeneity, and resistance. This strategy also shows potential for use before surgical resection of this aggressive brain tumor.

Methods
We evaluated a series of curcumin derivatives against three GSC lines. MTS assay was used to determine the IC50 of the compounds. Western blot analysis determined protein expression of targets and downstream effectors, including C/EBP Homologous Protein (CHOP), GRP78/Bip, caspase 7, and PARP.

Results
A trimethoxy bis-chalcone compound was identified that induces robust cell death in GSCs, with an average IC50 of just below 300 nM — 100-fold increase in potency compared to curcumin. This bis-chalcone showed minimal toxicity towards normal human mesenchymal stem cells. Western blot revealed that this compound effectively activated the pro-death UPR protein CHOP while reducing the expression of the pro-survival chaperone GRP78/Bip. It also induced caspase 7 expression and PARP cleavage, indicating apoptosis.

Conclusion
Most ER stress-inducing agents activate both survival pathways and cell death pathways of UPR. In contrast, our identified compound selectively promotes UPR cell death signaling and is highly toxic against radiation and temozolomide-resistant GSCs, while showing no toxicity to non-tumor cells. This bis-chalcone compound is a promising lead for developing UPR-targeted therapies, particularly for GBM. Its potential application in pre-surgical resection of GBM is noteworthy due to its ability to reduce cell migration and tumor size.

Identification of ER Stress-Inducing Agents for Glioblastoma: Advancing Therapeutic Strategies

Background 
The blood brain barrier (BBB) is a selective, semi-permeable boundary in the central nervous system (CNS) that plays a critical role in maintaining the homeostasis of the brain microenvironment. While clinical experiments can accurately determine which compounds effectively cross the BBB, these are time-consuming and labor-intensive. Machine Learning (ML) and computational techniques can alleviate this burden by rapidly screening a large set of potential drug candidates for those that should be prioritized for experimentation validation. In this study, we use ML to predict BBB permeability and then use Parallel Artificial Membrane Permeability Assays (PAMPA) as the in vitro method to assess the accuracy and reliability of these predictions. 

Methods 
The dataset used is B3DB, one of the largest public BBB datasets consisting of seven thousand compounds compiled from 50 published resources. We trained and validated models on this dataset with methods including support vector machines (SVMs), deep neural networks (DNNs), and graph convolutional neural networks (GCNNs). The transfer learning model consisted of an initial DNN trained to the task of quantum chemical property before retraining to the task of BBB permeability. After using these models to screen the Emory Enriched Bioactive Library, a test dataset of two thousand compounds, the top 50 compounds will be prioritized for in vitro experimental confirmation with PAMPA permeability assays. These assays consist of an artificial lipid membrane that simulates BBB properties. Samples will be collected from both sides at specific intervals and analyzed spectrophotometrically post-incubation. 

Results 
For fair comparison, the same 75/25 train/test split of the B3DB dataset was used consistently across the various models. The DNN had a similar performance to the SVM with an accuracy of 83.09% and 82.33% respectively. For transfer learning, the most predictive model was of the DNN with target domain of polarizability of 76.89%. The results indicate that the GCNN was the best performing model of 87.14%. 

Conclusion 
Currently, GCCN’s demonstrate the best predictive capability, highlighting their ability to learn relevant molecular features to the predictive task in the message passing phase. After then comparing these predictions with the in vitro experiments, the next step is to conduct in vivo validation using small and large animal models. The intent is for this hybrid computational and experimental approach to accelerate the understanding of drug therapeutic effects on neuro-oncology and provide insights into key chemical properties that will guide the design of future compounds.

Machine Learning Prediction and In Vitro Validation of Drug Blood Brain Barrier Permeability

Background: Meningiomas are the most common primary intracranial tumor, accounting for over 30% of all primary brain tumors. Although most are benign, up to 20% recur and can be considered aggressive. Based on RNA and whole-exome sequencing data, we identified three molecular subtypes of meningioma. Meningioma Group B and C (MenG B/C) are characterized by deletion of Neurofibromatosis Type 2 (NF2) gene. Simultaneously, the deactivation of the Hippo pathway has been linked to meningioma growth. NF2 inactivation has previously been linked to deregulating the Hippo signaling pathway; however, the mechanism of this relationship is unclear in meningioma. This study aims to investigate the impact of common meningiomal NF2 mutations on Hippo pathway activity and identify which specific NF2-Hippo signaling interactions contribute to these effects. 

Methods: We conducted an extensive literature review to identify the most common NF2 mutations in meningioma. Six of these mutants were cloned and overexpressed in 293 cells with a N-terminal mono-GFP tag. Localization was assessed via fluorescence imaging and compared to wild-type (WT) NF2 localization. We then generated a split-luciferase Hippo pathway biosensor to quantify mutant NF2’s effects on downstream Hippo pathway activity. In this biosensor, one of two luciferase domains is attached to a small amino acid stretch of the YAP protein that harbors the phosphorylation site for degradation. The other part is attached to the phospho-dependent interactor 14-3-3. When Hippo is on, the YAP site gets phosphorylated, binds to 14-3-3 and completes a functional luciferase, which emits a luminescence signal upon substrate binding. WT and mutant NF2s were co-transfected with this biosensor and corresponding signals were quantified. In a second line of experiments WT and mutants were co-expressed with Hippo pathway kinases, MST1 and LATS1, then immunoprecipitated to investigate if these interact. 

Results: We found 19 articles and one database describing 205 NF2 mutations in meningioma, most of which were nonsense mutations. We investigated six of the most common mutants along with the three most common WT NF2 variations (v1, v2, v7). NF2 var7 and NF2 mutants showed aberrant protein aggregation compared to var1 or var2 NF2 which correlated with severity of truncation. Luciferase assay revealed that var7 NF2 as well as mutant NF2 decreased YAP phosphorylation and subsequent Hippo pathway activity. Immunoprecipitation and Western Blot results found that although NF2 v1 and v2 show normal MST1 and LATS interaction, var7 lost interaction with MST1. Interestingly, the NF2 mutants lacked LATS1 interaction, but MST1 interacted even with the smallest truncation of NF2. 

Conclusion: We propose that NF2 forms a tertiary complex with MST1 and LATS1 to activate the Hippo kinase cascade and prevent proliferation. NF2 nonsense mutations create aberrant non-functional proteins that disrupt this complex formation and attenuate Hippo signaling. The naturally occurring var7 NF2 loses MST1 interaction while the NF2 mutations lose LATS1 interaction. These findings have strong therapeutic implications in targeting the MST1-LATS interaction to suppress oncogenicity. Future directions suggest investigating the role of TRAF7 in Hippo signaling, enabling a complete picture of the Hippo pathway in meningioma.

NF2 mutations found in meningioma disrupt the kinase cascade of the Hippo pathway

INTRODUCTION
Marine organisms produce a wide variety of metabolites with unique scaffolds that are biologically active. Manzamines are marine-derived polycyclic alkaloids. Approximately 100 manzamine-type alkaloids have been isolated from 16 marine sponge species. Some of these compounds are reported to have anti-inflammatory and neuritogenic properties. Previous neurite outgrowth screens have identified manzamine A and 8-methoxymanzamine A for further analyses. 
METHODS
To assess the neuritogenic potential, this study conducted neurite outgrowth assays to build dose-response curves for manzamine A and 8-methoxymanzamine A in murine primary cortical cultures. Sytox assays were performed in the Celloger to assess cell toxicity. 
RESULTS
The neurite outgrowth analyses showed that 8-methoxymanzamine A induced neurite outgrowth in a statistically significant manner at 1 micromolar dose. Likewise, that dose and others demonstrated a statistically significant increase in neuronal complexity via number of processes and number of branches. However, at 10 micromolar dosage, 8-methoxymanzamine appears to be largely toxic, with reduced neurite outgrowth, number of processes and number of branches, accompanied by some nuclear blebbing indicative of apoptosis. In contrast, manzamine A doesn’t show increased neurite outgrowth or complexity at any dose. Moreover, like 8-methoxymanzamine A, it appears potentially toxic at the 10 micromolar dose. Initial studies with Sytox indicate substantial toxicity for manzamine A and 8-mehthoxymanzamine A between 1 and 10 micromolar dosage.
CONCLUSIONS
Together, these data suggest 8-methoxymanzamine A has the potential to increase neurite outgrowth, and shows similar indications of toxicity at the 10 micromolar dose as manzamine A. Further studies will be performed with Sytox to refine the concentration at which it is toxic to 50% of cells (LC50). Together, these studies will help identify novel neuroactive manzamine compounds for the marine natural products preclinical pipeline.
Study conducted with intramural funding from Midwestern University.

8-methoxymanzamine A, but not manzamine A, induced neurite outgrowth in murine cortical cultures

Background: Stuttering is a neurodevelopmental disorder of unclear etiology characterized by involuntary disruptions in speech fluency. Recently, stuttering has been shown to be related to a point mutation in GNPTAB, a housekeeping gene involved in cellular lysosomal enzyme-targeting pathway, though it remained unclear how such a mutation might cause the stuttering phenotype. Transcranial ultrasounds in adults who stutter detected mesencephalic hyperechogenicity, likely associated with iron deposits. The current study aims to confirm, assess, and alleviate iron accumulation in a transgenic mouse model engineered with a mutation associated with Gnptab. 
Methods: 
Animal studies: All procedures in this study were approved by the Animal Care and Use Committee of the Intramural Research Program of the National Institute of Neurological Disorders and Stroke. 
Vocalization studies: Ultrasonic vocalizations (USVs) in male mice were recorded via Avisoft Bioacoustics and SASLab Pro software, and the complexity and geometry of USVs were analyzed in Sonotrack (Metris Inc). 
Iron chelation therapy: Adult male Gnptab-mutant (n = 10) and control littermates (n = 10) were treated by 3- Hydroxy-1,2-dimethyl-4(1H)-pyridone (Deferiprone; 0.1 g/L) orally for 1 month via water supply. Animals' vocal behaviors were recorded and compared before and after the treatment. 
Histological processing: Mice were transcardially perfused with 4% paraformaldehyde (PFA) fixative. The brain was extracted and post-fixed for 3-5 days in PFA. Iron was detected by iron-sensitive DAB-enhanced Perl’s staining on coronal brain sections. Bright field images were taken and imported to Fiji, where regional iron content was quantified. 
Statistical analysis: All data was exported to Prism 9.0. Statistical significance was taken at p < 0.05. 
Results: Gnptab-mutant animals show higher percentage of complex USVs and higher iron deposition in the medial lateral striatum, dorsolateral striatum, and central striatum when analyzed for percent area of iron deposition following Perl’s staining. Alleviation of this iron accumulation via chelation therapy was confirmed by Perl’s staining. Strikingly, the percentage of complex vocal bouts decreased following iron chelator therapy. 
Conclusion: The mutation in Gnptab gene, associated with stuttering, leads to an accumulation of CNS iron that may be alleviated with iron chelation, ultimately improving the stuttering phenotype in mice. Accordingly, we posit a novel therapeutic option and long-awaited etiology of the disorder.

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