United States

Background: Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is caused by mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied dual AAV-mediated gene therapy to a mouse model of USH1B, the shaker-1 (Myo7a4626SB/4626SB) mouse. The shaker-1 mouse has a nonsense mutation in Myo7a. It is profoundly deaf throughout life, and it has significant vestibular dysfunction, similar to patients with USH1B. 

Methods: Neonatal (P0 to P5) shaker-1 (Myo7a4626SB/4626SB) mutant mice were used in this study. Due to the ~6.7 kb of the Myo7a cDNA, the dual AAV approach was used for gene delivery via the posterior semicircular canal. Auditory brainstem response (ABR) was used to assess auditory function. Vestibular evoked potential (VsEP) and circling behavior recording were used to assess vestibular function. Immunohistochemistry was used to evaluate viral transduction efficiency, sensory hair cell viability, and stereocilia morphology. 

Results: The shaker-1 mice are profoundly deaf due to significant stereocilia disorganization and rapidly progressive cochlear hair cell loss. Even though vestibular hair cells do not undergo degeneration in shaker-1 mice, they also exhibit significant vestibular dysfunction, due to stereocilia disorganization of the vestibular hair cells as well. When Myo7a cDNA was delivered to shaker-1 inner ears using the dual AAV approach, cochlear hair cell survival was improved. However, stereocilia organization and auditory function were not improved. In the vestibular system, dual AAV-mediated Myo7a delivery caused an improvement in stereocilia organization. In addition, the treated shaker-1 mice also had improved vestibular function, reflected by reduced circling behavior and improved VsEP thresholds. 

Conclusions: Our results showed that dual AAV gene therapy was able to improve the vestibular function in the shaker-1 mutant mice.

2022 AMA Research Challenge

Medical Genetics and Genomics - Dual AAV-Mediated Gene Replacement Therapy Improves the Vestibular Function in a Mouse Model of USH1B - Medical Genetics and Genomics

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Charles Lopresto, DO

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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.


poster

Background
FMR1-related disorders is a classification of X-linked trinucleotide repeat conditions which includes Fragile X syndrome. Preimplantation genetic testing for monogenic diseases (PGT-M) is classically considered a linkage-based test for determination of the allele present in an at-risk embryo. FMR1 expansion size modifies interpretation of embryo disease risk, where >200 CGG repeats is consistent with a diagnosis of Fragile X syndrome. This study is the first collection of embryo outcomes that includes triplet repeat size determination.

Methods
Trophectoderm biopsies were sent to CooperSurgical for PGT-M for FMR1 between January 2018 and February 2021. Samples underwent linkage analysis via Karyomapping (lllumina, USA) and triplet repeat size determination. Patients with FMR1 expansions on both alleles and those with a full expansion (>200 repeats) on one allele were removed. A total of 1130 blastocysts from 318 IVF cycles underwent triplet repeat size determination and linkage analysis. 

Results
48.6% (549/1130) of embryos had the abnormal FMR1 allele. 69.4% (381/549) blastocyst samples inherited the abnormal allele but did not experience a significant expansion, with repeats remaining < 200. 202/381 of samples were euploid. 17.9% of the total study population (202/1130) was euploid, abnormal allele present, and expansion <200.

Conclusion
Embryo triplet repeat size determination increased the number of embryos that could be considered for transfer compared to linkage-only methods by 17.9%. Complete information about CGG sizes in embryos is an asset for genetic counseling about selection for embryo transfer, especially in a patient population where FMR1 carrier status confers a risk for premature ovarian insufficiency. Expanded information of embryo status may reduce the number of total IVF cycles a patient pursues if they choose to transfer a carrier embryo with a repeat size < 200.

Medical Genetics and Genomics - Observed outcomes of FMR1 PGT-M analysis with incorporation of CGG repeat expansion - Medical Genetics and Genomics

Background: Arteriovenous Fistula (AVF) stenosis is responsible for a very significant morbidity, mortality and economic cost. Despite the magnitude of the clinical problem there are currently no effective therapies for AVF stenosis. The focus of this project was therefore to identify uremia specific functional and histological differences in AVF stenosis in a mouse model.

Methods: Mice were made uremic through the removal of the upper and lower poles of one kidney followed by a nephrectomy of the contralateral kidney. AVFs were created through an end (vein) to side (artery) anastomosis using standard techniques. Functional parameters included flow mediated dilation (FMD) and the area enclosed within the elastic lamina at 100 micron intervals x 12 from the AV anastomosis. The histological end point was the ratio of thrombus area/perimeter at 100 micron intervals x 12 from the AV anastomosis (with the first 600 microns being considered proximal and the second 600 microns as distal).

Results: A total of 22 mice were used in these experiments. The mean +/- SE for BUN in the uremic animals was 109+/-4.8 mg/dL as compared to 17.4 +/- 1.0 mg/dL in the control animals (p<0.05). FMD at 60 seconds was decreased significantly in uremic animals (13.4 +/- 1.5%) as compared to the control animals (19.5 +/- 1.1%), although there was no difference at the 90 second peak. There was a trend towards a smaller area enclosed by the elastic lamina (an indicator of inward remodeling) in the uremic animals, although this achieved statistical significance only in the distal segments (at 1000-1200 microns beyond the AV anastomosis). Finally, uremic animals had significantly more attached thrombus (thrombus area/perimeter) in the distal portions of the AVF (9414.7 +/- 7187.5) as compared to the proximal portions (1055.0 +/- 529.5).

Conclusion: These data document impairment of endothelial linked, functional (FMD and inward remodeling) and histological (thrombus formation) pathways in a mouse model of AVF stenosis. Further elucidation of the uremia specific mechanistic pathways responsible for these differences, could result in the development of novel drugs and devices for the treatment of AVF stenosis in patients with CKD and ESRD.

Uremia Induces Functional and Histological Changes in a Mouse Model of AVF Stenosis

Traumatic brain injury (TBI) is characterized by diffuse axonal injury that leads to neurodegeneration mediated by glial-driven neuroinflammation and neurotoxicity. Several chronic morbidities associated with TBI are psychiatric and stress-related, such as post-traumatic stress disorder. The basolateral (BLA) and central amygdala (CeA) are regulators of emotion, behavior, and autonomic function, and disruption of the amygdala due to experimental TBI has been shown to induce chronic anxiety-like behaviors, dysfunction of neurotransmission, hypertrophy of glutamatergic neurons, and dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. Beyond their role in stress and inflammation, changes in glucocorticoid receptor (GR) expression can mediate behavior, neurotransmission, neuroplasticity, and reflect dysregulation of the HPA axis. To evaluate these processes, 64 young adult, age-matched male and naturally cycling female Sprague-Dawley underwent sham surgery or midline fluid percussion injury (mFPI), and were sacrificed at 7-, 56-, or 168-days post injury (DPI). Image processing was used to analyze ionized calcium binding adaptor molecule 1 (IBA1) and glial fibrillary acid protein (GFAP)-stained photomicrographs to quantify microglia and astrocyte activation, respectively. Visual interpretation of amino-cupric-silver (AmCuAg) staining patterns were subjectively done to assess neuropathology. Simple western blotting was performed in 58 animals (25 from histology cohort) using whole amygdala biopsies to evaluate GR protein at 7- and 56-DPI. Time post-surgery, despite sporadic sex and nuclei-specific instances, was a primary driver of microglia activation and astrogliosis by 168-DPI within the BLA/CeA. The chronic, mild increase in AmCuAg staining intensity visually observed in all groups indicated that age-related neurodegeneration was likely behind histopathologic findings. Midline FPI decreased GR protein in males only at 7-DPI. These data indicate that age and HPA axis dysregulation may be primarily responsible for previously reported behavioral, neurochemical, cellular, and molecular outcomes, ruling out TBI-induced axonal injury and chronic neurodegeneration in the BLA/CeA underlying the pathophysiology of affective-like symptoms after mFPI.

Neurological Surgery - Chronic Histopathological and Glucocorticoid Receptor Assessment of the Amygdala Post-TBI - Neurological Surgery

Background Traumatic brain injury (TBI) impacts 42 million people per year and is especially common among veterans and individuals experiencing homelessness. Effective treatments for TBI remain a major unmet clinical need. Enriched housing environments have been shown to ameliorate TBI symptoms, however, little to no data is available relating TBI pathology to homelessness, despite TBI being two to seven times more prevalent among those experiencing homelessness than the general population. Currently, many trials for TBI therapeutics aim to mitigate neuroinflammation. Individuals experiencing homelessness likely display greater neuroinflammation due to stresses of daily living, potentially exacerbating TBI symptoms. This unique TBI patient population must be considered to design optimal treatments for all patients. 

Methods We developed a rat model of homelessness using a novel cage switching housing environment. We rotated animals (nhomeless = 18, nstandard = 18) into dirty cages daily for 37 days, simulating housing instability and unsanitary conditions. We then induced TBI with moderate controlled cortical impact TBI surgery (nhomeless = 9, nstandard = 9). Following TBI surgery, we performed 4 days of behavioral testing including the beam walk (BW), elevated body swing test (EBST), forelimb akinesia (FA), paw grasp (PG), rotarod, and elevated-T maze. 

Results Averaged over 4 days, significant differences in motor function, demonstrated impaired function in rats with TBI compared to controls, regardless of housing (BW 85.0%, FA 104.7%, and PG 100% greater deficit in TBI). Notably, however, homelessness exacerbated the functional deficits in TBI rats (BW 93.3%, FA 40.5%, and PG 50% greater deficit). TBI rats in the homelessness model also showed a surprising recovery in functional deficits on day 4, while control housing rats continued to decline. 

Conclusion These findings exhibit the magnification of certain motor insufficiencies shortly after TBI due to homelessness, possibly due to an underlying pathophysiology. Interestingly, however, TBI rats in the homeless simulation showed more robust recovery by day 4, suggesting a form of resilience to stressful events like TBI. That exacerbation of TBI symptoms accompanies homelessness suggests that the environmental condition of the patient must be considered in disease diagnosis and to aptly treat all TBI patient populations.


Neurological Surgery - Forgotten veterans: Pathological implications of homelessness on traumatic brain injury - Neurological Surgery

Background
Elucidating the role of perivascular clearance has been a current focus in the study of neurodegenerative disorders. Multiple imaging modalities have been used, in both clinical and preclinical settings, to investigate the clearance and drainage of waste from the brain; however, we still only have a limited understanding of how to enhance perivascular clearance. In this study, we show for the first time the reduction of perivascular flow in a murine model of mucopolysaccharidosis type VII (MPS7), a neurodegenerative lysosomal storage disease, and show the reversal of this perivascular dysfunction via chronic retinoic acid treatment. 

Methods
8-month-old MPS7 (β-glucuronidase knockout) and wild-type (WT) mice received weekly treatments of either 0.3 or 3.0 mg/kg of intraperitoneal retinoic acid injections for 5 weeks. Two-photon imaging was used to quantify the perivascular flow 30 min after intracisternal injection of Cascade blue dextran (2%, 2 uL). Similarly, blood vessels were visualized by intravenous injection of Tomato lectin AF649 (2%, 200 uL). Additionally, cortical perivascular collagen I matrix levels were determined by second harmonic generation imaging. Image analyses were performed by 3D reconstituted volumetric data and statistical evaluations were performed.

Results
Untreated MPS7 mice had an 82.8% (p<0.01) decrease in perivascular flow and a 46.2% (p<0.001) decrease in collagen I expression compared to WT mice. Whereas MPS7 mice treated with 0.3 mg/kg of retinoic acid had an 83.4% (p<0.01) decrease in the perivascular flow and 37% (p<0.05) decrease in collagen expression compared to WT mice, MPS7 mice treated with 3.0 mg/kg recovered the perivascular flow (41.4%) and collagen I (35.0%) expression to levels statistically comparable to WT mice, suggesting chronic retinoic acid mitigated the perivascular dysfunction in MPS7 mice.

Conclusion
We show the dysfunction of perivascular flow in MPS7 mice, and that retinoic acid treatment can recover both the perivascular flow and collagen I matrix at the cerebral vasculature. These results suggest that restructuring the vascular basement membrane is associated with restoring perivascular clearance, and that retinoic acid plays a signaling role, possibly opening a novel treatment method that could be generalized for other neurodegenerative diseases.

Neurology - A Novel Approach to Ameliorate Perivascular Flow Dysfunction in Neurodegenerative Disease - Neurology

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.



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

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.

Medical Genetics and Genomics - Dual AAV-Mediated Gene Replacement Therapy Improves the Vestibular Function in a Mouse Model of USH1B - Medical Genetics and Genomics

Next from 2022 AMA Research Challenge

Medical Genetics and Genomics - Observed outcomes of FMR1 PGT-M analysis with incorporation of CGG repeat expansion - Medical Genetics and Genomics

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