Abstract List for 2024 meeting

#803: Chemogenetic Manipulation of Astrocytic Calcium Signaling in Demyelinating Disease

Angeliu, Christy G ¹; Garbarini, Karissa K ¹; Cheli, Veronica T ²; Paez, Pablo M ²;

¹Neuroscience Program, ²Department of Pharmacology and Toxicology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Profound astrogliosis is a hallmark of the most common demyelinating disease, Multiple Sclerosis (MS), in which demyelinated lesions develop in the central nervous system (CNS). While this astrogliosis can be beneficial in some contexts, it can be detrimental in others. Recent work shows that astrocyte reactivity can be stimulated by the Gq-GPCR hM3Dq and attenuated by the Gi-GPCR hM4Di. We hypothesize that in demyelinating disease, activation of astrocytic hM3Dq will promote reactivity, worsening neuroinflammation and demyelination, while hM4Di will have opposing effects. Calcium imaging in primary cortical astrocytes revealed that hM4Di is inhibitory via suppression of Ca2+-permeable ion channels. Unexpectedly, stimulation also seems to promote astrocyte proliferation. Next we employed the cuprizone and experimental autoimmune encephalomyelitis (EAE) models to investigate the consequences of astrocytic hM4Di stimulation in demyelinating disease, using mice that express hM4Di in astrocytes. In a preliminary study, activation of hM4Di during the acute phase reduced clinical severity and motor impairment. We found reduced neuroinflammation and immune cell infiltration in the brain along with reduced astrogliosis and demyelination in the spinal cord. In the cuprizone model, when hM4Di was activated at the peak of astrogliosis and demyelination, we found suppressed demyelination. We also found an increase in GFAP, which is consistent with increased proliferation seen in vitro. We have demonstrated that hM4Di activation alters calcium signaling as anticipated and found previously unreported effects on astrocyte proliferation. Presumably through suppression of calcium signaling, reduction of reactivity, and stimulation of proliferation, hM4Di activation in astrocytes seems to attenuate the severity of demyelinating disease.

#818: Estrous-cycle dependent regulation of catecholamine signaling in response to food and drug reward

Bhimani, Rohan V ¹; Pauly, Ryan C ¹; Mietlicki-Baase, Elizabeth ²; Park, Jinwoo ¹;

¹Biotechnical and Clinical Laboratory Sciences, SUNY University at Buffalo, School of Medicine and Biomedical Sciences; ²Exercise and Nutritional Sciences, SUNY University at Buffalo;

The central catecholamine systems, dopamine and norepinephrine, play a critical role in encoding the valence of environmental stimuli to promote engagement in behaviors that potentiate an organism’s survival. Furthermore, both systems in limbic brain areas such as the ventral striatum and bed nucleus of the stria terminals (BNST) are major targets of stimulant drugs. Canonically, limbic dopamine transmission is generally considered to increase in response to appetitive or rewarding stimuli whereas norepinephrine signaling is enhanced in the presence of aversive or noxious stimuli. However, it remains to be elucidated whether sex differences, especially at different stages of the estrous cycle, distinctly modulate such responses. In this study we (i) identified estrous cycle-dependent changes in catecholamine regulation via their transporters and autoreceptors in the ventral striatum and BNST of anesthetized rats and (ii) determined how natural (sucrose) and stimulant (methamphetamine) reward impacts norepinephrine and dopamine transmission in the ventral striatum and BNST, respectively, in male and female rats using in vivo fast-scan cyclic voltammetry. Our results demonstrate opposing limbic catecholamine regulation throughout different stages of the estrous cycle wherein dopamine responses are heightened during the estrus stages of the cycle. In contrast, norepinephrine levels are impacted the greatest during the diestrus/proestrus stages, suggesting a critical role of plasma estrogens. These findings offer new insights into the role of estrous cycle stage on how the brain encodes environmental stimuli and provides a new framework for therapeutically targeting the central catecholamine systems in health and disease ranging from drug use disorders to obesity.

#838: Characterizing the Chemo, Mechano, and Crawling Behavior of Larvae Expressing Human Alpha-Synuclein 

Boukari, Donaldine D ; Rathnayake, Rasika ; Gunawardena, Shermali ;

Department of Biological Sciences, SUNY University at Buffalo;

Parkinson’s disease (PD) is a neurodegenerative disease characterized by loss of motor function, uncontrollable movements as well as balance and coordination. Previously we found that Drosophila larvae expressing human Alpha-Synuclein (a-syn), the gene mutated in PD and the protein found in Lewy bodies, the aggregates seen in all PD cases showed axonal transport defects (axonal blockages) and synaptic defects. Alpha-synuclein is enriched in the brain and seems to play a critical role in neurotransmitter release. We also found that the NAC region of an a-syn which is essential for protein aggregation, rescued axonal transport defects. To identify how defects in axonal transport and synaptic dysfunction translate clinical behaviors seen in PD we tested the hypothesis that larvae expressing a-syn will show defects in larval crawling, chemosensory and mechanosensory behaviors. We examined six genotypes (wild type, a-syn-WT, a-syn-NAC, a-syn 1-120, and two PD mutants a-syn A30P and a-syn A53T) for changes in larval crawling velocities and contractions, and for defects in response to attractants (Octanol), repellents (quinine) and heat. Our findings suggest that excess human a-syn affects neuronal pathways involved in crawling, chemo-sensation, and mechano-sensation.

#844: Mechanisms of Mismatch Repair Promoted Trinucleotide Repeat Expansions in Saccharomyces cerevisiae

Casazza, Katherine M ; Williams, Gregory M ; Johengen, Lauren ; McDougall, Ashley ; Kumar, Charanya ; Bukhari, Sarah ; Twoey, Gavin ; Surtees, Jennifer A ;

Department of Biochemistry, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Mismatch repair (MMR) is an important DNA repair mechanism that protects genomic stability through correcting polymerase errors at the replication fork. In most eukaryotes, including Saccharomyces cerevisiae and humans, MMR is initiated by MutS homolog (MSH) complexes, Msh2-Msh3 and Msh2-Msh6, recognizing and binding misincorporations and insertion/deletion loops, respectively. MSH-DNA complexes recruit and activate endonuclease activity of MutL homolog (MLH) complexes to nick the DNA and recruit downstream repair factors. Deficiencies in the MMR system can lead to Lynch syndrome and a predisposition to cancer. While MMR is critical for protecting the genome by preventing mutagenesis, components of the MMR system have been implicated in promoting trinucleotide repeat (TNR) expansions. TNR expansions are the cause of over 40 neurodegenerative and neuromuscular diseases, including Huntington’s disease and myotonic dystrophy which are caused by CAG and CTG repeats respectively. In stark contrast to their function in promoting replication fidelity, components of the MMR system exhibit a pathogenic role through promoting TNR expansions. We have previously demonstrated that Msh2-Msh3 can promote genome instability through both CAG and CTG expansions in vivo. This is consistent with work in mice and humans, including GWAS studies that identified Msh3 as a genetic modifier of expansions. Our work aims to elucidate mechanistic details of Msh3 promoted expansions of threshold length TNR tracts and their distinctions from Msh3-mediated repair.

#799: Exploring developmental and age-related pathologies in the brain using the Nrmt1^-/- mouse model

Catlin, James P ¹; Marziali, Leandro N ²; Rein, Benjamin ³; Yan, Zhen ³; Feltri, M Laura ²; Schaner Tooley, Christine E ¹;

¹Department of Biochemistry, ²Department of Neurology, ³Department of Physiology and Biophysics, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

It is widely thought that age-related damage is the single biggest contributing factor to neurodegenerative diseases. However, recent studies are beginning to indicate that many of these diseases may actually have developmental origins that become unmasked overtime. It has been difficult to prove these developmental origins because there are still very few known links between defective embryonic neurogenesis and progressive neurodegeneration. We have created a constitutive knockout mouse for the N-terminal methyltransferase NRMT1 (Nrmt1^-/- mice). N-terminal methylation is an important post-translational modification that regulates protein/DNA interactions and other crucial cellular functions such as DNA damage repair, mitosis, and transcriptional regulation. Nrmt1^-/- mice display phenotypes associated with premature aging, including early graying, kyphosis, fibrotic skin, and shortened lifespan. Specifically in the brain, they exhibit lateral ventricle enlargement and striatal and hippocampal degeneration, which is accompanied by impaired short and long-term memory and hyperactivity. We have found that the appearance of apoptotic cells corresponds with a significant increase in astrogliosis and pro-inflammatory cytokine signaling and are now determining if this is an acute response or the beginning of chronic neuroinflammation. Interestingly, these phenotypes are preceded by defects in postnatal neural stem cell (NSC) quiescence and differentiation, and these defects appear to originate embryonically. We are now interested in characterizing the embryonic neurogenesis defects in Nrmt1^-/- mice and identifying which type of neurons are undergoing apoptosis as the animals age. These studies will help link defects in embryonic neurogenesis with progressive neurodegeneration and provide a model for how human neurodegenerative diseases could be of developmental origin.

#798: The Neurosurgical Management of Pancoast Tumors

Chan, Deana G ; Okai, Bernard K ;

Department of Neuro-Oncology, Roswell Park Comprehensive Cancer Center;

Objective: The surgical management of Pancoast tumors requires collaboration between thoracic surgeons and neurosurgeons. We present a case series on the neurosurgical management including neurologic function and survival outcomes for these patients at an NCI/NCCN-designated cancer center highlighting the perioperative management, operative techniques, and outcomes. Methods: Patients at an NCI-designated cancer center were prospectively enrolled in an IRB-approved study.  Those patients surgically treated for Pancoast tumors were identified. Patient demographics, tumor characteristics, and preoperative and postoperative measurements were collected. Outcome measures included neurologic function and overall survival.  Basic descriptive statistics (i.e., mean, standard deviation, range) summarized cumulative data for each measurement recorded.  Results: Nineteen patients were included in the study. Mean age was 59+7.88 years and 42.11% (n=8) were female. 43.75% (n=7) were staged as T3 and 56.25% (n=9) as T4. 89.47% (n=17) had preoperative chemoradiotherapy (Table 1). The chief presenting symptom was pain in 89.47% (n=17) of patients (Table 2). 100% underwent resection of apical tumor and chest wall, with complete removal achieved in 89.47% (n=17). Complications occurred in of 84.21% (n=16) patients. Average follow-up was 3.83+3.11 years. 10.53% (n=2) had decrease in motor function from baseline (p=0.162). 42.11% (n=8) had tumor recurrence, with an average of 0.91 years from tumor resection. There were 26.32% (n=5) patients who were deceased at an average of 1.65+1.15 years (Table 3). Table 4 shows outcomes specific to the 12 patients whose care involved neurosurgical intervention. Conclusions: Our case series highlights the importance of neurosurgical involvement and expected outcomes in managing Pancoast tumors. Given the neurosurgeon’s critical role in tumor resection, further studies highlighting the neurosurgical management of Pancoast tumors are warranted. 

#802: Inhibiting endocytosis in nociceptors alters MIA-Induced Osteoarthritis pain behavior

Cooper, Aubrey J ¹; Joshi, Ojasi M ¹; Tabman, Jessica ¹; Rodriguez, Raider ²; Bhatacharjee, Arin ²;

¹Neuroscience Program, ²Department of Pharmacology and Toxicology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Osteoarthritis (OA) is a degenerative joint condition that leads to chronic pain and a need for pain relief.  Our previous work has indicated that inhibition of the nociceptor endocytotic adaptor protein complex 2 (AP2) inhibits neuronal hyperexcitability and inflammatory pain behavior. Here we show the AP2A2 subunit localized to CGRP-containing large dense core vesicles in human and mouse DRG neurons. IHC was performed on human synovium to demonstrate AP2 co-localization with CGRP in synovial afferents. We demonstrate that pain behavior in OA is reduced by local pharmacological inhibition of the nociceptor AP2 complex using a lipidated AP2 peptide inhibitor. Monoiodoacetate (MIA) was used to induce knee joint OA in Sprague-Dawley rats. Upon verification of pain, a one-time intra-articular injection to the arthritic knee of either the AP2 inhibitor peptide or a scrambled peptide control was administered. Dynamic weight-bearing (DWB) assay assessed joint pain. Effects of the peptides on central sensitization were seen via the von Frey (vF) method. DWB analyses showed a decrease in weight bearing in the scrambled peptide group; however, an increase in weight bearing, persisting up to 24 days, was observed in the AP2 peptide injected group. The scrambled peptide group showed a decrease in paw withdrawal threshold (PWT) when subjected to the vF filaments post MIA-induced OA; whereas the AP2 inhibitor peptide prevented such change. Micro-computed tomography analysis was performed on MIA injected knee joints as well as the contralateral healthy knee joints at end of 28 days to evaluate the effects on disease progression. Pathological analyses indicate  reduction of subchondral bone content and cartilage formation in scrambled peptide-injected MIA-treated joints, intra-articular AP2 inhibitor peptide injections resulted in a significant retention of bone content and cartilage. This data suggests that inhibition of AP2 can decrease OA pain and modify disease.

#811: Chemogenetic Manipulation of Schwann Cells Development and Myelination

Corral, Jazmin G ; Garbarini, Karissa ; Cheli, Veronica T ; Paez, Pablo M ;

Department of Pharmacology and Toxicology, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Schwann cells are the major glial cells in the peripheral nervous system (PNS), and they are essential for the maintenance and myelination of peripheral nerves. In the central nervous system, the importance of G-Protein coupled receptors (GPCRs) modulating Ca²⁺ signaling in oligodendrocytes and neurons has previously been shown. However, there is a lack of information of how the modulation of Ca²⁺ signaling by GPCRs in Schwann cells affect myelination of the PNS. Using Cre-mediated recombination we specifically expressed the excitatory hM3Dq and the inhibitory hM4Di GPCRs in Schwann cells. These receptors were created from different subtypes of human muscarinic receptors and are exclusively activated by clozapine N-oxide (CNO). We performed Ca²⁺ imaging, immunocytochemistry, and electron microscopy experiments to assess Schwann cell development and function after hM3Dq and hM4Di activation in vitro as well as in vivo. Our results show that hM3Dq activation during early development significantly delays the myelination of the sciatic nerve and the maturation of Schwann cells. Furthermore, hM3Dq activity in mature Schwann cells disrupts the myelin sheath and induces a severe demyelination in the adult sciatic nerve. We have conducted Ca²⁺ imaging experiments to determine how hM3Dq affects the activity of Ca²⁺ channels and receptors in Schwann cells and we have also performed behavioral test, such as Rotarod and Catwalk, to study how changes in myelination induced by hM3Dq impact motor coordination in young and adult mice.

#816: Investigating Neurodegenerative Pathophysiology: Is there a link between glutamate and ferroptosis?

Cruz, Sara ¹; Bailey, Danielle K ²; Kosman, Daniel J ²;

¹Neuroscience Program, ²Department of Biochemistry, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Glutamate is a major excitatory neurotransmitter in the human brain involved in neurological functions such as learning, memory, and synaptic plasticity. It is a positive regulator of the N-methyl-D-aspartate (NMDA) receptor, stimulating calcium flux and activating calcium-mediated signaling pathways. However, excess glutamate can overstimulate these receptors leading to toxic effects such as mitochondrial dysfunction, lactate dehydrogenase leakage, and oxidative stress. These excitotoxic events have been associated with neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease. Ferroptosis is a programmed cell death resulting from pathophysiological stress from the mis-regulation of iron trafficking, accompanied by lipid peroxidation. Although the role of ferroptosis in neurodegenerative diseases has been explored, the specific regulatory mechanisms remain poorly understood. Due to having similar results, we propose NMDAR cytotoxicity is a calcium-calmodulin-driven ferrous iron-dependent ferroptotic cell death that contributes to neurodegenerative mechanisms. The immortalized murine hippocampal neuronal HT22 cell line serves as a model to study glutamate-induced toxicity. However, when differentiated, our data show they lack characteristic neuronal phenotypes such as NMDA receptors and conductive activity. Human induced pluripotent stem cells (iPSCs) can be differentiated into glutamatergic-like neurons, iNeurons, showing to be a promising cellular model to study cytotoxicity and ferroptosis in neurons. Techniques such as quantitative polymerase chain reaction (qPCR) and Western Blots are being performed to characterize these cells. Furthermore, we will use pharmacological agents such as calcium chelators to investigate the role of calcium conductance in glutamatergic cytotoxicity and ferroptosis in neurons.

#830: Ramanomics for Molecular Understanding of Fentanyl impact in Central Nervous System (CNS) Cells

Das, Rahul K¹; Kuzmin, Andrey ¹; Pliss, Artem ¹; Shukla, Shobha ²; Prasad, Paras N¹; Mahajan, Supriya D³;

¹Department of Chemistry, SUNY University at Buffalo, College of Arts and Sciences; ²Department of Metallurgical Engineering & Materials Science, Indian Institute of Technology Bombay; ³Department of Medicine, SUNY University at Buffalo, School of Medicine and Biomedical Sciences;

Fentanyl is a synthetic opioid with analgesic properties, but its abuse imposes devastating social, economic, and health burdens. Fentanyl driven chemical changes at molecular level can guide decoding neuroscience of drug accumulation and its pathophysiological effects in the human brain. Understanding the molecular changes in response to fentanyl overdose in key cells of the Central Nervous System (CNS), namely microglia and astrocytes can provide insight into regulation of neuroinflammatory response in the scenario of opiate drug addiction. Lipid droplets (LD) act as hydrophobic depot for Fentanyl in terms of increased drug accumulation and metabolism alteration. Ramanomics technique facilitates ultrasensitive quantification of biomolecules in live cells and organelles using biomolecular component analysis (BCA). LDs within fentanyl dosed human astrocytes and microglia were studied using Ramanomics to gain novel information on multiple changes in chemical identities associated to a single organelle level. Our data exhibits Fentanyl induced significant changes in degree of lipid saturation, lipid peroxidation, sterol imbalance, sphingomyelin concentration and phosphocholine release. Thus, favours neurologic understanding for the drug’s potency in alteration of cellular metabolism and toxic potential. Distinct changes in neurochemical features for LDs of microglia and astrocyte with response to fentanyl treatment demonstrates the utility of Ramanomics as a non-invasive and real time cellular/molecular analysis. We speculate that our data inspires therapeutic advancements in fentanyl addiction and identifying its molecular dynamics.