Young Investigators

Joaquín Pardo^{1 2}, Martino Avallone², Sara Palo², Tomas Björklund²

¹Instituto de Investigaciones Bioquímicas de La Plata “Profesor Doctor Rodolfo R. Brenner”. Facultad de Ciencias Médicas. Universidad Nacional de La Plata. Buenos Aires, Argentina.
²Molecular Neuromodulation, Wallenberg Neuroscience Center, Lund University, Lund, Sweden

Abstract
Parkinson’s Disease (PD) is a neurodegenerative condition affecting motor function without a cure. PD is characterized by proteinaceous aggregates in the brain, the Lewy Bodies (LB), mainly composed of alpha-Synuclein protein (aSyn). In PD, LBs primarily affect dopaminergic (DA) neurons of the midbrain substantia nigra (SN), which project to the Striatum. Hence, detecting transcriptomic responses in these neurons at the single cell level according to their aSyn load can provide insights into the disease progression biology.
PD has been modeled in animals by overexpressing aSyn in DA neurons by Adeno-Associated Virus (AAV) nigral injection. However, it was not possible to distinguish aSyn effects from injection damage and cellular response to the AAV transduction or protein overload. To tackle these obstacles, we devised novel AAVs expressing CRE-dependent aSyn or BFP followed by a molecular barcode. Since these AAVs were packaged into the recently developed MNM008 capsid, injection in the TH CRE rats’ striatum is followed by retrograde transport to the SN, where single neurons can be sorted by cytometry and sequenced. To enrich for DA cells, we tagged them with an AAV expressing CRE-dependent H2B-GFP.
In summary, viral barcodes provided a marker for viral particle count and a pseudo marker for transgene expression. These unique data provide solid ground for discoveries in PD biology and the development of novel therapies.

Medina C, Ojea Ramos S, Depino AM, Romano AG, Krawzcyk MC and Boccia MM.

The claustrum is a brain structure that remains shrouded in mystery due to the limited understanding of its cellular structure, neural pathways, functionality and physiological aspects. Significant research has unveiled connections spanning from the claustrum to the entire cortex as well as subcortical areas. This widespread connectivity has led to speculations of its role in integrating information from different brain
regions, possibly contributing to processes such as attention, consciousness, learning and memory. Our working hypothesis posits that claustrum neural activity contributes to the formation, stabilization and updating of long-term memories in mice. We found evidence in CF-1 mice of a decline in behavioral performance in an inhibitory avoidance task due to intra-claustral administration of 2% lidocaine immediately after a training session or memory recall. Nevertheless, this does not seem to be the case for the acquisition or retrieval of this type of memory, although its neural activity is significantly increased after training, evaluated through c-Fos expression. Moreover, inhibition of the claustrum’s synaptic activity appears to impair stabilization but not the acquisition or retrieval of an unconditioned memory formed in a nose-poke habituation task.

María Bárbara Eizaguirre^1°, Natalia Ciufia^1°, Aldana Marinangeli^1°, Lucia Bacigalupe^1°, Lucia Ibarra^1°, Lucas Lapalma^1°, Matias Shulz^2°, Gerardo Fernandez^2°, Danilo Verge^2°, Ricardo Alonso^1°

^1° Multiple Sclerosis University Center (CUEM), Neurology, Ramos Mejia Hospital, Buenos Aires, Argentina
^2° View Mind Inc, New York, NY, USA.

Background: Previous research has demonstrated that eye movements can reveal brain alterations and provide insights into neurodegeneration and cognitive impairment. Objective: This study aimed to evaluate the relationship between motor and cognitive functions and eye movement parameters during the n-back task (NBKT) in people with multiple sclerosis (pwMS). Methods: A cross-sectional study was conducted involving 71 pwMS. Participants completed the n-back task using a head-mounted display equipped with eye-tracking technology to record eye movements. Motor and cognitive functions were assessed using the Expanded Disability Status Scale (EDSS), Nine Hole Peg Test (NHPT), Timed 25-Foot Walk (T25FW), and Symbol Digit Modalities Test (SDMT). Parametric and non-parametric statistical analyses were performed. Results: Participants had a mean age of 40.2±12.03 years, 13.6±3.7 years of education, and a mean disease duration of 9.7±6.8 years. The median EDSS was 3.0 (IQR 2-4.5). Significant correlations were found between gaze duration, number of fixations, saccade amplitude, and motor and cognitive impairments as measured by EDSS, NHPT, T25FW, and SDMT (p<0.05).Conclusion: This study demonstrates significant associations between eye movement patterns and motor and cognitive impairments in pwMS, suggesting that eye movements could serve as a potential biomarker for monitoring MS progression.

I am tDaniela Alejandra Cassano^1°, Franco Barrile^1°,Mirta Reynaldo^1°, Nathalia Ferreira^2°, María Paula Cornejo^1°, Higor Fideles Silva^2°, Rodrigo Rorato^2°, Helgi Schioth^3°, Mario Perelló^1°3°,

^1° Laboratory of Neurophysiology of the Multidisciplinary Institute of Cell Biology (IMBICE) (CONICET – CICPBA – UNLP). La Plata, Argentina
^2° Department of Biophysics, Paulista Medical School, Federal University of Sao Paulo. São Paulo, Brasil
^3° Department of Surgical Sciences, Functional Pharmacology and Neuroscience, University of Uppsala. Uppsala, Sweden
^4°

Ghrelin is a stomach-derived hormone that acts via growth hormone secretagogue receptor (GHSR). GHSR has ligand dependent and independent actions and is highly expressed in Agouti-related protein (AgRP) expressing neurons located in the hypothalamic arcuate nucleus (ARH). Ghrelin rises during energy deficit condition, and leads to the activation of AgRP neurons. GHSR signalling and AgRP neurons are known to modulate reward-related behaviors. We studied the role of AgRP neurons and GHSR in the enhancement of reward-related behaviours in calorie-restricted (CR) mice. Male mice were fed with the 40% of their daily food intake for 5 days and daily exposed to a non-caloric sweetener solution, saccharine, for 4 hours before each meal. We characterized the ghrelin-GHSR system and we found that CR wildtype mice showed an increase in GHSR mRNA levels in the ARH, an increase in plasma ghrelin levels and an increase of saccharine intake. Using two transgenic mouse model with lack of GHSR or a reduction of GHSR ligand independent activity we found that GHSR is required for the enhancement of reward-related behavior. Using DREADDs technology we, 1) selectively inhibited AgRP neurons and found a reduction of CR-induced enhancement of saccharine intake, and 2) selectively activated AgRP neurons in ad libitum fed mice and found an increase of saccharin intake. In conclusion, GHSR expression and activation of AgRP neurons are required for the enhanced saccharine intake during CR.

Federico Gascue^1°2°, Nicolás Pírez^1°2°, Fernando Locatelli^1°2°

^1° Instituto de Fisiología, Biología Molecular y Neurociencias; UBA-CONICET
^2° Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, UBA.

The olfactory system is continuously exposed to an extraordinary array of chemical stimuli. To keep its sensitivity within a functional and adaptive range, the system must be adjusted based on the animal’s experience. One of the main phenomena that contribute to this adjustment is sensory adaptation, which is defined as a decrease in sensitivity or response to a stimulus after a sustained exposure to it. In this work, we investigate the role and mechanisms involved in olfactory sensory adaptation using honey bees. We measured the activity of olfactory receptor neurons (ORNs) by means of electroantennograms and characterized specificity and temporal aspects of this phenomenon. Furthermore, to study the behavioral implications for the animal of the adaptation, we conducted classical conditioning experiments and found that this phenomenon reduces appetitive learning of the adapted stimuli in a mixture, while facilitates learning of the other component in cases where they would normally stay occluded. We also conducted calcium imaging experiments to measure odor-evoked signals in projection neurons (PNs) of the antennal lobe, the first olfactory neuropil in the insect brain. This allowed us to observe how adaptation changes the neural representation of odors. Overall, our results emphasize that sensory adaptation is critical in maintaining the olfactory system unsaturated and ready to detect changes in the olfactory context.

Victoria Rozés-Salvador¹; Cecilia Alvarez¹

¹ Facultad de Ciencias Químicas, CIBICI-CONICET-UNC

victoria.rozes@unc.edu.ar

CREB3L1 belongs to the CREB3 family of transcription factors involved in ER and Golgi stress responses as regulators of cellular secretory capacity and cell-specific cargoes. In response to different signals, CREB3 proteins are transported from the ER to the Golgi, where they are cleaved (activated) by S1P and S2P proteases sequentially. Although CREB3 factors have a wide range of biological functions, their role in neuronal development is poorly understood. Our study showed that CREB3L1 localizes to the basal bodies of primary cilia during early neuronal development. Primary cilia are sensory organelles that project from the plasma membrane of many cell types, including neurons. They are essential in intracellular signaling pathways and act as sensory organelles for extracellular and intracellular signals. Our preliminary results show that CREB3L1 colocalizes with γ-tubulin and Inversin (INVS), proteins located in the basal body of primary cilia at early times of culture (3DIV). This basal body localization is lost when CREB3L1 activation by S1P and S2P proteases is inhibited. Furthermore, CREB3L1 knockdown neurons present shorter primary cilium than controls. Our findings suggest that CREB3L1 may have a non-canonical function in primary cilia relevant to neuronal development and function. Additional research could provide new insights into the mechanisms underlying neuronal function.

Abstract:
BORC is an hetero-octameric complex that couples lysosomes to ARL8 and kinesin-1 and -3 for anterograde transport along microtubules. The ability of lysosomes to move within the cytoplasm is critical for many cellular functions, including maintenance of axonal health. KO of the Borcs5 or Borcs7 in mice causes neonatal lethality, however, the pathological importance of BORC in humans remained unknown. We recently identified biallelic BORCS8 variants in five children from three unrelated families, exhibiting severe intellectual disability, limb spasticity, hypomyelination, and neurodegenerative features. To further analyze the role of BORC in neuron physiology, we examined axonal mRNA transport in BORC-KO human iPSC-derived neurons and found a dramatic depletion of many mitochondrial and ribosomal mRNAs that were common with those involved in pathways of neurodegeneration. We also observed decreased synthesis of mitochondrial and ribosomal proteins in the axon. These affected mitochondria had reduced membrane potential, and were targeted for mitophagy. Finally, we found that BORC-KO axons developed swellings filled with autophagosomes and Tau aggregates, and eventually degenerated. These findings demonstrated a critical role of lysosome-coupled mRNA transport into the axon for the maintenance of mitochondrial homeostasis and could explain the pathogenesis of BORCS8 patients, and, more generally, of neurodegenerative disorders characterized by defective lysosomal transport.

Jessica Lorena Presa^1°2°, Carlos Pomilio^1°2°, Ángeles Vinuesa,  M. Eugenia Matzkin^1°, Mariano Soiza-Relly^4°, Agustina Alaimo^3°, Soledad Gori^2°,  Juan Beauquis^1°2°, Gabriel A. Rabinovich^1°, Flavia E. Saravia^1°2°

^1° IBYME-CONICET
^2° Depto de Química Biológica, FCEN, UBA
^3° INQUIBICEN, UBA-CONICET
^4° IFYBINE, UBA-CONICET

Alzheimer’s disease (AD) is a major public health challenge, with no cure and increasing prevalence. Vascular changes in AD correlate with disease progression, making them a key target for intervention. Galectins, a family of galactoside-binding proteins, are involved in survival, immune, and vascular pathways. We treated 12 m.o PDAPPJ20 mice, an AD model, with 9 i.p. injections of Gal1 (100 µg/dose) or vehicle. Tg mice showed high vascular amyloid deposits in the hippocampal hilus, a vulnerable region in AD. Gal1 reduced these deposits by 35% (p<0.05) without altering vascular density. Astrocyte-endothelial contact, crucial for blood-brain barrier integrity and Aβ clearance, was reduced in Tg mice but restored in Tg-Gal1 mice (lectin staining and GFAP IF). AQP4, an astrocytic endfeet protein necessary for fluid exchange through the BBB, also showed recovery in Tg-Gal1 mice which was diminished in Tg (p<0.02) in an array tomography analysis. We also assessed BBB integrity with i.v. Evans blue. Tg-Gal1 mice showed less vascular permeability to the dye than Tg-Veh mice(p<0.05). In vitro, we used human brain endothelial cells to model the blood-brain barrier. Exposure to 24h of Aβ 1-40 0.1 µM reduced the monolayer’s electrical resistance, while Gal1(15 µg/µl) prevented this disruption. Gal1 also mitigated proteostasis alterations in the UPR pathway and proinflammatory activation in endothelial cells caused by Aβ. Our results suggest Gal1 as a potential therapeutic agent for AD.

DANTE DANIEL GOMEZ CUAUTLE1°, ALICIA ROSSI1°, ALEJANDRO VILLAREAL2°, LUCIANA D´ALESSIO3°, ALBERTO JAVIER RAMOS1°

1° LABORATORIO DE NEUROPATOLOGIA MOLECULAR – FACULTAD DE MEDICINA
2° IBCN – FACULTAD DE MEDICINA
3° LABORATORIO DE EPILEPSIA, CONDUCTA Y NEUROPATOLOGIA

Temporal lobe epilepsy (TLE) is the most prevalent epilepsy in humans. Retrospective studies in TLE patients show an initial precipitating event (IPE) in early childhood followed by a silent period, ultimately leading to chronic epilepsy. We hypothesized that epigenetics may be involved in epileptogenesis, particularly affecting astrocytes. To study this, we used the lithium-pilocarpine model of TLE in rats, primary astroglial cultures, and resected samples from TLE patients. We found that astrocytes from TLE patients showed reactive astrogliosis, increased DNA methylation, and downregulation of homeostatic genes Kir4.1, Glutamine Synthetase and AQP4 by immunohistochemistry. In Wistar rats, the IPE induced by lithium-pilocarpine treatment (30 mg/kg IP) caused hypermethylation of astrocytes at 7, 21, and 35 days post-IPE, indicating persistent epigenetic alterations. Additionally, we observed the downregulation of homeostatic astroglial genes AQP4, glutamine synthase (GS), and Kir4.1, along with an increased proinflammatory response (C3, MAFG) and elevated DNMT expression by qPCR. These alterations were mimicked in primary astrocyte cultures exposed to DAMP HMGB1 (500 ng/ml; 18 hours) and PAMP LPS (25 ng/ml; 18 hours) and were reversed by the DNMT inhibitor decitabine (100µM). These findings show that astrocytes are pathologically altered, potentially sustaining the long-term changes underlying epilepsy. Grants PICT 2021-0760/2019-0851; UBACYT, PIP Conicet.

Magdalena Antonino^1°, Ángela Debiagge^1°, Romina Almirón^1°, Tiago Miguel Piconêz Pereira^2°, Alfredo Lorenzo^1°, Claudia Guimas Almeida^2°, Anahí Elena Bignante^1°

^1° Instituto de Investigación Médica Mercedes y Martín Ferreyra
^2° iNOVA4Health, Chronic Diseases Research Center (CEDOC), NOVA Medical School (NMS)

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by cerebral amyloid-β (Aβ) deposition. We recently found that Aβ increases APP and BACE1 convergence and interaction in recycling endosomes (RE) of human neurons derived from iPSCs (HN) through Go/Gβγ signaling, leading to enhanced APP processing by BACE1 and intracellular Aβ1-42 accumulation. In this study, we focus on the effects of Aβ on APP trafficking in the endocytic pathway.
N2A cells were transfected with APP or APP-VN/BACE1-VC (BiFC) and intracellular compartment markers, then treated with gallein (GAL), a Gβγ inhibitor, and Aβ. We found that Aβ increased APP levels and its interaction with BACE1 in RE and the Golgi apparatus while decreasing its presence and interaction in lysosomes; both effects were abrogated by GAL. Pulse-chase assays revealed that Aβ enhances APP endocytosis and reduces its recycling via Gβγ-dependent signaling. Further, we found that Aβ induces dramatic enlargement of RE and early endosomes in HN, both effects prevented by GAL.
In conclusion, Aβ increases APP endocytosis, reduces recycling, and redirects it to endosomes via Gβγ signaling, avoiding lysosomal degradation. This leads to APP accumulation in endosomes, where it interacts with BACE1, promoting amyloidogenic processing and endosomal enlargement—an early pathological hallmark of AD.