Young Investigators

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.

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.

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.

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.