Cleide Dos Santos Souza

Background/Objectives: Protein–carbohydrate interactions are implicated in amyloid aggregation pathways associated with Alzheimer’s disease (AD). Designing glycomimetics that modulate amyloid assembly represents a promising strategy. In addition, the interaction of Aβ_1–42 oligomers (Aβo) with prion protein (PrP^C) activates Fyn kinase and leads to Tau hyperphosphorylation, another process characterizing AD. Thus, we generated a library of phenyl 2-acetamidoselenogalactoside mimetics to evaluate their interactions with Aβo and disruption of Aβo–PrP^C binding, and consequently their potential to inhibit Fyn kinase activation. Methods: The synthetic approach comprised azidophenylselenylation, a modified one-pot Staudinger reduction–acylation, a selective α-glycosylation, and deacetylation. Structural diversity was achieved mainly via acylation or ureation. The compounds were screened for binding to Aβo using STD-NMR, ¹⁹F-NMR, and rapid equilibrium dialysis (RED). ADME properties were assessed through microsomal metabolism and solubility assays, while cytotoxicity was evaluated by MTT assays in human embryonic kidney (HEK) cells. Results: Several compounds bound Aβo in STD-NMR experiments, mainly through aromatic and anomeric protons, and phenyl 2-deoxy-2-phenylureido-1-seleno-α-d-galactopyranoside (34) showed the most consistent response, with >50% increase in relative binding signal in competition assays, demonstrating also some inhibition of Aβo–PrP^C interactions (12%). Selenium at the anomeric position enhanced binding compared to sulphur and oxygen analogs. RED experiments confirmed weak binding interactions, consistent with STD-NMR results. ADME revealed that acetylated compounds undergo microsomal metabolism, whereas deacetylated derivatives displayed high aqueous solubility (>100 μM) and showed no cytotoxicity. Conclusions: Phenyl 2-acetamidoselenogalactosides are a novel class of amyloid-binding glycomimetics. Among them, 34 emerges as the most promising compound, combining favorable solubility, metabolic stability, low toxicity, and measurable interference with Aβo and Aβo–PrP^C interactions, thus supporting further developments toward therapeutic applications in AD.

Three-dimensional (3D) cultures, so-called organoids, have emerged as an attractive tool for disease modeling and therapeutic innovations. Here, we aim to determine if boundary cap neural crest stem cells (BC) can survive and differentiate in gelatin-based 3D bioprinted bioink scaffolds in order to establish an enabling technology for the fabrication of spinal cord organoids on a chip. BC previously demonstrated the ability to support survival and differentiation of co-implanted or co-cultured cells and supported motor neuron survival in excitotoxically challenged spinal cord slice cultures. We tested different combinations of bioink and cross-linked material, analyzed the survival of BC on the surface and inside the scaffolds, and then tested if human iPSC-derived neural cells (motor neuron precursors and astrocytes) can be printed with the same protocol, which was developed for BC. We showed that this protocol is applicable for human cells. Neural differentiation was more prominent in the peripheral compared to central parts of the printed construct, presumably because of easier access to differentiation-promoting factors in the medium. These findings show that the gelatin-based and enzymatically cross-linked hydrogel is a suitable bioink for building a multicellular, bioprinted spinal cord organoid, but that further measures are still required to achieve uniform neural differentiation.

Background: Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. Methods: Here, we investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila, as well as upon neuroprotection taking advantage of our gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72-repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. Results: Our study shows that manipulation of 362 transcripts out of 2257 pathological changes, in addition to inhibiting the nuclear export of repeat transcripts, is sufficient to confer neuroprotection in C9ORF72-ALS patient-derived neurons. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. We also functionally validated the high neuroprotective potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1–3 (Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons and Drosophila motor deficits. Conclusions: Strikingly, the partial depletion of SRSF1 leads to expression changes in only a small proportion of disease-altered transcripts, indicating that not all RNA alterations need normalization and that the gene therapeutic approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with transcripts modulated in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.