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Abstract Parkinson’s disease (PD) is a neurodegenerative condition featured by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (αSyn) aggregates. Growing evidence suggests that PD diagnosis happens late in the disease progression and that the pathology may originate much earlier in the enteric nervous system (ENS) before advancing to the brain, via autonomic fibers. It was recently described that a specific cell type from the gut epithelium named enteroendocrine cells (EECs) possess many neuron-like properties including αSyn expression. By facing the gut lumen and being directly connected with αSyn-containing enteric neurons in a synaptic manner, EECs form a neural circuit between the gastrointestinal tract and the ENS, thereby being a possible key player in the outcome of PD in the gut. We have characterized the progression and the cellular mechanisms involved in αSyn pre-formed fibrils (PFFs) transfer from EECs to neuronal cells. We show that brain organoids efficiently internalize αSyn PFF seeds which triggers the formation of larger intracellular inclusions. In addition, in the enteroendocrine cell line STC-1 and in the neuronal cell line SH-SY5Y, αSyn PFFs induced intracellular calcium (Ca2+) oscillations on an extracellular Ca2+ source-dependent manner and triggered αSyn fibrils internalization by endocytosis. We characterized the spread of αSyn PFFs from enteroendocrine to neuronal cells and showed that this process is dependent on physical cell-to-cell contact and on Rab35 GTPase. Lastly, inhibition of Rab35 increases the clearance of αSyn fibrils by redirecting them to the lysosomal compartment. Therefore, our results reveal mechanisms that contribute to the understanding of how seeded αSyn fibrils promote the progression of αSyn pathology from EECs to neuronal cells shifting the focus of PD etiology to the ENS.

Summary: The gut microbiota influence neurodevelopment, modulate behavior, and contribute to neurodegenerative disorders. Several studies have consistently reported a greater abundance of Akkermansia muciniphila in Parkinson disease (PD) fecal samples. Therefore, we investigated whether A.muciniphila-conditioned medium (CM) could initiate α-synuclein (αSyn) misfolding in enteroendocrine cells (EEC) — a component of the gut epithelium featuring neuron-like properties. We found that A. muciniphila CM composition is influenced by the ability of the strain to degrade mucin. Our in vitro experiments showed that the protein-enriched fraction of mucin-free CM induces RyR-mediated Ca2+ release and increased mitochondrial Ca2+ uptake leading to ROS generation and αSyn aggregation. Oral administration of A. muciniphila cultivated in the absence of mucin to mice led to αSyn aggregation in cholecystokinin (CCK)-positive EECs but no motor deficits were observed. Noteworthy, buffering mitochondrial Ca2+ reverted the damaging effects observed. These molecular insights offer evidence that bacterial proteins can induce αSyn aggregation in EECs.

Summary: Current studies estimate that 1–3% of females with unexplained intellectual disability (ID) present de novo splice site, nonsense, frameshift, or missense mutations in the DDX3X protein (DEAD-Box Helicase 3 X-Linked). However, the cellular and molecular mechanisms by which DDX3X mutations impair brain development are not fully comprehended. Here, we show that the ID-linked missense mutation L556S renders DDX3X prone to aggregation. By using a combination of biophysical assays and imaging approaches, we demonstrate that this mutant assembles solid-like condensates and amyloid-like fibrils. Although we observed greatly reduced expression of the mutant allele in a patient who exhibits skewed X inactivation, this appears to be enough to sequestrate healthy proteins into solid-like ectopic granules, compromising cell function. Therefore, our data suggest ID-linked DDX3X L556S mutation as a disorder arising from protein misfolding and aggregation.

The assessment of three-dimensional (3D) brain cytoarchitecture at a cellular resolution remains a great challenge in the field of neuroscience and constant development of imaging techniques has become crucial, particularly when it comes to offering direct and clear obtention of data from macro to nano scales. Magnetic resonance imaging (MRI) and electron or optical microscopy, although valuable, still face some issues such as the lack of contrast and extensive sample preparation protocols. In this context, x-ray microtomography (μCT) has become a promising non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens. It is a new supplemental method to be explored for deciphering the cytoarchitecture and connectivity of the brain. This review aims to bring together published works using x-ray μCT in neurobiology in order to discuss the achievements made so far and the future of this technique for neuroscience.