Biology subjects

Sousa, M. M.

Publications and source records attributed to Sousa, M. M..

5 recordsLinked to original sources

bioRxiv2026

In vitro programming and pseudounipolarization of human iPSC-derived sensory neurons

Ectopic expression of NGN1, BRN3A and ISLET1 (NBI) from a safe harbor locus in induced pluripotent stem cells (iPSCs) yielded robust differentiation into functional sensory neurons (iNBI-SNs) within seven days. Single nucleus transcriptomics identified peripheral sensory neuron profiles of nociceptors and mechanoreceptors. Electrophysiological studies showed that more than 98 % of iNBI-SNs display TTX-resistant sodium currents and that they establish functional connections to excitatory CNS neurons. iNBI-SNs derived from patients with inherited erythromelalgia, a pain disorder associated with gain-of-function mutations in the Nav1.7 sodium channel showed pathologically increased firing rates which could be partially rescued with a Nav1.7 inhibitor. Notably, iNBI-SNs acquire a characteristic pseudounipolar morphology upon co-culture with embryonic rodent DRG cells. Taken together, NBI-based forward programming of iPSCs represents a robust approach for the generation of human sensory neurons suitable for developmental, disease- and therapy-related studies.

neuroscience↗

bioRxiv2024

Axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons

Sensory dorsal root ganglion (DRG) neurons have a unique pseudo-unipolar morphology in which a stem axon bifurcates into a peripheral and a central axon, with different regenerative abilities. Whereas peripheral DRG axons regenerate, central axons are unable to regrow. Central axon regeneration can however be elicited by a prior conditioning lesion to the peripheral axon. How DRG axon asymmetry is established, remains unknown. Here we developed a rodent in vitro system replicating DRG pseudo-unipolarization and asymmetric axon regeneration. Using this model, we observed that from early development, central DRG axons have a higher density of growing microtubules. This asymmetry was also present in vivo and was abolished by a conditioning lesion that decreased microtubule polymerization of central DRG axons. An axon-specific microtubule-associated protein (MAP) signature, including the severases spastin and katanin and the microtubule regulators CRMP5 and tau, was found and shown to adapt upon conditioning lesion. Supporting its significance, interfering with the DRG MAP signature either in vitro or in vivo, readily abolished central-peripheral asymmetries in microtubule dynamics and regenerative ability. In summary, our data unveil that axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons. Impact statementSensory neurons have a stem axon that bifurcates originating two axons with different properties. This work shows that DRG axons have a specific protein signature underlying microtubule and regeneration asymmetries. It also provides an in vitro system replicating DRG biology.

neuroscience↗

bioRxiv2023

HDAC-6 inhibition ameliorates the early neuropathology in a mouse model of Krabbe disease

In Krabbe disease (KD), mutations in {beta}-galactosylceramidase (GALC), a lysosomal enzyme responsible for the catabolism of galactolipids, lead to the accumulation of its substrates galactocerebroside and psychosine. This neurologic condition is characterized by a severe and progressive demyelination together with neuron-autonomous defects and degeneration. Twitcher mice mimic the infantile form of KD, which is the most common form of the human disease. The Twitcher CNS and PNS present demyelination, axonal loss and neuronal defects including decreased levels of acetylated tubulin, decreased microtubule stability and impaired axonal transport. Here, we tested whether inhibiting the -tubulin deacetylase HDAC6 with a specific inhibitor, ACY-738, was able to counteract the early neuropathology and neuronal defects of Twitcher mice. Our data show that delivery of ACY-738 corrects the low levels of acetylated tubulin in the Twitcher nervous system. Furthermore, it reverts the loss myelinated axons in the sciatic nerve and in the optic nerve when administered from birth to postnatal day 9, suggesting that the drug holds neuroprotective properties. The extended delivery of ACY-738 to Twitcher mice delayed axonal degeneration in the CNS and ameliorated the general presentation of the disease. ACY-738 was effective in rescuing neuronal defects of Twitcher neurons, stabilizing microtubule dynamics and increasing the axonal transport of mitochondria. Overall, our results support that ACY-738 has a neuroprotective effect in KD and should be considered as an add-on therapy combined with strategies targeting metabolic correction.

neuroscience↗

bioRxiv2021

Transthyretin promotes axon growth via regulation of microtubule dynamics and tubulin acetylation

Transthyretin (TTR), a plasma and cerebrospinal fluid protein, increases axon growth and organelle transport in sensory neurons. These TTR functions were suggested to underlie its activity in promoting nerve regeneration. While neurons extend their axons, the microtubule (MT) cytoskeleton is crucial for the segregation of functional compartments and axonal outgrowth. Herein, we investigated the hypothesis that TTR promotes axon elongation and regeneration by modulating MT dynamics. Indeed, we found that TTR KO mice have an intrinsic increase in dynamic MTs and reduced levels of acetylated -tubulin in uninjured peripheral axons, and fail to modulate microtubule dynamics in response to sciatic nerve injury. Importantly, restoring acetylated -tubulin levels of TTR KO DRG neurons using an HDAC6 inhibitor was sufficient to completely revert defective MT dynamics and neurite outgrowth. In summary, our results revealed a new role for TTR in the modulation of MT dynamics by regulating -tubulin acetylation and support that this activity underlies TTR neuritogenic function.

neuroscience↗

bioRxiv2018

Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast

Resolution in microscopy is not limited by diffraction as long as a nonlinear sample response is exploited. In a paradigmatic example, stimulated-emission depletion (STED) fluorescence microscopy fundamentally breaks the diffraction limit by using a structured optical pattern to saturate depletion on a previously excited sample area. Two-dimensional (2D) STED, the canonical low-noise STED mode, structures the STED beam by using a vortex phase mask, achieving a significant lateral resolution improvement over confocal fluorescence microscopy. However, axial resolution and optical sectioning remain bound to diffraction. Here we use a tunable coherent-hybrid (CH) beam to improve optical sectioning, markedly reducing background fluorescence. CH-STED, which inherits the 2D-STED immunity to spherical aberration, diversifies the depletion strategy, allowing an optimal balance between two key metrics (lateral resolution and background suppression) to be found. CH-STED is used to perform high-contrast imaging of complex biological structures, such as the mitotic spindle and the neuron cell body.

biophysics↗