Instituto de Química Médica

6-(Methylsulfinyl)hexyl isothiocyanate, here referred to as hexaraphane, is a bioactive compound found in wasabi, which exhibits cytoprotective, anti-inflammatory, and chemopreventive properties. The beneficial effects of hexaraphane are largely mediated through derepression of transcription factor Nrf2, which is typically repressed via proteasomal degradation mediated by its two-site interaction with the ubiquitin E3 ligase adapter Keap1. Like other isothiocyanates, hexaraphane increases Nrf2 activity by perturbing the Nrf2-Keap1 interaction and stalling Keap1-directed ubiquitination. However, we found that hexaraphane modestly increases Nrf2 levels in Keap1-deficient cells, suggesting an additional Keap1-independent mechanism. Unlike other electrophilic Nrf2 activators, hexaraphane did not significantly impact Erk, p38^Mapk, Jnk, or Pten/Pi3k/Akt signaling. Instead, our data reveal that hexaraphane inhibits Gsk-3β, a kinase that targets Nrf2 for proteasomal degradation by phosphorylating a DSGIS degron in the transcription factor through which it interacts with the β-TrCP E3 ligase adaptor. Hexaraphane reduced Nrf2 ubiquitination and its binding to Gsk-3β, mimicking the effects of the Gsk-3β inhibitor SB216763. In vitro kinase assays confirmed that hexaraphane suppresses Nrf2 phosphorylation. Furthermore, simulations of molecular docking and dynamics predict a specific interaction between hexaraphane and the catalytic groove of Gsk-3β. These findings identify hexaraphane as a previously unrecognized dual-acting Nrf2 activator that stabilizes Nrf2 both by disturbing Keap1 binding and by inhibiting Gsk-3β that forms the DSGIpS³³⁸ phosphodegron.

West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bites of infected mosquitoes. Severe forms of the disease include meningitis, encephalitis, acute flaccid paralysis, or even death, and survivors develop long-lasting sequelae. The damage induced by WNV does not only stem from viral multiplication but also arises from immune-related pathology linked to neuroinflammation. Certain sphingolipids are key players in WNV infection, neurodegenerative diseases and inflammatory disorders. Neutral sphingomyelinase 2 (nSMase2), catalyzes the conversion of sphingomyelin into ceramide and is essential for flavivirus multiplication. Thus, nSMase2 constitutes a promising therapeutic target for the development of dual-acting antiviral and anti-inflammatory therapies against WNV. The effect of an orally bioavailable and brain-penetrating prodrug of the potent nSMase2 inhibitor DPTIP (DPTIP-P1) was studied in WNV-infected mice. While no reduction in the viral load in the brain of infected animals was observed, WNV-induced expression of inflammatory markers was modulated, resulting in a reduced IL-1β expression in brain. Transcriptomic analyses revealed that treatment with the DPTIP prodrug also modulated the expression of various genes related to immune cell function without altering antiviral innate response. Among others, DPTIP-P1 reduced the expression of Lyz2, an inducible genetic marker associated with macrophage infiltration, and modified the expression of genes related to T cell activation such as Trbc1 and Ptnp22. These results identify nSMase2 inhibitors as a new type of immune modulators of WNV infection. The neuroprotective effects exerted by DPTIP-P1 could contribute to mitigate WNV-induced neuroinflammation and sequelae.

Frontotemporal dementia (FTD) comprises a group of disorders characterized by a progressive decline in behavior or language linked to the degeneration of the frontal and anterior temporal lobes followed by hippocampal atrophy. There are no effective treatments for FTD and for this reason, novel pharmacological targets, such as the endocannabinoid system (ECS), are being explored. Previous results from our laboratory showed a TAU^P301L-dependent increase in CB₂ receptor expression in hippocampal neurons of a FTD mouse model, alongside the neuroprotective impact of CB₂ ablation. In this study, we evaluated the therapeutic potential of a new CB₂ antagonist (PGN36) in our TAU-dependent FTD mouse model. Six-month-old mice received stereotaxic injections of an adeno-associated virus expressing human TAU^P301L protein (AAV-TAU^P301L) into the right hippocampus and were treated daily with PGN36 (5 mg/kg, i.p.) or vehicle for three weeks. By integrating behavioral tests, RNA-seq, qPCR expression analysis, and immunofluorescence in the AAV expressing TAU mouse model, we found that PGN36 treatment reverses key features of the neurodegenerative process triggered by TAU^P301L overexpression. PGN36 treatment effectively countered TAU^P301L-induced cognitive decline by reducing TAU protein expression levels and restoring markers of synaptic plasticity. Notably, we observed neuroprotection in the dentate gyrus granular layer, which we attribute to the modulation of pyroptosis. This programmed cell death pathway, is triggered by TAU^P301L overexpression. PGN36 appears to modulate the pyroptotic cascade, thereby preventing the pyroptosis-induced neuronal loss. These findings collectively underscore the neuroprotective potential of this novel CB₂ antagonist treatment against TAU-associated FTD.