Gasdermin D activation in oligodendrocytes and microglia drives inflammatory demyelination in progressive multiple sclerosis.

Neuroinflammation coupled with demyelination and neuro-axonal damage in the central nervous system (CNS) contribute to disease advancement in progressive multiple sclerosis (P-MS). Inflammasome activation accompanied by proteolytic cleavage of gasdermin D (GSDMD) results in cellular hyperactivation and lytic death. Using multiple experimental platforms, we investigated the actions of GSDMD within the CNS and its contributions to P-MS. Brain tissues from persons with P-MS showed significantly increased expression of GSDMD, NINJ1, IL-1ß, and -18 within chronic active demyelinating lesions compared to MS normal appearing white matter and nonMS (control) white matter. Conditioned media (CM) from stimulated GSDMD+/+ human macrophages caused significantly greater cytotoxicity of oligodendroglial and neuronal cells, compared to CM from GSDMD-/- macrophages. Oligodendrocytes and CNS macrophages displayed increased Gsdmd immunoreactivity in the central corpus callosum (CCC) of cuprizone (CPZ)-exposed Gsdmd+/+ mice, associated with greater demyelination and reduced oligodendrocyte precursor cell proliferation, compared to CPZ-exposed Gsdmd-/- animals. CPZ-exposed Gsdmd+/+ mice exhibited significantly increased G-ratios and reduced axonal densities in the CCC compared to CPZ-exposed Gsdmd-/- mice. Proteomic analyses revealed increased brain complement C1q proteins and hexokinases in CPZ-exposed Gsdmd-/- animals. [18F]FDG PET imaging showed increased glucose metabolism in the hippocampus and whole brain with intact neurobehavioral performance in Gsdmd-/- animals after CPZ exposure. GSDMD activation in CNS macrophages and oligodendrocytes contributes to inflammatory demyelination and neuroaxonal injury, offering mechanistic and potential therapeutic insights into P-MS pathogenesis.

Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity.

Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and can also be activated by some Group 9/10 transition metals, which is believed to mediate immune hypersensitivity reactions. In this work, we test whether TLR4 can be activated by the Group 10 metal platinum and the platinum-based chemotherapeutic cisplatin. Cisplatin is invaluable in childhood cancer treatment but its use is limited due to a permanent hearing loss (cisplatin-induced ototoxicity, CIO) adverse effect. We demonstrate that platinum and cisplatin activate pathways downstream of TLR4 to a similar extent as the known TLR4 agonists LPS and nickel. We further show that TLR4 is required for cisplatin-induced inflammatory, oxidative, and cell death responses in hair cells in vitro and for hair cell damage in vivo. Finally, we identify a TLR4 small molecule inhibitor able to curtail cisplatin toxicity in vitro. Thus, our findings indicate that TLR4 is a promising therapeutic target to mitigate CIO.

Prion gene paralogs are dispensable for early zebrafish development and have nonadditive roles in seizure susceptibility.

Normally folded prion protein (PrPC) and its functions in healthy brains remain underappreciated compared with the intense study of its misfolded forms ("prions," PrPSc) during the pathobiology of prion diseases. This impedes the development of therapeutic strategies in Alzheimer's and prion diseases. Disrupting the zebrafish homologs of PrPC has provided novel insights; however, mutagenesis of the zebrafish paralog prp2 did not recapitulate previous dramatic developmental phenotypes, suggesting redundancy with the prp1 paralog. Here, we generated zebrafish prp1 loss-of-function mutant alleles and dual prp1-/-;prp2-/- mutants. Zebrafish prp1-/- and dual prp1-/-;prp2-/- mutants resemble mammalian Prnp knockouts insofar as they lack overt phenotypes, which surprisingly contrasts with reports of severe developmental phenotypes when either prp1 or prp2 is knocked down acutely. Previous studies suggest that PrPC participates in neural cell development/adhesion, including in zebrafish where loss of prp2 affects adhesion and deposition patterns of lateral line neuromasts. In contrast with the expectation that prp1's functions would be redundant to prp2, they appear to have opposing functions in lateral line neurodevelopment. Similarly, loss of prp1 blunted the seizure susceptibility phenotypes observed in prp2 mutants, contrasting the expected exacerbation of phenotypes if these prion gene paralogs were serving redundant roles. In summary, prion mutant fish lack the overt phenotypes previously predicted, and instead they have subtle phenotypes similar to mammals. No evidence was found for functional redundancy in the zebrafish prion gene paralogs, and the phenotypes observed when each gene is disrupted individually are consistent with ancient functions of prion proteins in neurodevelopment and modulation of neural activity.

A label-free electrical impedimetric biosensor for the specific detection of Alzheimer's amyloid-beta oligomers.

Alzheimer's disease (AD) is the most common form of dementia, with over 37 million sufferers worldwide and a global cost of over $600 billion. There is currently no cure for AD and no reliable method of diagnosis other than post-mortem brain examination. The development of a point-of-care test for AD is an urgent requirement in order to provide earlier diagnosis and, thus, useful therapeutic intervention. Here, we present a novel, label-free impedimetric biosensor for the specific detection of amyloid-beta oligomers (AßO), which are the primary neurotoxic species in AD. AßO have been proposed as the best biomarker for AD and levels of AßO in the blood have been found to correlate with cerebrospinal fluid load. The biorecognition element of our biosensor is a fragment of the cellular prion protein (PrP(C), residues 95-110), a highly expressed synaptic protein which mediates the neuronal binding and toxicity of AßO. During the layer-by-layer sensor construction, biotinylated PrP(C) (95-110) was attached via a biotin/NeutrAvidin bridge to polymer-functionalised gold screen-printed electrodes. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry and scanning electron microscopy were used to validate biosensor assembly and functionality. EIS was employed for biosensor interrogation in the presence of Aß oligomers or monomers. The biosensor was specific for the detection of synthetic AßO and gave a linear response, without significant detection of monomeric Aß, down to an equivalent AßO concentration of ~0.5 pM. The biosensor was also able to detect natural, cell-derived AßO present in conditioned medium. The eventual commercialisation of this biosensor system could allow for the early diagnosis and disease monitoring of AD.