LXR/CD38 activation drives cholesterol-induced macrophage senescence and neurodegeneration via NAD+ depletion.

Although dysregulated cholesterol metabolism predisposes aging tissues to inflammation and a plethora of diseases, the underlying molecular mechanism remains poorly defined. Here, we show that metabolic and genotoxic stresses, convergently acting through liver X nuclear receptor, upregulate CD38 to promote lysosomal cholesterol efflux, leading to nicotinamide adenine dinucleotide (NAD+) depletion in macrophages. Cholesterol-mediated NAD+ depletion induces macrophage senescence, promoting key features of age-related macular degeneration (AMD), including subretinal lipid deposition and neurodegeneration. NAD+ augmentation reverses cellular senescence and macrophage dysfunction, preventing the development of AMD phenotype. Genetic and pharmacological senolysis protect against the development of AMD and neurodegeneration. Subretinal administration of healthy macrophages promotes the clearance of senescent macrophages, reversing the AMD disease burden. Thus, NAD+ deficit induced by excess intracellular cholesterol is the converging mechanism of macrophage senescence and a causal process underlying age-related neurodegeneration.

Probing the drivers of Staphylococcus aureus biofilm protein amyloidogenesis and disrupting biofilms with engineered protein disaggregases.

Phenol-soluble modulins (PSMs) are the primary proteinaceous component of Staphylococcus aureus biofilms. Residence in the protective environment of biofilms allows bacteria to rapidly evolve and acquire antimicrobial resistance, which can lead to persistent infections such as those caused by methicillin-resistant S. aureus (MRSA). In their soluble form, PSMs hinder the immune response of the host and can increase the virulence potential of MRSA. PSMs also self-assemble into insoluble functional amyloids that contribute to the structural scaffold of biofilms. The specific roles of PSM peptides in biofilms remain poorly understood. Here, we report the development of a genetically tractable yeast model system for studying the properties of PSMα peptides. Expression of PSMα peptides in yeast drives the formation of toxic insoluble aggregates that adopt vesicle-like structures. Using this system, we probed the molecular drivers of PSMα aggregation to delineate key similarities and differences among the PSMs and identified a crucial residue that drives PSM features. Biofilms are a major public health threat; thus, biofilm disruption is a key goal. To solubilize aggregates comprised of a diverse range of amyloid and amyloid-like species, we have developed engineered variants of Hsp104, a hexameric AAA+ protein disaggregase from yeast. Here, we demonstrate that potentiated Hsp104 variants counter the toxicity and aggregation of PSMα peptides. Further, we demonstrate that a potentiated Hsp104 variant can drive the disassembly of preformed S. aureus biofilms. We suggest that this new yeast model can be a powerful platform for screening for agents that disrupt PSM aggregation and that Hsp104 disaggregases could be a promising tool for the safe enzymatic disruption of biofilms. IMPORTANCE Biofilms are complex mixtures secreted by bacteria that form a material in which the bacteria can become embedded. This process transforms the properties of the bacteria, and they become more resistant to removal, which can give rise to multidrug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA). Here, we study phenol-soluble modulins (PSMs), which are amyloidogenic proteins secreted by S. aureus, that become incorporated into biofilms. Biofilms are challenging to study, so we have developed a new genetically tractable yeast model to study the PSMs. We used our system to learn about several key features of the PSMs. We also demonstrate that variants of an amyloid disaggregase, Hsp104, can disrupt the PSMs and, more importantly, dissolve preformed S. aureus biofilms. We propose that our system can be a powerful screening tool and that Hsp104 disaggregases may be a new avenue to explore for biofilm disruption agents.

Molecular determinants and modifiers of Matrin-3 toxicity, condensate dynamics, and droplet morphology.

Matrin-3 (MATR3) is a DNA- and RNA-binding protein implicated in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and distal myopathy. Here, we report the development of a yeast model of MATR3 proteotoxicity and aggregation. MATR3 is toxic and forms dynamic shell-like nuclear condensates in yeast. Disease-associated mutations in MATR3 impair condensate dynamics and disrupt condensate morphology. MATR3 toxicity is largely driven by its RNA-recognitions motifs (RRMs). Further, deletion of one or both RRMs drives coalescence of these condensates. Aberrant phase separation of several different RBPs underpins ALS/FTD, and we have engineered Hsp104 variants to reverse this misfolding. Here, we demonstrate that these same variants also counter MATR3 toxicity. We suggest that these Hsp104 variants which rescue MATR3, TDP-43, and FUS toxicity might be employed against a range of ALS/FTD-associated proteins. We anticipate that our yeast model could be a useful platform to screen for modulators of MATR3 misfolding.

Functional analysis of proposed substrate-binding residues of Hsp104.

Hsp104 is a hexameric AAA+ yeast disaggregase capable of solubilizing disordered aggregates and amyloid. Hsp104 couples ATP hydrolysis to polypeptide translocation through its central channel. Substrate binding by Hsp104 is mediated primarily by two conserved tyrosine residues in nucleotide binding domain (NBD) 1 and NBD2. Recent structural studies have revealed that an additional tyrosine residue (Y650) located in NBD2 appears to contact substrate and may play an important role in Hsp104 function. Here, we functionally analyze the properties of this proposed Hsp104 -substrate interaction. We find that Y650 is not essential for Hsp104 to confer thermotolerance. Supporting these findings, in a potentiated Hsp104 variant background, the Y650A mutation does not abolish potentiation. However, modulation of this site does have subtle effects on the activity of this potentiated Hsp104 variant. We therefore suggest that while Y650 is not essential for Hsp104 function, its modulation may be useful for fine-tuning Hsp104 properties.