Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI +][PIN +] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI +], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI +]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN +]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI +], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN +]-independent pathway.
Pathological changes involving TDP-43 protein (‘TDP-43 proteinopathy’) are typical for several neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). FTLD-TDP cases are characterized by increased binding of TDP-43 to an abundant lncRNA, NEAT1, in the cortex. However it is unclear whether enhanced TDP-43-NEAT1 interaction represents a protective mechanism. We show that accumulation of human TDP-43 leads to upregulation of the constitutive NEAT1 isoform, NEAT1_1, in cultured cells and in the brains of transgenic mice. Further, we demonstrate that overexpression of NEAT1_1 ameliorates TDP-43 toxicity in Drosophila and yeast models of TDP-43 proteinopathy. Thus, NEAT1_1 upregulation may be protective in TDP-43 proteinopathies affecting the brain. Approaches to boost NEAT1_1 expression in the CNS may prove useful in the treatment of these conditions.
The formation of small Aβ42 oligomers has been implicated as a toxic species in Alzheimer disease (AD). In strong support of this hypothesis we found that overexpression of Yap1802, the yeast ortholog of the human AD risk factor, phosphatidylinositol binding clathrin assembly protein (PICALM), reduced oligomerization of Aβ42 fused to a reporter in yeast. Thus we used the Aβ42-reporter system to identify drugs that could be developed into therapies that prevent or arrest AD. From a screen of 1,200 FDA approved drugs and drug-like small compounds we identified 7 drugs that reduce Aβ42 oligomerization in yeast: 3 antipsychotics (bromperidol, haloperidol and azaperone), 2 anesthetics (pramoxine HCl and dyclonine HCl), tamoxifen citrate, and minocycline HCl. Also, all 7 drugs caused Aβ42 to be less toxic to PC12 cells and to relieve toxicity of another yeast AD model in which Aβ42 aggregates targeted to the secretory pathway are toxic. Our results identify drugs that inhibit Aβ42 oligomers from forming in yeast. It remains to be determined if these drugs inhibit Aβ42 oligomerization in mammals and could be developed as a therapeutic treatment for AD.
The trans-activating response DNA-binding protein 43 (TDP-43) is a transcriptional repressor and splicing factor. TDP-43 is normally mostly in the nucleus, although it shuttles to the cytoplasm. Mutations in TDP-43 are one cause of familial amyotrophic lateral sclerosis (ALS). In neurons of these patients, TDP-43 forms cytoplasmic aggregates. In addition, wild-type TDP-43 is also frequently found in neuronal cytoplasmic aggregates in patients with neurodegenerative diseases not caused by TDP-43 mutations. TDP-43 expressed in yeast causes toxicity and forms cytoplasmic aggregates. This disease model has been validated because genetic modifiers of TDP-43 toxicity in yeast have led to the discovery that their conserved genes in humans are ALS genetic risk factors. While how TDP-43 is associated with toxicity is unknown, several studies find that TDP-43 alters mitochondrial function. We now report that TDP-43 is much more toxic when yeast are respiring than when grown on a carbon source where respiration is inhibited. However, respiration is not the unique target of TDP-43 toxicity because we found that TDP-43 retains some toxicity even in the absence of respiration. We found that H 2 O 2 increases the toxicity of TDP-43, suggesting that the reactive oxygen species associated with respiration could likewise enhance the toxicity of TDP-43. In this case, the TDP-43 toxicity targets in the presence or absence of respiration could be identical, with the reactive oxygen species produced by respiration activating TDP-43 to become more toxic or making TDP-43 targets more vulnerable.
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