Recapitulation of endogenous 4R tau expression and formation of insoluble tau in directly reprogrammed human neurons.

Tau is a microtubule-binding protein expressed in neurons, and the equal ratios between 4-repeat (4R) and 3-repeat (3R) isoforms are maintained in normal adult brain function. Dysregulation of 3R:4R ratio causes tauopathy, and human neurons that recapitulate tau isoforms in health and disease will provide a platform for elucidating pathogenic processes involving tau pathology. We carried out extensive characterizations of tau isoforms expressed in human neurons derived by microRNA-induced neuronal reprogramming of adult fibroblasts. Transcript and protein analyses showed that miR neurons expressed all six isoforms with the 3R:4R isoform ratio equivalent to that detected in human adult brains. Also, miR neurons derived from familial tauopathy patients with a 3R:4R ratio altering mutation showed increased 4R tau and the formation of insoluble tau with seeding activity. Our results collectively demonstrate the utility of miRNA-induced neuronal reprogramming to recapitulate endogenous tau regulation comparable with the adult brain in health and disease.

Tau protein aggregates inhibit the protein-folding and vesicular trafficking arms of the cellular proteostasis network.

Tauopathies are a diverse class of neurodegenerative diseases characterized by the formation of insoluble tau aggregates and the loss of cellular function and neuronal death. Tau inclusions have been shown to contain a number of proteins, including molecular chaperones, but the consequences of these entrapments are not well established. Here, using a human cell system for seeding-dependent tau aggregation, we demonstrate that the molecular chaperones heat-shock cognate 71-kDa protein (HSC70)/heat-shock protein 70 (HSP70), HSP90, and J-domain co-chaperones are sequestered by tau aggregates. By employing single-cell analysis of protein-folding and clathrin-mediated endocytosis, we show that both chaperone-dependent cellular activities are significantly impaired by tau aggregation and can be reversed by treatment with small-molecule regulators of heat-shock transcription factor 1 (HSF1) proteostasis that induce the expression of cytosolic chaperones. These results reveal that the sequestration of cytoplasmic molecular chaperones by tau aggregates interferes with two arms of the proteostasis network, likely having profound negative consequences for cellular function.

Tertiary Amine Pyrazolones and Their Salts as Inhibitors of Mutant Superoxide Dismutase 1-Dependent Protein Aggregation for the Treatment of Amyotrophic Lateral Sclerosis.

Pyrazolone derivatives have previously been found to be inhibitors of Cu/Zn superoxide dismutase 1 (SOD1)-dependent protein aggregation, which extended survival of an amyotrophic lateral sclerosis (ALS) mouse model. On the basis of ADME analysis, we describe herein a new series of tertiary amine-containing pyrazolones and their structure-activity relationships. Further conversion to the conjugate salts greatly improved their solubility. Phosphate compound 17 exhibited numerous benefits both to cellular activity and to CNS-related drug-like properties in vitro and in vivo, including microsomal stability, tolerated toxicity, and blood-brain barrier permeation. These results indicate that tertiary amine pyrazolones comprise a valuable class of ALS drug candidates.

Proteasome activation is a mechanism for pyrazolone small molecules displaying therapeutic potential in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1(G93A) cells. PC12-SOD1(G93A) cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1(G93A) cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS.