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Aberrant activity of cyclin-dependent kinase (Cdk5) has been implicated in various neurodegenerative diseases. This deleterious effect is mediated by pathological cleavage of the Cdk5 activator p35 into the truncated product p25, leading to prolonged Cdk5 activation and altered substrate specificity. Elevated p25 levels have been reported in humans and rodents with neurodegeneration, and the benefit of genetically blocking p25 production has been demonstrated previously in rodent and human neurodegenerative models. Here, we report a 12-amino-acid-long peptide fragment derived from Cdk5 (Cdk5i) that is considerably smaller than existing peptide inhibitors of Cdk5 (P5 and CIP) but shows high binding affinity toward the Cdk5/p25 complex, disrupts the interaction of Cdk5 with p25, and lowers Cdk5/p25 kinase activity. When tagged with a fluorophore (FITC) and the cell-penetrating transactivator of transcription (TAT) sequence, the Cdk5i-FT peptide exhibits cell- and brain-penetrant properties and confers protection against neurodegenerative phenotypes associated with Cdk5 hyperactivity in cell and mouse models of neurodegeneration, highlighting Cdk5i’s therapeutic potential.

Tauopathies are neurodegenerative diseases that are characterized by accumulation of hyperphosphorylated tau protein, higher-order aggregates, and tau filaments. Protein phosphatase 2A (PP2A) is a major tau dephosphorylating phosphatase, and a decrease in its activity has been demonstrated in tauopathies, including Alzheimer’s disease. Prolyl oligopeptidase is a serine protease that is associated with neurodegeneration, and its inhibition normalizes PP2A activity without toxicity under pathological conditions. Here, we assessed whether prolyl oligopeptidase inhibition could protect against tau-mediated toxicity in cellular models in vitro and in the PS19 transgenic mouse model of tauopathy carrying the human tau-P301S mutation. We show that inhibition of prolyl oligopeptidase with the inhibitor KYP-2047 reduced tau aggregation in tau-transfected HEK-293 cells and N2A cells as well as in human iPSC–derived neurons carrying either the P301L or tau-A152T mutation. Treatment with KYP-2047 resulted in increased PP2A activity and activation of autophagic flux in HEK-293 cells and N2A cells and in patient-derived iNeurons, as indicated by changes in autophagosome and autophagy receptor markers; this contributed to clearance of insoluble tau. Furthermore, treatment of PS19 transgenic mice for 1 month with KYP-2047 reduced tau burden in the brain and cerebrospinal fluid and slowed cognitive decline according to several behavioral tests. In addition, a reduction in an oxidative stress marker was seen in mouse brains after KYP-2047 treatment. This study suggests that inhibition of prolyl oligopeptidase could help to ameliorate tau-dependent neurodegeneration.

SUMMARYMicroRNAs (miRNAs) are short RNAs that regulate fundamental biological processes. miR-132, a key miRNA with established functions in Tau homeostasis and neuroprotection, is consistently downregulated in Alzheimer’s disease (AD) and other tauopathies. miR-132 overexpression rescues neurodegenerative phenotypes in several AD models. To complement research on miRNA-mimicking oligonucleotides targeting the central nervous system, we developed a high-throughput-screen coupled high-throughput-sequencing (HTS-HTS) in human induced pluripotent stem cell (iPSC)-derived neurons to identify small molecule inducers of miR-132. We discovered that cardiac glycosides, which are canonical sodium-potassium ATPase inhibitors, selectively upregulated miR-132 in the sub-μM range. Coordinately, cardiac glycoside treatment downregulated total and phosphorylated Tau in rodent and human neurons and protected against toxicity by glutamate, N-methyl-D-aspartate, rotenone, and Aβ oligomers. In conclusion, we identified small-molecule drugs that upregulated the neuroprotective miR-132 and ameliorated neurodegenerative phenotypes. Our dataset also represents a comprehensive resource for discovering small molecules that modulate specific miRNAs for therapeutic purposes.

SummaryAutophagy activation has the potential to ameliorate neurodegenerative disease phenotypes, including protein aggregation, lipid level perturbations and axonal trafficking defects. We performed a high content imaging-based screen assessing 940,000 small molecules to identify those that accelerate lipid droplet clearance. Hits were validated in diverse cell lines and by counter-screening. Of the 77 validated structurally diverse hits, 24 increase autophagy flux. Herein, we highlight CCT020312 as a mammalian target of rapamycin (mTOR) inhibitor-independent autophagy activator, which should function without suppressing the immune response. CCT020312 dose-dependently reduces cytotoxic axonal mutant prion protein aggregate levels within endosomes of primary murine hippocampal neurons and normalizes axonal trafficking deficiencies. Moreover, CCT020312 robustly clears phosphorylated insoluble tau, while reducing tau-mediated neuronal stress vulnerability in patient-derived neuronal models. CCT020312 also restores lysosomal function in neurons differentiated from sporadic Alzheimer’s patients fibroblasts bearing epigenetic marks of aging. Taken together, we describe a promising strategy to uncover novel pharmacological agents that normalize cellular neurodegenerative disease pathology.

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable.

A major obstacle in studying human central nervous system (CNS) diseases is inaccessibility to the affected tissue and cells. Even in limited cases where tissue is available through surgical interventions, differentiated neurons cannot be maintained for extended time frames, which is prohibitive for experimental repetition and scalability. Advances in methodologies for reprogramming human somatic cells into induced pluripotent stem cells (iPSC) and directed differentiation of human neurons in culture now allow access to physiological and disease relevant cell types. In particular, patient iPSC-derived neurons represent unique ex vivo neuronal networks that allow investigating disease genetic and molecular pathways in physiologically accurate cellular microenvironments, importantly recapitulating molecular and cellular phenotypic aspects of disease. Generation of functional neural cells from iPSCs relies on manipulation of culture formats in the presence of specific factors that promote the conversion of pluripotent stem cells into neurons. To this end, several experimental protocols have been developed. Direct differentiation of stem cells into post-mitotic neurons is usually associated with low throughput, low yield, and high technical variability. Instead, methods relying on expansion of the intermediate neural progenitor cells (NPCs) show incredible potential for posterior generation of suitable neuronal cultures for cellular and biochemical assays, as well as drug screening. NPCs are expandable, self-renewable multipotent cells that can differentiate into astrocytes, oligodendrocytes, and electrically active neurons. Here, we describe a protocol for generating iPSC-derived NPCs via formation of neural aggregates and selection of NPC precursor neural rosettes, followed by a simple and reproducible method for generating a mixed population of cortical-like neurons through growth factor withdrawal. Implementation of this protocol has the potential to advance the goals of precision medicine research for both neurological and psychiatric disorders.

Although circular RNAs (circRNAs) are enriched in the brain, their relevance for brain function and psychiatric disorders is poorly understood. Here, we show that circHomer1 is inversely associated with relative HOMER1B mRNA isoform levels in both the orbitofrontal cortex (OFC) and stem-cell-derived neuronal cultures of subjects with psychiatric disorders. We further demonstrate that in vivo circHomer1 knockdown (KD) within the OFC can inhibit the synaptic expression of Homer1b mRNA. Furthermore, we show that circHomer1 directly binds to Homer1b mRNA and that Homer1b-specific KD increases synaptic circHomer1 levels and improves OFC-mediated behavioral flexibility. Importantly, double circHomer1 and Homer1b in vivo co-KD results in a complete rescue in circHomer1-associated alterations in both chance reversal learning and synaptic gene expression. Lastly, we uncover an RNA-binding protein that can directly bind to circHomer1 and promote its biogenesis. Taken together, our data provide mechanistic insights into the importance of circRNAs in brain function and disease.