Defining RNA oligonucleotides that reverse deleterious phase transitions of RNA-binding proteins with prion-like domains.

RNA-binding proteins with prion-like domains, such as FUS and TDP-43, condense into functional liquids, which can transform into pathological fibrils that underpin fatal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Here, we define short RNAs (24-48 nucleotides) that prevent FUS fibrillization by promoting liquid phases, and distinct short RNAs that prevent and, remarkably, reverse FUS condensation and fibrillization. These activities require interactions with multiple RNA-binding domains of FUS and are encoded by RNA sequence, length, and structure. Importantly, we define a short RNA that dissolves aberrant cytoplasmic FUS condensates, restores nuclear FUS, and mitigates FUS proteotoxicity in optogenetic models and human motor neurons. Another short RNA dissolves aberrant cytoplasmic TDP-43 condensates, restores nuclear TDP-43, and mitigates TDP-43 proteotoxicity. Since short RNAs can be effectively delivered to the human brain, these oligonucleotides could have therapeutic utility for ALS/FTD and related disorders.

Loss of HIPK2 Protects Neurons from Mitochondrial Toxins by Regulating Parkin Protein Turnover.

Mitochondria are important sources of energy, but they are also the target of cellular stress, toxin exposure, and aging-related injury. Persistent accumulation of damaged mitochondria has been implicated in many neurodegenerative diseases. One highly conserved mechanism to clear damaged mitochondria involves the E3 ubiquitin ligase Parkin and PTEN-induced kinase 1 (PINK1), which cooperatively initiate the process called mitophagy that identifies and eliminates damaged mitochondria through the autophagosome and lysosome pathways. Parkin is a mostly cytosolic protein, but is rapidly recruited to damaged mitochondria and target them for mitophagy. Moreover, Parkin interactomes also involve signaling pathways and transcriptional machinery critical for survival and cell death. However, the mechanism that regulates Parkin protein level remains poorly understood. Here, we show that the loss of homeodomain interacting protein kinase 2 (HIPK2) in neurons and mouse embryonic fibroblasts (MEFs) has a broad protective effect from cell death induced by mitochondrial toxins. The mechanism by which Hipk2-/- neurons and MEFs are more resistant to mitochondrial toxins is in part due to the role of HIPK2 and its kinase activity in promoting Parkin degradation via the proteasome-mediated mechanism. The loss of HIPK2 leads to higher cytosolic Parkin protein levels at basal conditions and upon exposure to mitochondrial toxins, which protects mitochondria from toxin-induced damage. In addition, Hipk2-/- neurons and MEFs show increased expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1), a Parkin downstream target that can provide additional benefits via transcriptional activation of mitochondrial genes. Together, these results reveal a previously unrecognized avenue to target HIPK2 in neuroprotection via the Parkin-mediated pathway.SIGNIFICANCE STATEMENT In this study, we provide evidence that homeodomain interacting protein kinase 2 (HIPK2) and its kinase activity promote Parkin degradation via the proteasome-mediated pathway. The loss of HIPK2 increases cytosolic and mitochondrial Parkin protein levels under basal conditions and upon exposure to mitochondrial toxins, which protect mitochondria from toxin-induced damage. In addition, Hipk2-/- neurons and mouse embryonic fibroblasts also show increased expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1), a Parkin downstream target that can provide additional benefits via transcriptional activation of mitochondrial genes. These results indicate that targeting HIPK2 and its kinase activity can have neuroprotective effects by elevating Parkin protein levels.

HIPK2-Mediated Transcriptional Control of NMDA Receptor Subunit Expression Regulates Neuronal Survival and Cell Death.

NMDA receptors are critical for neuronal communication. Dysfunction in NMDA receptors has been implicated in neuropsychiatric diseases. While it is well recognized that the composition of NMDA receptors undergoes a GluN2B-to-GluN2A switch in early postnatal life, the mechanism regulating this switch remains unclear. Using transcriptomic and functional analyses in brain tissues from male and female Hipk2+/+ and Hipk2-/- mice, we showed that the HIPK2-JNK-c-Jun pathway is important in suppressing the transcription of Grin2a and Grin2c, which encodes the GluN2A and GluN2C subunits of the NMDA receptors, respectively. Loss of HIPK2 leads to a significant decrease in JNK-c-Jun signaling, which in turn derepresses the transcription of Grin2a and Grin2c mRNA and upregulates GluN2A and GluN2C protein levels. These changes result in a significant increase of GluN2A/GluN2B ratio in synapse and mitochondria, a persistent activation of the ERK-CREB pathway and the upregulation of synaptic activity-regulated genes, which collectively contribute to the resistance of Hipk2-/- neurons to cell death induced by mitochondrial toxins.SIGNIFICANCE STATEMENT We identify HIPK2-JNK-c-Jun signaling as a key mechanism that regulates the transcription of NMDA receptor subunits GluN2A and GluN2C in vivo Our results provide insights into a previously unrecognized molecular mechanism that control the switch of NMDA receptor subunits in early postnatal brain development. Furthermore, we provide evidence that changes in the ratio of NMDA subunits GluN2A/GluN2B can also be detected in the synapse and mitochondria, which contributes to a persistent activation of the prosurvival ERK-CREB pathway and its downstream target genes. Collectively, these changes protect HIPK2 deficient neurons from mitochondrial toxins.

TGF-ß Signaling in Dopaminergic Neurons Regulates Dendritic Growth, Excitatory-Inhibitory Synaptic Balance, and Reversal Learning.

Neural circuits involving midbrain dopaminergic (DA) neurons regulate reward and goal-directed behaviors. Although local GABAergic input is known to modulate DA circuits, the mechanism that controls excitatory/inhibitory synaptic balance in DA neurons remains unclear. Here, we show that DA neurons use autocrine transforming growth factor ß (TGF-ß) signaling to promote the growth of axons and dendrites. Surprisingly, removing TGF-ß type II receptor in DA neurons also disrupts the balance in TGF-ß1 expression in DA neurons and neighboring GABAergic neurons, which increases inhibitory input, reduces excitatory synaptic input, and alters phasic firing patterns in DA neurons. Mice lacking TGF-ß signaling in DA neurons are hyperactive and exhibit inflexibility in relinquishing learned behaviors and re-establishing new stimulus-reward associations. These results support a role for TGF-ß in regulating the delicate balance of excitatory/inhibitory synaptic input in local microcircuits involving DA and GABAergic neurons and its potential contributions to neuropsychiatric disorders.

Activation of HIPK2 Promotes ER Stress-Mediated Neurodegeneration in Amyotrophic Lateral Sclerosis.

Persistent accumulation of misfolded proteins causes endoplasmic reticulum (ER) stress, a prominent feature in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here we report the identification of homeodomain interacting protein kinase 2 (HIPK2) as the essential link that promotes ER-stress-induced cell death via the IRE1α-ASK1-JNK pathway. ER stress, induced by tunicamycin or SOD1(G93A), activates HIPK2 by phosphorylating highly conserved serine and threonine residues (S359/T360) within the activation loop of the HIPK2 kinase domain. In SOD1(G93A) mice, loss of HIPK2 delays disease onset, reduces cell death in spinal motor neurons, mitigates glial pathology, and improves survival. Remarkably, HIPK2 activation positively correlates with TDP-43 proteinopathy in NEFH-tTA/tetO-hTDP-43ΔNLS mice, sporadic ALS and C9ORF72 ALS, and blocking HIPK2 kinase activity protects motor neurons from TDP-43 cytotoxicity. These results reveal a previously unrecognized role of HIPK2 activation in ER-stress-mediated neurodegeneration and its potential role as a biomarker and therapeutic target for ALS. VIDEO ABSTRACT.

Progranulin Deficiency Promotes Circuit-Specific Synaptic Pruning by Microglia via Complement Activation.

Microglia maintain homeostasis in the brain, but whether aberrant microglial activation can cause neurodegeneration remains controversial. Here, we use transcriptome profiling to demonstrate that deficiency in frontotemporal dementia (FTD) gene progranulin (Grn) leads to an age-dependent, progressive upregulation of lysosomal and innate immunity genes, increased complement production, and enhanced synaptic pruning in microglia. During aging, Grn(-/-) mice show profound microglia infiltration and preferential elimination of inhibitory synapses in the ventral thalamus, which lead to hyperexcitability in the thalamocortical circuits and obsessive-compulsive disorder (OCD)-like grooming behaviors. Remarkably, deleting C1qa gene significantly reduces synaptic pruning by Grn(-/-) microglia and mitigates neurodegeneration, behavioral phenotypes, and premature mortality in Grn(-/-) mice. Together, our results uncover a previously unrecognized role of progranulin in suppressing aberrant microglia activation during aging. These results represent an important conceptual advance that complement activation and microglia-mediated synaptic pruning are major drivers, rather than consequences, of neurodegeneration caused by progranulin deficiency.

Mechanisms of FUS mutations in familial amyotrophic lateral sclerosis.

Recent advances in the genetics of amyotrophic lateral sclerosis (ALS) have provided key mechanistic insights to the pathogenesis of this devastating neurodegenerative disease. Among many etiologies for ALS, the identification of mutations and proteinopathies in two RNA binding proteins, TDP-43 (TARDBP or TAR DNA binding protein 43) and its closely related RNA/DNA binding protein FUS (fused in sarcoma), raises the intriguing possibility that perturbations to the RNA homeostasis and metabolism in neurons may contribute to the pathogenesis of these diseases. Although the similarities between TDP-43 and FUS suggest that mutations and proteinopathy involving these two proteins may converge on the same mechanisms leading to neurodegeneration, there is increasing evidence that FUS mutations target distinct mechanisms to cause early disease onset and aggressive progression of disease. This review focuses on the recent advances on the molecular, cellular and genetic approaches to uncover the mechanisms of wild type and mutant FUS proteins during development and in neurodegeneration. These findings provide important insights to understand how FUS mutations may perturb the maintenance of dendrites through fundamental processes in RNA splicing, RNA transport and DNA damage response/repair. These results contribute to the understanding of phenotypic manifestations in neurodegeneration related to FUS mutations, and to identify important directions for future investigations. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.

ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects.

Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf) as a target of FUS-R521C-associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions.

Transcriptional corepressors HIPK1 and HIPK2 control angiogenesis via TGF-ß-TAK1-dependent mechanism.

Several critical events dictate the successful establishment of nascent vasculature in yolk sac and in the developing embryos. These include aggregation of angioblasts to form the primitive vascular plexus, followed by the proliferation, differentiation, migration, and coalescence of endothelial cells. Although transforming growth factor-ß (TGF-ß) is known to regulate various aspects of vascular development, the signaling mechanism of TGF-ß remains unclear. Here we show that homeodomain interacting protein kinases, HIPK1 and HIPK2, are transcriptional corepressors that regulate TGF-ß-dependent angiogenesis during embryonic development. Loss of HIPK1 and HIPK2 leads to marked up-regulations of several potent angiogenic genes, including Mmp10 and Vegf, which result in excessive endothelial proliferation and poor adherens junction formation. This robust phenotype can be recapitulated by siRNA knockdown of Hipk1 and Hipk2 in human umbilical vein endothelial cells, as well as in endothelial cell-specific TGF-ß type II receptor (TßRII) conditional mutants. The effects of HIPK proteins are mediated through its interaction with MEF2C, and this interaction can be further enhanced by TGF-ß in a TAK1-dependent manner. Remarkably, TGF-ß-TAK1 signaling activates HIPK2 by phosphorylating a highly conserved tyrosine residue Y-361 within the kinase domain. Point mutation in this tyrosine completely eliminates the effect of HIPK2 as a transcriptional corepressor in luciferase assays. Our results reveal a previously unrecognized role of HIPK proteins in connecting TGF-ß signaling pathway with the transcriptional programs critical for angiogenesis in early embryonic development.

Homeodomain interacting protein kinase 2 regulates postnatal development of enteric dopaminergic neurons and glia via BMP signaling.

Trophic factor signaling is important for the migration, differentiation, and survival of enteric neurons during development. The mechanisms that regulate the maturation of enteric neurons in postnatal life, however, are poorly understood. Here, we show that transcriptional cofactor HIPK2 (homeodomain interacting protein kinase 2) is required for the maturation of enteric neurons and for regulating gliogenesis during postnatal development. Mice lacking HIPK2 display a spectrum of gastrointestinal (GI) phenotypes, including distention of colon and slowed GI transit time. Although loss of HIPK2 does not affect the enteric neurons in prenatal development, a progressive loss of enteric neurons occurs during postnatal life in Hipk2(-/-) mutant mice that preferentially affects the dopaminergic population of neurons in the caudal region of the intestine. The mechanism by which HIPK2 regulates postnatal enteric neuron development appears to involve the response of enteric neurons to bone morphogenetic proteins (BMPs). Specifically, compared to wild type mice, a larger proportion of enteric neurons in Hipk2(-/-) mutants have an abnormally high level of phosphorylated Smad1/5/8. Consistent with the ability of BMP signaling to promote gliogenesis, Hipk2(-/-) mutants show a significant increase in glia in the enteric nervous system. In addition, numbers of autophagosomes are increased in enteric neurons in Hipk2(-/-) mutants, and synaptic maturation is arrested. These results reveal a new role for HIPK2 as an important transcriptional cofactor that regulates the BMP signaling pathway in the maintenance of enteric neurons and glia, and further suggest that HIPK2 and its associated signaling mechanisms may be therapeutically altered to promote postnatal neuronal maturation.

Firefly luciferase complementation imaging assay for protein-protein interactions in plants.

The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The previously described techniques, fluorescence resonance energy transfer and bimolecular fluorescence complementation, which are used widely for protein-protein interaction studies in plants, require extensive instrumentation. To facilitate protein-protein interaction studies in plants, we adopted the luciferase complementation imaging assay. The amino-terminal and carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins, and that can be visualized with a low-light imaging system. A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants. We tested nine pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, components of disease resistance protein complex, and transcription factors. In each case, strong luciferase complementation was observed for positive interactions. Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity. Thus, the assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants.

RAR1, a central player in plant immunity, is targeted by Pseudomonas syringae effector AvrB.

Pathogenic bacterial effectors suppress pathogen-associated molecular pattern (PAMP)-triggered host immunity, thereby promoting parasitism. In the presence of cognate resistance genes, it is proposed that plants detect the virulence activity of bacterial effectors and trigger a defense response, referred to here as effector-triggered immunity (ETI). However, the link between effector virulence and ETI at the molecular level is unknown. Here, we show that the Pseudomonas syringae effector AvrB suppresses PAMP-triggered immunity (PTI) through RAR1, a co-chaperone of HSP90 required for ETI. AvrB expressed in plants lacking the cognate resistance gene RPM1 suppresses cell wall defense induced by the flagellar peptide flg22, a well known PAMP, and promotes the growth of nonpathogenic bacteria in a RAR1-dependent manner. rar1 mutants display enhanced cell wall defense in response to flg22, indicating that RAR1 negatively regulates PTI. Furthermore, coimmunoprecipitation experiments indicated that RAR1 and AvrB interact in the plant. The results demonstrate that RAR1 molecularly links PTI, effector virulence, and ETI. The study supports that both pathogen virulence and plant disease resistance have evolved around PTI.