Amy A. Tang

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.

HIPK2 represses β-catenin-mediated transcription, epidermal stem cell expansion, and skin tumorigenesis

Transcriptional control by β-catenin and lymphoid enhancer-binding factor 1 (LEF1)/T cell factor regulates proliferation in stem cells and tumorigenesis. Here we provide evidence that transcriptional co repressor homeodomain interacting protein kinase 2 (HIPK2) controls the number of stem and progenitor cells in the skin and the susceptibility to develop squamous cell carcinoma. Loss of HIPK2 leads to increased proliferative potential, more rapid G1–S transition in cell cycle, and expansion of the epidermal stem cell compartment. Among the critical regulators of G1–S transition in the cell cycle, only cyclin D1 is selectively up-regulated in cells lacking HIPK2. Conversely, overexpression of HIPK2 suppresses LEF1/β-catenin-mediated transcriptional activation of cyclin D1 expression. However, deletion of the C-terminal YH domain of HIPK2 completely abolishes its ability to recruit another transcriptional corepressor CtBP and suppress LEF1/β-catenin-mediated transcription. To determine whether loss of HIPK2 leads to increased susceptibility to tumorigenesis, we treat wild-type, Hipk2+/−, andHipk2−/− mice with the two-stage carcinogenesis protocol. Our results indicate that more skin tumors are induced in Hipk2+/− and Hipk2−/− mutants, with most of the tumors showing shortened incubation time and malignant progression. Together, our results indicate that HIPK2 is a tumor suppressor that controls proliferation by antagonizing LEF1/β-catenin-mediated transcription. Loss of HIPK2 synergizes with activation of H-ras to induce tumorigenesis.

Interaction of Brn3a and HIPK2 mediates transcriptional repression of sensory neuron survival.

The Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-x L. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

Activity-dependent FUS dysregulation disrupts synaptic homeostasis

Significance Both overexpression of wild-type fused in sarcoma (FUS) protein and missense mutations can be pathogenic in a group of related neurodegenerative disorders that includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. It is unclear how FUS overexpression and missense mutations cause disease in human patients. In this work, we generated novel transgenic mouse models expressing low levels of wild-type and mutant human FUS, both of which recapitulate aspects of the human diseases. We found a profound difference in the underlying mechanisms by which missense mutation and wild-type overexpression cause disease. Overexpression of wild-type FUS protein alters its nuclear function at the level of gene expression. In contrast, missense mutation disrupts activity-dependent synaptic homeostasis to gain a toxic function at dendritic spines. The RNA-binding protein fused-in-sarcoma (FUS) has been associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two neurodegenerative disorders that share similar clinical and pathological features. Both missense mutations and overexpression of wild-type FUS protein can be pathogenic in human patients. To study the molecular and cellular basis by which FUS mutations and overexpression cause disease, we generated novel transgenic mice globally expressing low levels of human wild-type protein (FUSWT) and a pathological mutation (FUSR521G). FUSWT and FUSR521G mice that develop severe motor deficits also show neuroinflammation, denervated neuromuscular junctions, and premature death, phenocopying the human diseases. A portion of FUSR521G mice escape early lethality; these escapers have modest motor impairments and altered sociability, which correspond with a reduction of dendritic arbors and mature spines. Remarkably, only FUSR521G mice show dendritic defects; FUSWT mice do not. Activation of metabotropic glutamate receptors 1/5 in neocortical slices and isolated synaptoneurosomes increases endogenous mouse FUS and FUSWT protein levels but decreases the FUSR521G protein, providing a potential biochemical basis for the dendritic spine differences between FUSWT and FUSR521G mice.

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.

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.

Dysmyelination not demyelination causes neurological symptoms in preweaned mice in a murine model of Cockayne syndrome

Cockayne syndrome (CS) is a rare autosomal recessive neurodegenerative disease that is associated with mutations in either of two transcription-coupled DNA repair genes, CSA or CSB. Mice with a targeted mutation in the Csb gene (Cs-bm/m) exhibit a milder phenotype compared with human patients with mutations in the orthologous CSB gene. Mice mutated in Csb were crossed with mice lacking Xpc (Xp-c−/−), the global genome repair gene, to enhance the pathological symptoms. These Cs-bm/m.Xp-c−/− mice were normal at birth but exhibited progressive failure to thrive, whole-body wasting, and ataxia and died at approximately postnatal day 21. Characterization of Cs-bm/m.Xp-c−/− brains at postnatal stages demonstrated widespread reduction of myelin basic protein (MBP) and myelin in the sensorimotor cortex, the stratum radiatum, the corpus callosum, and the anterior commissure. Quantification of individual axons by electron microscopy showed a reduction in both the number of myelinated axons and the average diameter of myelin surrounding the axons. There were no significant differences in proliferation or oligodendrocyte differentiation between Cs-bm/m.Xp-c−/− and Cs-bm/+.Xp-c−/− mice. Rather, Cs-bm/m.Xp-c−/− oligodendrocytes were unable to generate sufficient MBP or to maintain the proper myelination during early development. Csb is a multifunctional protein regulating both repair and the transcriptional response to reactive oxygen through its interaction with histone acetylase p300 and the hypoxia-inducible factor (HIF)1 pathway. On the basis of our results, combined with that of others, we suggest that in Csb the transcriptional response predominates during early development, whereas a neurodegenerative response associated with repair deficits predominates in later life.

Transcriptional Corepressors HIPK1 and HIPK2 Control Angiogenesis Via TGF-β–TAK1–Dependent Mechanism

During angiogenesis, endothelial cells in nascent blood vessels use the transcriptional cofactors HIPK1 and HIPK2 to transduce TGF-β signals and control cell proliferation and adhesion.

Suppression of C9orf72 RNA repeat-induced neurotoxicity by the ALS-associated RNA-binding protein Zfp106

Expanded GGGGCC repeats in the first intron of the C9orf72 gene represent the most common cause of familial amyotrophic lateral sclerosis (ALS), but the mechanisms underlying repeat-induced disease remain incompletely resolved. One proposed gain-of-function mechanism is that repeat-containing RNA forms aggregates that sequester RNA binding proteins, leading to altered RNA metabolism in motor neurons. Here, we identify the zinc finger protein Zfp106 as a specific GGGGCC RNA repeat-binding protein, and using affinity purification-mass spectrometry, we show that Zfp106 interacts with multiple other RNA binding proteins, including the ALS-associated factors TDP-43 and FUS. We also show that Zfp106 knockout mice develop severe motor neuron degeneration, which can be suppressed by transgenic restoration of Zfp106 specifically in motor neurons. Finally, we show that Zfp106 potently suppresses neurotoxicity in a Drosophila model of C9orf72 ALS. Thus, these studies identify Zfp106 as an RNA binding protein with important implications for ALS. DOI: http://dx.doi.org/10.7554/eLife.19032.001

An RNA interference screen identifies druggable regulators of MeCP2 stability

Inhibition of proteins that stabilize MeCP2 is a potential strategy to treat MECP2 duplication syndrome. An HIP strategy for treating MeCP2 disorders The role of altered gene dosage is increasingly being recognized in neuropsychiatric disorders and intellectual disability (ID). Even a twofold change in the dose of methyl-CpG–binding protein 2 (MeCP2)—either increased or decreased—results in distinct disorders with overlapping features including ID, autistic behavior, and severe motor dysfunction. In a new work, Lombardi et al. identified four regulators of MeCP2 stability and validated regulation of MeCP2 by PP2A and HIPK2 in vivo. They then demonstrated that pharmacological inhibition of PP2A was sufficient to partially rescue MeCP2 overexpression and certain motor abnormalities in a mouse model of MECP2 duplication syndrome. Alterations in gene dosage due to copy number variation are associated with autism spectrum disorder, intellectual disability (ID), and other psychiatric disorders. The nervous system is so acutely sensitive to the dose of methyl-CpG–binding protein 2 (MeCP2) that even a twofold change in MeCP2 protein—either increased or decreased—results in distinct disorders with overlapping features including ID, autistic behavior, and severe motor dysfunction. Rett syndrome is caused by loss-of-function mutations in MECP2, whereas duplications spanning the MECP2 locus result in MECP2 duplication syndrome (MDS), which accounts for ~1% of X-linked ID. Despite evidence from mouse models that restoring MeCP2 can reverse the course of disease, there are currently no U.S. Food and Drug Administration–approved therapies available to clinically modulate MeCP2 abundance. We used a forward genetic screen against all known human kinases and phosphatases to identify druggable regulators of MeCP2 stability. Two putative modulators of MeCP2, HIPK2 (homeodomain-interacting protein kinase 2) and PP2A (protein phosphatase 2A), were validated as stabilizers of MeCP2 in vivo. Further, pharmacological inhibition of PP2A in vivo reduced MeCP2 in the nervous system and rescued both overexpression and motor abnormalities in a mouse model of MDS. Our findings reveal potential therapeutic targets for treating disorders of altered MECP2 dosage.