Patient-derived neuron model: Capturing age-dependent adult-onset degenerative pathology in Huntington's disease.

MicroRNAs play a crucial role in directly reprogramming (converting) human fibroblasts into neurons. Specifically, miR-9/9* and miR-124 (miR-9/9*-124) display neurogenic and cell fate-switching activities when ectopically expressed in human fibroblasts by erasing fibroblast identity and inducing a pan-neuronal state. These converted neurons maintain the biological age of the starting fibroblasts and thus provide a human neuron-based platform to study cellular properties in aged neurons and model adult-onset neurodegenerative disorders using patient-derived cells. Furthermore, the expression of striatal-enriched transcription factors in conjunction with miR-9/9*-124 guides the identity of medium spiny neurons (MSNs), the primary targets in Huntington's disease (HD). Converted MSNs from HD patient-derived fibroblasts (HD-MSNs) can replicate HD-related phenotypes including neurodegeneration associated with age-related declines in critical cellular functions such as autophagy. Here, we review the role of microRNAs in the direct conversion of patient-derived fibroblasts into MSNs and the practical application of converted HD-MSNs as a model for studying adult-onset neuropathology in HD. We provide valuable insights into age-related, cell-intrinsic changes contributing to neurodegeneration in HD-MSNs. Ultimately, we address a comprehensive understanding of the complex molecular landscape underlying HD pathology, offering potential avenues for therapeutic application.

Longitudinal modeling of human neuronal aging reveals the contribution of the RCAN1-TFEB pathway to Huntington's disease neurodegeneration.

Aging is a common risk factor in neurodegenerative disorders. Investigating neuronal aging in an isogenic background stands to facilitate analysis of the interplay between neuronal aging and neurodegeneration. Here we perform direct neuronal reprogramming of longitudinally collected human fibroblasts to reveal genetic pathways altered at different ages. Comparative transcriptome analysis of longitudinally aged striatal medium spiny neurons (MSNs) in Huntington's disease identified pathways involving RCAN1, a negative regulator of calcineurin. Notably, RCAN1 protein increased with age in reprogrammed MSNs as well as in human postmortem striatum and RCAN1 knockdown rescued patient-derived MSNs of Huntington's disease from degeneration. RCAN1 knockdown enhanced chromatin accessibility of genes involved in longevity and autophagy, mediated through enhanced calcineurin activity, leading to TFEB's nuclear localization by dephosphorylation. Furthermore, G2-115, an analog of glibenclamide with autophagy-enhancing activities, reduced the RCAN1-calcineurin interaction, phenocopying the effect of RCAN1 knockdown. Our results demonstrate that targeting RCAN1 genetically or pharmacologically can increase neuronal resilience in Huntington's disease.

Longitudinal modeling of human neuronal aging identifies RCAN1-TFEB pathway contributing to neurodegeneration of Huntington's disease.

Aging is a common risk factor in neurodegenerative disorders and the ability to investigate aging of neurons in an isogenic background would facilitate discovering the interplay between neuronal aging and onset of neurodegeneration. Here, we perform direct neuronal reprogramming of longitudinally collected human fibroblasts to reveal genetic pathways altered at different ages. Comparative transcriptome analysis of longitudinally aged striatal medium spiny neurons (MSNs), a primary neuronal subtype affected in Huntington's disease (HD), identified pathways associated with RCAN1, a negative regulator of calcineurin. Notably, RCAN1 undergoes age-dependent increase at the protein level detected in reprogrammed MSNs as well as in human postmortem striatum. In patient-derived MSNs of adult-onset HD (HD-MSNs), counteracting RCAN1 by gene knockdown (KD) rescued HD-MSNs from degeneration. The protective effect of RCAN1 KD was associated with enhanced chromatin accessibility of genes involved in longevity and autophagy, mediated through enhanced calcineurin activity, which in turn dephosphorylates and promotes nuclear localization of TFEB transcription factor. Furthermore, we reveal that G2-115 compound, an analog of glibenclamide with autophagy-enhancing activities, reduces the RCAN1-Calcineurin interaction, phenocopying the effect of RCAN1 KD. Our results demonstrate that RCAN1 is a potential genetic or pharmacological target whose reduction-of-function increases neuronal resilience to neurodegeneration in HD through chromatin reconfiguration.

Modeling Huntington disease through microRNA-mediated neuronal reprogramming identifies age-associated autophagy dysfunction driving the onset of neurodegeneration.

Huntington disease (HD) is an inherited neurodegenerative disease with adult-onset clinical symptoms. However, the mechanism by which aging triggers the onset of neurodegeneration in HD patients remains unclear. Modeling the age-dependent progression of HD with striatal medium spiny neurons (MSNs) generated by direct reprogramming of fibroblasts from HD patients at different disease stages identifies age-dependent decline in critical cellular functions such as autophagy/macroautophagy and onset of neurodegeneration. Mechanistically, MSNs derived from symptomatic HD patients (HD-MSNs) are characterized by increased chromatin accessibility proximal to the MIR29B-3p host gene and its upregulation compared to MSNs from younger pre-symptomatic patients. MIR29B-3p in turn targets and represses STAT3 (signal transducer and activator of transcription 3) that controls the biogenesis of autophagosomes, leading to HD-MSN degeneration. Our recent study demonstrates age-associated microRNA (miRNA) and autophagy dysregulation linked to MSN degeneration, and potential approaches for protecting MSNs by enhancing autophagy in HD.Abbreviations: HD: Huntington disease; mHTT: mutant HTT; MIR9/9*-124: MIR9/9* and MIR124; miRNA: microRNA; MSN: medium spiny neuron; STAT3: signal transducer and activator of transcription 3.

Age-related Huntington's disease progression modeled in directly reprogrammed patient-derived striatal neurons highlights impaired autophagy.

Huntington's disease (HD) is an inherited neurodegenerative disorder with adult-onset clinical symptoms, but the mechanism by which aging drives the onset of neurodegeneration in patients with HD remains unclear. In this study we examined striatal medium spiny neurons (MSNs) directly reprogrammed from fibroblasts of patients with HD to model the age-dependent onset of pathology. We found that pronounced neuronal death occurred selectively in reprogrammed MSNs from symptomatic patients with HD (HD-MSNs) compared to MSNs derived from younger, pre-symptomatic patients (pre-HD-MSNs) and control MSNs from age-matched healthy individuals. We observed age-associated alterations in chromatin accessibility between HD-MSNs and pre-HD-MSNs and identified miR-29b-3p, whose age-associated upregulation promotes HD-MSN degeneration by impairing autophagic function through human-specific targeting of the STAT3 3' untranslated region. Reducing miR-29b-3p or chemically promoting autophagy increased the resilience of HD-MSNs against neurodegeneration. Our results demonstrate miRNA upregulation with aging in HD as a detrimental process driving MSN degeneration and potential approaches for enhancing autophagy and resilience of HD-MSNs.

Voltage-Independent SK-Channel Dysfunction Causes Neuronal Hyperexcitability in the Hippocampus of Fmr1 Knock-Out Mice.

Neuronal hyperexcitability is one of the major characteristics of fragile X syndrome (FXS), yet the molecular mechanisms of this critical dysfunction remain poorly understood. Here we report a major role of voltage-independent potassium (K+)-channel dysfunction in hyperexcitability of CA3 pyramidal neurons in Fmr1 knock-out (KO) mice. We observed a reduction of voltage-independent small conductance calcium (Ca2+)-activated K+ (SK) currents in both male and female mice, leading to decreased action potential (AP) threshold and reduced medium afterhyperpolarization. These SK-channel-dependent deficits led to markedly increased AP firing and abnormal input-output signal transmission of CA3 pyramidal neurons. The SK-current defect was mediated, at least in part, by loss of FMRP interaction with the SK channels (specifically the SK2 isoform), without changes in channel expression. Intracellular application of selective SK-channel openers or a genetic reintroduction of an N-terminal FMRP fragment lacking the ability to associate with polyribosomes normalized all observed excitability defects in CA3 pyramidal neurons of Fmr1 KO mice. These results suggest that dysfunction of voltage-independent SK channels is the primary cause of CA3 neuronal hyperexcitability in Fmr1 KO mice and support the critical translation-independent role for the fragile X mental retardation protein as a regulator of neural excitability. Our findings may thus provide a new avenue to ameliorate hippocampal excitability defects in FXS.SIGNIFICANCE STATEMENT Despite two decades of research, no effective treatment is currently available for fragile X syndrome (FXS). Neuronal hyperexcitability is widely considered one of the hallmarks of FXS. Excitability research in the FXS field has thus far focused primarily on voltage-gated ion channels, while contributions from voltage-independent channels have been largely overlooked. Here we report that voltage-independent small conductance calcium-activated potassium (SK)-channel dysfunction causes hippocampal neuron hyperexcitability in the FXS mouse model. Our results support the idea that translation-independent function of fragile X mental retardation protein has a major role in regulating ion-channel activity, specifically the SK channels, in hyperexcitability defects in FXS. Our findings may thus open a new direction to ameliorate hippocampal excitability defects in FXS.

Epigenetic regulator UHRF1 inactivates REST and growth suppressor gene expression via DNA methylation to promote axon regeneration.

Injured peripheral sensory neurons switch to a regenerative state after axon injury, which requires transcriptional and epigenetic changes. However, the roles and mechanisms of gene inactivation after injury are poorly understood. Here, we show that DNA methylation, which generally leads to gene silencing, is required for robust axon regeneration after peripheral nerve lesion. Ubiquitin-like containing PHD ring finger 1 (UHRF1), a critical epigenetic regulator involved in DNA methylation, increases upon axon injury and is required for robust axon regeneration. The increased level of UHRF1 results from a decrease in miR-9. The level of another target of miR-9, the transcriptional regulator RE1 silencing transcription factor (REST), transiently increases after injury and is required for axon regeneration. Mechanistically, UHRF1 interacts with DNA methyltransferases (DNMTs) and H3K9me3 at the promoter region to repress the expression of the tumor suppressor gene phosphatase and tensin homolog (PTEN) and REST. Our study reveals an epigenetic mechanism that silences tumor suppressor genes and restricts REST expression in time after injury to promote axon regeneration.

MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts.

The ability to convert human somatic cells efficiently to neurons facilitates the utility of patient-derived neurons for studying neurological disorders. As such, ectopic expression of neuronal microRNAs (miRNAs), miR-9/9∗ and miR-124 (miR-9/9∗-124) in adult human fibroblasts has been found to evoke extensive reconfigurations of the chromatin and direct the fate conversion to neurons. However, how miR-9/9∗-124 break the cell fate barrier to activate the neuronal program remains to be defined. Here, we identified an anti-neurogenic function of EZH2 in fibroblasts that acts outside its role as a subunit of Polycomb Repressive Complex 2 to directly methylate and stabilize REST, a transcriptional repressor of neuronal genes. During neuronal conversion, miR-9/9∗-124 induced the repression of the EZH2-REST axis by downregulating USP14, accounting for the opening of chromatin regions harboring REST binding sites. Our findings underscore the interplay between miRNAs and protein stability cascade underlying the activation of neuronal program.

Erratum: Modulation of mRNA and lncRNA expression dynamics by the Set2-Rpd3S pathway.

This corrects the article DOI: 10.1038/ncomms13534.

Modulation of mRNA and lncRNA expression dynamics by the Set2-Rpd3S pathway.

H3K36 methylation by Set2 targets Rpd3S histone deacetylase to transcribed regions of mRNA genes, repressing internal cryptic promoters and slowing elongation. Here we explore the function of this pathway by analysing transcription in yeast undergoing a series of carbon source shifts. Approximately 80 mRNA genes show increased induction upon SET2 deletion. A majority of these promoters have overlapping lncRNA transcription that targets H3K36me3 and deacetylation by Rpd3S to the mRNA promoter. We previously reported a similar mechanism for H3K4me2-mediated repression via recruitment of the Set3C histone deacetylase. Here we show that the distance between an mRNA and overlapping lncRNA promoter determines whether Set2-Rpd3S or Set3C represses. This analysis also reveals many previously unreported cryptic ncRNAs induced by specific carbon sources, showing that cryptic promoters can be environmentally regulated. Therefore, in addition to repression of cryptic transcription and modulation of elongation, H3K36 methylation maintains optimal expression dynamics of many mRNAs and ncRNAs.

Activating Injury-Responsive Genes with Hypoxia Enhances Axon Regeneration through Neuronal HIF-1α.

Injured peripheral neurons successfully activate a proregenerative transcriptional program to enable axon regeneration and functional recovery. How transcriptional regulators coordinate the expression of such program remains unclear. Here we show that hypoxia-inducible factor 1α (HIF-1α) controls multiple injury-induced genes in sensory neurons and contribute to the preconditioning lesion effect. Knockdown of HIF-1α in vitro or conditional knock out in vivo impairs sensory axon regeneration. The HIF-1α target gene Vascular Endothelial Growth Factor A (VEGFA) is expressed in injured neurons and contributes to stimulate axon regeneration. Induction of HIF-1α using hypoxia enhances axon regeneration in vitro and in vivo in sensory neurons. Hypoxia also stimulates motor neuron regeneration and accelerates neuromuscular junction re-innervation. This study demonstrates that HIF-1α represents a critical transcriptional regulator in regenerating neurons and suggests hypoxia as a tool to stimulate axon regeneration.

Independent role for presynaptic FMRP revealed by an FMR1 missense mutation associated with intellectual disability and seizures.

Fragile X syndrome (FXS) results in intellectual disability (ID) most often caused by silencing of the fragile X mental retardation 1 (FMR1) gene. The resulting absence of fragile X mental retardation protein 1 (FMRP) leads to both pre- and postsynaptic defects, yet whether the pre- and postsynaptic functions of FMRP are independent and have distinct roles in FXS neuropathology remain poorly understood. Here, we demonstrate an independent presynaptic function for FMRP through the study of an ID patient with an FMR1 missense mutation. This mutation, c.413G > A (R138Q), preserves FMRP's canonical functions in RNA binding and translational regulation, which are traditionally associated with postsynaptic compartments. However, neuronally driven expression of the mutant FMRP is unable to rescue structural defects at the neuromuscular junction in fragile x mental retardation 1 (dfmr1)-deficient Drosophila, suggesting a presynaptic-specific impairment. Furthermore, mutant FMRP loses the ability to rescue presynaptic action potential (AP) broadening in Fmr1 KO mice. The R138Q mutation also disrupts FMRP's interaction with the large-conductance calcium-activated potassium (BK) channels that modulate AP width. These results reveal a presynaptic- and translation-independent function of FMRP that is linked to a specific subset of FXS phenotypes.

USP8 modulates ubiquitination of LRIG1 for Met degradation.

The Met receptor tyrosine kinase is an attractive target for cancer therapy as it promotes invasive tumor growth. SAIT301 is a novel anti-Met antibody, which induces LRIG1-mediated Met degradation and inhibits tumor growth. However, detailed downstream mechanism by which LRIG1 mediates target protein down-regulation is unknown. In the present study, we discovered that SAIT301 induces ubiquitination of LRIG1, which in turn promotes recruitment of Met and LRIG1 complex to the lysosome through its interaction with Hrs, resulting in concomitant degradation of both LRIG1 and Met. We also identified USP8 as a LRIG1-specific deubiquitinating enzyme, reporting the interaction between USP8 and LRIG1 for the first time. SAIT301 triggers degradation of LRIG1 by inhibiting the interaction of LRIG1 and USP8, which regulates ubiquitin modification and stability of LRIG1. In summary, SAIT301 employs ubiquitination of LRIG1 for its highly effective Met degradation. This unique feature of SAIT301 enables it to function as a fully antagonistic antibody without Met activation. We found that USP8 is involved in deubiquitination of LRIG1, influencing the efficiency of Met degradation. The relation of Met, LRIG1 and USP8 strongly supports the potential clinical benefit of a combination treatment of a USP8 inhibitor and a Met inhibitor, such as SAIT301.

A new anti-c-Met antibody selected by a mechanism-based dual-screening method: therapeutic potential in cancer.

c-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Furthermore, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers.

SUMOylation of hnRNP-K is required for p53-mediated cell-cycle arrest in response to DNA damage.

Heterogeneous ribonucleoprotein-K (hnRNP-K) is normally ubiquitinated by HDM2 for proteasome-mediated degradation. Under DNA-damage conditions, hnRNP-K is transiently stabilized and serves as a transcriptional co-activator of p53 for cell-cycle arrest. However, how the stability and function of hnRNP-K is regulated remained unknown. Here, we demonstrated that UV-induced SUMOylation of hnRNP-K prevents its ubiquitination for stabilization. Using SUMOylation-defective mutant and purified SUMOylated hnRNP-K, SUMOylation was shown to reduce hnRNP-K's affinity to HDM2 with an increase in that to p53 for p21-mediated cell-cycle arrest. PIAS3 served as a small ubiquitin-related modifier (SUMO) E3 ligase for hnRNP-K in an ATR-dependent manner. During later periods after UV exposure, however, SENP2 removed SUMO from hnRNP-K for its destabilization and in turn for release from cell-cycle arrest. Consistent with the rise-and-fall of both SUMOylation and stability of hnRNP-K, its ability to interact with PIAS3 was inversely correlated to that with SENP2 during the time course after UV exposure. These findings indicate that SUMO modification plays a crucial role in the control of hnRNP-K's function as a p53 co-activator in response to DNA damage by UV.

Bilateral vestibulopathy: clinical characteristics and diagnostic criteria.

OBJECTIVES: To define clinical and laboratory characteristics of bilateral vestibulopathy (BV) and to propose diagnostic criteria of this disorder based on clinical and laboratory findings. STUDY DESIGN: Retrospective case series review. MATERIALS AND METHODS: We recruited 108 patients with a clinical suspicion of BV based on presenting symptoms (unsteadiness or oscillopsia during locomotion) and bedside (dynamic visual acuity or head impulse tests) and laboratory (bithermal caloric or rotatory chair tests) findings after excluding the patients with other disorders that may explain the symptoms. Definite diagnosis of BV was made when the patients showed abnormal findings on both bedside and laboratory tests in addition to the symptoms, whereas probable diagnosis was obtained when either the bedside or laboratory findings were abnormal along with the symptoms. RESULTS: All patients had unsteadiness, and 36 (33%) reported oscillopsia. Diminished vestibulo-ocular responses to head impulse in both horizontal directions were present in 45 of the 100 patients evaluated. Dynamic visual acuity was impaired in 65 (95%) of the 68 patients who underwent testing. Fifty-one (57%) patients showed bilateral hyporesponsiveness during bithermal caloric tests. Forty-eight (53%) patients had reduced gain of the vestibulo-ocular reflex during rotatory chair test. By adopting our diagnostic criteria, 93 patients (86%) were diagnosed as having BV, definite in 49 (45%), and probable in 44 (41%). CONCLUSION: The proposed diagnostic criteria encompass the symptoms and findings of both bedside and laboratory evaluations and may provide a valuable tool for investigating BV.

Nuclear localization of Chfr is crucial for its checkpoint function.

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257-259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.

Chfr is linked to tumour metastasis through the downregulation of HDAC1.

Chfr is a ubiquitin ligase that functions in the mitotic checkpoint by delaying entry into metaphase in response to mitotic stress. It has been suggested that Chfr is a tumour suppressor as Chfr is frequently silenced in human cancers. To better understand how Chfr activity relates to cell-cycle progression and tumorigenesis, we sought to identify Chfr-interacting proteins using affinity purification combined with mass spectrometry. Histone deacetylase 1 (HDAC1), which represses transcription by deacetylating histones, was newly isolated as a Chfr-interacting protein. Chfr binds and downregulates HDAC1 by inducing its polyubiquitylation, both in vitro and in vivo. Ectopic expression of Chfr in cancer cells that normally do not express it results in downregulation of HDAC1, leading to upregulation of the Cdk inhibitor p21(CIP1/WAF1) and the metastasis suppressors KAI1 and E-cadherin. Coincident with these changes, cells arrest in the G1 phase of the cell cycle and become less invasive. Collectively, our data suggest that Chfr functions as a tumour suppressor by regulating HDAC1.

Bimedial rectus hypermetabolism in convergence spasm as observed on positron emission tomography.

A 52-year-old man developed vertical gaze palsy, convergence spasm, and convergence-retraction nystagmus due to glioblastoma of the right thalamus. 18F-fluorodeoxyglucose positron emission tomography (PET) inadvertently demonstrated markedly increased metabolism in the medial rectus muscles. The hypermetabolism indicates active contraction of these extraocular muscles due to excessive convergence drive attributed to inappropriate activation or disrupted inhibition of convergence neurons by the diencephalic lesion.

Sequential bilateral hearing loss in multiple sclerosis.

OBJECTIVES: We describe a case of multiple sclerosis presenting with sequential bilateral hearing loss. METHODS: A 46-year-old woman underwent a series of audiological and neurologic evaluations for sequential bilateral hearing losses that occurred 6 months apart. RESULTS: Initially, the patient suffered from sudden left hearing loss, and magnetic resonance imaging documented an enhancing lesion in the left middle cerebellar peduncle. Six months later, another episode of sudden vertigo, right hearing loss, and right facial palsy developed. Magnetic resonance imaging disclosed a new lesion in the right middle cerebellar peduncle. CONCLUSIONS: Sequential bilateral hearing loss may be a manifestation of multiple sclerosis. In younger patients with sudden hearing loss, multiple sclerosis should be included in the differential diagnosis.