Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins.

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

Transplanted human neural stem cells rescue phenotypes in zQ175 Huntington's disease mice and innervate the striatum.

Huntington's disease (HD), a genetic neurodegenerative disorder, primarily affects the striatum and cortex with progressive loss of medium-sized spiny neurons (MSNs) and pyramidal neurons, disrupting cortico-striatal circuitry. A promising regenerative therapeutic strategy of transplanting human neural stem cells (hNSCs) is challenged by the need for long-term functional integration. We previously described that, with short-term hNSC transplantation into the striatum of HD R6/2 mice, human cells differentiated into electrophysiologically active immature neurons, improving behavior and biochemical deficits. Here, we show that long-term (8 months) implantation of hNSCs into the striatum of HD zQ175 mice ameliorates behavioral deficits, increases brain-derived neurotrophic factor (BDNF) levels, and reduces mutant huntingtin (mHTT) accumulation. Patch clamp recordings, immunohistochemistry, single-nucleus RNA sequencing (RNA-seq), and electron microscopy demonstrate that hNSCs differentiate into diverse neuronal populations, including MSN- and interneuron-like cells, and form connections. Single-nucleus RNA-seq analysis also shows restoration of several mHTT-mediated transcriptional changes of endogenous striatal HD mouse cells. Remarkably, engrafted cells receive synaptic inputs, innervate host neurons, and improve membrane and synaptic properties. Overall, the findings support hNSC transplantation for further evaluation and clinical development for HD.

Treatment with JQ1, a BET bromodomain inhibitor, is selectively detrimental to R6/2 Huntington's disease mice.

Transcriptional and epigenetic alterations occur early in Huntington's disease (HD), and treatment with epigenetic modulators is beneficial in several HD animal models. The drug JQ1, which inhibits histone acetyl-lysine reader bromodomains, has shown promise for multiple cancers and neurodegenerative disease. We tested whether JQ1 could improve behavioral phenotypes in the R6/2 mouse model of HD and modulate HD-associated changes in transcription and epigenomics. R6/2 and non-transgenic (NT) mice were treated with JQ1 daily from 5 to 11 weeks of age and behavioral phenotypes evaluated over this period. Following the trial, cortex and striatum were isolated and subjected to mRNA-seq and ChIP-seq for the histone marks H3K4me3 and H3K27ac. Initially, JQ1 enhanced motor performance in NT mice. In R6/2 mice, however, JQ1 had no effect on rotarod or grip strength but exacerbated weight loss and worsened performance on the pole test. JQ1-induced gene expression changes in NT mice were distinct from those in R6/2 and primarily involved protein translation and bioenergetics pathways. Dysregulation of HD-related pathways in striatum was exacerbated by JQ1 in R6/2 mice, but not in NTs, and JQ1 caused a corresponding increase in the formation of a mutant huntingtin protein-dependent high molecular weight species associated with pathogenesis. This study suggests that drugs predicted to be beneficial based on their mode of action and effects in wild-type or in other neurodegenerative disease models may have an altered impact in the HD context. These observations have important implications in the development of epigenetic modulators as therapies for HD.

Therapeutic effects of stem cells in rodent models of Huntington's disease: Review and electrophysiological findings.

The principal symptoms of Huntington's disease (HD), chorea, cognitive deficits, and psychiatric symptoms are associated with the massive loss of striatal and cortical projection neurons. As current drug therapies only partially alleviate symptoms, finding alternative treatments has become peremptory. Cell replacement using stem cells is a rapidly expanding field that offers such an alternative. In this review, we examine recent studies that use mesenchymal cells, as well as pluripotent, cell-derived products in animal models of HD. Additionally, we provide further electrophysiological characterization of a human neural stem cell line, ESI-017, which has already demonstrated disease-modifying properties in two mouse models of HD. Overall, the field of regenerative medicine represents a viable and promising avenue for the treatment of neurodegenerative disorders including HD.

Human Neural Stem Cell Transplantation Rescues Functional Deficits in R6/2 and Q140 Huntington's Disease Mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder with no disease-modifying treatment. Expansion of the glutamine-encoding repeat in the Huntingtin (HTT) gene causes broad effects that are a challenge for single treatment strategies. Strategies based on human stem cells offer a promising option. We evaluated efficacy of transplanting a good manufacturing practice (GMP)-grade human embryonic stem cell-derived neural stem cell (hNSC) line into striatum of HD modeled mice. In HD fragment model R6/2 mice, transplants improve motor deficits, rescue synaptic alterations, and are contacted by nerve terminals from mouse cells. Furthermore, implanted hNSCs are electrophysiologically active. hNSCs also improved motor and late-stage cognitive impairment in a second HD model, Q140 knockin mice. Disease-modifying activity is suggested by the reduction of aberrant accumulation of mutant HTT protein and expression of brain-derived neurotrophic factor (BDNF) in both models. These findings hold promise for future development of stem cell-based therapies.

Promoter analysis of the human ascorbic acid transporters SVCT1 and 2: mechanisms of adaptive regulation in liver epithelial cells.

Ascorbic acid, the active form of vitamin C, is a vital antioxidant in the human liver, yet the molecular mechanisms involved in the regulation of ascorbic acid transporters [human sodium-dependent vitamin C transporters (hSVCT) 1 and 2] in liver cells are poorly understood. Therefore, we characterized the minimal promoter regions of hSVCT1 and 2 in cultured human liver epithelial cells (HepG2) and examined the effects of ascorbic acid deprivation and supplementation on activity and regulation of the transport systems. Identified minimal promoters required for basal activity were found to include multiple cis regulatory elements, whereas mutational analysis demonstrated that HNF-1 sites in the hSVCT1 promoter and KLF/Sp1 sites in the hSVCT2 promoter were essential for activities. When cultured in ascorbic acid deficient or supplemented media, HepG2 cells demonstrated significant (P<.01) and specific reciprocal changes in [(14)C]-Ascorbic acid uptake, and in hSVCT1 mRNA and protein levels as well as hSVCT1 promoter activity. However, no significant changes in hSVCT2 expression or promoter activity were observed during ascorbic acid deficient or supplemented conditions. We mapped the ascorbic acid responsive region in the hSVCT1 promoter and determined that HNF-1 sites are important for the adaptive regulation response. The results of these studies further characterize the hSVCT1 and 2 promoters establish that ascorbic acid uptake by human liver epithelial cells is adaptively regulated and show that transcriptional mechanisms via HNF-1 in the hSVCT1 promoter may, in part, be involved in this regulation.

Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells.

Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na(+) gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca(2+)/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.

Differentiation-dependent regulation of the intestinal folate uptake process: studies with Caco-2 cells and native mouse intestine.

Differentiation of intestinal epithelial cells is accompanied by alterations in levels of expression of many genes, including those involved in nutrient uptake. Effects of differentiation of intestinal epithelial cells on the physiological and molecular parameters of the intestinal folate uptake process are not well characterized. To address this issue, we used two models, Caco-2 cells and native mouse intestine. Studies with Caco-2 cells showed a significant increase in the initial rate of carrier-mediated folic acid uptake during differentiation (i.e., as the cells transitioned from preconfluent to confluent and then to postconfluent stages). This increase was associated with an increase in the level of expression of the human reduced folate carrier (hRFC) and the human proton-coupled folate transporter (hPCFT) both at the protein and mRNA levels with differentiation; it was also associated with a significant increase in activity of the hRFC and hPCFT promoters. Studies with native mouse intestine showed a significantly higher folate uptake in villus compared with crypt cells, which was again associated with a significantly higher level of expression of the mouse RFC and PCFT at the protein and mRNA levels. Together, these studies demonstrate that the intestinal folate uptake process undergoes differentiation-dependent regulation and that this regulation is mediated via changes in the level of expression of both the RFC and PCFT. In addition, the studies suggest the possible involvement (at least in part) of a transcriptional mechanism(s) in this type of regulation of the intestinal folate uptake process.

Molecular mechanisms involved in the adaptive regulation of human intestinal biotin uptake: A study of the hSMVT system.

Biotin, a water-soluble micronutrient, is vital for cellular functions, including growth and development. The human intestine utilizes the human sodium-dependent multivitamin transporter (hSMVT) for biotin uptake. Evidence exists showing that the intestinal biotin uptake process is adaptively regulated during biotin deficiency. Nothing, however, is known about molecular mechanism(s) involved during this adaptive regulation. This study compared two human-derived intestinal epithelial cell lines (HuTu-80 and Caco-2) during biotin-deficient or biotin-sufficient states and with an approach that assessed carrier-mediated biotin uptake, hSMVT protein and RNA levels, RNA stability, and hSMVT promoter activity. The results showed that during biotin deficiency, a significant and specific upregulation in carrier-mediated biotin uptake occurred in both human intestinal epithelial cell lines and that this increase was associated with an induction in protein and mRNA levels of hSMVT. The increase in mRNA levels was not due to an increase in RNA stability but was associated with an increase in activity of the hSMVT promoter in transfected human intestinal cells. Using promoter deletion constructs and mutational analysis in transiently transfected HuTu-80 and Caco-2 cells, a biotin deficiency-responsive region was mapped to a 103-bp area within the hSMVT promoter that contains gut-enriched Kruppel-like factor (GKLF) sites that confer the response to biotin deficiency. These results confirm that human intestinal biotin uptake is adaptively regulated and provide novel evidence demonstrating that the upregulation is not mediated via changes in hSMVT RNA stability but rather is due to transcriptional regulatory mechanism(s) that likely involve GKLF sites in the hSMVT promoter.

Regulation of the human biotin transporter hSMVT promoter by KLF-4 and AP-2: confirmation of promoter activity in vivo.

The mechanism of biotin uptake in human intestine has been well characterized and involves the human sodium-dependent multivitamin transporter (hSMVT), yet little is known about the molecular/transcriptional regulation of the system. Previous investigations cloned the 5' regulatory region of the hSMVT gene and identified the minimal promoter. To expand these investigations, we compared activity of the hSMVT promoter in three human intestinal epithelial cell lines (NCM460, Caco-2, and HuTu-80) and contrasted a renal epithelial cell line (HEK-293). We analyzed the role of putative cis-elements in regulating promoter activity and confirmed activity of the cloned hSMVT promoter in vivo. In vitro studies demonstrated that all cell lines utilized the same minimal promoter region, and mutation of specific cis-regulatory elements [Kruppel-like factor 4 (KLF-4) and activator protein-2 (AP-2)] led to a decrease in promoter activity in all intestinal cell types but not in renal cells. Using electrophoretic mobility shift assays, we identified two specific DNA/protein complexes. Using oligonucleotide competition and antibody supershift analysis, we determined that KLF-4 and AP-2 were involved in forming the complexes. In HEK-293 cells, overexpressing KLF-4 increased the endogenous hSMVT message levels threefold and activated a cotransfected hSMVT promoter-reporter construct. In vivo studies using hSMVT promoter-luciferase transgenic mice established physiological relevance and showed the pattern of hSMVT promoter expression to be similar to endogenous mouse SMVT mRNA expression. The results demonstrate, for the first time, the importance of KLF-4 and AP-2 in regulating the activity of the hSMVT promoter in the intestine and provide direct in vivo confirmation of hSMVT promoter activity.

Differentiation-dependent up-regulation of intestinal thiamin uptake: cellular and molecular mechanisms.

Differentiation of intestinal epithelial cells is associated with up-and-down regulation of expression of a variety of genes including those involved in nutrient uptake. Nothing is known about possible differentiation-dependent regulation of the intestinal thiamin uptake process and the cellular and molecular mechanisms involved in such regulation. Using as models human-derived intestinal epithelial Caco-2 cells and crypt/villus epithelial cells isolated from wild-type and transgenic mice carrying promoters for human thiamin transporter-1 and -2 (hTHTR-1 and hTHTR-2), we addressed this issue. Our results showed that differentiation of Caco-2 cells is associated with a significant up-regulation in carrier-mediated thiamin uptake. Up-regulation was associated with a significant increase in the level of expression of hTHTR-1 and hTHTR-2 protein and mRNA as well as in activity of the corresponding transfected human thiamin transporter-1 (SLC19A2) and -2 (SLC19A3) promoters. Deletion analysis identified the differentiation-responsive region to be at position -356 to -275 bp for the SLC19A2 promoter and at position -77 to -13 bp for the SLC19A3 promoter. In addition, a critical and specific role in the differentiation-mediated effects for an NF1 binding site (-348 to -345 bp) in the SLC19A2 promoter and a SP1/GC-box binding site (-48 to -45 bp) in the SLC19A3 promoter were established using mutational analysis. The physiological relevance of in vitro findings with Caco-2 cells was confirmed in wild-type and transgenic mice by demonstrating that thiamin uptake and mRNA levels of the mouse THTR-1 and THTR-2, as well as activity of human SLC19A2 and SLC19A3 promoters expressed in transgenic mice, were all significantly higher in intestinal villus compared with crypt epithelial cells. These studies demonstrate for the first time that differentiation of intestinal epithelial cells is associated with an up-regulation in thiamin uptake process and that this up-regulation appears to be mediated via transcriptional regulatory mechanisms that involve the SLC19A2 and SLC19A3 genes.

In vitro and in vivo characterization of the minimal promoter region of the human thiamin transporter SLC19A2.

The molecular mechanisms involved in the regulation of thiamin transport in mammalian cells are poorly understood. Previous studies established that a human thiamin transporter, SLC19A2, plays a role in thiamin uptake in human tissues. We cloned the 5' regulatory region of the SLC19A2 gene, identified the minimal promoter required for basal activity, and located multiple putative cis elements. To further characterize the SLC19A2 promoter, we investigated, in the present study, the role of the putative cis elements in regulating the activity of the SLC19A2 promoter in vitro and confirmed the activity of the SLC19A2 promoter in vivo. In vitro studies demonstrated that mutation of specific cis elements in the SLC19A2 minimal promoter [Gut-enriched Krupple-like factor (GKLF), nuclear factor-1 (NF-1), and stimulating protein-1 (SP-1)] led to a decrease in activity. Using electrophoretic mobility shift assays, four specific DNA/protein complexes were identified. The interacting factors were determined by oligonucleotide competition and antibody supershift analysis and shown to be GKLF, NF-1, and SP-1. Cotransfection studies of the SLC19A2 promoter with an SP-1 containing vector in Drosophila SL2 cells further confirmed a role for SP-1 in regulating SLC19A2 promoter activity. In vivo studies using transgenic mice established the functionality of the full-length and minimal SLC19A2 promoters. Furthermore, our studies revealed that the pattern of expression of the SLC19A2 promoter-Luciferase constructs in transgenic mice was similar to the reported SLC19A2 RNA expression pattern in native human tissues. The results demonstrate the importance of GKLF, NF-1, and SP-1 in regulating the activity of the SLC19A2 promoter and provide direct in vivo confirmation of promoter activity.