HIPK3 modulates autophagy and HTT protein levels in neuronal and mouse models of Huntington disease

Macroautophagy/autophagy is an important cellular protein quality control process that clears intracellular aggregate-prone proteins. These proteins may cause neurodegenerative disorders such as Huntington disease (HD), which is mainly caused by the cytotoxicity of the mutant HTT/Hdh protein (mHTT). Thus, autophagy modulators may regulate mHTT levels and provide potential drug targets for HD and similar diseases. Meanwhile, autophagy function is also impaired in HD and other neurodegenerative disorders via unknown mechanisms. In a recent study, we identified a positive feedback mechanism that may contribute to mHTT accumulation and autophagy impairment in HD. Through genome-scale screening, we identified a kinase gene, HIPK3, as a negative modulator of autophagy and a positive regulator of mHTT levels in HD cells. Knocking down or knocking out HIPK3 reduces mHTT levels via enhancing autophagy in HD cells and in vivo in an HD knock-in mouse model. Interestingly, mHTT positively regulates HIPK3 mRNA levels in both HD cells and HD mouse brains, and this forms a positive feedback loop between mHTT and HIPK3. This loop potentially contributes to autophagy inhibition, mHTT accumulation, and disease progression in HD. The modulation of mHTT by HIPK3 is dependent on its kinase activity and its known substrate DAXX, providing potential HD drug targets. Collectively, our data reveal a novel kinase modulator of autophagy in HD cells, providing therapeutic entry points for HD and similar diseases.     

Morbus Huntington und Huntington-ähnliche Erkrankungen

Huntington disease (HD) is the main cause for hereditary chorea and is characterized by the clinical triad of motor abnormalities, psychiatric symptoms and cognitive decline. Several other genetic disorders, named Huntington disease-like (HDL) syndromes, are known to present with an HD phenotype, and it is difficult to distinguish them from HD. These rare diseases have been named HDL1 to HDL4 and pathogenic mutations in the PRNP gene have been identified for HDL1, whereas mutations in the JPH3 gene cause HDL2 and mutations in the TBP gene cause HDL4 (SCA17). Furthermore, a multitude of diseases can present with a HD phenotype, although other symptoms are usually predominant. These disorders (e.g. SCA, DRPLA, neuroferritinopathy, chorea-acanthocytosis, benign chorea) have to be considered in the differential diagnoses if an HD mutation cannot be confirmed in an index patient with characteristic HD symptoms. In this article an overview of the main disorders that can present with an HD phenotype are given. Diagnostic indicators that may help with the differential diagnosis are also highlighted.     

Mutant Huntingtin induces activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein (BNip3)

Huntington''s disease (HD) is a neurodegenerative disorder characterized by progressive neuronal death in the basal ganglia and cortex. Although increasing evidence supports a pivotal role of mitochondrial dysfunction in the death of patients'' neurons, the molecular bases for mitochondrial impairment have not been elucidated. We provide the first evidence of an abnormal activation of the Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNip3) in cells expressing mutant Huntingtin. In this study, we show an abnormal accumulation and dimerization of BNip3 in the mitochondria extracted from human HD muscle cells, HD model cell cultures and brain tissues from HD model mice. Importantly, we have shown that blocking BNip3 expression and dimerization restores normal mitochondrial potential in human HD muscle cells. Our data shed light on the molecular mechanisms underlying mitochondrial dysfunction in HD and point to BNip3 as a new potential target for neuroprotective therapy in HD.     

Establishment of lal-/- Myeloid Lineage Cell Line That Resembles Myeloid-Derived Suppressive Cells

Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out (lal-/-) mice, we have established a wild type (HD1A) and a lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4⁺ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions.     

Selective inhibitors of death in mutant huntingtin cells

Huntington disease (HD) is an inherited neurodegenerative disorder with unclear pathophysiology. We developed a high-throughput assay in a neuronal cell culture model of HD, screened 43,685 compounds and identified 29 novel selective inhibitors of cell death in mutant huntingtin-expressing cells. Four compounds were active in diverse HD models, which suggests a role for cell death in HD; these compounds are mechanistic probes and potential drug leads for HD.     

Urgent-Start Peritoneal Dialysis and Hemodialysis in ESRD Patients: Complications and Outcomes

BackgroundSeveral studies have suggested that urgent-start peritoneal dialysis (PD) is a feasible alternative to hemodialysis (HD) in patients with end-stage renal disease (ESRD), but the impact of the dialysis modality on outcome, especially on short-term complications, in urgent-start dialysis has not been directly evaluated. The aim of the current study was to compare the complications and outcomes of PD and HD in urgent-start dialysis ESRD patients.MethodsIn this retrospective study, ESRD patients who initiated dialysis urgently without a pre-established functional vascular access or PD catheter at a single center from January 2013 to December 2014 were included. Patients were grouped according to their dialysis modality (PD and HD). Each patient was followed for at least 30 days after catheter insertion (until January 2016). Dialysis-related complications and patient survival were compared between the two groups.ResultsOur study enrolled 178 patients (56.2% male), of whom 96 and 82 patients were in the PD and HD groups, respectively. Compared with HD patients, PD patients had more cardiovascular disease, less heart failure, higher levels of serum potassium, hemoglobin, serum albumin, serum pre-albumin, and lower levels of brain natriuretic peptide. There were no significant differences in gender, age, use of steroids, early referral to a nephrologist, prevalence of primary renal diseases, prevalence of co-morbidities, and other laboratory characteristics between the groups. The incidence of dialysis-related complications during the first 30 days was significantly higher in HD than PD patients. HD patients had a significantly higher probability of bacteremia compared to PD patients. HD was an independent predictor of short-term (30-day) dialysis-related complications. There was no significant difference between PD and HD patients with respect to patient survival rate.ConclusionIn an experienced center, PD is a safe and feasible dialysis alternative to HD for ESRD patients with an urgent need for dialysis.     

A mating-type factors of Coprinus cinereus have variable numbers of specificity genes encoding two classes of homeodomain proteins

We have identified the seven genes that constitute the A43 mating-type factor of Coprinus cinereus and compare the organisation of A43 with the previously characterised A42 factor. In both, the genes that trigger clamp cell development, the so-called specificity genes, are separated into α and β loci by 7 kb of noncoding sequence and are flanked by homologous genes α-fg and β-fg. The specificity genes are known to encode two classes of dissimilar homeodomain (HD1 and HD2) proteins and have different allelic forms which show little or no cross-hybridisation. By partial sequencing we identified a divergently transcribed HD1 (a1-2) and HD2 (a2-2) gene in the A43 α locus. a2-2 failed to elicit clamp cell development in three different hosts, suggesting that it is non-functional. a1-2 elicited clamp cells in an A42 host that has only an HD2 gene (a2-1) in its α locus, thus demonstrating that the compatible Aα mating interaction is between an HD1 and an HD2 protein. The A43 β locus contains three specificity genes, the divergently transcribed HD1 and HD2 genes b1-2 and b2-2 and a third HD1 gene (d1-1) that was shown by hybridisation and transformation analyses to be functionally equivalent to d1-1 in A42. An untranscribed footprint of a third A42 HD1 gene, c1-1, was detected between the A43 b2-2 and d1-1 genes by Southern hybridisation.     

The V471A Polymorphism in Autophagy-Related Gene ATG7 Modifies Age at Onset Specifically in Italian Huntington Disease Patients

The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently, we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the “REGISTRY” cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients). In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis.     

Three Huntington’s Disease Specific Mutation-Carrying Human Embryonic Stem Cell Lines Have Stable Number of CAG Repeats upon In Vitro Differentiation into Cardiomyocytes

Huntington disease (HD; OMIM 143100), a progressive neurodegenerative disorder, is caused by an expanded trinucleotide CAG (polyQ) motif in the HTT gene. Cardiovascular symptoms, often present in early stage HD patients, are, in general, ascribed to dysautonomia. However, cardio-specific expression of polyQ peptides caused pathological response in murine models, suggesting the presence of a nervous system-independent heart phenotype in HD patients. A positive correlation between the CAG repeat size and severity of symptoms observed in HD patients has also been observed in in vitro HD cellular models. Here, we test the suitability of human embryonic stem cell (hESC) lines carrying HD-specific mutation as in vitro models for understanding molecular mechanisms of cardiac pathology seen in HD patients. We have differentiated three HD-hESC lines into cardiomyocytes and investigated CAG stability up to 60 days after starting differentiation. To assess CAG stability in other tissues, the lines were also subjected to in vivo differentiation into teratomas for 10 weeks. Neither directed differentiation into cardiomyocytes in vitro nor in vivo differentiation into teratomas, rich in immature neuronal tissue, led to an increase in the number of CAG repeats. Although the CAG stability might be cell line-dependent, induced pluripotent stem cells generated from patients with larger numbers of CAG repeats could have an advantage as a research tool for understanding cardiac symptoms of HD patients.     

Azadiradione Restores Protein Quality Control and Ameliorates the Disease Pathogenesis in a Mouse Model of Huntington’s Disease

Huntington’s disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by expansion of CAG repeats in the coding area of huntingtin gene. In the HD brain, mutant huntingtin protein goes through proteolysis, and its amino-terminal portion consisting of polyglutamine repeats accumulate as inclusions that result in progressive impairment of cellular protein quality control system. Here, we demonstrate that partial rescue of the defective protein quality control in HD model mouse by azadiradione (a bioactive limonoids found in the seed of Azadirachta indica) could potentially improve the disease pathology. Prolonged treatment of azadiradione to HD mice significantly improved the progressive deterioration in body weight, motor functioning along with extension of lifespan. Azadiradione-treated HD mice brain also exhibited considerable decrease in mutant huntingtin aggregates load and improvement of striatal pathology in comparison with age-matched saline-treated HD controls. Biochemical analysis further revealed upregulation and activation of not only HSF1 (master regulator of protein folding) but also Ube3a (an ubiquitin ligase involved in the clearance of mutant huntingtin) in azadiradione-treated mice. Our results indicate that azadiradione-mediated enhanced folding and clearance of mutant huntingtin might underlie improved disease pathology in HD mice and suggests that it could be a potential therapeutic molecule to delay the progression of HD.     

Influence of pN2 and pD2 on HD formation by various nitrogenases

Formation of HD from D2 has been demonstrated with nitrogenase preparations from Azotobacter vinelandii, Clostridium pasteurianum, Klebsiella pneumoniae, and Azospirillum sp. We conclude that the formation of HD from D2 is a general property of nitrogenases. However, the nitrogenases differ in their Ki values for D2 (N2 fixation) and in their rates of catalyzing HD formation; among the nitrogenases tested, C. pasteurianum nitrogenase had the lowest activity for formation of HD. When contaminating N2 was removed from the atmospheres above reaction mixtures, less than 1% of the total electron flux in the system was directed to HD formation; hence, we doubt that N2-independent HD formation is significant. A working hypothesis is suggested that operates without invoking an N2-independent reaction for forming HD.     

The role of drought as a determinant of hemorrhagic disease in the eastern United States

Bluetongue virus and epizootic hemorrhagic disease (HD) virus are globally distributed, vector‐borne viruses that infect and cause disease in domestic and wild ruminant species. The forces driving increases in resulting HD may be linked to weather conditions and increasing severity has been noted in northerly latitudes. We evaluated the role of drought severity in both space and time on changes in HD reports across the eastern United States for a recent 15 year period. The objectives of this study were to: (a) develop a spatiotemporal model to evaluate if drought severity explains changing patterns of HD presence; and (b) determine whether this potential risk factor varies in importance over the present range of HD in the eastern United States. Historic data (2000–2014) from an annual HD presence–absence survey conducted by the Southeastern Cooperative Wildlife Disease Study and from the United States Drought Monitor were used for this analysis. For every county in 23 states and for each of 15 years, data were based on reported drought status for August, wetland cover, the physiographic region, and the status of HD in the previous year. We used a generalized linear mixed model to explain HD presence and evaluated spatiotemporal predictors across the region. We found that drought severity was a significant predictor of HD presence and the significance of this relationship was dependent on latitude. In more northerly latitudes, where immunological naivety is most likely, we demonstrated the increasing strength of drought severity as a determinant of reported HD and established the importance of variation in drought severity as a risk factor over the present range of HD in the eastern United States. Our research provides spatially explicit evidence for the link between climate forces and emerging disease patterns across latitude for a globally distributed disease.     

Enhancement of brain-type creatine kinase activity ameliorates neuronal deficits in Huntington's disease

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene. Brain-type creatine kinase (CKB) is an enzyme involved in energy homeostasis via the phosphocreatine–creatine kinase system. Although downregulation of CKB was previously reported in brains of HD mouse models and patients, such regulation and its functional consequence in HD are not fully understood. In the present study, we demonstrated that levels of CKB found in both the soma and processes were markedly reduced in primary neurons and brains of HD mice. We show for the first time that mutant HTT (mHTT) suppressed the activity of the promoter of the CKB gene, which contributes to the lowered CKB expression in HD. Exogenous expression of wild-type CKB, but not a dominant negative CKB mutant, rescued the ATP depletion, aggregate formation, impaired proteasome activity, and shortened neurites induced by mHTT. These findings suggest that negative regulation of CKB by mHTT is a key event in the pathogenesis of HD and contributes to the neuronal dysfunction associated with HD. In addition, besides dietary supplementation with the CKB substrate, strategies aimed at increasing CKB expression might lead to the development of therapeutic treatments for HD.     

Deranged neuronal calcium signaling and Huntington disease

Huntington disease (HD) is an autosomal-dominant neurodegenerative disorder that primarily affects medium spiny striatal neurons (MSN). HD is caused by polyglutamine (polyQ) expansion (exp) in the amino-terminal region of a protein huntingtin (Htt). The connection between polyQ expansion in Httexp and MSN neurodegeneration remains elusive. Here we discuss recent data that link polyQ expansion in Httexp and deranged Ca2+ signaling in MSN neurons. Experimental evidence indicates that (1) Ca2+ homeostasis is abnormal in mitochondria isolated from lymphoblasts of HD patients and from brains of the YAC72 HD mouse model; (2) Httexp leads to potentiation of NR1/NR2B NMDA receptor activity in heterologous expression systems and in MSN from YAC72 HD mouse model; and (3) Httexp binds to the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) carboxy-terminus and causes sensitization of InsP3R1 to activation by InsP3 in planar lipid bilayers and in MSN. Based on these results we propose that Httexp-induced cytosolic and mitochondrial Ca2+ overload of MSN plays an important role in the pathogenesis of HD and that Ca2+ signaling blockers may play a beneficial role in treatment of HD.     

Filtration of Macrophage Migration Inhibitory Factor (MIF) in Patients with End Stage Renal Disease Undergoing Hemodialysis

BackgroundEnd stage renal disease (ESRD) patients are characterized by increased morbidity and mortality due to highest prevalence of cardiovascular disease. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that controls cellular signaling in human physiology, pathophysiology, and diseases. Increased MIF plasma levels promote vascular inflammation and development of atherosclerosis. We have shown that MIF is associated with vascular dysfunction in ESRD patients. Whether hemodialysis (HD) affects circulating MIF plasma levels is unknown. We here aimed to investigate whether HD influences the circulating MIF pool in ESRD patients.ConclusionMIF is a dialyzable plasma component that is effectively filtrated during HD from the patient blood pool in large amounts. After removal of remarkable amounts of MIF during a single HD session, MIF plasma pool is early reconstituted after termination of HD from unknown sources.     

Iron Accumulates in Huntington’s Disease Neurons: Protection by Deferoxamine

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull’s staining for ferrous iron (II) combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2) and accordingly decreased expression of iron uptake transferrin receptor (TfR) and increased levels of neuronal iron export protein ferroportin (FPN). Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.