Deregulated Transcriptome as a Platform for Adrenal Huntington's Disease-Related Pathology.

Huntington's disease (HD) is a neurodegenerative disorder that affects mainly the central nervous system (CNS) by inducing progressive deterioration in both its structure and function. In recent years, there has been growing interest in the impact of HD on peripheral tissue function. Herein, we used the R6/2 mouse model of HD to investigate the influence of the disease on adrenal gland functioning. A transcriptomic analysis conducted using a well-established quantitative method, an Affymetrix array, revealed changes in gene expression in the R6/2 model compared to genetic background controls. For the first time, we identified disruptions in cholesterol and sterol metabolism, blood coagulation, and xenobiotic metabolism in HD adrenal glands. This study showed that the disrupted expression of these genes may contribute to the underlying mechanisms of Huntington's disease. Our findings may contribute to developing a better understanding of Huntington's disease progression and aid in the development of novel diagnostic or therapeutic approaches.

A minimal region of the HSP90AB1 promoter is suitable for ubiquitous expression in different somatic tissues with applicability for gene therapy.

Huntington's disease (HD) is a multi-tissue failure disorder for which there is no cure. We have previously shown an effective therapeutic approach limited mainly to the central nervous system, based on a synthetic zinc finger (ZF) transcription repressor gene therapy, but it would be important to target other tissues as well. In this study, we identify a novel minimal HSP90AB1 promoter region that can efficiently control expression not only in the CNS but also in other affected HD tissues. This promoter-enhancer is effective in driving expression of ZF therapeutic molecules in both HD skeletal muscles and the heart, in the symptomatic R6/1 mouse model. Moreover, for the first time we show that ZF molecules repressing mutant HTT reverse transcriptional pathological remodelling in HD hearts. We conclude that this HSP90AB1 minimal promoter may be used to target multiple HD organs with therapeutic genes. The new promoter has the potential to be added to the portfolio of gene therapy promoters, for use where ubiquitous expression is needed.

Structural Abnormalities of the Optic Nerve and Retina in Huntington's Disease Pre-Clinical and Clinical Settings.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. HD-related pathological remodelling has been reported in HD mouse models and HD carriers. In this study, we studied structural abnormalities in the optic nerve by employing Spectral Domain Optical Coherence Tomography (SD-OCT) in pre-symptomatic HD carriers of Caucasian origin. Transmission Electron Microscopy (TEM) was used to investigate ultrastructural changes in the optic nerve of the well-established R6/2 mouse model at the symptomatic stage of the disease. We found that pre-symptomatic HD carriers displayed a significant reduction in the retinal nerve fibre layer (RNFL) thickness, including specific quadrants: superior, inferior and temporal, but not nasal. There were no other significant irregularities in the GCC layer, at the macula level and in the optic disc morphology. The ultrastructural analysis of the optic nerve in R6/2 mice revealed a significant thinning of the myelin sheaths, with a lamellar separation of the myelin, and a presence of myelonoid bodies. We also found a significant reduction in the thickness of myelin sheaths in peripheral nerves within the choroids area. Those ultrastructural abnormalities were also observed in HD photoreceptor cells that contained severely damaged membrane disks, with evident vacuolisation and swelling. Moreover, the outer segment of retinal layers showed a progressive disintegration. Our study explored structural changes of the optic nerve in pre- and clinical settings and opens new avenues for the potential development of biomarkers that would be of great interest in HD gene therapies.

Identification of the Transcriptional Biomarkers Panel Linked to Pathological Remodelling of the Eye Tissues in Various HD Mouse Models.

Ocular abnormalities are becoming associated with a spectrum of pathological events in various neurodegenerative diseases. Huntington's disease (HD) is just such an example of a fatal neurological disorder, where mutated genes (CAG trinucleotide expansions in the Huntingtin gene) have widespread expression, leading to the production of mutant Huntingtin (mHTT) protein. It is well known that mutant HTT protein is prone to form toxic aggregates, which are a typical pathological feature, along with global transcriptome alterations. In this study, we employed well-established quantitative methods such as Affymetrix arrays and quantitative PCR (qPCR) to identify a set of transcriptional biomarkers that will track HD progression in three well-established mouse models: R6/2, R6/1, and HdhQ150. Our array analysis revealed significantly deregulated networks that are related to visual processes and muscle contractions. Furthermore, our targeted quantitative analysis identified a panel of biomarkers with some being dysregulated even at the presymptomatic stage of the disease, e.g., Opn1mw, Opn1sw, and Pfkfb2. Some of the deregulated genes identified in this study have been linked to other genetic ocular disorders such as: GNAT2, a source of achromatopsia, and REEP6, linked to Retinitis pigmentosa. It may thus be a useful platform for preclinical evaluations of therapeutic interventions.

Kinetin stimulates differentiation of C2C12 myoblasts.

Kinetin or N6-furfuryladenine (K) belongs to a class of plant hormones called cytokinins, which are biologically active molecules modulating many aspects of plant growth and development. However, biological activities of cytokinins are not only limited to plants; their effects on animals have been widely reported in the literature. Here, we found that Kinetin is a potent small molecule that efficiently stimulates differentiation of C2C12 myoblasts into myotubes in vitro. The highest efficacy was achieved at 1µM and 10µM Kinetin concentrations, in both mitogen-poor and rich media. More importantly, Kinetin was able to strongly stimulate the MyoD-dependent conversion of fibroblasts into myotubes. Kinetin alone did not give rise to fibroblast conversion and required MyoD; this demonstrates that Kinetin augments the molecular repertoire of necessary key regulatory factors to facilitate MyoD-mediated myogenic differentiation. This novel Kinetin pro-myogenic function may be explained by its ability to alter intracellular calcium levels and by its potential to impact on Reactive Oxygen Species (ROS) signalling. Taken together, our findings unravel the effects of a new class of small molecules with potent pro-myogenic activities. This opens up new therapeutic avenues with potential for treating skeletal muscle diseases related to muscle aging and wasting.

Gene Therapy Advances: A Meta-Analysis of AAV Usage in Clinical Settings.

Adeno-associated viruses (AAVs) are the safest and most effective gene delivery vehicles to drive long-term transgene expression in gene therapy. While animal studies have shown promising results, the translatability of AAVs into clinical settings has been partly limited due to their restricted gene packaging capacities, off-target transduction, and immunogenicity. In this study, we analysed over two decades of AAV applications, in 136 clinical trials. This meta-analysis aims to provide an up-to-date overview of the use and successes of AAVs in clinical trials, while evaluating the approaches used to address the above challenges. First, this study reveals that the speed of novel AAV development has varied between therapeutic areas, with particular room for improvement in Central Nervous System disorders, where development has been slow. Second, the lack of dose-dependent toxicity and efficacy data indicates that optimal dosing regimes remain elusive. Third, more clinical data on the effectiveness of various immune-modulation strategies and gene editing approaches are required to direct future research and to accelerate the translation of AAV-mediated gene therapy into human applications.

Cross-Sectional Transcriptional Analysis of the Aging Murine Heart.

Cardiovascular disease accounts for millions of deaths each year and is currently the leading cause of mortality worldwide. The aging process is clearly linked to cardiovascular disease, however, the exact relationship between aging and heart function is not fully understood. Furthermore, a holistic view of cardiac aging, linking features of early life development to changes observed in old age, has not been synthesized. Here, we re-purpose RNA-sequencing data previously-collected by our group, investigating gene expression differences between wild-type mice of different age groups that represent key developmental milestones in the murine lifespan. DESeq2's generalized linear model was applied with two hypothesis testing approaches to identify differentially-expressed (DE) genes, both between pairs of age groups and across mice of all ages. Pairwise comparisons identified genes associated with specific age transitions, while comparisons across all age groups identified a large set of genes associated with the aging process more broadly. An unsupervised machine learning approach was then applied to extract common expression patterns from this set of age-associated genes. Sets of genes with both linear and non-linear expression trajectories were identified, suggesting that aging not only involves the activation of gene expression programs unique to different age groups, but also the re-activation of gene expression programs from earlier ages. Overall, we present a comprehensive transcriptomic analysis of cardiac gene expression patterns across the entirety of the murine lifespan.

Prevalence of Non-psychiatric Comorbidities in Pre-symptomatic and Symptomatic Huntington's Disease Gene Carriers in Poland.

Huntington's disease (HD) is monogenic neurodegenerative disorder caused by CAG expansions within the Huntingtin gene (Htt); it has a prevalence of 1 in 10,000 worldwide and is invariably fatal. Typically, healthy individuals have fewer than 35 CAG repeats, while the CAG expansions range from 36 to ~200 in HD patients. The hallmark of HD is neurodegeneration, especially in the striatal nuclei, basal ganglia and cerebral cortex, leading to neurological symptoms that involve motor, cognitive, and psychiatric events. However, HD is a complex disorder that may also affect peripheral organs, so it is possible that HD patients could be affected by comorbidities. Hence, we investigated the prevalence of comorbid conditions in HD patients (pre-symptomatic and symptomatic groups) and compared the frequency of those conditions to a control group. Our groups represent 65% of HD gene carriers registered in Poland. We identified 8 clusters of comorbid conditions in both HD groups, namely: musculoskeletal, allergies, cardiovascular, neurological, gastrointestinal, thyroid, psychiatric, and ophthalmologic. We found that HD patients have a significantly higher percentage of co-existing conditions in comparison to the control group. Among the 8 clusters of diseases, musculoskeletal, psychiatric, and cardiovascular events were significantly more frequent in both pre- and symptomatic HD patients, while neurological and gastrointestinal clusters showed significantly higher occurrence in the HD symptomatic group. A greater recognition of comorbidity in HD might help to better understand health outcomes and improve clinical management.

Neuro-Cardio Mechanisms in Huntington's Disease and Other Neurodegenerative Disorders.

Although Huntington's disease is generally considered to be a neurological disorder, there is mounting evidence that heart malfunction plays an important role in disease progression. This is perhaps not unexpected since both cardiovascular and nervous systems are strongly connected - both developmentally and subsequently in health and disease. This connection occurs through a system of central and peripheral neurons that control cardiovascular performance, while in return the cardiovascular system works as a sensor for the nervous system to react to physiological events. Hence, given their permanent interconnectivity, any pathological events occurring in one system might affect the second. In addition, some pathological signals from Huntington's disease might occur simultaneously in both the cardiovascular and nervous systems, since mutant huntingtin protein is expressed in both. Here we aim to review the source of HD-related cardiomyopathy in the light of recently published studies, and to identify similarities between HD-related cardiomyopathy and other neuro-cardio disorders.

Early transcriptional alteration of histone deacetylases in a murine model of doxorubicin-induced cardiomyopathy.

Doxorubicin is a potent chemotherapeutic agent that is widely-used to treat a variety of cancers but causes acute and chronic cardiac injury, severely limiting its use. Clinically, the acute side effects of doxorubicin are mostly manageable, whereas the delayed consequences can lead to life-threatening heart failure, even decades after cancer treatment. The cardiotoxicity of doxorubicin is subject to a critical cumulative dose and so dosage limitation is considered to be the best way to reduce these effects. Hence, a number of studies have defined a "safe dose" of the drug, both in animal models and clinical settings, with the aim of avoiding long-term cardiac effects. Here we show that a dose generally considered as safe in a mouse model can induce harmful changes in the myocardium, as early as 2 weeks after infusion. The adverse changes include the development of fibrotic lesions, disarray of cardiomyocytes and a major transcription dysregulation. Importantly, low-dose doxorubicin caused specific changes in the transcriptional profile of several histone deacetylases (HDACs) which are epigenetic regulators of cardiac remodelling. This suggests that cardioprotective therapies, aimed at modulating HDACs during doxorubicin treatment, deserve further exploration.

Transcriptional Signature of an Altered Purine Metabolism in the Skeletal Muscle of a Huntington's Disease Mouse Model.

Huntington's disease (HD) is a fatal neurodegenerative disorder, caused by a polyglutamine expansion in the huntingtin protein (HTT). HD has a peripheral component to its pathology: skeletal muscles are severely affected, leading to atrophy, and malfunction in both pre-clinical and clinical settings. We previously used two symptomatic HD mouse models to demonstrate the impairment of the contractile characteristics of the hind limb muscles, which was accompanied by a significant loss of function of motor units. The mice displayed a significant reduction in muscle force, likely because of deteriorations in energy metabolism, decreased oxidation, and altered purine metabolism. There is growing evidence suggesting that HD-related skeletal muscle malfunction might be partially or completely independent of CNS degeneration. The pathology might arise from mutant HTT within muscle (loss or gain of function). Hence, it is vital to identify novel peripheral biomarkers that will reflect HD skeletal muscle atrophy. These will be important for upcoming clinical trials that may target HD peripherally. In order to identify potential biomarkers that might reflect muscle metabolic changes, we used qPCR to validate key gene transcripts in different skeletal muscle types. Consequently, we report a number of transcript alterations that are linked to HD muscle pathology.

Oxidized low-density lipoproteins enhance expression and activity of CD39 and CD73 in the human aortic valve endothelium.

Extracellular nucleotides regulate thrombosis, inflammation, and immune response. Ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and ecto-5'-nucleotidase (CD73) convert extracellular nucleotides in a sequential order: ATP to ADP, AMP, and then to adenosine. In this study, we aimed to test an effect of oxidized low-density lipoprotein (ox-LDL) on CD39 and CD73 in endothelial cells. Human aortic valve endothelial cells were exposed to ox-LDL for 24-48 h. Next, the activity, protein expression, and mRNA transcripts level of CD39 and CD73 were characterized by an incubation with ATP or AMP followed by high-performance liquid chromatography analysis of media as well as western blots and qPCR. CD73 activity in human valve endothelial cells was increased in presence of ox-LDL (4.04 ± 0.32 nmol/mg prot./min, mean +/- SEM) as compared with control (2.75 ± 0.21 nmol/mg prot/min). There was almost no effect of ox-LDL on CD39 activity. A similar effect was observed for mRNA and protein expression. In conclusion, we found that ox-LDL modulated CD39 and CD73 activity in the endothelium, which may contribute to relevant pathologies and featured treatments.

Changes in cardiac nucleotide metabolism in Huntington's disease.

Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5'NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5'NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling.

Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin repression in mice.

BACKGROUND: Synthetic zinc finger (ZF) proteins can be targeted to desired DNA sequences and are useful tools for gene therapy. We recently developed a ZF transcription repressor (ZF-KOX1) able to bind to expanded DNA CAG-repeats in the huntingtin (HTT) gene, which are found in Huntington's disease (HD). This ZF acutely repressed mutant HTT expression in a mouse model of HD and delayed neurological symptoms (clasping) for up to 3 weeks. In the present work, we sought to develop a long-term single-injection gene therapy approach in the brain. METHOD: Since non-self proteins can elicit immune and inflammatory responses, we designed a host-matched analogue of ZF-KOX1 (called mZF-KRAB), to treat mice more safely in combination with rAAV vector delivery. We also tested a neuron-specific enolase promoter (pNSE), which has been reported as enabling long-term transgene expression, to see whether HTT repression could be observed for up to 6 months after AAV injection in the brain. RESULTS: After rAAV vector delivery, we found that non-self proteins induce significant inflammatory responses in the brain, in agreement with previous studies. Specifically, microglial cells were activated at 4 and 6 weeks after treatment with non-host-matched ZF-KOX1 or GFP, respectively, and this was accompanied by a moderate neuronal loss. In contrast, the host-matched mZF-KRAB did not provoke these effects. Nonetheless, we found that using a pCAG promoter (CMV early enhancer element and the chicken ß-actin promoter) led to a strong reduction in ZF expression by 6 weeks after injection. We therefore tested a new non-viral promoter to see whether the host-adapted ZF expression could be sustained for a longer time. Vectorising mZF-KRAB with a promoter-enhancer from neuron-specific enolase (Eno2, rat) resulted in up to 77 % repression of mutant HTT in whole brain, 3 weeks after bilateral intraventricular injection of 10(10) virions. Importantly, repressions of 48 % and 23 % were still detected after 12 and 24 weeks, respectively, indicating that longer term effects are possible. CONCLUSION: Host-adapted ZF-AAV constructs displayed a reduced toxicity and a non-viral pNSE promoter improved long-term ZF protein expression and target gene repression. The optimized constructs presented here have potential for treating HD.

An impaired metabolism of nucleotides underpins a novel mechanism of cardiac remodeling leading to Huntington's disease related cardiomyopathy.

Huntington's disease (HD) is mainly thought of as a neurological disease, but multiple epidemiological studies have demonstrated a number of cardiovascular events leading to heart failure in HD patients. Our recent studies showed an increased risk of heart contractile dysfunction and dilated cardiomyopathy in HD pre-clinical models. This could potentially involve metabolic remodeling, that is a typical feature of the failing heart, with reduced activities of high energy phosphate generating pathways. In this study, we sought to identify metabolic abnormalities leading to HD-related cardiomyopathy in pre-clinical and clinical settings. We found that HD mouse models developed a profound deterioration in cardiac energy equilibrium, despite AMP-activated protein kinase hyperphosphorylation. This was accompanied by a reduced glucose usage and a significant deregulation of genes involved in de novo purine biosynthesis, in conversion of adenine nucleotides, and in adenosine metabolism. Consequently, we observed increased levels of nucleotide catabolites such as inosine, hypoxanthine, xanthine and uric acid, in murine and human HD serum. These effects may be caused locally by mutant HTT, via gain or loss of function effects, or distally by a lack of trophic signals from central nerve stimulation. Either may lead to energy equilibrium imbalances in cardiac cells, with activation of nucleotide catabolism plus an inhibition of re-synthesis. Our study suggests that future therapies should target cardiac mitochondrial dysfunction to ameliorate energetic dysfunction. Importantly, we describe the first set of biomarkers related to heart and skeletal muscle dysfunction in both pre-clinical and clinical HD settings.

A shared mechanism of muscle wasting in cancer and Huntington's disease.

Skeletal muscle loss and dysfunction in aging and chronic diseases is one of the major causes of mortality in patients, and is relevant for a wide variety of diseases such as neurodegeneration and cancer. Muscle loss is accompanied by changes in gene expression and metabolism that lead to contractile impairment and likely affect whole-body metabolism and function. The changes may be caused by inactivity, inflammation, age-related factors or unbalanced nutrition. Although links with skeletal muscle loss have been found in diseases with disparate aetiologies, for example both in Huntington's disease (HD) and cancer cachexia, the outcome is a similar impairment and mortality. This short commentary aims to summarize recent achievements in the identification of common mechanisms leading to the skeletal muscle wasting syndrome seen in diseases as different as cancer and HD. The latter is the most common hereditary neurodegenerative disorder and muscle wasting is an important component of its pathology. In addition, possible therapeutic strategies for anti-cachectic treatment will be also discussed in the light of their translation into possible therapeutic approaches for HD.

Huntington's disease is a multi-system disorder.

Huntington's disease (HD) is one of the most common non-curable rare diseases and is characterized by choreic movements, psychiatric symptoms, and slowly progressive dementia. HD is inherited as an autosomal dominant disorder with complete penetrance. Although brain pathology has become a hallmark of HD, there is a critical mass of new studies suggesting peripheral tissue pathology as an important factor in disease progression. In particular, recently published studies about skeletal muscle malfunction and HD-related cardiomyopathy in HD mouse models strongly suggest their important roles, leading to upcoming preclinical and clinical trials. One might conclude that therapeutic approaches in HD should not be restricted only to the brain pathology but instead major efforts should also be made to understand the cross-talk between diseased tissues like the CNS-Heart or CNS-skeletal muscle axes.

HDAC4-myogenin axis as an important marker of HD-related skeletal muscle atrophy.

Skeletal muscle remodelling and contractile dysfunction occur through both acute and chronic disease processes. These include the accumulation of insoluble aggregates of misfolded amyloid proteins that is a pathological feature of Huntington's disease (HD). While HD has been described primarily as a neurological disease, HD patients' exhibit pronounced skeletal muscle atrophy. Given that huntingtin is a ubiquitously expressed protein, skeletal muscle fibres may be at risk of a cell autonomous HD-related dysfunction. However the mechanism leading to skeletal muscle abnormalities in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that symptomatic animals developed a progressive impairment of the contractile characteristics of the hind limb muscles tibialis anterior (TA) and extensor digitorum longus (EDL), accompanied by a significant loss of motor units in the EDL. In symptomatic animals, these pronounced functional changes were accompanied by an aberrant deregulation of contractile protein transcripts and their up-stream transcriptional regulators. In addition, HD mouse models develop a significant reduction in muscle force, possibly as a result of a deterioration in energy metabolism and decreased oxidation that is accompanied by the re-expression of the HDAC4-DACH2-myogenin axis. These results show that muscle dysfunction is a key pathological feature of HD.

HDAC4 as a potential therapeutic target in neurodegenerative diseases: a summary of recent achievements.

For the past decade protein acetylation has been shown to be a crucial post-transcriptional modification involved in the regulation of protein functions. Histone acetyltransferases (HATs) mediate acetylation of histones which results in the nucleosomal relaxation associated with gene expression. The reverse reaction, histone deacetylation, is mediated by histone deacetylases (HDACs) leading to chromatin condensation followed by transcriptional repression. HDACs are divided into distinct classes: I, IIa, IIb, III, and IV, on the basis of size and sequence homology, as well as formation of distinct repressor complexes. Implications of HDACs in many diseases, such as cancer, heart failure, and neurodegeneration, have identified these molecules as unique and attractive therapeutic targets. The emergence of HDAC4 among the members of class IIa family as a major player in synaptic plasticity raises important questions about its functions in the brain. The characterization of HDAC4 specific substrates and molecular partners in the brain will not only provide a better understanding of HDAC4 biological functions but also might help to develop new therapeutic strategies to target numerous malignancies. In this review we highlight and summarize recent achievements in understanding the biological role of HDAC4 in neurodegenerative processes.