Reduction in PA28αβ activation in HD mouse brain correlates to increased mHTT aggregation in cell models

Huntington’s disease is an autosomal dominant heritable disorder caused by an expanded CAG trinucleotide repeat at the N-terminus of the Huntingtin (HTT) gene. Lowering the levels of soluble mutant HTT protein prior to aggregation through increased degradation by the proteasome would be a therapeutic strategy to prevent or delay the onset of disease. Native PAGE experiments in HdhQ150 mice and R6/2 mice showed that PA28αβ disassembles from the 20S proteasome during disease progression in the affected cortex, striatum and hippocampus but not in cerebellum and brainstem. Modulating PA28αβ activated proteasomes in various in vitro models showed that PA28αβ improved polyQ degradation, but decreased the turnover of mutant HTT. Silencing of PA28αβ in cells lead to an increase in mutant HTT aggregates, suggesting that PA28αβ is critical for overall proteostasis, but only indirectly affects mutant HTT aggregation

Alternative processing of human HTT mRNA with implications for Huntington's disease therapeutics

Huntington disease is caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT) that is translated into a polyglutamine stretch in the huntingtin protein (HTT). We previously showed that HTT mRNA carrying an expanded CAG repeat was incompletely spliced to generate HTT1a, an exon 1 only transcript, which was translated to produce the highly aggregation-prone and pathogenic exon 1 HTT protein. This occurred in all knock-in mouse models of Huntington's disease and could be detected in patient cell lines and post-mortem brains. To extend these findings to a model system expressing human HTT, we took advantage of YAC128 mice that are transgenic for a yeast artificial chromosome carrying human HTT with an expanded CAG repeat. We discovered that the HTT1a transcript could be detected throughout the brains of YAC128 mice. We implemented RNAscope to visualise HTT transcripts at the single molecule level and found that full-length HTT and HTT1a were retained together in large nuclear RNA clusters, as well as being present as single transcripts in the cytoplasm. Homogeneous time-resolved fluorescence analysis demonstrated that the HTT1a transcript had been translated to produce the exon 1 HTT protein. The levels of exon 1 HTT in YAC128 mice, correlated with HTT aggregation, supportive of the hypothesis that exon 1 HTT initiates the aggregation process. Huntingtin-lowering strategies are a major focus of therapeutic development for Huntington's disease. These approaches often target full-length HTT alone and would not be expected to reduce pathogenic exon 1 HTT levels. We have established YAC128 mouse embryonic fibroblast lines and shown that, together with our QuantiGene multiplex assay, these provide an effective screening tool for agents that target HTT transcripts. The effects of current targeting strategies on nuclear RNA clusters are unknown, structures that may have a pathogenic role, or alternatively could be protective by retaining HTT1a in the nucleus and preventing it from being translated. In light of recently halted antisense oligonucleotide trials, it is vital that agents targeting HTT1a are developed, and that the effects of HTT-lowering strategies on the subcellular levels of all HTT transcripts and their various HTT protein isoforms are understood

Hippocampal neurophysiology is modified by a disease-associated C-terminal fragment of tau protein

The accumulation of cleaved tau fragments in the brain is associated with several tauopathies. For this reason, we recently developed a transgenic mouse that selectively accumulates a C-Terminal 35 kDa human tau fragment (Tau35). These animals develop progressive motor and spatial memory impairment, paralleled by increased hippocampal glycogen synthase kinase 3b activity. In this neurophysiological study, we focused on the CA1 subfield of the hippocampus, a brain area involved in memory encoding. The accumulation of Tau35 results in a significant increase of short-term facilitation of the synaptic response in the theta frequency range (10 Hz), without affecting basal synaptic transmission and long-term synaptic plasticity. Tau35 expression also alters the intrinsic excitability of CA1 pyramidal neurons. Thus, Tau35 presence is associated with increased and decreased excitability at hyperpolarized and depolarized potentials, respectively. These observations are paralleled by a hyperpolarization of the voltage-sensitivity of noninactivating Kþ currents. Further investigation is needed to assess the causal link between such functional alterations and the cognitive and motor impairments previously observed in this model

The Huntington's disease-related cardiomyopathy prevents a hypertrophic response in the R6/2 mouse model

<div><p>Huntington's disease (HD) is neurodegenerative disorder for which the mutation results in an extra-long tract of glutamines that causes the huntingtin protein to aggregate. It is characterized by neurological symptoms and brain pathology that is associated with nuclear and cytoplasmic aggregates and with transcriptional deregulation. Despite the fact that HD has been recognized principally as a neurological disease, there are multiple epidemiological studies showing that HD patients exhibit a high rate of cardiovascular events leading to heart failure. To unravel the mechanistic basis of cardiac dysfunction in HD, we employed a wide range of molecular techniques using the well-established genetic R6/2 mouse model that develop a considerable degree of the cardiac atrophy at end stage disease. We found that chronic treatment with isoproterenol, a potent beta-adrenoreceptor agonist, did not change the overall gross morphology of the HD murine hearts. However, there was a partial response to the beta-adrenergenic stimulation by the further re-expression of foetal genes. In addition we have profiled the expression level of <i>Hdacs</i> in the R6/2 murine hearts and found that the isoproterenol stimulation of <i>Hdac</i> expression was partially blocked. For the first time we established the <i>Hdac</i> transcriptional profile under hypertrophic conditions and found 10 out of 18 <i>Hdacs</i> to be markedly deregulated. Therefore, we conclude that R6/2 murine hearts are not able to respond to the chronic isoproterenol treatment to the same degree as wild type hearts and some of the hypertrophic signals are likely attenuated in the symptomatic HD animals.</p></div

Tauopathy induced by low level expression of a human brain-derived tau fragment in mice is rescued by phenylbutyrate

Human neurodegenerative tauopathies exhibit pathological tau aggregates in the brain along with diverse clinical features including cognitive and motor dysfunction. Post-translational modifications including phosphorylation, ubiquitination and truncation, are characteristic features of tau present in the brain in human tauopathy. We have previously reported an N-terminally truncated form of tau in human brain that is associated with the development of tauopathy and is highly phosphorylated. We have generated a new mouse model of tauopathy in which this human brain-derived, 35 kDa tau fragment (Tau35) is expressed in the absence of any mutation and under the control of the human tau promoter. Most existing mouse models of tauopathy overexpress mutant tau at levels that do not occur in human neurodegenerative disease, whereas Tau35 transgene expression is equivalent to less than 10% of that of endogenous mouse tau. Tau35 mice recapitulate key features of human tauopathies, including aggregated and abnormally phosphorylated tau, progressive cognitive and motor deficits, autophagic/lysosomal dysfunction, loss of synaptic protein, and reduced life-span. Importantly, we found that sodium 4-phenylbutyrate (Buphenyl®), a drug used to treat urea cycle disorders and currently in clinical trials for a range of neurodegenerative diseases, reverses the observed abnormalities in tau and autophagy, behavioural deficits, and loss of synapsin 1 in Tau35 mice. Our results show for the first time that, unlike other tau transgenic mouse models, minimal expression of a human disease-associated tau fragment in Tau35 mice causes a profound and progressive tauopathy and cognitive changes, which are rescued by pharmacological intervention using a clinically approved drug. These novel Tau35 mice therefore represent a highly disease-relevant animal model in which to investigate molecular mechanisms and to develop novel treatments for human tauopathies.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Chronic administration of isoproterenol causes a significant transcriptional deregulation of many <i>Hdacs</i> and <i>Sirtuins</i>.

<p>(A) <i>Hdac1</i>, <i>Hdac2</i>, <i>Hdac4</i>, <i>Hdac6</i>, <i>Hdac7</i> and <i>Hdac8</i> transcript levels were increased while <i>Hdac3</i> mRNA was significantly reduced in the heart of WT mice treated with Isoproterenol. Only <i>Hdac4</i> and <i>Hdac6</i> were significantly increased in the hearts of R6/2 mice. (B) <i>Sirt3</i> and <i>Sirt5</i> transcript levels were significantly increased in the heart of WT mice treated with Isoproterenol. <i>Sirt 1</i> and <i>Sirt 2</i> were decreased in the hearts of WT mice, but only <i>Sirt2</i> was decreased in the hearts of R6/2 mice. All Taqman qPCR values were normalized to the geometric mean of three housekeeping genes: <i>Actb</i>, <i>Cyc1</i> and <i>Gapdh</i>. Error bars are SEM (n = 6). Two-way ANOVA with Bonferroni <i>post-hoc</i> test: *<i>p</i><0.05, **<i>p</i><0.01; ***<i>p</i><0.001.</p

Gross cardiac morphology of the hearts treated with isoproterenol.

<p>Representative phalloidin staining (green) shows left ventricle myocyte hypertrophy in WT mice but not in 12 week old R6/2 mice. Nuclei (blue) were visualized with DAPI. Scale bar 30 µm.</p

Partial re-activation of foetal gene markers in the hearts of R6/2 treated with isoproterenol.

<p><i>Anp</i> (atrial natriuretic peptide), <i>Bnp</i> (brain natriuretic protein) and members of the four and half LIM family <i>Fhl1</i> and <i>Fhl2 were</i> elevated in the heart of WT and R6/2 mice. All Taqman qPCR values were normalized to the geometric mean of three housekeeping genes: <i>Actb</i>, <i>Cyc1</i> and <i>Gapdh</i>. Error bars are SEM (n = 6). Two-way ANOVA with Bonferroni <i>post-hoc</i> test: *<i>p</i><0.05, **<i>p</i><0.01; ***<i>p</i><0.001.</p

Transcriptional deregulation of HD markers involved in heart failure.

<p>(A) <i>S100A4</i> (S100 calcium binding protein A4), Vgl-3 (vestigial related factor 3), <i>Vgl-4</i> (vestigial related factor 4) were up-regulated while <i>Mck</i> (muscle creatine kinase) and <i>Bdnf</i> (brain derived neurotophic factor) transcripts were significantly decreased in the heart of R6/2 mice. (B) Transcripts of Transcriptional Enhancer Family members (TEAD or TEFs) were significantly deregulated in isoproterenol treated mice. All Taqman qPCR values were normalized to the geometric mean of three housekeeping genes: <i>Actb</i>, <i>Cyc1</i> and <i>Gapdh</i>. Error bars are SEM (n = 6). Two-way ANOVA with Bonferroni <i>post-hoc</i> test: *<i>p</i><0.05, **<i>p</i><0.01; ***<i>p</i><0.001.</p

Moderate fibrosis level based on collagen VI deposits is not attenuated in the hearts of R6/2 mice.

<p>(A) Representative confocal pictograms of whole heart sections from 12 week old WT and R6/2 mice. (B) Quantification of the collagen VI staining area. Fibrosis was detected with the anti-collagen VI antibody (green) and nuclei (blue) were visualised with DAPI. Scale bar 30 µm. Values are mean ± SEM (<i>n</i> = 3). Student's <i>t</i> test: *<i>p</i><0.05, ***<i>p</i><0.001.</p