Rrs1 is involved in endoplasmic reticulum stress response in Huntington disease

The induction of Rrs1 expression is one of the earliest events detected in a presymptomatic knock-in mouse model of Huntington disease (HD). Rrs1 up-regulation fulfills the HD criteria of dominance, striatal specificity, and polyglutamine dependence. Here we show that mammalian Rrs1 is localized both in the nucleolus as well as in the endoplasmic reticulum (ER) of neurons. This dual localization is shared with its newly identified molecular partner 3D3/lyric. We then show that both genes are induced by ER stress in neurons. Interestingly, we demonstrate that ER stress is an early event in a presymptomatic HD mouse model that persists throughout the life span of the rodent. We further show that ER stress also occurs in postmortem brains of HD patients

HdhQ111 Mice Exhibit Tissue Specific Metabolite Profiles that Include Striatal Lipid Accumulation

The HTT CAG expansion mutation causes Huntington’s Disease and is associated with a wide range of cellular consequences, including altered metabolism. The mutant allele is expressed widely, in all tissues, but the striatum and cortex are especially vulnerable to its effects. To more fully understand this tissue-specificity, early in the disease process, we asked whether the metabolic impact of the mutant CAG expanded allele in heterozygous B6.HdhQ111/+ mice would be common across tissues, or whether tissues would have tissue-specific responses and whether such changes may be affected by diet. Specifically, we cross-sectionally examined steady state metabolite concentrations from a range of tissues (plasma, brown adipose tissue, cerebellum, striatum, liver, white adipose tissue), using an established liquid chromatography-mass spectrometry pipeline, from cohorts of 8 month old mutant and wild-type littermate mice that were fed one of two different high-fat diets. The differential response to diet highlighted a proportion of metabolites in all tissues, ranging from 3% (7/219) in the striatum to 12% (25/212) in white adipose tissue. By contrast, the mutant CAG-expanded allele primarily affected brain metabolites, with 14% (30/219) of metabolites significantly altered, compared to wild-type, in striatum and 11% (25/224) in the cerebellum. In general, diet and the CAG-expanded allele both elicited metabolite changes that were predominantly tissue-specific and non-overlapping, with evidence for mutation-by-diet interaction in peripheral tissues most affected by diet. Machine-learning approaches highlighted the accumulation of diverse lipid species as the most genotype-predictive metabolite changes in the striatum. Validation experiments in cell culture demonstrated that lipid accumulation was also a defining feature of mutant HdhQ111 striatal progenitor cells. Thus, metabolite-level responses to the CAG expansion mutation in vivo were tissue specific and most evident in brain, where the striatum featured signature accumulation of a set of lipids including sphingomyelin, phosphatidylcholine, cholesterol ester and triglyceride species. Importantly, in the presence of the CAG mutation, metabolite changes were unmasked in peripheral tissues by an interaction with dietary fat, implying that the design of studies to discover metabolic changes in HD mutation carriers should include metabolic perturbations

Membrane trafficking and mitochondrial abnormalities precede subunit c deposition in a cerebellar cell model of juvenile neuronal ceroid lipofuscinosis

<p>Abstract</p> <p>Background</p> <p>JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a ~1 kb <it>CLN3 </it>mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated <it>Cln3</it>^Δex7/8knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology.</p> <p>Results</p> <p>To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized Cb<it>Cln3 </it>wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous Cb<it>Cln3</it>^Δex7/8precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced.</p> <p>Conclusions</p> <p>These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival.</p

Increased lipid concentrations are common in <i>STHdh</i>^Q111/Q111 cell pellets.

<p>A) Strip-plots summarize the logarithm of the concentration ratios of metabolites altered in mutant, compared to wild-type, in cell pellets (left) or growth media (right) at a false-discovery rate of 10% and fold-change cut-off of +/- 30%. Metabolites whose concentrations are increased in mutant samples are indicated with a blue square, while those metabolites that are decreased in mutant samples are shown in a green. B) Normalized lipid peak values in replicate <i>STHdh</i>^+/+ and <i>STHdh</i>^Q111/Q111 cell pellets are depicted in the heat-map with higher concentrations in red and lower concentrations in blue. For comparison across lipid species, LC-MS peak heights were converted to a Z-score by subtracting the mean concentration of each species and dividing the remainder by the standard deviation. Each lipid class includes species with variable chain lengths and number of saturated bonds (columns). With the exception of lysophosphatidylcholine species, most lipid species are increased in mutant cells. C) The distribution of the actual mutant / wild type peak ratios for all measured lipid concentrations is shown in red. To test for lipid accumulation in mutant cells, the same distribution was calculated 1,000 using the same data with permuted genotype labels; those distributions are shown in gray.</p

Coherently increased lipids in striatal cells and striatal tissue from <i>Hdh</i>^{<i>Q111/+</i>} mice.

<p>A. The mutant/wild-type ratio of each lipid species is indicated on the heatmap (red = 0.5, yellow = 1.0 and green = 1.5). In the panel on the left, lipid species (rows, labels omitted for clarity) were sorted by their genotype p-value in the striatum. Sorting in this way reveals a clustering of green boxes near the top of the heatmap, suggesting an enrichment of accumulated lipids in the striatum. In the panel on the right, the same data was sorted by the genotype p-value in the cerebellum—no such clustering is observed. Metabolites nominally significant in a given tissue are outlined in black. The class of each lipid is indicated in the text, with neutral lipid species indicated in bold. B. The mutant/wild-type ratio of each lipid species that was quantified in both striatal tissue and striatal cells is graphed for striatal tissue (horizontal axis) and immortalized striatal cells (vertical axis); values greater than 1 indicate increased concentrations in mutant samples, while values less than 1 indicate decreased concentrations in mutant samples. The ratios in both cells and tissue for each lipid species are indicated by the position of its label. Lipids whose levels are increased in both striatal cells and tissue predominate, and are indicated by the green rectangle. Lipids with inconsistent changes between cells and tissue are in gray rectangles, while only 1 measured lipid species (C18.0.LPC) is decreased in both samples. Lipid species are indicated as CX.Y.Z, where X = total acyl chain length, Y = total number of unsaturated bonds and Z = lipid species. The lipid species graphed are D/TAG = Di-/Tri-glyceride; LPE/C = Lysophosphatidyl-ethanolamine/choline; PC = phosphatidylcholine.</p

Alterations in tissue metabolite levels in response to CAG-expansion in <i>Htt</i> and increased dietary fat.

<p>A) Summary of the number of nominally significant metabolites per tissue (as a percentage of total measured metabolites) for each tissue—heat map color indicates the number of metabolites significant at p < 0.05. B) A relatively limited number of metabolites show concordant (green text) or discordant (red text) concentration changes in response to CAG-expansion in <i>Htt</i> in the striatum and cerebellum. The striatum and cerebellum also show the largest number (4) of overlapping diet-responsive metabolites amongst tissue pairs. C) Determination of genotype using metabolite concentrations using random forests—the error rate for each tissue is shown over 500 iterations. Below, the contribution of individual metabolites to model accuracy is shown for striatum and cerebellum, the two tissues with accurate models. The y-axis indicates the increase in errors in genotype prediction (in %) after permuting the genotype labels for the indicated metabolite. D) The concentrations of lipid species important for genotype prediction in the striatum are increased in the <i>Htt</i>^{<i>Q111/Q7</i>} samples (PC 32:2–49% increase, p = 0.024; CE 18:0–54% increase, p = 0.004; SM 24:0–33% increase, p = 0.02; 65% increase, p = 0.06).</p

Metabolite profiles clearly discriminate tissue types.

<p>A—Linear discriminant analysis was conducted to derive linear combinations of metabolite concentrations that separate tissue types. In this approach, linear combinations of factors (here, metabolites) are constructed that maximally differentiate a factor of interest (here, tissue type). Scatter-plot matrix of the top 3 linear discriminants (linear combinations of variables) constructed reveals that they very effectively separate tissue types; the tissue type of each sample is indicated by text and color. To test the robustness of this finding, the tissue labels of each sample were randomly permuted, and the LDA analysis repeated. Increased scatter suggests that tissue-separation is much less accurate using permuted data, consistent with the hypothesis that inter-tissue variability is lower than intra-tissue variability in these samples. Linear discriminants derived from actual data are shown on the left, those derived from tissue-label permuted data are shown on the right. B—Sample concentrations of metabolites with clear tissue-specific roles; GABA is high in CNS tissues (striatum and cerebellum), and absent in peripheral tissues. The conjugated bile acid taurochenodeoxycholate are present in the liver, and to a smaller extent the plasma, but absent from other tissues.</p

Striatal enrichment of the 10 most genotype-sensitive metabolites in the striatum of <i>Htt</i>^{<i>Q111/+</i>} mice.

<p>Top ranked metabolites by genotype ANOVA F-statistic are included in order of magnitude. The relative integrated peak height of each metabolite in the striatum compared to the liver and cerebellum is indicated by the rank of each metabolite on lists of 243 total metabolites.</p