Inducible mutant huntingtin expression in HN10 cells reproduces Huntington's disease-like neuronal dysfunction

<p>Abstract</p> <p>Background</p> <p>Expansion of a polyglutamine repeat at the amino-terminus of huntingtin is the probable cause for Huntington's disease, a lethal progressive autosomal-dominant neurodegenerative disorders characterized by impaired motor performance and severe brain atrophy. The expanded polyglutamine repeat changes the conformation of huntingtin and initiates a range of pathogenic mechanisms in neurons including intracellular huntingtin aggregates, transcriptional dysregulation, energy metabolism deficits, synaptic dystrophy and ultimately neurodegeneration. It is unclear how these events relate to each other or if they can be reversed by pharmacological intervention. Here, we describe neuronal cell lines expressing inducible fragments of normal and mutant huntingtin.</p> <p>Results</p> <p>In HN10 cells, the expression of wild type and mutant huntingtin fragments was dependent on the induction time as well as on the concentration of the RheoSwitch^®inducing ligand. In order to analyze the effect of mutant huntingtin expression on cellular functions we concentrated on the 72Q exon1 huntingtin expressing cell line and found that upon induction, it was possible to carefully dissect mutant huntingtin-induced phenotypes as they developed over time. Dysregulation of transcription as a result of mutant huntingtin expression showed a transcription signature replicating that reported in animal models and Huntington's disease patients. Crucially, triggering of neuronal differentiation in mutant huntingtin expressing cell resulted in the appearance of additional pathological hallmarks of Huntington's disease including cell death.</p> <p>Conclusion</p> <p>We developed neuronal cell lines with inducible expression of wild type and mutant huntingtin. These new cell lines represent a reliable <it>in vitro </it>system for modeling Huntington's disease and should find wide use for high-throughput screening application and for investigating the biology of mutant huntingtin.</p

Induction of autophagy with catalytic mTOR inhibitors reduces huntingtin aggregates in a neuronal cell model

Huntington's disease is a progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. This expansion produces a mutant form of the huntingtin protein, which contains an elongated polyglutamine stretch at its amino-terminus. Mutant huntingtin may adopt an aberrant, aggregation-prone conformation predicted to start the pathogenic process leading to neuronal dysfunction and cell death. Thus, strategies reducing mutant huntingtin may lead to disease-modifying therapies. We investigated the mechanisms and molecular targets regulating huntingtin degradation in a neuronal cell model. We first found that mutant and wild-type huntingtin displayed strikingly diverse turn-over kinetics and sensitivity to proteasome inhibition. Then, we show that autophagy induction led to accelerate degradation of mutant huntingtin aggregates. In our neuronal cell model, allosteric inhibition of mTORC1 by everolimus, a rapamycin analogue, did not induce autophagy or affect aggregate degradation. In contrast, this occurred in the presence of catalytic inhibitors of both mTOR complexes mTORC1 and mTORC2. Our data demonstrate the existence of an mTOR-dependent but everolimus-independent mechanism regulating autophagy and huntingtin-aggregate degradation in cells of neuronal origin

PML tumor suppressor is regulated by HIPK2-mediated phosphorylation in response to DNA damage.

The promyelocytic leukemia (PML) tumor suppressor protein, a central regulator of cell proliferation and apoptosis, is frequently fused to the retinoic acid receptor-alpha (RARalpha) in acute PML. Here we show the interaction of PML with another tumor suppressor protein, the serine/threonine kinase homeodomain-interacting protein kinase (HIPK2). In response to DNA damage, HIPK2 phosphorylates PML at serines 8 and 38. Although HIPK2-mediated phosphorylation of PML occurs early during the DNA damage response, the oncogenic PML-RARalpha fusion protein is phosphorylated with significantly delayed kinetics. DNA damage or HIPK2 expression leads to the stabilization of PML and PML-RARalpha proteins. The N-terminal phosphorylation sites contribute to the DNA damage-induced PML SUMOylation and are required for the ability of PML to cooperate with HIPK2 for the induction of cell death

Atg4b-dependent Autophagic Flux Alleviates Huntington’s Disease Progression

The accumulation of aggregated mutant huntingtin (mHtt) inclusion bodies is involved in Huntigton’s disease (HD) progression. Medium sized-spiny neurons (MSNs) in the corpus striatum are highly vulnerable to mHtt aggregate accumulation and degeneration, but the mechanisms and pathways involved have remained elusive. Here we show that cortico-striatal organotypic slice cultures from HD transgenic mice mimic specific features of HD progression. We then show that induction of autophagy using catalytic inhibitors of mTOR prevents MSNs degeneration in HD cortico-striatal slice cultures. Furthermore, disrupting autophagic flux by overexpressing dominant negative Atg4b in neurons and slice cultures, accelerated mHtt aggregation and neuronal death, suggesting that Atg4b-dependent autophagic flux influences HD progression. Under these circumstances induction of autophagy by inhibiting mTOR was inefficient and did not affect mHtt aggregate accumulation, indicating that mTOR inhibition alleviates HD progression by inducing Atg4b-dependent autophagic flux. These results establish modulators of ATG4b-dependent autophagic flux as new potential targets in the treatment of proteinopathies such as HD

CStS recapitulate MSNs degeneration observed in R6/2 mouse model.

<p>A) Single confocal planes for the immunohistochemistry of CStS derived from R6/2 show progressive mHtt accumulation in the striatum at DIV14 and DIV21. B) Higher magnification of MSNs in R6/2 slices visualized with DAPI (blue), DARPP-32 (red) and mHtt (green) at DIV21. Note the presence of cytosolic and nuclear mHtt accumulation (arrows). C) Quantification of mHtt density (left) and size (right) throughout development. Aggregates are primarily increasing in size after DIV14 and density stabilizes. D) Biochemical detection of mHtt accumulation using AGERA at DIV21. 10 µg from the total lysate of WT and R6/2 slices were loaded and selective signals were detected in R6/2 slices; each lane represents an aliquot of a distinct slice. N = 5 independent slices. E) Selective degeneration of MSNs in R6/2 slices visualized by immunohistochemistry with the striatal marker DARPP-32 (red) and cortical NeuF (green). Neurodegeneration is observed as a decrease in marker intensity. F) Left: quantification for the ratio of striatal NeuN and DAPI positive nuclei in WT and R6/2 slices at DIV21. Right: quantification of DARPP-32 and NeuF intensity per area in WT and R6/2 slices at DIV21. N = 10 images from 5 independent slices; median values ± SEM; Students t test: **p<0.01 and ***p<0.001 Bars: (A) 30 µm, (B) 10 µm, (D) 25 µm.</p

CStS recapitulate MSNs degeneration observed in (CAG)150-het mouse model.

<p>A) Single confocal planes for the immunohistochemistry of CStS derived from (CAG)150-het mice show progressive mHtt accumulation in the striatum at DIV14 and DIV21. B) Higher magnification of MSNs in (CAG)150-het slices show selective mHtt accumulation at DIV21. Note the extranuclear mHtt accumulation and few small nuclear inclusions (arrows). C) Time course quantification of mHtt intensity per area shows progressive accumulation in (CAG)150-het slices. D) Western blot showing mHtt is selective to (CAG)150-het slices. Biochemical detection of soluble mHtt in (CAG)150-het slices; each lane represents an aliquot of 10 µg from total lysates of distinct slices. N = 10 images from 5 independent slices; median values ± SEM; ***p<0.001 Bars: (A) 30 µm, (B) 10 µm.</p

MSNs degeneration in R6/2 mice.

<p>A) Immunohistochemistry against DAPI (blue) and mHtt (green) of R6/2 brain sections shows progressive mHtt accumulation in striatum. B) Quantification of mHtt staining from A) showing mHtt aggregation in terms of density (left) and size (right). R6/2 mice develop progressive mHtt aggregation. C) Progressive striatal neurodegeneration in R6/2 mice. Brain sections from adult R6/2 mice and WT controls were stained for a striatal marker DARPP-32 (red), neurofilament (NeuF, green) and NeuN (green) at 10 weeks of age. By this time the R6/2 mice have developed mHtt accumulation and they have decreased DARPP-32 staining as well as NeuF, whereas the neuronal marker NeuN is unchanged. D) Left: the ratio of striatal NeuN and DAPI positive nuclei is unchanged between WT and R6/2 mice at 10 weeks of age, in line with the limited cell death characteristic of the R6/2 mouse model. Right: quantification of C) shows a highly significant decrease in both NeuF and DARPP-32 intensity. Three mice per condition; N = 5 images/each; median values ± SEM; Students t test:*p<0.05, **p<0.01 Bars and ***p<0.001: (A) 200 µm, (C) 50 µm.</p

Atg4b dependent-autophagy flux attenuates HD progression.

<p>A) Representative images for tTa/Exon1-72Q and Atg4b/Exon1-72Q cortical neurons treated or not with AZD8055 at 8 days post lentiviral infection (dpi). Note the enhanced mHtt accumulation (green) in both Atg4b/Exon1-72Q +/− AZD8055 neurons. B) Quantitative analysis of mHtt intensity per area. Note that AZD8055 reduced mHtt accumulation in tTa/Exon1-72Q neurons but had no effect in Atg4b/Exon1-72Q neurons. C) Representative images of neurons in CStS transfected with gene gun expressing Atg4b or GFP at DIV7 and DIV28. Note how Atg4b accelerates the appearance of mHtt accumulation in neurons of R6/2 slices at DIV7, but it is not inducing toxicity in WT slices. D) Left: quantitative distribution of mHtt accumulation in R6/2 Atg4b and R6/2 GFP expressing neurons at DIV7 and 28. Note how the percentage of mHtt accumulation is almost similar between R6/2 Atg4b neurons at DIV7 and R6/2 GFP neurons at DIV28. Middle: distribution of positive neurons in R6/2 GFP and R6/2 Atg4b slices at DIV 7, 10 and 28. No R6/2 Atg4b neurons were detected at DIV10 and 28. On the other hand WT Atg4b neurons were detected at DIV28, suggesting mHtt-dependent toxicity in the R6/2 Atg4b neurons. Right: AZD8055 is not rescuing the loss of Atg4b neurons in R6/2 slices at DIV10. A) N = 50 images from 3 independent preparations; (C) N = 10 images from 5 independent slices; median values ± SEM; Student’s t test: *p<0.05, **p<0.01 Bars: 10 µm.</p

MSNs can be cultured for weeks in organotypic cortico-striatal slice cultures.

<p>A) Schematic of the preparation for the oganotypic cortico-striatal slice cultures used in this study. Cortico-striatal slices (CStS) were prepared at postnatal day 6 (P6) and maintained for several weeks <i>in vitro</i>. For time course analysis, CStS were typically collected at DIV7, DIV14 and DIV21. B) Analysis of cortico-striatal slices prepared from P6 WT mice and maintained in culture for 4 weeks (DIV28). Immunohistochemistry with neuronal markers DARPP-32 (red) and NeuF (green) show strong stainning in the striatum and cortex, respectively. C) Quantification of DARPP-32 and NeuF intensity in WT CStS. Note, a progressive increase over time <i>in vitro</i>. D) Left: representative single confocal plane for the immunohistochemistry of CStS with neuronal markers DARPP-32 (red), NeuF (green), VGLUT1 (magenta) and DAPI (blue) at DIV7. Right: zoom in highlighting VGLUT1 positive glutamate vesicles (arrows) within the cortex and striatum. E) Quantitative analysis of VGLUT1 area normalized to DARPP-32 at DIV7 and DIV14. Note how VGLUT1 significantly increases with development from DIV7 to DIV14. Str = Striatum; Cx = Cortex; N = 10 images from 5 independent slices; median values ± SEM; Students t test:*p<0.05 and ***p<0.001 Bars: (A) 1 mm, (B, D left) 200 µm, (D right) 40 µm.</p