Identification of a karyopherin ß1/ß2 proline-tyrosine nuclear localization signal in huntingtin protein.

Among the known pathways of protein nuclear import, the karyopherin ß2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin ß2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a ß sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin ß1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.

Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease.

Huntington's disease (HD) is caused by an expanded CAG tract in the Interesting transcript 15 (IT15) gene encoding the 350 kDa huntingtin protein. Cellular stresses can trigger the release of huntingtin from the endoplasmic reticulum, allowing huntingtin nuclear entry. Here, we show that endogenous, full-length huntingtin localizes to nuclear cofilin-actin rods during stress and is required for the proper stress response involving actin remodeling. Mutant huntingtin induces a dominant, persistent nuclear rod phenotype similar to that described in Alzheimer's disease for cytoplasmic cofilin-actin rods. Using live cell temporal studies, we show that this stress response is similarly impaired when mutant huntingtin is present, or when normal huntingtin levels are reduced. In clinical lymphocyte samples from HD patients, we have quantitatively detected cross-linked complexes of actin and cofilin with complex formation varying in correlation with disease progression. By live cell fluorescence lifetime imaging measurement-Förster resonant energy transfer studies and western blot assays, we quantitatively observed that stress-activated tissue transglutaminase 2 (TG2) is responsible for the actin-cofilin covalent cross-linking observed in HD. These data support a direct role for huntingtin in nuclear actin re-organization, and describe a new pathogenic mechanism for aberrant TG2 enzymatic hyperactivity in neurodegenerative diseases.

Kinase inhibitors modulate huntingtin cell localization and toxicity.

Two serine residues within the first 17 amino acid residues of huntingtin (N17) are crucial for modulation of mutant huntingtin toxicity in cell and mouse genetic models of Huntington's disease. Here we show that the stress-dependent phosphorylation of huntingtin at Ser13 and Ser16 affects N17 conformation and targets full-length huntingtin to chromatin-dependent subregions of the nucleus, the mitotic spindle and cleavage furrow during cell division. Polyglutamine-expanded mutant huntingtin is hypophosphorylated in N17 in both homozygous and heterozygous cell contexts. By high-content screening in live cells, we identified kinase inhibitors that modulated N17 phosphorylation and hence huntingtin subcellular localization. N17 phosphorylation was reduced by casein kinase-2 inhibitors. Paradoxically, IKKß kinase inhibition increased N17 phosphorylation, affecting huntingtin nuclear and subnuclear localization. These data indicate that huntingtin phosphorylation at Ser13 and Ser16 can be modulated by small-molecule drugs, which may have therapeutic potential in Huntington's disease.

nuPRISM: Microfluidic Genome-Wide Phenotypic Screening Platform for Cellular Nuclei.

Genome-wide loss-of-function screens are critical tools to identify novel genetic regulators of intracellular proteins. However, studying the changes in the organelle-specific expression profile of intracellular proteins can be challenging due to protein localization differences across the whole cell, hindering context-dependent protein expression and activity analyses. Here, we describe nuPRISM, a microfluidics chip specifically designed for large-scale isolated nuclei sorting. The new device enables rapid genome-wide loss-of-function phenotypic CRISPR-Cas9 screens directed at intranuclear targets. We deployed this technology to identify novel genetic regulators of ß-catenin nuclear accumulation, a phenotypic hallmark of APC-mutated colorectal cancer. nuPRISM expands our ability to capture aberrant nuclear morphological and functional traits associated with distinctive signal transduction and subcellular localization-driven functional processes with substantial resolution and high throughput.

Nucleocytoplasmic trafficking and transcription effects of huntingtin in Huntington's disease.

There are nine genetic neurodegenerative diseases caused by a similar genetic defect, a CAG DNA triplet-repeat expansion in the disease gene's open reading frame resulting in a polyglutamine expansion in the disease proteins. Despite the commonality of polyglutamine expansion, each of the polyglutamine diseases manifest as unique diseases, with some similarities, but important differences. These differences suggest that the context of the polyglutamine expansion is important to the mechanism of pathology of the disease proteins. Therefore, it is becoming increasingly paramount to understand the normal functions of these polyglutamine disease proteins, which include huntingtin, the polyglutamine-expanded protein in Huntington's disease (HD). Transcriptional dysregulation is seen in HD. Here we discuss the role of normal huntingtin in transcriptional regulation and misregulation in Huntington's disease in relation to potentially analogous model systems, and to other polyglutamine disease proteins. Huntingtin has functional roles in both the cytoplasm and the nucleus. One commonality of activity of polyglutamine disease proteins is at the level of protein dynamics and ability to import and export to and from the nucleus. Knowing the temporal location of huntingtin protein in response to signaling and neuronal communication could lead to valuable insights into an important trigger of HD pathology.

Huntington's disease: revisiting the aggregation hypothesis in polyglutamine neurodegenerative diseases.

After the successful cloning of the first gene for a polyglutamine disease in 1991, the expanded polyglutamine tract in the nine polyglutamine disease proteins became an obvious therapeutic target. Early hypotheses were that misfolded, precipitated protein could be a universal pathogenic mechanism. However, new data are accumulating on Huntington's disease and other polyglutamine diseases that appear to contradict the toxic aggregate hypothesis. Recent data suggest that the toxic species of protein in these diseases may be soluble mutant conformers, and that the protein context of expanded polyglutamine is critical to understanding disease specificity. Here we discuss recent publications that define other important therapeutic targets for polyglutamine-mediated neurodegeneration related to the context of the expanded polyglutamine tract in the disease protein.

Canadian Association of Neurosciences Review: polyglutamine expansion neurodegenerative diseases.

Since the early 1990s, DNA triplet repeat expansions have been found to be the cause in an ever increasing number of genetic neurologic diseases. A subset of this large family of genetic diseases has the expansion of a CAG DNA triplet in the open reading frame of a coding exon. The result of this DNA expansion is the expression of expanded glutamine amino acid repeat tracts in the affected proteins, leading to the term, Polyglutamine Diseases, which is applied to this sub-family of diseases. To date, nine distinct genes are known to be linked to polyglutamine diseases, including Huntington's disease, Machado-Joseph Disease and spinobulbar muscular atrophy or Kennedy's disease. Most of the polyglutamine diseases are characterized clinically as spinocerebellar ataxias. Here we discuss recent successes and advancements in polyglutamine disease research, comparing these different diseases with a common genetic flaw at the level of molecular biology and early drug design for a family of diseases where many new research tools for these genetic disorders have been developed. Polyglutamine disease research has successfully used interdisciplinary collaborative efforts, informative multiple mouse genetic models and advanced tools of pharmaceutical industry research to potentially serve as the prototype model of therapeutic research and development for rare neurodegenerative diseases.

A stress sensitive ER membrane-association domain in Huntingtin protein defines a potential role for Huntingtin in the regulation of autophagy.

We have recently published the precise definition of an aminoterminal membrane association domain in huntingtin, capable of targeting to the endoplasmic reticulum and late endosomes as well as autophagic vesicles. In response to ER stress induced by several pathways, huntingtin releases from membranes and rapidly translocates into the nucleus. Huntingtin is then capable of nuclear export and re-association with the ER in the absence of stress. This release is inhibited when huntingtin contains the polyglutamine expansion seen in Huntington's disease. As a result, mutant huntingtin expressing cells have a perturbed ER and an increase in autophagic vesicles. Here, we discuss the potential function of the huntingtin protein as an ER sentinel, potentially regulating autophagy in response to ER stress. We compare these recent findings to the well characterized mammalian target of rapamycin, mTor, a protein described over a decade ago as related to huntingtin structurally by leucine-rich, repetitive HEAT sequences. Since then, the described functional similarities between Huntingtin and mTor are striking, and this new information about huntingtin's direct association with autophagic vesicles indicates that this structural similarity may extend to functional similarities having an impact upon ER functionality and autophagy.

Huntingtin has a membrane association signal that can modulate huntingtin aggregation, nuclear entry and toxicity.

Huntington's disease is caused by an expanded polyglutamine tract in huntingtin protein, leading to accumulation of huntingtin in the nuclei of striatal neurons. The 18 amino-acid amino-terminus of huntingtin is an amphipathic alpha helical membrane-binding domain that can reversibly target to vesicles and the endoplasmic reticulum (ER). The association of huntingtin to the ER is affected by ER stress. A single point mutation in huntingtin 1-18 predicted to disrupt this helical structure displayed striking phenotypes of complete inhibition of polyglutamine-mediated aggregation, increased huntingtin nuclear accumulation and greatly increased mutant huntingtin toxicity in a striatal-derived mouse cell line. Huntingtin vesicular interaction mediated by 1-18 is specific to late endosomes and autophagic vesicles. We propose that huntingtin has a normal biological function as an ER-associated protein that can translocate to the nucleus and back out in response to ER stress or other events. The increased nuclear entry of mutant huntingtin due to loss of ER-targeting results in increased toxicity.