Nrf2 mitigates LRRK2- and α-synuclein-induced neurodegeneration by modulating proteostasis.

Mutations in leucine-rich repeat kinase 2 (LRRK2) and α-synuclein lead to Parkinson's disease (PD). Disruption of protein homeostasis is an emerging theme in PD pathogenesis, making mechanisms to reduce the accumulation of misfolded proteins an attractive therapeutic strategy. We determined if activating nuclear factor erythroid 2-related factor (Nrf2), a potential therapeutic target for neurodegeneration, could reduce PD-associated neuron toxicity by modulating the protein homeostasis network. Using a longitudinal imaging platform, we visualized the metabolism and location of mutant LRRK2 and α-synuclein in living neurons at the single-cell level. Nrf2 reduced PD-associated protein toxicity by a cell-autonomous mechanism that was time-dependent. Furthermore, Nrf2 activated distinct mechanisms to handle different misfolded proteins. Nrf2 decreased steady-state levels of α-synuclein in part by increasing α-synuclein degradation. In contrast, Nrf2 sequestered misfolded diffuse LRRK2 into more insoluble and homogeneous inclusion bodies. By identifying the stress response strategies activated by Nrf2, we also highlight endogenous coping responses that might be therapeutically bolstered to treat PD.

Sequence-Level Analysis of the Major European Huntington Disease Haplotype.

Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry.

Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have distinct clinical features but a common pathology--cytoplasmic inclusions rich in transactive response element DNA-binding protein of 43 kDa (TDP43). Rare TDP43 mutations cause ALS or FTD, but abnormal TDP43 levels and localization may cause disease even if TDP43 lacks a mutation. Here we show that individual neurons vary in their ability to clear TDP43 and are exquisitely sensitive to TDP43 levels. To measure TDP43 clearance, we developed and validated a single-cell optical method that overcomes the confounding effects of aggregation and toxicity and discovered that pathogenic mutations shorten TDP43 half-life. New compounds that stimulate autophagy improved TDP43 clearance and localization and enhanced survival in primary murine neurons and in human stem cell-derived neurons and astrocytes harboring mutant TDP43. These findings indicate that the levels and localization of TDP43 critically determine neurotoxicity and show that autophagy induction mitigates neurodegeneration by acting directly on TDP43 clearance.

Transient arterial insufficiency and neurologic deficit following external iliac vein stent reconstruction for malignant compression.

Acute iliofemoral deep vein thrombosis may present with pain and swelling or phlegmasia cerulea dolens. When thrombosis occurs in the setting of an underlying venous obstruction, stent reconstruction should be performed after thrombus clearance to prevent rethrombosis. Stent reconstruction after thrombus clearance is associated with high technical success rates and durable patency. This report describes transient lower extremity arterial insufficiency and neurologic deficit after external iliac vein stent expansion and reconstruction within a confined space resulting from a malignant obstruction. It serves as a cautionary tale that, in rare cases, aggressive venous stenting within a confined space can transfer clinically significant forces to adjacent arteries and nerves.

Author Correction: Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease.

The original version of this Article incorrectly gave a publication date of 8 October 2018; this should have been 28 September 2018. This has now been corrected in the PDF and HTML versions of the Article.

Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington's disease.

Huntington's disease is a progressive neurodegenerative disorder caused by polyglutamine-expanded mutant huntingtin (mHTT). Here, we show that the deubiquitinase Usp12 rescues mHTT-mediated neurodegeneration in Huntington's disease rodent and patient-derived human neurons, and in Drosophila. The neuroprotective role of Usp12 may be specific amongst related deubiquitinases, as the closely related homolog Usp46 does not suppress mHTT-mediated toxicity. Mechanistically, we identify Usp12 as a potent inducer of neuronal autophagy. Usp12 overexpression accelerates autophagic flux and induces an approximately sixfold increase in autophagic structures as determined by ultrastructural analyses, while suppression of endogenous Usp12 slows autophagy. Surprisingly, the catalytic activity of Usp12 is not required to protect against neurodegeneration or induce autophagy. These findings identify the deubiquitinase Usp12 as a regulator of neuronal proteostasis and mHTT-mediated neurodegeneration.

Serine 421 regulates mutant huntingtin toxicity and clearance in mice.

Huntington's disease (HD) is a progressive, adult-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminal region of the protein huntingtin (HTT). There are no cures or disease-modifying therapies for HD. HTT has a highly conserved Akt phosphorylation site at serine 421, and prior work in HD models found that phosphorylation at S421 (S421-P) diminishes the toxicity of mutant HTT (mHTT) fragments in neuronal cultures. However, whether S421-P affects the toxicity of mHTT in vivo remains unknown. In this work, we used murine models to investigate the role of S421-P in HTT-induced neurodegeneration. Specifically, we mutated the human mHTT gene within a BAC to express either an aspartic acid or an alanine at position 421, mimicking tonic phosphorylation (mHTT-S421D mice) or preventing phosphorylation (mHTT-S421A mice), respectively. Mimicking HTT phosphorylation strongly ameliorated mHTT-induced behavioral dysfunction and striatal neurodegeneration, whereas neuronal dysfunction persisted when S421 phosphorylation was blocked. We found that S421 phosphorylation mitigates neurodegeneration by increasing proteasome-dependent turnover of mHTT and reducing the presence of a toxic mHTT conformer. These data indicate that S421 is a potent modifier of mHTT toxicity and offer in vivo validation for S421 as a therapeutic target in HD.

High-throughput screening in primary neurons.

Despite years of incremental progress in our understanding of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), there are still no disease-modifying therapeutics. The discrepancy between the number of lead compounds and approved drugs may partially be a result of the methods used to generate the leads and highlights the need for new technology to obtain more detailed and physiologically relevant information on cellular processes in normal and diseased states. Our high-throughput screening (HTS) system in a primary neuron model can help address this unmet need. HTS allows scientists to assay thousands of conditions in a short period of time which can reveal completely new aspects of biology and identify potential therapeutics in the span of a few months when conventional methods could take years or fail all together. HTS in primary neurons combines the advantages of HTS with the biological relevance of intact, fully differentiated neurons which can capture the critical cellular events or homeostatic states that make neurons uniquely susceptible to disease-associated proteins. We detail methodologies of our primary neuron HTS assay workflow from sample preparation to data reporting. We also discuss the adaptation of our HTS system into high-content screening (HCS), a type of HTS that uses multichannel fluorescence images to capture biological events in situ, and is uniquely suited to study dynamical processes in living cells.

Tau reduction prevents Abeta-induced defects in axonal transport.

Amyloid-ß (Aß) peptides, derived from the amyloid precursor protein, and the microtubule-associated protein tau are key pathogenic factors in Alzheimer's disease (AD). How exactly they impair cognitive functions is unknown. We assessed the effects of Aß and tau on axonal transport of mitochondria and the neurotrophin receptor TrkA, cargoes that are critical for neuronal function and survival and whose distributions are altered in AD. Aß oligomers rapidly inhibited axonal transport of these cargoes in wild-type neurons. Lowering tau levels prevented these defects without affecting baseline axonal transport. Thus, Aß requires tau to impair axonal transport, and tau reduction protects against Aß-induced axonal transport defects.

High-content screening of primary neurons: ready for prime time.

High-content screening (HCS), historically limited to drug-development companies, is now a powerful and affordable technology for academic researchers. Through automated routines, this technology acquires large datasets of fluorescence images depicting the functional states of thousands to millions of cells. Information on shapes, textures, intensities, and localizations is then used to create unique representations, or 'phenotypic signatures,' of each cell. These signatures quantify physiologic or diseased states, for example, dendritic arborization, drug response, or cell coping strategies. Live-cell imaging in HCS adds the ability to correlate cellular events at different points in time, thereby allowing sensitivities and observations not possible with fixed endpoint analysis. HCS with live-cell imaging therefore provides an unprecedented capability to detect spatiotemporal changes in cells and is particularly suited for time-dependent, stochastic processes such as neurodegenerative disorders.

Evaluation of methods that locate the center of the ankle for computer-assisted total knee arthroplasty.

Accurate alignment of the mechanical axis of the limb is important to the success of a total knee arthroplasty. Although computer-assisted navigation systems can align implants more accurately than traditional mechanical guides, the ideal technique to determine the distal end point of the mechanical axis, the center of the ankle, is unknown. In this study, we evaluated the accuracy, precision, objectivity, and speed of five anatomic methods and two kinematic methods for estimating the ankle center in 11 healthy subjects. Magnetic resonance images were used to characterize the shape of the ankle and establish the true ankle center. The most accurate and precise anatomic method was establishing the midpoint of the most medial and most lateral aspects of the malleoli (4.5 +/- 4.1 mm lateral error; 2.7 +/- 4.5 mm posterior error). A biaxial model of the ankle (2.0 +/- 6.4 mm medial error; 0.3 +/- 7.6 mm anterior error) was the most accurate kinematic method. Establishing the midpoint of the most medial and most lateral aspects of the malleoli was an accurate, precise, objective, and fast method for establishing the center of the ankle.