No abstract

The protein mutated in Huntington's disease is phosphorylated by the inflammatory kinase IKK, which promotes other post-translational modifications, and protein degradation.

Autophagy is an intracellular turnover pathway. It has special relevance for neurodegenerative proteinopathies, such as Alzheimer disease, Parkinson disease, and Huntington disease (HD), which are characterized by the accumulation of misfolded proteins. Although induction of autophagy enhances clearance of misfolded protein and has therefore been suggested as a therapy for proteinopathies, neurons appear to be less responsive to classic autophagy inducers than nonneuronal cells. Searching for improved inducers of neuronal autophagy, we discovered an N 10 -substituted phenoxazine that, at proper doses, potently and safely up-regulated autophagy in neurons in an Akt-and mTOR-independent fashion. In a neuron model of HD, this compound was neuroprotective and decreased the accumulation of diffuse and aggregated misfolded protein. A structure/ activity analysis with structurally similar compounds approved by the US Food and Drug Administration revealed a defined pharmacophore for inducing neuronal autophagy. This pharmacophore should prove useful in studying autophagy in neurons and in developing therapies for neurodegenerative proteinopathies.Akt | light-chain 3 | neuronal autophagy | phenoxazine M acroautophagy (hereafter referred to as autophagy) is used by cells to remove long-lived proteins, organelles, or parasites (1) and involves sequestration of material inside doublemembrane vesicles called autophagosomes, which fuse to lysosomes to degrade vesicle contents for reuse in cellular processes. Autophagy is important in neuronal survival: Mice deficient in autophagy develop neurodegeneration (2). Deficient autophagy might allow accumulation of misfolded proteins that lead to neurodegenerative proteinopathies (3). In cell lines, clearance of mutated α-synuclein, implicated in Parkinson disease, depends on autophagy (4). Other examples of autophagy's involvement in neurodegeneration are amyotrophic lateral sclerosis (5), Huntington disease (HD) (6), and Alzheimer disease (7).Studies in cell lines and fly models of neurodegenerative diseases suggest that autophagy can be enhanced to remove misfolded proteins and might be therapeutical (1). One of the best-studied pharmacological inducers of autophagy is rapamycin, which inhibits mTOR, a key regulator of cell growth and proliferation. In cell line and fly models, rapamycin slows accumulation of misfolded proteins implicated in several neurodegenerative diseases (1). In a mouse model of HD, everolimus, a rapamycin analogue, induced autophagy in muscle but not in brain (8). Compounds that decrease inositol levels also induce autophagy (5, 9). Screens for small-molecule enhancers of autophagy in nonneuronal cells yielded a diverse set of structurally unrelated autophagy activators, some of which might act via similar pathways (9-11).Autophagy stimulators have been studied mainly in nonneuronal cells and cell lines. However, autophagy might be regulated differently in primary neurons and other cell types (12). Chemical inhibition of the proteasome affects lipidated ligh...

Huntington’s disease (HD) is an incurable neurodegenerative disease characterized by abnormal motor movements, personality changes, and early death. HD is caused by a mutation in the IT-15 gene that expands abnormally the number of CAG nucleotide repeats. As a result, the translated protein huntingtin contains disease-causing expansions of glutamines (polyQ) that make it prone to misfold and aggregate. While the gene and mutations that cause HD are known, the mechanisms underlying HD pathogenesis are not. Here we will review the state of knowledge of HD, focusing especially on a hallmark pathological feature—intracellular aggregates of mutant Htt called inclusion bodies (IBs). We will describe the role of IBs in the disease. We speculate that IB formation could be just one component of a broader coping response triggered by misfolded Htt whose efficacy may depend on the extent to which it clears toxic forms of mutant Htt. We will describe how IB formation might be regulated and which factors could determine different coping responses in different subsets of neurons. A differential regulation of IB formation as a function of the cellular context could, eventually, explain part of the neuronal vulnerability observed in HD.

In polyglutamine (polyQ) diseases, only certain neurons die, despite widespread expression of the offending protein. PolyQ expansion may induce neurodegeneration by impairing proteostasis, but protein aggregation and toxicity tend to confound conventional measurements of protein stability. Here, we used optical pulse labeling to measure effects of polyQ expansions on the mean lifetime of a fragment of huntingtin, the protein that causes Huntington's disease, in living neurons. We show that polyQ expansion reduced the mean lifetime of mutant huntingtin within a given neuron and that the mean lifetime varied among neurons, indicating differences in their capacity to clear the polypeptide. We found that neuronal longevity is predicted by the mean lifetime of huntingtin, as cortical neurons cleared mutant huntingtin faster and lived longer than striatal neurons. Thus, cell type–specific differences in turnover capacity may contribute to cellular susceptibility to toxic proteins, and efforts to bolster proteostasis in Huntington's disease, such as protein clearance, could be neuroprotective.

SUMMARY Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function on proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies (IBs), IB formation may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that monoclonal antibody (mAb) 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular weight conformational states expanded polyQ assumes and disappears in higher molecular-weight aggregated forms, such as IBs. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

Unraveling cause-and-effect relationships in the nervous system is challenging because some biological processes begin stochastically, take a significant amount of time to unfold, and affect small neuronal subpopulations that can be difficult to isolate and measure. Single-cell approaches are slow, subject to user bias, and sometimes too laborious to achieve sample sizes large enough to detect important effects. Here, we describe an automated imaging and analysis system that enables us to follow the fates of individual cells and intracellular proteins over time. Observations can be quantified in a high-throughput manner with minimal user bias. We have adapted survival analysis methods to determine whether and how factors measured during longitudinal analysis predict a particular biological outcome. The ability to monitor complex processes at single-cell resolution quickly, quantitatively, and over long intervals should have wide applications for biology.Akt ͉ proportional hazard analysis ͉ survival analysis

An expanded polyglutamine (polyQ) stretch in the protein huntingtin (htt) induces self-aggregation into inclusion bodies (IBs) and causes Huntington's disease (HD). Defining precise relationships between early observable variables and neuronal death at the molecular and cellular levels should improve our understanding of HD pathogenesis. Here, we used an automated microscope that tracks thousands of neurons individually over their entire lifetime to quantify interconnected relationships between early variables, such as htt levels, polyQ length, and IB formation, and neuronal death in a primary striatal model of HD. The resulting model revealed that mutant htt increases the risk of death by tonically interfering with homeostatic coping mechanisms rather than producing accumulated damage to the neuron, htt toxicity is saturable, the rate-limiting steps for inclusion body formation and death can be traced to different conformational changes in monomeric htt, and IB formation reduces the impact of the starting levels of htt of a neuron on its risk of death. Finally, the model that emerges from our quantitative measurements places critical limits on the potential mechanisms by which mutant htt might induce neurodegeneration, which should help direct future research.