Lysosomal ceramides regulate cathepsin B-mediated processing of saposin C and glucocerebrosidase activity.

Variants in multiple lysosomal enzymes increase Parkinson's disease (PD) risk, including the genes encoding glucocerebrosidase (GCase), acid sphingomyelinase (ASMase) and galactosylceramidase. Each of these enzymes generates ceramide by hydrolysis of sphingolipids in lysosomes, but the role of this common pathway in PD pathogenesis has not yet been explored. Variations in GBA1, the gene encoding GCase, are the most common genetic risk factor for PD. The lysosomal enzyme cathepsin B has recently been implicated as an important genetic modifier of disease penetrance in individuals harboring GBA1 variants, suggesting a mechanistic link between these enzymes. Here, we found that ceramide activates cathepsin B, and identified a novel role for cathepsin B in mediating prosaposin cleavage to form saposin C, the lysosomal coactivator of GCase. Interestingly, this pathway was disrupted in Parkin-linked PD models, and upon treatment with inhibitor of ASMase which resulted in decreased ceramide production. Conversely, increasing ceramide production by inhibiting acid ceramidase activity was sufficient to upregulate cathepsin B- and saposin C-mediated activation of GCase. These results highlight a mechanistic link between ceramide and cathepsin B in regulating GCase activity and suggest that targeting lysosomal ceramide or cathepsin B represents an important therapeutic strategy for activating GCase in PD and related disorders.

Acetylation targets mutant huntingtin to autophagosomes for degradation.

Huntington's disease (HD) is an incurable neurodegenerative disease caused by neuronal accumulation of the mutant protein huntingtin. Improving clearance of the mutant protein is expected to prevent cellular dysfunction and neurodegeneration in HD. We report here that such clearance can be achieved by posttranslational modification of the mutant Huntingtin (Htt) by acetylation at lysine residue 444 (K444). Increased acetylation at K444 facilitates trafficking of mutant Htt into autophagosomes, significantly improves clearance of the mutant protein by macroautophagy, and reverses the toxic effects of mutant huntingtin in primary striatal and cortical neurons and in a transgenic C. elegans model of HD. In contrast, mutant Htt that is rendered resistant to acetylation dramatically accumulates and leads to neurodegeneration in cultured neurons and in mouse brain. These studies identify acetylation as a mechanism for removing accumulated protein in HD, and more broadly for actively targeting proteins for degradation by autophagy.

Mutant Huntingtin Is Secreted via a Late Endosomal/Lysosomal Unconventional Secretory Pathway.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by the expansion of a CAG triplet in the gene encoding for huntingtin (Htt). The resulting mutant protein (mHtt) with extended polyglutamine (polyQ) sequence at the N terminus leads to neuronal degeneration both in a cell-autonomous and a non-cell-autonomous manner. Recent studies identified mHtt in the extracellular environment and suggested that its spreading contributes to toxicity, but the mechanism of mHtt release from the cell of origin remains unknown. In this study, we performed a comprehensive, unbiased analysis of secretory pathways and identified an unconventional lysosomal pathway as an important mechanism for mHtt secretion in mouse neuroblastoma and striatal cell lines, as well as in primary neurons. mHtt secretion was dependent on synaptotagmin 7, a regulator of lysosomal secretion, and inhibited by chemical ablation of late endosomes/lysosomes, suggesting a lysosomal secretory pattern. mHtt was targeted preferentially to the late endosomes/lysosomes compared with wild-type Htt. Importantly, we found that late endosomal/lysosomal targeting and secretion of mHtt could be inhibited efficiently by the phosphatidylinositol 3-kinase and neutral sphingomyelinase chemical inhibitors, Ly294002 and GW4869, respectively. Together, our data suggest a lysosomal mechanism of mHtt secretion and offer potential strategies for pharmacological modulation of its neuronal secretion.SIGNIFICANCE STATEMENT This is the first study examining the mechanism of mutant huntingtin (mHTT) secretion in an unbiased manner. We found that the protein is secreted via a late endosomal/lysosomal unconventional secretory pathway. Moreover, mHtt secretion can be reduced significantly by phosphatidylinositol 3-kinase and neutral sphingomyelinase inhibitors. Understanding and manipulating the secretion of mHtt is important because of its potentially harmful propagation in the brain.

Peroxisome-proliferator-activated receptor gamma coactivator 1 α contributes to dysmyelination in experimental models of Huntington's disease.

The peroxisome-proliferator-activated receptor gamma coactivator 1 α (PGC1α) has been implicated in the pathogenesis of several neurodegenerative disorders, including Huntington's disease (HD). Recent data demonstrating white matter abnormalities in PGC1α knock-out (KO) mice prompted us to examine the role of PGC1α in CNS myelination and its relevance to HD pathogenesis. We found deficient postnatal myelination in the striatum of PGC1α KO mice, accompanied by a decrease in myelin basic protein (MBP). In addition, brain cholesterol, its precursors, and the rate-limiting enzymes for cholesterol synthesis, HMG CoA synthase (HMGCS1) and HMG CoA reductase (HMGCR), were also reduced in PGC1α KO mice. Moreover, knockdown of PGC1α in oligodendrocytes by lentiviral shRNA led to a decrease in MBP, HMGCS1, and Hmgcr mRNAs. Chromatin immunoprecipitations revealed the recruitment of PGC1α to MBP promoter in mouse brain, and PGC1α over-expression increased MBP and SREBP-2 promoter activity, suggesting that PGC1α regulates MBP and cholesterol synthesis at the transcriptional level. Importantly, expression of mutant huntingtin (Htt) in primary oligodendrocytes resulted in decreased expression of PGC1α and its targets HmgcS1, Hmgcr, and MBP. Decreased expression of MBP and deficient myelination were found postnatally and in adult R6/2 mouse model of HD. Diffusion tensor imaging detected white matter abnormalities in the corpus callosum of R6/2 mice, and electron microscopy revealed thinner myelin sheaths and increased myelin periodicity in BACHD [bacterial artificial chromosome (BAC)-mediated transgenic model for Huntington's disease] mice expressing full-length mutant Htt. Together, these data suggest that PGC1α plays a role in postnatal myelination and that deficient PGC1α activity in oligodendrocytes may contribute to abnormal myelination in HD.

Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons.

Quantitative analysis of proteins secreted from the cells poses a challenge due to their low abundance and the interfering presence of a large amount of bovine serum albumin (BSA) in the cell culture media. We established assays for detection of mutant huntingtin (mHtt) secreted from Neuro2A cell line stably expressing mHtt and rat primary cortical neurons by Western blotting. Our protocol is based on reducing the amounts of BSA in the media while maintaining cell viability and secretory potential, and concentrating the media prior to analysis by means of ultrafiltration.

Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein.

The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington's disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington's disease, and potentially for other neurodegenerative disorders.

Indocyanine green encapsulated nanogels for hyaluronidase activatable and selective near infrared imaging of tumors and lymph nodes.

Indocyanine green (ICG) encapsulated hyaluronic acid (HA) nanogels were first studied for highly selective detection of specific cancers and lymph nodes via hyaluronidase sensitive switch-on of near infrared fluorescence as a long-lasting and stimuli-responsive imaging probe.

Label-free electrochemical detection of human α-thrombin in blood serum using ferrocene-coated gold nanoparticles.

This paper describes a novel approach to label-free electrochemical detection of human α-thrombin in human blood serum that utilizes ferrocene-coated gold nanoparticles (Fc-AuNPs). Human α-thrombin was specifically bound by the thiolated aptamers immobilized on the electrode. Positively charged Fc-AuNPs were electrostatically bound to the negatively charged aptamers. In principle, a high current peak should be observed in the absence of interactions between the aptamers and the human α-thrombin. This behavior indicates maximum adsorption of Fc-AuNPs by the negatively charged aptamers on the electrode surface. In contrast, when the thrombin-aptamer complex is formed, a low signal is expected because of the blocking capacities of the protein, which hinders the electrostatic binding of the Fc-AuNPs. The electrochemical signal, recorded by cyclic voltammetry and differential pulse voltammetry, indicates whether interactions between aptamers and proteins have occurred. There is a good correlation between the ferrocene oxidation peak intensity readings from our thrombin sensing system and the thrombin concentration, within the range of 1.2 µM-12 pM.

Sirt1 mediates neuroprotection from mutant huntingtin by activation of the TORC1 and CREB transcriptional pathway.

Sirt1, a NAD-dependent protein deacetylase, has emerged as a key regulator of mammalian transcription in response to cellular metabolic status and stress. Here we show that Sirt1 has a neuroprotective role in models of Huntington's disease, an inherited neurodegenerative disorder caused by a glutamine repeat expansion in huntingtin protein (HTT). Brain-specific knockout of Sirt1 results in exacerbation of brain pathology in a mouse model of Huntington's disease, whereas overexpression of Sirt1 improves survival, neuropathology and the expression of brain-derived neurotrophic factor (BDNF) in Huntington's disease mice. We show that Sirt1 deacetylase activity directly targets neurons to mediate neuroprotection from mutant HTT. The neuroprotective effect of Sirt1 requires the presence of CREB-regulated transcription coactivator 1 (TORC1), a brain-specific modulator of CREB activity. We show that under normal conditions, Sirt1 deacetylates and activates TORC1 by promoting its dephosphorylation and its interaction with CREB. We identified BDNF as a key target of Sirt1 and TORC1 transcriptional activity in both normal and Huntington's disease neurons. Mutant HTT interferes with the TORC1-CREB interaction to repress BDNF transcription, and Sirt1 rescues this defect in vitro and in vivo. These studies suggest a key role for Sirt1 in transcriptional networks in both the normal and Huntington's disease brain and offer an opportunity for therapeutic development.

Neuroprotective role of Sirt1 in mammalian models of Huntington's disease through activation of multiple Sirt1 targets.

Huntington's disease is a fatal neurodegenerative disorder caused by an expanded polyglutamine repeat in huntingtin (HTT) protein. We previously showed that calorie restriction ameliorated Huntington's disease pathogenesis and slowed disease progression in mice that model Huntington's disease (Huntington's disease mice). We now report that overexpression of sirtuin 1 (Sirt1), a mediator of the beneficial metabolic effects of calorie restriction, protects neurons against mutant HTT toxicity, whereas reduction of Sirt1 exacerbates mutant HTT toxicity. Overexpression of Sirt1 improves motor function, reduces brain atrophy and attenuates mutant-HTT-mediated metabolic abnormalities in Huntington's disease mice. Further mechanistic studies suggested that Sirt1 prevents the mutant-HTT-induced decline in brain-derived neurotrophic factor (BDNF) concentrations and the signaling of its receptor, TrkB, and restores dopamine- and cAMP-regulated phosphoprotein, 32 kDa (DARPP32) concentrations in the striatum. Sirt1 deacetylase activity is required for Sirt1-mediated neuroprotection in Huntington's disease cell models. Notably, we show that mutant HTT interacts with Sirt1 and inhibits Sirt1 deacetylase activity, which results in hyperacetylation of Sirt1 substrates such as forkhead box O3A (Foxo3a), thereby inhibiting its pro-survival function. Overexpression of Sirt1 counteracts the mutant-HTT-induced deacetylase deficit, enhances the deacetylation of Foxo3a and facilitates cell survival. These findings show a neuroprotective role for Sirt1 in mammalian Huntington's disease models and open new avenues for the development of neuroprotective strategies in Huntington's disease.

Huntingtin cleavage product A forms in neurons and is reduced by gamma-secretase inhibitors.

BACKGROUND: The mutation in Huntington's disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB). CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown. RESULTS: Delivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec) or selectively (LY-411,575 or DAPT) reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin. CONCLUSION: We show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin-D like properties in immortalized neurons and gamma secretase-like properties in primary neurons, suggesting that cell type may be a critical factor that specifies the aspartyl protease responsible for cpA. Since gamma secretase inhibitors were also protective in primary neurons, further study of the role of gamma-secretase activity in HD neurons is justified.

Transcriptional repression of PGC-1alpha by mutant huntingtin leads to mitochondrial dysfunction and neurodegeneration.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by a glutamine repeat expansion in huntingtin protein. Transcriptional deregulation and altered energy metabolism have been implicated in HD pathogenesis. We report here that mutant huntingtin causes disruption of mitochondrial function by inhibiting expression of PGC-1alpha, a transcriptional coactivator that regulates several metabolic processes, including mitochondrial biogenesis and respiration. Mutant huntingtin represses PGC-1alpha gene transcription by associating with the promoter and interfering with the CREB/TAF4-dependent transcriptional pathway critical for the regulation of PGC-1alpha gene expression. Crossbreeding of PGC-1alpha knockout (KO) mice with HD knockin (KI) mice leads to increased neurodegeneration of striatal neurons and motor abnormalities in the HD mice. Importantly, expression of PGC-1alpha partially reverses the toxic effects of mutant huntingtin in cultured striatal neurons. Moreover, lentiviral-mediated delivery of PGC-1alpha in the striatum provides neuroprotection in the transgenic HD mice. These studies suggest a key role for PGC-1alpha in the control of energy metabolism in the early stages of HD pathogenesis.

In vitro analysis of huntingtin-mediated transcriptional repression reveals multiple transcription factor targets.

Transcriptional dysregulation has emerged as a potentially important pathogenic mechanism in Huntington's disease, a neurodegenerative disorder associated with polyglutamine expansion in the huntingtin (htt) protein. Here, we report the development of a biochemically defined in vitro transcription assay that is responsive to mutant htt. We demonstrate that both gene-specific activator protein Sp1 and selective components of the core transcription apparatus, including TFIID and TFIIF, are direct targets inhibited by mutant htt in a polyglutamine-dependent manner. The RAP30 subunit of TFIIF specifically interacts with mutant htt both in vitro and in vivo to interfere with formation of the RAP30-RAP74 native complex. Importantly, overexpression of RAP30 in cultured primary striatal cells protects neurons from mutant htt-induced cellular toxicity and alleviates the transcriptional inhibition of the dopamine D2 receptor gene by mutant htt. Our results suggest a mutant htt-directed repression mechanism involving multiple specific components of the basal transcription apparatus.

Sp1 and TAFII130 transcriptional activity disrupted in early Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract in the huntingtin protein. Transcriptional dysregulation has been implicated in HD pathogenesis. Here, we report that huntingtin interacts with the transcriptional activator Sp1 and coactivator TAFII130. Coexpression of Sp1 and TAFII130 in cultured striatal cells from wild-type and HD transgenic mice reverses the transcriptional inhibition of the dopamine D2 receptor gene caused by mutant huntingtin, as well as protects neurons from huntingtin-induced cellular toxicity. Furthermore, soluble mutant huntingtin inhibits Sp1 binding to DNA in postmortem brain tissues of both presymptomatic and affected HD patients. Understanding these early molecular events in HD may provide an opportunity to interfere with the effects of mutant huntingtin before the development of disease symptoms.