Preventing mutant huntingtin proteolysis and intermittent fasting promote autophagy in models of Huntington disease.

Huntington disease (HD) is caused by the expression of mutant huntingtin (mHTT) bearing a polyglutamine expansion. In HD, mHTT accumulation is accompanied by a dysfunction in basal autophagy, which manifests as specific defects in cargo loading during selective autophagy. Here we show that the expression of mHTT resistant to proteolysis at the caspase cleavage site D586 (C6R mHTT) increases autophagy, which may be due to its increased binding to the autophagy adapter p62. This is accompanied by faster degradation of C6R mHTT in vitro and a lack of mHTT accumulation the C6R mouse model with age. These findings may explain the previously observed neuroprotective properties of C6R mHTT. As the C6R mutation cannot be easily translated into a therapeutic approach, we show that a scheduled feeding paradigm is sufficient to lower mHTT levels in YAC128 mice expressing cleavable mHTT. This is consistent with a previous model, where the presence of cleavable mHTT impairs basal autophagy, while fasting-induced autophagy remains functional. In HD, mHTT clearance and autophagy may become increasingly impaired as a function of age and disease stage, because of gradually increased activity of mHTT-processing enzymes. Our findings imply that mHTT clearance could be enhanced by a regulated dietary schedule that promotes autophagy.

Palmitoylation of caspase-6 by HIP14 regulates its activation.

Caspase-6 (CASP6) has an important role in axonal degeneration during neuronal apoptosis and in the neurodegenerative diseases Alzheimer and Huntington disease. Decreasing CASP6 activity may help to restore neuronal function in these and other diseases such as stroke and ischemia, where increased CASP6 activity has been implicated. The key to finding approaches to decrease CASP6 activity is a deeper understanding of the mechanisms regulating CASP6 activation. We show that CASP6 is posttranslationally palmitoylated by the palmitoyl acyltransferase HIP14 and that the palmitoylation of CASP6 inhibits its activation. Palmitoylation of CASP6 is decreased both in Hip14-/- mice, where HIP14 is absent, and in YAC128 mice, a model of Huntington disease, where HIP14 is dysfunctional and where CASP6 activity is increased. Molecular modeling suggests that palmitoylation of CASP6 may inhibit its activation via steric blockage of the substrate-binding groove and inhibition of CASP6 dimerization, both essential for CASP6 function. Our studies identify palmitoylation as a novel CASP6 modification and as a key regulator of CASP6 activity.

Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

In vivo evaluation of candidate allele-specific mutant huntingtin gene silencing antisense oligonucleotides.

Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD.

The palmitoyl acyltransferase HIP14 shares a high proportion of interactors with huntingtin: implications for a role in the pathogenesis of Huntington's disease.

HIP14 is the most highly conserved of 23 human palmitoyl acyltransferases (PATs) that catalyze the post-translational addition of palmitate to proteins, including huntingtin (HTT). HIP14 is dysfunctional in the presence of mutant HTT (mHTT), the causative gene for Huntington disease (HD), and we hypothesize that reduced palmitoylation of HTT and other HIP14 substrates contributes to the pathogenesis of the disease. Here we describe the yeast two-hybrid (Y2H) interactors of HIP14 in the first comprehensive study of interactors of a mammalian PAT. Unexpectedly, we discovered a highly significant overlap between HIP14 interactors and 370 published interactors of HTT, 4-fold greater than for control proteins (P = 8 × 10(-5)). Nearly half of the 36 shared interactors are already implicated in HD, supporting a direct link between HIP14 and the disease. The HIP14 Y2H interaction set is significantly enriched for palmitoylated proteins that are candidate substrates. We confirmed that three of them, GPM6A, and the Sprouty domain-containing proteins SPRED1 and SPRED3, are indeed palmitoylated by HIP14; the first enzyme known to palmitoylate these proteins. These novel substrates functions might be affected by reduced palmitoylation in HD. We also show that the vesicular cargo adapter optineurin, an established HTT-binding protein, co-immunoprecipitates with HIP14 but is not palmitoylated. mHTT leads to mislocalization of optineurin and aberrant cargo trafficking. Therefore, it is possible that optineurin regulates trafficking of HIP14 to its substrates. Taken together, our data raise the possibility that defective palmitoylation by HIP14 might be an important mechanism that contributes to the pathogenesis of HD.

Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS.

Autosomal dominant diseases such as Huntington's disease (HD) are caused by a gain of function mutant protein and/or RNA. An ideal treatment for these diseases is to selectively suppress expression of the mutant allele while preserving expression of the wild-type variant. RNase H active antisense oligonucleotides (ASOs) or small interfering RNAs can achieve allele selective suppression of gene expression by targeting single nucleotide polymorphisms (SNPs) associated with the repeat expansion. ASOs have been previously shown to discriminate single nucleotide changes in targeted RNAs with ∼5-fold selectivity. Based on RNase H enzymology, we enhanced single nucleotide discrimination by positional incorporation of chemical modifications within the oligonucleotide to limit RNase H cleavage of the non-targeted transcript. The resulting oligonucleotides demonstrate >100-fold discrimination for a single nucleotide change at an SNP site in the disease causing huntingtin mRNA, in patient cells and in a completely humanized mouse model of HD. The modified ASOs were also well tolerated after injection into the central nervous system of wild-type animals, suggesting that their tolerability profile is suitable for advancement as potential allele-selective HD therapeutics. Our findings lay the foundation for efficient allele-selective downregulation of gene expression using ASOs-an outcome with broad application to HD and other dominant genetic disorders.

Rescue from excitotoxicity and axonal degeneration accompanied by age-dependent behavioral and neuroanatomical alterations in caspase-6-deficient mice.

Apoptosis, or programmed cell death, is a cellular pathway involved in normal cell turnover, developmental tissue remodeling, embryonic development, cellular homeostasis maintenance and chemical-induced cell death. Caspases are a family of intracellular proteases that play a key role in apoptosis. Aberrant activation of caspases has been implicated in human diseases. In particular, numerous findings implicate Caspase-6 (Casp6) in neurodegenerative diseases, including Alzheimer disease (AD) and Huntington disease (HD), highlighting the need for a deeper understanding of Casp6 biology and its role in brain development. The use of targeted caspase-deficient mice has been instrumental for studying the involvement of caspases in apoptosis. The goal of this study was to perform an in-depth neuroanatomical and behavioral characterization of constitutive Casp6-deficient (Casp6-/-) mice in order to understand the physiological function of Casp6 in brain development, structure and function. We demonstrate that Casp6-/- neurons are protected against excitotoxicity, nerve growth factor deprivation and myelin-induced axonal degeneration. Furthermore, Casp6-deficient mice show an age-dependent increase in cortical and striatal volume. In addition, these mice show a hypoactive phenotype and display learning deficits. The age-dependent behavioral and region-specific neuroanatomical changes observed in the Casp6-/- mice suggest that Casp6 deficiency has a more pronounced effect in brain regions that are involved in neurodegenerative diseases, such as the striatum in HD and the cortex in AD.

Caspase-6-Resistant Mutant Huntingtin Does not Rescue the Toxic Effects of Caspase-Cleavable Mutant Huntingtin in vivo.

BACKGROUND: The amelioration of behavioral and neuropathological deficits in mice expressing caspase-6-resistant (C6R) mutant huntingtin (mhtt), despite the presence of an expanded polyglutamine tract, highlights proteolysis of htt at the 586aa caspase-6 (casp6) site may be an important mechanism in the pathogenesis of Huntington disease (HD). One possible explanation of these effects is that C6R mhtt could act as a dominant negative on mhtt. OBJECTIVE AND METHODS: To determine if the neuroprotective effect observed in the C6R mice is due to dominant negative effects, we crossed the C6R mice to the YAC128 HD mouse model to generate mice expressing both caspase-cleavable and C6R mhtt (YAC/C6R) concurrently and assessed previously defined behavioral and neuropathological endpoints. RESULTS: Our results demonstrate that YAC/C6R animals exhibit similar motor abnormalities and learning deficits as the YAC128 mice. Neuropathological analysis reveals a significant decrease in brain weight and striatal volume in the YAC/C6R mice comparable to the YAC128 mice. In contrast, and similar to previous findings, C6R mice demonstrate preserved brain weight and striatal volume. As expected, body weight is significantly increased in the YAC/C6R mice due to the increased levels of htt. CONCLUSIONS: The results of this study suggest that the lack of an HD phenotype in the C6R mice is most likely due to the absence of cleavage of htt and not due to suppression of expression of mhtt.

Altered palmitoylation and neuropathological deficits in mice lacking HIP14.

Huntingtin interacting protein 14 (HIP14, ZDHHC17) is a huntingtin (HTT) interacting protein with palmitoyl transferase activity. In order to interrogate the function of Hip14, we generated mice with disruption in their Hip14 gene. Hip14-/- mice displayed behavioral, biochemical and neuropathological defects that are reminiscent of Huntington disease (HD). Palmitoylation of other HIP14 substrates, but not Htt, was reduced in the Hip14-/- mice. Hip14 is dysfunctional in the presence of mutant htt in the YAC128 mouse model of HD, suggesting that altered palmitoylation mediated by HIP14 may contribute to HD.

Cleavage at the 586 amino acid caspase-6 site in mutant huntingtin influences caspase-6 activation in vivo.

Caspase cleavage of huntingtin (htt) and nuclear htt accumulation represent early neuropathological changes in brains of patients with Huntington's disease (HD). However, the relationship between caspase cleavage of htt and caspase activation patterns in the pathogenesis of HD remains poorly understood. The lack of a phenotype in YAC mice expressing caspase-6-resistant (C6R) mutant htt (mhtt) highlights proteolysis of htt at the 586 aa caspase-6 (casp6) site as a key mechanism in the pathology of HD. The goal of this study was to investigate how proteolysis of htt at residue 586 plays a role in the pathogenesis of HD and determine whether inhibiting casp6 cleavage of mhtt alters cell-death pathways in vivo. Here we demonstrate that activation of casp6, and not caspase-3, is observed before onset of motor abnormalities in human and murine HD brain. Active casp6 levels correlate directly with CAG size and inversely with age of onset. In contrast, in vivo expression of C6R mhtt attenuates caspase activation. Increased casp6 activity and apoptotic cell death is evident in primary striatal neurons expressing caspase-cleavable, but not C6R, mhtt after NMDA application. Pretreatment with a casp6 inhibitor rescues the apoptotic cell death observed in this paradigm. These data demonstrate that activation of casp6 is an early marker of disease in HD. Furthermore, these data provide a clear link between excitotoxic pathways and proteolysis and suggest that C6R mhtt protects against neurodegeneration by influencing the activation of neuronal cell-death and excitotoxic pathways operative in HD.

Palmitoylation of ATP-binding cassette transporter A1 is essential for its trafficking and function.

ATP-binding cassette transporter (ABC)A1 lipidates apolipoprotein A-I both directly at the plasma membrane and also uses lipids from the late endosomal or lysosomal compartment in the internal lipidation of apolipoprotein A-I. However, how ABCA1 targeting to these specific membranes is regulated remains unknown. Palmitoylation is a dynamically regulated lipid modification that targets many proteins to specific membrane domains. We hypothesized that palmitoylation may also regulate ABCA1 transport and function. Indeed, ABCA1 is robustly palmitoylated at cysteines 3, -23, -1110, and -1111. Abrogation of palmitoylation of ABCA1 by mutation of the cysteines results in a reduction of ABCA1 localization at the plasma membranes and a reduction in the ability of ABCA1 to efflux lipids to apolipoprotein A-I. ABCA1 is palmitoylated by the palmitoyl transferase DHHC8, and increasing DHHC8 protein results in increased ABCA1-mediated lipid efflux. Thus, palmitoylation regulates ABCA1 localization at the plasma membrane, and regulates its lipid efflux ability.

Cortactin (CTTN), N-WASP (WASL), and clathrin (CLTC) are present at podosome-like tubulobulbar complexes in the rat testis.

Tubulobulbar complexes are actin filament-rich plasma membrane protrusions that form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. Tubulobulbar complexes morphologically resemble podosomes found at cell/substrate attachments in other systems. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat testis for two key actin-related components found at podosomes, and for the endocytosis-related protein clathrin. N-WASP and cortactin, two regulators of actin network assembly known to be components of podosomes, are concentrated at tubulobulbar complexes. Clathrin-positive structures occur in Sertoli cell regions containing tubulobulbar complexes when analyzed by immunofluorescence microscopy and occur at the ends of the complexes when evaluated by immunoelectron microscopy. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures. We propose that the formation of tubulobulbar complexes may be clathrin initiated and that their growth is due to the dendritic assembly of a membrane-related actin network.

Tubulobulbar complexes are intercellular podosome-like structures that internalize intact intercellular junctions during epithelial remodeling events in the rat testis.

Tubulobulbar complexes are actin-related double-membrane projections that resemble podosomes in other systems and form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat and mouse testes for junction molecules reported to be present at apical sites of attachment (ectoplasmic specializations) between Sertoli cells and spermatids. The adhesion molecules nectin 2 (PVRL2), nectin 3 (PVRL3) and alpha 6 integrin (ITGA6) are present in the elongate parts of tubulobulbar complexes and concentrated at their distal ends. Tubulobulbar complexes contain cortactin (CTTN), a key component of podosomes, and vesicles at the distal ends of tubulobulbar complexes that contain junction molecules are related to early endosome antigen (EEA1). N-cadherin (CDH2), a protein reported to be present at ectoplasmic specializations, is not localized to these unique junctions or to tubulobulbar complexes but, rather, is primarily concentrated at desmosomes in basal regions of the epithelium. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures that are responsible for internalizing intact intercellular junctions during spermatogenesis.

The Sertoli cell cytoskeleton.

The cytoskeleton of terminally differentiated mammalian Sertoli cells is one of the most elaborate of those that have been described for cells in tissues. Actin filaments, intermediate filaments and microtubules have distinct patterns of distribution that change during the cyclic process of spermatogenesis. Each of the three major cytoskeletal elements is either concentrated at or related in part to intercellular junctions. Actin filaments are concentrated in unique structures termed ectoplasmic specializations that function in intercellular adhesion, and at tubulobulbar complexes that are thought to be involved with junction internalization during sperm release and movement of spermatocytes through basal junctions between neighboring Sertoi cells. Intermediate filaments occur in a perinuclear network which has peripheral extensions to desmosome-like junctions with adjacent cells and to small hemidesmosome-like attachments to the basal lamina. Unlike in most other epithelia where the intermediate filaments are of the keratin type, intermediate filaments in mature Sertoli cells are of the vimentin type. The function of intermediate filaments in Sertoli cells in not entirely clear; however, the pattern of filament distribution and the limited experimental data available are consistent with a role in maintaining tissue integrity when the epithelium is mechanically stressed. Microtubules are abundant in Sertoli cells and are predominantly oriented parallel to the long axis of the cell. Microtubules are involved with maintaining the columnar shape of Sertoli cells, with transporting and positioning organelles in the cytoplasm, and with secreting seminiferous tubule fluid. In addition, microtubule-based transport machinery is coupled to intercellular junctions to translocate and position adjacent spermatids in the epithelium. Although the cytoskeleton of Sertoli cells has structural and functional properties common to cells generally, there are a number of properties that are unique and that appear related to processes fundamental to spermatogenesis and to interfacing somatic cells both with similar neighboring somatic cells and with differentiating cells of the germ cell line.

Enrichment and disassembly of ectoplasmic specializations in the rat testis.

Ectoplasmic specializations are testis specific intercellular adhesion junctions found in Sertoli cells. They are tripartite structures consisting of the plasma membrane of the Sertoli cell, a submembrane layer of actin filaments and an attached cistern of endoplasmic reticulum. Ectoplasmic specializations occur in areas of attachment to spermatids and as part of the basal junction complex between neighboring Sertoli cells. They are functionally related to a number of fundamental events that occur during spermatogenesis, including attachment and then release of developing sperm cells and the translocation of spermatocytes through the blood-testis barrier. The structures may contain viable molecular targets for the development of contraceptives. Here we describe techniques for obtaining, from rat testes, testicular fractions enriched for spermatids with attached ectoplasmic specializations and for disassembling the complexes with gelsolin to obtain supernatants enriched for plaque components. The techniques involve stripping the epithelium from tubule walls, mechanically fragmenting the epithelium, using step sucrose gradients to enrich for spermatids with attached junction plaques, and then incubating with exogenous gelsolin to release plaque components into solution.

A kinesin is present at unique sertoli/spermatid adherens junctions in rat and mouse testes.

During spermatogenesis, spermatids undergo a "down and up" translocation event in the seminiferous epithelium. This event has been proposed to result from the movement of ectoplasmic specializations, which are formed in Sertoli cells at sites of adhesion to spermatids, along adjacent microtubule tracts. To test the hypothesis that a kinesin is associated with ectoplasmic specializations, we generated antibodies to conserved kinesin sequences and detected kinesins on fixed frozen testis sections and fixed seminiferous epithelial fragments. The antibodies reacted with ectoplasmic specializations related to spermatids, in addition to reacting with other structures in the epithelium known to contain kinesins. At the electron microscopy level, the antibodies reacted with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. Based on mRNA transcript screens using mouse GeneChip arrays of testis and Sertoli cells, we identified KIF20 as a candidate kinesin at ectoplasmic specializations. Antibodies generated against a peptide sequence unique to this kinesin reacted at ectoplasmic specializations in testis sections and epithelial fragments, as well as with the endoplasmic reticulum component of ectoplasmic specializations when analyzed by electron microscopy. The antibody reacted on Western blots with full-length KIF20. On Western blots of testis lysates, the antibody reacted with a protein that is not present in other tissues and which migrates at a higher molecular weight than that predicted for KIF20. Our results demonstrate that a kinesin is associated with apical ectoplasmic specializations in Sertoli cells and that the motor may be an isoform of KIF20.

A re-evaluation of gelsolin at ectoplasmic specializations in sertoli cells: the influence of serum in blocking buffers on staining patterns.

In this study, we test the hypothesis that gelsolin immunolocalized in actin filament-rich ectoplasmic specializations may be exogenous gelsolin present in normal serum used in blocking buffers, and that binds to the intercellular adhesion plaques during tissue processing. Fixed frozen sections of rat and rabbit testis were pre-treated with standard blocking buffers containing 5% normal goat serum (NGS) and then incubated with anti-gelsolin antibodies in the presence of 1% NGS. Other sections were treated in a similar fashion, but in buffers not containing NGS. Sections were then labeled with secondary antibody conjugated to a fluorochrome. Localized staining at ectoplasmic specializations occurred only in sections treated with NGS. The only positive staining in sections not treated with NGS was associated with seminiferous tubule walls and blood vessels in rabbit tissue. The antibodies reacted with a single band at the appropriate molecular weight for gelsolin on immunoblots of NGS, but did not react on immunoblots of testis or seminiferous epithelium. We conclude that gelsolin localized at ectoplasmic specializations using current commercially available antibodies is a result of non-specific binding to the fixed tissues of gelsolin present in blocking buffers.

Absence of behavioral abnormalities and neurodegeneration in vivo despite widespread neuronal huntingtin inclusions.

We have serendipitously established a mouse that expresses an N-terminal human huntingtin (htt) fragment with an expanded polyglutamine repeat (approximately 120) under the control of the endogenous human promoter (shortstop). Frequent and widespread htt inclusions occur early in shortstop mice. Despite these inclusions, shortstop mice display no clinical evidence of neuronal dysfunction and no neuronal degeneration as determined by brain weight, striatal volume, and striatal neuronal count. These results indicate that htt inclusions are not pathogenic in vivo. In contrast, the full-length yeast artificial chromosome (YAC) 128 model with the identical polyglutamine length and same level of transgenic protein expression as the shortstop demonstrates significant neuronal dysfunction and loss. In contrast to the YAC128 mouse, which demonstrates enhanced susceptibility to excitotoxic death, the shortstop mouse is protected from excitotoxicity, providing in vivo evidence suggesting that neurodegeneration in Huntington disease is mediated by excitotoxic mechanisms.