Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo

We have developed yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 or YAC72) human huntingtin (htt), in a developmental- and tissue-specific manner, that is identical to endogenous htt. YAC72 mice develop selective degeneration of medium spiny projection neurons in the lateral striatum, similar to what is observed in Huntington disease. Mutant human htt expressed by YAC transgenes can compensate for the absence of endogenous htt and can rescue the embryonic lethality that characterizes mice homozygous for targeted disruption of the endogenous Hdh gene (-/-). YAC72 mice lacking endogenous htt (YAC72 -/-) manifest a novel phenotype characterized by infertility, testicular atrophy, aspermia, and massive apoptotic cell death in the testes. The testicular cell death in YAC72 -/- mice can be markedly reduced by increasing endogenous htt levels. YAC72 mice with equivalent levels of both wild-type and mutant htt (YAC72 +/+) breed normally and have no evidence of increased testicular cell death. Similar findings are seen in YAC46 -/- mice compared with YAC46 +/+ mice, in which wild-type htt can completely counteract the proapoptotic effects of mutant htt. YAC18 -/- mice display no evidence of increased cellular apoptosis, even in the complete absence of endogenous htt, demonstrating that the massive cellular apoptosis observed in YAC46 -/- mice and YAC72 -/- mice is polyglutamine-mediated toxicity from the mutant transgene. These data provide the first direct in vivo evidence of a role for wild-type htt in decreasing the cellular toxicity of mutant htt.     

Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.     

Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice

Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.     

Wild-type huntingtin protects neurons from excitotoxicity

Huntingtin is a caspase substrate, and loss of normal huntingtin function resulting from caspase-mediated proteolysis may play a role in the pathogenesis of Huntington disease. Here we tested the hypothesis that increasing huntingtin levels protect striatal neurons from NMDA receptor-mediated excitotoxicity. Cultured striatal neurons from yeast artificial chromosome (YAC)18 transgenic mice over-expressing full-length wild-type huntingtin were dramatically protected from apoptosis and caspase-3 activation compared with cultured striatal neurons from non-transgenic FVB/N littermates and YAC72 mice expressing mutant human huntingtin. NMDA receptor activation induced by intrastriatal injection of quinolinic acid initiated a form of apoptotic neurodegeneration within the striatum of mice that was associated with caspase-3 cleavage of huntingtin in neurons and astrocytes, decreased levels of full-length huntingtin, and the generation of a specific N-terminal caspase cleavage product of huntingtin. In vivo, over-expression of wild-type huntingtin in YAC18 transgenic mice conferred significant protection against NMDA receptor-mediated apoptotic neurodegeneration. These data provide in vitro and in vivo evidence that huntingtin may regulate the balance between neuronal survival and death following acute excitotoxic stress, and that the levels of huntingtin may modulate neuronal sensitivity to excitotoxic neurodegeneration. We suggest that further study of huntingtin's anti-apoptotic function will contribute to our understanding of the pathogenesis of Huntingdon's disease and provide insights into the selective vulnerability of striatal neurons to excitotoxic cell death.     

Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)(n) repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.     

Dissociation of progressive dopaminergic neuronal death and behavioral impairments by Bax deletion in a mouse model of Parkinson's diseases

Parkinson's disease (PD) is a common, late-onset movement disorder with selective degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Although the neurotoxin 6-hydroxydopamine (6-OHDA) has been used to induce progressive degeneration of DA neurons in various animal models of PD, the precise molecular pathway and the impact of anti-apoptotic treatment on this neurodegeneration are less understood. Following a striatal injection of 6-OHDA, we observed atrophy and progressive death of DA neurons in wild-type mice. These degenerating DA neurons never exhibited signs of apoptosis (i.e., caspase-3 activation and cytoplasmic release of cytochrome C), but rather show nuclear translocation of apoptosis-inducing factor (AIF), a hallmark of regulated necrosis. However, mice with genetic deletion of the proapoptotic gene Bax (Bax-KO) exhibited a complete absence of 6-OHDA-induced DA neuron death and nuclear translocation of AIF, indicating that 6-OHDA-induced DA neuronal death is mediated by Bax-dependent AIF activation. On the other hand, DA neurons that survived in Bax-KO mice exhibited marked neuronal atrophy, without significant improvement of PD-related behavioral deficits. These findings suggest that anti-apoptotic therapy may not be sufficient for PD treatment, and the prevention of Bax-independent neuronal atrophy may be an important therapeutic target.     

Inducing huntingtin inclusion formation in primary neuronal cell culture and in vivo by high-capacity adenoviral vectors expressing truncated and full-length huntingtin with polyglutamine expansion

BACKGROUND: Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (htt) gene. Vector-mediated delivery of N-terminal fragments of mutant htt has been used to study htt function in vitro and to establish HD models in rats. Due to the large size of the htt cDNA vector-mediated delivery of full-length htt has not been achieved so far. METHODS: High-capacity adenoviral (HC-Ad) vectors were generated expressing mutant and wild-type versions of N-terminal truncated and full-length htt either in vitro in primary neuronal cells or in the striatum of mice. RESULTS: In vitro these vectors were used for transduction of primary neuronal cells isolated from E17 mouse embryos. Expression of mutant htt resulted in the formation of htt inclusions, a surrogate marker of the HD pathology. Kinetics of generation and localization of htt inclusions differed between truncated and full-length htt carrying identical mutations. Following injection into the striatum vector-mediated expression of mutant truncated htt led to prominent accumulation of htt inclusions in cell nuclei, while inclusions formed upon expression of mutant full-length htt localized to the cytoplasm. CONCLUSIONS: These results indicate that HC-Ad vector-mediated in vitro and in vivo delivery of truncated and full-length mutant htt results in prominent inclusion formation in neuronal cells but in different cell compartments. These vectors will be useful tools for studying HD and may be used to generate large animal HD models.     

Mutant huntingtin promotes the fibrillogenesis of wild-type huntingtin: a potential mechanism for loss of huntingtin function in Huntington's disease

Aggregation of huntingtin (htt) in neuronal inclusions is associated with the development of Huntington's disease (HD). Previously, we have shown that mutant htt fragments with polyglutamine (polyQ) tracts in the pathological range (>37 glutamines) form SDS-resistant aggregates with a fibrillar morphology, whereas wild-type htt fragments with normal polyQ domains do not aggregate. In this study we have investigated the co-aggregation of mutant and wild-type htt fragments. We found that mutant htt promotes the aggregation of wild-type htt, causing the formation of SDS-resistant co-aggregates with a fibrillar morphology. Conversely, mutant htt does not promote the fibrillogenesis of the polyQ-containing protein NOCT3 or the polyQ-binding protein PQBP1, although these proteins are recruited into inclusions containing mutant htt aggregates in mammalian cells. The formation of mixed htt fibrils is a highly selective process that not only depends on polyQ tract length but also on the surrounding amino acid sequence. Our data suggest that mutant and wild-type htt fragments may also co-aggregate in neurons of HD patients and that a loss of wild-type htt function may contribute to HD pathogenesis.     

Essential sequence of the N-terminal cytoplasmic localization-related domain of huntingtin and its effect on huntingtin aggregates

Huntington’s disease (HD) is caused by abnormal CAG repeat expansion in the 5′-end of the Huntingtin (HTT) gene. In addition to the canonical C-terminal full-length huntingtin (htt) nuclear export signal, a cytoplasmic localization-related domain (CLRD) in the N-terminus of htt has recently been reported. Here, we analyzed this domain by introducing deletion and substitution mutations in a truncated N-terminal htt protein and subsequently monitored htt expression, aggregation and subcellular localization by immunocytochemistry and Western blot analysis. We demonstrated that Htt_4–17 was the essential sequence for htt cytoplasmic localization. We also found that the subcellular distribution of htt was altered when Htt_1–17 was mutated to contain amino acids of different charges, suggesting a structural requirement of Htt_1–17 for the cytoplasmic localization of htt. Deletion of the first three amino acids did not affect its association with mitochondria. We observed that defective cytoplasmic localization resulted in a reduction of total htt aggregates and increased nuclear aggregates, indicating that the subcellular distribution of the protein might influence the aggregation process. These studies provide new insight into the molecular mechanism of htt aggregation in HD.     

Huntingtin disrupts lipid bilayers in a polyQ-length dependent manner

Huntington's Disease (HD) is a neurodegenerative disorder that is defined by the accumulation of nanoscale aggregates comprised of the huntingtin (htt) protein. Aggregation is directly caused by an expanded polyglutamine (polyQ) domain in htt, leading to a diverse population of aggregate species, such as oligomers, fibrils, and annular aggregates. Furthermore, the length of this polyQ domain is directly related to onset and severity of disease. The first 17 N-terminal amino acids of htt have been shown to further modulate aggregation. Additionally, these 17 amino acids appear to have lipid binding properties as htt interacts with a variety of membrane-containing structures present in cells, such as organelles, and interactions with these membrane surfaces may further modulate htt aggregation. To investigate the interaction between htt exon1 and lipid bilayers, in situ atomic force microscopy (AFM) was used to directly monitor the aggregation of htt exon1 constructs with varying Q-lengths (35Q, 46Q, 51Q, and myc-53Q) on supported lipid membranes comprised of total brain lipid extract. The exon1 fragments accumulated on the lipid membranes, causing disruption of the membrane, in a polyQ dependent manner. Furthermore, the addition of an N-terminal myc-tag to the htt exon1 fragments impeded the interaction of htt with the bilayer.     

Huntingtin forms toxic NH2-terminal fragment complexes that are promoted by the age-dependent decrease in proteasome activity

Although NH2-terminal mutant huntingtin (htt) fragments cause neurological disorders in Huntington's disease (HD), it is unclear how toxic htt fragments are generated and contribute to the disease process. Here, we report that complex NH2-terminal mutant htt fragments smaller than the first 508 amino acids were generated in htt-transfected cells and HD knockin mouse brains. These fragments constituted neuronal nuclear inclusions and appeared before neurological symptoms. The accumulation and aggregation of these htt fragments were associated with an age-dependent decrease in proteasome activity and were promoted by inhibition of proteasome activity. These results suggest that decreased proteasome activity contributes to late onset htt toxicity and that restoring the ability to remove NH2-terminal fragments will provide a more effective therapy for HD than inhibiting their production.     

Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

Huntington disease (HD) is caused by expansion of a polyglutamine (polyQ) domain in the protein known as huntingtin (htt), and the disease is characterized by selective neurodegeneration. Expansion of the polyQ domain is not exclusive to HD, but occurs in eight other inherited neurodegenerative disorders that show distinct neuropathology. Yet in spite of the clear genetic defects and associated neurodegeneration seen with all the polyQ diseases, their pathogenesis remains elusive. The present review focuses on HD, outlining the effects of mutant htt in the nucleus and neuronal processes as well as the role of cell-cell interactions in HD pathology. The widespread expression and localization of mutant htt and its interactions with a variety of proteins suggest that mutant htt engages multiple pathogenic pathways. Understanding these pathways will help us to elucidate the pathogenesis of HD and to target therapies effectively.     

Distinct aggregation and cell death patterns among different types of primary neurons induced by mutant huntingtin protein

Aggregation of disease proteins is believed to be a central event in the pathology of polyglutamine diseases, whereas the relationship between aggregation and neuronal death remains controversial. We investigated this question by expressing mutant huntingtin (htt) with a defective adenovirus in different types of neurons prepared from rat cerebral cortex, striatum or cerebellum. The distribution pattern of inclusions is not identical among different types of primary neurons. On day 2 after infection, cytoplasmic inclusions are dominant in cortical and striatal neurons, whereas at day 4 the ratio of nuclear inclusions overtakes that of cytoplasmic inclusions. Meanwhile, nuclear inclusions are always predominantly present in cerebellar neurons. The percentage of inclusion-positive cells is highest in cerebellar neurons, whereas mutant htt induces cell death most remarkably in cortical neurons. As our system uses htt exon 1 protein and thus aggregation occurs independently from cleavage of the full-length htt, our observations indicate that the aggregation process is distinct among different neurons. Most of the neurons containing intracellular (either nuclear or cytoplasmic) aggregates are viable. Our findings suggest that the process of mutant htt aggregation rather than the resulting inclusion body is critical for neuronal cell death.     

Huntingtin spheroids and protofibrils as precursors in polyglutamine fibrilization

The pathology of Huntington's disease is characterized by neuronal degeneration and inclusions containing N-terminal fragments of mutant huntingtin (htt). To study htt aggregation, we examined purified htt fragments in vitro, finding globular and protofibrillar intermediates participating in the genesis of mature fibrils. These intermediates were high in beta-structure. Furthermore, Congo Red, a dye that stains amyloid fibrils, prevented the assembly of mutant htt into mature fibrils, but not the formation of protofibrils. Other proteins capable of forming ordered aggregates, such as amyloid beta and alpha-synuclein, form similar intermediates, suggesting that the mechanisms of mutant htt aggregation and possibly htt toxicity may overlap with other neurodegenerative disorders.     

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.     

N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy

The Huntington’s disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1–208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD.     

Huntingtin fragments that aggregate go their separate ways

N-terminal region of mutant huntingtin forms intranuclear and cytoplasmic aggregates in neurons that may contribute to neuronal death in Huntington's disease. show that different endoprotease-cleaved huntingtin fragments form nuclear and cytoplasmic inclusions.