Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits.

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

Maintenance of susceptibility to neurodegeneration following intrastriatal injections of quinolinic acid in a new transgenic mouse model of Huntington's disease.

A transgenic mouse model of Huntington's disease (R6/1 and R6/2 lines) expressing exon 1 of the HD gene with 115-150 CAG repeats resisted striatal damage following injection of quinolinic acid and other neurotoxins. We examined whether excitotoxin resistance characterizes mice with mutant huntingtin transgenes. In a new transgenic mouse with 3 kb of mutant human huntingtin cDNA with 18, 46, or 100 CAG repeats, we found no change in susceptibility to intrastriatal injections of the excitotoxin quinolinic acid, compared to wild-type littermates. The new transgenic mice were injected with the same dose of quinolinic acid (30 nmol) as had been the R6 mice. Our findings highlight the importance of studying pathogenetic mechanisms in different transgenic models of a disease.

Mice transgenic for exon 1 of the Huntington's disease gene display reduced striatal sensitivity to neurotoxicity induced by dopamine and 6-hydroxydopamine.

Huntington's disease is an autosomal dominant hereditary neurodegenerative disorder characterized by severe striatal cell loss. Dopamine (DA) has been suggested to play a role in the pathogenesis of the disease. We have previously reported that transgenic mice expressing exon 1 of the human Huntington gene (R6 lines) are resistant to quinolinic acid-induced striatal toxicity. In this study we show that with increasing age, R6/1 and R6/2 mice develop partial resistance to DA- and 6-hydroxydopamine-mediated toxicity in the striatum. Using electron microscopy, we found that the resistance is localized to the cell bodies and not to the neuropil. The reduction of dopamine and cAMP regulated phosphoprotein of a molecular weight of 32 kDa (DARPP-32) in R6/2 mice does not provide the resistance, as DA-induced striatal lesions are not reduced in size in DARPP-32 knockout mice. Neither DA receptor antagonists nor a N-methyl-d-aspartate (NMDA) receptor blocker reduce the size of DA-induced striatal lesions, suggesting that DA toxicity is not dependent upon DA- or NMDA receptor-mediated pathways. Moreover, superoxide dismutase-1 overexpression, monoamine oxidase inhibition and the treatment with the free radical scavenging spin-trap agent phenyl-butyl-tert-nitrone (PBN) also did not block DA toxicity. Levels of the antioxidant molecules, glutathione and ascorbate were not increased in R6/1 mice. Because damage to striatal neurons following intrastriatal injection of 6-hydroxydopamine was also reduced in R6 mice, a yet-to-be identified antioxidant mechanism may provide neuroprotection in these animals. We conclude that striatal neurons of R6 mice develop resistance to DA-induced toxicity with age.

Changes in cortical and striatal neurons predict behavioral and electrophysiological abnormalities in a transgenic murine model of Huntington's disease.

Neurons in Huntington's disease exhibit selective morphological and subcellular alterations in the striatum and cortex. The link between these neuronal changes and behavioral abnormalities is unclear. We investigated relationships between essential neuronal changes that predict motor impairment and possible involvement of the corticostriatal pathway in developing behavioral phenotypes. We therefore generated heterozygote mice expressing the N-terminal one-third of huntingtin with normal (CT18) or expanded (HD46, HD100) glutamine repeats. The HD mice exhibited motor deficits between 3 and 10 months. The age of onset depended on an expanded polyglutamine length; phenotype severity correlated with increasing age. Neuronal changes in the striatum (nuclear inclusions) preceded the onset of phenotype, whereas cortical changes, especially the accumulation of huntingtin in the nucleus and cytoplasm and the appearance of dysmorphic dendrites, predicted the onset and severity of behavioral deficits. Striatal neurons in the HD mice displayed altered responses to cortical stimulation and to activation by the excitotoxic agent NMDA. Application of NMDA increased intracellular Ca(2+) levels in HD100 neurons compared with wild-type neurons. Results suggest that motor deficits in Huntington's disease arise from cumulative morphological and physiological changes in neurons that impair corticostriatal circuitry.

Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis.

The Huntington's disease (HD) mutation is a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). How neurons die in HD is unclear. Mutant N-htt aggregates in neurons in the HD brain; expression of mutant N-htt in vitro causes cell death. Other in vitro studies show that proteolysis by caspase 3 could be important in regulating mutant N-htt function, but there has been no direct evidence for caspase 3-cleaved N-htt fragments in brain. Here, we show that N-htt fragments consistent with the size produced by caspase 3 cleavage in vitro are resident in the cortex, striatum, and cerebellum of normal and adult onset HD brain and are similar in size to the fragments seen after exogenous expression of human huntingtin in mouse clonal striatal neurons. HD brain extracts treated with active caspase 3 had increased levels of N-htt fragments. Compared with the full-length huntingtin, the caspase 3-cleaved N-htt fragments, especially the mutant fragment, preferentially segregated with the membrane fraction. Partial proteolysis of the human caspase 3-cleaved N-htt fragment by calpain occurred in vitro and resulted in smaller N-terminal products; products of similar size appeared when mouse brain protein extracts were treated with calpain. Results support the idea that sequential proteolysis by caspase 3 and calpain may regulate huntingtin function at membranes and produce N-terminal mutant fragments that aggregate and cause cellular dysfunction in HD.

Early and progressive accumulation of reactive microglia in the Huntington disease brain.

Microglia may contribute to cell death in neurodegenerative diseases. We studied the activation of microglia in affected regions of Huntington disease (HD) brain by localizing thymosin beta-4 (Tbeta4), which is increased in reactive microglia. Activated microglia appeared in the neostriatum, cortex, and globus pallidus and the adjoining white matter of the HD brain, but not in control brain. In the striatum and cortex, reactive microglia occurred in all grades of pathology, accumulated with increasing grade, and grew in density in relation to degree of neuronal loss. The predominant morphology of activated microglia differed in the striatum and cortex. Processes of reactive microglia were conspicuous in low-grade HD, suggesting an early microglia response to changes in neuropil and axons and in the grade 2 and grade 3 cortex, were aligned with the apical dendrites of pyramidal neurons. Some reactive microglia contacted pyramidal neurons with huntingtin-positive nuclear inclusions. The early and proximate association of activated microglia with degenerating neurons in the HD brain implicates a role for activated microglia in HD pathogenesis.

Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation.

The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was colocalized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in nuclei. Pro-caspase-8 was found in the intermembrane space, inner membrane, and matrix of mitochondria after limited digestion of mitochondrial fractions, and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside, which opens the mitochondria permeability transition pore. Release was blocked by the mitochondria permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 h to an apoptotic inducer tumor necrosis factor-alpha (TNF-alpha), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8 cleavage products, in the cytoplasm of the TNF-alpha-treated striatal cells. CsA blocked the TNF-alpha-induced release of pro-caspase 8 but not cytochrome c. Internucleosomal DNA fragmentation started at 6 h and peaked 12 h after TNF-alpha treatment. These results suggest that pro-caspase-8 is predominantly localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.

Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation, and autophagy.

An expansion of polyglutamines in the N terminus of huntingtin causes Huntington's disease (HD) and results in the accrual of mutant protein in the nucleus and cytoplasm of affected neurons. How mutant huntingtin causes neurons to die is unclear, but some recent observations suggest that an autophagic process may occur. We showed previously that huntingtin markedly accumulates in endosomal-lysosomal organelles of affected HD neurons and, when exogenously expressed in clonal striatal neurons, huntingtin appears in cytoplasmic vacuoles causing cells to shrink. Here we show that the huntingtin-enriched cytoplasmic vacuoles formed in vitro internalized the lysosomal enzyme cathepsin D in proportion to the polyglutamine-length in huntingtin. Huntingtin-labeled vacuoles displayed the ultrastructural features of early and late autophagosomes (autolysosomes), had little or no overlap with ubiquitin, proteasome, and heat shock protein 70/heat shock cognate 70 immunoreactivities, and altered the arrangement of Golgi membranes, mitochondria, and nuclear membranes. Neurons with excess cytoplasmic huntingtin also exhibited increased tubulation of endosomal membranes. Exogenously expressed human full-length wild-type and mutant huntingtin codistributed with endogenous mouse huntingtin in soluble and membrane fractions, whereas human N-terminal huntingtin products were found only in membrane fractions that contained lysosomal organelles. We speculate that mutant huntingtin accumulation in HD activates the endosomal-lysosomal system, which contributes to huntingtin proteolysis and to an autophagic process of cell death.

Are there multiple pathways in the pathogenesis of Huntington's disease?

Studies of huntingtin localization in human post-mortem brain offer insights and a framework for basic experiments in the pathogenesis of Huntington's disease. In neurons of cortex and striatum, we identified changes in the cytoplasmic localization of huntingtin including a marked perinuclear accumulation of huntingtin and formation of multivesicular bodies, changes conceivably pointing to an altered handling of huntingtin in neurons. In Huntington's disease, huntingtin also accumulates in aberrant subcellular compartments such as nuclear and neuritic aggregates co-localized with ubiquitin. The site of protein aggregation is polyglutamine-dependent, both in juvenile-onset patients having more aggregates in the nucleus and in adult-onset patients presenting more neuritic aggregates. Studies in vitro reveal that the genesis of these aggregates and cell death are tied to cleavage of mutant huntingtin. However, we found that the aggregation of mutant huntingtin can be dissociated from the extent of cell death. Thus properties of mutant huntingtin more subtle than its aggregation, such as its proteolysis and protein interactions that affect vesicle trafficking and nuclear transport, might suffice to cause neurodegeneration in the striatum and cortex. We propose that mutant huntingtin engages multiple pathogenic pathways leading to neuronal death.

Forskolin and dopamine D1 receptor activation increase huntingtin's association with endosomes in immortalized neuronal cells of striatal origin.

Huntingtin is a cytoplasmic protein of unknown function that associates with vesicle membranes and microtubules. Its protein interactions suggest that huntingtin has a role in endocytosis and organelle transport. In this study we sought to identify factors that regulate the transport of huntingtin in striatal neurons, which are the cells most affected in Huntington's disease. In clonal striatal cells derived from fusions of neuroblastoma and embryonic striatal neurons, huntingtin localization is diffuse and slightly punctate in the cytoplasm. When these neurons were differentiated by treatment with forskolin, huntingtin redistributed to perinuclear regions, discrete puncta along plasma membranes, and branch points and terminal growth cones in neurites. Huntingtin staining overlapped with clathrin, a coat protein involved in endocytosis. Immunoblot analysis of subcellular membrane fractions separated by differential centrifugation confirmed that huntingtin immunoreactivity in differentiated neurons markedly increased in membrane fractions enriched with clathrin and with huntingtin-interacting protein 1. Dopamine treatment altered the subcellular localization of huntingtin and increased its expression in clathrin-enriched membrane fractions. The dopamine-induced changes were blocked by the D1 antagonist SCH 23390 and were absent in a clonal cell line lacking D1 receptors. Results suggest that the transport of huntingtin and its co-expression in clathrin and huntingtin-interacting protein 1-enriched membranes is influenced by activation of adenylyl cyclase and stimulation of dopamine D1 receptors.

Axonal transport of N-terminal huntingtin suggests early pathology of corticostriatal projections in Huntington disease.

Aggregation of N-terminal mutant huntingtin within nuclear inclusions and dystrophic neurites occurs in the cortex and striatum of Huntington disease (HD) patients and may be involved in neurodegeneration. We examined the prevalence of inclusions and dystrophic neurites in the cortex and striatum of 15 adult onset HD patients who had mild to severe striatal cell loss (grades 1, 2 or 3) using an antibody that detects the N-terminal region of huntingtin. Nuclear inclusions were more frequent in the cortex than the striatum and were sparse or absent in the striatum of patients with low-grade striatal pathology. Dystrophic neurites occurred in both regions. Patients with low-grade striatal pathology had numerous fibers with immunoreactive puncta and large swellings within the striatal neuropil, the subcortical white matter, and the internal and external capsules. In the globus pallidus of 3 grade 1 cases, N-terminal huntingtin markedly accumulated in the perinuclear cytoplasm and in some axons but not in the nucleus. Findings suggest that in the earlier stages of HD, accumulation of N-terminal mutant huntingtin occurs in the cytoplasm and is associated with degeneration of the corticostriatal pathway.

Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition.

Neuronal intranuclear inclusions are found in the brains of patients with Huntington's disease and form from the polyglutamine-expanded N-terminal region of mutant huntingtin. To explore the properties of inclusions and their involvement in cell death, mouse clonal striatal cells were transiently transfected with truncated and full-length human wild-type and mutant huntingtin cDNAs. Both normal and mutant proteins localized in the cytoplasm, and infrequently, in dispersed and perinuclear vacuoles. Only mutant huntingtin formed nuclear and cytoplasmic inclusions, which increased with polyglutamine expansion and with time after transfection. Nuclear inclusions contained primarily cleaved N-terminal products, whereas cytoplasmic inclusions contained cleaved and larger intact proteins. Cells with wild-type or mutant protein had distinct apoptotic features (membrane blebbing, shrinkage, cellular fragmentation), but those with mutant huntingtin generated the most cell fragments (apoptotic bodies). The caspase inhibitor Z-VAD-FMK significantly increased cell survival but did not diminish nuclear and cytoplasmic inclusions. In contrast, Z-DEVD-FMK significantly reduced nuclear and cytoplasmic inclusions but did not increase survival. A series of N-terminal products was formed from truncated normal and mutant proteins and from full-length mutant huntingtin but not from full-length wild-type huntingtin. One prominent N-terminal product was blocked by Z-VAD-FMK. In summary, the formation of inclusions in clonal striatal cells corresponds to that seen in the HD brain and is separable from events that regulate cell death. N-terminal cleavage may be linked to mutant huntingtin's role in cell death.

Analysis of Huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons.

Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin-associated protein 1 (HAP-1), a brain-enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP-1 is unknown, but recent evidence supports a role in microtubule-dependent organelle transport. We examined the subcellular localization of HAP-1 with an antibody made against the NH2-terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP-1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP-1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP-1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP-1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP-1 localized to membrane-bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP-1 was also strongly associated with an unusual large "dense" organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP-1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP-1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons.

Wild-type and mutant huntingtins function in vesicle trafficking in the secretory and endocytic pathways.

Huntingtin is a cytoplasmic protein that is found in neurons and somatic cells. In patients with Huntington's disease (HD), the NH2-terminal region of huntingtin has an expanded polyglutamine tract. An abnormal protein interaction by mutant huntingtin has been proposed as a mechanism for HD pathogenesis. Huntingtin associates with vesicle membranes and interacts with proteins involved in vesicle trafficking. It is unclear where along vesicle transport pathways wild-type and mutant huntingtins are found and whether polyglutamine expansion affects this localization. To distinguish wild-type and mutant huntingtin, fibroblasts from normals and HD patients with two mutant alleles (homozygotes) were examined. Immunofluorescence confocal microscopy showed that mutant huntingtin localized with clathrin in membranes of the trans Golgi network and in clathrin-coated and noncoated endosomal vesicles in the cytoplasm and along plasma membranes. Separation of organelles in Nycodenz gradients showed that in normal and HD homozygote patient cells, huntingtin was present in membrane fractions enriched in clathrin. Similar results were obtained in fibroblasts from heterozyote juvenile HD patients who had a highly expanded polyglutamine tract in the HD allele. Western blot analysis of membrane fractions from rat brain showed that wild-type huntingtin was present in fractions that contained purified clathrin-coated membranes or a mixture of clathrin-coated and noncoated membranes. Electron microscopy of huntingtin immunoreactivity in rat brain revealed labeling along dendritic plasma membranes in association with clathrin-coated pits and clusters of noncoated endosomal vesicles 40-60 nm in diameter. These data suggest that wild-type and mutant huntingtin can influence vesicle transport in the secretory and endocytic pathways through associations with clathrin-coated vesicles.

Are neuronal intranuclear inclusions the common neuropathology of triplet-repeat disorders with polyglutamine-repeat expansions?

Neuronal intranuclear inclusions have been found in the brain of a transgenic mouse model of Huntington's disease and in necropsy brain tissue of patients with Huntington's disease. We suggest that neuronal intranuclear inclusions are the common neuropathology for all inherited diseases caused by expansion of polyglutamine repeats. We also suggest that patients with a pathological diagnosis of neuronal intranuclear hyaline inclusion disease may also have polyglutamine repeat expansions.

Huntingtin localization in brains of normal and Huntington's disease patients.

The immunohistochemical localization of huntingtin was examined in the Huntington's disease (HD) brain with an antibody that recognizes the wild-type and mutant proteins. Neuronal staining was reduced in areas of the HD striatum depleted of medium-sized neurons; large striatal neurons, which are spared in HD, retained normal levels of huntingtin expression. Neuronal labeling was markedly reduced in both segments of the globus pallidus including in brains with minimal loss of pallidal neurons. In some HD cortical and striatal neurons with normal looking morphology, huntingtin was associated with punctate cytoplasmic granules that at the ultrastructural level resembled the multivesicular body, an organelle involved in retrograde transport and protein degradation. Some immunoreactive processes showed blebbing and segmentation similar to that induced experimentally by hypoxic-ischemic or excitotoxic injury. Huntingtin staining was more concentrated in the perinuclear cytoplasm and reduced or absent in processes of atrophic cortical neurons. Nuclear staining was also evident. Fibers in the subcortical white matter of HD patients had significantly increased huntingtin immunoreactivity compared with those of controls. Results suggest that there may be changes in the neuronal expression and transport of wild-type and/or mutant huntingtin at early and late stages of neuronal degeneration in affected areas of the HD brain.

Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.

The cause of neurodegeneration in Huntington's disease (HD) is unknown. Patients with HD have an expanded NH2-terminal polyglutamine region in huntingtin. An NH2-terminal fragment of mutant huntingtin was localized to neuronal intranuclear inclusions (NIIs) and dystrophic neurites (DNs) in the HD cortex and striatum, which are affected in HD, and polyglutamine length influenced the extent of huntingtin accumulation in these structures. Ubiquitin was also found in NIIs and DNs, which suggests that abnormal huntingtin is targeted for proteolysis but is resistant to removal. The aggregation of mutant huntingtin may be part of the pathogenic mechanism in HD.