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.     

From neuronal inclusions to neurodegeneration: neuropathological investigation of a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease caused by the expansion of a polyglutamine repeat sequence within a novel protein. Recent work has shown that abnormal intranuclear inclusions of aggregated mutant protein within neurons is a characteristic feature shared by HD and several other diseases involving glutamine repeat expansion. This suggests that in each of the these disorders the affected nerve cells degenerate as a result of these abnormal inclusions. A transgenic mouse model of HD has been generated by introducing exon 1 of the HD gene containing a highly expanded CAG sequence into the mouse germline. These mice develop widespread neuronal intranuclear inclusions and neurodegeneration specifically within those areas of the brain known to degenerate in HD. We have investigated the sequence of pathological changes that occur after the formation of nuclear inclusions and that precede neuronal cell death in these cells. Although the relation between inclusion formation and neurodegeneration has recently been questioned, a full characterization of the pathways linking protein aggregation and cell death will resolve some of these controversies and will additionally provide new targets for potential therapies.     

Mouse models of Alzheimer's disease: a quest for plaques and tangles

Many genetically altered mice have been designed to help understand the role of specific gene mutations in the pathogenesis of Alzheimer's disease (AD) based on the realization that specific mutations in the genes for amyloid precursor protein--the presenilins and tau--are associated with early-onset familial AD or, in the case of tau mutations, other neurodegenerative diseases with neurofibrillary tangles. However, attempts to reproduce the neuropathology of AD in the mouse have been frustrating. Transgenic designs emphasizing amyloid precursor protein produced mice that develop amyloid plaques, but neurodegeneration and neurofibrillary tangles failed to form. Strategies emphasizing tau resulted in increased phosphorylation of tau and tangle formation, although amyloid plaques were absent. Nevertheless, crossing transgenic animals expressing mutated tau and amyloid precursor protein has produced a mouse that closely recapitulates the neuropathology of AD. A review of the various murine models, their role in understanding the pathogenesis of AD and their use in testing therapeutic regimens, is provided.     

Transgenic mice expressing mutated full-length HD cDNA: a paradigm for locomotor changes and selective neuronal loss in Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized clinically by motor and psychiatric disturbances and pathologically by neuronal loss and gliosis (reactive astrocytosis) particularly in the striatum and cerebral cortex. We have recently created HD full-length cDNA transgenic mouse models that may serve as a paradigm for HD. A more detailed characterization of these models is presented here. The transgene encoding normal huntingtin consists of 9417 bp of the huntingtin coding sequences including 16 tandem CAGs coding for polyglutamines as part of exon 1. The transgene is driven by a heterologous cytomegalovirus promoter. Five independent transgenic mouse lines were obtained using this construct. An additional six transgenic lines were obtained using full-length HD constructs that have been modified to include either 48 or 89 CAG repeat expansions. Southern blot and densitometric analyses indicated unique integration sites for the transgene in each of the lines with a copy number ranging from two to 22 copies. Widespread expression of the transgene in brain, heart, spleen, kidney, lung, liver and gonads from each line was determined by Western blot analyses. In the brain, transgene expression was found in cerebral cortex, striatum, hippocampus and cerebellum. Expression of the transgene was as much as five times the endogenous mouse huntingtin level. Phenotypically, only mice expressing 48 or 89 CAG repeats manifested progressive behavioural and motor dysfunction. Early behavioural abnormalities were characterized by trunk curling and clasping of both fore- and hindlimbs when the animals were suspended by their tails. Subsequently, these mice exhibited hyperkinetic movements, including heightened exploratory activities, unidirectional rotational behaviour, backflipping and excessive grooming that lasted for several weeks. Eventually, the animals progressed to a hypokinetic phase consisting of slowed movements and lack of response to sensory stimuli. Urine retention or incontinence was also a prominent feature of the hypokinetic phase. At the end stage of the disease process, HD48(B,D) and HD89(A-C) mice became akinetic just prior to death. Neuropathological examination of mice at various stages indicated that it was only during the hypokinetic phase and thereafter when selective neuronal loss was most apparent. Regions of neurodegeneration and loss included the striatum, cerebral cortex, thalamus and hippocampus. TUNEL staining indicated an apoptotic mode of cell death in these brain regions. Comparative neuronal counts after Nissl staining showed as much as 20% loss of small and medium neurons in the striatum in mice at the hypokinetic and akinetic stages. Reactive astrocytosis accompanied the areas of neurodegeneration and loss. Polyglutamine aggregates in the form of neuronal intranuclear inclusions and diffuse nuclear and perinuclear aggregations were found in a small percentage of neurons, including those in brain regions that are typically spared in HD. This observation suggests that polyglutamine aggregates may not be sufficient to cause neuronal loss in HD. In both behavioural and neuropathological analyses, wild-type and transgenic animals with 16 CAG repeats were indistinguishable from each other and do not exhibit the changes observed for mice carrying the 48 and 89 CAG repeat mutations. Thus, animals expressing the CAG repeat expansions appear to represent clinically analogous models for HD pathogenesis, and may also provide insights into the underlying pathophysiological mechanisms of other triplet repeat disorders.     

Molecular mediators, environmental modulators and experience-dependent synaptic dysfunction in Huntington's disease

Huntington's disease (HD) is an autosomal dominant disorder in which there is progressive neurodegeneration producing motor, cognitive and psychiatric symptoms. HD is caused by a trinucleotide (CAG) repeat mutation, encoding an expanded polyglutamine tract in the huntingtin protein. At least eight other neurodegenerative diseases are caused by CAG/glutamine repeat expansions in different genes. Recent evidence suggests that environmental factors can modify the onset and progression of Huntington's disease and possibly other neurodegenerative disorders. This review outlines possible molecular and cellular mechanisms mediating the polyglutamine-induced toxic 'gain of function' and associated gene-environment interactions in HD. Key aspects of pathogenesis shared with other neurodegenerative diseases may include abnormal protein-protein interactions, selective disruption of gene expression and 'pathological plasticity' of synapses in specific brain regions. Recent discoveries regarding molecular mechanisms of pathogenesis are guiding the development of new therapeutic approaches. Knowledge of gene-environment interactions, for example, could lead to development of 'enviromimetics' which mimic the beneficial effects of specific environmental stimuli. The effects of environmental enrichment on brain and behaviour will also be discussed, together with the general implications for neuroscience research involving animal models.     

Changes in the striatal proteome of YAC128Q mice exhibit gene-environment interactions between mutant huntingtin and manganese

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat within the Huntingtin (HTT) gene, though the clinical presentation of disease and age-of-onset are strongly influenced by ill-defined environmental factors. We recently reported a gene-environment interaction wherein expression of mutant HTT is associated with neuroprotection against manganese (Mn) toxicity. Here, we are testing the hypothesis that this interaction may be manifested by altered protein expression patterns in striatum, a primary target of both neurodegeneration in HD and neurotoxicity of Mn. To this end, we compared striatal proteomes of wild-type and HD (YAC128Q) mice exposed to vehicle or Mn. Principal component analysis of proteomic data revealed that Mn exposure disrupted a segregation of WT versus mutant proteomes by the major principal component observed in vehicle-exposed mice. Identification of altered proteins revealed novel markers of Mn toxicity, particularly proteins involved in glycolysis, excitotoxicity, and cytoskeletal dynamics. In addition, YAC128Q-dependent changes suggest that axonal pathology may be an early feature in HD pathogenesis. Finally, for several proteins, genotype-specific responses to Mn were observed. These differences include increased sensitivity to exposure in YAC128Q mice (UBQLN1) and amelioration of some mutant HTT-induced alterations (SAE1, ENO1). We conclude that the interaction of Mn and mutant HTT may suppress proteomic phenotypes of YAC128Q mice, which could reveal potential targets in novel treatment strategies for HD.     

Protein misfolding detected early in pathogenesis of transgenic mouse model of Huntington disease using amyloid seeding assay

Huntington disease (HD) is one of several fatal neurodegenerative disorders associated with misfolded proteins. Here, we report a novel method for the sensitive detection of misfolded huntingtin (HTT) isolated from the brains of transgenic (Tg) mouse models of HD and humans with HD using an amyloid seeding assay (ASA), which is based on the propensity of misfolded proteins to act as a seed and shorten the nucleation-associated lag phase in the kinetics of amyloid formation in vitro. Using synthetic polyglutamine peptides as the substrate for amyloid formation, we found that partially purified misfolded HTT obtained from end-stage brain tissue of two Tg HD mouse models and brain tissue of post-mortem human HD patients was capable of specifically accelerating polyglutamine amyloid formation compared with unseeded reactions and controls. Alzheimer and prion disease brain tissues did not do so, demonstrating the specificity of the ASA. It is unclear whether early intermediates or later conformational species in the protein misfolding process act as seeds in the ASA for HD. However, we were able to detect misfolded protein in the brains of YAC128 mice early in disease pathogenesis (11 weeks of age), whereas large inclusion bodies have not been observed in the brains of these mice by histology until 78 weeks of age, much later in the pathogenic process. The sensitive detection of misfolded HTT protein early in the disease pathogenesis in the YAC128 Tg mouse model strengthens the argument for a causative role of protein misfolding in HD.     

Mitochondrial DNA damage is a hallmark of chemically induced and the R6/2 transgenic model of Huntington's disease

Many forms of neurodegeneration are associated with oxidative stress and mitochondrial dysfunction. Mitochondria are prominent targets of oxidative damage, however, it is not clear whether mitochondrial DNA (mtDNA) damage and/or its lack of repair are primary events in the delayed onset observed in Huntington's disease (HD). We hypothesize that an age-dependent increase in mtDNA damage contributes to mitochondrial dysfunction in HD. Two HD mouse models were studied, the 3-nitropropionic acid (3-NPA) chemically induced model and the HD transgenic mice of the R6/2 strain containing 115-150 CAG repeats in the huntingtin gene. The mitochondrial toxin 3-NPA inhibits complex II of the electron transport system and causes neurodegeneration that resembles HD in the striatum of human and experimental animals. We measured nuclear and mtDNA damage by quantitative PCR (QPCR) in striatum of 5- and 24-month-old untreated and 3-NPA treated C57BL/6 mice. Aging caused an increase in damage in both nuclear and mitochondrial genomes. 3-NPA induced 4-6 more damage in mtDNA than nuclear DNA in 5-month-old mice, and this damage was repaired by 48h in the mtDNA. In 24-month-old mice 3NPA caused equal amounts of nuclear and mitochondrial damage and this damage persistent in both genomes for 48h. QPCR analysis showed a progressive increase in the levels of mtDNA damage in the striatum and cerebral cortex of 7-12-week-old R6/2 mice. Striatum exhibited eight-fold more damage to the mtDNA compared with a nuclear gene. These data suggest that mtDNA damage is an early biomarker for HD-associated neurodegeneration and supports the hypothesis that mtDNA lesions may contribute to the pathogenesis observed in HD.     

Msh2 Acts in Medium-Spiny Striatal Neurons as an Enhancer of CAG Instability and Mutant Huntingtin Phenotypes in Huntington's Disease Knock-In Mice

The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT) exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR) pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111) with mice carrying a conditional (floxed) Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit.     

Integration-independent Transgenic Huntington Disease Fragment Mouse Models Reveal Distinct Phenotypes and Life Span in Vivo

The cascade of events that lead to cognitive decline, motor deficits, and psychiatric symptoms in patients with Huntington disease (HD) is triggered by a polyglutamine expansion in the N-terminal region of the huntingtin (HTT) protein. A significant mechanism in HD is the generation of mutant HTT fragments, which are generally more toxic than the full-length HTT. The protein fragments observed in human HD tissue and mouse models of HD are formed by proteolysis or aberrant splicing of HTT. To systematically investigate the relative contribution of the various HTT protein proteolysis events observed in vivo, we generated transgenic mouse models of HD representing five distinct proteolysis fragments ending at amino acids 171, 463, 536, 552, and 586 with a polyglutamine length of 148. All lines contain a single integration at the ROSA26 locus, with expression of the fragments driven by the chicken β-actin promoter at nearly identical levels. The transgenic mice N171-Q148 and N552-Q148 display significantly accelerated phenotypes and a shortened life span when compared with N463-Q148, N536-Q148, and N586-Q148 transgenic mice. We hypothesized that the accelerated phenotype was due to altered HTT protein interactions/complexes that accumulate with age. We found evidence for altered HTT complexes in caspase-2 fragment transgenic mice (N552-Q148) and a stronger interaction with the endogenous HTT protein. These findings correlate with an altered HTT molecular complex and distinct proteins in the HTT interactome set identified by mass spectrometry. In particular, we identified HSP90AA1 (HSP86) as a potential modulator of the distinct neurotoxicity of the caspase-2 fragment mice (N552-Q148) when compared with the caspase-6 transgenic mice (N586-Q148).     

Synaptic mutant huntingtin inhibits synapsin-1 phosphorylation and causes neurological symptoms

Many genetic mouse models of Huntington’s disease (HD) have established that mutant huntingtin (htt) accumulates in various subcellular regions to affect a variety of cellular functions, but whether and how synaptic mutant htt directly mediates HD neuropathology remains to be determined. We generated transgenic mice that selectively express mutant htt in the presynaptic terminals. Although it was not overexpressed, synaptic mutant htt caused age-dependent neurological symptoms and early death in mice as well as defects in synaptic neurotransmitter release. Mass spectrometry analysis of synaptic fractions and immunoprecipitation of synapsin-1 from HD CAG150 knockin mouse brains revealed that mutant htt binds to synapsin-1, a protein whose phosphorylation is critical for neurotransmitter release. We found that polyglutamine-expanded exon1 htt binds to the C-terminal region of synapsin-1 to reduce synapsin-1 phosphorylation. Our findings point to a critical role for synaptic htt in the neurological symptoms of HD, providing a new therapeutic target.     

Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington''s disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.     

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.     

Enhancement of brain-type creatine kinase activity ameliorates neuronal deficits in Huntington's disease

Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin (HTT) gene. Brain-type creatine kinase (CKB) is an enzyme involved in energy homeostasis via the phosphocreatine–creatine kinase system. Although downregulation of CKB was previously reported in brains of HD mouse models and patients, such regulation and its functional consequence in HD are not fully understood. In the present study, we demonstrated that levels of CKB found in both the soma and processes were markedly reduced in primary neurons and brains of HD mice. We show for the first time that mutant HTT (mHTT) suppressed the activity of the promoter of the CKB gene, which contributes to the lowered CKB expression in HD. Exogenous expression of wild-type CKB, but not a dominant negative CKB mutant, rescued the ATP depletion, aggregate formation, impaired proteasome activity, and shortened neurites induced by mHTT. These findings suggest that negative regulation of CKB by mHTT is a key event in the pathogenesis of HD and contributes to the neuronal dysfunction associated with HD. In addition, besides dietary supplementation with the CKB substrate, strategies aimed at increasing CKB expression might lead to the development of therapeutic treatments for HD.     

Striatal Synaptic Dysfunction and Hippocampal Plasticity Deficits in the Hu97/18 Mouse Model of Huntington Disease

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene (HTT) encoding the huntingtin protein (HTT). This mutation leads to multiple cellular and synaptic alterations that are mimicked in many current HD animal models. However, the most commonly used, well-characterized HD models do not accurately reproduce the genetics of human disease. Recently, a new ‘humanized’ mouse model, termed Hu97/18, has been developed that genetically recapitulates human HD, including two human HTT alleles, no mouse Hdh alleles and heterozygosity of the HD mutation. Previously, behavioral and neuropathological testing in Hu97/18 mice revealed many features of HD, yet no electrophysiological measures were employed to investigate possible synaptic alterations. Here, we describe electrophysiological changes in the striatum and hippocampus of the Hu97/18 mice. At 9 months of age, a stage when cognitive deficits are fully developed and motor dysfunction is also evident, Hu97/18 striatal spiny projection neurons (SPNs) exhibited small changes in membrane properties and lower amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSCs); however, release probability from presynaptic terminals was unaltered. Strikingly, these mice also exhibited a profound deficiency in long-term potentiation (LTP) at CA3-to-CA1 synapses. In contrast, at 6 months of age we found only subtle alterations in SPN synaptic transmission, while 3-month old animals did not display any electrophysiologically detectable changes in the striatum and CA1 LTP was intact. Together, these data reveal robust, progressive deficits in synaptic function and plasticity in Hu97/18 mice, consistent with previously reported behavioral abnormalities, and suggest an optimal age (9 months) for future electrophysiological assessment in preclinical studies of HD.     

Polyglutamine pathogenesis.

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.     

Small interfering RNAs based on huntingtin trinucleotide repeats are highly toxic to cancer cells

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA‐sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complementary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the six members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR‐based siRNAs induce cell death in vitro in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR‐based siRNAs as a novel form of anticancer reagents.