Similar striatal gene expression profiles in the striatum of the YAC128 and HdhQ150 mouse models of Huntington's disease are not reflected in mutant Huntingtin inclusion prevalence.

BACKGROUND: The YAC128 model of Huntington's disease (HD) shows substantial deficits in motor, learning and memory tasks and alterations in its transcriptional profile. We examined the changes in the transcriptional profile in the YAC128 mouse model of HD at 6, 12 and 18 months and compared these with those seen in other models and human HD caudate. RESULTS: Differential gene expression by genotype showed that genes related to neuronal function, projection outgrowth and cell adhesion were altered in expression. A Time-course ANOVA revealed that genes downregulated with increased age in wild-type striata were likely to be downregulated in the YAC128 striata. There was a substantial overlap of concordant gene expression changes in the YAC128 striata compared with those in human HD brain. Changes in gene expression over time showed fewer striatal YAC128 RNAs altered in abundance than in the HdhQ150 striata but there was a very marked overlap in transcriptional changes at all time points. Despite the similarities in striatal expression changes at 18 months the HdhQ150 mice showed widespread mHTT and ubiquitin positive inclusion staining in the striatum whereas this was absent in the YAC128 striatum. CONCLUSIONS: The gene expression changes in YAC128 striata show a very closely matched profile to that of HdhQ150 striata and are already significantly different between genotypes by six months of age, implying that the temporal molecular gene expression profiles of these models match very closely, despite differences in the prevalence of brain inclusion formation between the models. The YAC128 gene expression changes appear to correlate well with gene expression differences caused by ageing. A relatively small number of genes showed significant differences in expression between the striata of the two models and these could explain some of the phenotypic differences between the models.

Tests to assess motor phenotype in mice: a user's guide.

The characterization of mouse models of human disease is essential for understanding the underlying pathophysiology and developing new therapeutics. Many diseases are often associated with more than one model, and so there is a need to determine which model most closely represents the disease state or is most suited to the therapeutic approach under investigation. In the case of neurological disease, motor tests provide a good read-out of neurological function. This overview of available motor tasks aims to aid researchers in making the correct choice of test when attempting to tease out a transgenic phenotype or when assessing the recovery of motor function following therapeutic intervention.

Another look at follicular lymphoma: immunophenotypic and molecular analyses identify distinct follicular lymphoma subgroups.

AIMS: The aim of this study was to analyse the immunophenotypic and molecular features of a large series of follicular lymphomas, focusing in particular on atypical cases that fail to express CD10 and/or bcl-2. Such cases present diagnostic pitfalls, especially with regard to the differential diagnosis from follicular hyperplasia and marginal zone B-cell lymphoma. Therefore, we also included an immunohistochemical evaluation of stathmin, which is strongly expressed by germinal centre B cells, as a putative new marker for follicular lymphomas, particularly those with an atypical phenotype. METHODS AND RESULTS: Two hundred and five follicular lymphomas were investigated with immunohistochemistry and fluorescence in-situ hybridization (FISH). The use of three distinct anti-bcl-2 antibodies together with CD10 expression data and FISH analysis for bcl-2 and bcl-6 rearrangements allowed subclassification of follicular lymphoma into four distinct subgroups: (i) CD10-positive/bcl-2-positive, (ii) CD10-positive/bcl-2-negative, (iii) CD10-negative/bcl-2-positive, and (iv) CD10-negative/bcl-2-negative. All cases were bcl-6-positive. STMN1 (stathmin) was shown to be helpful in diagnosing bcl-2-negative and/or CD10-negative follicular lymphomas, and in their distinction from marginal zone B-cell lymphoma. CONCLUSIONS: Combined immunohistological and molecular analyses reveal that follicular lymphomas showing an atypical immunophenotypic and molecular profile exist, and we demonstrate that STMN1 represents a novel useful diagnostic marker for these.

The Nance-Horan syndrome protein encodes a functional WAVE homology domain (WHD) and is important for co-ordinating actin remodelling and maintaining cell morphology.

Nance-Horan syndrome (NHS) is an X-linked developmental disorder, characterized by bilateral congenital cataracts, dental anomalies, facial dysmorphism and mental retardation. Null mutations in a novel gene, NHS, cause the syndrome. The NHS gene appears to have multiple isoforms as a result of alternative transcription, but a cellular function for the NHS protein has yet to be defined. We describe NHS as a founder member of a new protein family (NHS, NHSL1 and NHSL2). Here, we demonstrate that NHS is a novel regulator of actin remodelling and cell morphology. NHS localizes to sites of cell-cell contact, the leading edge of lamellipodia and focal adhesions. The N-terminus of isoforms NHS-A and NHS-1A, implicated in the pathogenesis of NHS, have a functional WAVE homology domain that interacts with the Abi protein family, haematopoietic stem/progenitor cell protein 300 (HSPC300), Nap1 and Sra1. NHS knockdown resulted in the disruption of the actin cytoskeleton. We show that NHS controls cell morphology by maintaining the integrity of the circumferential actin ring and controlling lamellipod formation. NHS knockdown led to a striking increase in cell spreading. Conversely, ectopic overexpression of NHS inhibited lamellipod formation. Remodelling of the actin cytoskeleton and localized actin polymerization into branched actin filaments at the plasma membrane are essential for mediating changes in cell shape, migration and cell contact. Our data identify NHS as a new regulator of actin remodelling. We suggest that NHS orchestrates actin regulatory protein function in response to signalling events during development.

Age-dependent maintenance of motor control and corticostriatal innervation by death receptor 3.

Death receptor 3 is a proinflammatory member of the immunomodulatory tumor necrosis factor receptor superfamily, which has been implicated in several inflammatory diseases such as arthritis and inflammatory bowel disease. Intriguingly however, constitutive DR3 expression has been detected in the brains of mice, rats, and humans, although its neurological function remains unknown. By mapping the normal brain expression pattern of DR3, we found that DR3 is expressed specifically by cells of the neuron lineage in a developmentally regulated and region-specific pattern. Behavioral studies on DR3-deficient (DR3(ko)) mice showed that constitutive neuronal DR3 expression was required for stable motor control function in the aging adult. DR3(ko) mice progressively developed behavioral defects characterized by altered gait, dyskinesia, and hyperactivity, which were associated with elevated dopamine and lower serotonin levels in the striatum. Importantly, retrograde tracing showed that absence of DR3 expression led to the loss of corticostriatal innervation without significant neuronal loss in aged DR3(ko) mice. These studies indicate that DR3 plays a key nonredundant role in the retention of normal motor control function during aging in mice and implicate DR3 in progressive neurological disease.

X-linked cataract and Nance-Horan syndrome are allelic disorders.

Nance-Horan syndrome (NHS) is an X-linked developmental disorder characterized by congenital cataract, dental anomalies, facial dysmorphism and, in some cases, mental retardation. Protein truncation mutations in a novel gene (NHS) have been identified in patients with this syndrome. We previously mapped X-linked congenital cataract (CXN) in one family to an interval on chromosome Xp22.13 which encompasses the NHS locus; however, no mutations were identified in the NHS gene. In this study, we show that NHS and X-linked cataract are allelic diseases. Two CXN families, which were negative for mutations in the NHS gene, were further analysed using array comparative genomic hybridization. CXN was found to be caused by novel copy number variations: a complex duplication-triplication re-arrangement and an intragenic deletion, predicted to result in altered transcriptional regulation of the NHS gene. Furthermore, we also describe the clinical and molecular analysis of seven families diagnosed with NHS, identifying four novel protein truncation mutations and a novel large deletion encompassing the majority of the NHS gene, all leading to no functional protein. We therefore show that different mechanisms, aberrant transcription of the NHS gene or no functional NHS protein, lead to different diseases. Our data highlight the importance of copy number variation and non-recurrent re-arrangements leading to different severity of disease and describe the potential mechanisms involved.

CTIP2-Regulated Reduction in PKA-Dependent DARPP32 Phosphorylation in Human Medium Spiny Neurons: Implications for Huntington Disease.

The mechanisms underlying the selective degeneration of medium spiny neurons (MSNs) in Huntington disease (HD) remain largely unknown. CTIP2, a transcription factor expressed by all MSNs, is implicated in HD pathogenesis because of its interactions with mutant huntingtin. Here, we report a key role for CTIP2 in protein phosphorylation via governing protein kinase A (PKA) signaling in human striatal neurons. Transcriptomic analysis of CTIP2-deficient MSNs implicates CTIP2 target genes at the heart of cAMP-Ca2+ signal integration in the PKA pathway. These findings are further supported by experimental evidence of a substantial reduction in phosphorylation of DARPP32 and GLUR1, two PKA targets in CTIP2-deficient MSNs. Moreover, we show that CTIP2-dependent dysregulation of protein phosphorylation is shared by HD hPSC-derived MSNs and striatal tissues of two HD mouse models. This study therefore establishes an essential role for CTIP2 in human MSN homeostasis and provides mechanistic and potential therapeutic insight into striatal neurodegeneration.

Loss of CRMP2 O-GlcNAcylation leads to reduced novel object recognition performance in mice.

O-GlcNAcylation is an abundant post-translational modification in the nervous system, linked to both neurodevelopmental and neurodegenerative disease. However, the mechanistic links between these phenotypes and site-specific O-GlcNAcylation remain largely unexplored. Here, we show that Ser517 O-GlcNAcylation of the microtubule-binding protein Collapsin Response Mediator Protein-2 (CRMP2) increases with age. By generating and characterizing a Crmp2S517A knock-in mouse model, we demonstrate that loss of O-GlcNAcylation leads to a small decrease in body weight and mild memory impairment, suggesting that Ser517 O-GlcNAcylation has a small but detectable impact on mouse physiology and cognitive function.

Time course of choice reaction time deficits in the Hdh(Q92) knock-in mouse model of Huntington's disease in the operant serial implicit learning task (SILT).

A range of transgenic and knock-in mouse models of Huntington's disease have been created since identification in 1993 of the disease mutation in the HD gene. Knock-in models that express the full-length mutant protein tend to exhibit less severe behavioural deficits than transgenic models and so require more sensitive tasks in order to reveal impairments. To achieve this, we therefore used a Serial Implicit Learning Task (SILT), which measures serial reaction times to visual stimuli, requiring detection and responding in both predictable and unpredictable locations in the 9-hole operant chamber. We have previously reported that knock-in Hdh(Q92/Q92) mice exhibit a modest impairment in learning the SILT tasks at 4 months of age, prior to the formation of overt neuronal nuclear inclusions. In the present study we have explored the time course of the development of impairments from 5 to 14 months of age. The deficit previously found in accuracy and reaction time was present at all ages examined in these Hdh(Q92/Q92) mice; the deficit was not progressive, and did not correlate with the evolution of neuronal nuclear inclusions.

Motor Assessment in Huntington's Disease Mice.

Motor deficits are a characteristic consequence of striatal damage, whether induced by experimental lesions, or in genetic models of Huntington's disease involving polyglutamine expansion in the huntingtin protein. With the growing power of genetic models and genetic tools for analysis, mice are increasingly the animal model of choice, and objective quantitative measures of motor performance are in demand for experimental analysis of disease pathophysiology, progression, and treatment. We present methodological protocols for six of the most common tests of motor function-ranging from spontaneous activity, locomotor coordination, balance, and skilled limb use-that are simple, effective, efficient, and widely used for motor assessment in Huntington's disease research in experimental mice.

The Effect of Tissue Preparation and Donor Age on Striatal Graft Morphology in the Mouse.

Huntington's disease (HD) is a progressive neurodegenerative disease in which striatal medium spiny neurons (MSNs) are lost. Neuronal replacement therapies aim to replace MSNs through striatal transplantation of donor MSN progenitors, which successfully improve HD-like deficits in rat HD models and have provided functional improvement in patients. Transplants in mouse models of HD are more variable and have lower cell survival than equivalent rat grafts, yet mice constitute the majority of transgenic HD models. Improving the quality and consistency of mouse transplants would open up access to this wider range of rodent models and facilitate research to increase understanding of graft mechanisms, which is essential to progress transplantation as a therapy for HD. Here we determined how donor age, cell preparation, and donor/host strain choice influenced the quality of primary embryonic grafts in quinolinic acid lesion mouse models of HD. Both a within-strain (W-S) and a between-strain (B-S) donor/host paradigm were used to compare transplants of donor tissues derived from mice at embryonic day E12 and E14 prepared either as dissociated suspensions or as minimally manipulated tissue pieces (TP). Good graft survival was observed, although graft volume and cellular composition were highly variable. The effect of cell preparation on grafts differed significantly depending on donor age, with E14 cell suspensions yielding larger grafts compared to TP. Conversely, TP were more effective when derived from E12 donor tissue. A W-S model produced larger grafts with greater MSN content, and while high levels of activated microglia were observed across all groups, a greater number was found in B-S transplants. In summary, we show that the effect of tissue preparation on graft morphology is contingent on the age of donor tissue used. The presence of microglial activation in all groups highlights the host immune response as an important consideration in mouse transplantation.

Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand.

Dysregulated mitophagy has been linked to Parkinson's disease (PD) due to the role of PTEN-induced kinase 1 (PINK1) in mediating depolarization-induced mitophagy in vitro. Elegant mouse reporters have revealed the pervasive nature of basal mitophagy in vivo, yet the role of PINK1 and tissue metabolic context remains unknown. Using mito-QC, we investigated the contribution of PINK1 to mitophagy in metabolically active tissues. We observed a high degree of mitophagy in neural cells, including PD-relevant mesencephalic dopaminergic neurons and microglia. In all tissues apart from pancreatic islets, loss of Pink1 did not influence basal mitophagy, despite disrupting depolarization-induced Parkin activation. Our findings provide the first in vivo evidence that PINK1 is detectable at basal levels and that basal mammalian mitophagy occurs independently of PINK1. This suggests multiple, yet-to-be-discovered pathways orchestrating mammalian mitochondrial integrity in a context-dependent fashion, and this has profound implications for our molecular understanding of vertebrate mitophagy.

Phosphorylation of Parkin at serine 65 is essential for its activation in vivo.

Mutations in PINK1 and Parkin result in autosomal recessive Parkinson's disease (PD). Cell culture and in vitro studies have elaborated the PINK1-dependent regulation of Parkin and defined how this dyad orchestrates the elimination of damaged mitochondria via mitophagy. PINK1 phosphorylates ubiquitin at serine 65 (Ser65) and Parkin at an equivalent Ser65 residue located within its N-terminal ubiquitin-like domain, resulting in activation; however, the physiological significance of Parkin Ser65 phosphorylation in vivo in mammals remains unknown. To address this, we generated a Parkin Ser65Ala (S65A) knock-in mouse model. We observe endogenous Parkin Ser65 phosphorylation and activation in mature primary neurons following mitochondrial depolarization and reveal this is disrupted in ParkinS65A/S65A neurons. Phenotypically, ParkinS65A/S65A mice exhibit selective motor dysfunction in the absence of any overt neurodegeneration or alterations in nigrostriatal mitophagy. The clinical relevance of our findings is substantiated by the discovery of homozygous PARKIN (PARK2) p.S65N mutations in two unrelated patients with PD. Moreover, biochemical and structural analysis demonstrates that the ParkinS65N/S65N mutant is pathogenic and cannot be activated by PINK1. Our findings highlight the central role of Parkin Ser65 phosphorylation in health and disease.

Identification of three novel NHS mutations in families with Nance-Horan syndrome.

PURPOSE: Nance-Horan Syndrome (NHS) is an infrequent and often overlooked X-linked disorder characterized by dense congenital cataracts, microphthalmia, and dental abnormalities. The syndrome is caused by mutations in the NHS gene, whose function is not known. The purpose of this study was to identify the frequency and distribution of NHS gene mutations and compare genotype with Nance-Horan phenotype in five North American NHS families. METHODS: Genomic DNA was isolated from white blood cells from NHS patients and family members. The NHS gene coding region and its splice site donor and acceptor regions were amplified from genomic DNA by PCR, and the amplicons were sequenced directly. RESULTS: We identified three unique NHS coding region mutations in these NHS families. CONCLUSIONS: This report extends the number of unique identified NHS mutations to 14.

Striatal lesions in the mouse disrupt acquisition and retention, but not implicit learning, in the SILT procedural motor learning task.

People with Huntington's disease (HD) have been found to have an implicit learning deficit whereby they are typically unable to detect repeated sequences embedded within randomly presented stimuli. The operant serial implicit learning task (SILT) was designed to probe animal models of HD for implicit learning deficits using the 9-hole box apparatus. The present study used mice to determine whether "early" striatal lesions would prevent SILT acquisition and to confirm previous findings that post-training "late lesions" would impair the retention of task performance. The SILT is a two-phase task whereby an initial stimulus light (S1) presentation was presented in one of five possible locations. A correct nose-poke response to the S1 resulted in this light being extinguished and a second, apparently random light presentation (S2). A correct nose-poke to S2 resulted in a reward. Within the apparently random stimulus light presentations, a predictable S1/S2 combination was embedded. Both lesion groups ("early" pre-acquisition and "late" post-acquisition lesions) demonstrated increased reaction times to S1, with the late-lesion group also recording reduced task accuracy when compared with the sham control group. The early-lesion group also demonstrated increased response latencies for the S2 stimuli during task acquisition, this was also true for task retention in the late-lesion group. No difference between the control group and early-lesion group was found for the S2 response accuracy during the acquisition period. After the lesioning of the late-lesion group, both lesion groups demonstrated reduced accuracy to the S2 stimuli as the control group improved their performance throughout the test period, while the accuracy of both lesion groups remained stable at a lower performance level. All three experimental groups were able to utilize the embedded predictable information. The present data suggest that the striatum is important for the acquisition and retention of motor learning tasks, but does not play a role in the learning of implicit information.

Motivational, proteostatic and transcriptional deficits precede synapse loss, gliosis and neurodegeneration in the B6.HttQ111/+ model of Huntington's disease.

We investigated the appearance and progression of disease-relevant signs in the B6.HttQ111/+ mouse, a genetically precise model of the mutation that causes Huntington's disease (HD). We find that B6.HttQ111/+ mice are healthy, show no overt signs of central or peripheral inflammation, and no gross motor impairment as late as 12 months of age. Behaviorally, we find that 4-9 month old B6.HttQ111/+ mice have normal activity levels and show no clear signs of anxiety or depression, but do show clear signs of reduced motivation. The neuronal density, neuronal size, synaptic density and number of glia is normal in B6.HttQ111/+ striatum, the most vulnerable brain region in HD, up to 12 months of age. Despite this preservation of the synaptic and cellular composition of the striatum, we observe clear progressive, striatal-specific transcriptional dysregulation and accumulation of neuronal intranuclear inclusions (NIIs). Simulation studies suggest these molecular endpoints are sufficiently robust for future preclinical studies, and that B6.HttQ111/+ mice are a useful tool for modeling disease-modifying or neuroprotective strategies for disease processes before the onset of overt phenotypes.

New mutations in the NHS gene in Nance-Horan Syndrome families from the Netherlands.

Mutations in the NHS gene cause Nance-Horan Syndrome (NHS), a rare X-chromosomal recessive disorder with variable features, including congenital cataract, microphthalmia, a peculiar form of the ear and dental anomalies. We investigated the NHS gene in four additional families with NHS from the Netherlands, by dHPLC and direct sequencing. We identified an unique mutation in each family. Three out of these four mutations were not reported before. We report here the first splice site sequence alteration mutation and three protein truncating mutations. Our results suggest that X-linked cataract and NHS are allelic disorders.

The utilisation of operant delayed matching and non-matching to position for probing cognitive flexibility and working memory in mouse models of Huntington's disease.

BACKGROUND: Operant behavioural testing provides a highly sensitive and automated method of exploring the behavioural deficits seen in rodent models of neurodegenerative diseases, including Huntington's disease (HD). The delayed matching to position (DMTP) and delayed non-matching to position (DNMTP) tasks probe spatial learning and working memory and when applied serially they can be used to measure reversal learning, which has been shown to be an early symptom of executive dysfunction in HD. NEW METHOD: The DMTP and DNMTP tasks were conducted in two configurations of operant apparatus; the conventional 9-hole operant apparatus, and a Skinner-like operant apparatus, to compare, contrast and optimise the DMTP and DNMTP operant protocols for use in mice. The optimised tasks were then tested in the Hdh(Q111) mouse model of HD. RESULTS: Optimisation of the operant apparatus demonstrated that the mice learned the DMTP and DNMTP tasks more rapidly and effectively in the Skinner-like apparatus configuration in comparison to the conventional 9-hole apparatus configuration. When tested in the Hdh(Q111) mouse model of HD, the DMTP and DNMTP tasks revealed significant deficits in reversal learning. COMPARISON WITH EXISTING METHOD: We found that mice were capable of performing the DMTP and DNMTP tasks in both apparatus configurations, but in comparison to the 9-hole configuration, the Skinner-like configuration produced more efficient, robust and reliable results. CONCLUSIONS: The results presented here suggest that DMTP and DNMTP tasks, incorporating a reversal learning manipulation, are valid and robust methods for probing selected cognitive deficits in mouse models of neurodegenerative diseases.

A Longitudinal Motor Characterisation of the HdhQ111 Mouse Model of Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is a rare, incurable neurodegenerative disorder caused by a CAG trinucleotide expansion with the first exon of the huntingtin gene. Numerous knock-in mouse models are currently available for modelling HD. However, before their use in scientific research, these models must be characterised to determine their face and predictive validity as models of the disease and their reliability in recapitulating HD symptoms. OBJECTIVE: Manifest HD is currently diagnosed upon the onset of motor symptoms, thus we sought to longitudinally characterise the progression and severity of motor signs in the HdhQ111 knock-in mouse model of HD, in heterozygous mice. METHODS: An extensive battery of motor tests including: rotarod, inverted lid test, balance beam, spontaneous locomotor activity and gait analysis were applied longitudinally to a cohort of HdhQ111 heterozygous mice in order to progressively assess motor function. RESULTS: A progressive failure to gain body weight was demonstrated from 11 months of age and motor problems in all measures of balance beam performance were shown in HdhQ111 heterozygous animals in comparison to wild type control animals from 9 months of age. A decreased latency to fall from the rotarod was demonstrated in HdhQ111 heterozygous animals in comparison to wild type animals, although this was not progressive with time. No genotype specific differences were demonstrated in any of the other motor tests included in the test battery. CONCLUSIONS: The HdhQ111 heterozygous mouse demonstrates a subtle and progressive motor phenotype that begins at 9 months of age. This mouse model represents an early disease stage and would be ideal for testing therapeutic strategies that require elongated lead-in times, such as viral gene therapies or striatal transplantation.

A Longitudinal Operant Assessment of Cognitive and Behavioural Changes in the HdhQ111 Mouse Model of Huntington's Disease.

Huntington's disease (HD) is characterised by motor symptoms which are often preceded by cognitive and behavioural changes, that can significantly contribute to disease burden for people living with HD. Numerous knock-in mouse models of HD are currently available for scientific research. However, before their use, they must be behaviourally characterised to determine their suitability in recapitulating the symptoms of the human condition. Thus, we sought to longitudinally characterise the nature, severity and time course of cognitive and behavioural changes observed in HdhQ111 heterozygous knock-in mice.To determine changes in cognition and behaviour an extensive battery of operant tests including: fixed ratio, progressive ratio, the five choice serial reaction time task and the serial implicit learning task, were applied longitudinally to HdhQ111 and wild type mice. The operant test battery was conducted at 6, 12 and 18 months of age. Significant deficits were observed in HdhQ111 animals in comparison to wild type animals in all operant tests indicating altered cognition (attentional and executive function) and motivation. However, the cognitive and behavioural deficits observed were not shown to be progressive over time in the longitudinal testing paradigm that was utilised. The results therefore demonstrate that the HdhQ111 mouse model of HD reflects some features of the cognitive and behavioural changes shown in the human condition of HD. Although, the cognitive and behavioural deficits demonstrated were not shown to be progressive over time.