ABSTRACT
Loss-of-function mutations in the GNAL gene are responsible for DYT-GNAL dystonia. However, how GNAL mutations contribute to synaptic dysfunction is still unclear. The GNAL gene encodes the Gαolf protein, an isoform of stimulatory Gαs enriched in the striatum, with a key role in the regulation of cAMP signaling. Here, we used a combined biochemical and electrophysiological approach to study GPCR-mediated AC-cAMP cascade in the striatum of the heterozygous GNAL (GNAL+/-) rat model. We first analyzed adenosine type 2 (A2AR), and dopamine type 1 (D1R) receptors, which are directly coupled to Gαolf, and observed that the total levels of A2AR were increased, whereas D1R level was unaltered in GNAL+/- rats. In addition, the striatal isoform of adenylyl cyclase (AC5) was reduced, despite unaltered basal cAMP levels. Notably, the protein expression level of dopamine type 2 receptor (D2R), that inhibits the AC5-cAMP signaling pathway, was also reduced, similar to what observed in different DYT-TOR1A dystonia models. Accordingly, in the GNAL+/- rat striatum we found altered levels of the D2R regulatory proteins, RGS9-2, spinophilin, Gß5 and ß-arrestin2, suggesting a downregulation of D2R signaling cascade. Additionally, by analyzing the responses of striatal cholinergic interneurons to D2R activation, we found that the receptor-mediated inhibitory effect is significantly attenuated in GNAL+/- interneurons. Altogether, our findings demonstrate a profound alteration in the A2AR/D2R-AC-cAMP cascade in the striatum of the rat DYT-GNAL dystonia model, and provide a plausible explanation for our previous findings on the loss of dopamine D2R-dependent corticostriatal long-term depression.
Subject(s)
Dystonia , Dystonic Disorders , Rats , Animals , Adenylyl Cyclases/genetics , Adenylyl Cyclases/metabolism , Dopamine/metabolism , Cyclic AMP/metabolism , Dystonia/genetics , Signal Transduction/physiology , Corpus Striatum/metabolism , Receptors, Dopamine/metabolism , Protein Isoforms/metabolismABSTRACT
DYT6 dystonia is caused by mutations in the transcription factor THAP1. THAP1 knock-out or knock-in mouse models revealed complex gene expression changes, which are potentially responsible for the pathogenesis of DYT6 dystonia. However, how THAP1 mutations lead to these gene expression alterations and whether the gene expression changes are also reflected in the brain of THAP1 patients are still unclear. In this study we used epigenetic and transcriptomic approaches combined with multiple model systems [THAP1 patients' frontal cortex, THAP1 patients' induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons, THAP1 heterozygous knock-out rat model, and THAP1 heterozygous knock-out SH-SY5Y cell lines] to uncover a novel function of THAP1 and the potential pathogenesis of DYT6 dystonia. We observed that THAP1 targeted only a minority of differentially expressed genes caused by its mutation. THAP1 mutations lead to dysregulation of genes mainly through regulation of SP1 family members, SP1 and SP4, in a cell type dependent manner. Comparing global differentially expressed genes detected in THAP1 patients' iPSC-derived midbrain dopaminergic neurons and THAP1 heterozygous knock-out rat striatum, we observed many common dysregulated genes and 61 of them were involved in dystonic syndrome-related pathways, like synaptic transmission, nervous system development, and locomotor behaviour. Further behavioural and electrophysiological studies confirmed the involvement of these pathways in THAP1 knock-out rats. Taken together, our study characterized the function of THAP1 and contributes to the understanding of the pathogenesis of primary dystonia in humans and rats. As SP1 family members were dysregulated in some neurodegenerative diseases, our data may link THAP1 dystonia to multiple neurological diseases and may thus provide common treatment targets.
Subject(s)
Dystonia , Dystonic Disorders , Neuroblastoma , Humans , Mice , Animals , Rats , Dystonia/genetics , Nuclear Proteins/genetics , DNA-Binding Proteins/metabolism , Apoptosis Regulatory Proteins/genetics , Dystonic Disorders/genetics , Mutation/genetics , Sp1 Transcription Factor/geneticsABSTRACT
Huntington's disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a cytosine-adenine-guanine triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine stretch in the protein product which becomes aggregation prone. The mutant Htt (mHtt) aggregates are associated with components of the ubiquitin-proteasome system, suggesting that mHtt is marked for proteasomal degradation and that, for reasons still debated, are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and subsequent cellular responses. We identified two lysine residues, 6 and 9, in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Proteomic analysis indicated that cellular reactions to mHtt expression were weaker in cells expressing the lysineless protein, possibly implying a reduced capacity to cope with the proteotoxic stress. Taken together, the findings suggest a novel role for ubiquitination-attenuation of the pathogenic effect of mHtt.
Subject(s)
Huntingtin Protein , Huntington Disease , Ubiquitination/physiology , Animals , Brain/cytology , Brain/metabolism , Cell Death/physiology , Cells, Cultured , Disease Models, Animal , Humans , Huntingtin Protein/chemistry , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Huntington Disease/genetics , Huntington Disease/metabolism , Huntington Disease/pathology , Lysine/chemistry , Lysine/metabolism , Neurons/metabolism , Proteasome Endopeptidase Complex , Protein Aggregation, Pathological/metabolism , Rats , Rats, TransgenicABSTRACT
Background. For neurodegenerative diseases such as Huntington's disease (HD), early diagnosis is essential to treat patients and delay symptoms. Impaired olfaction, as observed as an early symptom in Parkinson´s disease, may also constitute a key symptom in HD. However, there are few reports on olfactory deficits in HD. Therefore, we aimed to investigate, in a transgenic rat model of HD: (1) whether general olfactory impairment exists and (2) whether there are disease-specific dynamics of olfactory dysfunction when the vomeronasal (VNE) and main olfactory epithelium (MOE) are compared. Methods. We used male rats of transgenic line 22 (TG22) of the bacterial artificial chromosome Huntington disease model (BACHD), aged 3 days or 6 months. Cell proliferation, apoptosis and macrophage activity were examined with immunohistochemistry in the VNE and MOE. Results. No differences were observed in cellular parameters in the VNE between the groups. However, the MOE of the 6-month-old HD animals showed a significantly increased number of mature olfactory receptor neurons. Other cellular parameters were not affected. Conclusions. The results obtained in the TG22 line suggest a relative stability in the VNE, whereas the MOE seems at least temporarily affected.
Subject(s)
Huntington Disease , Olfaction Disorders , Olfactory Receptor Neurons , Animals , Chromosomes, Artificial, Bacterial , Disease Models, Animal , Huntington Disease/metabolism , Male , Olfaction Disorders/metabolism , Olfactory Mucosa/metabolism , Olfactory Receptor Neurons/metabolism , Rats , Rats, TransgenicABSTRACT
Dystonia is a neurological movement disorder characterized by sustained or intermittent involuntary muscle contractions. Loss-of-function mutations in the GNAL gene have been identified to be the cause of "isolated" dystonia DYT25. The GNAL gene encodes for the guanine nucleotide-binding protein G(olf) subunit alpha (Gαolf), which is mainly expressed in the olfactory bulb and the striatum and functions as a modulator during neurotransmission coupling with D1R and A2AR. Previously, heterozygous Gαolf -deficient mice (Gnal+/-) have been generated and showed a mild phenotype at basal condition. In contrast, homozygous deletion of Gnal in mice (Gnal-/-) resulted in a significantly reduced survival rate. In this study, using the CRISPR-Cas9 system we generated and characterized heterozygous Gnal knockout rats (Gnal+/-) with a 13 base pair deletion in the first exon of the rat Gnal splicing variant 2, a major isoform in both human and rat striatum. Gnal+/- rats showed early-onset phenotypes associated with impaired dopamine transmission, including reduction in locomotor activity, deficits in rotarod performance and an abnormal motor skill learning ability. At cellular and molecular level, we found down-regulated Arc expression, increased cell surface distribution of AMPA receptors, and the loss of D2R-dependent corticostriatal long-term depression (LTD) in Gnal+/- rats. Based on the evidence that D2R activity is normally inhibited by adenosine A2ARs, co-localized on the same population of striatal neurons, we show that blockade of A2ARs restores physiological LTD. This animal model may be a valuable tool for investigating Gαolf function and finding a suitable treatment for dystonia associated with deficient dopamine transmission.
Subject(s)
Adenosine/metabolism , Disease Models, Animal , Dopamine/metabolism , Dystonia , Long-Term Synaptic Depression/physiology , Animals , Dystonia/metabolism , Dystonia/physiopathology , GTP-Binding Protein alpha Subunits/genetics , Gene Knockout Techniques , Male , Rats , Rats, Sprague-Dawley , Receptor, Adenosine A2A/metabolism , Signal Transduction/physiologyABSTRACT
White matter (WM) atrophy is a significant feature of Huntington disease (HD), although its aetiology and early pathological manifestations remain poorly defined. In this study, we aimed to characterize WM-related features in the transgenic YAC128 and BACHD models of HD. Using diffusion tensor magnetic resonance imaging (DT-MRI), we demonstrate that microstructural WM abnormalities occur from an early age in YAC128 mice. Similarly, electron microscopy analysis of myelinated fibres of the corpus callosum indicated that myelin sheaths are thinner in YAC128 mice as early as 1.5 months of age, well before any neuronal loss can be detected. Transcript levels of myelin-related genes in striatal and cortical tissues were significantly lower in YAC128 mice from 2 weeks of age, and these findings were replicated in differentiated primary oligodendrocytes from YAC128 mice, suggesting a possible mechanistic explanation for the observed structural deficits. Concordant with these observations, we demonstrate reduced expression of myelin-related genes at 3 months of age and WM microstructural abnormalities using DT-MRI at 12 months of age in the BACHD rats. These findings indicate that WM deficits in HD are an early phenotype associated with cell-intrinsic effects of mutant huntingtin on myelin-related transcripts in oligodendrocytes, and raise the possibility that WM abnormalities may be an early contributing factor to the pathogenesis of HD.
Subject(s)
Huntington Disease/genetics , Myelin Sheath/physiology , White Matter/physiopathology , Animals , Atrophy/pathology , Brain/metabolism , Corpus Callosum/metabolism , Corpus Striatum/metabolism , Diffusion Tensor Imaging/methods , Disease Models, Animal , Gene Expression , Humans , Huntington Disease/etiology , Mice , Mice, Transgenic , Myelin Sheath/genetics , Myelin Sheath/metabolism , Neostriatum/metabolism , Nerve Tissue Proteins/genetics , Neurons/metabolism , Oligodendroglia/metabolism , RatsABSTRACT
Huntington's disease is a fatal human neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene, which translates into a mutant huntingtin protein. A key event in the molecular pathogenesis of Huntington's disease is the proteolytic cleavage of mutant huntingtin, leading to the accumulation of toxic protein fragments. Mutant huntingtin cleavage has been linked to the overactivation of proteases due to mitochondrial dysfunction and calcium derangements. Here, we investigated the therapeutic potential of olesoxime, a mitochondria-targeting, neuroprotective compound, in the BACHD rat model of Huntington's disease. BACHD rats were treated with olesoxime via the food for 12 months. In vivo analysis covered motor impairments, cognitive deficits, mood disturbances and brain atrophy. Ex vivo analyses addressed olesoxime's effect on mutant huntingtin aggregation and cleavage, as well as brain mitochondria function. Olesoxime improved cognitive and psychiatric phenotypes, and ameliorated cortical thinning in the BACHD rat. The treatment reduced cerebral mutant huntingtin aggregates and nuclear accumulation. Further analysis revealed a cortex-specific overactivation of calpain in untreated BACHD rats. Treated BACHD rats instead showed significantly reduced levels of mutant huntingtin fragments due to the suppression of calpain-mediated cleavage. In addition, olesoxime reduced the amount of mutant huntingtin fragments associated with mitochondria, restored a respiration deficit, and enhanced the expression of fusion and outer-membrane transport proteins. In conclusion, we discovered the calpain proteolytic system, a key player in Huntington's disease and other neurodegenerative disorders, as a target of olesoxime. Our findings suggest that olesoxime exerts its beneficial effects by improving mitochondrial function, which results in reduced calpain activation. The observed alleviation of behavioural and neuropathological phenotypes encourages further investigations on the use of olesoxime as a therapeutic for Huntington's disease.
Subject(s)
Calpain/metabolism , Cholestenones/pharmacology , Cholestenones/therapeutic use , Huntington Disease/drug therapy , Huntington Disease/metabolism , Mutant Proteins/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Proteolysis/drug effects , Animals , Behavior, Animal/drug effects , Brain/drug effects , Brain/metabolism , Brain/pathology , Calpain/antagonists & inhibitors , Cholestenones/blood , Cholestenones/metabolism , Disease Models, Animal , Enzyme Activation/drug effects , Huntingtin Protein , Huntington Disease/enzymology , Huntington Disease/genetics , Male , Mitochondria/drug effects , Mitochondria/metabolism , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Rats , Rats, TransgenicABSTRACT
Executive dysfunction and psychiatric symptoms are hallmarks of Huntington disease (HD), a neurodegenerative disorder genetically characterized by expanded CAG repeats in the HTT gene. Using the BACHD rat model of HD (97 CAG-CAA repeats), the present research seeks to characterize the progressive emergence of decision-making impairments in a rat version of the Iowa Gambling Task (RGT) and the impact of emotional modulation, whether positive or negative, on choice behavior. The choice efficiency shown both by WT rats (independent of their age) and the youngest BACHD rats (2 and 8months old) evidenced that they are able to integrate outcomes of past decisions to determine expected reward values for each option. However, 18months old BACHD rats made fewer choices during the RGT session and were less efficient in choosing advantageous options than younger animals. Presenting either chocolate pellets or electrical footshocks half-way through a second RGT session reduced exploratory activity (inefficient nose-poking) and choices with a weaker effect on BACHD animals than on WT. Choice efficiency was left intact in transgenic rats. Our results bring new knowledge on executive impairments and impact of emotional state on decision-making at different stages of the disease, increasing the face-validity of the BACHD rat model.
Subject(s)
Choice Behavior/physiology , Emotions/physiology , Huntington Disease/psychology , Animals , Behavior, Animal/physiology , Disease Models, Animal , Electroshock , Huntington Disease/genetics , Motor Activity/physiology , Rats , Rats, TransgenicABSTRACT
Spinocerebellar ataxia 17 (SCA17) is an autosomal-dominant, late-onset neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat in the TATA-box-binding protein (TBP). To further investigate this devastating disease, we sought to create a first transgenic rat model for SCA17 that carries a full human cDNA fragment of the TBP gene with 64 CAA/CAG repeats (TBPQ64). In line with previous observations in mouse models for SCA17, TBPQ64 rats show a severe neurological phenotype including ataxia, impairment of postural reflexes, and hyperactivity in early stages followed by reduced activity, loss of body weight, and early death. Neuropathologically, the severe phenotype of SCA17 rats was associated with neuronal loss, particularly in the cerebellum. Degeneration of Purkinje, basket, and stellate cells, changes in the morphology of the dendrites, nuclear TBP-positive immunoreactivity, and axonal torpedos were readily found by light and electron microscopy. While some of these changes are well recapitulated in existing mouse models for SCA17, we provide evidence that some crucial characteristics of SCA17 are better mirrored in TBPQ64 rats. Thus, this SCA17 model represents a valuable tool to pursue experimentation and therapeutic approaches that may be difficult or impossible to perform with SCA17 transgenic mice. We show for the first time positron emission tomography (PET) and diffusion tensor imaging (DTI) data of a SCA animal model that replicate recent PET studies in human SCA17 patients. Our results also confirm that DTI are potentially useful correlates of neuropathological changes in TBPQ64 rats and raise hope that DTI imaging could provide a biomarker for SCA17 patients.
Subject(s)
Diffusion Tensor Imaging , Disease Models, Animal , Spinocerebellar Ataxias , TATA-Box Binding Protein/genetics , Trinucleotide Repeat Expansion/genetics , Animals , Anxiety/etiology , Anxiety/genetics , Body Weight/genetics , Brain/metabolism , Brain/pathology , Brain/ultrastructure , Electronic Data Processing , Female , Genotype , Humans , Male , Maze Learning , Motor Activity , Neurologic Examination , Positron-Emission Tomography , Psychomotor Performance/physiology , Raclopride/pharmacokinetics , Rats , Rats, Transgenic , Rotarod Performance Test , Severity of Illness Index , Spinocerebellar Ataxias/diagnosis , Spinocerebellar Ataxias/genetics , Spinocerebellar Ataxias/physiopathology , Tubulin/metabolismABSTRACT
Huntington's disease (HD), a congenital neurodegenerative disorder, extends its pathological damages beyond the nervous system. The systematic manifestation of HD has been extensively described in numerous studies, including dysfunction in peripheral organs and peripheral inflammation. Gut dysbiosis and the gut-liver-brain axis have garnered greater emphasis in neurodegenerative research, and increased plasma levels of pro-inflammatory cytokines have been identified in HD patients and various in vivo models, correlating with disease progression. In the present study, we investigated hepatic pathological markers in the liver of R6/2 mice which convey exon 1 of the human mutant huntingtin gene. Furthermore, we evaluated the impact of intravenously administered Mesenchymal Stromal Cells (MSCs) on the liver enzymes, changes in hepatic inflammatory markers, as well as brain pathology and behavioral deficits in R6/2 mice. Our results revealed altered enzyme expression and increased levels of inflammatory mediators in the liver of R6/2 mice, which were significantly attenuated in the MSC-treated R6/2 mice. Remarkably, neuronal pathology and altered motor activities in the MSC-treated R6/2 mice were significantly ameliorated, despite the absence of MSCs in the postmortem brain. Our data highlight the importance of hepatic pathological changes in HD, providing a potential therapeutic approach. Moreover, the data open new perspectives for the search in blood biomarkers correlating with liver pathology in HD.
Subject(s)
Huntington Disease , Mice , Humans , Animals , Huntington Disease/metabolism , Mice, Transgenic , Disease Models, Animal , Brain/metabolism , Liver/metabolismABSTRACT
Huntington disease (HD) is an inherited progressive neurodegenerative disorder, characterized by motor, cognitive, and psychiatric deficits as well as neurodegeneration and brain atrophy beginning in the striatum and the cortex and extending to other subcortical brain regions. The genetic cause is an expansion of the CAG repeat stretch in the HTT gene encoding huntingtin protein (htt). Here, we generated an HD transgenic rat model using a human bacterial artificial chromosome (BAC), which contains the full-length HTT genomic sequence with 97 CAG/CAA repeats and all regulatory elements. BACHD transgenic rats display a robust, early onset and progressive HD-like phenotype including motor deficits and anxiety-related symptoms. In contrast to BAC and yeast artificial chromosome HD mouse models that express full-length mutant huntingtin, BACHD rats do not exhibit an increased body weight. Neuropathologically, the distribution of neuropil aggregates and nuclear accumulation of N-terminal mutant huntingtin in BACHD rats is similar to the observations in human HD brains. Aggregates occur more frequently in the cortex than in the striatum and neuropil aggregates appear earlier than mutant htt accumulation in the nucleus. Furthermore, we found an imbalance in the striatal striosome and matrix compartments in early stages of the disease. In addition, reduced dopamine receptor binding was detectable by in vivo imaging. Our data demonstrate that this transgenic BACHD rat line may be a valuable model for further understanding the disease mechanisms and for preclinical pharmacological studies.
Subject(s)
Chromosomes, Artificial, Bacterial/genetics , Huntington Disease/genetics , Huntington Disease/pathology , Nerve Tissue Proteins/genetics , Alternative Splicing , Animals , Anxiety/genetics , Anxiety/psychology , Behavior, Animal/physiology , Blotting, Western , Body Weight/physiology , Eating/physiology , Gait Disorders, Neurologic/psychology , Gene Dosage , Humans , Huntingtin Protein , Huntington Disease/psychology , Immunohistochemistry , Motor Activity/physiology , Positron-Emission Tomography , Postural Balance/physiology , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Rats, Transgenic , Real-Time Polymerase Chain ReactionABSTRACT
Body weight (BW) loss and reduced body mass index (BMI) are the most common peripheral alterations in Huntington disease (HD) and have been found in HD mutation carriers and HD animal models before the manifestation of neurological symptoms. This suggests that, at least in the early disease stage, these changes could be due to abnormal tissue growth rather than tissue atrophy. Moreover, BW and BMI are reported to be more affected in males than females in HD animal models and patients. Here, we confirmed sex-dependent growth alterations in the BACHD rat model for HD and investigated the associated contributing factors. Our results showed growth abnormalities along with decreased plasma testosterone and insulin-like growth factor 1 (IGF-1) levels only in males. Moreover, we demonstrated correlations between growth parameters, IGF-1, and testosterone. Our analyses further revealed an aberrant transcription of testosterone biosynthesis-related genes in the testes of BACHD rats with undisturbed luteinizing hormone (LH)/cAMP/PKA signaling, which plays a key role in regulating the transcription process of some of these genes. In line with the findings in BACHD rats, analyses in the R6/2 mouse model of HD showed similar results. Our findings support the view that mutant huntingtin may induce abnormal growth in males via the dysregulation of gene transcription in the testis, which in turn can affect testosterone biosynthesis.
Subject(s)
Huntingtin Protein , Huntington Disease , Testosterone , Animals , Female , Male , Mice , Rats , Brain/metabolism , Disease Models, Animal , Huntington Disease/genetics , Huntington Disease/metabolism , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Testosterone/biosynthesis , Huntingtin Protein/geneticsABSTRACT
Evidence from patients with Parkinson's disease (PD) and our previously reported α-synuclein (SNCA) transgenic rat model support the idea that increased SNCA protein is a substantial risk factor of PD pathogenesis. However, little is known about the transcription control of the human SNCA gene in the brain in vivo. Here, we identified that the DYT6 gene product THAP1 (THAP domain-containing apoptosis-associated protein 1) and its interaction partner CTCF (CCCTC-binding factor) act as transcription regulators of SNCA. THAP1 controls SNCA intronic enhancers' activities, while CTCF regulates its enhancer-promoter loop formation. The SNCA intronic enhancers present neurodevelopment-dependent activities and form enhancer clusters similar to "super-enhancers" in the brain, in which the PD-associated single-nucleotide polymorphisms are enriched. Deletion of the SNCA intronic enhancer clusters prevents the release of paused RNA polymerase II from its promoter and subsequently reduces its expression drastically in the brain, which may provide new therapeutic approaches to prevent its accumulation and thus related neurodegenerative diseases defined as synucleinopathies.
Subject(s)
Brain , Parkinson Disease , Humans , alpha-Synuclein/genetics , Apoptosis Regulatory Proteins/metabolism , Brain/metabolism , DNA-Binding Proteins/metabolism , Introns/genetics , Parkinson Disease/metabolism , Polymorphism, Single Nucleotide , Regulatory Sequences, Nucleic AcidABSTRACT
Intrastriatal administration of mesenchymal stem cells (MSCs) has shown beneficial effects in rodent models of Huntington disease (HD). However, the invasive nature of surgical procedure and its potential to trigger the host immune response may limit its clinical use. Hence, we sought to evaluate the non-invasive intranasal administration (INA) of MSC delivery as an effective alternative route in HD. GFP-expressing MSCs derived from bone marrow were intranasally administered to 4-week-old R6/2 HD transgenic mice. MSCs were detected in the olfactory bulb, midbrain and striatum five days post-delivery. Compared to phosphate-buffered saline (PBS)-treated littermates, MSC-treated R6/2 mice showed an increased survival rate and attenuated circadian activity disruption assessed by locomotor activity. MSCs increased the protein expression of DARPP-32 and tyrosine hydroxylase (TH) and downregulated gene expression of inflammatory modulators in the brain 7.5 weeks after INA. While vehicle treated R6/2 mice displayed decreased Iba1 expression and altered microglial morphology in comparison to the wild type littermates, MSCs restored both, Iba1 level and the thickness of microglial processes in the striatum of R6/2 mice. Our results demonstrate significantly ameliorated phenotypes of R6/2 mice after MSCs administration via INA, suggesting this method as an effective delivering route of cells to the brain for HD therapy.
Subject(s)
Dopamine/metabolism , Huntington Disease/physiopathology , Huntington Disease/therapy , Inflammation/pathology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Synaptic Transmission , Administration, Intranasal , Animals , Brain/pathology , Brain/physiopathology , Cell Tracking , Circadian Rhythm , Disease Models, Animal , Dopamine and cAMP-Regulated Phosphoprotein 32/metabolism , Gene Expression Regulation , Humans , Huntington Disease/genetics , Inflammation/genetics , Male , Mice, Inbred C57BL , Mice, Transgenic , Microglia/metabolism , Microglia/pathology , Motor Activity , Nerve Growth Factors/metabolism , Sleep , Survival Analysis , Tyrosine 3-Monooxygenase/metabolismABSTRACT
BACKGROUND: Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene, which results in brain neurodegeneration and peripheral pathology affecting different organs including testis. Patients with HD suffer from motor and cognitive impairment, and multiple psychiatric symptoms. Among behavioral abnormalities in HD, sexual disturbances have often been reported, but scarcely investigated in animal models. The BACHD rat model of HD carries the human full-length mutated HTT (mHTT) genomic sequence with 97 CAG-CAA repeats and displays HD-like alterations at neuropathological and behavioral level. OBJECTIVE: This study aims to phenotype the BACHD rats' sexual behavior and performance as well as testis morphology because alterations in these aspects have been associated to HD. METHODS: Two rat cohorts at the age of 3 and 7 months were subjected to mating tests to assess different parameters of sexual behavior. Histological analyses for testis morphology were performed in different rat cohorts at 1.5, 7 and 12 months of age whereas immunohistochemical analyses were carried out at 7 and 12 months of age to visualize the presence of mHTT in testicular tissue. Furthermore, western blot analyses were used to assess HTT and mHTT expression levels in striatum and testis at three months of age. RESULTS: At 3 months, BACHD rats showed a decreased time exploring the female anogenital area (AGA), decreased latency to mount, increased number of intromissions and ejaculations and enhanced hit rate. At 7 months, all sexual parameters were comparable between genotypes with the exception that BACHD rats explored the AGA less than wild type rats. Testis analyses did not reveal any morphological alteration at any of the examined ages, but showed presence of mHTT limited to Sertoli cells in transgenic rats at both 7 and 12 months. BACHD rat HTT and mHTT expression levels in testis were lower than striatum at 3 months of age. CONCLUSIONS: The testis phenotype in the BACHD rat model does not mimic the changes observed in human HD testis. The altered sexual behavior in BACHD rats at three months of age could be to a certain extent representative of and share common underlying pathways with some of the sexual disturbances in HD patients. Further investigating the biological causes of the sexual phenotype in BACHD rats may therefore contribute to clarifying the mechanisms at the base of sexual behavior changes in HD.
Subject(s)
Huntingtin Protein/genetics , Huntington Disease/psychology , Sexual Behavior, Animal/physiology , Testis/anatomy & histology , Animals , Corpus Striatum/metabolism , Disease Models, Animal , Female , Humans , Huntingtin Protein/metabolism , Huntington Disease/genetics , Huntington Disease/metabolism , Male , Mutation , Rats , Rats, Transgenic , Testis/metabolismABSTRACT
Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene which results in progressive neurodegeneration in the striatum, cortex, and eventually most brain areas. Despite being a monogenic disorder, environmental factors influence HD characteristics. Both human and mouse studies suggest that mutant HTT (mHTT) leads to gene expression changes that harbor potential to be modulated by the environment. Yet, the underlying mechanisms integrating environmental cues into the gene regulatory program have remained largely unclear. To better understand gene-environment interactions in the context of mHTT, we employed RNA-seq to examine effects of maternal separation (MS) and environmental enrichment (EE) on striatal gene expression during development of BACHD rats. We integrated our results with striatal consensus modules defined on HTT-CAG length and age-dependent co-expression gene networks to relate the environmental factors with disease progression. While mHTT was the main determinant of expression changes, both MS and EE were capable of modulating these disturbances, resulting in distinctive and in several cases opposing effects of MS and EE on consensus modules. This bivalent response to maternal separation and environmental enrichment may aid in explaining their distinct effects observed on disease phenotypes in animal models of HD and related neurodegenerative disorders.
Subject(s)
Corpus Striatum/pathology , Environmental Exposure , Gene Expression , Huntington Disease/pathology , Animals , Disease Models, Animal , Disease Progression , Gene Expression Profiling , Gene Regulatory Networks , Huntingtin Protein/deficiency , Mice , Rats , Sequence Analysis, RNAABSTRACT
A three-base-pair deletion in the human TOR1A gene is causative for the most common form of primary dystonia: the early-onset dystonia type 1 (DYT1 dystonia). The pathophysiological consequences of this mutation are still unknown. To study the pathology of the mutant torsinA (TOR1A) protein, we have generated a transgenic rat line that overexpresses the human mutant protein under the control of the human TOR1A promoter. This new animal model was phenotyped with several approaches, including behavioral tests and neuropathological analyses. Motor phenotype, cellular and ultrastructural key features of torsinA pathology were found in this new transgenic rat line, supporting that it can be used as a model system for investigating the disease's development. Analyses of mutant TOR1A protein expression in various brain regions also showed a dynamic expression pattern and a reversible nuclear envelope pathology. These findings suggest the differential vulnerabilities of distinct neuronal subpopulations. Furthermore, the reversibility of the nuclear envelope pathology might be a therapeutic target to treat the disease.
ABSTRACT
In Huntington's disease (HD), dysfunctional affective processes emerge as key symptoms of disturbances. In human HD and transgenic rat models of the disease, the amygdala was previously shown to have a reduced volume and to carry a high load of mutant huntingtin (mHTT) aggregates. In search of the pathophysiology of affective dysregulation in HD, we hypothesized a specific role of the central amygdala (CeA), known to be particularly involved in emotional regulation. Using transgenic BACHD rats carrying full-length human mHTT, we compared behavioral consequences of pharmacological modulation of CeA function by infusing GABAA receptor (GABAAR) antagonist picrotoxin into â¼4.5 month old BACHD and WT rats before confronting them to potentially threatening situations. Our results show that disinhibition of the CeA induced differential behaviors in WT and BACHD rats in our tasks: it increased social contacts and responses to the threatening warning signal in an avoidance task in BACHD rats but not in WT animals. At the cellular level, analyzes of amygdala alteration/dysfunction showed (1) an age-dependent increase in number and size of mHTT aggregates specifically in the CeA of BACHD rats; (2) no alteration of GABA and GABAAR expression level, but (3) an increased neuronal reactivity (Arc labelling) to a threatening stimulus in the medial part of this nucleus in 4.5 months old BACHD rats. These results suggest a basal pathological hyper-reactivity in the CeA (in particular its medial part) in the transgenic animals. Such amygdala dysfunction could account, at least in part, for affective symptoms in HD patients.
Subject(s)
Central Amygdaloid Nucleus/drug effects , Emotions/drug effects , GABA-A Receptor Antagonists/pharmacology , Huntington Disease/metabolism , Picrotoxin/pharmacology , Receptors, GABA-A/metabolism , Aging/metabolism , Aging/pathology , Animals , Avoidance Learning/drug effects , Avoidance Learning/physiology , Basolateral Nuclear Complex/drug effects , Basolateral Nuclear Complex/metabolism , Basolateral Nuclear Complex/pathology , Central Amygdaloid Nucleus/metabolism , Central Amygdaloid Nucleus/pathology , Cytoskeletal Proteins/metabolism , Disease Models, Animal , Emotions/physiology , Humans , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Huntington Disease/pathology , Male , Nerve Tissue Proteins/metabolism , Neural Inhibition/drug effects , Neural Inhibition/physiology , Protein Aggregation, Pathological/metabolism , Protein Aggregation, Pathological/pathology , Rats, Transgenic , Social Behavior , gamma-Aminobutyric Acid/metabolismABSTRACT
Huntington disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for the huntingtin protein (HTT). Mutant HTT (mHTT) has been proposed to cause neuronal dysfunction and neuronal loss through multiple mechanisms. Transcriptional changes may be a core pathogenic feature of HD. Utilizing the Affymetrix platform we performed a genome-wide RNA expression analysis in two BACHD transgenic rat lines (TG5 and TG9) at 12 months of age, both of which carry full-length human mHTT but with different expression levels. By defining the threshold of significance at p < 0.01, we found 1608 genes and 871 genes differentially expressed in both TG5 and TG9 rats when compared to the wild type littermates, respectively. We only chose the highly up-/down-regulated genes for further analysis by setting an additional threshold of 1.5 fold change. Comparing gene expression profiles of human HD brains and BACHD rats revealed a high concordance in both functional and IPA (Ingenuity Pathway Analysis) canonical pathways relevant to HD. In addition, we investigated the causes leading to gene expression changes at molecular and protein levels in BACHD rats including the involvement of polyQ-containing transcription factors TATA box-binding protein (TBP), Sp1 and CBP as well as the chromatin structure. We demonstrate that the BACHD rat model recapitulates the gene expression changes of the human disease supporting its role as a preclinical research animal model. We also show for the first time that TFIID complex formation is reduced, while soluble TBP is increased in an HD model. This finding suggests that mHTT is a competitor instead of a recruiter of polyQ-containing transcription factors in the transcription process in HD.
Subject(s)
Corpus Striatum/metabolism , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Mutation , Animals , Chromatin/metabolism , Corpus Striatum/pathology , Disease Models, Animal , Electrophoresis, Agar Gel , Gene Expression , Gene Expression Profiling , Humans , Immunoblotting , Immunohistochemistry , Microarray Analysis , Rats, Sprague-Dawley , Rats, Transgenic , Real-Time Polymerase Chain Reaction , TATA-Box Binding Protein/metabolismABSTRACT
Cognitive deficits associated with Huntington disease (HD) are generally dominated by executive function disorders often associated with disinhibition and impulsivity/compulsivity. Few studies have directly examined symptoms and consequences of behavioral disinhibition in HD and its relation with decision-making. To assess the different forms of impulsivity in a transgenic model of HD (tgHD rats), two tasks assessing cognitive/choice impulsivity were used: risky decision-making with a rat gambling task (RGT) and intertemporal choices with a delay discounting task (DD). To assess waiting or action impulsivity the differential reinforcement of low rate of responding task (DRL) was used. In parallel, the volume as well as cellular activity of the amygdala was analyzed. In contrast to WT rats, 15 months old tgHD rats exhibited a poor efficiency in the RGT task with difficulties to choose advantageous options, a steep DD curve as delays increased in the DD task and a high rate of premature and bursts responses in the DRL task. tgHD rats also demonstrated a concomitant and correlated presence of both action and cognitive/choice impulsivity in contrast to wild type (WT) animals. Moreover, a reduced volume associated with an increased basal cellular activity of the central nucleus of amygdala indicated a dysfunctional amygdala in tgHD rats, which could underlie inhibitory dyscontrol. In conclusion, tgHD rats are a good model for impulsivity disorder that could be used more widely to identify potential pharmacotherapies to treat these invasive symptoms in HD.