Huntington disease: new insights into molecular pathogenesis and therapeutic opportunities

Huntington disease (HD) is a neurodegenerative disease caused by CAG repeat expansion in the huntingtin gene (HTT) and involves a complex web of pathogenic mechanisms. Mutant HTT (mHTT) disrupts transcription, interferes with immune and mitochondrial function, and is aberrantly modified post-translationally. Evidence suggests that the mHTT RNA is toxic, and at the DNA level, somatic CAG repeat expansion in vulnerable cells influences the disease course. Genome-wide association studies have identified DNA repair pathways as modifiers of somatic instability and disease course in HD and other repeat expansion diseases. In animal models of HD, nucleocytoplasmic transport is disrupted and its restoration is neuroprotective. Novel cerebrospinal fluid (CSF) and plasma biomarkers are among the earliest detectable changes in individuals with premanifest HD and have the sensitivity to detect therapeutic benefit. Therapeutically, the first human trial of an HTT-lowering antisense oligonucleotide successfully, and safely, reduced the CSF concentration of mHTT in individuals with HD. A larger trial, powered to detect clinical efficacy, is underway, along with trials of other HTT-lowering approaches. In this Review, we discuss new insights into the molecular pathogenesis of HD and future therapeutic strategies, including the modulation of DNA repair and targeting the DNA mutation itself

ALTERED EXPRESSION AND FUNCTIONALITY OF A2A ADENOSINE RECEPTORS IN HUNTINGTON’S DISEASE AND OTHER POLYGLUTAMINE DISORDERS

Several studies have suggested the possible involvement of A2A adenosine receptors in the pathogenesis of neuronal disorders, including Huntington’s disease. Huntington’s disease is an inherited neurodegenerative disease clinically characterized by motor, cognitive and behavioural impairments. The genetic cause of the disease is the expanded CAG triplet in a gene coding for huntingtin, a protein involved in several physiological processes. Huntington’s disease affects primarly GABAergic neurons in the basal ganglia that express adenosine A2A and dopamine D2 receptors. The present study describes a functional alteration of A2A adenosine receptor in striatal cells engineerized to express full length or truncated, wild type or mutant huntingtin. The data obtained demonstrate that the presence of mutant huntingtin induce an amplification of the transduction signal mediated by adenylyl cyclase and an aberrant coupling of A2A receptor to this transduction pathway. The expression and functionality of A2A adenosine receptor were subsequently evaluated in transgenic mice R6/2, an animal model of Huntington’s disease that express exon 1 of the human huntingtin gene. Saturation binding experiments revealed an increase of A2A receptor levels in striatum of R6/2 mice until 14 post natal days. In addition, also the potency of a typical A2A agonist was increased in striatal membranes of R6/2 mice when compared to wild type mice. The subsequent study aimed the evaluation of the presence and functionality of A2A adenosine receptors in peripheral blood cells from patients affected by Huntington’s disease compared with control subjects. The results revealed a statistically significant increase of the A2A receptor density in platelets, lymphocytes and neutrophils of Huntington’s disease patients and presymptomatic carriers of the mutation when compared to control subjects. In order to verify the specificity of A2A receptor alteration in polyglutamine disease, the same study was conducted in blood cells from patients affected by Spinocerebellar ataxia, characterized by an expanded CAG triplet in the ataxin gene and in patients affected by Friedreich’s ataxia, characterized by an expansion of the GAA triplet. Saturation binding experiments in peripheral blood cells from Spinocerebellar ataxia showed altered A2A binding parameters similar to those obtained in Huntington’s disease patients. In addition, data obtained in Friedreich’s ataxia patients showed affinity and density values for A2A receptors similar to those obtained from control subjects, demonstrating the involvement of the CAG but not of the GAA triplet. Overall these data demonstrate that an aberrant A2A receptor phenotype is present in polyglutamine disorders and this seems to be related with the expanded CAG triplet. The amplification of the signal transduction system of A2A receptors suggests that the use of selective A2A antagonists could be beneficial in the treatment of Huntington’s disease as well as in other related polyglutamine diseases. In addition, the alteration of A2A receptors in peripheral blood cells of patients with polyglutamine diseases suggests that this receptor could be an easily accessible biomarker for the evaluation of the efficacy of potential new therapies

Therapeutic Silencing of Mutant \u3cem\u3eHuntingtin\u3c/em\u3e by Targeting Single Nucleotide Polymorphisms: A Dissertation

Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disorder. Invariably fatal, HD is caused by expansion of the CAG repeat region in exon 1 of the Huntingtin gene which creates a toxic protein with an extended polyglutamine tract 1. Silencing mutant Huntingtin messenger RNA (mRNA) is a promising therapeutic approach 2-6. The ideal silencing strategy would reduce mutant Huntingtin while leaving the wild-type mRNA intact. Unfortunately, targeting the disease causing CAG repeat expansion is difficult and risks targeting other CAG repeat containing genes. We examined an alternative strategy, targeting single nucleotide polymorphisms (SNPs) in the Huntingtin mRNA. The feasibility of this approach hinges on the presence of a few common highly heterozygous SNPs which are amenable to SNP-specific targeting. In a population of HD patients from Europe and the United states, forty-eight percent were heterozygous at a single SNP site; one isoform of this SNP is associated with HD. Seventy-five percent of patients are heterozygous at least one of three frequently heterozygous SNPs. Consequently, only five allele-specific siRNAs are required to treat three-quarters of the patients in the European and U.S. patient populations. We have designed and validated siRNAs targeting these SNPs. We also developed artificial microRNAs (miRNAs) targeting Huntingtin SNPs for delivery using recombinant adeno-associated viruses (rAAVs). Both U6 promoter driven and CMV promoter driven miRNAs can discriminate between matched and mismatched targets in cell culture but the U6 promoter driven miRNAs produce the mature miRNA at levels exceeding those of the vast majority of endogenous miRNAs. The U6 promoter driven miRNAs can produce a number of unwanted processing products, most likely due to a combination of overexpression and unintended export of the pri-miRNA from the nucleus. In contrast, CMV-promoter driven miRNAs produce predominantly a single species at levels comparable to endogenous miRNAs. Injection of recombinant self complementary AAV9 viruses carrying polymerase II driven Huntingtin SNP targeting miRNAs into the striatum results in expression of the mature miRNA sequence in the brain and has no significant effect on endogenous miRNAs. Matched, but not mismatched SNP-targeting miRNAs reduce inclusions in a knock-in mouse model of HD. These studies bring us closer to an allele-specific therapy for Huntington’s disease

Emerging Roles of Signal Transduction Pathways in Neurodegenerative Diseases. Hunting New Possible Therapeutic Molecular Targets

Illnesses following the degeneration of the nervous system can occur due to aging or genetic mutations and represent a clinical concern. In neurodegenerative diseases, loss of neuronal structure and functions mainly causes cognitive impairment, representing an increasing social burden. In neurodegenerative diseases, the progressive loss of vulnerable populations of neurons in specific regions of the central nervous system was traced to different pathological events, such as misfolded proteins’ accumulation, abnormalities in proteasomes or phagosomes, as well as anomalies in lysosomes or mitochondria. Many research efforts identified important events involved in neurodegeneration, but the complex pathogenesis of neurodegenerative diseases is far from being fully elucidated. More recently, insights into the signal transduction pathways acting in the nervous system contributed to unveiling some molecular mechanisms triggering neurodegeneration. Abnormalities in the intra- or inter-cellular signaling were described to be involved in the pathogenesis of neurodegenerative disease. Understanding the signal transduction pathways that impact the nervous system homeostasis can offer a wide panel of potential targets for modulating therapeutic approaches. The present review will discuss the main signal transduction pathways involved in neurodegenerative disorders

Rastreio de estratégias terapêuticas num modelo de ratinho da doença de Machado-Joseph: foco na proteostase

Tese de Doutoramento em Ciências da SaúdeMachado-Joseph Disease (MJD) belongs to the group of nine Polyglutamine (PolyQ) diseases, which are characterized by the appearance of misfolded protein species, protein aggregates, neuronal dysfunction and ultimately cell death, of which is triggered by the presence of an expanded CAG repeat in the respective disease-causing genes. Along with the pathogenic motif, all these disease-associated proteins are widely expressed but the diseases only affect specific subsets of neurons. So far there is no effective treatment available for these disorders. Because the pathogenic mechanism(s) underlying MJD are poorly understood, which might be hampering the progression of effective therapeutical targets, this work focused on the search for new therapies based on hypotheses, that additionally allow the study of molecular mechanism(s) of disease. For this, we used the CMVMJD135 mouse model, which recapitulates the human disease both at the phenotypic and neuropathologic levels. We tested the efficacy of several compounds acting on different aspects of cellular function: (i) molecular chaperone load, (Hsp90 inhibitor - 17-dimethyl aminoethylamino-17-demethoxygeldanamycin hydrochloride - 17-DMAG); (ii) autophagy (lithium chloride and a combination of lithium chloride – LiCl - and temsirolimus - CCI-779); (iii) Endoplasmic reticulum (ER) stress response, (taroursodeoxycholic acid - TUDCA), also known to have multiple actions in the cells, and (iv) the energy status in the cell (creatine). Chronic treatment of CMVMJD mice with 17-DMAG resulted in a delay in progression of the motor coordination deficits and a reduction in neuropathology. While we observed limited induction of heat-shock proteins with treatment, we found evidence that 17-DMAG could be acting through autophagy, as levels of LC3-II and beclin-1 were induced in the brain of treated animals. This resulted in decreased levels of the mutant ataxin-3 (ATXN3) and reduced intranuclear aggregation of this protein. To further test autophagy induction as potential therapeutic approach for MJD, we tested LiCl alone and a combination of LiCl with CCI-779 (m-TOR independent and dependent inducers, respectively). In spite of activating autophagy, as suggested by the increased levels of Beclin-1, Atg7, and LC3- II, and a reduction in the p62 protein levels, lithium had no overall beneficial effects in MJD model concerning motor performance. Furthermore, the combined therapy using LiCl and CCI-779 proved to be deleterious to both WT and transgenic animals, failing to rescue their neurological symptoms in the first and exerting in neurotoxic effects in the latter. TUDCA is an orally bioavailable and BBB permeable bile acid with known neuroprotective action, through its anti-amyloidogenic and chemical chaperone activities and its ability to modulate apoptotic pathways. In addition, TUDCA is FDA-approved for chronic use in humans to treat liver disorders, but has also been shown to be beneficial in several models of different neurodegenerative diseases. In the CMVMJD135 model, TUDCA delayed the onset of disease and improved the motor phenotype, including balance, motor coordination and gait parameters, and ameliorated neurological reflexes, exploratory movement deficits and muscular strength problems. Although TUDCA treatment did not reduce the ATXN3 aggregate load in the brain, it normalized the RNA levels of TNF-α, Il1β and Il10 in the brainstem and reduced the astrogliosis in the substantia nigra and brainstem. These results demonstrated the therapeutic efficacy of TUDCA supplementation for MJD, most likely mediated by its anti-inflammatory properties, making this compound an attractive for the treatment of neurodegenerative diseases, including MJD.Lastly, we tested the efficacy of creatine, another naturally-occurring compound known to balance energy status in the cell. Creatine-supplemented diet led to an overall improvement in the motor phenotype of CMVMJD135 mice, namely improved motor coordination, limb strength and gait quality, and amelioration of some neurological parameters such as tremors and abnormal reflexes. Remarkably, chronic creatine treatment delayed the onset of several symptoms and, in some cases, completely abolished their appearance, supporting creatine supplementation as a useful strategy to slow the progression of MJD. In summary in this work we validated a new MJD mouse model for use in preclinical trials as a powerful tool to study simultaneously pathogenic mechanisms associated with the disease and to develop novel therapeutic strategies. Furthermore, we found that Hsp90 inhibitors, TUDCA and creatine are relevant therapeutic candidates for MJD, some of which could be advanced to clinical trials in MJD patients.A doença de Machado Joseph (DMJ) pertence ao grupo de nove doenças poliglutaminas em que o gene causador da doença possui uma expansão anómala do codão CAG. A presença desta expansão conduz ao aparecimento de espécies proteicas com conformação incorreta, agregados proteicos, disfunção neuronal e consequentemente, morte celular. Para além da expansão, todos os genes associados a este grupo de doenças possuem expressão ubíqua apesar de só afetarem subconjuntos específicos de neurónios. Atualmente, não existe tratamento eficaz para a DMJ. Uma das razões pelas quais há um atraso no desenvolvimento de alvos terapêuticos está relacionada com o conhecimento incompleto do(s) mecanismo(s) de patogénese associados à DMJ. Assim sendo, o objetivo deste estudo centrou-se na procura de novas terapias, usando uma abordagem baseada em hipóteses, que pode também permitir o estudo dos mecanismo(s) moleculares da doença. Para tal, foi usado o modelo de ratinho CMVMJD135 que recapitula a doença humana a vários níveis, nomeadamente fenotípico e neuropatológico. Testámos a eficácia de diversos compostos com alvos terapêuticos relacionados com diferentes funções celulares: (i) níveis de chaperones moleculares (inibidor de Hsp90 – 17-dimetil aminoetillamino 17- demetoxigeldanamicina - 17-DMAG); (ii) autofagia (cloreto de lítio e combinação de cloreto de lítico –LiCL e temsirolimus - CCI-779); (iii) resposta ao stress do retículo endoplasmático (ácido tauroursodesoxicólico – TUDCA), com múltiplas acções celulares, e (iv) estado energético celular (creatina). O tratamento crónico com 17-DMAG no ratinho CMVMJD135 resultou num atraso da progressão da descoordenação motora e redução da neuropatologia. Apesar de ter sido observada uma indução limitada das chaperonas moleculares, este tratamento poderá ter atuado a nível da autofagia na medida em que houve indução dos níveis de LC3-II e beclina-1 no cérebro de animais tratados. Esta indução resultou na diminuição dos níveis de ataxina-3 (ATXN3) e uma redução da agregação intranuclear desta proteína. Para testar mais aprofundadamente a indução da autofagia como potencial terapia para a DMJ, foi testado LiCl isoladamente e em combinação com CCI-779 (atuando de forma independente e dependente da sinalização mTOR, respectivamente). Apesar da ativação da autofagia, sugerida pelos níveis aumentados de beclina-1, Atg7, LC3-II e redução dos níveis proteicos de p62, o tratamento do modelo DMJ com lítio foi de uma forma geral ineficaz ao nível do desempenho motor. Além disso, a terapia combinada de LiCl e CCI-779 revelou-se prejudicial em ambos os ratinhos WT e transgénicos, falhando na recuperação de sintomas neurológicos e sendo neurotóxico. O TUDCA é um ácido biliar com administração oral e permeável à BBB com funções neuroprotectoras conhecidas, através da sua capacidade anti-amiloidogénica, actividade de chaperona química e de modulação de vias apoptóticas. Adicionalmente, o TUDCA é um composto aprovado pela FDA para administração crónica no tratamento de doenças hepáticas, sendo também benéfico em diversos modelos de doenças neurodegenerativas. No modelo CMVMJD135, o tratamento com TUDCA atrasou o início da doença e melhorou o fenótipo motor incluindo equilíbrio, coordenação motora, e parâmetros da marcha, reduziu os reflexos neurológicos anómalos, melhorou os défices no movimento exploratório e força muscular. Apesar de o tratamento com este composto não ter reduzido os níveis de agregação da proteína ATXN3, foi eficaz na redução dos níveis de expressão de TNF-α, Il1β e Il10, tendo também reduzido a astrogliose no tronco cerebral e substantia nigra. Estes resultados demonstram a eficácia terapêutica da suplementação com TUDCA na DMJ, muito provavelmente através das suas capacidades anti-inflamatórias, o que torna este composto atrativo no tratamento de doenças neurodegenerativas, incluindo a DMJ. Por fim foi testada a creatina, outro composto natural reconhecido pela eficácia em restabelecer o equilíbrio energético na célula. A dieta suplementada com creatina conduziu a uma melhoria geral no fenótipo motor do ratinho CMVMJD135; nomeadamente, melhorou a coordenação motora, força nos membros e qualidade da marcha. Também teve impacto positivo em parâmetros neurológicos como os tremores e reflexos anormais. Surpreendentemente, o tratamento crónico com creatina atrasou o início do aparecimento de diversos sintomas e até aboliu a presença de alguns, o que reinforça a suplementação com creatina como uma estratégica eficaz no atraso na progressão de DMJ. Em conclusão, este estudo validou o novo modelo DMJ de ratinho para ensaios pré-clínicos como uma ferramenta valiosa para estudar simultaneamente mecanismos patogénicos associado à doença e para desenvolver novas estratégias terapêuticas. Além disso foi demonstrado que inibidores da Hsp90, TUDCA e creatina são candidatos terapêuticos relevantes para a DMJ, alguns dos quais com possibilidade de avançarem para ensaios clínicos em doentes.Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/78388/201

Recent advances in the manipulation of murine gene expression and its utility for the study of human neurological disease

Transgenic mouse models have vastly contributed to our knowledge of the genetic and molecular pathways underlying the pathogenesis of neurological disorders that affect millions of people worldwide. Not only they have allow the generation of disease models mimicking the human pathological state but they have also permit the exploration of the pathological role of specific genes through the generation of knock-out and knock-in models. Classical constitutive transgenic mice have several limitations however, due to behavioral adaptation process occurring and conditional mouse models are time-consuming and often lack of extensive spatial ortemporal control of gene manipulation. These limitations could be overcome by means of innovative methods that are now available such as RNAi, viral vectors and large cloning DNA vectors. These tools have been extensively used for the generation of mouse models and are characterized by the superior control of transgene expression that has proved invaluable in the assessment of novel treatments for neurological diseases and to further investigate the molecular processes underlying the etiopathology of neurological disorders. Furthermore, in association with classical transgenic mouse models, they have allowed the validation of innovative therapeutic strategies for the treatment of human neurological disorders. This review describes how these tools have overcome the limitations of classical transgenic mouse models and how they have been of value for the study of human neurological diseases

Huntington’s disease in Finland. Epidemiologic, genetic and clinical studies

Huntington’s disease (HD) is a lethal, dominantly inherited neurodegenerative disorder reported to be unusually rare in Finland. The overall HD prevalence and the proportion of late-onset cases (LOHD) are increasing in many populations. The characteristics of LOHD are nevertheless poorly understood. Information on neurological comorbidity in patients with HD is also scarce. These retrospective studies analyzed a national Finnish HD cohort in the time frame 1987-2010 by searching national registries and archives. Data was extracted from medical records. Population genotypes were obtained from the 1000 Genomes project. The prevalence of HD in Finland was found to be 2.12/100,000, or over four times more common than reported previously. Nonetheless, HD is more uncommon than in other Western European countries. The national cohort of 207 patients included 52 (25%) patients with LOHD; they had poorer motor status at the time of diagnosis than patients with mid-age onset, possibly because of the diagnostic delay. No other differences were detected between these groups. Interestingly, only one individual (0.5% of all HD patients in Finland) with juvenile-onset HD was identified. The length of the affected CAG repeat or its intergenerational stability did not differ from those reported in other populations. However, the high risk chromosome 4 haplogroup A was relatively uncommon in the Finnish general population (39.2%), possibly partly explaining the relative rarity of HD in Finland. Patients with adult-onset HD had epilepsy and strokes as often as reported in the general population. HD patients were, however, at an increased risk of suffering subdural haematomas.Huntingtonin tauti Suomessa. Epidemiologisia, perinnöllisyystieteellisiä ja kliinisiä tutkimuksia. Huntingtonin tauti (HD) on autosomaalisesti vallitsevasti periytyvä, kuolemaan johtava hermoston rappeumasairaus. Taudin on todettu olevan poikkeuksellisen harvinainen Suomessa. Monissa väestöissä HD:n esiintyvyyden sekä myöhäisiällä alkavan HD:n (LOHD) osuuden on havaittu lisääntyneen. LOHD:n ominaispiirteet tunnetaan kuitenkin huonosti. Myös HD-potilaiden neurologisesta oheissairastavuudesta on käytettävissä vain hyvin vähän tietoja. Näissä takautuvissa tutkimuksissa analysoitiin kansallinen suomalainen HD-potilaiden kohortti vuosilta 1987-2010. Potilaat tunnistettiin kansallisista rekistereistä sekä tietyistä arkistoista. Tutkimustiedot kerättiin sairauskertomuksista. Väestön genotyyppitiedot saatiin 1000 Genomes –projektista. Huntingtonin taudin vallitsevuuden (2,12/100’000) havaittiin olevan Suomessa yli nelinkertainen aiempaan tutkimustietoon nähden. Silti HD on selvästi harvinaisempi Suomessa kuin muissa läntisen Euroopan maissa. Kansallisesta 207 HD-potilaan kohortista 52 (25%) oli LOHD-potilaita ja heidän motoriset oireensa ja löydöksensä olivat diagnoosinteon hetkellä vaikeampia kuin aiemmalla aikuisiällä sairastuneiden, mikä mahdollisesti johtui diagnoosien viivästymisestä LOHD-potilailla. Muuten näiden ryhmien välillä ei havaittu eroja. Yllättäen löysimme vain yhden (0,5%) potilaan, jolla HD alkoi nuoruusiällä. Tautialleelin CAG-toistojakson pituus tai sen sukupolvien välinen vakaus eivät eronneet muissa väestöissä raportoiduista. Kromosomin 4 korkean riskin haploryhmä A:n havaittiin kuitenkin olevan suomalaisessa väestössä verrattain harvinainen (39,2%), mikä saattaa osittain selittää HD:n suhteellista harvinaisuutta Suomessa. Aikuisiällä HD-diagnoosin saaneilla potilailla oli epilepsiaa ja aivoverenkiertohäiriöitä samassa määrin kuin valtaväestöllä. Kovakalvon alaisten verenvuotojen riski havaittiin HD-potilailla suurentuneeksi.Siirretty Doriast

Synaptic dysfunction in the perirhinal cortex of the R6/1 mouse model of Huntington's disease

Huntington's disease (HD) is a fatal brain disorder characterised by a progressive motor, psychiatric and cognitive decline, often manifesting in midlife. Since the identification of the gene responsible for HD and the development of predictive testing, it has become apparent that asymptomatic patients often exhibit early cognitive deficits. In particular, an impairment in recognition memory that is evident prior to the onset of classical symptoms and trank cell death. The perirhinal cortex is believed to be involved in processing aspects of recognition memory, specifically the discrimination between novel and familiar cues. Evidence suggests that activity-dependent decrements in neuronal activity within the perirhinal region could underlie this cognitive process and that the synaptic mechanism may be one of long-term depression (LTD). In the R6/1 mouse model of HD, synaptic plasticity is progressively altered in the perirhinal cortex in vitro. LTD expression is enhanced at one month, but then declines and is absent by 7 months of age. Dopamine is shown here to be a neuromodulator at normal perirhinal cortical synapses. Fluorescent immunohistochemical techniques are used to demonstrate altered dopamine receptor expression in the R6/1 perirhinal cortex that correlates with the aberrant synaptic plasticity. By exposing normal mouse slices to a D2 dopamine receptor antagonist, it is possible to recapitulate the R6/1 perirhinal synaptic phenotype. Importantly, the alteration of synaptic fiinction and loss of LTD in R6/1 mice can be restored by applying an agonist of dopamine receptors to brain slices. These data suggest that impaired perirhinal LTD may contribute to poor recognition memory and that the defect is at the level of the dopaminergic system. Moreover, dopaminergic therapy, targeted to the cortex, may be beneficial in HD