Anabela Silva-Fernandes

Evidence for abnormal early development in a mouse model of Rett syndrome

Rett syndrome (RTT) is a neurodevelopmental disorder that affects mainly females, associated in most cases to mutations in the MECP2 gene. After an apparently normal prenatal and perinatal period, patients display an arrest in growth and in psychomotor development, with autistic behaviour, hand stereotypies and mental retardation. Despite this classical description, researchers always questioned whether RTT patients did have subtle manifestations soon after birth. This issue was recently brought to light by several studies using different approaches that revealed abnormalities in the early development of RTT patients. Our hypothesis was that, in the mouse models of RTT as in patients, early neurodevelopment might be abnormal, but in a subtle manner, given the first descriptions of these models as initially normal. To address this issue, we performed a postnatal neurodevelopmental study in the Mecp2tm1.1Bird mouse. These animals are born healthy, and overt symptoms start to establish a few weeks later, including features of neurological disorder (tremors, hind limb clasping, weight loss). Different maturational parameters and neurological reflexes were analysed in the pre‐weaning period in the Mecp2‐mutant mice and compared to wild‐type littermate controls. We found subtle but significant sex‐dependent differences between mutant and wild‐type animals, namely a delay in the acquisition of the surface and postural reflexes, and impaired growth maturation. The mutant animals also show altered negative geotaxis and wire suspension behaviours, which may be early manifestations of later neurological symptoms. In the post‐weaning period the juvenile mice presented hypoactivity that was probably the result of motor impairments. The early anomalies identified in this model of RTT mimic the early motor abnormalities reported in the RTT patients, making this a good model for the study of the early disease process.

Chronic Treatment with 17-DMAG Improves Balance and Coordination in A New Mouse Model of Machado-Joseph Disease

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease currently with no treatment. We describe a novel mouse model of MJD which expresses mutant human ataxin-3 at near endogenous levels and manifests MJD-like motor symptoms that appear gradually and progress over time. CMVMJD135 mice show ataxin-3 intranuclear inclusions in the CNS and neurodegenerative changes in key disease regions, such as the pontine and dentate nuclei. Hsp90 inhibition has shown promising outcomes in some neurodegenerative diseases, but nothing is known about its effects in MJD. Chronic treatment of CMVMJD mice with Hsp90 inhibitor 17-DMAG resulted in a delay in the progression of their motor coordination deficits and, at 22 and 24 weeks of age, was able to rescue the uncoordination phenotype to wild-type levels; in parallel, a reduction in neuropathology was observed in treated animals. We observed limited induction of heat-shock proteins with treatment, but found evidence that 17-DMAG may be acting through autophagy, as LC3-II (both at mRNA and protein levels) and beclin-1 were induced in the brain of treated animals. This resulted in decreased levels of the mutant ataxin-3 and reduced intranuclear aggregation of this protein. Our data validate this novel mouse model as a relevant tool for the study of MJD pathogenesis and for pre-clinical studies, and show that Hsp90 inhibition is a promising therapeutic strategy for MJD.

Motor uncoordination and neuropathology in a transgenic mouse model of Machado-Joseph disease lacking intranuclear inclusions and ataxin-3 cleavage products

Machado-Joseph disease (MJD) is a late-onset neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in the ataxin-3 protein. We generated two transgenic mouse lineages expressing the expanded human ataxin-3 under the control of the CMV promoter: CMVMJD83 and CMVMJD94, carrying Q83 and Q94 stretches, respectively. Behavioral analysis revealed that the CMVMJD94 transgenic mice developed motor uncoordination, intergenerational instability of the CAG repeat and a tissue-specific increase in the somatic mosaicism of the repeat with aging. Histopathological analysis of MJD mice at early and late stages of the disease revealed neuronal atrophy and astrogliosis in several brain regions; however, we found no signs of microglial activation or neuroinflammatory response prior to the appearance of an overt phenotype. In our model, the appearance of MJD-like symptoms was also not associated with the presence of ataxin-3 cleavage products or intranuclear aggregates. We propose the transgenic CMVMJD94 mice as a useful model to study the early stages in the pathogenesis of MJD and to explore the molecular mechanisms involved in CAG repeat instability.

The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Monoamine deficits in the brain of methyl-CpG binding protein 2 null mice suggest the involvement of the cerebral cortex in early stages of Rett syndrome

Rett syndrome is a neurodevelopmental disorder caused by mutations in the methyl-CpG binding protein 2 gene (MECP2). Several neural systems are affected in Rett, resulting in an autonomic dysfunction, a movement disorder with characteristic loss of locomotor abilities and profound cognitive impairments. A deregulation of monoamines has been detected in the brain and cerebrospinal fluid of both Rett patients and a Rett syndrome murine model, the Mecp2 knock-out mouse. Our goal was to characterize the onset and progression of motor dysfunction in Mecp2(tm1.1Bird) knock-out mice and the possible neurochemical alterations in different brain regions potentially playing a role in Rett-like pathophysiology, at two different time-points, at weaning (3 weeks old) and in young adults when overt symptoms are observed (8 weeks old). Our results revealed significant age- and region-dependent impairments in these modulatory neurotransmitter systems that correspond well with the motor phenotype observed in these mice. At 3 weeks of age, male Mecp2 knock-out mice exhibited ataxia and delayed motor initiation. At this stage, noradrenergic and serotonergic transmission was mainly altered in the prefrontal and motor cortices, whereas during disease progression the neurochemical changes were also observed in hippocampus and cerebellum. Our data suggest that the deregulation of norepinephrine and serotonin systems in brain regions that participate in motor control are involved in the pathophysiology of Rett syndrome motor phenotypes. Moreover, we highlight the contribution of cortical regions along with the brainstem to be in the origin of the pathology and the role of hippocampus and cerebellum in the progression of the disease rather than in its establishment.

Serotonergic signalling suppresses ataxin 3 aggregation and neurotoxicity in animal models of Machado-Joseph disease

Polyglutamine diseases are a class of dominantly inherited neurodegenerative disorders for which there is no effective treatment. Here we provide evidence that activation of serotonergic signalling is beneficial in animal models of Machado-Joseph disease. We identified citalopram, a selective serotonin reuptake inhibitor, in a small molecule screen of FDA-approved drugs that rescued neuronal dysfunction and reduced aggregation using a Caenorhabditis elegans model of mutant ataxin 3-induced neurotoxicity. MOD-5, the C. elegans orthologue of the serotonin transporter and cellular target of citalopram, and the serotonin receptors SER-1 and SER-4 were strong genetic modifiers of ataxin 3 neurotoxicity and necessary for therapeutic efficacy. Moreover, chronic treatment of CMVMJD135 mice with citalopram significantly reduced ataxin 3 neuronal inclusions and astrogliosis, rescued diminished body weight and strikingly ameliorated motor symptoms. These results suggest that small molecule modulation of serotonergic signalling represents a promising therapeutic target for Machado-Joseph disease.

Patterns of Mitochondrial DNA Damage in Blood and Brain Tissues of a Transgenic Mouse Model of Machado-Joseph Disease

Background: Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar ataxia caused by a CAG tract expansions in the ATXN3 gene. Patterns of mitochondrial damage associated with pathological findings of brain tissues could provide molecular biomarkers of this disorder. Objective: The potential of mitochondrial DNA (mtDNA) damage as a biomarker of MJD progression was investigated using a transgenic mouse model. Methods: DNA was obtained from affected (pontine nuclei) and nonaffected tissues (hippocampus and blood) of transgenic animals of three distinct age groups: 8 weeks, before onset of the phenotype; 16 weeks, at onset, and 24 weeks, at well-established phenotype. Wild-type littermate mice, serving as controls, were analyzed for the same tissues and age groups. mtDNA damage was studied by fluorescence-based quantitative PCR in 84 transgenic and 93 wild-type samples. Results: A clear pattern of decrease in mtDNA copy number with age and accumulation of 3,867-bp deletions at the initial stages (both being more pronounced in transgenic mice) was observed. Pontine nuclei, the affected tissue in transgenic mice, displayed 1.5 times less copies of mtDNA than nonaffected brain tissue hippocampus (odds ratio = 1.21). Pontine nuclei displayed the highest percentage of mtDNA deletions (6.05% more in transgenic mice). Conclusion: These results suggest that mtDNA damage is related to the initiation of the phenotype in transgenic mice; mtDNA 3,867-bp deletions may be a biomarker of the initial stages of the disease.

Inactivation of PNKP by Mutant ATXN3 Triggers Apoptosis by Activating the DNA Damage-Response Pathway in SCA3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3’-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

Mutational analysis of MSX1 and PAX9 genes in Portuguese families with maxillary lateral incisor agenesis

The observation that certain patterns of tooth agenesis occur more frequently in individuals of the same family may suggest the existence of predisposing genetic factors. The aim of this study was to search for mutations in the PAX9 and MSX1 genes and to investigate their potential association with the maxillary lateral incisor agenesis (MLIA) phenotype in 12 Portuguese families, a total of 52 individuals, 12 probands and 40 relatives (eight of which had MLIA). Twenty-three of the subjects were male and 29 female with an age range of 10-75 years. The control group comprised random DNA samples of 91 Portuguese individuals. Nucleotide alterations were not detected in the coding regions of the MSX1 gene, analysed by single-strand conformation polymorphism and sequencing; in the PAX9 gene, a polymorphism was found that led to transition of G718 to C, implying a change of alanine 240 for proline. However, the differences in the frequencies of the PAX9 gene polymorphism between the probands (67 per cent) and the control population (56 per cent carrying the c allele) were not statistically significant as determined by chi-square test, and the polymorphism did not clearly segregate with the trait in the families. Aggregating the available data, there does not seem to exist a clear association between the alanine 240 for proline variant in the PAX9 gene and the MLIA phenotype. Further studies are required to clarify the basic genetics of MLIA.

Combined therapy with m-TOR-dependent and -independent autophagy inducers causes neurotoxicity in a mouse model of Machado–Joseph disease

A major pathological hallmark in several neurodegenerative disorders, like polyglutamine disorders (polyQ), including Machado-Joseph disease (MJD), is the formation of protein aggregates. MJD is caused by a CAG repeat expansion in the ATXN3 gene, resulting in an abnormal protein, which is prone to misfolding and forms cytoplasmic and nuclear aggregates within neurons, ultimately inducing neurodegeneration. Treatment of proteinopathies with drugs that up-regulate autophagy has shown promising results in models of polyQ diseases. Temsirolimus (CCI-779) inhibits the mammalian target of rapamycin (m-TOR), while lithium chloride (LiCl) acts by inhibiting inositol monophosphatase, both being able to induce autophagy. We have previously shown that chronic treatment with LiCl (10.4 mg/kg) had limited effects in a transgenic MJD mouse model. Also, others have shown that CCI-779 had mild positive effects in a different mouse model of the disease. It has been suggested that the combination of mTOR-dependent and -independent autophagy inducers could be a more effective therapeutic approach. To further explore this avenue toward therapy, we treated CMVMJD135 transgenic mice with a conjugation of CCI-779 and LiCl, both at concentrations known to induce autophagy and not to be toxic. Surprisingly, this combined treatment proved to be deleterious to both wild-type (wt) and transgenic animals, failing to rescue their neurological symptoms and actually exerting neurotoxic effects. These results highlight the possible dangers of manipulating autophagy in the nervous system and suggest that a better understanding of the potential disruption in the autophagy pathway in MJD is required before successful long-term autophagy modulating therapies can be developed.