Marta Ruiz

The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases

Astrocyte reactivity is a hallmark of neurodegenerative diseases (ND), but its effects on disease outcomes remain highly debated. Elucidation of the signaling cascades inducing reactivity in astrocytes during ND would help characterize the function of these cells and identify novel molecular targets to modulate disease progression. The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is associated with reactive astrocytes in models of acute injury, but it is unknown whether this pathway is directly responsible for astrocyte reactivity in progressive pathological conditions such as ND. In this study, we examined whether the JAK/STAT3 pathway promotes astrocyte reactivity in several animal models of ND. The JAK/STAT3 pathway was activated in reactive astrocytes in two transgenic mouse models of Alzheimers disease and in a mouse and a nonhuman primate lentiviral vector-based model of Huntingtons disease (HD). To determine whether this cascade was instrumental for astrocyte reactivity, we used a lentiviral vector that specifically targets astrocytes in vivo to overexpress the endogenous inhibitor of the JAK/STAT3 pathway [suppressor of cytokine signaling 3 (SOCS3)]. SOCS3 significantly inhibited this pathway in astrocytes, prevented astrocyte reactivity, and decreased microglial activation in models of both diseases. Inhibition of the JAK/STAT3 pathway within reactive astrocytes also increased the number of huntingtin aggregates, a neuropathological hallmark of HD, but did not influence neuronal death. Our data demonstrate that the JAK/STAT3 pathway is a common mediator of astrocyte reactivity that is highly conserved between disease states, species, and brain regions. This universal signaling cascade represents a potent target to study the role of reactive astrocytes in ND.

Early transcriptional changes linked to naturally occurring Huntington's disease mutations in neural derivatives of human embryonic stem cells

Huntingtons disease (HD) is characterized by a late clinical onset despite ubiquitous expression of the mutant gene at all developmental stages. How mutant huntingtin impacts on signalling pathways in the pre-symptomatic period has remained essentially unexplored in humans due to a lack of appropriate models. Using multiple human embryonic stem cell lines derived from blastocysts diagnosed as carrying the mutant huntingtin gene by pre-implantation genetic diagnosis, we explored early developmental changes in gene expression using differential transcriptomics, combined with gain and loss of function strategies. We demonstrated a down-regulation of the HTT gene itself in HD neural cells and identified three genes, the expression of which differs significantly in HD cells when compared with wild-type controls, namely CHCHD2, TRIM4 and PKIB. Similar dysregulation had been observed previously for CHCDH2 and TRIM4 in blood cells from patients. CHCHD2 is involved in mitochondrial function and PKIB in protein kinase A-dependent pathway regulation, which suggests that these functions may be precociously impacted in HD.

Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells.

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disorder resulting from polyglutamine expansion in the huntingtin (HTT) protein and for which there is no cure. Although suppression of both wild type and mutant HTT expression by RNA interference is a promising therapeutic strategy, a selective silencing of mutant HTT represents the safest approach preserving WT HTT expression and functions. We developed small hairpin RNAs (shRNAs) targeting single nucleotide polymorphisms (SNP) present in the HTT gene to selectively target the disease HTT isoform. Most of these shRNAs silenced, efficiently and selectively, mutant HTT in vitro. Lentiviral-mediated infection with the shRNAs led to selective degradation of mutant HTT mRNA and prevented the apparition of neuropathology in HD rats striatum expressing mutant HTT containing the various SNPs. In transgenic BACHD mice, the mutant HTT allele was also silenced by this approach, further demonstrating the potential for allele-specific silencing. Finally, the allele-specific silencing of mutant HTT in human embryonic stem cells was accompanied by functional recovery of the vesicular transport of BDNF along microtubules. These findings provide evidence of the therapeutic potential of allele-specific RNA interference for HD.

Viral-mediated overexpression of mutant huntingtin to model HD in various species

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expansion of CAG repeats in the huntingtin (Htt) gene. Despite intensive efforts devoted to investigating the mechanisms of its pathogenesis, effective treatments for this devastating disease remain unavailable. The lack of suitable models recapitulating the entire spectrum of the degenerative process has severely hindered the identification and validation of therapeutic strategies. The discovery that the degeneration in HD is caused by a mutation in a single gene has offered new opportunities to develop experimental models of HD, ranging from in vitro models to transgenic primates. However, recent advances in viral-vector technology provide promising alternatives based on the direct transfer of genes to selected sub-regions of the brain. Rodent studies have shown that overexpression of mutant human Htt in the striatum using adeno-associated virus or lentivirus vectors induces progressive neurodegeneration, which resembles that seen in HD. This article highlights progress made in modeling HD using viral vector gene transfer. We describe data obtained with of this highly flexible approach for the targeted overexpression of a disease-causing gene. The ability to deliver mutant Htt to specific tissues has opened pathological processes to experimental analysis and allowed targeted therapeutic development in rodent and primate pre-clinical models.

Abnormalities of motor function, transcription and cerebellar structure in mouse models of THAP1 dystonia

DYT6 dystonia is caused by mutations in THAP1 [Thanatos-associated (THAP) domain-containing apoptosis-associated protein] and is autosomal dominant and partially penetrant. Like other genetic primary dystonias, DYT6 patients have no characteristic neuropathology, and mechanisms by which mutations in THAP1 cause dystonia are unknown. Thap1 is a zinc-finger transcription factor, and most pathogenic THAP1 mutations are missense and are located in the DNA-binding domain. There are also nonsense mutations, which act as the equivalent of a null allele because they result in the generation of small mRNA species that are likely rapidly degraded via nonsense-mediated decay. The function of Thap1 in neurons is unknown, but there is a unique, neuronal 50-kDa Thap1 species, and Thap1 levels are auto-regulated on the mRNA level. Herein, we present the first characterization of two mouse models of DYT6, including a pathogenic knockin mutation, C54Y and a null mutation. Alterations in motor behaviors, transcription and brain structure are demonstrated. The projection neurons of the deep cerebellar nuclei are especially altered. Abnormalities vary according to genotype, sex, age and/or brain region, but importantly, overlap with those of other dystonia mouse models. These data highlight the similarities and differences in age- and cell-specific effects of a Thap1 mutation, indicating that the pathophysiology of THAP1 mutations should be assayed at multiple ages and neuronal types and support the notion of final common pathways in the pathophysiology of dystonia arising from disparate mutations.

Non cell-autonomous role of DCC in the guidance of the corticospinal tract at the midline

DCC, a NETRIN-1 receptor, is considered as a cell-autonomous regulator for midline guidance of many commissural populations in the central nervous system. The corticospinal tract (CST), the principal motor pathway for voluntary movements, crosses the anatomic midline at the pyramidal decussation. CST fails to cross the midline in Kanga mice expressing a truncated DCC protein. Humans with heterozygous DCC mutations have congenital mirror movements (CMM). As CMM has been associated, in some cases, with malformations of the pyramidal decussation, DCC might also be involved in this process in human. Here, we investigated the role of DCC in CST midline crossing both in human and mice. First, we demonstrate by multimodal approaches, that patients with CMM due to DCC mutations have an increased proportion of ipsilateral CST projections. Second, we show that in contrast to Kanga mice, the anatomy of the CST is not altered in mice with a deletion of DCC in the CST. Altogether, these results indicate that DCC controls CST midline crossing in both humans and mice, and that this process is non cell-autonomous in mice. Our data unravel a new level of complexity in the role of DCC in CST guidance at the midline.

Tetraploidy Enhances Boron-Excess Tolerance in Carrizo Citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.)

Tetraploidy modifies root anatomy which may lead to differentiated capacity to uptake and transport mineral elements. This work provides insights into physiological and molecular characters involved in boron (B) toxicity responses in diploid (2x) and tetraploid (4x) plants of Carrizo citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.), a widely used citrus rootstock. With B excess, 2x plants accumulated more B in leaves than 4x plants, which accounted for their higher B uptake and root-to-shoot transport rates. Ploidy did not modify the expression of membrane transporters NIP5 and BOR1 in roots. The cellular allocation of B excess differed between ploidy levels in the soluble fraction, which was lower in 4x leaves, while cell wall-linked B was similar in 2x and 4x genotypes. This correlates with the increased damage and stunted growth recorded in the 2x plants. The 4x roots were found to have fewer root tips, shorter specific root length, longer diameter, thicker exodermis and earlier tissue maturation in root tips, where the Casparian strip was detected at a shorter distance from the root apex than in the 2x roots. The results presented herein suggest that the root anatomical characters of the 4x plants play a key role in their lower B uptake capacity and root-to-shoot transport. Highlights Tetraploidy enhances B excess tolerance in citrange Carrizo Expression of NIP5 and BOR1 transporters and cell wall-bounded B are similar between ploidies B tolerance is attributed to root anatomical modifications induced by genome duplication The rootstock 4x citrange carrizo may prevent citrus trees from B excess.

Behavioral and transcriptome alterations in male and female mice with postnatal deletion of TrkB in dorsal striatal medium spiny neurons

BackgroundThe high affinity tyrosine kinase receptor, TrkB, is the primary receptor for brain derived neurotrophic factor (BDNF) and plays an important role in development, maintenance and plasticity of the striatal output medium size spiny neuron. The striatal BDNF/TrkB system is thereby implicated in many physiologic and pathophysiologic processes, the latter including mood disorders, addiction, and Huntington’s disease. We crossed a mouse harboring a transgene directing cre-recombinase expression primarily to postnatal, dorsal striatal medium spiny neurons, to a mouse containing a floxed TrkB allele (fB) mouse designed for deletion of TrkB to determine its role in the adult striatum.ResultsWe found that there were sexually dimorphic alterations in behaviors in response to stressful situations and drugs of abuse. Significant sex and/or genotype differences were found in the forced swim test of depression-like behaviors, anxiety-like behaviors on the elevated plus maze, and cocaine conditioned reward. Microarray analysis of dorsal striatum revealed significant dysregulation in individual and groups of genes that may contribute to the observed behavioral responses and in some cases, represent previously unidentified downstream targets of TrkB.ConclusionsThe data point to a set of behaviors and changes in gene expression following postnatal deletion of TrkB in the dorsal striatum distinct from those in other brain regions.

Dystonia type 6 gene product Thap1: identification of a 50 kDa DNA-binding species in neuronal nuclear fractions

Mutations in THAP1 result in dystonia type 6, with partial penetrance and variable phenotype. The goal of this study was to examine the nature and expression pattern of the protein product(s) of the Thap1 transcription factor (DYT6 gene) in mouse neurons, and to study the regional and developmental distribution, and subcellular localization of Thap1 protein. The goal was accomplished via overexpression and knock-down of Thap1 in the HEK293T cell line and in mouse striatal primary cultures and western blotting of embryonic Thap1-null tissue. The endogenous and transduced Thap1 isoforms were characterized using three different commercially available anti-Thap1 antibodies and validated by immunoprecipitation and DNA oligonucleotide affinity chromatography. We identified multiple, novel Thap1 species of apparent Mr 32 kDa, 47 kDa, and 50–52 kDa in vitro and in vivo, and verified the previously identified species at 29–30 kDa in neurons. The Thap1 species at the 50 kDa size range was exclusively detected in murine brain and testes and were located in the nuclear compartment. Thus, in addition to the predicted 25 kDa apparent Mr, we identified Thap1 species with greater apparent Mr that we speculate may be a result of posttranslational modifications. The neural localization of the 50 kDa species and its nuclear compartmentalization suggests that these may be key Thap1 species controlling neuronal gene transcription. Dysfunction of the neuronal 50 kDa species may therefore be implicated in the pathogenesis of DYT6.

Tetraploidy enhances the ability to exclude chloride from leaves in carrizo citrange seedlings

Tetraploid citrus seedlings are more tolerant to salt stress than diploid genotypes. To provide insight into the causes of differences in salt tolerance due to ploidy and thus to better understand Cl- exclusion mechanisms in citrus, diploid and tetraploid seedlings of Carrizo citrange (CC) were grown at 0 (control) and 40mM NaCl (salt-treated) medium for 20 days. Chloride uptake and root-to-shoot translocation rates were on average 1.4-fold higher in diploid than in tetraploid salt-treated plants, which resulted in a greater (1.6-fold) Cl- build up in the leaves of the former. Root hydraulic conductance and leaf transpiration rate were 58% and 17% lower, respectively, in tetraploid than in diploid control plants. Differences remained after salt treatment which reduced these parameters by 30-40% in both genotypes. Morphology of the root system was significantly influenced by ploidy. Tetraploid roots were less branched and with lower number of root tips than those of diploid plants. The cross-section diameter and area were lower in the diploid, and consequently specific root length was higher (1.7-fold) than in tetraploid plants. The exodermis in sections close to the root apex was broader and with higher deposition of suberin in cell walls in the tetraploid than in the diploid genotype. Net CO2 assimilation rate in tetraploid salt-treated seedlings was 1.5-fold higher than in diploid salt-treated plants, likely due to the loss of photosynthetic capacity of diploid plants induced by Cl- toxicity. Leaf damage was much higher, in terms of burnt area and defoliation, in diploid than in tetraploid salt-treated plants (8- and 6-fold, respectively). Salt treatment significantly reduced (37%) the dry weight of the diploid plants, but did not affect the tetraploids. In conclusion, tetraploid CC plants appear more tolerant to salinization and this effect seems mainly due to differences in morphological and histological traits of roots affecting hydraulic conductance and transpiration rate. These results may suggest that tetraploid CC used as rootstock could improve salt tolerance in citrus trees.