Thomas Ott

Transgenic mice for cGMP imaging.

Rationale: Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown. Objective: To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer–based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues. Methods and Results: Mouse lines with smooth muscle–specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22&agr; promoter fragment or by targeted integration of a Cre recombinase–activatable expression cassette driven by the cytomegalovirus early enhancer/chicken &bgr;-actin/&bgr;-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were <100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ≈3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide–induced cGMP transients with peak concentrations of ≈1 to >3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide–stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation. Conclusions: These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions or during pharmacotherapy with cGMP-elevating drugs.

Generation of a novel rodent model for DYT1 dystonia

A mutation in the coding region of the Tor1A gene, resulting in a deletion of a glutamic acid residue in the torsinA protein (∆ETorA), is the major cause of the inherited autosomal-dominant early onset torsion dystonia (DYT1). The pathophysiological consequences of this amino acid loss are still not understood. Currently available animal models for DYT1 dystonia provided important insights into the disease; however, they differ with respect to key features of torsinA associated pathology. We developed transgenic rat models harboring the full length human mutant and wildtype Tor1A gene. A complex phenotyping approach including classical behavioral tests, electrophysiology and neuropathology revealed a progressive neurological phenotype in ∆ETorA expressing rats. Furthermore, we were able to replicate key pathological features of torsinA associated pathology in a second species, such as nuclear envelope pathology, behavioral abnormalities and plasticity changes. We therefore suggest that this rat model represents an appropriate new model suitable to further investigate the pathophysiology of ∆ETorA and to test for therapeutic approaches.

Phenotype of single hepatocytes expressing an activated version of β-catenin in liver of transgenic mice

The gene CTNNB1 encoding β-catenin is mutated in about 30% of hepatocellular carcinoma, generally often combined with other genetic alterations. In transgenic mice, it has been shown that activation of β-catenin in more than 70% of all hepatocytes causes immediate proliferation leading to hepatomegaly. In this study we established a novel mouse model where β-catenin is activated only in individual, dispersed hepatocytes. Hepatocyte-specific expression of activated point-mutated β-catenin (human β-cateninS33Y) was established using the Cre/loxP system. Expression of several downstream targets of β-catenin signaling such as glutamine synthetase and several cytochrome P450 isoforms was confirmed by immunostaining. Only a minor portion of hepatocytes expressed the β-cateninS33Y transgene, which were mainly positioned as dispersed individual cells within the normal liver parenchyma. The hepatocytes with activated β-catenin did not show increased proliferation and the mice did not develop hepatomegaly. In conclusion, activated β-catenin in single hepatocytes induces a gene expression pattern in hepatocytes which is similar to that of Ctnnb1-mutated mouse liver tumors, but is apparently not sufficient to induce increased cell proliferation. Therefore, onset of proliferation seems to require concomitant activation of β-catenin in clusters of hepatocytes, suggesting a role of cell–cell communication in this process.

Screening of mutations in GNAL in sporadic dystonia patients.

GNAL mutations have been shown to cause adult‐onset isolated dystonia, a disabling movement disorder characterized by involuntary muscle contractions causing twisting and repetitive movements or abnormal postures.

Behavioral deficits and striatal DA signaling in LRRK2 p.G2019S transgenic rats: a multimodal investigation including PET neuroimaging.

BACKGROUND A major risk-factor for developing Parkinsons disease (PD) is genetic variability in leucine-rich repeat kinase 2 (LRRK2), most notably the p.G2019S mutation. Examination of the effects of this mutation is necessary to determine the etiology of PD and to guide therapeutic development. OBJECTIVE Assess the behavioral consequences of LRRK2 p.G2019S overexpression in transgenic rats as they age and test the functional integrity of the nigro-striatal dopamine system. Conduct positron emission tomography (PET) neuroimaging to compare transgenic rats with previous data from human LRRK2 mutation carriers. METHODS Rats overexpressing human LRRK2 p.G2019S were generated by BAC transgenesis and compared to non-transgenic (NT) littermates. Motor skill tests were performed at 3, 6 and 12 months-of-age. PET, performed at 12 months, assessed the density of dopamine and vesicular monoamine transporters (DAT and VMAT2, respectively) and measured dopamine synthesis, storage and availability. Brain tissue was assayed for D2, DAT, dopamine and cAMP-regulated phosphoprotein (DARPP32) and tyrosine hydroxylase (TH) expression by Western blot, and TH by immunohistochemistry. RESULTS Transgenic rats had no abnormalities in measures of striatal dopamine function at 12 months. A behavioral phenotype was present, with LRRK2 p.G2019S rats performing significantly worse on the rotarod than non-transgenic littermates (26% reduction in average running duration at 6 months), but with normal performance in other motor tests. CONCLUSIONS Neuroimaging using dopaminergic PET did not recapitulate prior studies in human LRRK2 mutation carriers. Consistently, LRRK2 p.G2019S rats do not develop overt neurodegeneration; however, they do exhibit behavioral abnormalities.

Combined occurrence of a novel TOR1A and a THAP1 mutation in primary dystonia

The ΔGAG deletion of the TOR1A gene (DYT1) is responsible for DYT1 dystonia. However, no other TOR1A mutation has been reported in the Chinese population.

Mitochondrial Defects and Neurodegeneration in Mice Overexpressing Wild Type or G399S Mutant HtrA2

The protease HtrA2 has a protective role inside mitochondria, but promotes apoptosis under stress. We previously identified the G399S HtrA2 mutation in Parkinsons disease (PD) patients and reported mitochondrial dysfunction in vitro. Mitochondrial dysfunction is a common feature of PD and related to neurodegeneration. Complete loss of HtrA2 has been shown to cause neurodegeneration in mice. However, the full impact of HtrA2 overexpression or the G399S mutation is still to be determined in vivo. Here, we report the first HtrA2 G399S transgenic mouse model. Our data suggest that the mutation has a dominant-negative effect. We also describe a toxic effect of wild-type (WT) HtrA2 overexpression. Only low overexpression of the G399S mutation allowed viable animals and we suggest that the mutant protein is likely unstable. This is accompanied by reduced mitochondrial respiratory capacity and sensitivity to apoptotic cell death. Mice overexpressing WT HtrA2 were viable, yet these animals have inhibited mitochondrial respiration and significant induction of apoptosis in the brain leading to motor dysfunction, highlighting the opposing roles of HtrA2. Our data further underscore the importance of HtrA2 as a key mediator of mitochondrial function and its fine regulatory role in cell fate. The location and abundance of HtrA2 is tightly controlled and, therefore, human mutations leading to gain- or loss of function could provide significant risk for PD-related neurodegeneration.

Mutations in CIZ1 are not a major cause for dystonia in Germany.

Primary dystonias are a clinically and genetically heterogeneous group of movement disorders, characterized by involuntary, sustained muscle contractions, frequently causing twisting and repetitive movements or abnormal postures. Whereas most cases of early-onset generalized dystonia are caused by a 3-bp deletion in TOR1A, the exact genetic contribution for the more prevalent adult-onset and localized subtypes of dystonia is unknown. Apart from THAP1, mutations in CIZ1 have been reported to be responsible for autosomal dominantly inherited cervical dystonia in a large Caucasian pedigree. We aimed to evaluate the role of CIZ1 mutations in a series of 683 patients with sporadic and familial forms of predominantly cervical dystonia from movement disorder clinics in Germany (see Supplemental Data Table S1). The CIZ1 coding sequence (ENSG00000148337/ ENST00000393608) and adjacent intronic bases were analyzed by an amplicon-based Next Generation Sequencing approach (n 5 153 patients) and Sanger sequencing (n 5 530 patients). (For Methods, Primers. and NGS quality parameters, see Supplemental Data Tables S2–S5). In the overall combined cohort, we identified mostly annotated single-nucleotide polymorphisms detected at frequencies reported in the Exom variant server database (EVS). (For a summary of variants, see Table 1).

Lack of sequence variations in THAP1 gene and THAP1-binding sites in TOR1A promoter of DYT1 patients.

Pilar Gomez-Garre, F´tima Carrillo, and Pablo Mir were supported by grants from the Ministerio de Ciencia e Innovacion de Espana (SAF2007-60700), the Instituto de Salud Carlos III (PI10/01674), the Consejeria de Innovacion, Ciencia y Empresa de la Junta de Andalucia (CVI-02526), the Consejeria de Salud de la Junta de Andalucia (PI-0377/2007, PI-0741/2010), the Sociedad Andaluza de Neurologia, and the Jaques and Gloria Gossweiler Foundation. Pilar Gomez-Garre was supported by the “Miguel Servet” program from the Instituto de Salud Carlos III.

Hsp72 protects against liver injury via attenuation of hepatocellular death, oxidative stress, and JNK signaling

BACKGROUND & AIMS Heat shock protein (Hsp) 72 is a molecular chaperone that has broad cytoprotective functions and is upregulated in response to stress. To determine its hepatic functions, we studied its expression in human liver disorders and its biological significance in newly generated transgenic animals. METHODS Double transgenic mice overexpressing Hsp72 (gene Hspa1a) under the control of a tissue-specific tetracycline-inducible system (Hsp72-LAP mice) were produced. Acute liver injury was induced by a single injection of acetaminophen (APAP). Feeding with either a methionine choline-deficient (MCD; 8 weeks) or a 3,5-diethoxycarbonyl-1,4-dihydrocollidine-supplemented diet (DDC; 12 weeks) was used to induce lipotoxic injury and Mallory-Denk body (MDB) formation, respectively. Primary hepatocytes were treated with palmitic acid. RESULTS Patients with non-alcoholic steatohepatitis and chronic hepatitis C infection displayed elevated HSP72 levels. These levels increased with the extent of hepatic inflammation and HSP72 expression was induced after treatment with either interleukin (IL)-1β or IL-6. Hsp72-LAP mice exhibited robust, hepatocyte-specific Hsp72 overexpression. Primary hepatocytes from these animals were more resistant to isolation-induced stress and Hsp72-LAP mice displayed lower levels of hepatic injury in vivo. Mice overexpressing Hsp72 had fewer APAP protein adducts and were protected from oxidative stress and APAP-/MCD-induced cell death. Hsp72-LAP mice and/or hepatocytes displayed significantly attenuated Jnk activation. Overexpression of Hsp72 did not affect steatosis or the extent of MDB formation. CONCLUSIONS Our results demonstrate that HSP72 induction occurs in human liver disease, thus, HSP72 represents an attractive therapeutic target owing to its broad hepatoprotective functions. LAY SUMMARY HSP72 constitutes a stress-inducible, protective protein. Our data demonstrate that it is upregulated in patients with chronic hepatitis C and non-alcoholic steatohepatitis. Moreover, Hsp72-overexpressing mice are protected from various forms of liver stress.