Protein-protein-interactions (PPIs) are fundamental to all cellular processes. Recently, large human interaction maps have been generated using high throughput technologies such as the yeast two hybrid system (Y2H) or affinity chromatography and mass spectrometry. However, the resulting networks are static, incomplete and not disease specific. To address these limitations, we have developed an integrated functional genomics strategy that combinesY2H interaction and gene expression studies in order to create tissue specific disease networks. A network for Huntington’s disease was generated and subsequently validated in vitro and in vivo (cell and Drosophila models). In this way, the huntingtin interaction partner CRMP1 (collapsin response mediator protein1) was identified as a novel modulator of Huntington’s disease pathogenesis. We propose that tissue specific PPI networks are valuable resources for disease research, providing essential clues to protein function. Also, they do not provide immediate clues about disease processes and modifiers.

6th German Parkinson Congress of the German Parkinson Society (DPG) Marburg, Germany, March 5–7, 2009 Published online: 24 January 2009 Springer-Verlag 2009

Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph-Disease (MJD) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG stretch in the MJD1 gene encoding a polyglutamine repeat in the respective ataxin-3-protein. In order to study the course of the disease, we generated an inducible transgenic mouse model using the “Tet-Off-System“. This system is based on two constructs: The promoter construct controls the expression of the so called tTA (Tetracycline transactivator) gene product. The binding of this protein to a Tetracycline responsive element (TRE) in the responder construct induces the transcription of the gene of interest. The expression can be blocked by the addition of Tetracycline or Doxycycline which allosterically inhibits the tTA protein. For the ataxin-3-responder mouse lines, a full length constructs containing an expanded repeat with the pathological length of 77 glutamine repeats was used. The use of two different promoter mouse lines with known expression in the brain (Prion protein (Prp) promoter, Ca2+/Calmoduline-dependent protein kinase II (CamKII) promoter) allows us to target the transgene expression to different brain regions. In preceding promoter analyses we identified and characterized these brain regions in detail. Using the CamKII promoter, the trangene is targeted mainly to the frontal brain and mice display first symptoms at the age of six months. However, when we use the Prp promoter, the transgene is widely expressed in the brain with pronounced expression in the cerebellum and the brain stem. In these mice, first motor symptoms can be detected at the age of two months. We then stopped the expression of the transgene by treatment with doxycyclin and compared treated and untreated mice at different levels: We first analysed the phenotype in behavioural studies. Afterwards we performed immunohistochemical analyses of brain sections to study the expression of the transgene and neurodegenerative processes in the brain. These analyses revealed that turning off the pathogenic transgene at least slows down the progression of symptoms but did not reverse the phenotype. Comparable approaches in human patients -if available- would therefore require early initiation.

Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph disease (MJD), is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG stretch in the MJD1 gene resulting in an expanded polyglutamine repeat in the encoded ataxin-3 protein. SCA3 therefore belongs to the group of the so called polyglutamine diseases.

alpha-Synuclein has been implicated in the pathophysiology of many neurodegenerative disorders, including Parkinson's disease (PD). In order to elucidate the role of this protein in the pathogenesis of PD, transgenic mice were created by using the tet-regulatable system driving the expression of human wildtype and mutated [A30P] alpha-synuclein in CNS neurons. Our studies demonstrate that the expression pattern depends on both the neuron-specific promoter and the integration site of the transgene constructs. Mice expressing high levels of human wildtype alpha-synuclein developed neuropathology and progressive motor impairment which could be stopped after blockade of expression in symptomatic mice. Mice expressing the mutated [A30P] alpha-synuclein restricted to the olfactory bulb showed significantly lower levels of dopamine and metabolites in this brain region. Thus our conditional mouse-model may help to define the role of human alpha-synuclein in synucleinopathies and might be used to demonstrate at which stage neuropathological symptoms of these diseases are reversible.

Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph-Disease (MJD) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG stretch in the MJD1 gene encoding a polyglutamine repeat in the respective ataxin-3-protein. In order to study the course of the disease we generated an inducible transgenic mouse model using the inducible “Tet-Off-System“. This system is based on two constructs: The promoter construct controls the expression of the so called tTA (Tetracycline transactivator) gene product. The binding of this protein to a Tetracycline responsive element (TRE) in the responder construct induces the transcription of the gene of interest. The expression can be blocked by the addition of Tetracycline which allosterically inhibits the tTA protein.

Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph-Disease (MJD) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG stretch in the MJD1 gene encoding a polyglutamine repeat in the respective ataxin-3-protein. In order to study the course of the disease we generated an inducible transgenic mouse model using the „Tet-Off-System“ developed by Dr. Bujard (Heidelberg). This system is based on two constructs: The promoter construct controls the expression of the so called tTA (Tetracycline transactivator) gene product. The binding of this protein to a Tetracycline responsive element (TRE) in the responder construct induces the transcription of the gene of interest. The expression can be blocked by the addition of Tetracycline which allosterically inhibits the tTA protein.

Alpha-synuclein, a small soluble protein is expressed primarily at presynaptic terminals in the central nervous system. It has been implicated in the pathophysiology of many neurodegenerative disorders, including Parkinson's disease and dementia with Lewy-bodies, collectively referred to as synucleinopathies. As neurodegenerative disorders like Parkinson's disease are charecterized by progressive neuropathological and clinical symptoms, conditional models of these neurodegenerative diseases are powerful means to analyze the relationship between transgene expression and progression of the disease. We have created a conditional mouse model of synucleinopathies by using the tet-off gene system combined with expression of human wildtype or mutated (A30P) alpha-synuclein. We investigated the behavioral and neuropathological effects of overexpression of human alpha-synuclein under conditional control of both the PrP promotor and the CaMKIIalpha promotor. Western blot analyses revealed that double-transgenic mice express alpha-synuclein at different levels in specific brain regions. The expression-pattern and level of human alpha-synuclein in the brain of double-transgenic mice depend on both the neuron-specific promotor and the integration site of the human alpha-synuclein construct. Histological analysis of the brainsof transgenic mice showed aberrant expression of the protein in cell soma, axons and synapses, but no Lewy body-like alpha-synuclein inclusions could be identified, yet. We currently investigate, whether focal neurodegeneration, which we observed in one 9 month-old CaMKIIalpha-h[wt]alpha-synuclein double-transgenic mouse could be reproduced. Double-transgenic males of this line performerd significantly weaker on accelerated rotarod than age-matched control animals. HPLC-analyses of double-transgenic mice that showed expression of the transgene limited to the olfactory bulb, demonstrate significantly lower levels of Dopamin, DOPAC and HVA in this brain region. In all lines administration of doxycycline downregulates alpha-synuclein expression to basal level. Our conditional mouse-model may help to define the role of human alpha-synuclein in synucleinopathies and might be used to demonstrate whether neuropathological symptoms of these diseases are reversible.

Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Disease (MJD) is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG stretch in the MJD1 gene encoding a polyglutamine repeat in the respective Ataxin-3 protein. SCA3 therefore belongs to the group of the so called polyglutamine diseases. In order to study the course of the disease we generated transgenic mouse models for this disorder. We used the prion protein (PrP) promotor to control the expression of the full length Ataxin-3 gene containing 70 or 148 CAG repeats, respectively. As controls transgenic mice expressing ataxin-3 with 15 CAG repeats were used. Western blot analyses confirmed the expression of the transgenic Ataxin-3 proteins in the brain. The mice carrying 148 polyglutamine repeats were severely affected by a remarkable neurological phenotype with aberrant behaviour, conspicuous footprint pattern as well as poor rotarod performance. 148 polyglutamine repeats in the Ataxin-3 protein lead to early death at about 15 weeks of age. Neuropathological examination by immunohistochemical staining revealed Ubiquitin- and Ataxin-3-positive intranuclear inclusion bodies in a multitude of neurons. The transgene containing 70 polyglutamine repeats give rise to a comparable but significant milder phenotype resulting in death at about 30 weeks of age. The phenotypical observations as well as the neuropathological results correspond to the mouse model with 148 CAG repeats. On the other hand mice carrying the Ataxin-3 transgene with a normal repeat length (15 CAG repeats) were phenotypically normal with no neuropathological findings. We believe that our mouse models will be a very helpful tool to study the progression of pathology in SCA3. In addition the different repeat numbers allow choosing from a more severe (148 repeats) or milder (77 repeats) phenotype.

Das alpha-Synuklein ist ein niedermolekulares, lösliches Protein, welches primär in präsynaptischen Nervenendigungen exprimiert wird. Es ist an der Pathophysiologie einer ganzen Gruppe neurodegenerativer Erkrankungen wie bspw. dem Morbus Parkinson und der Demenz mit Lewy-Körperchen beteiligt, die man heute unter dem Begriff "Synukleinopathien" zusammenfasst. Neurodegenerative Erkrankungen, wie der Morbus Parkinson, sind durch progrediente neuropathologische und klinische Symptome charakterisiert. Deshalb stellen konditionelle Modelle solcher Erkrankungen ein wichtiges Hilfsmittel zur Analyse der Beziehung zwischen der Expression des Transgens und dem progressiven Verlauf der Neurodegeneration, dar. Aus diesem Grund erzeugten wir ein konditionelles Mausmodell für Synukleinopathien mithilfe des sog. Tet-off-Systems. Hierfür wurden Konstrukte für das humane Wildtyp-alpha-Synuklein und das mutierte (A30P) alpha-Synuklein eingesetzt. Wir analysierten Verhaltensauffälligkeiten und neuropathologische Effekte der sowohl durch den PrP-Promotor als auch den CaMKIIalpha-Promotor induzierten Überexpression des humanen alpha-Synukleins im transgenen Mausgehirn. Die Western-Blot-Analyse transgener Mäusen zeigte, dass doppelt-transgene Tiere humanes alpha-Synuklein in unterschiedlicher Stärke in spezifischen Hirnregionen exprimierten. Dabei ist sowohl der neuronenspezifische Promotor als auch die Integrationsstelle des humanen alpha-Synukleins im Mausgenom für das jeweilige Expressionsmuster entscheidend. Die histologische Untersuchung des transgenen Mausgehirns ergab eine starke Proteinexpression im neuronalen Zellsoma, Axonen und Synapsen. Eine Lewy-Körperchen ähnliche Pathologie konnte jedoch bisher noch nicht nachgewiesen werden. Momentan untersuchen wir, ob eine fokale Neurodegeneration, die in einer für CaMKIIalpha-Promotor und das humane Wildtyp-alpha-Synuklein doppelt-transgenen Maus beobachtet wurde, in einer zweiten Maus derselben Linie ebenfalls auftritt. Die Gabe von Doxyzyklin regulierte die alpha-Synuklein Expression im Gehirn doppelt-transgener Mäuse auf einen basalen Level herab. Ebenso führten wir Microarray-Expressionsanalysen durch, um Einblicke in die Pathomechanismen zu bekommen, die einer Überexpression des humanen alpha-Synukleins im Gehirn der transgenen Mäuse zu Grunde liegen. Unser konditionelles Mausmodell könnte für die Aufklärung der Rolle des alpha-Synukleins bei Entstehung von Synukleinopathien entscheidend sein und aufzeigen, ob neuropathologische Symptome dieser Erkrankungen reversibel sind.

In traditional transgenic mouse models for neurological diseases, the transgene is constitutively expressed in invariable brain regions. The „Tet-Off-System“ developed by Dr. H. Bujard (ZMBH Heidelberg) however allows the generation of inducible transgenic mouse models and flexibility in the selection of the targeted brain regions. This system is based on two constructs: The promoter construct controls the expression of the so called tTA (Tetracycline transactivator) gene product. The binding of this protein to a Tetracycline responsive element (TRE) in the responder construct induces the transcription of the gene of interest. The expression can be blocked by the addition of Tetracycline which allosterically inhibits the tTA protein. In order to assess whether a specific promoter mouse line is suitable for the generation of a disease model the knowledge of the brain regions in which the transgene will be expressed is indispensable. The expression pattern of the promoter mouse line states whether the transgene will be targeted to the desired brain regions and in which brain regions a phenotype or pathology is to be expected, respectively. For this reason we studied the expression pattern of available Tet-Off promoter mouse lines with known expression in the brain (Prion protein (Prp) promoter, Ca2+/Calmoduline-dependent protein kinase II (CamKII) promoter). We crossbred these mouse lines with responder mice transgenic for the lacZ reporter gene. The expression of beta-galactosidase in brain regions with promoter activity was detected using X-Gal as beta-galactosidase substrate resulting in a blue staining. For an overall view entire mouse brains were stained. Brains were sectioned for a detailed analysis of the localization of beta-galactosidase, and composite images of whole sections were generated. The visualization of this expression data in a 3-D brain atlas facilitates the future goal-directed generation of inducible mouse models using the Tet-Off-System.