Carsten Holzmann

14-3-3 proteins in the nervous system.

14-3-3 proteins are abundantly expressed in the brain and have been detected in the cerebrospinal fluid of patients with different neurological disorders. Although the function of this family of highly conserved proteins is not completely known, recent evidence indicates their involvement in multiple cellular processes. By their interaction with more than 100 binding partners, 14-3-3 proteins modulate the action of proteins that are involved in cell cycle and transcriptional control, signal transduction, intracellular trafficking and regulation of ion channels. The study of some of these interactions is sheding light on the role of 14-3-3 proteins in processes such as apoptosis and neurodegeneration.

Neurodegeneration and Motor Dysfunction in a Conditional Model of Parkinson's Disease

α-Synuclein (α-syn) has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinsons disease. These disorders are characterized by various neurological and psychiatric symptoms based on progressive neuropathological alterations. Whether the neurodegenerative process might be halted or even reversed is presently unknown. Therefore, conditional mouse models are powerful tools to analyze the relationship between transgene expression and progression of the disease. To explore whether α-syn solely originates and further incites these alterations, we generated conditional mouse models by using the tet-regulatable system. Mice expressing high levels of human wild-type α-syn in midbrain and forebrain regions developed nigral and hippocampal neuropathology, including reduced neurogenesis and neurodegeneration in absence of fibrillary inclusions, leading to cognitive impairment and progressive motor decline. Turning off transgene expression in symptomatic mice halted progression but did not reverse the symptoms. Thus, our data suggest that approaches targeting α-syn-induced pathological pathways might be of benefit rather in early disease stages. Furthermore, α-syn-associated cytotoxicity is independent of filamentous inclusion body formation in our conditional mouse model.

Selective striatal neuron loss and alterations in behavior correlate with impaired striatal function in Huntington's disease transgenic rats

Huntingtons disease (HD) is an inherited neurodegenerative disorder characterized by selective striatal neuron loss and motor, cognitive and affective disturbances. The present study aimed to test the hypothesis of adult-onset neuron loss in striatum and frontal cortical layer V as well as alterations in behavior pointing to impaired striatal function in a recently developed transgenic rat model of HD (tgHD rats) exhibiting enlarged ventricles, striatal atrophy and pycnotic pyramidal cells in frontal cortical layer V. High-precision design-based stereological analysis revealed a reduced mean total number of neurons in the striatum but not in frontal cortical layer V of 12-month-old tgHD rats compared with age-matched wild-type controls. No alterations in mean total numbers of striatal neurons were found in 6-month-old animals. Testing 14-month-old animals in a choice reaction time task indicated impaired striatal function of tgHD rats compared with controls.

Reversibility of symptoms in a conditional mouse model of spinocerebellar ataxia type 3

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a CAG repeat tract that affects the MJD1 gene which encodes the ataxin-3 protein. In order to analyze whether symptoms caused by ataxin-3 with an expanded repeat are reversible in vivo, we generated a conditional mouse model of SCA3 using the Tet-Off system. We used a full-length human ataxin-3 cDNA with 77 repeats in order to generate the responder mouse line. After crossbreeding with a PrP promoter mouse line, double transgenic mice developed a progressive neurological phenotype characterized by neuronal dysfunction in the cerebellum, reduced anxiety, hyperactivity, impaired Rotarod performance and lower body weight gain. When ataxin-3 expression was turned off in symptomatic mice in an early disease state, the transgenic mice were indistinguishable from negative controls after 5 months of treatment. These results show that reducing the production of pathogenic ataxin-3 indeed may be a promising approach to treat SCA3, provided that such treatment is applied before irreversible damage has taken place and that it is continued for a sufficiently long time.

14-3-3 protein is a component of Lewy bodies in Parkinson's disease-mutation analysis and association studies of 14-3-3 eta.

Mutations in alpha-synuclein have been identified in some rare families with autosomal dominant Parkinsons disease (PD). The synuclein gene family shares physical and functional homology with 14-3-3 proteins and binds to 14-3-3 proteins and to its ligands. We therefore investigated whether 14-3-3 proteins are also involved in the pathogenesis of PD. Here we demonstrate that 14-3-3 proteins are colocalized with Lewy bodies in PD. We investigated the 14-3-3 eta (YWHAH) gene by mutation analysis and association studies as it maps to human chromosome 22q12.1-q13.1, a region which has been recently implicated in PD and carried out immunohistochemical studies of Lewy bodies with two different 14-3-3 eta antibodies. In 358 sporadic and familial PD patients, disease causing mutations were not identified. Furthermore, association studies with intragenic polymorphisms do not provide evidence for an involvement of 14-3-3 eta in the pathogenesis of PD. In accordance with these findings, there was no staining of substantia nigra Lewy bodies with antibodies specific for the 14-3-3 eta subunit.

Extended mutation analysis and association studies of Nurr1 (NR4A2) in Parkinson disease

Mutations in four genes (α -synuclein, UCH-L1, parkin , and DJ-1 ) have been identified in familial Parkinson disease (PD). Recently, two mutations in the 5′-region of the orphan nuclear receptor Nurr1 ( NR4A2 ) gene have been found in 10% of familial PD cases, of which one-third were of German ancestry.1 Furthermore, a polymorphism in intron 6 of the Nurr1 gene was associated with PD in two independent studies.2,3⇓ The transcription factor Nurr1 is highly expressed in dopaminergic cells of the substantia nigra. Deficiency of Nurr1 in mice leads to a lack of dopaminergic neurons.4 Based on these data, we explored the 7048G7049 polymorphism in intron 6 as well as two novel sequence variations, −291Tdel and −245T→G, in exon 1 of Nurr1 in a large cohort of PD patients. For analysis of the 7048G7049 polymorphism, 424 PD patients were investigated (mean age at disease onset 55.4 ± 12.1 years; 54.6% male, 45.4% female; with 55 patients [13.0%] having a positive family history). For analysis of the two novel sequence variations, −291Tdel and −245T→G, …

Polymorphisms of the alpha-synuclein promoter: expression analyses and association studies in Parkinson's disease.

Summary. Mutations of the α-synuclein gene have shown to be relevant in some rare families with autosomal dominant Parkinsons disease (PD). Furthermore, α-synuclein protein is a major component of the Lewy bodies also in sporadic PD patients. Increased levels of wildtype α-synuclein in the cell leads to increased intracellular hydrogen peroxide levels and causes death of dopaminergic neurons in rat primary culture. Subsequently, oxidative stress has been directly linked with α-synuclein aggregation in vitro. This raises the question whether increased α-synuclein expression might be linked to higher susceptibility to PD and whether α-synuclein promoter polymorphisms are associated with PD. Here, two polymorphisms (−116C>G and −668T>C) of the α-synuclein promoter defining four haplotypes have been characterized in 315 German PD patients. The influence of the four haplotypes on gene expression was studied by CAT reporter gene assays in neuronal SK-N-AS cells. The −668C/−116G haplotype revealed significant higher CAT expression than the −668T/−116G or the −668T/−116C haplotype, respectively. Although the −668C/−116G haplotype was more common in PD patients, this difference was not significant.

Regional and subtype selective changes of neurotransmitter receptor density in a rat transgenic for the Huntington's disease mutation

Huntingtons disease (HD) is an autosomal dominantly inherited progressive neurodegenerative disorder caused by a CAG/polyglutamine repeat expansion in the gene encoding the huntingtin protein. We have recently generated a rat model transgenic for HD, which displays a slowly progressive phenotype resembling the human adult‐onset type of disease. In this study we systematically assessed the distribution and density of 17 transmitter receptors in the brains of 2‐year‐old rats using quantitative multi‐tracer autoradiography and high‐resolution positron emission tomography. Heterozygous animals expressed increased densities of M2 acetylcholine (increase of 148 ± 16% of controls; p > 0.001; n = 7), nicotine (increase of 149 ± 16% of controls; p > 0.01; n = 6), and α2 noradrenergic receptors (increase of 141 ± 15% of controls; p > 0.001; n = 6), respectively. Densities of these receptors were decreased in homozygous animals. Decreases of receptor density in both hetero‐ and homozygous animals were found for M1 acetylcholine, 5‐HT2A serotonin, A2A adenosine, D1 and D2 dopamine, and GABAA receptors, respectively. Other investigated receptor systems showed small changes or were not affected. The present data suggest that the moderate increase of CAG/polyglutamine repeat expansions in the present rat model of Huntingtons disease is characterized by subtype‐selective and region‐specific changes of neuroreceptor densities. In particular, there is evidence for a contribution of predominantly presynaptically localized cholinergic and noradrenergic receptors in the response to Huntingtons disease pathology.

A transgenic mouse model of spinocerebellar ataxia type 3 resembling late disease onset and gender-specific instability of CAG repeats

Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is caused by the expansion of a polyglutamine repeat in the ataxin-3 protein. We generated a mouse model of SCA3 expressing ataxin-3 with 148 CAG repeats under the control of the huntingtin promoter, resulting in ubiquitous expression throughout the whole brain. The model resembles many features of the disease in humans, including a late onset of symptoms and CAG repeat instability in transmission to offspring. We observed a biphasic progression of the disease, with hyperactivity during the first months and decline of motor coordination after about 1 year of age; however, intranuclear aggregates were not visible at this age. Few and small intranuclear aggregates appeared first at the age of 18 months, further supporting the claim that neuronal dysfunction precedes the formation of intranuclear aggregates.

Lifelong Caloric Restriction Increases Working Memory in Mice

Caloric restriction (CR) is argued to positively affect general health, longevity and the normally occurring age-related reduction of cognition. This issue is well examined, but most studies investigated the effect of short-term periods of CR. Herein, 4 weeks old female mice were fed caloric restricted for 4, 20 and especially for 74 weeks. CR mice received 60% of food eaten by their ad libitum (AL) fed littermates, and all age-matched groups were behaviorally analyzed. The motor coordination, which was tested by rotarod/accelerod, decreased age-related, but was not influenced by the different periods of CR. In contrast, the age-related impairment of spontaneous locomotor activity and anxiety, both being evaluated by open field and by elevated plus maze test, was found aggravated by a lifelong CR. Measurement of cognitive performance with morris water maze showed that the working memory decreased age-related in AL mice, while a lifelong CR caused a better cognitive performance and resulted in a significantly better spatial memory upon 74 weeks CR feeding. However, a late-onset CR feeding in 66 weeks old mice did not ameliorate the working memory. Therefore, a lifelong CR seems to be necessary to improve working memory.