Non-motor symptoms associated with progressive loss of dopaminergic neurons in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is characterized by three main motor symptoms: bradykinesia, rigidity and tremor. PD is also associated with diverse non-motor symptoms that may develop in parallel or precede motor dysfunctions, ranging from autonomic system dysfunctions and impaired sensory perception to cognitive deficits and depression. Here, we examine the role of the progressive loss of dopaminergic transmission in behaviors related to the non-motor symptoms of PD in a mouse model of the disease (the TIF-IADATCreERT2 strain). We found that in the period from 5 to 12 weeks after the induction of a gradual loss of dopaminergic neurons, mild motor symptoms became detectable, including changes in the distance between paws while standing as well as the swing speed and step sequence. Male mutant mice showed no apparent changes in olfactory acuity, no anhedonia-like behaviors, and normal learning in an instrumental task; however, a pronounced increase in the number of operant responses performed was noted. Similarly, female mice with progressive dopaminergic neuron degeneration showed normal learning in the probabilistic reversal learning task and no loss of sweet-taste preference, but again, a robustly higher number of choices were performed in the task. In both males and females, the higher number of instrumental responses did not affect the accuracy or the fraction of rewarded responses. Taken together, these data reveal discrete, dopamine-dependent non-motor symptoms that emerge in the early stages of dopaminergic neuron degeneration.

Genetic lesions of the noradrenergic system trigger induction of oxidative stress and inflammation in the ventral midbrain.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor deficits caused by the loss of dopaminergic neurons in the substantia nigra (SN) and ventral tegmental area (VTA). However, clinical data revealed that not only the dopaminergic system is affected in PD. Postmortem studies showed degeneration of noradrenergic cells in the locus coeruleus (LC) to an even greater extent than that observed in the SN/VTA. Pharmacological models support the concept that modification of noradrenergic transmission can influence the PD-like phenotype induced by neurotoxins. Nevertheless, there are no existing data on animal models regarding the distant impact of noradrenergic degeneration on intact SN/VTA neurons. The aim of this study was to create a transgenic mouse model with endogenously evoked progressive degeneration restricted to noradrenergic neurons and investigate its long-term impact on the dopaminergic system. To this end, we selectively ablated the transcription initiation factor-IA (TIF-IA) in neurons expressing dopamine ß-hydroxylase (DBH) by the Cre-loxP system. This mutation mimics a condition of nucleolar stress affecting neuronal survival. TIF-IADbhCre mice were characterized by selective, progressive degeneration of noradrenergic neurons, followed by phenotypic alterations associated with sympathetic system impairment. Our studies did not show any loss of tyrosine hydroxylase (TH)-positive cells in the SN/VTA of mutant mice; however, we observed increased indices of oxidative stress, enhanced markers of glial cell activation, inflammatory processes and isolated degenerating cells positive for FluoroJade C. These results were supported by gene expression profiling of VTA and SN from TIF-IADbhCre mice, revealing that 34 out of 246 significantly regulated genes in the SN/VTA were related to PD. Overall, our results shed new light on the possible negative influence of noradrenergic degeneration on dopaminergic neurons, reinforcing the neuroprotective role of noradrenaline.

The influence of CaMKII and ERK phosphorylation on BDNF changes observed in mice selectively devoid of CREB in serotonergic or noradrenergic neurons.

BACKGROUND: The transcription factor CREB and the neurotrophin BDNF are important mood regulators due to their profound role in controlling the neuronal plasticity. Our previously published results from transgenic mice functionally lacking CREB in chosen neural populations have shown that BDNF upregulation evoked by chronic treatment with fluoxetine seems to be dependent on CREB residing exclusively in serotonergic neurons. To further elucidate this observation, we focused on the representative signaling cascades engaged in the regulation of BDNF production. METHODS: The study was carried out on mice lacking CREB in noradrenergic (Creb1DBHCre) or serotonergic (Creb1TPH2CreERT2) neurons in CREM deficient background. Animals received fluoxetine (10 mg/kg, ip) or desipramine (20 mg/kg, ip) for 21 days. The expression of following proteins and their phosphorylated forms was assessed by Western blot: CREB, BDNF, CaMKIIα, ERK1/2. RESULTS: We showed that consistent with previously observed BDNF upregulation, chronic treatment with fluoxetine causes an increase in the pool of active CaMKIIα in w/t males, while in Creb1TPH2CreERT2 mutants, this effect ceased along with the observed decrease in ERK1/2 phosphorylation. These effects were region- and sex-specific. We did not observe a similar pattern of changes regarding the levels of BDNF expression and the CaMKIIα, ERK1/2 kinases in Creb1DBHCre mice exposed to desipramine. However, sex-dependent changes in the regulation of CaMKIIα and ERK1/2 activity were also observed. CONCLUSIONS: Our study highlights the pivotal role of CREB in response to antidepressants, emphasizing different sex-dependent vulnerabilities to particular drugs and the important impact of CREM on the effects of CREB deletion.

Stimulation of noradrenergic transmission by reboxetine is beneficial for a mouse model of progressive parkinsonism.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by motor deficits such as tremor, rigidity and bradykinesia. These symptoms are directly caused by the loss of dopaminergic neurons. However, a wealth of clinical evidence indicates that the dopaminergic system is not the only system affected in PD. Postmortem studies of brains from PD patients have revealed the degeneration of noradrenergic neurons in the locus coeruleus (LC) to the same or even greater extent than that observed in the dopaminergic neurons of substantia nigra (SN) and ventral tegmental area (VTA). Moreover, studies performed on rodent models suggest that enhancement of noradrenergic transmission may attenuate the PD-like phenotype induced by MPTP administration, a neurotoxin-based PD model. The aim of this study was to investigate whether chronic treatment with either of two compounds targeting the noradrenergic system (reboxetine or atipamezole) possess the ability to reduce the progression of a PD-like phenotype in a novel mouse model of progressive dopaminergic neurodegeneration induced by the genetic inhibition of rRNA synthesis in dopaminergic neurons, mimicking a PD-like phenotype. The results showed that reboxetine improved the parkinsonian phenotype associated with delayed progression of SN/VTA dopaminergic neurodegeneration and higher dopamine content in the striatum. Moreover, the alpha1-adrenergic agonist phenylephrine enhanced survival of TH+ neurons in primary cell cultures, supporting the putative neuroprotective effects of noradrenergic stimulation. Our results provide new insights regarding the possible influence of the noradrenergic system on dopaminergic neuron survival and strongly support the hypothesis regarding the neuroprotective role of noradrenaline.

Selective Depletion of CREB in Serotonergic Neurons Affects the Upregulation of Brain-Derived Neurotrophic Factor Evoked by Chronic Fluoxetine Treatment.

Neurotrophic factors are regarded as crucial regulatory components in neuronal plasticity and are postulated to play an important role in depression pathology. The abundant expression of brain-derived neurotrophic factor (BDNF) in various brain structures seems to be of particular interest in this context, as downregulation of BDNF is postulated to be correlated with depression and its upregulation is often observed after chronic treatment with common antidepressants. It is well-known that BDNF expression is regulated by cyclic AMP response element-binding protein (CREB). In our previous study using mice lacking CREB in serotonergic neurons (Creb1TPH2CreERT2 mice), we showed that selective CREB ablation in these particular neuronal populations is crucial for drug-resistant phenotypes in the tail suspension test observed after fluoxetine administration in Creb1TPH2CreERT2 mice. The aim of this study was to investigate the molecular changes in the expression of neurotrophins in Creb1TPH2CreERT2 mice after chronic fluoxetine treatment, restricted to the brain structures implicated in depression pathology with profound serotonergic innervation including the prefrontal cortex (PFC) and hippocampus. Here, we show for the first time that BDNF upregulation observed after fluoxetine in the hippocampus or PFC might be dependent on the transcription factor CREB residing, not within these particular structures targeted by serotonergic projections, but exclusively in serotonergic neurons. This observation may shed new light on the neurotrophic hypothesis of depression, where the effects of BDNF observed after antidepressants in the hippocampus and other brain structures were rather thought to be regulated by CREB residing within the same brain structures. Overall, these results provide further evidence for the pivotal role of CREB in serotonergic neurons in maintaining mechanisms of antidepressant drug action by regulation of BDNF levels.

Transgenic mice lacking CREB and CREM in noradrenergic and serotonergic neurons respond differently to common antidepressants on tail suspension test.

Evidence exists that chronic antidepressant therapy enhances CREB levels and activity. Nevertheless, the data are not conclusive, as previous analysis of transgenic mouse models has suggested that CREB inactivation in fact contributes to antidepressant-like behavior. The aim of this study was to evaluate the role of CREB in this context by exploiting novel transgenic mouse models, characterized by selective ablation of CREB restricted to noradrenergic (Creb1DBHCre/Crem-/-) or serotonergic (Creb1TPH2CreERT2/Crem-/-) neurons in a CREM-deficient background to avoid possible compensatory effects of CREM. Selective and functional ablation of CREB affected antidepressant-like behavior in a tail suspension test (TST) after antidepressant treatment. Contrary to single Creb1DBHCre mutants, Creb1DBHCre/Crem-/- mice did not respond to acute desipramine administration (20 mg/kg) on the TST. On the other hand, single Creb1TPH2CreERT2 mutants displayed reduced responses to fluoxetine (10 mg/kg) on the TST, while the effects in Creb1TPH2CreERT2/Crem-/- mice differed by gender. Our results provide further evidence for the important role of CREM as a compensatory factor. Additionally, the results indicate that new models based on the functional ablation of CREB in select neuronal populations may represent a valuable tool for investigating the role of CREB in the mechanism of antidepressant therapy.

Lack of riluzole efficacy in the progression of the neurodegenerative phenotype in a new conditional mouse model of striatal degeneration.

BACKGROUND: Huntington's disease (HD) is a rare familial autosomal dominant neurodegenerative disorder characterized by progressive degeneration of medium spiny neurons (MSNs) located in the striatum. Currently available treatments of HD are only limited to alleviating symptoms; therefore, high expectations for an effective therapy are associated with potential replacement of lost neurons through stimulation of postnatal neurogenesis. One of the drugs of potential interest for the treatment of HD is riluzole, which may act as a positive modulator of adult neurogenesis, promoting replacement of damaged MSNs. The aim of this study was to evaluate the effects of chronic riluzole treatment on a novel HD-like transgenic mouse model, based on the genetic ablation of the transcription factor TIF-IA. This model is characterized by selective and progressive degeneration of MSNs. METHODS: Selective ablation of TIF-IA in MSNs (TIF-IAD1RCre mice) was achieved by Cre-based recombination driven by the dopamine 1 receptor (D1R) promoter in the C57Bl/6N mouse strain. Riluzole was administered for 14 consecutive days (5 mg/kg, i.p.; 1× daily) starting at six weeks of age. Behavioral analysis included a motor coordination test performed on 13-week-old animals on an accelerated rotarod (4-40 r.p.m.; 5 min). To visualize the potential effects of riluzole treatment, the striata of the animals were stained by immunohistochemistry (IHC) and/or immunofluorescence (IF) with Ki67 (marker of proliferating cells), neuronal markers (NeuN, MAP2, DCX), and markers associated with neurodegeneration (GFAP, 8OHdG, FluoroJade C). Additionally, the morphology of dendritic spines of neurons was assessed by a commercially available FD Rapid Golgi Stain™ Kit. RESULTS: A comparative analysis of IHC staining patterns with chosen markers for the neurodegeneration process in MSNs did not show an effect of riluzole on delaying the progression of MSN cell death despite an observed enhancement of cell proliferation as visualized by the Ki67 marker. A lack of a riluzole effect was also reflected by the behavioral phenotype associated with MSN degeneration. Moreover, the analysis of dendritic spine morphology did not show differences between mutant and control animals. DISCUSSION: Despite the observed increase in newborn cells in the subventricular zone (SVZ) after riluzole administration, our study did not show any differences between riluzole-treated and non-treated mutants, revealing a similar extent of the neurodegenerative phenotype evaluated in 13-week-old TIF-IAD1RCre animals. This could be due to either the treatment paradigm (relatively low dose of riluzole used for this study) or the possibility that the effects were simply too weak to have any functional meaning. Nevertheless, this study is in line with others that question the effectiveness of riluzole in animal models and raise concerns about the utility of this drug due to its rather modest clinical efficacy.

Disruption of glucocorticoid receptors in the noradrenergic system leads to BDNF up-regulation and altered serotonergic transmission associated with a depressive-like phenotype in female GR(DBHCre) mice.

Recently, we have demonstrated that conditional inactivation of glucocorticoid receptors (GRs) in the noradrenergic system, may evoke depressive-like behavior in female but not male mutant mice (GR(DBHCre) mice). The aim of the current study was to dissect how selective ablation of glucocorticoid signaling in the noradrenergic system influences the previously reported depressive-like phenotype and whether it might be linked to neurotrophic alterations or secondary changes in the serotonergic system. We demonstrated that selective depletion of GRs enhances brain derived neurotrophic factor (BDNF) expression in female but not male GR(DBHCre) mice on both the mRNA and protein levels. The possible impact of the mutation on brain noradrenergic and serotonergic systems was addressed by investigating the tissue neurotransmitter levels under basal conditions and after acute restraint stress. The findings indicated a stress-provoked differential response in tissue noradrenaline content in the GR(DBHCre) female but not male mutant mice. An analogous gender-specific effect was identified in the diminished content of 5-hydroxyindoleacetic acid, the main metabolite of serotonin, in the prefrontal cortex, which suggests down-regulation of this monoamine system in female GR(DBHCre) mice. The lack of GR also resulted in an up-regulation of alpha2-adrenergic receptor (α2-AR) density in the female but not male mutants in the locus coeruleus. We have also confirmed the utility of the investigated model in pharmacological studies, which demonstrates that the depressive-like phenotype of GR(DBHCre) female mice can be reversed by antidepressant treatment with desipramine or fluoxetine, with the latter drug evoking more pronounced effects. Overall, our study validates the use of female GR(DBHCre) mice as an interesting and novel genetic tool for the investigation of the cross-connected mechanisms of depression that is not only based on behavioral phenotypes.