Mouse and Rat Models of Dystonia

TP6 Team

SP6: Mouse and Rat Models of Dystonia

Principle Investigator: Michael Sendtner

This Sub-Project pursues two goals:

1. Generation of new mouse models for Dystonia, based on known mutations in the target genes Dyt1, Dyt6 and Dyt25.

2. Analyses of altered BDNF-signaling in corticostriatal neurons, using these mouse models. The central hypothesis is that dysfunction of BDNF signaling at corticostriatal synapses leads to altered motoric circuit responses which define the clinical symptoms of dystonia.

In the frist year of the project, transgenic mice were generated which carry both the wildtypic THAP1 as well as the THAP1F81L which is mutated in Dystonia patients. By now the first mice carrying a mutation in GNALS293X are also available. Further mouse models will be generated by conventional techniques in 2017 and 2018. These mice will be analyzed within the Dystract Network for alterations in phenotypic and pathological characteristics to validate their suitability as dystonia model system.

BDNF is an important neurotrophic factor for the regulation of synaptic plasticity at corticostriatal synapses. It is produced in cortical projection neurons and anterogradely transported along their axons towards the striatum where it is secreted from axon terminals. Since the levels of endogenous BDNF are very low, aims to visualize BDNF in corticostriatal axon terminals failed to so far.

For this reason methods are currently optimized to characterize the role of BDNF in these synaptic structures. Based on optimized visualization techniques for observing BDNF in the CNS (Fig. 1 BDNF-IR in hippocampus) efforts for reliable detection of BDNF in the striatum will be established until 2018.

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Fig. 1: Localization of BDNF-IR (DyeLight550, red) together with immunoreactivity for VGluT1 in the hippocampal CA3 region (Alexa488, green). BDNF is localized in axon terminals of mossy fibers which project towards the CA3 region.
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Earlier studies have shown that the genetic inactivation of BDNF in the CNS via Tau-Cre conditional recombination leads to severe morphological and functional alterations within the striatum. These transgenic mice show a decrease in striatal volume by 40% caused by significant reduction in dendritc arboritzation, combined with severe spine pruning (Fig. 2; Rauskolb et al., J. Neuroscience 2010). These observations are the basis for the idea that alterations in either the anterograde transport of BDNF or synaptic release from corticostriatal terminals as well as disruption of postsynaptic TrkB activation lead to motoric deficits in the pathogenesis of dystonia.

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Fig. 2: Depletion of BDNF in neuron specific gene knockout mice leads to massive reduction of dendritic arborization in striatal medium spiny neurons. (Rauskolb et al., J. Neuroscience 2010)

Corticostriatal synapses are central gateways for the direct and indirect signaling pathway, regulating motor function and could thus play an important role in the pathology of dystonia. To follow this hypothesis, histological techniques for the identification of direct and indirect pathway MSNs were optimized (Fig. 3) which are now the basis for the investigation of corresponding synapses and circuitry regarding motor functions and potential alterations in BDNF deficient mice as well as newly generated mouse models for Dystonia.

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Fig. 3: Specific detection of DRD2 positive indirect pathway medium spiny neurons in striatum of transgenic mice (GFP, green) (Gerfen et. al. Nat. Neuroscience, 2006). Immunostaining against dopamine and cyclic AMP regulated phosphoprotein of 32kDa (DARPP-32, Cy3, red) stains all striatal medium spiny neurons, including DRD1 positive neurons of the direct pathway within the basal ganglia.
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To investigate potential changes in BDNF-signaling on the postsynaptic side, cell culture methods were established and optimized which enable the purification, enrichment and subsequent separate investigation of direct pathway DRD1 pos. (dMSNs) or indirect pathway DRD2 pos. medium spiny neurons (iMSNs) (Fig. 4). These techniques were not available so far and will be further optimized until 2018, for application regarding cell-biological and molecular experiments addressing changes in BDNF / TrkB signaling in striatal medium spiny neurons.

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Fig. 4: Isolated indirect pathway medium spiny neurons (iMSNs) from striatum of reporter mice using FACS. DRD2 positive medium spiny neurons are indicated in green (GFP), also expressing the general MSN marker DARPP-32. By FACS of either dMSNs or iMSNs from specific reporter mice - high density monocultures can be obtained for investigation of MSNs subtypes in vitro.
(click the figure to open in higher resoluation ...)