Current Research

Functional characterization of the Engrailed transcription factors

(The Engrailed genes are homeobox transcription factors which participate in neuronal specification. There are expressed in mesencephalic dopaminergic (mesDA) briefly after they become postmitotic and critically determine their survival. In mouse embryos deficient of the two genes (En1 and En2), mesDA neurons are generated, start to express their neurotransmitter phenotype, but then disappear (Alberi et al., 2004; Simon et al., 2001). The Engrailed requirement for the survival of these neurons is gene-dose dependent and cell-autonomous. Interestingly, in one of the genotypes (En1-/+;En2-/-), which is viable and fertile, the nigral DA neurons are gradually and specifically lost during the first two postnatal months (Fig.1). The disappearance of the cells leads to diminished storage and release of dopamine in the dorsal striatum (caudate putamen) and to motor deficiencies which are reminiscent of akinesia and bradykinesia, two cardinal symptoms of Parkinson’s disease (PD) (Sgado et al., 2006). Death of Engrailed-deficient mesDA neurons is caused by the activation of the mitochondrial pathway of apoptosis, a molecular mechanism, which is shared with in neurotoxin based models for PD, intoxication with 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone and 6-hydroxydopamine (6-OHDA). An over-expression experiment in a neuronal cell line demonstrated that the Engrailed genes repress p75NTR, a neurotrophin receptor. Several lines of evidence now suggest that elevated p75NTR expression is causal to the death of engrailed deficient mesDA neurons. Direct inference with p75NTR by a function-blocking antibody or by RNA interference using penetratin-coupled oligos rescues the mutant neurons in vitro. Markedly, the cell death induced by P75 is mediated by the suppression of the Erk1/2 MAPK signalling pathway. (Alavian et al. submitted). We currently investigate this issue further in vivo and vitro, by using mutant mice, viruses and pharmacological approaches.

The role of K-ATP channel activity for the sensitivity of mesencephalic dopaminergic to mitochondrial insult.

K-ATP channels couple the metabolic state of the neuron to its membrane potential by sensing the ATP/ADP ratio. The channel opens, when the intracellular ATP concentration decreases, leading to an outflux of potassium ions and to hyperpolarization of the membrane, thus reducing the excitability of the cells. In mesDA neurons, the channel consists of four inner-core forming Kir6.2 molecules and four surrounding Sur1 molecules. We have shown that genetic inactivation of the channel does not change the survival rate of mesDA neurons during the entire lifespan of null mutants for Sur1 and Kir6.2 (Fig.2). On the contrary, the in vivo loss of function of the channel results in rescue of nigral DA neurons in two mechanistically distinct mouse models of nigral degeneration, intoxication with MPTP and the weaver mouse (Liss et al., 2005). In our own experiments, we have demonstrated that the same can be achieved by employing channel blockers like the sulfonylurea tolbutamide. Sulfonylureas are widely used to treat diabetes type 2 in humans. We are currently examining whether these pharmacological findings can be employed to rescue nigral DA neurons from degeneration in models for PD. For this purpose, we use Alzet mini-pumps to chronically administer K-ATP channel blocking compounds in mouse and rat.

Simon Fig1
Fig 1 Slow progressive loss of nigral DA neurons in mutant mice heterozygous null for En1 and homozygous null for En2 (EnHT). P0 (A,B), P30 (C,D) and 3 months old wild-type (A-F) and EnHT (A’-F’) mice. TH immunostaining on the level of SN (A,C,E) and VTA (B,D,F).

 

Simon Fig2
Fig. 2. K-ATP channel component Kir6.2 and Sur1. (A) Scheme of K-ATP channel. (B) Cell count of 18 months old wild type and null mutants for Kir6.2 and Sur1. (C) Expression of tyrosine hydroxylase (TH), Sur1 and Kir6.2 in nigral DA neurons
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Latest Revision: 2012-09-11
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