Gremlin-1, a Putative VTA-derived Neuroprotective Factor in Parkinson's Disease (Supplement)

Promising Outcomes of Original Grant:
We have previously postulated that the glycoprotein Gremlin has a neuroprotective role in PD. To test this hypothesis, we sought to develop a gremlin knockout pre-clinical model. Since homozygous null mutations of the grem1 gene result in embryonic lethality, our goal in the first year of the MJFF award was to generate a conditional knockout in which a pre-clinical model with a tamoxifen-inducible En1Cre pre-clinical model [En1(tm7 (Cre/ESR1)Alj/J- from Jackson Labs] were crossed with Grem-1loxP/loxP pre-clinical model generated at Regeneron Inc. In allowing visualization of DA neurons, these bitransgenic pre-clinical models were further crossed with our hTHGFP pre-clinical model (Kessler et al., 2003). We then characterized the triple transgenic pre-clinical model: after treatment with 2mg tamoxifen (tam) for 5 days, we found a significant 77% decrease in grem expression by Q-PCR and western. Controls were single mutation hTHGFP pre-clinical models or bitransgenic  En1CreERT1 pre-clinical models treated with tam and triple tg pre-clinical models treated with corn oil and no tam showed no change in gremlin expression. Because both genetic manipulation and tamoxifen can produce toxicity, we further showed that the expression levels of TH and the cell death gene, caspase 3, were the same in Grem KO and control pre-clinical models. Moreover, midbrain neurons in Grem KO pre-clinical model treated with tamoxifen appeared healthy and their number was nearly identical to those in control models.

Objectives for Supplemental Investigation:
Our first goal in year 2 is the determination of whether the absence of Gremlin increases the vulnerability of DA neurons to MPTP toxicity, particularly in the VTA using a progressive pre-clinical model of PD in which low doses of MPTP are given over an extended time course to mimic the advancing cell death seen at various stages of the disease. In addition, we will test whether the increased vulnerability of DA neurons to MPTP in cGrem KO pre-clinical model is mediated by the loss of Gremlin signaling through its downstream mediators (VEGFR, MAPK), using pharmacological inhibitors of these intermediaries to mimic the effects of genetic knockout of Gremlin.

Our second goal is the determination of whether Gremlin plays a neurorestorative/neuroregenerative role in PD. Our previous work showing that the co-administration of Gremlin (intracerebral infusion) with MPTP (IP injection) will rescue SN neurons from cell death indicates that Gremlin plays a neuroprotective role in PD. However, since PD is generally diagnosed after considerable damage to midbrain DA neurons has already occurred, the search for factors that can repair and restore function to the nigrostriatal system remains a high priority.  We will therefore infuse Gremlin by Alzet pump into the SN at various times following MPTP damage and test for its ability to rescue dying DA neurons.  In addition, we will verify that these effects are due to Gremlin’s action at the VEGF receptor.

Importance of This Research for the Development of a New PD Therapy
With wild type and triple transgenic Gremlin KO pre-clinical model in hand, these studies will explore Gremlin’s potential role in Parkinson’s disease (PD) by testing 1) whether dopamine (DA) neurons of the Ventral Tegmental Area (VTA) and Substantia nigra (SN) are less neuroprotected (ie. exhibit increased vulnerability to MPTP) when Gremlin gene expression is decreased in a conditional knockout (cGrem KO) pre-clinical model; and 2) whether Gremlin also plays a role in neurorestoration/neuroregeneration once PD damage has begun (early stage disease) and/or progressed (late stage disease) in the brain. Our goal in these studies is to validate Gremlin as a novel biological target with potential impact in the treatment of PD.