Role of Matrix Metalloproteinases in Fragile X

With a $220,000 grant from FRAXA Research Foundation over 3 years, Dr. Iryna Ethell from the University of California at Riverside studied the regulation of dendritic structure by matrix metalloproteinases and other extracellular signaling pathways. This work identified a major treatment strategy for Fragile X with the available MMP-9 inhibitor, minocycline.

Iryna Ethell, PhD, at University of California
$220,000 Grant
Iryna Ethell, PhD
Principal Investigator
University of California at Riverside
2007-2009 FRAXA Research Grant
$220,000 over 3 Years

Targeting Matrix Metalloproteinases to Treat Dendritic Spine Malformation and Behavioral Defects in Fragile X Mice

by Iryna Ethell, 5/1/2009

This project will determine if the beneficial effects of minocycline result from its ability to regulate MMP-9 expression and activity, with an emphasis on the role MMPs play in Fragile-X pathophysiology. There are three directions these studies will follow:

1. We will study the specific effects of inhibiting or depleting MMP-9 have on dendritic spine development and behavior in Fmr1 KO mice. This will be done with specific MMP-9 inhibitors and through genetic crosses with Fmr1 KO mice with mmp9 KO mice. We anticipate that reduced MMP-9 expression or activity in Fmr1 KO neurons will lead to similar behavioral and morphological effects as are seen with minocycline treatment. We will investigate the effects of MMP-9 depletion on dendritic spine/synapse development in Fmr1 KO hippocampal neurons by crossing mmp-9 KO mice (FVB.Cg-MMP9tm1Tvu/J; stock number 004104) with Fmr1 KO mice. Further, we will screen some new tetracycline derivatives for their ability to promote dendritic spine maturation in Fmr1 KO hippocampal neurons.

2. We will examine whether higher levels of MMP expression/activity can be detected in Fragile X patients. Since enhanced MMP-9 expression and activity have been found in the brains of Fmr1 KO mice, and may underlie the morphological and behavioral abnormalities in these mice, it is important to determine if MMP-2 and MMP-9 activity is higher in FXS patients. FXS may also affect MMP regulation outside the central nervous system (CNS) as patients display several characteristics of modified extracellular matrix (ECM) including large ears, long faces, hyperextensible joints and flat feet. We will use gelatinase assays, gelatin gel zymography and ELISAs to analyze the levels of MMP-9 expression and activity in blood and tissue samples from FXS patients and controls. We will also analyze the levels and activities of other MMPs as well as the activities of endogenous MMP inhibitors, the TIMPs, using specific ELISA and activity assays.

Discovery of Minocyline as a Treatment for Fragile X

This team made the original discovery that led to minocycline as a potential treatment

By Iryna Ethell, 6/1/2008

Our investigation led to the discovery that minocycline treatment can accelerate dendritic spine maturation in hippocampal neurons of young Fmr1 KO mice both in vitro and in vivo. These changes were accompanied by behavioral improvements. Untreated Fmr1 knockout mice show higher anxiety in the elevated plus-maze and decreased hippocampal function in the Y-maze, compared to wild-type mice; however, minocycline treatment significantly improved the behavioral performance of one-month-old Fmr1 KO mice. Our findings establish that minocycline induces dendritic spine maturation and improves behavior in Fmr1 KO mice. We hypothesize that these effects of minocycline may relate to its inhibitory action on MMP-9 expression and activity that are up-regulated in FMRP-deficient hippocampal neurons.

Directions:

1. We will test whether the mechanisms responsible for higher MMP-9 activity in the hippocampus of Fmr1 KO mice are related to metabotropic glutamate receptor (mGluR) hyperactivity and if ERK1/2 activation is involved.

2. We will determine if higher levels of MMPs expression/activity can be detected in Fragile X patients. FXS patients may also be affected by MMP dysregulation outside the CNS as they display several characteristics of modified ECM regulation including large ears, long faces, hyperextensible joints and extremely soft skin.

3. We will investigate the effects of mmp-9 KO on dendritic spine/synapse development in Fmr1 KO hippocampal neurons using a genetic approach by crossing mmp-9 KO and Fmr1 KO mice.

4. We will determine the relative efficacy of minocycline on behavior in older Fmr1 KO mice, when treatment is started at 2, 4 and 6 months of age.

Role for Matrix Metalloproteinases (MMPs) in the Development of Abnormal Spines in Fragile X

by Iryna Ethell, 5/1/2007

In our laboratories we are interested in understanding how the neuronal network develops in the brain and how perturbations in this process can manifest in cognitive impairment. Toward this end, we are currently working to clearly define the mechanisms governing the abnormal development of dendritic spines in Fragile X.

Dendritic spines are small protrusions on the surface of the dendrite that receive the majority of excitatory synapses in the brain and play a critical role in cognitive development. Defects in dendritic spine formation have been found in the brains of patients with some autistic spectrum disorders, such as Fragile X and Rett Syndrome (Fiala et al., 2002). Therefore, abnormal dendritic spine development may represent an anatomical and physiological basis for the cognitive deficits in these disorders.

This FRAXA-funded project will investigate a role for matrix metalloproteinases (MMPs) in the development of abnormal spines in Fragile X, with an emphasis on using an MMP inhibitor, minocycline, to accelerate dendritic spine development in a mouse model for this disorder, Fragile X mental retardation (FMR1) knockout mice. FMR1 knockout mice show social behaviors and abnormalities in brain development that are similar to those of patients with Fragile X and some other forms of autism, which makes these mice an excellent animal model to study the underlying mechanisms. We hypothesize that higher than normal levels of MMP activity in the brains of FMR1 knockout mice contribute to dendritic spine abnormalities and cognitive dysfunction. Further, we propose that inhibiting this excess MMP activity with minocycline will prevent the abnormal dendritic spine development and related behavioral defects, thus bypassing the genetic causes of these abnormalities, and effectively treat Fragile X.

Tina Bilousova, PhD
FRAXA Postdoctoral Fellow

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