Peter Vanderklish, Ph.D.
Principal Investigator
Sowmya Venkata Yelamanchili, PhD
Postdoctoral Fellow
Scripps Research Institute, San Diego, CA

FRAXA Awards:
  $40,000 in 2008
  $50,000 in 2007
  $80,000 in 2006
  $50,000 in 2005
  $95,000 in 2004

Molecular mechanisms underlying changes in dendritic spine morphology in Fragile X Syndrome

By Peter Vanderklish 5/2008

Elegant studies by many investigators working on Fragile X syndrome have determined that FMRP is an important regulator of mRNA translation in dendrites.  A critical observation to come out of these studies is that FMRP may regulate a very large set of mRNAs, with variable effects in terms of enhancement or inhibition depending on the particular mRNA.  The question of what proteins, and how many, show altered expression in the absence of FMRP has been open for some time.  Answering it is essential for a better understanding of how loss of FMRP alters synaptic function, and for a more comprehensive view of potential therapeutic approaches.  During the last funding period, our laboratory conducted collaborative proteomic studies with Dr.s Lujian Liao and John Yates at the Scripps Research Institute to identify synaptic proteins that are expressed at different levels in the mouse model of Fragile X syndrome (Fmr1 KO).  The data we are obtaining will be an important complement to studies by others in the field on protein changes and the identity of FMRP target mRNAs. 

Currently, we are investigating how several classes of proteins that exhibit expression changes in the Fmr1 KO mouse act to regulate synaptic structure and function.  Many of the identified proteins have been characterized previously in other systems as having morphoregulatory functions at synapses.  Adhesion molecules – proteins that mediate physical association of pre and postsynaptic elements – are a good example of this, as are proteases that cleave extracellular proteins at the synaptic junction.  We are conducting basic studies to determine whether altered levels of these and many other proteins are involved in, or perhaps sufficient for, the production of dendritic spine changes seen in Fmr1 KO mouse brain.  Our proteomic studies also revealed changes in proteins that have no obvious functions in the regulation of synaptic structure, but do have important influences in the firing characteristics of neurons.  Electrophysiological techniques are being used to determine if predicted changes in the number and timing of action potentials the neurons generate are evident in the Fmr1 KO mouse.  Such studies could uncover a previously unrecognized level of dysfunction in neuronal activity, one that may be amenable to drug therapy.  Our overarching goal for the coming year and beyond is to contribute to an understanding of the basic mechanisms producing synaptic dysfunction in Fragile X syndrome, and to hopefully broaden the set of potential therapeutic strategies.

by Michael Tranfaglia, FRAXA, 3/2007

Specific Tests of the mGluR Hypothesis

This research group at Scripps is using the most sophisticated proteomic techniques currently available to understand exactly how the absence of FMRP affects levels of other proteins in neurons. "Our proteomic work is motivated by the fact that the most critical gap in our understanding of the molecular basis of FXS resides in the identification of proteins that are dysregulated in the absence of FMRP. Studies of the mRNAs associated with FMRP suggest that it regulates the synthesis of several proteins that can affect synaptic structure and function. However, the number of validated changes in proteins is relatively small compared to the estimated number of FMRP target mRNAs, and there is as yet no reliable way to estimate protein changes from mRNA data. In addition, mRNA methods are not sensitive to potential changes in proteins encoded by mRNAs that are not FMRP targets. Given these and other considerations, it is likely that many more protein changes are critically involved in producing the synaptic alterations seen in FXS."

This investigation has already led to identification of several proteins that may contribute to the abnormalities in synaptic shape seen in fragile X and also a few proteins which have been implicated in autism. One protein presents a potential opportunity for therapeutic intervention with drugs taht are currently in clinical trials for unrelated indications.

Before he became a Fragile X investigator, Dr. Peter Vanderklish had demonstrated that activation of group I metabotropic glutamate receptors (mGluR1 and mGluR5) could cause rapid changes in dendritic spine shape. In as little as 15 minutes, spines of cultured neurons could become long, thin, and immaturelooking. Since this shape is characteristic of the spine shape previously seen in Fragile X brains, this would appear to support the notion that excessive function of these mGluR pathways might be the primary pathology in Fragile X.

Since 2003 when Dr.Vanderklish attended a Fragile X Banbury meeting, he has been studying neurons from the Fragile X knockout mouse in great depth. Initial studies in his lab have shown therapeutic effects of MPEP (the prototype mGluR5 antagonist) in his model system. One of the most intriguing aspects of this line of inquiry is that it strongly suggests that some structural changes seen in Fragile X brains may not only be treatable, but may reverse surprisingly rapidly with specific treatment.

by Pete Vanderklish, 3/2004

Our Previous Work

Consistent with the mGluR theory (see Mark Bear's abstract ), we observed that stimulation of mGluRs leads to elongation of dendritic spines. These changes in dendritic spine shape are dependent on protein synthesis and resemble those that occur in the Fragile X brain. Interestingly, multiple lines of evidence indicate that LTD and spine elongation are mechanistically linked; that is, that longer, thinner spines express the depressed synaptic state. Thus, altered synaptic plasticity and morphology (shape) may result from the same translation-dependent process that, once induced, is not properly limited in the Fragile X brain. As the saying goes, form follows function.

Our 2004-5 Project

We are testing three predictions of the mGluR theory:

1. Mark Bear and Kim Huber have already shown that LTD is enhanced in mice lacking FMRP. We predict that mGluR-induced spine elongation should be exaggerated in these mice. We are using live-cell imaging techniques to test this possibility and the ability of candidate pharmacological therapies for Fragile X, such as Ampakines and MPEP, to correct any imbalances.

2.We have evidence that two modes of translation initiation operate in dendrites (CAP-dependent and IRESdependent), and that stimulation of mGluRs primarily activates just one of these (cap-dependent). The mGluR hypothesis predicts that lack of FMRP increases CAP-dependent translation; we are testing to see if this is true.

3. Finally, we are testing whether preponderance of long, thin, and presumably lower efficacy, synapses in the Fragile X brain leads to compensatory changes in neurons. Recent research has shown that neurons adapt to deficits in net input by lowering firing thresholds and altering a number of intrinsic properties. If long, thin spines reduce net synaptic input, we would expect to see such changes, and they could underlie a number of symptoms of Fragile X. We are characterizing the intrinsic properties of neurons in the cortex and hippocampus of mice lacking FMRP. If differences are found with respect to control animals, this system could provide a testbed to see if potential therapeutic drugs (such as Ampakines and MPEP) can restore basic neuronal properties to their normal state.