Our goal is to identify new molecular pathways that can be exploited for therapeutic purposes in neurodevelopmental disorders characterized by autism and mental retardation. Fragile X syndrome is the most common inherited form of mental retardation and autism. The vast majority of affected individuals have an expanded CGG repeat within the Fmr1 gene, leading to a loss of function phenotype. How this translates into the complex neuropsychiatric phenotype of fragile X is not yet understood.
Interestingly, brains of children with fragile X have abnormally long and densely packed dendritic spines, and the same defect has also been observed in Fmr1 knockout mice. These bizarre spines are reminiscent of dendritic protrusions normally present in the developing brain: filopodia. It has therefore been proposed that fragile X may be caused by a failure in the normal transition from filopodia to spines. Yet, the development of dendritic filopodia in the first postnatal days, at the peak of their expression, has never been examined in fragile X mice.
We recently described the developmental maturation of dendritic filopodia in Layer 5 pyramidal neurons in acute slices of mouse primary visual cortex. We established that filopodia in dendritic shafts elongate in response to the neurotransmitter glutamate. This observation suggests that glutamate released by nearby axons may recruit filopodia to form early synaptic contacts. These filopodial synapses presumably stabilize filopodia and allow them to mature into spines. It is conceivable that impaired filopodial dynamics that interfere with their ability to make synapses, might lead to defects in spine elaboration similar to those seen in fragile X. The notion that glutamate neurotransmission might be perturbed in fragile X is further supported by a recent theory about fragile X known as the metabotropic glutamate receptor hypothesis.
We therefore want to test the hypothesis that a defect in filopodia, linked to abnormal glutamate signaling or downstream molecular pathways, impairs their ability to mature into spines, leading to the dendritic abnormalities found in fragile X. Because filopodia are motile, exploring this would require imaging neurons in real time, preferably with preserved connectivity. Therefore, the experiments should be done in vivo, because the disruption of circuits that occurs after preparing acute or cultured slices is likely to have an impact on these phenomena. Sadly, it has not yet been possible to image neurons in the intact brain of living newborn mice.
To overcome this challenge, we have developed a novel and sophisticated method for imaging filopodia in neonatal mice using two-photon microscopy. We intend to use this state-of-the-art brain imaging technique to examine the maturation of dendritic filopodia and their role in synapse and spine formation during normal development and in fragile X mice. First, we will look for abnormalities in filopodia dynamics in the mutant mice, using two-photon microscopy through a small optical 'window' in the skull. Second, we will examine whether the ability of filopodia to respond to glutamate is impaired in the mutant mice, using two-photon glutamate uncaging together with pharmacology to interfere with various signaling pathways. Third, we will test whether the filopodia to spine transition is affected in fragile X.
The experiments proposed combine the use of innovative imaging techniques, pharmacology, and molecular approaches to unravel the defective signaling pathways that lead to aberrant spine maturation in fragile X. This research may uncover new strategies for treating autism.
Dr. Portera-Cailliau's laboratory has been funded by the Fragile X Research Foundation (FRAXA). Grants are also pending from the NIH (RO1) and other private foundations, including the March of Dimes Foundation.