Precise control of protein translation in neurons, particularly translation occurring in dendrites near synaptic sites, is critical for the proper regulation of synaptic strength. The most direct way to affect the strength of glutamatergic synapses is to alter the abundance of AMPA-type glutamate receptors (AMPARs). Here I demonstrate the critical role that two separate translational regulators play in controlling the translation of the AMPAR subunit GluR1, both under steady-state conditions and during synaptic plasticity.

Homeostatic synaptic plasticity adjusts the strength of synapses during global changes in neural activity, thereby stabilizing the overall activity of neural networks. Suppression of synaptic activity increases synaptic strength by inducing synthesis of retinoic acid (RA), which activates postsynaptic synthesis and insertion of AMPARs. Here, I show that the Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein that regulates dendritic protein synthesis, is essential for increases in synaptic strength induced by RA or by blockade of neural activity in the mouse hippocampus. Although activity-dependent RA synthesis is maintained in Fmr1 knockout neurons, RA-dependent dendritic translation of GluR1-type AMPARs is impaired. Intriguingly, FMRP is only required for the form of homeostatic plasticity which is dependent on both RA signaling and local protein synthesis. Expression of FMRP in knockout neurons reduced the total, surface, and synaptic levels of AMPARs, implying a role for FMRP in regulating AMPAR abundance. Critically, postsynaptic expression in knockout neurons of wild-type FMRP, but not two different mutant forms of the protein, was able to fully restore synaptic scaling.

microRNAs (miRNAs) are small RNA molecules which bind to the untranslated regions of mRNAs and inhibit translation. Using a bioinformatics approach, I identified a pair of miRNAs, miR-96 and miR-182, which bind specifically to a known sequence in the GluR1 mRNA and prevent its translation. When overexpressed, these miRNAs reduce total and extrasynaptic levels of GluR1 protein in neurons, and prevent the induction of homeostatic plasticity by activity blockade. Both miR-96 and miR-182 are expressed in cortex throughout postnatal development, although we were unable to detect activity-dependent changes in the abundance of either miRNA. Attempted knockdown of both miRNAs revealed no significant effect on the abundance of GluR1 or the ability of neurons to undergo homeostatic plasticity.

Taken together, these data offer significant insight into the regulation of local translation of glutamate receptors at the synapse, particularly during specific forms of synaptic plasticity. These results also suggest that some of the symptoms of Fragile-X syndrome may be attributed to defects in the induction of homeostatic plasticity.

Lymphatic dysfunction is associated with a wide array of disorders from transplant rejection to cancer metastasis. There are currently few effective treatments for lymphatic diseases. The transparent and easily accessible cornea provides an ideal tissue for lymphatic research because the growth of new lymphatics can be readily induced in a normal, healthy alymphatic cornea. While most studies have focused on preventing lymphangiogenesis (LG) before it is initiated, a new research direction of our lab is to define critical factors involved in lymphatic maturation and regression. This is important because many patients who are in need of lymphatic treatment are already at the middle or late stage of LG. Results from our studies have shown for the first time that luminal valves are formed inside corneal lymphatics as LG proceeds, and this process is mediated by an up-regulation of integrin alpha-9. Moreover, we have defined the peak of valvulogenesis in relation to LG, and the functional relevance of the valves in directing lymph flow. We also show that it is possible to suppress valvulogenesis with anti-integrin alpha-9 treatment in a transplantation model. Lastly, we provide strong evidence that corneal distribution of lymphatic vessels and valves are predominantly localized in the nasal region. Taken together, our studies not only reveal new mechanisms underlying lymphatic maturation but also provide novel therapeutic targets for lymphatic-related diseases occurring inside or outside the eye.

Excitatory transmission in the brain is largely mediated by synapses containing the neurotransmitter glutamate. Neuronal circuitry is first established early in brain development requiring the formation of vast numbers of glutamatergic synapses at individual sites of contact made between presynaptic axons and postsynaptic dendrites. Despite mounting efforts in the last decade to identify the complex molecular mechanisms underlying initial synaptogenesis and the subsequent steps of synapse maturation and stabilization, this complex process is still not well understood. Interestingly newly formed glutamatergic synapses in the young brain often lack postsynaptic AMPA-type glutamate receptors (AMPARs). As development progresses, AMPARs are trafficked into synaptic sites but the significance of this event to the functional maturation of synapses remains unclear.

To investigate the role of postsynaptic AMPAR insertion in synapse maturation we used RNA interference (RNAi) to knockdown AMPARs in young cultured hippocampal neurons. Surprisingly, loss of postsynaptic AMPARs caused a concurrent reduction in synaptic responses mediated by NMDA-type glutamate receptors (NMDARs), without an apparent change in their synaptic expression. Strikingly, heterologous synapses formed between axons and co-cultured non-neuronal cells expressing AMPARs develop significantly fewer inactive presynaptic terminals, suggesting that AMPARs mediate a retrograde signal to promote presynaptic function. Indeed, the extracellular domain of the AMPAR subunit GluA2 was sufficient to reproduce this effect at heterologous synapses, indicating that this retrograde signaling is independent of AMPAR channel function. Our findings suggest that postsynaptic AMPARs perform an organizational function at synapses that exceeds their standard role as ionotropic receptors by conveying a retrograde trans-synaptic signal that increases the transmission efficacy at a synapse.

The cornea is the outermost layer of the eye serving as the first barrier from the outside environment which plays a crucial role in a clear vision. It is maintained by keeping transparency and avascularity under the normal state. This nature makes the cornea an ideal site to study lymphangiogenesis (LG), new lymphatic vessel growth, that can be induced by inflammation and assessed without any background vessels. The lymphatic vessels have long been neglected over blood vessels due to its invisibility, but its importance and involvement in inflammation, cancer metastasis, and transplant rejection are now well known since the discovery of several lymphatic-specific markers in recent years. Another important event that occurs during LG is valvulogenesis (VG). Previous studies from our lab have shown that lymphatic vessels develop luminal valves in the cornea during LG, and these valves express integrin alpha 9 (Itga-9) that plays a critical role in directing lymph flow.

My study consists of three parts: 1) to investigate the dynamic changes of LG and VG during corneal inflammation. This was revealed by our newly developed intravital imaging system; 2) to assess the role of VG itself or VG and LG together in corneal transplant rejection; and 3) to explore the cellular origin of corneal lymphatic endothelial cells that comprises lymphatic vessel walls.

First, using the intravital imaging technology, I demonstrated the multifaceted dynamics of LG and VG associated with corneal transplantation, from the initiation to regression phases, and reported several novel and critical phenomena and mechanisms that couldn’t be detected by conventional ex vivo immunohistochemistry.

Secondly, I investigated corneal transplant rejection reaction after blocking VG alone or in combination of LG. The results showed that anti–Itga-9 treatment alone suppressed corneal VG after transplantation. While this treatment did not affect LG, it promoted corneal graft survival in the low-risk setting. This study has provided the first evidence on the critical role of VG in mediating graft rejection. Furthermore, I have shown that combined blockade of VEGFR-3 (vascular endothelial growth factor receptor-3) and Itga-9 significantly suppressed both LG and VG after corneal transplantation, and this treatment led to a markedly promoted survival rate in the high-risk setting.

Lastly, I have provided the first in vivo evidence showing that transplanted bone marrow (BM) derived cells integrated into the newly formed lymphatic vessels in inflamed host corneas with the live imaging system.

In summary, my study reveals novel molecular and cellular mechanisms that contribute to LG and VG in the cornea. It is our hope that understanding these mechanisms would lead to novel therapies not only for corneal transplant rejection and inflammation, but also for lymphatic-related disease occurring outside the eye, such as cancer metastasis and lymphedema.

Homeostatic synaptic plasticity (HSP) is a form on non-Hebbian plasticity that allows neurons to sense their global level of activity and modulate their own function to keep firing rate within a working range. In particular, chronic elevation or reduction of network activity activates compensatory mechanisms that modulate synaptic strength in the opposite direction (i.e. reduced network activity leads to increased synaptic strength). Among the many mechanisms that mediate homeostatic synaptic plasticity, retinoic acid (RA) has emerged as a novel signaling molecule that is critically involved in homeostatic synaptic plasticity induced by blockade of activity. In neurons, global silencing of synaptic transmission triggers RA synthesis. RA then acts at synapses by a non-genomic mechanism that is independent of its well-known function as a transcriptional regulator, but operates through direct activation of protein translation in neuronal dendrites. Protein synthesis is activated by RA-binding to its receptor RARα, which functions locally in dendrites in a non-canonical manner as an RNA-binding protein that mediate RA's effect on translation.

In this dissertation I demonstrate the critical role that translational regulation operated by RA plays in synaptic scaling, a post-synaptic form of homeostatic plasticity which consists in the regulation of receptor abundance on the post-synaptic membrane in response to activity blockade. In this context, my work has focused on the regulation of both excitatory and inhibitory transmission operated by RA in order to elucidate whether this molecule, normally only associated with neural development, plays a pivotal role in the modulation of excitation and inhibition balance in the adult brain. Since, abnormalities in this balance have been linked to several pathological conditions such as autism and Fragile-X mental retardation, my work described here emphasizes the primary role of the dynamic regulation of the excitation/inhibition balance in neural networks mediated by RA.

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