Now a research team from UHealth’s Bascom Palmer Eye Institute at the Miller School demonstrates for the first time how these disease processes cause important changes to neurons in the retina. The discoveries are not only important scientifically, but they will facilitate clinical interventions to potentially reverse – or ideally prevent — vision loss before patients become symptomatic.
Sanjoy K. Bhattacharya, Ph.D., M.Tech., associate professor of ophthalmology, study lead co-authors Di Ding, Ph.D., post-doctoral associate, and graduate student Mabel Enriquez-Algeciras, and their associates discovered fundamental changes to dendritic cells of neurons in the retina that correlated with loss of vision in experimental disease models. They report their findings in the January 2 issue of the Journal of Clinical Investigation.
The process of protein deimination proves to be central to both vision loss and its potential treatment. Dendrites with lower levels of protein deimination tend to be stunted, which impairs their connectivity and, the researchers theorize, contributes to progressive vision loss in demyelinating diseases. This discovery of posttranslational modifications in the proximal part of the neuron represents a major paradigm shift, said Bhattacharya.
Traditionally, researchers focused on changes to the soma and distal portions of the neuron in people with demyelinating diseases. “Now people should also realize that the proximal part is equally important. And if we have a specific way of targeting the proximal part, it can be very, very important for health and disease,” Bhattacharya said.
For some patients in the not-too-distant future, these advances could obviate the need for complicated cell replacement therapy, for example. “The importance of this is that we showed, with a very tiny posttranslational modification, that we could restore visual function,” Bhattacharya said. Although they studied multiple sclerosis, where optic neuritis and vision loss often precede other symptoms, their molecular pathway discoveries also could improve the outcome of patients with other neurodegenerative disorders such as glaucoma.
Location also is important, according to their multiple sclerosis model using transgenic ND4 mice. Protein deimination, it turns out, is a good thing in neurons but can be harmful at high levels in the astrocytes in the brain. This new finding clarifies earlier work that only implicated elevated deimination globally in the brain in disease.
However, with this important discovery comes the challenge of developing therapeutics that target one cell type while sparing the other. “Development of therapeutics will not be easy. You need a therapy targeted to specific cells. If you want to bring overexpression of the deimination to the neurons,” said Bhattacharya, “and it goes into the astroglial cells, instead of doing a good thing you will be doing a bad thing.”
Using mass spectrometry, Bhattacharya and his team not only identified the most important proteins involved in deimination, but also some important anatomic locations. “This is the first time loss of deimination has been shown” in neurons in the retinal ganglion cell layer (GCL), for example. Reduction of GCL deimination correlated with loss of inner retina visual function and defective neurite outgrowth. They verified these findings through evaluation of cell population specific markers in stained retinal sections that supported the decreased outgrowth of dendrites in the GCL.
To verify the physiologic changes associated with vision loss in the ND4 mice retina, the investigators also recorded and compared pattern electroretinogram (PERG) findings between affected mice and controls. As expected, the amplitude of the PERG readouts dropped over time in ND4 mice and remained stable in controls, supporting a decrease in visual function over time in their experimental model.
The next step was to demonstrate that hypodeimination was directly involved in this visual loss. To accomplish this, Bhattacharya and his colleagues generated ND4 mice with exogenous optic nerve expression of the peptidylarginine deiminase PAD2, an enzyme that deiminates proteins and is found in excess amounts in the brains of people with multiple sclerosis. The mice that expressed PAD2 showed a greater than 30 percent gain in PERG amplitude. Additionally, isolated neurons from these mice demonstrated a 48 percent increase in neurite length compared with controls.
Not yet satisfied, the investigators next sought to identify the major deiminated proteins playing an essential role in the GCL. Using mass spectrometry they identified the RNA binding and export protein REF and its murine equivalent, REFBP-2. They demonstrated that decreased levels of deiminated REF are associated with reduced neurite outgrowth, or conversely, that replacement of exogenous deiminated REF could potentially promote elongation of neurites in the GCL and promote visual restoration.
Their report in the Journal of Clinical Investigation also explains how REF/REFBP-2 binds to mRNA and allows its export from the neuron nucleus, at which point the complex becomes more deiminated. Specifically, SNAP-25 mRNA has a particular affinity for deiminated REF and binds to it to promote neurite outgrowth. In vitro experiments verified that SNAP-25 translation is much more enhanced in the presence of deiminated versus non-deiminated REF.
Important to development of targeted therapeutics is the finding that neurite outgrowth is a local phenomenon in the dendrites modulated by specific mRNA species. REF, in particular, demonstrated this local activity in their studies.
Bhattacharya said their findings add to a growing realization that posttranslational modifications such as deimination regulate local dendritic protein synthesis. Importantly, loss or reduction of protein deimination precedes the progressive loss of visual function. Reduced deimination in the GCL, therefore, could provide an early clinical clue to damage or dysfunction of neurons.
“Increased dendritic connections could mean increased visual regeneration,” he said.
Their research also is important because it supports a mechanism for visual loss in demyelinating disease outside of the traditional immune-activated inflammation research. “Our discovery of local dendritic protein synthesis regulated by deimination opens up a potential new avenue for intervention strategies for development of new treatment modalities for neuropathies,” the authors wrote.
University of Miami