Latrophilin-3 in Brain Function

According to the CDC, the prevalence of attention deficit hyperactivity disorder (ADHD) in the U.S. is 9.4% in children and 4.7% in adults. ADHD consists of three core symptoms: hyperactivity, inattention and impulsivity. ADHD shows 75% concordance among identical twins indicating genetic contributions, however, no single or few genes have been identified that account for the heritability. Instead, ADHD is polygenic. Multiple small-effect gene variants contribute collectively in ways that are poorly understood. The most consistent associations are with dopamine, such as the dopamine reuptake transporter (DAT) and several dopamine receptors. A new association was discovered in a Columbian population with high prevalence of ADHD. Genetic analyses revealed a novel association with latrophilin-3 (LPHN3). The protein is an adhesion G protein-coupled receptor. There are 3 latrophilins in mammals, but only latrophilin-3 is brain-specific and is expressed most abundantly in striatum and hippocampus in dopamine and glutamate positive neurons. Latrophilins are highly conserved in that they are found in all higher and lower animals including insects. Yet the function of LPHN3 in the brain is unknown. The gene has been knocked out in Drosophila, zebrafish, mice, and now in our new rat model using CRISPR/Cas9 genome editing. In all four species, knocking out the gene results in hyperactivity. However, in our model, Lphn3 KO rats also have cognitive deficits in forms of learning related specifically to the striatum and hippocampus. The rats also have impaired CA1 long-term potentiation, increases in striatal protein levels of tyrosine hydroxylase (TH) and DAT and compensatory decreases in the dopamine D1 receptor and DARPP-32. We found striking increases in striatal dopamine release by fast scan cyclic voltammetry. More recently, we developed a floxed Lphn3 rat which we are crossing with TH-Cre rats to create conditional Lphn3 rats with LPHN3 deleted only in dopamine and norepinephrine positive cells to isolate the effect of LPHN3 in dopamine signaling apart from its role in other neuronal types.

Manganese Neurotoxicity

Children exposed to excess manganese (Mn) develop learning problems and ADHD at higher rates than in typically developing children. Mn is an essential micronutrient used physiologically as a cofactor in enzymatic reactions, but excess Mn can be neurotoxic and children are more susceptible to Mn neurotoxicity than adults. However, the mechanism of Mn neurotoxicity is poorly understood. Cellular regulation of Mn depends on several importers, exporters, and transporters. The most important of these is solute carrier 30a10 (Slc30a10). There is a rare human genetic disorder in which SLC30A10 is null. These children develop Mn neurotoxicity gradually even if on a Mn free diet. Efforts to create models of Slc30a10 dysfunction have been unsuccessful because knocking out the gene constitutively results in death in mice within 7 weeks. To avoid this, we created a floxed Slc30a10 rat using CRISPR/Cas9. We crossed these rats with TH-Cre rats to create a line that has Slc30a10 deleted only in dopamine and norepinephrine positive cells. These rats are viable and we have new data that they have learning and memory deficits and other phenotypic abnormalities.  These results are a first step in determining the role of this transporter in regulating neuronal Mn. We are testing the idea that Mn in the diet causes the ion to slowly accumulate intracellularly since there is no Slc30a10 to transport Mn out of the cell, and then Mn reaches a threshold where it becomes neurotoxic.

Effects of Protons on Brain

Cranial irradiation is an essential curative treatment for many brain tumors, but it can impair neurocognitive function. Proton radiotherapy decreases toxicity by reducing radiation exposure of normal brain, but has equivalent efficacy compared with X-ray radiotherapy. A recent discovery is that high dose-rate, short duration irradiation (FLASH) causes less off-target toxicity compared with equivalent doses of conventional proton radiation. For example, electron beam FLASH induced less pulmonary fibrosis and intestinal mucositis with improved survival in mice compared with conventional dose-rate electrons. Electron and X-ray beam FLASH induced less brain injury and cognitive deficits in mice, but there are few data on more clinically relevant proton FLASH and no studies after brain irradiation. Furthermore, little is known about the optimal radiation dose rate to achieve tissue protection from FLASH radiation. Whether FLASH proton irradiation spares normal brain tissue and cognition better than conventional proton treatment has yet to be examined. In collaboration the Proton Center and physicists and oncologists, we are using rats to study the effects of protons on neurotransmitters, receptors, transporters, RNA expression, cytokines and behavior with special emphasis on effects on learning and memory, to identify the effects of FLASH vs. conventional protons and their mechanisms of action. We also plan to compare X-rays with protons during early brain development to determine of conventional and/or FLASH proton irradiation have advantages relative to X-rays.