A distinct microRNA expression profile is associated with α[11C]-methyl-L-tryptophan (AMT) PET uptake in epileptogenic cortical tubers resected from patients with tuberous sclerosis complex

Abstract

Tuberous sclerosis complex (TSC) is characterized by hamartomatous lesions in various organs and arises due to mutations in the TSC1 or TSC2 genes. TSC mutations lead to a range of neurological manifestations including epilepsy, cognitive impairment, autism spectrum disorders (ASD), and brain lesions that include cortical tubers. There is evidence that seizures arise at or near cortical tubers, but it is unknown why some tubers are epileptogenic while others are not. We have previously reported increased tryptophan metabolism measured with α[¹¹C]-methyl-l-tryptophan (AMT) positron emission tomography (PET) in epileptogenic tubers in approximately two-thirds of patients with tuberous sclerosis and intractable epilepsy. However, the underlying mechanisms leading to seizure onset in TSC remain poorly characterized. MicroRNAs are enriched in the brain and play important roles in neurodevelopment and brain function. Recent reports have shown aberrant microRNA expression in epilepsy and TSC. In this study, we performed microRNA expression profiling in brain specimens obtained from TSC patients undergoing epilepsy surgery for intractable epilepsy. Typically, in these resections several non-seizure onset tubers are resected together with the seizure-onset tubers because of their proximity. We directly compared seizure onset tubers, with and without increased tryptophan metabolism measured with PET, and non-onset tubers to assess the role of microRNAs in epileptogenesis associated with these lesions. Whether a particular tuber was epileptogenic or non-epileptogenic was determined with intracranial electrocorticography, and tryptophan metabolism was measured with AMT PET. We identified a set of five microRNAs (miR-142-3p, 142-5p, 223-3p, 200b-3p and 32-5p) that collectively distinguish among the three primary groups of tubers: non-onset/AMT-cold (NC), onset/AMT-cold (OC), and onset/AMT-hot (OH). These microRNAs were significantly upregulated in OH tubers compared to the other two groups, and microRNA expression was most significantly associated with AMT-PET uptake. The microRNAs target a group of genes enriched for synaptic signaling and epilepsy risk, including SLC12A5, SYT1, GRIN2A, GRIN2B, KCNB1, SCN2A, TSC1, and MEF2C. We confirmed the interaction between miR-32-5p and SLC12A5 using a luciferase reporter assay. Our findings provide a new avenue for subsequent mechanistic studies of tuber epileptogenesis in TSC.

Keywords: Epilepsy; Gene expression; Positron emission tomography; Seizure; Tryptophan; Tuberous sclerosis complex; microRNA; α[(11)C]-methyl-L-tryptophan.

Figure 1

qPCR validation of differentially expressed microRNAs. Five of the differentially expressed microRNAs (Table 2) were validated using qPCR. Blue circles indicate non-onset/AMT-cold tubers, blue triangles indicate onset/AMT-cold tubers, and red triangles indicate onset/AMT-hot tubers. Consistent with our microRNA array data, there was significant upregulation of these miRNAs in OH samples as compared to NC or OC. Relative expression is in log2 units. P values are shown for statistically significant comparisons.

Figure 2

The trend of microRNA induction is consistent in patient-matched pairs of tissues. OH/NC fold changes were calculated for patient-matched pairs of OH and NC tissues from three subjects (83002, 81603, L1412RK). Fold change values (log2) were derived from qPCR expression data. Error bars represent standard error of the mean.

Figure 3

Seizure onset/AMT-hot tubers have elevated levels of miRs 142-3p and 223-3p. Expression levels measured with qPCR reveal coincident induction of miRs-142-3p and −223-3p in seizure onset/AMT-hot TSC tubers (R2 = 0.931). All onset/AMT-hot tubers had elevated expression of the two microRNAs.

Figure 4

Linear discriminant analysis (LDA) demonstrates that expression profiles of the five microRNAs can distinguish cortical tubers by their seizure onset propensity and AMT-PET uptake status. The axes represent linear combinations of the microRNA expression values, optimally selected to discriminate the tuber categories. Each sample is represented by the respective tuber category symbol (onset/AMT-hot (OH): red triangles; onset/AMT-cold (OC): blue triangles; non-onset/AMT-cold (NC): blue circles). Colored ellipses represent the 95% confidence region for each category. The correct tuber category is predicted for 95% of samples.

Figure 5

miR-32-5p targets and inhibits SLC12A5 at the transcript level. Computational analysis revealed that the highest scoring microRNA predicted to target the SLC12A5 3’ UTR was miR-32-5p. A) Gene and microRNA expression profiles measured with qPCR demonstrate a significant anti-correlation between miR-32-5p and SLC12A5 expression in our set of TSC tubers (P≤ 0.0001). B) A luciferase reporter assay was used to confirm that miR-32 directly targets the 3’ UTR of SLC12A5 and causes transcript repression.