Temporal Lobe Epilepsy: Sex-Based Differences and Neuropeptide Locations

Temporal lobe epilepsy (TLE) is the most common variant of epilepsy. It is also the form of epilepsy least responsive to treatment. Many individuals with TLE demonstrate hippocampal sclerosis, which is the loss of significant neuronal cells and gliosis in specific areas of the Ammon’s horn (regions 1 and 3). This condition may arise following a brain injury, tumor, illness (meningitis or encephalitis) or childhood trauma. The underlying mechanisms of the disease are poorly understood.

 

Recently, Mériaux et al. used shotgun proteomics to map seven neuropeptides—neuropeptide Y (1–30), somatostatin-14, neurokinin B, galanin, cortistatin, chromogranin B fragment, and cocaine- and amphetamine-regulated transcript (CART) peptide fragment—located in the hippocampus of individuals with TLE.¹ They also revealed that the proteins detected differ based on the biological sex of the patient.

 

To do this, the researchers collected tissue from 20 individuals with TLE and applied a matrix-assisted laser desorption/ionization (MALDI) matrix prior to mass spectrometry imaging (MSI). They prepped the instrument to linear positive ion mode (mass range m/z 500–10,000) and established 50 μm as the distance between raster points for 300 laser shots at a repetition rate of 200 Hz. Using FlexAnalysis 3.2, FlexImaging 2.1 and ClinProTools 2.2, the team processed, visualized, and statistically evaluated the spectral data. The SwePep relational database served as a reference.

For tissue proteomics, the researchers then removed the matrix and manually microdissected the samples. They loaded the extracts on polyacrylamide gel and performed separation by electrophoresis before in-gel trypsin digestion. Then, they used online reversed-phase chromatography on the Easy-nLC system (Thermo Scientific) with a Proxeon 100 μm ID x 2 cm trap column and a 100 μm ID x 10 cm C18 packed-tip column to separate the prepared samples. The parameters for elution were an increasing gradient of AcN (5% to 30% over 120 minutes) and a 300 nL/min flow rate. The research team coupled the chromatography system to an LTQ Orbitrap XL Hybrid Ion Trap-Orbitrap mass spectrometer (Thermo Scientific) operating in data-dependent mode with 60,000 (FWHM) resolving power and a 300 to 2,000 m/z mass range. They selected precursor ions with an intensity over 500 counts for collision-induced dissociation fragmentation, analyzing the 20 most intense ions identified by survey scan with a dynamic exclusion of 20 sec. Finally, the team processed the mass spectra with Proteome Discoverer Software revision 1.4 and matched them against the Swiss-Prot human database with the SEQUEST algorithm (Thermo Scientific).

 

For specific localization of the neuropeptides, Mériaux et al. investigated the layers of the dentate gyrus of the hippocampus. In the molecular layer, they identified somatostatin-14 (1637.5 m/z), oxidized chromogranin B (357–374) + phosphate (2110.1 m/z), and thymosin beta 4 (4938.6 m/z). In the granular layer, they identified neurokinin B (1210.4 m/z) and galanin (3155.1 m/z). In the polymorphic layer, they identified CART (28–36) (1066.5 m/z), cortistatin 14 (1746 m/z), cortistatin 14 Na+ (1763 m/z), neuropeptide Y (1–30) (3454.4 m/z), and thymosin beta 4 (4938.6 m/z). The researchers posit that these data may serve as potential targets for the development of drug therapies.

 

The researchers achieved reproducibility of >98% with tissue proteomics. They identified 8,435 proteins involved in various functions, including outgrowth of neurites, neuronal differentiation, tubule polarization, cell migration, cytoskeleton networking, intracellular contacts, growth factors, cellular signaling and tumor suppressors (including previously identified LGI1).² Of the identified proteins, 1,450 were specific to male-type TLE, and 1,052 were specific to female-type TLE. Interestingly, only TLE samples from male patients contained tumor suppressors (DMBT1, TUSC2, MGEA5, GBAS, CNDP2 and LIGI1). According to the authors, this grouping, along with the presence of other specific proteins (PVRL1, FSTL4, GRIN1, BPIB1, SRGP2 and APBA1), forms a “signature” for male-type TLE. Among the samples taken from male patients, the individual variation ranged from 1% to 3.8%. Similarly, female-type TLE also bears a signature formed by the presence of specific proteins, mostly related to brain synaptic plasticity (OPTN, OPALIN, LIN7C), odorant receptors (REEP2), growth factors (SESN2, ALS, BMR1B, IRF2, ABI1), and actin-associated cytoskeleton proteins (SRC8, AP2S1).

 

The researchers note that the specific hormones active in male versus female patients play a role in the sex-based mechanisms of the disease.³ This, coupled with the sex-specific protein expression evident in this study, confirms the previous assumption that the TLE sexome contributes to the divergent expression of TLE in male and female patients.

 

References 

1. Mériaux, C., et al. (2014) “Human temporal lobe epilepsy analyses by tissue proteomics,” Hippocampus [e-pub ahead of print], doi: 10.1002/hipo.22246.

 

2. Kalachikov, S., et al. (2002) “Mutations in LGI1 cause autosomal dominant partial epilepsy with auditory features,” Nature Genetics, 30(3) (pp. 335–41).

 

3. Christensen, J., et al. (2005) “Gender differences in epilepsy,” Epilepsia 46(6) (pp. 956–60).

 

Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.