Probing Molecular Mechanisms of Epilepsy Progression

By Zhong Ying, MD, PhD, and Imad Najm, MD

This article is excerpted from the authors’ article in the July 2014 Annals of Clinical and Translational Neurology, which is copyrighted by the authors: Ying Z, Najm I, Nemes A, Pinheiro-Martins AP, Alexopoulos A, Gonzalez-Martinez J, Bingaman W. Growth-associated protein 43 and progressive epilepsy in cortical dysplasia. Ann Clin Transl Neurol. 2014;1(7):453-461.

Focal cortical dysplasias (FCDs) are the most common pathologic substrates in both adults and children with pharmacoresistant focal neocortical epilepsy. Postoperative seizure outcome has been less successful in patients with FCDs as compared with patients who have mesial temporal lobe epilepsy due to hippocampal sclerosis (mTLE/HS).

Previous studies suggest that the most important predictor of success following epilepsy surgery is complete resection of the epileptic focus. There has been increasing awareness, however, that epileptogenicity in FCDs encompasses a more complex network extending beyond the lesion. Moreover, epilepsy associated with FCDs is a progressive disease with compelling evidence of seizure worsening over time, change of EEG patterns and improved outcomes with early surgical resection.

Focusing on GAP-43

At Cleveland Clinic’s Epilepsy Center, we aim to discover the molecular mechanisms that underlie epilepsy progression in FCD. Our translational research has focused on growth-associated protein 43 (GAP-43) as a potential substrate contributing to epileptogenic networks and the progression of epileptogenesis.

GAP-43 has been known as a marker for axonal growth and synaptic plasticity and is maximally expressed during brain development. Once growing axons reach their targets and synaptogenesis is established, GAP-43 levels rapidly decline. Re-expression of GAP-43 in human adult brain occurs during axonal sprouting following stroke, mossy fiber sprouting in sclerotic hippocampi, and axonal regeneration in multiple sclerosis and post-traumatic brain injury lesions.

GAP-43 and Epileptogenicity

We investigated GAP-43 levels using western blot analysis in electrophysiologically defined epileptic vs. nonepileptic brain samples from three patients with FCD-associated intractable epilepsy (using extraoperative recordings from subdural grid electrodes or stereotactically implanted depth electrodes/SEEG).

As shown in Figure 1, the epileptic brain samples from all three patients (pathology-confirmed FCD type II lesions) exhibited clear within-patient increases of GAP-43 relative to each patient’s corresponding nonepileptic area. The optical densities of GAP-43 bands demonstrated that, in each patient, the epileptic area clearly showed higher gray values compared with the nonepileptic cortex.

Figure 1. Western blot analyses of GAP-43 in three patients. Each patient had samples taken from subdural grid-characterized epileptic areas (lanes A´, B´ and C´) and nonepileptic areas (lanes A, B and C). (A and A´ are from the first patient, B and B´ from the second patient, etc.) Note the greater GAP-43 levels in the epileptic areas relative to the nonepileptic areas in all three patients.

These findings show that GAP-43 is differentially upregulated in FCD epileptic cortex compared with adjacent nonepileptic cortex within the same patient, indicating that GAP-43 expression may contribute to (and be a potential biomarker of) epileptogenic mechanisms.

Increased GAP-43 Expression at the Cellular Level

GAP-43 is synthesized in the neuronal cell body as a soluble protein that is quickly bound to the membranes, packaged on vesicles and transported in the rapid phase of transport down axonal processes; therefore, GAP-43 accumulates in the somata.

We studied GAP-43-stained elements around cell surface (rim) structures and intercellular tubular punctate structures. The features of GAP-43 immunohistochemistry in normal and dysplastic cortex are shown in Figure 2.

Figure 2. Photomicrographs of cresyl echt violet (CV) and GAP-43 immunohistochemistry (IHC) staining from normal-appearing cortex (panels A, C and E) and dysplastic cortex (panels B, D, F, G, H and I). Normal-appearing cortex: A CV-stained section (A) shows well-laminated cortical pyramidal cells with dendrites appropriately positioned toward the pial surface. The adjacent section with GAP-43 IHC (C) shows only background staining. At higher magnification (E), no specific GAP-43 immunostaining is seen in cell bodies or intercellular space. Dysplastic cortex: A CV-stained section (B) shows that the vertical and horizontal laminations are disrupted and dysmorphic cells are darkly stained. In this area, GAP-43 (D) shows increased immunoreactivity. Higher magnification (F) reveals GAP-43 staining of the cell surface in a rim pattern as well as stained punctate clumps or tubular structures. Those GAP-43-stained patterns in dysplastic cortex are illustrated at higher magnification (I). The balloon cells appear as strikingly large opalescent cytoplasm with eccentric nuclei (G). Some of these balloon cells are faintly stained with GAP-43 in the cytoplasm (H).

The normal-appearing cortex is defined by well-preserved columnar organization and horizontal lamination with no dysmorphic neurons. In normal-appearing neocortex, there was an absence of GAP-43 immunoreactivity in the neuronal somata and cell surface as well as a lack of punctate tubular elements; GAP-43 immunohistochemistry showed homogeneous, nonspecific background staining.

In the dysplastic cortex, GAP-43 immunoreactivity was seen within the neuronal cell surface, resulting in a rim staining appearance, and was also present between neurons, giving the appearance of punctate tubular structures. This punctate tubular staining can have an intensely “clumped” appearance at higher magnification. Neuronal somata were negative for GAP-43 immunoreactivity.

GAP-43 and Epilepsy Duration

We also performed semiquantitative analyses of GAP-43 immunohistochemistry labeling by grading the percentage of cells with GAP-43-stained rim appearance and the intensity of GAP-43-stained punctate tubular structures. The final GAP-43 grading score for each brain specimen was obtained by adding scores of both rim and tubular staining. We studied GAP-43 immunohistochemistry in samples from three groups of patients:

• 12 non-temporal lobe specimens from patients with pathologically verified FCD type II (IIA or IIB)
• 9 non-temporal lobe specimens from patients with FCD type IA
• 20 histologically normal neocortical temporal lobe specimens from patients with mTLE/HS (used as an “epilepsy” control group)

As illustrated in Figure 3, higher GAP-43 immunoreactivity scores showed a significant correlation with longer epilepsy duration only in patients with FCD type IIA/B pathology (p < .0001; ordinal logistic regression). Despite the absence of a significant difference in epilepsy duration between FCD IIA/B and FCD IA groups, GAP-43 scores in patients with FCD IA were not correlated with epilepsy duration (Figure 3B), although it should be noted that the FCD IA group had a small sample size. Similarly, GAP-43 scores did not correlate with duration of epilepsy in the mTLE/HS group even though these patients had longer epilepsy duration (Figure 3C).

Figure 3. Analysis of GAP-43 immunoreactivity and epilepsy duration in three groups of patients: (A) 12 patients with FCD type IIA/B pathology, (B) nine patients with FCD type IA pathology and (C) 20 patients with mTLE/HS. There is no significant difference in epilepsy duration between the FCD IIA/B and FCD IA groups despite the presence of two patients with epilepsy of more than 20 years’ duration in the former group. Note that the association between epilepsy duration and GAP-43 score is significant only in the patients with FCD type IIA/B pathology (p < .0001).

Among many plausible epileptogenic mechanisms, our hypothesis is that dysplastic neurons in FCD type II retain their ability to continue upregulating GAP-43 expression, which may associate with synaptogenesis and the presence of positive feedback interplay between intrinsic epileptic discharges.

The mechanisms for increased expression of GAP-43 are likely multifactorial, including such factors as an intrinsic program-driven increase of GAP-43 mRNA in dysmorphic neurons (but not in normal pyramidal cells).

In addition, the increase of GAP-43 could be modulated by N-methyl-D-aspartate (NMDA) receptor activation. Our studies have demonstrated upregulation of the NMDA receptor complex in dysplastic neurons.

Together, the upregulation of NMDA subunits could provide a molecular-functional underpinning for seizure-dependent (and time-dependent) selective GAP-43 expression in FCD type II lesions. In turn, the plastic changes of GAP-43-associated synaptogenesis and rearrangement of gap junction channels could account for the in situ expansion of local epileptic networks that may underlie the temporal progression of epilepsy in patients with type II FCDs.

Clinical Implications: A Biomarker for Epileptogenicity and Epileptogenesis?

Our research results showed increased GAP-43 expression in dysplastic neurons and that this increase is differentially expressed within the epileptic focus vs. adjacent nonepileptic brain regions. This rather specific expression of GAP-43 indicates fundamental pathophysiologic mechanisms for expression of epileptogenicity in FCD.

Assuming that GAP-43 expression is a biological correlate of disease progression/epileptogenesis — and considering our current findings indicating an increase in GAP-43 expression in relationship with longer epilepsy duration in patients with FCD type II — it is possible that early timing of resection of FCD type II lesions may halt epileptogenesis.

Our preliminary findings suggest that GAP-43 may be considered as a pathology-specific biomarker for both epileptogenicity and the development/progression of epilepsy in patients with type II FCD. Further studies in a larger patient population are needed to confirm and validate these results.

Dr. Ying is a staff physician in the Epilepsy Center in Cleveland Clinic’s Neurological Institute.

Dr. Najm is Director of the Epilepsy Center.