The role of mTORC1 in TSC and epilepsy

Tuberous Sclerosis Complex (TSC) is a multi-system disease where benign tumors grow on organs such as the brain, kidneys, heart, eyes, skin, and lungs. It can also appear in the central nervous system and result in epilepsy, autism, or other brain issues. The biggest affect on patients are central nervous system disorders.

According to the National Institute of Neurological Disorders and Stroke, most people with TSC have seizures at some point in their lives.

However, there is evidence to support a role of mTORC1 inhibitors as signals for the neurological symptoms associated with TSC. These inhibitors are being tested as potential treatments for TSC. Understanding how these mechanisms work is important to identifying specific targets for therapy to minimize side effects found with inhibiting mTOR.

Michael Wong MD PhD, at Washington University in St. Louis, has been researching proteins indicating inflammation in brain tissue of TSC patients, and has won an Exploration Hypothesis Development Award.

Dr. Wong along with his colleagues, screened for inflammation markers in a mouse model of TSC that develops seizures around 3 to 4 weeks of age.

When the researchers checked at 2 weeks of age, they found mRNA levels were different for three molecules, CCL2, IL-1beta, and CXCL10.

The finding suggests increases in these three affect seizure increase:

CCL2 - a chemokine which induces protein chemotaxis (increases or decreases a substance) by nearby cells; is considered a pro-inflammatory and can recruit the immune system to fight infection or homeostatic and in control of migration of cells for maintenance or development.

IL-1beta or interleukin 1 beta - a cytokine (protein and signaling molecule) that functions in the immune systeml; rare deficiencies of it can cause autoimmune disease or immune deficiency; cytokines promote development and differentiation of T and B lymphocytes hematopoietic (blood) cells; interleukin receptors on astrocytes in the hippocampus of mice are known to be involved in spatial memory

CXCL10 - a G protein coupled gene which produces protein products such as C-X-C motif chemokine ligand 10, INP10, SCYB10,  or "small inducible cytokine subfamily B (Cys-X-Cys), member 10".

All three precede the advent of seizures. But, as expression differences also appear later in other molecules at 4 weeks of age, they do not explain how.

The mice were treated with either rapamycin, a known inhibitor of mTORC1, or epicatechin-3-gallate (ECG) which is reported to inhibit production of IL-1beta and CXCL10.

After treatment — both compounds reversed the increase in IL-1beta and CXCL10.

Since rapamycin has been previously studied in epilepsy, Dr. Wong focused on using epicatechin-3-gallate (ECG) in the mice.

He found it decreased seizure development and slightly improved their survival, while only minimally correcting some brain tissue abnormalities.

Dr. Wong and his laboratory have provided a proof-of-concept preclinical study which suggests that anti-inflammatory treatment could be a potential therapy for epilepsy in TSC patients.

Abstract
Epilepsy and other neurological deficits are common, disabling manifestations of the genetic disorder, tuberous sclerosis complex (TSC). Brain inflammation has been implicated in contributing to epileptogenesis in acquired epilepsy due to brain injury, but the potential role of inflammatory mechanisms in genetic epilepsies is relatively unexplored. In this study, we investigated activation of inflammatory mediators and tested the effects of anti-inflammatory treatment on epilepsy in the Tsc1-GFAP conditional knock-out mouse model of TSC (Tsc1GFAPCKO mice). Real-time quantitative RT-PCR, immunohistochemistry, and Western blotting demonstrated increased expression of specific cytokines and chemokines, particularly IL-1β and CXCL10, in the neocortex and hippocampus of Tsc1GFAPCKO mice, which was reversed by treatment with a mammalian target of rapamycin complex 1 (mTORC1) inhibitor. Double-labeling immunohistochemical studies indicated that the increased IL-1β was localized primarily to astrocytes. Importantly, the increase in inflammatory markers was also observed in astrocyte culture in vitro and at 2 weeks of age in Tsc1GFAPCKO mice before the onset of epilepsy in vivo, indicating that the inflammatory changes were not secondary to seizures. Epicatechin-3-gallate, an inhibitor of IL-1β and CXCL10, at least partially reversed the elevated cytokine and chemokine levels, reduced seizure frequency, and prolonged survival of Tsc1GFAPCKO mice. These findings suggest that mTOR-mediated inflammatory mechanisms may be involved in epileptogenesis in the genetic epilepsy, TSC.

Keywords
Epilepsy; Seizure; Tuberous sclerosis; Inflammation; Mice; Interleukin; Cytokine; Chemokine

Received 7 October 2014, Revised 16 April 2015, Accepted 21 April 2015, Available online 21 May 2015
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LEFT Healthy control mice (Panel A) ; RIGHT (Panel B) IL-1beta expression
appears as highly dispersed calcium (GREEN) and tissue (BLUE) disorganization.
Image Credit: Courtesy of Washington University in St. Louis