Abstracts

Rationale: Tuberous sclerosis complex (TSC) results from mutations in the TSC1 and TSC2 genes, causing hyperactivation of the mTOR signaling pathway. Up to 90% of TSC patients develop epilepsy, and approximately 50% present with cognitive and behavioral impairments. The mTOR hyperactivity observed in TSC affects both neuronal and glial development and function. Although glial abnormalities have been extensively documented in human TSC cortical tubers, their contribution to epilepsy and cognitive dysfunction is less understood. Normal astrocytes play a key role in the formation of glutamatergic synapses through the release of several secreted factors, including the secreted protein acidic and rich in cysteine (SPARC) family proteins and glypicans. We hypothesized that the strongly reactive astrocytes in TSC may lose their ability to promote synaptogenesis and therefore may contribute to synapse pathology and cognitive dysfunction in TSC. Methods: Autopsy cortical tubers and peri-tuberal cortex tissue from TSC patients (n=12; ages 3-36 years, mean 21 years) and region-matched control specimens from cases with normal neurological history (n=16; ages 4-37 years, mean 17 years) were obtained from the NIH Nuerobiobank at the University of Maryland, Baltimore, MD. Frozen and fixed samples were used for Western blotting and immunohistochemistry (n=4-12/group).  Blots were probed with antibodies against synaptic proteins synaptophysin and PSD-95, and astrocytic synaptogenic factors SPARC-like protein 1 (SPARCL1) and Glypican-4. Actin was used as loading control. Fixed and frozen sections were stained with neuronal and glial markers GFAP and MAP2 and double labeled with either SPARCL1 or Glypican-4. Statistical significance (p<0.05) was assessed by Kruskal-Wallis tests with Dunn's correction.  Results: Synaptophysin and PSD-95 levels were significantly reduced in the tuber (58% of control, n=5, p<0.05 for synaptophysin and 25% of control, n=10, p<0.01 for PSD-95), but not in the peri-tuber (129% of control, n=5 for synaptophysin and 59% of control, n=11 for PSD-95; p>0.05). SPARCL1 levels were significantly decreased in the tuber (61% of control, n=10, p<0.05), but not in the peri-tuber (86% of control, n=11, p>0.05). Glypican-4 levels were similarly reduced in the tuber (53% of control, n=5), but slightly increased in the peri-tuber (135% of control, n=5), however these changes did not reach statistical significance (p>0.05). Double label immunohistochemistry confirmed a marked decrease in SPARCL1 and Glypican-4 expression in most TSC tuber astrocytes. Conclusions: Our results are suggestive of impaired astrocyte synaptogenic function in human TSC cortical tubers, which may contribute to impaired synaptic development and overall pathogenesis in TSC. These data also provide a rationale for the exploration of astrocyte-based therapies to treat synaptic pathology in TSC. Funding: Supported by: Penn Medicine Orphan Disease Center, University Research Foundation and NIHR01NS031718.