Ksenia A. Orlova

The Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that results from mutations in the TSC1 or TSC2 genes and is associated with hamartoma formation in multiple organ systems. The neurological manifestations of TSC are particularly challenging and include infantile spasms, intractable epilepsy, cognitive disabilities, and autism. Progress over the past 15 years has demonstrated that the TSC1 or TSC2 encoded proteins modulate cell function via the mTOR signaling cascade and serve as keystones in regulating cell growth and proliferation. The mTOR pathway provides an intersection for an intricate network of protein cascades that respond to cellular nutrition, energy levels, and growth‐factor stimulation. In the brain, TSC1 and TSC2 have been implicated in cell body size, dendritic arborization, axonal outgrowth and targeting, neuronal migration, cortical lamination, and spine formation. Antagonism of the mTOR pathway with rapamycin and related compounds may provide new therapeutic options for TSC patients.

STRADα deficiency results in aberrant mTORC1 signaling during corticogenesis in humans and mice

Polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) is a rare human autosomal-recessive disorder characterized by abnormal brain development, cognitive disability, and intractable epilepsy. It is caused by homozygous deletions of STE20-related kinase adaptor alpha (STRADA). The underlying pathogenic mechanisms of PMSE and the role of STRADA in cortical development remain unknown. Here, we found that a human PMSE brain exhibits cytomegaly, neuronal heterotopia, and aberrant activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. STRADalpha normally binds and exports the protein kinase LKB1 out of the nucleus, leading to suppression of the mTORC1 pathway. We found that neurons in human PMSE cortex exhibited abnormal nuclear localization of LKB1. To investigate this further, we modeled PMSE in mouse neural progenitor cells (mNPCs) in vitro and in developing mouse cortex in vivo by knocking down STRADalpha expression. STRADalpha-deficient mNPCs were cytomegalic and showed aberrant rapamycin-dependent activation of mTORC1 in association with abnormal nuclear localization of LKB1. Consistent with the observations in human PMSE brain, knockdown of STRADalpha in vivo resulted in cortical malformation, enhanced mTORC1 activation, and abnormal nuclear localization of LKB1. Thus, we suggest that the aberrant nuclear accumulation of LKB1 caused by STRADalpha deficiency contributes to hyperactivation of mTORC1 signaling and disruption of neuronal lamination during corticogenesis, and thereby the neurological features associated with PMSE.

Fetal Brain mTOR Signaling Activation in Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is characterized by developmental malformations of the cerebral cortex known as tubers, comprised of cells that exhibit enhanced mammalian target of rapamycin (mTOR) signaling. To date, there are no reports of mTORC1 and mTORC2 activation in fetal tubers or in neural progenitor cells lacking Tsc2. We demonstrate mTORC1 activation by immunohistochemical detection of substrates phospho-p70S6K1 (T389) and phospho-S6 (S235/236), and mTORC2 activation by substrates phospho-PKCα (S657), phospho-Akt (Ser473), and phospho-SGK1 (S422) in fetal tubers. Then, we show that Tsc2 shRNA knockdown (KD) in mouse neural progenitor cells (mNPCs) in vitro results in enhanced mTORC1 (phospho-S6, phospho-4E-BP1) and mTORC2 (phospho-Akt and phospho-NDRG1) signaling, as well as a doubling of cell size that is rescued by rapamycin, an mTORC1 inhibitor. Tsc2 KD in vivo in the fetal mouse brain by in utero electroporation causes disorganized cortical lamination and increased cell volume that is prevented with rapamycin. We demonstrate for the first time that mTORC1 and mTORC2 signaling is activated in fetal tubers and in mNPCs following Tsc2 KD. These results suggest that inhibition of mTOR pathway signaling during embryogenesis could prevent abnormal brain development in TSC.

Early Progenitor Cell Marker Expression Distinguishes Type II from Type I Focal Cortical Dysplasias

Type I and type II focal cortical dysplasias (FCDs) exhibit distinct histopathologic features that suggest different pathogenic mechanisms. Type I FCDs are characterized by mild laminar disorganization and hypertrophic neurons, whereas type II FCDs exhibit dramatic laminar disorganization and cytomegalic cells (balloon cells). Both FCD types are associated with intractable epilepsy; therefore, identifying cellular or molecular differences between these lesion types that explains the histologic differences could provide new diagnostic and therapeutic insights. Type II FCDs express nestin, a neuroglial progenitor protein that is modulated in vitro by the stem cell proteins c-Myc, sex-determining region Y-box 2 (SOX2), and Octamer-4 (Oct-4) after activation of mammalian target of rapamycin complex 1 (mTORC1). Because mTORC1 activation has been demonstrated in type II FCDs, we hypothesized that c-Myc, SOX2, and Oct-4 expression would distinguish type II from type I FCDs. In addition, we assayed the expression of progenitor cell proteins forkhead box G1 (FOXG1), Kruppel-like factor 4 (KLF4), Nanog, and SOX3. Differential expression of 7 stem cellproteins and aberrant phosphorylation of2mTORC1 substrates, S6 andS6 kinase 1 proteins, clearly distinguished type II from type I FCDs(n = 10 each). Our results demonstrate new potential pathogenic pathways in type II FCDs and suggest biomarkers for diagnostic pathology in resected epilepsy specimens.

Rapamycin Prevents Seizures After Depletion of STRADA in a Rare Neurodevelopmental Disorder

Blocking mTORC1 rescues the neural progenitor cell migratory defect caused by depletion of the STRADA pseudokinase and reduces seizures in patients with a rare neurodevelopmental disorder. Preventing Seizures with Rapamycin The discovery of new treatments for rare neurodevelopmental disorders associated with epilepsy and intellectual disability is often limited by small patient sample sizes that delay initiation of clinical trials. Mutations in the gene STRADA cause brain malformations, seizures, and failure to develop social language in children, with no known successful treatment. In a new study, Parker and colleagues now show that the protein STRADA modulates the mammalian target of rapamycin (mTOR) signaling pathway and that loss of STRADA results in unchecked mTOR activity. Depletion of STRADA in mouse neural progenitor cells resulted in loss of polarity, impaired migration, and inability to form layers in the cerebral cortex. Impaired migration was also identified in fibroblasts from patients lacking STRADA, and all of these effects were prevented with the mTOR inhibitor rapamycin, an immunosuppressant drug in clinical use. The authors then treated five children with rapamycin (sirolimus) beginning at 3 to 8 months of age, and abatement of seizures was observed in all of the children. Early treatment with mTOR pathway inhibitors may be beneficial for children with this neurodevelopmental disorder or with other conditions associated with enhanced mTOR signaling such as tuberous sclerosis complex and fragile X syndrome. A rare neurodevelopmental disorder in the Old Order Mennonite population called PMSE (polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome; also called Pretzel syndrome) is characterized by infantile-onset epilepsy, neurocognitive delay, craniofacial dysmorphism, and histopathological evidence of heterotopic neurons in subcortical white matter and subependymal regions. PMSE is caused by a homozygous deletion of exons 9 to 13 of the LYK5/STRADA gene, which encodes the pseudokinase STRADA, an upstream inhibitor of mammalian target of rapamycin complex 1 (mTORC1). We show that disrupted pathfinding in migrating mouse neural progenitor cells in vitro caused by STRADA depletion is prevented by mTORC1 inhibition with rapamycin or inhibition of its downstream effector p70 S6 kinase (p70S6K) with the drug PF-4708671 (p70S6Ki). We demonstrate that rapamycin can rescue aberrant cortical lamination and heterotopia associated with STRADA depletion in the mouse cerebral cortex. Constitutive mTORC1 signaling and a migration defect observed in fibroblasts from patients with PMSE were also prevented by mTORC1 inhibition. On the basis of these preclinical findings, we treated five PMSE patients with sirolimus (rapamycin) without complication and observed a reduction in seizure frequency and an improvement in receptive language. Our findings demonstrate a mechanistic link between STRADA loss and mTORC1 hyperactivity in PMSE, and suggest that mTORC1 inhibition may be a potential treatment for PMSE as well as other mTOR-associated neurodevelopmental disorders.

Enhanced Epidermal Growth Factor, Hepatocyte Growth Factor, and Vascular Endothelial Growth Factor Expression in Tuberous Sclerosis Complex

Epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) regulate angiogenesis and cell growth in the developing brain. EGF, HGF, and VEGF modulate the activity of the mammalian target of rapamycin (mTOR) cascade, a pathway regulating cell growth that is aberrantly activated in tuberous sclerosis complex (TSC). We hypothesized that expression of EGF, HGF, VEGF, and their receptors EGFR, c-Met, and Flt-1, respectively, would be altered in TSC. We show by cDNA array and immunohistochemical analysis that EGF, EGFR, HGF, c-Met, and VEGF, but not Flt-1, mRNA, and protein expression was up-regulated in Tsc1 conditional knockout (Tsc1(GFAP)CKO) mouse cortex. Importantly, these alterations closely predicted enhanced expression of these proteins in tuber and subependymal giant cell astrocytoma (SEGA) specimens in TSC. Expression of EGF, EGFR, HGF, c-Met, and VEGF protein, as well as hypoxia inducible factor-1α, a transcription factor that regulates VEGF levels and is also modulated by mTOR cascade activity, was enhanced in SEGAs (n = 6) and tubers (n = 10) from 15 TSC patients. Enhanced expression of these growth factors and growth factor receptors in human SEGAs and tubers and in the Tsc1(GFAP)CKO mouse may account for enhanced cellular growth and proliferation in tubers and SEGAs and provides potential target molecules for therapeutic development in TSC.

Advanced Endovascular Approaches in the Management of Challenging Proximal Aortic Neck Anatomy: Traditional Endografts and the Snorkel Technique.

Advances in endovascular technology, and access to this technology, have significantly changed the field of vascular surgery. Nowhere is this more apparent than in the treatment of abdominal aortic aneurysms (AAAs), in which endovascular aneurysm repair (EVAR) has replaced the traditional open surgical approach in patients with suitable anatomy. However, approximately one-third of patients presenting with AAAs are deemed ineligible for standard EVAR because of anatomic constraints, the majority of which involve the proximal aneurysmal neck. To overcome these challenges, a bevy of endovascular approaches have been developed to either enhance stent graft fixation at the proximal neck or extend the proximal landing zone to allow adequate apposition to the aortic wall and thus aneurysm exclusion. This article is composed of two sections that together address new endovascular approaches for treating aortic aneurysms with difficult proximal neck anatomy. The first section will explore advancements in the traditional EVAR approach for hostile neck anatomy that maximize the use of the native proximal landing zone; the second section will discuss a technique that was developed to extend the native proximal landing zone and maintain perfusion to vital aortic branches using common, off-the-shelf components: the snorkel technique. While the techniques presented differ in terms of approach, the available clinical data, albeit limited, support the notion that they may both have roles in the treatment algorithm for patients with challenging proximal neck anatomy.

Results of carotid angioplasty and stenting are equivalent for critical versus high-grade lesions in patients deemed high risk for carotid endarterectomy

BACKGROUND To examine outcomes of carotid angioplasty and stenting (CAS) in patients with critical carotid stenosis who are deemed high risk for carotid endarterectomy. METHODS Medical records were retrospectively analyzed for patients undergoing CAS between September 2002 and March 2011 at a single institution. Patients were classified as having either critical (≥ 90%) or high-grade (70%-89%) carotid stenosis based on angiography. The primary composite end point was stroke, myocardial infarction, or death from any cause during the periprocedural period or any ipsilateral stroke during the follow-up period. RESULTS A total of 245 patients underwent 257 CAS procedures during the study period. Fifty-one percentage (n = 130) of cases involved critical stenosis (66.2% male; mean age, 71 ± 10 y), with the remaining group (n = 127) involving high-grade stenosis (67.7% male; mean age, 71 ± 9 y). Symptomatic carotid disease was present in 25% of the critical stenosis and 31% of the high-grade stenosis groups (P = 0.33). Chronic obstructive pulmonary disease was more commonly found in the high-grade stenosis group (20% versus 8%, P = 0.01). No difference was observed between the groups relative to other baseline demographic characteristics, presence of contralateral carotid occlusion, stent diameter or length, maximum balloon diameter or length, use of embolic protection device, or procedural duration. Technical success was achieved in all cases. There was no difference in the need to predilate before the introduction of the filter or stent based on the degree of stenosis. We found no difference in the primary composite end point between the high-grade or critical stenosis groups (7.1% versus 7.7%, P = 0.74), or there were no differences between the individual components of the composite end point. Mid-term survival was similar between the two groups at a mean follow-up period of 2.4 y. CONCLUSIONS Despite concerns regarding the potential for increased neurologic complications, our data demonstrate that patients with high-grade and critical stenosis are able to safely undergo CAS and achieve similar periprocedural outcomes and mid-term prognosis.

Intracranial Sarcoma Metastasizing along Distal Shunt Tubing

cific actin, CD31, D2–40, factor VIII, synaptophysin, chromogranin, neurofilament and glial fibrillary acidic protein. The histological and immunohistochemical findings are those typical of an extraskeletal myxoid chondrosarcoma. A magnetic resonance scan demonstrated a mass in the head extending into the neck along the shunt catheter. The child received palliative chemotherapy but subsequently died from her disease. Postmortem examination of the brain showed a white, firm, multinodular tumor with extensive replacement and infiltration of the residual cerebral tissue. The tumor also infiltrated through the dura in different areas. The autopsy also showed extracranial extension of the tumor into the right neck, supraclavicular region, abdominal wall, omentum, liver and pelvis ( fig. 1 c). Histologically and immunohistochemically, the tumor was similar to the previous biopsy ( fig. 1 d). The exact location of the primary tumor is difficult to determine. Clinically the lesion first appeared in the intracranial compartment, and then the patient developed neck swelling; however, it is possible that the tumor arose in the subcutaneous tissue and migrated intracranially along the shunt path. We illustrate a case of a child presenting with a neck mass tracking along her distal shunt tubing. The child had a ventriculoperitoneal shunt placed as an infant for treatment of hydranencephaly ( fig. 1 a). At 6 years of age, the patient presented with a new neck mass and several months of recurrent shunt malfunctions. A head computed tomography (CT) scan demonstrated some intracranial changes ( fig. 1 b) thought to be consistent with cerebral tissue breakdown. The patient also had neck swelling thought to be a pseudomeningocele resulting from cerebrospinal fluid leaking through the burr hole. At the shunt revision, viscous fluid and a gelatinous material were found near the shunt entrance site, and it tracked into the neck along the shunt tubing. Histopathological examination of the mass revealed a malignant tumor with variable cellularity and chondromyxoid stroma. The neoplastic cells showed pleomorphic round to spindle nuclei and a moderate amount of eosinophilic and vacuolated cytoplasm. The tumor cells were positive for S-100, CD56, vimentin and, focally, neuronal specific enolase. The neoplastic cells had retained expression of INI-1. They were negative for pancytokeratin, epithelial membrane antigen, desmin, myoD1, myogenin, muscle-speReceived: August 29, 2011 Accepted: November 24, 2011 Published online: August 21, 2012