Isolation and Culture of Neural Stem/Progenitor Cells from the Postnatal Periventricular Region.

Due to the complexity of the neural stem cell (NSC) niche organization, the lack of specific NSC markers, and the difficulty of long-term tracking these cells and their progeny in vivo, the functional properties of the endogenous NSCs remain largely unexplored. These limitations have led to the development of methodologies to efficiently isolate, expand, and differentiate NSCs ex vivo. We describe here the peculiarities of the neurosphere assay (NSA) as a methodology that allows to efficiently isolate, expand, and differentiate somatic NSCs derived from the postnatal and adult forebrain periventricular region while preserving proliferation, self-renewal, and multipotency, the main attributes that provide their functional identification.

In vitro Validation of Chimeric ß-Galactosylceramidase Enzymes With Improved Enzymatic Activity and Increased Secretion.

Globoid Cell Leukodystrophy (GLD) is a lysosomal storage disease (LSD) caused by inherited defects of the ß-galactosylceramidase (GALC) gene. The infantile forms display a rapid and aggressive central and peripheral nervous system (CNS and PNS) dysfunction. No treatments are available for GLD patients. Effective gene therapy (GT) strategies for GLD require a safe and widespread delivery of the functional GALC enzyme to all affected tissues/organs, and particularly to the CNS. The use of chimeric lysosomal enzymes with increased secretion and enhanced transport across the blood-brain barrier (BBB) that boost the efficacy of GT approaches in pre-clinical models of similar neurodegenerative LSDs may benefit GLD as well. Here, we tested the safety and biological efficacy of chimeric GALC enzymes engineered to express an alternative signal peptide (iduronate-2-sulfatase - IDSsp) and the low-density lipoprotein receptor (LDLr)-binding domain from the Apolipoprotein E II (ApoE II) in GLD murine neural and hematopoietic stem/progenitor cells and progeny, which are relevant cells types in the context of in vivo and ex vivo GT platforms. We show that the lentiviral vector-mediated expression of the chimeric GALC enzymes is safe and leads to supranormal enzymatic activity in both neural and hematopoietic cells. The IDSsp.GALC shows enhanced expression and secretion in comparison to the unmodified GALC. The chimeric GALC enzymes produced by LV-transduced cells reduce intracellular galactosylceramide (GalCer) storage and effectively cross-correct GLD murine neurons and glial cells, indicating that the transgenic enzymes are delivered to lysosomes, efficiently secreted, and functional. Of note, the expression of LDLr and LDLr-related proteins in GLD neurons and glial cells supports the exploitation of this system to enhance the GALC supply in affected CNS cells and tissues. These in vitro studies support the use of chimeric GALC enzymes to develop novel and more effective GT approaches for GLD.

Tuberous sclerosis complex-associated CNS abnormalities depend on hyperactivation of mTORC1 and Akt.

Tuberous sclerosis complex (TSC) is a dominantly inherited disease caused by hyperactivation of the mTORC1 pathway and characterized by the development of hamartomas and benign tumors, including in the brain. Among the neurological manifestations associated with TSC, the tumor progression of static subependymal nodules (SENs) into subependymal giant cell astrocytomas (SEGAs) is one of the major causes of morbidity and shortened life expectancy. To date, mouse modeling has failed in reproducing these 2 lesions. Here we report that simultaneous hyperactivation of mTORC1 and Akt pathways by codeletion of Tsc1 and Pten, selectively in postnatal neural stem cells (pNSCs), is required for the formation of bona fide SENs and SEGAs. Notably, both lesions closely recapitulate the pathognomonic morphological and molecular features of the corresponding human abnormalities. The establishment of long-term expanding pNSC lines from mouse SENs and SEGAs made possible the identification of mTORC2 as one of the mediators conferring tumorigenic potential to SEGA pNSCs. Notably, in spite of concurrent Akt hyperactivation in mouse brain lesions, single mTOR inhibition by rapamycin was sufficient to strongly impair mouse SEGA growth. This study provides evidence that, concomitant with mTORC1 hyperactivation, sustained activation of Akt and mTORC2 in pNSCs is a mandatory step for the induction of SENs and SEGAs, and, at the same time, makes available an unprecedented NSC-based in vivo/in vitro model to be exploited for identifying actionable targets in TSC.