A novel approach to label bone marrow-derived mesenchymal stem cells with mixed-surface PAMAM dendrimers.

BACKGROUND: Transplantation of mesenchymal stem cells has created enormous opportunities as a potential treatment for various diseases including neurodegenerative diseases. Given current techniques, such as Hoechst labeling, have safety and leakage issues, our study focused, as a proof-of-concept, on a new dendrimer-based technique for labeling these stem cells to ensure their efficacy and safety following transplantation into the brain of a healthy mice. METHODS AND RESULTS: The bone marrow-derived mesenchymal stem cells (BM-MSCs) were labeled using polyaminoamine (PAMAM) dendrimers following which their stemness based on their proliferation and differentiation ability were analyzed by gold standard methods. These labeled BM-MSCs were transplanted into the striatum of C57BL/6J mice and were tracked using in vivo imaging system (IVIS) and analyzed using tissue imaging, 2 weeks after transplantation. Our results showed that the dendrimer-labeled BM-MSCs were able to successfully maintain their stemness and were tracked in vivo following transplantation. Unlike Hoechst, we did not find the dendrimers to be leaking out of the cells and were very specific to the cells that up took the dendrimers. Moreover, no adverse events were found in the transplanted animals proving that this is a safer method. CONCLUSIONS: Labeling BM-MSCs using fluorescently tagged PAMAM dendrimers can be used as a potentially safe and efficient method for labeling cells, particularly stem cells, in vitro and in vivo following transplantation in rodents.

PAMAM Dendrimers Cross the Blood-Brain Barrier When Administered through the Carotid Artery in C57BL/6J Mice.

Drug delivery into the central nervous system (CNS) is challenging due to the blood-brain barrier (BBB) and drug delivery into the brain overcoming the BBB can be achieved using nanoparticles such as dendrimers. The conventional cationic dendrimers used are highly toxic. Therefore, the present study investigates the role of novel mixed surface dendrimers, which have potentially less toxicity and can cross the BBB when administered through the carotid artery in mice. In vitro experiments investigated the uptake of amine dendrimers (G1-NH2 and G4-NH2) and novel dendrimers (G1-90/10 and G4-90/10) by primary cortical cultures. In vivo experiments involved transplantation of G4-90/10 into mice through (1) invasive intracranial injections into the striatum; and (2) less invasive carotid injections. The animals were sacrificed 24-h and 1-week post-transplantations and their brains were analyzed. In vivo experiments proved that the G4-90/10 can cross the BBB when injected through the carotid artery and localize within neurons and glial cells. The dendrimers were found to migrate through the corpus callosum 1-week post intracranial injection. Immunohistochemistry showed that the migrating cells are the dendrimer-infected glial cells. Overall, our results suggest that poly-amidoamine (PAMAM) dendrimers may be used as a minimally invasive means to deliver biomolecules for treating neurological diseases or disorders.

Abnormal N-acetylglucosaminyltransferase expression in prefrontal cortex in schizophrenia.

Changes in the extent of the posttranslational modification glycosylation have been previously reported in several brain regions in schizophrenia. Quality control within the endoplasmic reticulum and Golgi, branching of glycans, intracellular trafficking and targeting, protein-protein interactions, and endocytosis are processes regulated by both N-linked and O-linked glycosylation. Previous studies in schizophrenia have found altered glycan biosynthesis and abnormal glycan levels in cerebrospinal fluid (CSF) and plasma, as well as altered expression in frontal cortex of glycosyltransferase transcripts encoding proteins associated with both N- and O-linked glycosylation. The N-acetylglucosaminyltransferases (GlcNAcTs) are glycosylating enzymes that play a key role in adding N-acetylglucosamine (GlcNAc) to substrates to facilitate their proper trafficking, intracellular targeting, and cellular function. Given previous results indicating abnormal glycosylation in schizophrenia, we hypothesized that these GlcNAcTs may be abnormally expressed in this illness. We measured protein expression of nine distinct GlcNAcTs by Western blot analysis in postmortem samples of dorsolateral prefrontal cortex (DLPFC) from twelve pairs of elderly patients with schizophrenia and comparison subjects. We found decreased protein expression of UDP-GlcNAc:BetaGal Beta-1,3 GlcNAcT 8 (B3GNT8) and mannosyl (alpha-1,3-)-glycoprotein beta-1,4 GlcNAcT (MGAT4A) expression in schizophrenia. These data provide further evidence that glycosylation is dysregulated in schizophrenia, and suggest a potential mechanism associated with alterations in protein function, trafficking, and intracellular targeting in this illness.

Hippocampal and cortical primary cilia are required for aversive memory in mice.

It has been known for decades that neurons throughout the brain possess solitary, immotile, microtubule based appendages called primary cilia. Only recently have studies tried to address the functions of these cilia and our current understanding remains poor. To determine if neuronal cilia have a role in behavior we specifically disrupted ciliogenesis in the cortex and hippocampus of mice through conditional deletion of the Intraflagellar Transport 88 (Ift88) gene. The effects on learning and memory were analyzed using both Morris Water Maze and fear conditioning paradigms. In comparison to wild type controls, cilia mutants displayed deficits in aversive learning and memory and novel object recognition. Furthermore, hippocampal neurons from mutants displayed an altered paired-pulse response, suggesting that loss of IFT88 can alter synaptic properties. A variety of other behavioral tests showed no significant differences between conditional cilia mutants and controls. This type of conditional allele approach could be used to distinguish which behavioral features of ciliopathies arise due to defects in neural development and which result from altered cell physiology. Ultimately, this could lead to an improved understanding of the basis for the cognitive deficits associated with human cilia disorders such as Bardet-Biedl syndrome, and possibly more common ailments including depression and schizophrenia.