Nanotherapeutic Modulation of Human Neural Cells and Glioblastoma in Organoids and Monocultures

Inflammatory processes in the brain are orchestrated by microglia and astrocytes in response to activators such as pathogen-associated molecular patterns, danger-associated molecular patterns and some nanostructures. Microglia are the primary immune responders in the brain and initiate responses amplified by astrocytes through intercellular signaling. Intercellular communication between neural cells can be studied in cerebral organoids, co-cultures or in vivo. We used human cerebral organoids and glioblastoma co-cultures to study glia modulation by dendritic polyglycerol sulfate (dPGS). dPGS is an extensively studied nanostructure with inherent anti-inflammatory properties. Under inflammatory conditions, lipocalin-2 levels in astrocytes are markedly increased and indirectly enhanced by soluble factors released from hyperactive microglia. dPGS is an effective anti-inflammatory modulator of these markers. Our results show that dPGS can enter neural cells in cerebral organoids and glial cells in monocultures in a time-dependent manner. dPGS markedly reduces lipocalin-2 abundance in the neural cells. Glioblastoma tumoroids of astrocytic origin respond to activated microglia with enhanced invasiveness, whereas conditioned media from dPGS-treated microglia reduce tumoroid invasiveness. Considering that many nanostructures have only been tested in cancer cells and rodent models, experiments in human 3D cerebral organoids and co-cultures are complementary in vitro models to evaluate nanotherapeutics in the pre-clinical setting. Thoroughly characterized organoids and standardized procedures for their preparation are prerequisites to gain information of translational value in nanomedicine. This study provides data for a well-characterized dendrimer (dPGS) that modulates the activation state of human microglia implicated in brain tumor invasiveness

D-Ribose Induces Cellular Protein Glycation and Impairs Mouse Spatial Cognition

BACKGROUND: D-ribose, an important reducing monosaccharide, is highly active in the glycation of proteins, and results in the rapid production of advanced glycation end products (AGEs) in vitro. However, whether D-ribose participates in glycation and leads to production of AGEs in vivo still requires investigation. METHODOLOGY/PRINCIPAL FINDINGS: Here we treated cultured cells and mice with D-ribose and D-glucose to compare ribosylation and glucosylation for production of AGEs. Treatment with D-ribose decreased cell viability and induced more AGE accumulation in cells. C57BL/6J mice intraperitoneally injected with D-ribose for 30 days showed high blood levels of glycated proteins and AGEs. Administration of high doses D-ribose also accelerated AGE formation in the mouse brain and induced impairment of spatial learning and memory ability according to the performance in Morris water maze test. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that D-ribose but not D-glucose reacts rapidly with proteins and produces significant amounts of AGEs in both cultured cells and the mouse brain, leading to accumulation of AGEs which may impair mouse spatial cognition

D-Ribose Induces Cellular Protein Glycation and Impairs Mouse Spatial Cognition

Background: D-Ribose, an important reducing monosaccharide, is highly active in the glycation of proteins, and results in the rapid production of advanced glycation end products (AGEs) in vitro. However, whether D-ribose participates in glycation and leads to production of AGEs in vivo still requires investigation

Open Science Meets Stem Cells: A New Drug Discovery Approach for Neurodegenerative Disorders

Neurodegenerative diseases are a challenge for drug discovery, as the biological mechanisms are complex and poorly understood, with a paucity of models that faithfully recapitulate these disorders. Recent advances in stem cell technology have provided a paradigm shift, providing researchers with tools to generate human induced pluripotent stem cells (iPSCs) from patient cells. With the potential to generate any human cell type, we can now generate human neurons and develop “first-of-their-kind” disease-relevant assays for small molecule screening. Now that the tools are in place, it is imperative that we accelerate discoveries from the bench to the clinic. Using traditional closed-door research systems raises barriers to discovery, by restricting access to cells, data and other research findings. Thus, a new strategy is required, and the Montreal Neurological Institute (MNI) and its partners are piloting an “Open Science” model. One signature initiative will be that the MNI biorepository will curate and disseminate patient samples in a more accessible manner through open transfer agreements. This feeds into the MNI open drug discovery platform, focused on developing industry-standard assays with iPSC-derived neurons. All cell lines, reagents and assay findings developed in this open fashion will be made available to academia and industry. By removing the obstacles many universities and companies face in distributing patient samples and assay results, our goal is to accelerate translational medical research and the development of new therapies for devastating neurodegenerative disorders

Ribosylation triggering Alzheimer's disease-like Tau hyperphosphorylation via activation of CaMKII

Type 2 diabetes mellitus (T2DM) is regarded as one of the serious risk factors for age-related cognitive impairment; however, a causal link between these two diseases has so far not been established. It was recently discovered that, apart from high D-glucose levels, T2DM patients also display abnormally high concentrations of uric D-ribose. Here, we show for the first time that the administration of D-ribose, the most active glycator among monosaccharides, produces high levels of advanced glycation end products (AGEs) and, importantly, triggers hyperphosphorylation of Tau in the brain of C57BL/6 mouse and neuroblastoma N2a cells. However, the administration of D-glucose showed no significant changes in Tau phosphorylation under the same experimental conditions. Crucially, suppression of AGE formation using an AGEs inhibitor (aminoguanidine) effectively prevents hyperphosphorylation of Tau protein. Further study shows AGEs resulted from ribosylation activate calcium-/calmodulin-dependent protein kinase type II (CaMKII), a key kinase responsible for Tau hyperphosphorylation. These data suggest that there is indeed a mechanistic link between ribosylation and Tau hyperphosphorylation. Targeting ribosylation by inhibiting AGE formation may be a promising therapeutic strategy to prevent Alzheimer's disease-like Tau hyperphosphorylation and diabetic encephalopathies

Serum ALT and AST activity and serum creatinine concentration.

<p>After 30 days of treatment, serum was collected to assay the activity of ALT and AST and creatinine concentration. All values are expressed as means ± S.E.M.</p

Immunohistochemical staining of AGEs in the hippocampus and cortex.

<p>Mice were injected (i.p.) with Rib at dose of 2 g/kg, or Glc at dose of 2 g/kg, or 0.9% saline (controls) for 30 days. AGEs in the mouse brain were detected by immunohistochemistry using anti-AGEs monoclonal antibody. The areas outlined with dashed lines are magnified in the lower panels (bar  =  50 µm).</p

Enhancement of brain AGEs injected with D-ribose.

<p>Conditions for the injection of Rib were the same as those given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024623#pone-0024623-g003" target="_blank">Fig 3</a>, except that AGEs in the mouse brain were detected by Western blotting using anti-AGEs (6D12 monoclonal antibody, panel A). β-Actin level was used as a loading control. Quantification results are shown in panel B. The saline control value was set as 1.0. All values are expressed as means ± S.E.M. * P<0.05, ** P<0.01.</p

Changes in cell viability in the presence of D-ribose.

<p>The morphology of SH-SY5Y cells was observed by inverted contrast microscopy after incubation with 50 mM Rib (A), or 50 mM Glc (B) for 3 days. Untreated cells were used as controls (C). HEK293T cells treated with the same concentration of Rib (D), Glc (E) and control cells (F) were imaged under the same conditions. SH-SY5Y (G and H) and HEK293T (I and J) cells were incubated with Rib or Glc as indicated and cell viability was measured using the MTT assay at day 2 (G and I), and day 3 (H and J) after addition of the monosaccharides.</p