Dysregulation of mTOR signaling mediates common neurite and migration defects in both idiopathic and 16p11.2 deletion autism neural precursor cells.

Autism spectrum disorder (ASD) is defined by common behavioral characteristics, raising the possibility of shared pathogenic mechanisms. Yet, vast clinical and etiological heterogeneity suggests personalized phenotypes. Surprisingly, our iPSC studies find that six individuals from two distinct ASD subtypes, idiopathic and 16p11.2 deletion, have common reductions in neural precursor cell (NPC) neurite outgrowth and migration even though whole genome sequencing demonstrates no genetic overlap between the datasets. To identify signaling differences that may contribute to these developmental defects, an unbiased phospho-(p)-proteome screen was performed. Surprisingly despite the genetic heterogeneity, hundreds of shared p-peptides were identified between autism subtypes including the mTOR pathway. mTOR signaling alterations were confirmed in all NPCs across both ASD subtypes, and mTOR modulation rescued ASD phenotypes and reproduced autism NPC-associated phenotypes in control NPCs. Thus, our studies demonstrate that genetically distinct ASD subtypes have common defects in neurite outgrowth and migration which are driven by the shared pathogenic mechanism of mTOR signaling dysregulation.

Although the clinical presentation of individuals with autism spectrum disorder (ASD) can vary widely, the core features are repetitive behaviors and difficulties with social interactions and communication. In most cases, the cause of autism is unknown. However, in some cases, such as a form of ASD known as 16p11.2 deletion syndrome, specific genetic changes are responsible. Despite this variability in possible causes and clinical manifestations, the similarity of the core behavioral symptoms across different forms of the disorder indicates that there could be a shared biological mechanism. Furthermore, genetic studies suggest that abnormalities in early fetal brain development could be a crucial underlying cause of ASD. In order to form the complex structure of the brain, fetal brain cells must migrate and start growing extensions that ultimately become key structures of neurons. To test for shared biological mechanisms, Prem et al. reprogrammed blood cells from people with either 16p11.2 deletion syndrome or ASD with an unknown cause to become fetal-like brain cells. Experiments showed that both migration of the cells and their growth of extensions were similarly disrupted in the cells derived from both groups of individuals with autism. These crucial developmental changes were driven by alterations to an important signaling molecule in a pathway involved in brain function, known as the mTOR pathway. However, in some cells the pathway was overactive, whereas in others it was underactive. To probe the potential of the mTOR pathway as a therapeutic target, Prem et al. tested drugs that manipulate the pathway, finding that they could successfully reverse the defects in cells derived from people with both types of ASD. The discovery that a shared biological process may underpin different forms of ASD is important for understanding the early brain changes that are involved. A common target, like the mTOR pathway, could offer hope for treatments for a wide range of ASDs. However, to translate these benefits to the clinic, further research is needed to understand whether a treatment that is effective in fetal cells would also benefit people with autism.

Autism NPCs from both idiopathic and CNV 16p11.2 deletion patients exhibit dysregulation of proliferation and mitogenic responses.

Neural precursor cell (NPC) dysfunction has been consistently implicated in autism. Induced pluripotent stem cell (iPSC)-derived NPCs from two autism groups (three idiopathic [I-ASD] and two 16p11.2 deletion [16pDel]) were used to investigate if proliferation is commonly disrupted. All five individuals display defects, with all three macrocephalic individuals (two 16pDel, one I-ASD) exhibiting hyperproliferation and the other two I-ASD subjects displaying hypoproliferation. NPCs were challenged with bFGF, and all hyperproliferative NPCs displayed blunted responses, while responses were increased in hypoproliferative cells. mRNA expression studies suggest that different pathways can result in similar proliferation phenotypes. Since 16pDel deletes MAPK3, P-ERK was measured. P-ERK is decreased in hyperproliferative but increased in hypoproliferative NPCs. While these P-ERK changes are not responsible for the phenotypes, P-ERK and bFGF response are inversely correlated with the defects. Finally, we analyzed iPSCs and discovered that 16pDel displays hyperproliferation, while idiopathic iPSCs were normal. These data suggest that NPC proliferation defects are common in ASD.

Using iPSC-Based Models to Understand the Signaling and Cellular Phenotypes in Idiopathic Autism and 16p11.2 Derived Neurons.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that is remarkably heterogeneous at the clinical, neurobiological, and genetic levels. ASD can also affect language, a uniquely human capability, and is caused by abnormalities in brain development. Traditionally obtaining biologically relevant human cells to study ASD has been extremely difficult, but new technologies including iPSC-derived neurons and high-throughput omic techniques now provide new, exciting tools to uncover the cellular and signaling basis of ASD etiology.

The glutamate release inhibitor riluzole increases DNA damage and enhances cytotoxicity in human glioma cells, in vitro and in vivo.

PURPOSE: High-grade gliomas are lethal malignancies that cause morbidity and mortality due to local progression rather than metastatic spread. Our group has previously demonstrated that human GRM1 (hGRM1) is ectopically expressed in melanocytes leading to a transformed phenotype. Riluzole, a glutamate release inhibitor, leads to apoptotic cell death via DNA damage. Recent work has demonstrated the pathological significance of the related mGluR3/GRM3 (protein or gene: hGRM3) in gliomas. We evaluated the effect of riluzole on glioma cells. EXPERIMENTAL DESIGN: Western blot analysis and immunofluorescence was performed to assess for GRM3 expression in commercially available and patient-derived glioma cells and for functional analysis of GRM3 using receptor agonist/antagonists and downstream effectors, ERK and AKT phosphorylation, as the read-out. Glutamate secretion by glioma cells was measured using ELISA. Flank and intracranial mouse xenograft models were used to assess growth delay with the glutamate release inhibitor, riluzole (RIL). Immunofluorescence was used to evaluate 53BP1 or γ-H2AX foci after RIL. RESULTS: GRM3 was expressed in most tested glioma samples, and strongly expressed in some. Glioma cells were found to secrete glutamate in the extracellular space and to respond to receptor stimulation by activating downstream ERK. This signaling was abrogated by pretreatment with RIL. Treatment with RIL caused an increase in DNA damage markers, and an increase in cellular cytotoxicity in vitro and in vivo. CONCLUSIONS: We have demonstrated that pretreatment with the glutamate-release inhibitor riluzole sensitizes glioma cells to radiation and leads to greater cytotoxicity; these results have clinical implications for patients with glioblastoma.

Non-canonical activation of CREB mediates neuroprotection in a Caenorhabditis elegans model of excitotoxic necrosis.

Excitotoxicity, caused by exaggerated neuronal stimulation by Glutamate (Glu), is a major cause of neurodegeneration in brain ischemia. While we know that neurodegeneration is triggered by overstimulation of Glu-receptors (GluRs), the subsequent mechanisms that lead to cellular demise remain controversial. Surprisingly, signaling downstream of GluRs can also activate neuroprotective pathways. The strongest evidence involves activation of the transcription factor cAMP response element-binding protein (CREB), widely recognized for its importance in synaptic plasticity. Canonical views describe CREB as a phosphorylation-triggered transcription factor, where transcriptional activation involves CREB phosphorylation and association with CREB-binding protein. However, given CREB's ubiquitous cross-tissue expression, the multitude of cascades leading to CREB phosphorylation, and its ability to regulate thousands of genes, it remains unclear how CREB exerts closely tailored, differential neuroprotective responses in excitotoxicity. A non-canonical, alternative cascade for activation of CREB-mediated transcription involves the CREB co-factor cAMP-regulated transcriptional co-activator (CRTC), and may be independent of CREB phosphorylation. To identify cascades that activate CREB in excitotoxicity we used a Caenorhabditis elegans model of neurodegeneration by excitotoxic necrosis. We demonstrated that CREB's neuroprotective effect was conserved, and seemed most effective in neurons with moderate Glu exposure. We found that factors mediating canonical CREB activation were not involved. Instead, phosphorylation-independent CREB activation in nematode excitotoxic necrosis hinged on CRTC. CREB-mediated transcription that depends on CRTC, but not on CREB phosphorylation, might lead to expression of a specific subset of neuroprotective genes. Elucidating conserved mechanisms of excitotoxicity-specific CREB activation can help us focus on core neuroprotective programs in excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.14494.

Radiosensitization of Primary Human Glioblastoma Stem-like Cells with Low-Dose AKT Inhibition.

Glioblastoma (GBM) is the most frequent and lethal brain cancer. The lack of early detection methods, the presence of rapidly growing tumor cells, and the high levels of recurrence due to chemo- and radioresistance make this cancer an extremely difficult disease to treat. Emerging studies have focused on inhibiting AKT activation; here, we demonstrate that in primary GBM tumor samples, full-dose inhibition of AKT activity leads to differential responses among samples in the context of cell death and self-renewal, reinforcing the notion that GBM is a heterogeneous disease. In contrast, low-dose AKT inhibition when combined with fractionation of radiation doses leads to a significant apoptosis-mediated cell death of primary patient-derived GBM cells. Therefore, low-dose-targeted therapies might be better for radiosensitization of primary GBM cells and further allow for reducing the clinical toxicities often associated with targeting the AKT/PI3K/mTOR pathway. This work emphasizes the discrepancies between cell lines and primary tumors in drug testing, and indicates that there are salient differences between patients, highlighting the need for personalized medicine in treating high-grade glioma.

Autophagy inhibition by chloroquine sensitizes HT-29 colorectal cancer cells to concurrent chemoradiation.

AIM: To investigate whether the inhibition of autophagy by chloroquine (CQ) sensitizes rectal tumors to radiation therapy (RT) or concurrent chemoradiation (chemoRT). METHODS: In vitro, HCT-116 and HT-29 colorectal cancer (CRC) cell lines were treated as following: (1) PBS; (2) CQ; (3) 5-fluorouracil (5-FU); (4) RT; (5) CQ and RT; (6) 5-FU and RT; (7) CQ and 5-FU; and (8) 5-FU and CQ and RT. Each group was then exposed to various doses of radiation (0-8 Gy) depending on the experiment. Cell viability and proliferative capacity were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Clonogenic survival curves were constructed and compared across treatment groups. Autophagy status was determined by assessing the LC3-II to LC3-I ratio on western blot analysis, autophagosome formation on electron microscopy and identification of a perinuclear punctate pattern with GFP-labeled LC3 on fluorescence microscopy. Cell cycle arrest and cell death were evaluated by FACS and Annexin V analysis. All experiments were performed in triplicate and statistical analysis was performed by the student's t test to compare means between treatment groups. RESULTS: RT (2-8 Gy) induced autophagy in HCT-116 and HT-29 CRC cell lines at 4 and 6 h post-radiation, respectively, as measured by increasing LC3-II to LC3-I ratio on western blot. Additionally, electron microscopy demonstrated autophagy induction in HT-29 cells 24 h following irradiation at a dose of 8 Gy. Drug treatment with 5-FU (25 µmol/L) induced autophagy and the combination of 5-FU and RT demonstrated synergism in autophagy induction. CQ (10 µmol/L) alone and in combination with RT effectively inhibited autophagy and sensitized both HCT-116 and HT-29 cells to treatment with radiation (8 Gy; P < 0.001 and 0.00001, respectively). Significant decrease in clonogenic survival was seen only in the HT-29 cell line, when CQ was combined with RT at doses of 2 and 8 Gy (P < 0.5 and P = 0.05, respectively). There were no differences in cell cycle progression or Annexin V staining upon CQ addition to RT. CONCLUSION: Autophagy inhibition by CQ increases CRC cell sensitivity to concurrent treatment with 5-FU and RT in vitro, suggesting that addition of CQ to chemoRT improves CRC treatment response.

PALB2 interacts with KEAP1 to promote NRF2 nuclear accumulation and function.

PALB2/FANCN is mutated in breast and pancreatic cancers and Fanconi anemia (FA). It controls the intranuclear localization, stability, and DNA repair function of BRCA2 and links BRCA1 and BRCA2 in DNA homologous recombination repair and breast cancer suppression. Here, we show that PALB2 directly interacts with KEAP1, an oxidative stress sensor that binds and represses the master antioxidant transcription factor NRF2. PALB2 shares with NRF2 a highly conserved ETGE-type KEAP1 binding motif and can effectively compete with NRF2 for KEAP1 binding. PALB2 promotes NRF2 accumulation and function in the nucleus and lowers the cellular reactive oxygen species (ROS) level. In addition, PALB2 also regulates the rate of NRF2 export from the nucleus following induction. Our findings identify PALB2 as a regulator of cellular redox homeostasis and provide a new link between oxidative stress and the development of cancer and FA.

Quantitative analysis of forward and backward second-harmonic images of collagen fibers using Fourier transform second-harmonic-generation microscopy.

Fourier transform second-harmonic generation (SHG) microscopy has been applied to quantitatively compare the information content between SHG images obtained from the forward and backward direction for three tissue types: porcine tendon, sclera, and ear cartilage. Both signal types yield consistent information on the preferred orientation of collagen fibers. For all specimens, the Fourier transform of the forward and backward SHG images produces several overlapping peaks in the magnitude spectrum at various depths into the tissues, indicating that some information present in the forward SHG images can be extracted from the backward SHG images. This study highlights the potential of backward SHG microscopy for medical diagnostics.

Fourier transform-second-harmonic generation imaging of biological tissues.

Fourier transform-second-harmonic generation imaging is employed to obtain quantitative metrics of collagen fibers in biological tissues. In particular, the preferred orientation and maximum spatial frequency of collagen fibers for selected regions of interest in porcine trachea, ear, and cornea are determined. These metrics remain consistent when applied to collagen fibers in the ear, which can be expected from observation. Collagen fibers in the trachea are more random with large standard deviations in orientation, and large variations in maximum spatial frequency. In addition, these metrics are used to investigate structural changes through a 3D stack of the cornea. This technique can be used as a quantitative marker to assess the structure of collagen fibers that may change due to damage from disease or physical injury.