The conversion of endogenous alpha-synuclein (asyn) to pathological asyn-enriched aggregates is a hallmark of Parkinson's disease (PD). These inclusions can be detected in the central and enteric nervous system (ENS). Moreover, gastrointestinal symptoms can appear up to 20 years before the diagnosis of PD. The dual-hit hypothesis posits that pathological asyn aggregation starts in the ENS, and retrogradely spreads to the brain. In this study, we tested this hypothesis by directly injecting preformed asyn fibrils into the duodenum wall of wild-type rats and transgenic rats with excess levels of human asyn. We provide a meticulous characterization of the bacterial artificial chromosome (BAC) transgenic rat model with respect to initial propagation of pathological asyn along the parasympathetic and sympathetic pathways to the brainstem, by performing immunohistochemistry at early time points post-injection. Induced pathology was observed in all key structures along the sympathetic and parasympathetic pathways (ENS, autonomic ganglia, intermediolateral nucleus of the spinal cord (IML), heart, dorsal motor nucleus of the vagus, and locus coeruleus (LC)) and persisted for at least 4 months post-injection. In contrast, asyn propagation was not detected in wild-type rats, nor in vehicle-injected BAC rats. The presence of pathology in the IML, LC, and heart indicate trans-synaptic spread of the pathology. Additionally, the observed asyn inclusions in the stomach and heart may indicate secondary anterograde propagation after initial retrograde spreading. In summary, trans-synaptic propagation of asyn in the BAC rat model is fully compatible with the "body-first hypothesis" of PD etiopathogenesis. To our knowledge, this is the first animal model evidence of asyn propagation to the heart, and the first indication of bidirectional asyn propagation via the vagus nerve, i.e., duodenum-to-brainstem-to-stomach. The BAC rat model could be very valuable for detailed mechanistic studies of the dual-hit hypothesis, and for studies of disease modifying therapies targeting early pathology in the gastrointestinal tract.

Adenosine monophosphate (AMP)‑activated protein kinase (AMPK) is a major cellular energy sensor that is activated by an increase in the AMP/adenosine triphosphate (ATP) ratio. This causes the initiation of adaptive cellular programs, leading to the inhibition of anabolic pathways and increasing ATP synthesis. AMPK indirectly inhibits mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a serine/threonine kinase and central regulator of cell growth and metabolism, which integrates various growth inhibitory signals, such as the depletion of glucose, amino acids, ATP and oxygen. While neuroprotective approaches by definition focus on neurons, that are more sensitive under cell stress conditions, astrocytes play an important role in the cerebral energy homeostasis during ischemia. Therefore, the protection of astrocytic cells or other glial cells may contribute to the preservation of neuronal integrity and function. In the present study, it was thus hypothesized that a preventive induction of energy deprivation‑activated signaling pathways via AMPK may protect astrocytes from hypoxia and glucose deprivation. Hypoxia‑induced cell death was measured in a paradigm of hypoxia and partial glucose deprivation in vitro in the immortalized human astrocytic cell line SVG. Both the glycolysis inhibitor 2‑deoxy‑d‑glucose (2DG) and the AMPK activator A‑769662 induced the phosphorylation of AMPK, resulting in mTORC1 inhibition, as evidenced by a decrease in the phosphorylation of the target ribosomal protein S6 (RPS6). Treatment with both 2DG and A‑769662 also decreased glucose consumption and lactate production. Furthermore, A‑769662, but not 2DG induced an increase in oxygen consumption, possibly indicating a more efficient glucose utilization through oxidative phosphorylation. Hypoxia‑induced cell death was profoundly reduced by treatment with 2DG or A‑769662. On the whole, the findings of the present study demonstrate, that AMPK activation via 2DG or A‑769662 protects astrocytes under hypoxic and glucose‑depleted conditions.

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus-mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.

Parkinson disease (PD) is the second most common neurodegenerative disorder and the leading neurodegenerative cause of motor disability. Pathologic accumulation of aggregated alpha synuclein (AS) protein in brain, and imbalance in the nigrostriatal system due to the loss of dopaminergic neurons in the substantia nigra- pars compacta, are hallmark features in PD. AS aggregation and propagation are considered to trigger neurotoxic mechanisms in PD, including mitochondrial deficits and oxidative stress. The eukaryotic elongation factor-2 kinase (eEF2K) mediates critical regulation of dendritic mRNA translation and is a crucial molecule in diverse forms of synaptic plasticity. Here we show that eEF2K activity, assessed by immuonohistochemical detection of eEF2 phosphorylation on serine residue 56, is increased in postmortem PD midbrain and hippocampus. Induction of aggressive, AS-related motor phenotypes in a transgenic PD M83 mouse model also increased brain eEF2K expression and activity. In cultures of dopaminergic N2A cells, overexpression of wild-type human AS or the A53T mutant increased eEF2K activity. eEF2K inhibition prevented the cytotoxicity associated with AS overexpression in N2A cells by improving mitochondrial function and reduced oxidative stress. Furthermore, genetic deletion of the eEF2K ortholog efk-1 in C. elegans attenuated human A53T AS induced defects in behavioural assays reliant on dopaminergic neuron function. These data suggest a role for eEF2K activity in AS toxicity, and support eEF2K inhibition as a potential target in reducing AS-induced oxidative stress in PD.

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma, a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of Ewing sarcoma cells. Mechanistically, IL1RAP binds the cell-surface system Xc - transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Therefore, IL1RAP maintains cyst(e)ine and glutathione pools, which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of Ewing sarcoma cells. Therefore, we define IL1RAP as a new cell-surface target in Ewing sarcoma, which is potentially exploitable for immunotherapy. SIGNIFICANCE: Here, we identify cell-surface protein IL1RAP as a key driver of metastasis in Ewing sarcoma, a highly aggressive childhood sarcoma. Minimal expression in pediatric and adult normal tissues nominates IL1RAP as a promising target for immunotherapy.See related commentary by Yoon and DeNicola, p. 2679.This article is highlighted in the In This Issue feature, p. 2659.

SORL1 is strongly associated with both sporadic and familial forms of Alzheimer's disease (AD), but a lack of information about alternatively spliced transcripts currently limits our understanding of the role of SORL1 in AD. Here, we describe a SORL1 transcript (SORL1-38b) characterized by inclusion of a novel exon (E38b) that encodes a truncated protein. We identified E38b-containing transcripts in several brain regions, with the highest expression in the cerebellum and showed that SORL1-38b is largely located in neuronal dendrites, which is in contrast to the somatic distribution of transcripts encoding the full-length SORLA protein (SORL1-fl). SORL1-38b transcript levels were significantly reduced in AD cerebellum in three independent cohorts of postmortem brains, whereas no changes were observed for SORL1-fl. A trend of lower 38b transcript level in cerebellum was found for individuals carrying the risk variant at rs2282649 (known as SNP24), although not reaching statistical significance. These findings suggest synaptic functions for SORL1-38b in the brain, uncovering novel aspects of SORL1 that can be further explored in AD research.

The classification of the Neotropical Cybistrinae Sharp, 1880 (Coleoptera: Adephaga: Dytiscidae) is extensively revised based on a phylogenetic analysis of morphological features of the group. A new genus, Nilssondytesgen. nov. is described for a unique new species, Nilssondytesdiversussp. nov. from Venezuela. The New World genus, Megadytes Sharp, 1882, with several subgenera, was found to not be monophyletic. The type species of Megadytes, Dytiscuslatus Fabricius, 1801 and the species Cybisterparvus Trémouilles, 1984 were found to be monophyletic together, and phylogenetically more closely related to Cybister Curtis, 1827 than to other species assigned to Megadytes sensu stricto, which were found to also be monophyletic. The name Megadytes is here restricted to include only Megadyteslatus and Megadytesparvus. These two species assigned to this newly restricted genus concept are reviewed and diagnosed. A new genus, Metaxydytesgen. nov., is erected to include all the other species currently assigned to Megadytes sensu stricto. The current subgenus names assigned to Megadytes, Bifurcitus Brinck, 1945, Paramegadytes Trémouilles & Bachmann, 1980, and Trifurcitus Brinck, 1945, are elevated to genus rank since they are variously paraphyletic. The two species assigned to Cybister (Neocybister) Miller, Bergsten & Whiting, 2007, Cybister (Neocybister) festae Griffini, 1895, and Cybister (Neocybister) puncticollis (Brullé, 1837) re reviewed and diagnosed with the former redescribed and its type specimens considered for the first time since its description. Another evidently new species and possible new genus, Megadytes species, IR57 (Ribera et al. 2008), from Peru, is also characterized, but not formally treated because of lack of important data for the single, partial specimen. Diagnostic features are illustrated for the entire group.

Variations in the α-synuclein-encoding SNCA gene represent the greatest genetic risk factor for Parkinson's disease (PD), and duplications/triplications of SNCA cause autosomal dominant familial PD. These facts closely link brain levels of α-synuclein with the risk of PD, and make lowering α-synuclein levels a therapeutic strategy for the treatment of PD and related synucleinopathies. In this paper, we corroborate previous findings on the ability of overexpressed Polo-like kinase 2 (PLK-2) to decrease cellular α-synuclein, but demonstrate that the process is independent of PLK-2 phosphorylating S129 in α-synuclein because a similar reduction is achieved with the non-phosphorable S129A mutant α-synuclein. Using a specific PLK-2 inhibitor (compound 37), we demonstrate that endogenous PLK-2 phosphorylates S129 only in some cells, but increases α-synuclein protein levels in all tested cell cultures and brain slices. PLK-2 is found to regulate the transcription of α-synuclein mRNA from both the endogenous mouse SNCA gene and transgenic vectors that only contain the open reading frame. Moreover, we are the first to show that regulation of α-synuclein by PLK-2 is of physiological importance since 10days' inhibition of endogenous PLK-2 in wt C57BL/6 mice increases endogenous α-synuclein protein levels. Our findings collectively demonstrate that PLK-2 regulates α-synuclein levels by a previously undescribed transcription-based mechanism. This mechanism is active in cells and brain tissue, opening up for alternative strategies for modulating α-synuclein levels and thereby for the possibility of modifying disease progression in synucleinopaties.

The transthyretin amyloidoses (ATTR) are devastating diseases characterized by progressive neuropathy and/or cardiomyopathy for which novel therapeutic strategies are needed. We have recently shown that curcumin (diferuloylmethane), the major bioactive polyphenol of turmeric, strongly suppresses TTR fibril formation in vitro, either by stabilization of TTR tetramer or by generating nonfibrillar small intermediates that are innocuous to cultured neuronal cells. In the present study, we aim to assess the effect of curcumin on TTR amyloidogenesis in vivo, using a well characterized mouse model for familial amyloidotic polyneuropathy (FAP). Mice were given 2% (w/w) dietary curcumin or control diet for a six week period. Curcumin supplementation resulted in micromolar steady-state levels in plasma as determined by LC/MS/MS. We show that curcumin binds selectively to the TTR thyroxine-binding sites of the tetramer over all the other plasma proteins. The effect on plasma TTR stability was determined by isoelectric focusing (IEF) and curcumin was found to significantly increase TTR tetramer resistance to dissociation. Most importantly, immunohistochemistry (IHC) analysis of mice tissues demonstrated that curcumin reduced TTR load in as much as 70% and lowered cytotoxicity associated with TTR aggregation by decreasing activation of death receptor Fas/CD95, endoplasmic reticulum (ER) chaperone BiP and 3-nitrotyrosine in tissues. Taken together, our results highlight the potential use of curcumin as a lead molecule for the prevention and treatment of TTR amyloidosis.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are expressed by microglia and infiltrating macrophages following ischemic stroke. Whereas IL-1beta is primarily neurotoxic in ischemic stroke, TNF-alpha may have neurotoxic and/or neuroprotective effects. We investigated whether IL-1beta and TNF-alpha are synthesized by overlapping or segregated populations of cells after ischemic stroke in mice.

Tauroursodeoxycholic acid (TUDCA) is a unique natural compound that acts as a potent anti-apoptotic and anti-oxidant agent, reducing cytotoxicity in several neurodegenerative diseases. Since oxidative stress, apoptosis and inflammation are associated with transthyretin (TTR) deposition in Familial Amyloidotic Polyneuropathy (FAP), we investigated the possible TUDCA therapeutical application in this disease. We show by semi-quantitative immunohistochemistry and western blotting that administration of TUDCA to a transgenic mouse model of FAP decreased apoptotic and oxidative biomarkers usually associated with TTR deposition, namely the ER stress markers BiP and eIF2alpha, the Fas death receptor and oxidation products such as 3-nitrotyrosine. Most important, TUDCA treatment significantly reduced TTR toxic aggregates in as much as 75%. Since TUDCA has no effect on TTR aggregation "in vitro", this finding points for the "in vivo" modulation of TTR aggregation by cellular responses, such as by oxidative stress, ER stress and apoptosis and prompts for the use of this safe drug in prophylactic and therapeutic measures in FAP.

To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRβ chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer.

Circumstantial evidence points to a pathological role of alpha-synuclein (aSyn; gene symbol SNCA), conferred by aSyn misfolding and aggregation, in Parkinson disease (PD) and related synucleinopathies. Several findings in experimental models implicate perturbations in the tissue homeostatic mechanisms triggered by pathological aSyn accumulation, including impaired redox homeostasis, as significant contributors in the pathogenesis of PD. The nuclear factor erythroid 2-related factor (NRF2/Nrf2) is recognized as 'the master regulator of cellular anti-oxidant response', both under physiological as well as in pathological conditions. Using immunohistochemical analyses, we show a robust nuclear NRF2 accumulation in post-mortem PD midbrain, detected by NRF2 phosphorylation on the serine residue 40 (nuclear active p-NRF2, S40). Curated gene expression analyses of four independent publicly available microarray datasets revealed considerable alterations in NRF2-responsive genes in the disease affected regions in PD, including substantia nigra, dorsal motor nucleus of vagus, locus coeruleus and globus pallidus. To further examine the putative role of pathological aSyn accumulation on nuclear NRF2 response, we employed a transgenic mouse model of synucleionopathy (M83 line, expressing the mutant human A53T aSyn), which manifests widespread aSyn pathology (phosphorylated aSyn; S129) in the nervous system following intramuscular inoculation of exogenous fibrillar aSyn. We observed strong immunodetection of nuclear NRF2 in neuronal populations harboring p-aSyn (S129), and found an aberrant anti-oxidant and inflammatory gene response in the affected neuraxis. Taken together, our data support the notion that pathological aSyn accumulation impairs the redox homeostasis in nervous system, and boosting neuronal anti-oxidant response is potentially a promising approach to mitigate neurodegeneration in PD and related diseases.

Atypical teratoid/rhabdoid tumors (ATRTs) typically arise in the central nervous system (CNS) of children under 3 years of age. Despite intensive multimodal therapy (surgery, chemotherapy and, if age permits, radiotherapy), median survival is 17 months1,2. We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs. 3,4), which drive oncogenesis in ATRT, but requires residual SWItch/Sucrose Non-Fermentable (SWI/SNF) activity mediated by BRG1/SMARCA4. Consistent with the embryonic origin of ATRT5,6, B7-H3 is highly expressed on the prenatal, but not postnatal, brain. B7-H3.BB.z-chimeric antigen receptor (CAR) T cells administered intracerebroventricularly or intratumorally mediate potent antitumor effects against cerebral ATRT xenografts in mice, with faster kinetics, greater potency and reduced systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. CAR T cells administered ICV also traffic from the CNS into the periphery; following clearance of ATRT xenografts, B7-H3.BB.z-CAR T cells administered intracerebroventricularly or intravenously mediate antigen-specific protection from tumor rechallenge, both in the brain and periphery. These results identify B7-H3 as a compelling therapeutic target for this largely incurable pediatric tumor and demonstrate important advantages of locoregional compared to systemic delivery of CAR T cells for the treatment of CNS malignancies.

Parkinson's disease (PD) is a currently incurable disease and the number of patients is expected to increase due to the extended human lifespan. α-Synuclein is a pathological hallmark of PD and variations and triplications of the gene encoding α-synuclein are strongly correlated with the risk of developing PD. Decreasing α-synuclein is therefore a promising therapeutic strategy for the treatment of PD. We have previously demonstrated that Polo-like kinase 2 (PLK-2) regulates α-synuclein protein levels by modulating the expression of α-synuclein mRNA. In this study, we further expand the knowledge on this pathway and show that it depends on down-stream modulation of Glycogen-synthase kinase 3 β (GSK-3β). We show that PLK-2 inhibition only increases α-synuclein levels in the presence of active GSK-3β in both cell lines and primary neuronal cultures. Furthermore, direct inhibition of GSK-3β decreases α-synuclein protein and mRNA levels in our cell model and overexpression of Leucine-rich repeat kinase 2, known to activate GSK-3β, increases α-synuclein levels. Finally, we show an increase in endogenous α-synuclein in primary neurons when increasing GSK-3β activity. Our findings demonstrate a not previously described role of endogenous GSK-3β activity in the PLK-2 mediated regulation of α-synuclein levels. This finding opens up the possibility of GSK-3β as a novel target for decreasing α-synuclein levels by the use of small molecule compounds, hereby serving as a disease modulating strategy.

Transthyretin amyloidoses encompass a variety of acquired and hereditary diseases triggered by systemic extracellular accumulation of toxic transthyretin aggregates and fibrils, particularly in the peripheral nervous system. Since transthyretin amyloidoses are typically complex progressive disorders, therapeutic approaches aiming multiple molecular targets simultaneously, might improve therapy efficacy and treatment outcome. In this study, we evaluate the protective effect of physiologically achievable doses of curcumin on the cytotoxicity induced by transthyretin oligomers in vitro by showing reduction of caspase-3 activity and the levels of endoplasmic reticulum-resident chaperone binding immunoglobulin protein. When given to an aged Familial Amyloidotic Polyneuropathy mouse model, curcumin not only reduced transthyretin aggregates deposition and toxicity in both gastrointestinal tract and dorsal root ganglia but also remodeled congophilic amyloid material in tissues. In addition, curcumin enhanced internalization, intracellular transport and degradation of transthyretin oligomers by primary macrophages from aged Familial Amyloidotic Polyneuropathy transgenic mice, suggesting an impaired activation of naïve phagocytic cells exposed to transthyretin toxic intermediate species. Overall, our results clearly support curcumin or optimized derivatives as promising multi-target disease-modifying agent for late-stage transthyretin amyloidosis.

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disease caused by the extracellular deposition of mutant transthyretin (TTR), with special involvement of the peripheral nervous system (PNS). Currently, hepatic transplantation is considered the most efficient therapy to halt the progression of clinical symptoms in FAP since more than 95% of TTR is produced by the liver. However, less invasive and more reliable therapeutic approaches have been proposed for FAP therapy, namely based on drugs acting as inhibitors of amyloid formation or as amyloid disruptors. We have recently reported that epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, is able to inhibit TTR aggregation and fibril formation, "in vitro" and in a cellular system, and is also able to disrupt pre-formed amyloid fibrils "in vitro".

Several natural polyphenols with potent inhibitory effects on amyloid fibril formation have been reported. Herein, we studied modulation of transthyretin (TTR) fibrillogenesis by selected polyphenols. We demonstrate that both curcumin and nordihydroguaiaretic acid (NDGA) bind to TTR and stabilize the TTR tetramer. However, while NDGA slightly reduced TTR aggregation, curcumin strongly suppressed TTR amyloid fibril formation by generating small "off-pathway" oligomers and EGCG maintained most of the protein in a non-aggregated soluble form. This indicates alternative mechanisms of action supported by the occurrence of different non-toxic intermediates. Moreover, EGCG and curcumin efficiently disaggregated pre-formed TTR amyloid fibrils. Our studies, together with the safe toxicological profile of these phytochemicals may guide a novel pharmacotherapy for TTR-related amyloidosis targeting different steps in fibrillogenesis.

Medulloblastomas (MB) molecularly designated as Group 3 (Grp 3) MB represent a more clinically aggressive tumor variant which, as a group, displays heterogeneous molecular characteristics and disease outcomes. Reliable risk stratification of Grp 3 MB would allow for appropriate assignment of patients to aggressive treatment protocols and, vice versa, for sparing adverse effects of high-dose radio-chemotherapy in patients with standard or low-risk tumors. Here we performed RNA-based analysis on an international cohort of 179 molecularly designated Grp 3 MB treated with HIT protocols. We analyzed the clinical significance of differentially expressed genes, thereby developing optimal prognostic subdivision of this MB molecular group. We compared the transcriptome profiles of two Grp 3 MB subsets with various outcomes (76 died within the first 60 months vs. 103 survived this period) and identified 224 differentially expressed genes (DEG) between these two clinical groups (Limma R algorithm, adjusted p-value < 0.05). We selected the top six DEG overexpressed in the unfavorable cohort for further survival analysis and found that expression of all six genes strongly correlated with poor outcomes. However, only high expression of KIRREL2 was identified as an independent molecular prognostic indicator of poor patients' survival. Based on clinical and molecular patterns, four risk categories were outlined for Grp 3 MB patients: i. low-risk: M0-1/MYC non-amplified/KIRREL2 low (n = 48; 5-year OS-95%); ii. standard-risk: M0-1/MYC non-amplified/KIRREL2 high or M2-3/MYC non-amplified/KIRREL2 low (n = 65; 5-year OS-70%); iii. high-risk: M2-3/MYC non-amplified/KIRREL2 high (n = 36; 5-year OS-30%); iv. very high risk-all MYC amplified tumors (n = 30; 5-year OS-0%). Cross-validated survival models incorporating KIRREL2 expression with clinical features allowed for the reclassification of up to 50% of Grp 3 MB patients into a more appropriate risk category. Finally, KIRREL2 immunopositivity was also identified as a predictive indicator of Grp 3 MB poor survival, thus suggesting its application as a possible prognostic marker in routine clinical settings. Our results indicate that integration of KIRREL2 expression in risk stratification models may improve Grp 3 MB outcome prediction. Therefore, simple gene and/or protein expression analyses for this molecular marker could be easily adopted for Grp 3 MB prognostication and may help in assigning patients to optimal therapeutic approaches in prospective clinical trials.

Recurrent medulloblastoma and ependymoma are universally lethal, with no approved targeted therapies and few candidates presently under clinical evaluation. Nearly all recurrent medulloblastomas and posterior fossa group A (PFA) ependymomas are located adjacent to and bathed by the cerebrospinal fluid, presenting an opportunity for locoregional therapy, bypassing the blood-brain barrier. We identify three cell-surface targets, EPHA2, HER2 and interleukin 13 receptor α2, expressed on medulloblastomas and ependymomas, but not expressed in the normal developing brain. We validate intrathecal delivery of EPHA2, HER2 and interleukin 13 receptor α2 chimeric antigen receptor T cells as an effective treatment for primary, metastatic and recurrent group 3 medulloblastoma and PFA ependymoma xenografts in mouse models. Finally, we demonstrate that administration of these chimeric antigen receptor T cells into the cerebrospinal fluid, alone or in combination with azacytidine, is a highly effective therapy for multiple metastatic mouse models of group 3 medulloblastoma and PFA ependymoma, thereby providing a rationale for clinical trials of these approaches in humans.