Faculty Bibliography

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is a classic example of dysregulation of developmental pathways leading to tumorogenesis. Despite advancements in multi-modal therapies, most patients suffer from long-term neurocognitive and neuroendocrine disabilities. The Sonic Hedgehog subgroup of MB (SHH-MB) accounts for ~30% of all cases and originates from ATOH1+ cerebellar granule cell precursors (GCPs). Experimental data in mice has shown that activating mutations in the SHH pathway induce tumors only in rare GCPs, suggesting that additional mutations and epigenetic changes are required to influence tumor progression. The KMT2D gene, encoding the histone-lysine N-methyltransferase 2D, is amongst the ten most frequently mutated genes in MB, with somatic mutations seen in ~15% of all SHH-MB patients. We developed sporadic mouse models of SHH-MB with a low penetrance to enable studies of secondary mutations (Tan, PNAS, 2018). Immunofluorescence staining for KMT2D on early-stage SHH-MB lesions, mid-stage and late-stage tumors revealed that a subset of lesions/tumors (16/98) do not express KMT2D and are negative for H3K4me3. Interestingly, P53 and KMT2D expression showed a positive correlation in ~94% of tumors/lesions and NeuN and KMT2D showed a positive correlation in ~92% of tumors/lesions. In order to determine the roles for KMT2D in GCP proliferation and differentiation, and uncover whether and how KMT2D promotes SHH-MB tumorigenesis, we are using genetic mouse-models whereby Kmt2d is heterozygously or homozygously deleted alone, or in conjunction with activation of the SHH pathway. Mice with SHH-MB tumors expressing SmoM2 and a loss of Kmt2d develop aggressive tumors at high penetrance, with metastatic leptomeningeal spread in the brain stem and spinal cord. Thus, loss of Kmt2d increases SHH-MB tumor progression and leads to malignancy. Ongoing studies are determining how the chromatin landscape and gene expression are changed when Kmt2d is deleted in GCPs

OBJECTIVE:Tumour necrosis factor alpha (TNF-α) signalling plays a central role in the pathogenesis of various autoimmune diseases, particularly inflammatory arthritis. This study aimed to repurpose clinically approved drugs as potential inhibitors of TNF-α signalling in treatment of inflammatory arthritis. METHODS:In vitro and in vivo screening of an Food and Drug Administration (FDA)-approved drug library; in vitro and in vivo assays for examining the blockade of TNF actions by fexofenadine: assays for defining the anti-inflammatory activity of fexofenadine using TNF-α transgenic (TNF-tg) mice and collagen-induced arthritis in DBA/1 mice. Identification and characterisation of the binding of fexofenadine to cytosolic phospholipase A2 (cPLA2) using drug affinity responsive target stability assay, proteomics, cellular thermal shift assay, information field dynamics and molecular dynamics; various assays for examining fexofenadine inhibition of cPLA2 as well as the dependence of fexofenadine's anti-TNF activity on cPLA2. RESULTS:Serial screenings of a library composed of FDA-approved drugs led to the identification of fexofenadine as an inhibitor of TNF-α signalling. Fexofenadine potently inhibited TNF/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) signalling in vitro and in vivo, and ameliorated disease symptoms in inflammatory arthritis models. cPLA2 was isolated as a novel target of fexofenadine. Fexofenadine blocked TNF-stimulated cPLA2 activity and arachidonic acid production through binding to catalytic domain 2 of cPLA2 and inhibition of its phosphorylation on Ser-505. Further, deletion of cPLA2 abolished fexofenadine's anti-TNF activity. CONCLUSION/CONCLUSIONS:Collectively, these findings not only provide new insights into the understanding of fexofenadine action and underlying mechanisms but also provide new therapeutic interventions for various TNF-α and cPLA2-associated pathologies and conditions, particularly inflammatory rheumatic diseases.

Dystonia is a disabling neurological syndrome characterized by abnormal movements and postures that result from intermittent or sustained involuntary muscle contractions; mutations of DYT1/TOR1A are the most common cause of childhood-onset, generalized, inherited dystonia. Patient and mouse model data strongly support dysregulation of the nigrostriatal dopamine neurotransmission circuit in the presence of the DYT1-causing mutation. To determine striatal medium spiny neuron (MSN) cell-autonomous and non-cell autonomous effects relevant to dopamine transmission, we created a transgenic mouse in which expression of mutant torsinA in forebrain is restricted to MSNs. We assayed electrically evoked and cocaine-enhanced dopamine release and locomotor activity, dopamine uptake, gene expression of dopamine-associated neuropeptides and receptors, and response to the muscarinic cholinergic antagonist, trihexyphenidyl. We found that over-expression of mutant torsinA in MSNs produces complex cell-autonomous and non-cell autonomous alterations in nigrostriatal dopaminergic and intrastriatal cholinergic function, similar to that found in pan-cellular DYT1 mouse models. These data introduce targets for future studies to identify which are causative and which are compensatory in DYT1 dystonia, and thereby aid in defining appropriate therapies.

The mitochondrial inner membrane consists of the inner boundary membrane and invaginations called cristae, which differ in protein composition and likely have distinct functions. In this issue of The EMBO Journal, Wolf et al (2019) report that the cristae carry a higher membrane potential than the intervening boundary membranes. Their data suggest electro-chemical discontinuity among segments of the inner membrane, implying that individual cristae may operate with some degree of independence.

Modulation of cannabinoid and neuropeptide Y (NPY) receptors may offer therapeutic benefits for post-traumatic stress disorder (PTSD). In this study, we aimed to investigate the functional interaction between these systems in the basolateral amygdala (BLA) in a rat model of PTSD. Rats were exposed to the shock and reminders model of PTSD and tested for hyper arousal/PTSD- and depression-like behaviors 3 weeks later. Immediately after shock exposure rats were microinjected into the BLA with URB597, a selective inhibitor of fatty acid amide hydrolase (FAAH) that increases the levels of the endocannabinoid anandamide or with the NPY1 receptor agonist Leu³¹,Pro³⁴-NPY (Leu). Intra-BLA URB597 prevented the shock/reminders-induced PTSD- behaviors (extinction, startle) and depression-behaviors (despair, social impairments). These preventing effects of URB597 on PTSD- and depression-like behaviors were shown to be mostly mediated by cannabinoid CB1 and NPY1 receptors, as they were blocked when URB597 was co-administered with a low dose of a CB1 or NPY1 receptor antagonist. Similarly, intra-BLA Leu prevented development of all the behaviors. Interestingly, a CB1 antagonist prevented the effects of Leu on despair and social behavior, but not the effects on extinction and startle. Moreover, exposure to shock and reminders upregulated CB1 and NPY1 receptors in the BLA and infralimbic prefrontal cortex and this upregulation was restored to normal with intra-BLA URB597 or Leu. The findings suggest that the functional interaction between the eCB and NPY1 systems is complex and provide a rationale for exploring novel therapeutic strategies that target the cannabinoid and NPY systems for stress-related diseases.

Recent research shows that potentially cancerous, somatic mutations can reside in normal cells. Pineda et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201907178) report on a unique management technique by hair follicle stem cells to evade tumorigenesis.

Microglia, the brain resident macrophages, critically shape forebrain neuronal circuits. However, their precise function in the cerebellum is unknown. Here we show that human and mouse cerebellar microglia express a unique molecular program distinct from forebrain microglia. Cerebellar microglial identity was driven by the CSF-1R ligand CSF-1, independently of the alternate CSF-1R ligand, IL-34. Accordingly, CSF-1 depletion from Nestin⁺ cells led to severe depletion and transcriptional alterations of cerebellar microglia, while microglia in the forebrain remained intact. Strikingly, CSF-1 deficiency and alteration of cerebellar microglia were associated with reduced Purkinje cells, altered neuronal function, and defects in motor learning and social novelty interactions. These findings reveal a novel CSF-1-CSF-1R signaling-mediated mechanism that contributes to motor function and social behavior.