Probes for LONG

Abstract Background A parallel downregulation of brain-derived neurotrophic factor (BDNF) and somatostatin (SST), a marker of inhibitory γ-amino-butyric acid (GABA) interneurons which target pyramidal cell dendrites, has been reported in several brain areas of subjects with major depressive disorder (MDD), and rodent genetic studies suggests they are linked and both contribute to the illness. However, the mechanism by which they contribute to the pathophysiology of the illness has remained elusive. Methods With qPCR, we determined the expression level of BDNF transcript variants and synaptic markers in the prefrontal cortex (PFC) of MDD patients and matched controls (n=19/group) and of C57BL/6J mice exposed to chronic stress or control conditions (n=12/group). We next suppressed BDNF transcripts with long 3’ untranslated region (L-3’-UTR) using small hairpin RNA (shRNA) and investigated changes in cell morphology, gene expression and behavior. Results L-3’-UTR containing BDNF mRNAs, which migrate to distal dendrites of pyramidal neurons, are selectively reduced and highly correlated with SST expression in the PFC of MDD subjects. A similar downregulation occurs in mice submitted to chronic stress. We next show that Bdnf L-3’-UTR knockdown is sufficient to induce (i) dendritic shrinkage in cortical neurons, (ii) cell-specific MDD-like gene changes (including Sst downregulation), and (iii) depressive-/anxiety-like behaviors. The translational validity of the Bdnf L-3’-UTR shRNA-treated mice was confirmed by significant cross-species correlation of changes in MDD-associated gene expression. Conclusions These findings provide evidence for a novel MDD-related pathological mechanism linking local neurotrophic support, pyramidal cell structure, dendritic inhibition and mood regulation.

The myelinated white matter tracts of the central nervous system (CNS) are essential for fast transmission of electrical impulses and are often differentially affected in human neurodegenerative diseases across CNS region, age and sex. We hypothesize that this selective vulnerability is underpinned by physiological variation in white matter glia. Using single nucleus RNA sequencing of human post-mortem white matter samples from the brain, cerebellum and spinal cord and subsequent tissue-based validation we found substantial glial heterogeneity with tissue region: we identified region-specific oligodendrocyte precursor cells (OPCs) that retain developmental origin markers into adulthood, distinguishing them from mouse OPCs. Region-specific OPCs give rise to similar oligodendrocyte populations, however spinal cord oligodendrocytes exhibit markers such as SKAP2 which are associated with increased myelin production and we found a spinal cord selective population particularly equipped for producing long and thick myelin sheaths based on the expression of genes/proteins such as HCN2. Spinal cord microglia exhibit a more activated phenotype compared to brain microglia, suggesting that the spinal cord is a more pro-inflammatory environment, a difference that intensifies with age. Astrocyte gene expression correlates strongly with CNS region, however, astrocytes do not show a more activated state with region or age. Across all glia, sex differences are subtle but the consistent increased expression of protein-folding genes in male donors hints at pathways that may contribute to sex differences in disease susceptibility. These findings are essential to consider for understanding selective CNS pathologies and developing tailored therapeutic strategies.

Ketamine, an N-methyl-D-aspartate (NMDA)-receptor antagonist, is a recently revitalized treatment for pain and depression, yet its actions at the molecular level remain incompletely defined. In this molecular-pharmacological investigation in the rat, we used short- and longer-term infusions of high dose ketamine to stimulate neuronal transcription processes. We hypothesized that a progressively stronger modulation of neuronal gene networks would occur over time in cortical and limbic pathways. A continuous intravenous administration paradigm for ketamine was developed in rat consisting of short (1 h) and long duration (10 h, and 10 h + 24 h recovery) infusions of anesthetic concentrations to activate or inhibit gene transcription in a pharmacokinetically controlled fashion. Transcription was measured by RNA-Seq in three brain regions: frontal cortex, hippocampus, and amygdala. Cellular level gene localization was performed with multiplex fluorescent in situ hybridization. Induction of a shared transcriptional regulatory network occurred within 1 h in all three brain regions consisting of (a) genes involved in stimulus-transcription factor coupling that are induced during altered synaptic activity (immediate early genes, IEGs, such as c-Fos, 9-12 significant genes per brain region, p < 0.01 per gene) and (b) the Nrf2 oxidative stress-antioxidant response pathway downstream from glutamate signaling (Nuclear Factor Erythroid-Derived 2-Like 2) containing 12-25 increasing genes (p < 0.01) per brain region. By 10 h of infusion, the acute results were further reinforced and consisted of more and stronger gene alterations reflecting a sustained and accentuated ketamine modulation of regional excitation and plasticity. At the cellular level, in situ hybridization localized up-regulation of the plasticity-associated gene Bdnf, and the transcription factors Nr4a1 and Fos, in cortical layers III and V. After 24 h recovery, we observed overshoot of transcriptional processes rather than a smooth return to homeostasis suggesting an oscillation of plasticity occurs during the transition to a new phase of neuronal regulation. These data elucidate critical molecular regulatory actions during and downstream of ketamine administration that may contribute to the unique drug actions of this anesthetic agent. These molecular investigations point to pathways linked to therapeutically useful attributes of ketamine.