Reverse-translational identification of a cerebellar satiation network.

The brain is the seat of body weight homeostasis. However, our inability to control the increasing prevalence of obesity highlights a need to look beyond canonical feeding pathways to broaden our understanding of body weight control1-3. Here we used a reverse-translational approach to identify and anatomically, molecularly and functionally characterize a neural ensemble that promotes satiation. Unbiased, task-based functional magnetic resonance imaging revealed marked differences in cerebellar responses to food in people with a genetic disorder characterized by insatiable appetite. Transcriptomic analyses in mice revealed molecularly and topographically -distinct neurons in the anterior deep cerebellar nuclei (aDCN) that are activated by feeding or nutrient infusion in the gut. Selective activation of aDCN neurons substantially decreased food intake by reducing meal size without compensatory changes to metabolic rate. We found that aDCN activity terminates food intake by increasing striatal dopamine levels and attenuating the phasic dopamine response to subsequent food consumption. Our study defines a conserved satiation centre that may represent a novel therapeutic target for the management of excessive eating, and underscores the utility of a 'bedside-to-bench' approach for the identification of neural circuits that influence behaviour.

Distinct contributions of ventral CA1/amygdala co-activation to the induction and maintenance of synaptic plasticity.

The amygdala is known to modulate hippocampal synaptic plasticity. One role could be an immediate effect of basolateral amygdala (BLA) in priming synaptic plasticity in the hippocampus. Another role could be through associative synaptic co-operation and competition that triggers events involved in the maintenance of synaptic potentiation. We present evidence that the timing and activity level of BLA stimulation are important factors for the induction and maintenance of long-term potentiation (LTP) in ventral hippocampal area CA1. A 100 Hz BLA co-stimulation facilitated the induction of LTP, whereas 200 Hz co-stimulation attenuated induction. A 100 Hz BLA co-stimulation also caused enhanced persistence, sufficient to prevent synaptic competition. This maintenance effect is likely through translational mechanisms, as mRNA expression of primary response genes was unaffected, whereas protein level of plasticity-related products was increased. Further understanding of the neural mechanisms of amygdala modulation on hippocampus could provide insights into the mechanisms of emotional disorders.

Cytokine enrichment in deep cerebellar nuclei is contributed by multiple glial populations and linked to reduced amyloid plaque pathology.

Alzheimer's disease (AD) pathology and amyloid-beta (Aß) plaque deposition progress slowly in the cerebellum compared to other brain regions, while the entorhinal cortex (EC) is one of the most vulnerable regions. Using a knock-in AD mouse model (App KI), we show that within the cerebellum, the deep cerebellar nuclei (DCN) has particularly low accumulation of Aß plaques. To identify factors that might underlie differences in the progression of AD-associated neuropathology across regions, we profiled gene expression in single nuclei (snRNAseq) across all cell types in the DCN and EC of wild-type (WT) and App KI male mice at age 7 months. We found differences in expression of genes associated with inflammatory activation, PI3K-AKT signalling, and neuron support functions between both regions and genotypes. In WT mice, the expression of interferon-response genes in microglia is higher in the DCN than the EC and this enrichment is confirmed by RNA in situ hybridisation, and measurement of inflammatory cytokines by protein array. Our analyses also revealed that multiple glial populations are responsible for establishing this cytokine-enriched niche. Furthermore, homogenates derived from the DCN induced inflammatory gene expression in BV2 microglia. We also assessed the relationship between the DCN microenvironment and Aß pathology by depleting microglia using a CSF1R inhibitor PLX5622 and saw that, surprisingly, the expression of a subset of inflammatory cytokines was increased while plaque abundance in the DCN was further reduced. Overall, our study revealed the presence of a cytokine-enriched microenvironment unique to the DCN that when modulated, can alter plaque deposition.

iCLIP identifies novel roles for SAFB1 in regulating RNA processing and neuronal function.

BACKGROUND: SAFB1 is a RNA binding protein implicated in the regulation of multiple cellular processes such as the regulation of transcription, stress response, DNA repair and RNA processing. To gain further insight into SAFB1 function we used iCLIP and mapped its interaction with RNA on a genome wide level. RESULTS: iCLIP analysis found SAFB1 binding was enriched, specifically in exons, ncRNAs, 3' and 5' untranslated regions. SAFB1 was found to recognise a purine-rich GAAGA motif with the highest frequency and it is therefore likely to bind core AGA, GAA, or AAG motifs. Confirmatory RT-PCR experiments showed that the expression of coding and non-coding genes with SAFB1 cross-link sites was altered by SAFB1 knockdown. For example, we found that the isoform-specific expression of neural cell adhesion molecule (NCAM1) and ASTN2 was influenced by SAFB1 and that the processing of miR-19a from the miR-17-92 cluster was regulated by SAFB1. These data suggest SAFB1 may influence alternative splicing and, using an NCAM1 minigene, we showed that SAFB1 knockdown altered the expression of two of the three NCAM1 alternative spliced isoforms. However, when the AGA, GAA, and AAG motifs were mutated, SAFB1 knockdown no longer mediated a decrease in the NCAM1 9-10 alternative spliced form. To further investigate the association of SAFB1 with splicing we used exon array analysis and found SAFB1 knockdown mediated the statistically significant up- and downregulation of alternative exons. Further analysis using RNAmotifs to investigate the frequency of association between the motif pairs (AGA followed by AGA, GAA or AAG) and alternative spliced exons found there was a highly significant correlation with downregulated exons. Together, our data suggest SAFB1 will play an important physiological role in the central nervous system regulating synaptic function. We found that SAFB1 regulates dendritic spine density in hippocampal neurons and hence provide empirical evidence supporting this conclusion. CONCLUSIONS: iCLIP showed that SAFB1 has previously uncharacterised specific RNA binding properties that help coordinate the isoform-specific expression of coding and non-coding genes. These genes regulate splicing, axonal and synaptic function, and are associated with neuropsychiatric disease, suggesting that SAFB1 is an important regulator of key neuronal processes.

Seizure enhances SUMOylation and zinc-finger transcriptional repression in neuronal nuclei.

A single episode of pilocarpine-induced status epilepticus can trigger the development of spontaneous recurrent seizures in a rodent model for epilepsy. The initial seizure-induced events in neuronal nuclei that lead to long-term changes in gene expression and cellular responses likely contribute toward epileptogenesis. Using a transgenic mouse model to specifically isolate excitatory neuronal nuclei, we profiled the seizure-induced nuclear proteome via tandem mass tag mass spectrometry and observed robust enrichment of nuclear proteins associated with the SUMOylation pathway. In parallel with nuclear proteome, we characterized nuclear gene expression by RNA sequencing which provided insights into seizure-driven transcriptional regulation and dynamics. Strikingly, we saw widespread downregulation of zinc-finger transcription factors, specifically proteins that harbor Krüppel-associated box (KRAB) domains. Our results provide a detailed snapshot of nuclear events induced by seizure activity and demonstrate a robust method for cell-type-specific nuclear profiling that can be applied to other cell types and models.

Sex-specific accelerated decay in time/activity-dependent plasticity and associative memory in an animal model of Alzheimer's disease.

Clinical studies have shown that female brains are more predisposed to neurodegenerative diseases such as Alzheimer's disease (AD), but the cellular and molecular mechanisms behind this disparity remain unknown. In several mouse models of AD, synaptic plasticity dysfunction is an early event and appears before significant accumulation of amyloid plaques and neuronal degeneration. However, it is unclear whether sexual dimorphism at the synaptic level contributes to the higher risk and prevalence of AD in females. Our studies on APP/PS1 (APPSwe/PS1dE9) mouse model show that AD impacts hippocampal long-term plasticity in a sex-specific manner. Long-term potentiation (LTP) induced by strong tetanic stimulation (STET), theta burst stimulation (TBS) and population spike timing-dependent plasticity (pSTDP) show a faster decay in AD females compared with age-matched AD males. In addition, behavioural tagging (BT), a model of associative memory, is specifically impaired in AD females with a faster decay in memory compared with males. Together with the plasticity and behavioural data, we also observed an upregulation of neuroinflammatory markers, along with downregulation of transcripts that regulate cellular processes associated with synaptic plasticity and memory in females. Immunohistochemistry of AD brains confirms that female APP/PS1 mice carry a higher amyloid plaque burden and have enhanced microglial activation compared with male APP/PS1 mice. Their presence in the diseased mice also suggests a link between the impairment of LTP and the upregulation of the inflammatory response. Overall, our data show that synaptic plasticity and associative memory impairments are more prominent in females and this might account for the faster progression of AD in females.