Panobinostat Induced Spatial In Situ Biomarkers Predictive of Anti-PD-1 Efficacy in Mouse Mammary Carcinoma.

Immunotherapies, including anti-PD-1 immune checkpoint blocking (ICB) antibodies, have revolutionized the treatment of many solid malignancies. However, their efficacy in breast cancer has been limited to a subset of patients with triple-negative breast cancer, where ICBs are routinely combined with a range of cytotoxic and targeted agents. Reliable biomarkers predictive of the therapeutic response to ICB in breast cancer are critically missing, though a combination response has been associated with immunogenic cell death (ICD). Here, we utilized a recently developed integrated analytical platform, the multiplex implantable microdevice assay (MIMA), to evaluate the presence and spatial cell relations of literature-based candidate markers predictive of ICB efficacy in luminal mouse mammary carcinoma. MIMA integrates (i) an implantable microdevice for the localized delivery of small amounts of drugs inside the tumor bed with (ii) sequential multiplex immunohistochemistry (mIHC) and spatial cell analysis pipelines to rapidly (within days) describe drug mechanisms of action and find predictive biomarkers in complex tumor tissue. We show that the expression of cleaved caspase-3, ICAM-1, neuropilin-1, myeloperoxidase, calreticulin, galectin-3, and PD-L1 were spatially associated with the efficacy of panobinostat, a pan-HDAC inhibitor that was previously shown to induce immunogenic cell death and synergize with anti-PD-1 in breast cancer. PD-L1 by itself, however, was not a reliable predictor. Instead, ICB efficacy was robustly identified through the in situ hotspot detection of galectin-3-positive non-proliferating tumor zones enriched in cell death and infiltrated by anti-tumor cytotoxic neutrophils positive for ICAM-1 and neuropilin-1. Such hotspots can be specifically detected using distance-based cluster analyses. Single-cell measurements of the functional states in the tumor microenvironment suggest that both qualitative and quantitative effects might drive effective therapy responses. Overall, the presented study provides (i) complementary biological knowledge about the earliest cell events of induced anti-tumor immunity in breast cancer, including the emergence of resistant cancer stem cells, and (ii) newly identified biomarkers in form of specific spatial cell associations. The approach used standard cell-type-, IHC-, and FFPE-based techniques, and therefore the identified spatial clustering of in situ biomarkers can be readily integrated into existing clinical or research workflows, including in luminal breast cancer. Since early drug responses were detected, the biomarkers could be especially applicable to window-of-opportunity clinical trials to rapidly discriminate between responding and resistant patients, thus limiting unnecessary treatment-associated toxicities.

The oligodendrocyte-enriched orphan G protein-coupled receptor Gpr62 is dispensable for central nervous system myelination.

BACKGROUND: Myelination is a highly regulated process in the vertebrate central nervous system (CNS) whereby oligodendrocytes wrap axons with multiple layers of insulating myelin in order to allow rapid electrical conduction. Establishing the proper pattern of myelin in neural circuits requires communicative axo-glial interactions, however, the molecular interactions that occur between oligodendrocytes and axons during developmental myelination and myelin maintenance remain to be fully elucidated. Our previous work identified G protein-coupled receptor 62 (Gpr62), an uncharacterized orphan g-protein coupled receptor, as being selectively expressed by mature oligodendrocytes within the CNS, suggesting a potential role in myelination or axoglial interactions. However, no studies to date have assessed the functional requirement for Gpr62 in oligodendrocyte development or CNS myelination. METHODS: To address this, we generated a knockout mouse strain lacking the Gpr62 gene. We assessed CNS myelination during both postnatal development and adulthood using immunohistochemistry, electron microscopy and western blot. In addition, we utilized AAV-mediated expression of a tagged Gpr62 in oligodendrocytes to determine the subcellular localization of the protein in vivo. RESULTS: We find that virally expressed Gpr62 protein is selectively expressed on the adaxonal myelin layer, suggestive of a potential role for Gpr62 in axo-myelinic signaling. Nevertheless, Gpr62 knockout mice display normal oligodendrocyte numbers and apparently normal myelination within the CNS during both postnatal development and adulthood. CONCLUSIONS: We conclude that in spite of being well-placed to mediate neuronal-oligodendrocyte communications, Gpr62 is overall dispensable for CNS myelination.

Short-Term Depression of Sprouted Mossy Fiber Synapses from Adult-Born Granule Cells.

Epileptic seizures potently modulate hippocampal adult neurogenesis, and adult-born dentate granule cells contribute to the pathologic retrograde sprouting of mossy fiber axons, both hallmarks of temporal lobe epilepsy. The characteristics of these sprouted synapses, however, have been largely unexplored, and the specific contribution of adult-born granule cells to functional mossy fiber sprouting is unknown, primarily due to technical barriers in isolating sprouted mossy fiber synapses for analysis. Here, we used DcxCreERT2 transgenic mice to permanently pulse-label age-defined cohorts of granule cells born either before or after pilocarpine-induced status epilepticus (SE). Using optogenetics, we demonstrate that adult-born granule cells born before SE form functional recurrent monosynaptic excitatory connections with other granule cells. Surprisingly, however, although healthy mossy fiber synapses in CA3 are well characterized "detonator" synapses that potently drive postsynaptic cell firing through their profound frequency-dependent facilitation, sprouted mossy fiber synapses from adult-born cells exhibited profound frequency-dependent depression, despite possessing some of the morphological hallmarks of mossy fiber terminals. Mature granule cells also contributed to functional mossy fiber sprouting, but exhibited less synaptic depression. Interestingly, granule cells born shortly after SE did not form functional excitatory synapses, despite robust sprouting. Our results suggest that, although sprouted mossy fibers form recurrent excitatory circuits with some of the morphological characteristics of typical mossy fiber terminals, the functional characteristics of sprouted synapses would limit the contribution of adult-born granule cells to hippocampal hyperexcitability in the epileptic hippocampus.SIGNIFICANCE STATEMENT In the hippocampal dentate gyrus, seizures drive retrograde sprouting of granule cell mossy fiber axons. We directly activated sprouted mossy fiber synapses from adult-born granule cells to study their synaptic properties. We reveal that sprouted synapses from adult-born granule cells have a diminished ability to sustain recurrent excitation in the epileptic hippocampus, which raises questions about the role of sprouting and adult neurogenesis in sustaining seizure-like activity.

Neuroligin-1 knockdown reduces survival of adult-generated newborn hippocampal neurons.

Survival of adult-born hippocampal granule cells is modulated by neural activity, and thought to be enhanced by excitatory synaptic signaling. Here, we report that a reduction in the synaptogenic protein neuroligin-1 in adult-born neurons in vivo decreased their survival, but surprisingly, this effect was independent of changes in excitatory synaptic function. Instead, the decreased survival was associated with unexpected changes in dendrite and spine morphology during granule cell maturation, suggesting a link between cell growth and survival.

Distinct modes of dopamine and GABA release in a dual transmitter neuron.

We now know of a surprising number of cases where single neurons contain multiple neurotransmitters. Neurons that contain a fast-acting neurotransmitter, such as glutamate or GABA, and a modulatory transmitter, such as dopamine, are a particularly interesting case because they presumably serve dual signaling functions. The olfactory bulb contains a large population of GABA- and dopamine-containing neurons that have been implicated in normal olfaction as well as in Parkinson's disease. Yet, they have been classified as nonexocytotic catecholamine neurons because of the apparent lack of vesicular monoamine transporters. Thus, we examined how dopamine is stored and released from tyrosine hydroxylase-positive GFP (TH(+)-GFP) mouse periglomerular neurons in vitro. TH(+) cells expressed both VMAT2 (vesicular monoamine transporter 2) and VGAT (vesicular GABA transporter), consistent with vesicular storage of both dopamine and GABA. Carbon fiber amperometry revealed that release of dopamine was quantal and calcium-dependent, but quantal size was much less than expected for large dense core vesicles, suggesting that release originated from small clear vesicles identified by electron microscopy. A single action potential in a TH(+) neuron evoked a brief GABA-mediated synaptic current, whereas evoked dopamine release was asynchronous, lasting for tens of seconds. Our data suggest that dopamine and GABA serve temporally distinct roles in these dual transmitter neurons.

Neuroligin-1 overexpression in newborn granule cells in vivo.

Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons.

miR-132 mediates the integration of newborn neurons into the adult dentate gyrus.

Neuronal activity enhances the elaboration of newborn neurons as they integrate into the synaptic circuitry of the adult brain. The role microRNAs play in the transduction of neuronal activity into growth and synapse formation is largely unknown. MicroRNAs can influence the expression of hundreds of genes and thus could regulate gene assemblies during processes like activity-dependent integration. Here, we developed viral-based methods for the in vivo detection and manipulation of the activity-dependent microRNA, miR-132, in the mouse hippocampus. We find, using lentiviral and retroviral reporters of miR-132 activity, that miR-132 is expressed at the right place and right time to influence the integration of newborn neurons. Retroviral knockdown of miR-132 using a specific 'sponge' containing multiple target sequences impaired the integration of newborn neurons into the excitatory synaptic circuitry of the adult brain. To assess potential miR-132 targets, we used a whole-genome microarray in PC12 cells, which have been used as a model of neuronal differentiation. miR-132 knockdown in PC12 cells resulted in the increased expression of hundreds of genes. Functional grouping indicated that genes involved in inflammatory/immune signaling were the most enriched class of genes induced by miR-132 knockdown. The correlation of miR-132 knockdown to increased proinflammatory molecular expression may indicate a mechanistic link whereby miR-132 functions as an endogenous mediator of activity-dependent integration in vivo.

Loss of olfactory cell adhesion molecule reduces the synchrony of mitral cell activity in olfactory glomeruli.

Odours generate activity in olfactory receptor neurons, whose axons contact the dendritic tufts of mitral cells within olfactory bulb glomeruli. These axodendritic synapses are anatomically separated from dendrodendritic synapses within each glomerulus. Mitral cells within a glomerulus show highly synchronized activity as assessed with whole-cell recording from pairs of mitral cells. We examined glomerular activity in mice lacking the olfactory cell adhesion molecule (OCAM). Glomeruli in mice lacking OCAM show a redistribution of synaptic subcompartments, but the total area occupied by axonal inputs was similar to wild-type mice. Stimulation of olfactory nerve bundles showed that excitatory synaptic input to mitral cells as well as dendrodendritic inhibition was unaffected in the knockout. However, correlated spiking in mitral cells was significantly reduced, as was electrical coupling between apical dendrites. To analyse slow network dynamics we induced slow oscillations with a glutamate uptake blocker. Evoked and spontaneous slow oscillations in mitral cells and external tufted cells were broader and had multiple peaks in OCAM knockout mice, indicating that synchrony of slow glomerular activity was also reduced. To assess the degree of shared activity between mitral cells under physiological conditions, we analysed spontaneous sub-threshold voltage oscillations using coherence analysis. Coherent activity was markedly reduced in cells from OCAM knockout mice across a broad range of frequencies consistent with a decrease in tightly time-locked activity. We suggest that synchronous activity within each glomerulus is dependent on segregation of synaptic subcompartments.

Pten knockdown in vivo increases excitatory drive onto dentate granule cells.

Some cases of autism spectrum disorder have mutations in the lipid phosphatase, phosphatase and tensin homolog on chromosome 10 (Pten). Tissue specific deletion of Pten in the hippocampus and cortex of mice causes anatomical and behavioral abnormalities similar to human autism. However, the impact of reductions in Pten on synaptic and circuit function remains unexplored. We used in vivo stereotaxic injections of lentivirus expressing a short hairpin RNA to knock down Pten in mouse neonatal and young adult dentate granule cells. We then assessed the morphology and synaptic physiology between 2 weeks and 4 months later. Confocal imaging of the hippocampus revealed a marked increase in granule cell size and an increase in dendritic spine density. The onset of morphological changes occurred earlier in neonatal mice than in young adults. We used whole-cell recordings from granule cells in acute slices to assess synaptic function after Pten knockdown. Consistent with the increase in dendritic spines, the frequency of excitatory miniature and spontaneous postsynaptic currents increased. However, there was little or no effect on IPSCs. Thus, Pten knockdown results in an imbalance between excitatory and inhibitory synaptic activity. Because reductions in Pten affected mature granule cells as well as developing granule cells, we suggest that the disruption of circuit function by Pten hypofunction may be ongoing well beyond early development.

Seizures accelerate functional integration of adult-generated granule cells.

In humans and experimental animals, structural and functional changes in neural circuits can accompany the development of epilepsy. In the dentate gyrus, seizures enhance adult neurogenesis, but it is unclear to what extent newborn granule cells participate in seizure-induced synaptic reorganization. During the first weeks of their existence, mouse newborn granule cells labeled with enhanced green fluorescent protein have only short dendrites that lack excitatory input. We report that pilocarpine-induced seizures accelerated the morphological development of labeled granule cells, causing their dendrites to extend through the molecular layer. In whole-cell recordings 5-16 d after seizure induction, perforant-path stimulation now evoked glutamatergic input to newborn granule cells. These synaptic responses were mediated by monosynaptic as well as recurrent polysynaptic input. Thus, seizures facilitated functional integration of adult-generated granule cells. One month later, subsequent generations of newborn cells also showed alterations in dendrite morphology, suggesting persistent effects of seizures on granule cell maturation. The sensitivity of newborn granule cells to seizures could contribute to hyperexcitability during the latent period.

Delayed development of adult-generated granule cells in dentate gyrus.

A substantial fraction of adult-generated granule cells in the dentate gyrus survive and integrate into the existing neuronal network. These newborn neurons must navigate the environment of the adult brain, a setting that is presumably less optimized for neuronal maturation compared with that in the developing brain. We used EGFP (enhanced green fluorescent protein) expression in newborn granule cells to compare the maturation of adult-generated granule cells to those generated in neonates. Labeled newborn granule cells had indistinguishable physiological properties in adults and neonates, indicating they were at the same functional stage. However, the maturation of adult-generated granule cells was slower than neonatal-generated granule cells. Depolarizing GABAergic network activity and transcription factor activation were reduced in adults relative to neonates, suggesting a role for neural activity in the maturation of newborn granule cells. Consistent with this idea, maturation was altered in mice lacking the GABA synthetic enzyme GAD65 (glutamic acid decarboxylase 65). Together, these results provide evidence that activity-dependent processes in the local environment influence the maturation of newborn granule cells.

GABAergic signaling to newborn neurons in dentate gyrus.

Neurogenesis in the dentate gyrus begins before birth but then continues into adulthood. Consequently, many newborn granule cells must integrate into a preexisting hippocampal network. Little is known about the timing of this process or the characteristics of the first established synapses. We used mice that transiently express enhanced green fluorescent protein in newborn granule cells to examine their synaptic input. Although newborn granule cells had functional glutamate receptors, evoked and spontaneous synaptic currents were exclusively GABAergic with immature characteristics including slow rise and decay phases and depolarized reversal potentials. Synaptic currents in newborn granule cells were relatively insensitive to the GABA(A) receptor modulator zolpidem compared with neighboring mature granule cells. Consistent with the kinetics and pharmacology, newborn granule cells isolated by fluorescent cell sorting lacked the alpha1 GABA(A) receptor subunit. Our results indicate that newborn granule cells initially receive only GABAergic synapses even in the adult.