CC-90009, a novel cereblon E3 ligase modulator, targets acute myeloid leukemia blasts and leukemia stem cells.

A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).

Enhanced Robustness of a Bridge-Type Rf-Mems Switch for Enabling Applications in 5G and 6G Communications.

In this paper, new suspended-membrane double-ohmic-contact RF-MEMS switch configurations are proposed. Double-diagonal (DDG) beam suspensions, with either two or three anchoring points, are designed and optimized to minimize membrane deformation due to residual fabrication stresses, thus exhibiting smaller mechanical deformation and a higher stiffness with more release force than previously designed single diagonal beam suspensions. The two-anchor DDGs are designed in two different orientations, in-line and 90°-rotated. The membrane may include a window to minimize the coupling to the lower electrode. The devices are integrated in a coplanar-waveguide transmission structure and fabricated using an eight-mask surface-micro-machining process on high-resistivity silicon, with dielectric-free actuation electrodes, and including glass protective caps. The RF-MEMS switch behavior is assessed from measurements of the device S parameters in ON and OFF states. The fabricated devices feature a measured pull-in voltage of 76.5 V/60 V for the windowed/not-windowed two-anchor DDG membranes, and 54 V/49.5 V for the windowed/not-windowed three-anchor DDG membranes, with a good agreement with mechanical 3D simulations. The measured ON-state insertion loss is better than 0.7 dB/0.8 dB and the isolation in the OFF state is better than 40 dB/31 dB up to 20 GHz for the in-line/90°-rotated devices, also in good agreement with 2.5D electromagnetic simulations.

Strengthening of the Efferent Olivocochlear System Leads to Synaptic Dysfunction and Tonotopy Disruption of a Central Auditory Nucleus.

The auditory system in many mammals is immature at birth but precisely organized in adults. Spontaneous activity in the inner ear plays a critical role in guiding this maturation process. This is shaped by an efferent pathway that descends from the brainstem and makes transient direct synaptic contacts with inner hair cells. In this work, we used an α9 cholinergic nicotinic receptor knock-in mouse model (of either sex) with enhanced medial efferent activity (Chrna9L9'T, L9'T) to further understand the role of the olivocochlear system in the correct establishment of auditory circuits. Wave III of auditory brainstem responses (which represents synchronized activity of synapses within the superior olivary complex) was smaller in L9'T mice, suggesting a central dysfunction. The mechanism underlying this functional alteration was analyzed in brain slices containing the medial nucleus of the trapezoid body (MNTB), where neurons are topographically organized along a mediolateral (ML) axis. The topographic organization of MNTB physiological properties observed in wildtype (WT) was abolished in L9'T mice. Additionally, electrophysiological recordings in slices indicated MNTB synaptic alterations. In vivo multielectrode recordings showed that the overall level of MNTB activity was reduced in the L9'T The present results indicate that the transient cochlear efferent innervation to inner hair cells during the critical period before the onset of hearing is involved in the refinement of topographic maps as well as in setting the properties of synaptic transmission at a central auditory nucleus.SIGNIFICANCE STATEMENT Cochlear inner hair cells of altricial mammals display spontaneous electrical activity before hearing onset. The pattern and firing rate of these cells are crucial for the correct maturation of the central auditory pathway. A descending efferent innervation from the CNS contacts the hair cells during this developmental window. The present work shows that genetic enhancement of efferent function disrupts the orderly topographic distribution of biophysical and synaptic properties in the auditory brainstem and causes severe synaptic dysfunction. This work adds to the notion that the transient efferent innervation to the cochlea is necessary for the correct establishment of the central auditory circuitry.

Effect of maternal care on hearing onset induced by developmental changes in the auditory periphery.

Handling (H) and cross-fostering (CF) rodent pups during postnatal development triggers changes in maternal behavior which in turn trigger long-term physiological changes in the offspring. However, less is known about the short-term effects of H and CF on infant development. In this study we hypothesized that manipulations of maternal care affect the onset of hearing in Wistar rats. To test this hypothesis we obtained auditory brainstem responses (ABRs) and micro-CT x-ray scans to measure changes in the development of the auditory periphery in H and CF pups manipulated at postnatal day (P)1, P5, or P9. We found evidence of changes in hearing development in H and CF pups compared with naive pups, including changes in the percentage of animals with ABRs during development, a decrease in ABR thresholds between P13 and P15, and anatomical results consistent with an accelerated formation of the middle ear cavity and opening of the ear canal. Biochemical measurements showed elevated levels of thyroid hormone in plasma from naive and CF pups. These results provide evidence that manipulations of maternal care accelerate hearing onset in Wistar rats. Understanding the mechanisms by which maternal care affects hearing onset opens new opportunities to study experience-dependent development of mammalian hearing.

N-myc-Mediated Translation Control Is a Therapeutic Vulnerability in Medulloblastoma.

Deregulation of neuroblastoma-derived myc (N-myc) is a leading cause of malignant brain tumors in children. To target N-myc-driven medulloblastoma, most research has focused on identifying genomic alterations or on the analysis of the medulloblastoma transcriptome. Here, we have broadly characterized the translatome of medulloblastoma and shown that N-myc unexpectedly drives selective translation of transcripts that promote protein homeostasis. Cancer cells are constantly exposed to proteotoxic stress associated with alterations in protein production or folding. It remains poorly understood how cancers cope with proteotoxic stress to promote their growth. Here, our data revealed that N-myc regulates the expression of specific components (∼5%) of the protein folding machinery at the translational level through the major cap binding protein, eukaryotic initiation factor eIF4E. Reducing eIF4E levels in mouse models of medulloblastoma blocked tumorigenesis. Importantly, targeting Hsp70, a protein folding chaperone translationally regulated by N-myc, suppressed tumor growth in mouse and human medulloblastoma xenograft models. These findings reveal a previously hidden molecular program that promotes medulloblastoma formation and identify new therapies that may have impact in the clinic. SIGNIFICANCE: Translatome analysis in medulloblastoma shows that N-myc drives selective translation of transcripts that promote protein homeostasis and that represent new therapeutic vulnerabilities.

Developmental changes in short-term plasticity at the rat calyx of Held synapse.

The calyx of Held synapse of the medial nucleus of the trapezoid body functions as a relay synapse in the auditory brainstem. In vivo recordings have shown that this synapse displays low release probability and that the average size of synaptic potentials does not depend on recent history. We used a ventral approach to make in vivo extracellular recordings from the calyx of Held synapse in rats aged postnatal day 4 (P4) to P29 to study the developmental changes that allow this synapse to function as a relay. Between P4 and P8, we observed evidence for the presence of large short-term depression, which was counteracted by short-term facilitation at short intervals. Major changes occurred in the last few days before the onset of hearing for air-borne sounds, which happened at P13. The bursting pattern changed into a primary-like pattern, the amount of depression and facilitation decreased strongly, and the decay of facilitation became much faster. Whereas short-term plasticity was the most important cause of variability in the size of the synaptic potentials in immature animals, its role became minor around hearing onset and afterward. Similar developmental changes were observed during stimulation experiments both in brain slices and in vivo following cochlear ablation. Our data suggest that the strong reduction in release probability and the speedup of the decay of synaptic facilitation that happen just before hearing onset are important events in the transformation of the calyx of Held synapse into an auditory relay synapse.

Neurovascular responses to neuronal activity during sensory development.

Understanding the development of intercellular communication in sensory regions is relevant to elucidate mechanisms of physiological and pathological responses to oxygen shortage in the newborn brain. Decades of studies in laboratory rodents show that neuronal activity impacts sensory maturation during two periods of postnatal development distinguished by the maturation of accessory structures at the sensory periphery. During the first of these developmental periods, angiogenesis is modulated by neuronal activity, and physiological levels of neuronal activity cause local tissue hypoxic events. This correlation suggests that neuronal activity is upstream of the production of angiogenic factors, a process that is mediated by intermittent hypoxia caused by neuronal oxygen consumption. In this perspective article we address three theoretical implications based on this hypothesis: first, that spontaneous activity of sensory neurons has properties that favor the generation of intermittent tissue hypoxia in neonate rodents; second, that intermittent hypoxia promotes the expression of hypoxia inducible transcription factors (HIFs) in sensory neurons and astrocytes; and third, that activity-dependent production of angiogenic factors is involved in pathological oxygen contexts.

Dynamic development of the calyx of Held synapse.

The calyx of Held is probably the largest synaptic terminal in the brain, forming a unique one-to-one connection in the auditory ventral brainstem. During early development, calyces have many collaterals, whose function is unknown. Using electrophysiological recordings and fast-calcium imaging in brain slices, we demonstrate that these collaterals are involved in synaptic transmission. We show evidence that the collaterals are pruned and that the pruning already begins 1 week before the onset of hearing. Using two-photon microscopy to image the calyx of Held in neonate rats, we report evidence that both axons and nascent calyces are structurally dynamic, showing the formation, elimination, extension, or retraction of up to 65% of their collaterals within 1 hour. The observed dynamic behavior of axons may add flexibility in the choice of postsynaptic partners and thereby contribute to ensuring that each principal cell eventually is contacted by a single calyx of Held.

Characterization of Developmental Changes in Spontaneous Electrical Activity of Medial Superior Olivary Neurons Before Hearing Onset With a Combination of Injectable and Volatile Anesthesia.

In this work the impact of two widely used anesthetics on the electrical activity of auditory brainstem neurons was studied during postnatal development. Spontaneous electrical activity in neonate rats of either sex was analyzed through a ventral craniotomy in mechanically ventilated pups to carry out patch clamp and multi-electrode electrophysiology recordings in the medial region of the superior olivary complex (SOC) between birth (postnatal day 0, P0) and P12. Recordings were obtained in pups anesthetized with the injectable mix of ketamine/xylazine (K/X mix), with the volatile anesthetic isoflurane (ISO), or in pups anesthetized with K/X mix that were also exposed to ISO. The results of patch clamp recordings demonstrate for the first time that olivary and periolivary neurons in the medial region of the SOC fire bursts of action potentials. The results of multielectrode recordings suggest that the firing pattern of single units recorded in K/X mix is similar to that recorded in ISO anesthetized rat pups. Taken together, the results of this study provide a framework to use injectable and volatile anesthetics for future studies to obtain functional information on the activity of medial superior olivary neurons in vivo.

Distinct Cellular Profiles of Hif1a and Vegf mRNA Localization in Microglia, Astrocytes and Neurons during a Period of Vascular Maturation in the Auditory Brainstem of Neonate Rats.

Defining the relationship between vascular development and the expression of hypoxia-inducible factors (Hifs) and vascular endothelial growth factor (Vegf) in the auditory brainstem is important to understand how tissue hypoxia caused by oxygen shortage contributes to sensory deficits in neonates. In this study, we used histology, molecular labeling, confocal microscopy and 3D image processing methods to test the hypothesis that significant maturation of the vascular bed in the medial nucleus of the trapezoid body (MNTB) occurs during the postnatal period that precedes hearing onset. Isolectin-B4 histochemistry experiments suggested that the MNTB vasculature becomes more elaborate between P5 and P10. When combined with a cell proliferation marker and immunohistochemistry, we found that vascular growth coincides with a switch in the localization of proliferating cells to perivascular locations, and an increase in the density of microglia within the MNTB. Furthermore, microglia were identified as perivascular cells with proliferative activity during the period of vascular maturation. Lastly, combined in situ hybridization and immunohistochemistry experiments showed distinct profiles of Hif1a and Vegf mRNA localization in microglia, astrocytes and MNTB principal neurons. These results suggest that different cells of the neuro-glio-vascular unit are likely targets of hypoxic insult in the auditory brainstem of neonate rats.

Release and elementary mechanisms of nitric oxide in hair cells.

The enzyme nitric oxide (NO) synthase, that produces the signaling molecule NO, has been identified in several cell types in the inner ear. However, it is unclear whether a measurable quantity of NO is released in the inner ear to confer specific functions. Indeed, the functional significance of NO and the elementary cellular mechanism thereof are most uncertain. Here, we demonstrate that the sensory epithelia of the frog saccule release NO and explore its release mechanisms by using self-referencing NO-selective electrodes. Additionally, we investigated the functional effects of NO on electrical properties of hair cells and determined their underlying cellular mechanism. We show detectable amounts of NO are released by hair cells (>50 nM). Furthermore, a hair-cell efferent modulator acetylcholine produces at least a threefold increase in NO release. NO not only attenuated the baseline membrane oscillations but it also increased the magnitude of current required to generate the characteristic membrane potential oscillations. This resulted in a rightward shift in the frequency-current relationship and altered the excitability of hair cells. Our data suggest that these effects ensue because NO reduces whole cell Ca(2+) current and drastically decreases the open probability of single-channel events of the L-type and non L-type Ca(2+) channels in hair cells, an effect that is mediated through direct nitrosylation of the channel and activation of protein kinase G. Finally, NO increases the magnitude of Ca(2+)-activated K(+) currents via direct NO nitrosylation. We conclude that NO-mediated inhibition serves as a component of efferent nerve modulation of hair cells.

Defining the relationship between maternal care behavior and sensory development in Wistar rats: Auditory periphery development, eye opening and brain gene expression.

Defining the relationship between maternal care, sensory development and brain gene expression in neonates is important to understand the impact of environmental challenges during sensitive periods in early life. In this study, we used a selection approach to test the hypothesis that variation in maternal licking and grooming (LG) during the first week of life influences sensory development in Wistar rat pups. We tracked the onset of the auditory brainstem response (ABR), the timing of eye opening (EO), middle ear development with micro-CT X-ray tomography, and used qRT-PCR to monitor changes in gene expression of the hypoxia-sensitive pathway and neurotrophin signaling in pups reared by low-LG or high-LG dams. The results show the first evidence that the transcription of genes involved in the hypoxia-sensitive pathway and neurotrophin signaling is regulated during separate sensitive periods that occur before and after hearing onset, respectively. Although the timing of ABR onset, EO, and the relative mRNA levels of genes involved in the hypoxia-sensitive pathway did not differ between pups from different LG groups, we found statistically significant increases in the relative mRNA levels of four genes involved in neurotrophin signaling in auditory brain regions from pups of different LG backgrounds. These results suggest that sensitivity to hypoxic challenge might be widespread in the auditory system of neonate rats before hearing onset, and that maternal LG may affect the transcription of genes involved in experience-dependent neuroplasticity.

Learning drives differential clustering of axodendritic contacts in the barn owl auditory system.

Computational models predict that experience-driven clustering of coactive synapses is a mechanism for information storage. This prediction has remained untested, because it is difficult to approach through time-lapse analysis. Here, we exploit a unique feature of the barn owl auditory localization pathway that permits retrospective analysis of prelearned and postlearned circuitry: owls reared wearing prismatic spectacles develop an adaptive microcircuit that coexists with the native one but can be analyzed independently based on topographic location. To visualize the clustering of axodendritic contacts (potential synapses) within these zones, coactive axons were labeled by focal injection of fluorescent tracer and their target dendrites labeled with an antibody directed against CaMKII (calcium/calmodulin-dependent protein kinase type II, alpha subunit). Using high-resolution confocal imaging, we measured the distance from each contact to its nearest neighbor on the same branch of dendrite. We found that the distribution of intercontact distances for the adaptive zone was shifted dramatically toward smaller values compared with distributions for either the maladaptive zone of the same animals or the adaptive zone of normal juveniles, which indicates that a dynamic clustering of contacts had occurred. Moreover, clustering in the normal zone was greater in normal juveniles than in prism-adapted owls, indicative of declustering. These data demonstrate that clustering is bidirectionally adjustable and tuned by behaviorally relevant experience. The microanatomical configurations in all zones of both experimental groups matched the functional circuit strengths that were assessed by in vivo electrophysiological mapping. Thus, the observed changes in clustering are appropriately positioned to contribute to the adaptive strengthening and weakening of auditory-driven responses.

A single H/ACA small nucleolar RNA mediates tumor suppression downstream of oncogenic RAS.

Small nucleolar RNAs (snoRNAs) are a diverse group of non-coding RNAs that direct chemical modifications at specific residues on other RNA molecules, primarily on ribosomal RNA (rRNA). SnoRNAs are altered in several cancers; however, their role in cell homeostasis as well as in cellular transformation remains poorly explored. Here, we show that specific subsets of snoRNAs are differentially regulated during the earliest cellular response to oncogenic RASG12V expression. We describe a novel function for one H/ACA snoRNA, SNORA24, which guides two pseudouridine modifications within the small ribosomal subunit, in RAS-induced senescence in vivo. We find that in mouse models, loss of Snora24 cooperates with RASG12V to promote the development of liver cancer that closely resembles human steatohepatitic hepatocellular carcinoma (HCC). From a clinical perspective, we further show that human HCCs with low SNORA24 expression display increased lipid content and are associated with poor patient survival. We next asked whether ribosomes lacking SNORA24-guided pseudouridine modifications on 18S rRNA have alterations in their biophysical properties. Single-molecule Fluorescence Resonance Energy Transfer (FRET) analyses revealed that these ribosomes exhibit perturbations in aminoacyl-transfer RNA (aa-tRNA) selection and altered pre-translocation ribosome complex dynamics. Furthermore, we find that HCC cells lacking SNORA24-guided pseudouridine modifications have increased translational miscoding and stop codon readthrough frequencies. These findings highlight a role for specific snoRNAs in safeguarding against oncogenic insult and demonstrate a functional link between H/ACA snoRNAs regulated by RAS and the biophysical properties of ribosomes in cancer.

Alzheimer mouse brain tissue measured by time resolved fluorescence spectroscopy using single- and multi-photon excitation of label free native molecules.

Time resolved spectroscopic measurements with single-photon and multi-photon excitation of native molecules were performed ex vivo on brain tissues from an Alzheimer's disease (AD) and a wild type (WT) mouse model using a streak camera. The fluorescence decay times of native NADH and FAD show a longer relaxation time in AD than in WT tissue, suggesting less non-radiative processes in AD. The longer emission time of AD may be attributed to the coupling of the key native building block molecules to the amyloid-tau and/or to the caging of the native fluorophores by the deposition of amyloid-beta or tau plaques and neurofibrillary tangles that affect the local non-radiative interactions.

Transmission of classically entangled beams through mouse brain tissue.

Light transmission of Laguerre-Gaussian vector vortex beams in different local regions in mouse brain tissue is investigated. Transmittance is measured in the ballistic and diffusive regions with various polarizations states and orbital angular momentums (OAM). The transmission change observed with structured light other than linear polarization is attributed to chiroptical phenomena from the chiral brain media and the handedness of the light. For instance, classically entangled beams showed higher transmittance and constant value dependency on OAM modes than linear modes did. Also, circular polarization beam transmittance showed strong increase with topical charge OAM ( ℓ), which could be attributed to chiroptical effect.

Propagation of Gaussian and Laguerre-Gaussian vortex beams through mouse brain tissue.

Light transmission of Gaussian (G) and Laguerre-Gaussian (LG) vortex beams in mouse brain tissue is investigated. Transmittance is measured with different orbital angular momentums (OAM) at various tissue thicknesses. In both ballistic and diffusive regions, transmittances of G and LG beams show no significant difference. The transition point from ballistic to diffusive region for the mouse brain tissue is determined at about 480 µm. The observed transmittances of the G and LG beams show independence on OAM modes, which may be attributed to poorly understood interference effects from brain tissue.

Label-Free Fluorescence Spectroscopy for Detecting Key Biomolecules in Brain Tissue from a Mouse Model of Alzheimer's Disease.

In this study, label-free fluorescence spectroscopy was used for the first time to determine spectral profiles of tryptophan, reduced nicotinamide adenine dinucleotide (NADH), and flavin denine dinucleotide (FAD) in fresh brain samples of a mouse model of Alzheimer's disease (AD). Our results showed that the emission spectral profile levels of tryptophan and NADH were higher in AD samples than normal samples. The intensity ratio of tryptophan to NADH and the change rate of fluorescence intensity with respect to wavelength also increased in AD brain. These results yield an optical method for detecting early stage of AD by comparing spectral profiles of biomolecules.

Axodendritic contacts onto calcium/calmodulin-dependent protein kinase type II-expressing neurons in the barn owl auditory space map.

In the owl midbrain, a map of auditory space is synthesized in the inferior colliculus (IC) and conveyed to the optic tectum (OT). Ascending auditory information courses through these structures via topographic axonal projections. Little is known about the molecular composition of projection neurons or their postsynaptic targets. To visualize axodendritic contacts between identified cell types, we used double-label immunohistochemistry, in vivo retrograde tracing, in vitro anterograde tracing, high-resolution confocal microscopy, three-dimensional reconstruction and fly-through visualization. We discovered a major class of IC neurons that strongly expressed calcium/calmodulin-dependent protein kinase type II, alpha subunit (CaMKII). The distribution of these cells within the IC was mostly restricted to the external nucleus of the IC (ICX), in which the auditory space map is assembled. A large proportion of ICX-OT projection neurons were CaMKII positive. In addition to being the principal outputs, CaMKII cells were in direct contact with axonal boutons emanating from the main source of input to ICX, the lateral shell of the central nucleus of the inferior colliculus (ICCls). Numerous sites of putative synaptic contact were found on the somata, proximal dendrites, and distal dendrites. Double-label immunoelectron microscopy confirmed the existence of synapses between ICCls axons and the dendrites of CaMKII cells. Collectively, our data indicate that CaMKII ICX neurons are a cellular locus for the computation of auditory space-specific responses. Because the ICCls-ICX projection is physically altered during experience-dependent plasticity, these results lay the groundwork for probing microanatomical rearrangements that may underlie plasticity and learning.