Faculty Bibliography

Alzheimer's disease (AD) affects an estimated 5.8 million Americans, and advanced age is the greatest risk factor. AD patients have altered intestinal microbiota. Accordingly, depleting intestinal microbiota in AD animal models reduces amyloid-beta (Aβ) plaque deposition. Age-related changes in the microbiota contribute to immunologic and physiologic decline. Translationally relevant dietary manipulations may be an effective approach to slow microbiota changes during aging. We previously showed that calorie restriction (CR) reduced brain Aβ deposition in the well-established Tg2576 mouse model of AD. Presently, we investigated whether CR alters the microbiome during aging. We found that female Tg2576 mice have more substantial age-related microbiome changes compared to wildtype (WT) mice, including an increase in Bacteroides, which were normalized by CR. Specific gut microbiota changes were linked to Aβ levels, with greater effects in females than in males. In the gut, Tg2576 female mice had an enhanced intestinal inflammatory transcriptional profile, which was reversed by CR. Furthermore, we demonstrate that Bacteroides colonization exacerbates Aβ deposition, which may be a mechanism whereby the gut impacts AD pathogenesis. These results suggest that long-term CR may alter the gut environment and prevent the expansion of microbes that contribute to age-related cognitive decline.

Functional brain connectivity is increasingly being seen as critical for cognition, perception and motor control. Magnetoencephalography and electroencephalography are modalities that offer noninvasive mapping of electrophysiological interactions among brain regions, yet suffer from signal leakage and signal cancellation when estimating brain activity. This leads to biased connectivity values which complicate interpretation. In this study, we test the hypothesis that a Multiple Constrained Minimum Variance beamformer (MCMV) outperforms the more traditional Linearly Constrained Minimum Variance beamformer (LCMV) for estimation of electrophysiological connectivity. To this end, MCMV and LCMV performance is compared in task related analyses with both simulated data and human MEG recordings of visual steady state signals, and in resting state analyses with simulated data and human MEG data of 89 subjects. In task related scenarios connectivity was estimated using coherence and phase locking values, whereas envelope correlations were used for the resting state data. We also introduce a novel Augmented Pairwise MCMV (APW-MCMV) approach for signal leakage suppression in resting state analyses and assess its performance against LCMV and more conventional MCMV approaches. We demonstrate that with MCMV effects of signal mixing and coherent source cancellation are greatly reduced in both task related and resting state conditions, while in contrast to other approaches 0- and short time lag interactions are preserved. In addition, we demonstrate that in resting state analyses, APW-MCMV strongly reduces spurious connections while better controlling for false negatives compared to more conservative measures such as symmetrical orthogonalization.

The sparse activity of hippocampal dentate gyrus (DG) granule cells (GCs) is thought to be critical for cognition and behavior, whereas excessive DG activity may contribute to disorders such as temporal lobe epilepsy (TLE). Glutamatergic mossy cells (MCs) of the DG are potentially critical to normal and pathological functions of the DG because they can regulate GC activity through innervation of GCs or indirectly through GABAergic neurons. Here, we test the hypothesis that MC excitation of GCs is normally weak, but under pathological conditions, MC excitation of GCs is dramatically strengthened. We show that selectively inhibiting MCs during severe seizures reduced manifestations of those seizures, hippocampal injury, and chronic epilepsy. In contrast, selectively activating MCs was pro-convulsant. Mechanistic in vitro studies using optogenetics further demonstrated the unanticipated ability of MC axons to excite GCs under pathological conditions. These results demonstrate an excitatory and epileptogenic effect of MCs in the DG.

For neural systems to function effectively, the numbers of each cell type must be proportioned properly during development. We found that conditional knockout of the mouse homeobox genes En1 and En2 in the excitatory cerebellar nuclei neurons (eCN) leads to reduced postnatal growth of the cerebellar cortex. A subset of medial and intermediate eCN are lost in the mutants, with an associated cell non-autonomous loss of their presynaptic partner Purkinje cells by birth leading to proportional scaling down of neuron production in the postnatal cerebellar cortex. Genetic killing of embryonic eCN throughout the cerebellum also leads to loss of Purkinje cells and reduced postnatal growth but throughout the cerebellar cortex. Thus, the eCN play a key role in scaling the size of the cerebellum by influencing the survival of their Purkinje cell partners, which in turn regulate production of granule cells and interneurons via the amount of sonic hedgehog secreted.

An improved version of the integrative optical imaging method has been developed that substantially increases the time resolution of diffusion measurements. We present a theory for time-resolved integrative optical imaging that incorporates a time-dependent effective diffusion coefficient in homogeneous anisotropic media and a time-dependent nonspecific linear clearance. The method was applied to measure the very fast changes in extracellular diffusion that occur during spreading depression in rat hippocampal slices. We were able to achieve a time resolution of approximately 1 s, an improvement of at least 10 times compared to the standard methods for extracellular diffusion measurement. We have found that diffusion of a small fluorescent extracellular marker (MW 3000) completely stopped during the maximum direct current shift associated with the spreading depression wave, then gradually resumed over several minutes afterward. The effect of spreading depression on extracellular space is much larger than previously estimated by other methods with lower time resolution.

The cerebellum undergoes major rapid growth during the third trimester and early neonatal stage in humans, making it vulnerable to injuries in pre-term babies. Experiments in mice have revealed a remarkable ability of the neonatal cerebellum to recover from injuries around birth. In particular, recovery following irradiation-induced ablation of granule cell precursors (GCPs) involves adaptive reprogramming of Nestin-expressing glial progenitors (NEPs). Sonic hedgehog signaling is required for the initial step in NEP reprogramming; however, the full spectrum of developmental signaling pathways that promote NEP-driven regeneration is not known. Since the growth regulatory Hippo pathway has been implicated in the repair of several tissue types, we tested whether Hippo signaling is involved in regeneration of the cerebellum. Using mouse models, we found that the Hippo pathway transcriptional co-activator YAP1 (Yes-associated protein 1) but not TAZ (transcriptional coactivator with PDZ binding motif, or WWTR1) is required in NEPs for full recovery of cerebellar growth following irradiation one day after birth. Although Yap1 plays only a minor role during normal development in differentiation of NEPs or GCPs, the size of the cerebellum, and in particular the internal granule cell layer produced by GCPs, is significantly reduced in Yap1 mutants after irradiation, and the organization of Purkinje cells and Bergmann glial fibers is disrupted. The initial proliferative response of Yap1 mutant NEPs to irradiation is normal and the cells migrate to the GCP niche, but subsequently there is increased cell death of GCPs and altered migration of granule cells, possibly due to defects in Bergmann glia. Moreover, loss of Taz along with Yap1 in NEPs does not abrogate regeneration or alter development of the cerebellum. Our study provides new insights into the molecular signaling underlying postnatal cerebellar development and regeneration.

Acute myeloid leukemia (AML) has remained one of the most treatment resistant and deadliest cancers. The survival of AML blast cells is controlled by the balance of anti- and pro-apoptotic proteins. Recently approved Bcl-2 targeted therapy of AML with the Bcl-2 specific inhibitor Venetoclax in combinations has improved patients outcomes. However, a priori and developing resistance to venetoclax combinations with hypomethylating agents (HMA) azacitidine and decitabine challenge this treatment. As such, novel therapies to overcome venetoclax-HMA resistance are urgently needed. We have identified a combination of DNA damage repair interference by WEE1 inhibition with AZD1775, combined with low dose cytarabine (AraC) as an effective strategy to overcome combined venetoclax-azacitidine resistance (VAR). AZD1775 with low dose AraC induced massive apoptosis (by Annexin V and cleaved caspase-3) and almost completely reduced viability and clonogenic growth of primary AML cells. To delineate the molecular mechanism of the synergistic effect of AZD1775/AraC we performed RNAseq analysis of single agent or the combination of AZD1775+AraC in AML cell lines and primary CD34+ selected AML patient cells with the goal to identify deferentially regulated genes indicating a mechanistic underpinning of the potent activity. Only 2 genes were deferentially regulated across cell lines and CD34+ selected cells under AZD1775+AraC treatment: one of these is NR4A1, an orphan nuclear receptor, which we went on to validate as a potential downstream target of Wee1 inhibition. The inactivation of NR4A1 in mice was previously shown to induce AML and to maintain leukemia stem cells. Using qPCR we confirmed that the expression of NR4A1 is upregulated after AZD1775/AraC combo treatment in human leukemic cells. We then demonstrated that activators of NR4A1 (cytosporone B and pPhOCH3) reduce viability of leukemic cells, while NR4A1 inhibitor pPhOH was able to abolish the effect of AZD1775/AraC combo treatment increasing leukemic cell viability]. To investigate the involvement of mitochondria in the effect of AZD1775/AraC treatment we performed the expression of mitochondrial genes and pathway analyses in RNAseq data and found that mitochondrial gene expression, including many genes involved in apoptosis, has most dramatic changes in the combo treatment if compared to the single agents. Subsequently, we have examined the expression of the main BCL-2 family apoptotic genes by qPCR and western blot analysis. We found that AZD1775/AraC induces the expression of Bim isoforms, whereas Bcl-2, Mcl-1 and Bcl-Xl were largely unaffected. NR4A1 was previously shown to translocate to mitochondria, release Bim from Bcl-2 protein binding, as well as convert Bcl-2 to an extreme potent pro-apoptotic form. Finally, we generated several additional VAR cell lines and cells with subclones and demonstrated that AZD1775/AraC combination treatment is able to overcome VAR in almost every clone. Our results show that DNA damage repair interference with Wee1 inhibition has the potential to overcome VAR through a novel mechanisms of AZD1775 increasing NR4A1, freeing pro-apoptotic Bim irrespective of anti-apoptotic Bcl-2 proteins leading to massive apoptotic cell death in AML cells. The precise molecular mechanisms and the involvement of NR4A1 in this phenomenon will be presented at the meeting. Our findings will help to develop new therapeutic strategies in AML treatment and a trial of AZD1775 + AraC in AML is currently ongoing. Disclosures: No relevant conflicts of interest to declare.XXCopyright

Sleep quality varies widely across individuals, especially during normal aging, with impaired sleep contributing to deficits in cognition and emotional regulation. Sleep can also be impacted by a variety of adverse events, including childhood adversity. Here we examined how early life adverse events impacted later life sleep structure and physiology using an animal model to test the relationship between early life adversity and sleep quality across the life span. Rat pups were exposed to an Adversity-Scarcity model from postnatal day 8-12, where insufficient bedding for nest building induces maternal maltreatment of pups. Polysomnography and sleep physiology were assessed in weaning, early adult and older adults. Early life adversity induced age-dependent disruptions in sleep and behavior, including lifelong spindle decreases and later life NREM sleep fragmentation. Given the importance of sleep in cognitive and emotional functions, these results highlight an important factor driving variation in sleep, cognition and emotion throughout the lifespan that suggest age-appropriate and trauma informed treatment of sleep problems.

Autism spectrum disorder (ASD) is associated with noise hypersensitivity, the suboptimal extraction of meaningful signals in noisy environments. Because sensory filtering can involve distinct automatic and executive circuit mechanisms, however, developing circuit-specific therapeutic strategies for ASD noise hypersensitivity can be challenging. Here, we find that both of these processes are individually perturbed in one monogenic form of ASD, Ptchd1 deletion. Although Ptchd1 is preferentially expressed in the thalamic reticular nucleus during development, pharmacological rescue of thalamic perturbations in knockout (KO) mice only normalized automatic sensory filtering. By discovering a separate prefrontal perturbation in these animals and adopting a combinatorial pharmacological approach that also rescued its associated goal-directed noise filtering deficit, we achieved full normalization of noise hypersensitivity in this model. Overall, our work highlights the importance of identifying large-scale functional circuit architectures and utilizing them as access points for behavioral disease correction.

Infant maltreatment increases vulnerability to physical and mental disorders, yet specific mechanisms embedded within this complex infant experience that induce this vulnerability remain elusive. To define critical features of maltreatment-induced vulnerability, rat pups were reared from postnatal day 8 (PN8) with a maltreating mother, which produced amygdala and hippocampal deficits and decreased social behavior at PN13. Next, we deconstructed the maltreatment experience to reveal sufficient and necessary conditions to induce this phenotype. Social behavior and amygdala deficits (volume, neurogenesis, c-Fos, local field potential) required combined chronic high corticosterone and maternal presence (not maternal behavior). Hippocampal deficits were induced by chronic high corticosterone regardless of social context. Causation was shown by blocking corticosterone during maltreatment and suppressing amygdala activity during social behavior testing. These results highlight (1) that early life maltreatment initiates multiple pathways to pathology, each with distinct causal mechanisms and outcomes, and (2) the importance of social presence on brain development.