Anna K. Gillespie

Reducing human apolipoprotein E levels attenuates age-dependent Aβ accumulation in mutant human amyloid precursor protein transgenic mice.

Apolipoprotein E4 (apoE4) plays a major role in the pathogenesis of Alzheimers disease. Brain amyloid-β (Aβ) accumulation depends on age and apoE isoforms (apoE4 > apoE3) both in humans and in transgenic mouse models. Brain apoE levels are also isoform dependent, but in the opposite direction (apoE4 < apoE3). Thus, one prevailing hypothesis is to increase brain apoE expression to reduce Aβ levels. To test this hypothesis, we generated mutant human amyloid precursor protein transgenic mice expressing one or two copies of the human APOE3 or APOE4 gene that was knocked in and flanked by LoxP sites. We report that reducing apoE3 or apoE4 expression by 50% in 6-month-old mice results in efficient Aβ clearance and does not increase Aβ accumulation. However, 12-month-old mice with one copy of the human APOE gene had significantly reduced Aβ levels and plaque loads compared with mice with two copies, regardless of which human apoE isoform was expressed, suggesting a gene dose-dependent effect of apoE on Aβ accumulation in aged mice. Additionally, 12-month-old mice expressing one or two copies of the human APOE4 gene had significantly higher levels of Aβ accumulation and plaque loads than age-matched mice expressing one or two copies of the human APOE3 gene, suggesting an isoform-dependent effect of apoE on Aβ accumulation in aged mice. Moreover, Cre-mediated APOE4 gene excision in hippocampal astrocytes significantly reduced insoluble Aβ in adult mice. Thus, reducing, rather than increasing, apoE expression is an attractive approach to lowering brain Aβ levels.

Wilms tumor 1 (WT1) regulates KRAS-driven oncogenesis and senescence in mouse and human models

KRAS is one of the most frequently mutated human oncogenes. In some settings, oncogenic KRAS can trigger cellular senescence, whereas in others it produces hyperproliferation. Elucidating the mechanisms regulating these 2 drastically distinct outcomes would help identify novel therapeutic approaches in RAS-driven cancers. Using a combination of functional genomics and mouse genetics, we identified a role for the transcription factor Wilms tumor 1 (WT1) as a critical regulator of senescence and proliferation downstream of oncogenic KRAS signaling. Deletion or suppression of Wt1 led to senescence of mouse primary cells expressing physiological levels of oncogenic Kras but had no effect on wild-type cells, and Wt1 loss decreased tumor burden in a mouse model of Kras-driven lung cancer. In human lung cancer cell lines dependent on oncogenic KRAS, WT1 loss decreased proliferation and induced senescence. Furthermore, WT1 inactivation defined a gene expression signature that was prognostic of survival only in lung cancer patients exhibiting evidence of oncogenic KRAS activation. These findings reveal an unexpected role for WT1 as a key regulator of the genetic network of oncogenic KRAS and provide important insight into the mechanisms that regulate proliferation or senescence in response to oncogenic signals.

Inhibitory Interneuron Progenitor Transplantation Restores Normal Learning and Memory in ApoE4 Knock-In Mice without or with Aβ Accumulation

Excitatory and inhibitory balance of neuronal network activity is essential for normal brain function and may be of particular importance to memory. Apolipoprotein (apo) E4 and amyloid-β (Aβ) peptides, two major players in Alzheimers disease (AD), cause inhibitory interneuron impairments and aberrant neuronal activity in the hippocampal dentate gyrus in AD-related mouse models and humans, leading to learning and memory deficits. To determine whether replacing the lost or impaired interneurons rescues neuronal signaling and behavioral deficits, we transplanted embryonic interneuron progenitors into the hippocampal hilus of aged apoE4 knock-in mice without or with Aβ accumulation. In both conditions, the transplanted cells developed into mature interneurons, functionally integrated into the hippocampal circuitry, and restored normal learning and memory. Thus, restricted hilar transplantation of inhibitory interneurons restores normal cognitive function in two widely used AD-related mouse models, highlighting the importance of interneuron impairments in AD pathogenesis and the potential of cell replacement therapy for AD. More broadly, it demonstrates that excitatory and inhibitory balance are crucial for learning and memory, and suggests an avenue for investigating the processes of learning and memory and their alterations in healthy aging and diseases.

Hilar GABAergic Interneuron Activity Controls Spatial Learning and Memory Retrieval

Background Although extensive research has demonstrated the importance of excitatory granule neurons in the dentate gyrus of the hippocampus in normal learning and memory and in the pathogenesis of amnesia in Alzheimers disease (AD), the role of hilar GABAergic inhibitory interneurons, which control the granule neuron activity, remains unclear. Methodology and Principal Findings We explored the function of hilar GABAergic interneurons in spatial learning and memory by inhibiting their activity through Cre-dependent viral expression of enhanced halorhodopsin (eNpHR3.0)—a light-driven chloride pump. Hilar GABAergic interneuron-specific expression of eNpHR3.0 was achieved by bilaterally injecting adeno-associated virus containing a double-floxed inverted open-reading frame encoding eNpHR3.0 into the hilus of the dentate gyrus of mice expressing Cre recombinase under the control of an enhancer specific for GABAergic interneurons. In vitro and in vivo illumination with a yellow laser elicited inhibition of hilar GABAergic interneurons and consequent activation of dentate granule neurons, without affecting pyramidal neurons in the CA3 and CA1 regions of the hippocampus. We found that optogenetic inhibition of hilar GABAergic interneuron activity impaired spatial learning and memory retrieval, without affecting memory retention, as determined in the Morris water maze test. Importantly, optogenetic inhibition of hilar GABAergic interneuron activity did not alter short-term working memory, motor coordination, or exploratory activity. Conclusions and Significance Our findings establish a critical role for hilar GABAergic interneuron activity in controlling spatial learning and memory retrieval and provide evidence for the potential contribution of GABAergic interneuron impairment to the pathogenesis of amnesia in AD.

A novel zebrafish model of hyperthermia-induced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors

Febrile seizures are the most common seizure type in children under the age of five, but mechanisms underlying seizure generation in vivo remain unclear. Animal models to address this issue primarily focus on immature rodents heated indirectly using a controlled water bath or air blower. Here we describe an in vivo model of hyperthermia-induced seizures in larval zebrafish aged 3 to 7 days post-fertilization (dpf). Bath controlled changes in temperature are rapid and reversible in this model. Acute electrographic seizures following transient hyperthermia showed age-dependence, strain independence, and absence of mortality. Electrographic seizures recorded in the larval zebrafish forebrain were blocked by adding antagonists to the transient receptor potential vanilloid (TRPV4) channel or N-methyl-d-aspartate (NMDA) glutamate receptor to the bathing medium. Application of GABA, GABA re-uptake inhibitors, or TRPV1 antagonist had no effect on hyperthermic seizures. Expression of vanilloid channel and glutamate receptor mRNA was confirmed by quantitative PCR analysis at each developmental stage in larval zebrafish. Taken together, our findings suggest a role of heat-activation of TRPV4 channels and enhanced NMDA receptor-mediated glutamatergic transmission in hyperthermia-induced seizures.

Apolipoprotein E4 produced in GABAergic interneurons causes learning and memory deficits in mice.

Apolipoprotein (apo) E4 is expressed in many types of brain cells, is associated with age-dependent decline of learning and memory in humans, and is the major genetic risk factor for AD. To determine whether the detrimental effects of apoE4 depend on its cellular sources, we generated human apoE knock-in mouse models in which the human APOE gene is conditionally deleted in astrocytes, neurons, or GABAergic interneurons. Here we report that deletion of apoE4 in astrocytes does not protect aged mice from apoE4-induced GABAergic interneuron loss and learning and memory deficits. In contrast, deletion of apoE4 in neurons does protect aged mice from both deficits. Furthermore, deletion of apoE4 in GABAergic interneurons is sufficient to gain similar protection. This study demonstrates a detrimental effect of endogenously produced apoE4 on GABAergic interneurons that leads to learning and memory deficits in mice and provides a novel target for drug development for AD related to apoE4.

Neural Activity Patterns Underlying Spatial Coding in the Hippocampus

The hippocampus is well known as a central site for memory processing-critical for storing and later retrieving the experiences events of daily life so they can be used to shape future behavior. Much of what we know about the physiology underlying hippocampal function comes from spatial navigation studies in rodents, which have allowed great strides in understanding how the hippocampus represents experience at the cellular level. However, it remains a challenge to reconcile our knowledge of spatial encoding in the hippocampus with its demonstrated role in memory-dependent tasks in both humans and other animals. Moreover, our understanding of how networks of neurons coordinate their activity within and across hippocampal subregions to enable the encoding, consolidation, and retrieval of memories is incomplete. In this chapter, we explore how information may be represented at the cellular level and processed via coordinated patterns of activity throughout the subregions of the hippocampal network.

Approaching Alzheimer’s disease from a network level

www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 6), pp: 9003-9004 Editorial: Neuroscience Approaching Alzheimer’s disease from a network level Anna K. Gillespie, Emily A. Jones and Yadong Huang Despite decades of research, the root “cause” of Alzheimer’s disease (AD) remains elusive. Historically, most AD research has focused on genes implicated in early onset AD, such as amyloid precursor protein and the presenilins. However, these cases comprise less than 1% of all AD cases. The vast majority of AD cases are late onset and of unknown etiology, and in these cases, another genetic risk factor dominates: apolipoprotein E4 (apoE4). ApoE4 carriers, especially women, have an increased risk of developing AD and a lower average age of onset compared to carriers of the more common isoform, apoE3 [1]. Although the impact of apoE4 is clear, the pathological mechanisms underlying the increased AD risk are not well understood. On a molecular level, a single amino acid substitution differentiates apoE4 from apoE3, altering its structure, interfering with its normal function, and rendering it vulnerable to proteolytic cleavage into toxic fragments. To study the detrimental effects of apoE4 in an animal model, our lab and others use knock-in (KI) mice that express human apoE4 at the endogenous mouse apoE locus. Using these mice, we have shown that inhibitory interneuron populations in the dentate gyrus of the hippocampus of aged, female apoE4-KI mice are particularly vulnerable to apoE4 expression. In addition to this cellular phenotype, aged female apoE4-KI mice also show impairment on a hippocampus-dependent learning and memory task, which is correlated with the extent of interneuron loss [2]. Further studies provided additional evidence for a causal relationship between interneuron loss and behavioral impairment[3, 4], however, the functional link between the two was unclear. To address this question, we looked to the network activity in which these interneurons likely participate. While many studies have linked patterns of hippocampal activity to normal memory processes, the characterization of these hippocampal network-level patterns in disease models has been relatively rare. We were particularly interested in sharp-wave ripples (SWRs), brief high frequency oscillations in the hippocampal local field potential signal. These events are notable because they coincide with hippocampal replay, a phenomenon in which ensembles of hippocampal neurons are reactivated in a manner that recapitulates prior experience. As such, hippocampal replay during SWRs is thought to be critical for memory consolidation and potentially retrieval [5]. We www.impactjournals.com/oncotarget hypothesized that apoE4-induced interneuron malfunction might alter SWR activity, disrupting mnemonic processing and thus causing memory impairment. Indeed, we found that compared to apoE3-KI mice, apoE4-KI mice have fewer SWRs. Beyond that, the remaining apoE4-KI SWRs showed reduced power in a coincident lower frequency oscillation called slow gamma. As slow gamma during SWRs is thought to be a critical coordinator of accurate replay [6], both the reduction in replay events and the potentially degraded quality of replay due to slow gamma attenuation were promising candidate mechanisms for the apoE4-induced behavioral deficit. To further link the functional phenotype with our prior findings of interneuron loss in the dentate gyrus, we used a line of mice with the apoE4 gene removed specifically in inhibitory interneurons (apoE4-KI/Dlx- Cre mice). This genetic manipulation prevents both interneuron loss and learning and memory impairment [7]. Interestingly, the apoE4-induced reduction in SWR events was maintained in these mice, while the attenuation of slow gamma activity during SWRs was prevented. This suggests that the slow gamma activity during SWRs is dependent on intact interneuron activity and is critical for learning and memory processes. Studies of young apoE4-KI mice - prior to extensive interneuron loss and behavioral deficits - also showed a reduction of SWR events, further corroborating that the reduction of SWR events alone is insufficient to disrupt behavior. In contrast, the slow gamma during SWRs showed an age-dependent, progressive decline, aligned with the development of behavioral impairment and loss of interneurons [8]. Together, these results provide important insights to both normal hippocampal mnemonic processing as well as the pathological mechanisms of apoE4 in AD pathogenesis. We suggest that attenuated slow gamma activity during SWRs, rather than the reduction in SWR events, is likely to drive learning and memory impairment in apoE4-KI mice; the quality of SWRs may be more critical than their quantity in maintaining mnemonic function. Further, we conclude that interneuron populations, particularly those in the dentate gyrus, may be integral to maintaining slow gamma oscillations throughout the hippocampal circuit. These findings have important implications for designing therapeutic strategies for combating AD and evaluating current therapeutic candidates. One appealing Oncotarget

Abstract A41: SOX11 transcriptionally regulates a cytokine-based gene signature in lung cancer-associated fibroblasts (LCAF) to promote tumor growth

Increasing evidence points to an important role for the tumor microenvironment in cancer progression. However, the role of tumor stroma in the pathogenesis of lung cancer is poorly understood. Desmoplasia is a common pathologic feature of many NSCLC. We noted a similar phenotype in a mouse model of lung cancer driven by expression of oncogenic Kras. Lung cancer-associated fibroblasts (LCAFs) and normal lung fibroblasts (NLFs) from the mouse model of Kras-driven lung cancer were isolated and queried at the functional level using a xenograft model. First, we observed that LCAFs promote tumor growth more favorably than NLFs in vivo when co-injected with mouse tumor cells. Moreover, NLFs passaged in vivo acquire features of LCAFs as indicated by an increased tumor-promoting capacity on mouse and human NSCLC xenografts. In addition, conditioned media from LCAFs stimulated cell proliferation in vitro more significantly than that of NLFs. Expression profiling of NLFs and LCAFs using microarrays uncovered a gene set that includes multiple secreted proteins and genes related to inflammatory processes. Moreover, the SRY-related HMG transcription factor Sox11 was highly up-regulated in LCAFs compared to NLFs. Over-expression and knock-down experiments have confirmed that Sox11 regulates the expression of many of the secreted proteins expressed in LCAFs. The relevance of these findings for human NSCLC is suggested by the observation that the mouse LCAF gene expression signature was predictive of survival in a large cohort of human NSCLC. Besides, Sox11 and several of the secreted proteins expressed in LCAFs, along with their putative receptors, were up-regulated in human NSCLC samples. In conclusion, we have identified a potentially novel regulator of the transition between NLFs and LCAFs and, thus, uncovered a new signaling hub that could be used for therapeutic intervention.