Mice require proprioception to establish long-term visuospatial memory.

Because the retina moves constantly, the retinotopic representation of the visual world is spatially inaccurate and the brain must transform this spatially inaccurate retinal signal to a spatially accurate signal usable for perception and action. One of the salient discoveries of modern neuroscience is the role of the hippocampus in establishing gaze-independent, long-term visuospatial memories. The rat hippocampus has neurons which report the animal's position in space regardless of its angle of gaze. Rats with hippocampal lesions are unable to find the location of an escape platform hidden in a pool of opaque fluid, the Morris Water Maze (MWM) based on the visual aspects of their surrounding environment. Here we show that the representation of proprioception in the dysgranular zone of primary somatosensory cortex is equivalently necessary for mice to learn the location of the hidden platform, presumably because without it they cannot create a long-term gaze-independent visuospatial representation of their environment from the retinal signal. They have no trouble finding the platform when it is marked by a flag, and they have no motor or vestibular deficits.

Adult-born neurons maintain hippocampal cholinergic inputs and support working memory during aging.

Adult neurogenesis is reduced during aging and impaired in disorders of stress, memory, and cognition though its normal function remains unclear. Moreover, a systems level understanding of how a small number of young hippocampal neurons could dramatically influence brain function is lacking. We examined whether adult neurogenesis sustains hippocampal connections cumulatively across the life span. Long-term suppression of neurogenesis as occurs during stress and aging resulted in an accelerated decline in hippocampal acetylcholine signaling and a slow and progressing emergence of profound working memory deficits. These deficits were accompanied by compensatory reorganization of cholinergic dentate gyrus inputs with increased cholinergic innervation to the ventral hippocampus and recruitment of ventrally projecting neurons by the dorsal projection. While increased cholinergic innervation was dysfunctional and corresponded to overall decreases in cholinergic levels and signaling, it could be recruited to correct the resulting memory dysfunction even in old animals. Our study demonstrates that hippocampal neurogenesis supports memory by maintaining the septohippocampal cholinergic circuit across the lifespan.  It also provides a systems level explanation for the progressive nature of memory deterioration during normal and pathological aging and indicates that the brain connectome is malleable by experience.

Adult-born neurons maintain hippocampal cholinergic inputs and support working memory during aging.

Adult neurogenesis is reduced during aging and impaired in disorders of stress, memory, and cognition though its normal function remains unclear. Moreover, a systems level understanding of how a small number of young hippocampal neurons could dramatically influence brain function is lacking. We examined whether adult neurogenesis sustains hippocampal connections cumulatively across the life span. Long-term suppression of neurogenesis as occurs during stress and aging resulted in an accelerated decline in hippocampal acetylcholine signaling and a slow and progressing emergence of profound working memory deficits. These deficits were accompanied by compensatory reorganization of cholinergic dentate gyrus inputs with increased cholinergic innervation to the ventral hippocampus and recruitment of ventrally projecting neurons by the dorsal projection. While increased cholinergic innervation was dysfunctional and corresponded to overall decreases in cholinergic levels and signaling, it could be recruited to correct the resulting memory dysfunction even in old animals. Our study demonstrates that hippocampal neurogenesis supports memory by maintaining the septohippocampal cholinergic circuit across the lifespan. It also provides a systems level explanation for the progressive nature of memory deterioration during normal and pathological aging and indicates that the brain connectome is malleable by experience.

CyclinD2-mediated regulation of neurogenic output from the retinal ciliary margin is perturbed in albinism.

In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. Here, we focus on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ generates predominantly ipsilaterally projecting RGCs, but this output is altered in the albino visual system because of CyclinD2 downregulation and disturbed timing of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit diminished ipsilateral retinogeniculate projection and poor depth perception. In albino mice, pharmacological stimulation of calcium channels, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit.

Tumor cell metabolic reprogramming and hypoxic immunosuppression: driving carcinogenesis to metastatic colonization.

A significant factor in the antitumor immune response is the increased metabolic reprogramming of immunological and malignant cells. Increasing data points to the fact that cancer metabolism affects not just cancer signaling, which is essential for maintaining carcinogenesis and survival, but also the expression of immune cells and immune-related factors such as lactate, PGE2, arginine, IDO, which regulate the antitumor immune signaling mechanism. In reality, this energetic interaction between the immune system and the tumor results in metabolic competition in the tumor ecosystem, limiting the amount of nutrients available and causing microenvironmental acidosis, which impairs the ability of immune cells to operate. More intriguingly, different types of immune cells use metabolic reprogramming to keep the body and self in a state of homeostasis. The process of immune cell proliferation, differentiation, and performance of effector functions, which is crucial to the immune response, are currently being linked to metabolic reprogramming. Here, we cover the regulation of the antitumor immune response by metabolic reprogramming in cancer cells and immune cells as well as potential strategies for metabolic pathway targeting in the context of anticancer immunotherapy. We also discuss prospective immunotherapy-metabolic intervention combinations that might be utilized to maximize the effectiveness of current immunotherapy regimes.

A fast, aqueous, reversible three-day tissue clearing method for adult and embryonic mouse brain and whole body.

Optical clearing methods serve as powerful tools to study intact organs and neuronal circuits. We developed an aqueous clearing protocol, Fast 3D Clear, that relies on tetrahydrofuran for tissue delipidation and iohexol for clearing, such that tissues can be imaged under immersion oil in light-sheet imaging systems. Fast 3D Clear requires 3 days to achieve high transparency of adult and embryonic mouse tissues while maintaining their anatomical integrity and preserving a vast array of transgenic and viral/dye fluorophores. A unique advantage of Fast 3D Clear is its complete reversibility and thus compatibility with tissue sectioning and immunohistochemistry. Fast 3D Clear can be easily and quickly applied to a wide range of biomedical studies, facilitating the acquisition of high-resolution two- and three-dimensional images.

WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva.

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (~4 viral particles per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

3D neuronal mitochondrial morphology in axons, dendrites, and somata of the aging mouse hippocampus.

The brain's ability to process complex information relies on the constant supply of energy through aerobic respiration by mitochondria. Neurons contain three anatomically distinct compartments-the soma, dendrites, and projecting axons-which have different energetic and biochemical requirements, as well as different mitochondrial morphologies in cultured systems. In this study, we apply quantitative three-dimensional electron microscopy to map mitochondrial network morphology and complexity in the mouse brain. We examine somatic, dendritic, and axonal mitochondria in the dentate gyrus and cornu ammonis 1 (CA1) of the mouse hippocampus, two subregions with distinct principal cell types and functions. We also establish compartment-specific differences in mitochondrial morphology across these cell types between young and old mice, highlighting differences in age-related morphological recalibrations. Overall, these data define the nature of the neuronal mitochondrial network in the mouse hippocampus, providing a foundation to examine the role of mitochondrial morpho-function in the aging brain.

Sex Differences in Remote Contextual Fear Generalization in Mice.

The generalization of fear is adaptive in that it allows an animal to respond appropriately to novel threats that are not identical to previous experiences. In contrast, the overgeneralization of fear is maladaptive and is a hallmark of post-traumatic stress disorder (PTSD), a psychiatric illness that is characterized by chronic symptomatology and a higher incidence in women compared to men. Therefore, understanding the neural basis of fear generalization at remote time-points in female animals is of particular translational relevance. However, our understanding of the neurobiology of fear generalization is largely restricted to studies employing male mice and focusing on recent time-points (i.e., within 24-48 h following conditioning). To address these limitations, we examined how male and female mice generalize contextual fear at remote time intervals (i.e., 3 weeks after conditioning). In agreement with earlier studies of fear generalization at proximal time-points, we find that the test order of training and generalization contexts is a critical determinant of generalization and context discrimination, particularly for female mice. However, tactile elements that are present during fear conditioning are more salient for male mice. Our study highlights long-term sex differences in defensive behavior between male and female mice and may provide insight into sex differences in the processing and retrieval of remote fear memory observed in humans.

Early life stress delays hippocampal development and diminishes the adult stem cell pool in mice.

Early life stress predisposes to mental illness and behavioral dysfunction in adulthood, but the mechanisms underlying these persistent effects are poorly understood. Stress throughout life impairs the structure and function of the hippocampus, a brain system undergoing considerable development in early life. The long-term behavioral consequences of early life stress may therefore be due in part to interference with hippocampal development, in particular with assembly of the dentate gyrus (DG) region of the hippocampus. We investigated how early life stress produces long-term alterations in DG structure by examining DG assembly and the generation of a stable adult stem cell pool in routine housing and after stress induced by the limited bedding/nesting paradigm in mice. We found that early life stress leads to a more immature, proliferative DG than would be expected for the animal's age immediately after stress exposure, suggesting that early life stress delays DG development. Adult animals exposed to early life stress exhibited a reduction in the number of DG stem cells, but unchanged neurogenesis suggesting a depletion of the stem cell pool with compensation in the birth and survival of adult-born neurons. These results suggest a developmental mechanism by which early life stress can induce long-term changes in hippocampal function by interfering with DG assembly and ultimately diminishing the adult stem cell pool.

Anterograde and retrograde signaling by an Aplysia neurotrophin forms a transsynaptic functional unit.

Whereas short-term synaptic plasticity is often either pre- or postsynaptic, intermediate- and long-term plasticity generally require coordinated pre- and postsynaptic mechanisms. Thus, the transition from presynaptic short-term facilitation (STF) to intermediate-term facilitation (ITF) induced by 5HT at Aplysia sensory-to-motor neuron synapses requires the recruitment of postsynaptic mechanisms and activation of protein synthesis in both neurons. In the companion paper to this report, we found that presynaptic autocrine signaling by an Aplysia neurotrophin (ApNT) forms a positive feedback loop that drives the synapses from STF to ITF. Here we report that ApNT also acts through both anterograde and retrograde signaling to form a transsynaptic positive feedback loop that orchestrates cellular functions in both the presynaptic and postsynaptic neurons during the induction of ITF. These two feedback loops activate protein synthesis in each synaptic compartment, which in both cases depends on signaling from the other synaptic compartment. These results suggest that the pre- and postsynaptic compartments act as one functional unit during the consolidation of learning-related facilitation induced by 5HT.

RbAp48 Protein Is a Critical Component of GPR158/OCN Signaling and Ameliorates Age-Related Memory Loss.

Precisely deciphering the molecular mechanisms of age-related memory loss is crucial to create appropriate therapeutic interventions. We have previously shown that the histone-binding protein RbAp48/Rbbp4 is a molecular determinant of Age-Related Memory Loss. By exploring how this protein regulates the genomic landscape of the hippocampal circuit, we find that RbAp48 controls the expression of BDNF and GPR158 proteins, both critical components of osteocalcin (OCN) signaling in the mouse hippocampus. We show that inhibition of RbAp48 in the hippocampal formation inhibits OCN's beneficial functions in cognition and causes deficits in discrimination memory. In turn, disruption of OCN/GPR158 signaling leads to the downregulation of RbAp48 protein, mimicking the discrimination memory deficits observed in the aged hippocampus. We also show that activation of the OCN/GPR158 pathway increases the expression of RbAp48 in the aged dentate gyrus and rescues age-related memory loss.

Suppression of connexin 43 phosphorylation promotes astrocyte survival and vascular regeneration in proliferative retinopathy.

Degeneration of retinal astrocytes precedes hypoxia-driven pathologic neovascularization and vascular leakage in ischemic retinopathies. However, the molecular events that underlie astrocyte loss remain unclear. Astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and hemichannels. Cx channels can transfer toxic signals from dying cells to healthy neighbors under pathologic conditions. Here we show that Cx43 plays a critical role in astrocyte apoptosis and the resulting preretinal neovascularization in a mouse model of oxygen-induced retinopathy. Opening of Cx43 hemichannels was not observed following hypoxia. In contrast, GJ coupling between astrocytes increased, which could lead to amplification of injury. Accordingly, conditional deletion of Cx43 maintained a higher density of astrocytes in the hypoxic retina. We also identify a role for Cx43 phosphorylation in mediating these processes. Increased coupling in response to hypoxia is due to phosphorylation of Cx43 by casein kinase 1δ (CK1δ). Suppression of this phosphorylation using an inhibitor of CK1δ or in site-specific phosphorylation-deficient mice similarly protected astrocytes from hypoxic damage. Rescue of astrocytes led to restoration of a functional retinal vasculature and lowered the hypoxic burden, thereby curtailing neovascularization and neuroretinal dysfunction. We also find that absence of astrocytic Cx43 does not affect developmental angiogenesis or neuronal function in normoxic retinas. Our in vivo work directly links phosphorylation of Cx43 to astrocytic coupling and apoptosis and ultimately to vascular regeneration in retinal ischemia. This study reveals that targeting Cx43 phosphorylation in astrocytes is a potential direction for the treatment of proliferative retinopathies.

Gpr158 mediates osteocalcin's regulation of cognition.

That osteocalcin (OCN) is necessary for hippocampal-dependent memory and to prevent anxiety-like behaviors raises novel questions. One question is to determine whether OCN is also sufficient to improve these behaviors in wild-type mice, when circulating levels of OCN decline as they do with age. Here we show that the presence of OCN is necessary for the beneficial influence of plasma from young mice when injected into older mice on memory and that peripheral delivery of OCN is sufficient to improve memory and decrease anxiety-like behaviors in 16-mo-old mice. A second question is to identify a receptor transducing OCN signal in neurons. Genetic, electrophysiological, molecular, and behavioral assays identify Gpr158, an orphan G protein-coupled receptor expressed in neurons of the CA3 region of the hippocampus, as transducing OCN's regulation of hippocampal-dependent memory in part through inositol 1,4,5-trisphosphate and brain-derived neurotrophic factor. These results indicate that exogenous OCN can improve hippocampal-dependent memory in mice and identify molecular tools to harness this pathway for therapeutic purposes.

The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3.

Consolidation of long-term memories depends on de novo protein synthesis. Several translational regulators have been identified, and their contribution to the formation of memory has been assessed in the mouse hippocampus. None of them, however, has been implicated in the persistence of memory. Although persistence is a key feature of long-term memory, how this occurs, despite the rapid turnover of its molecular substrates, is poorly understood. Here we find that both memory storage and its underlying synaptic plasticity are mediated by the increase in level and in the aggregation of the prion-like translational regulator CPEB3 (cytoplasmic polyadenylation element-binding protein). Genetic ablation of CPEB3 impairs the maintenance of both hippocampal long-term potentiation and hippocampus-dependent spatial memory. We propose a model whereby persistence of long-term memory results from the assembly of CPEB3 into aggregates. These aggregates serve as functional prions and regulate local protein synthesis necessary for the maintenance of long-term memory.

MicroRNA-22 Gates Long-Term Heterosynaptic Plasticity in Aplysia through Presynaptic Regulation of CPEB and Downstream Targets.

The maintenance phase of memory-related long-term facilitation (LTF) of synapses between sensory and motor neurons of the gill-withdrawal reflex of Aplysia depends on a serotonin (5-HT)-triggered presynaptic upregulation of CPEB, a functional prion that regulates local protein synthesis at the synapse. The mechanisms whereby serotonin regulates CPEB levels in presynaptic sensory neurons are not known. Here, we describe a sensory neuron-specific microRNA 22 (miR-22) that has multiple binding sites on the mRNA of CPEB and inhibits it in the basal state. Serotonin triggers MAPK/Erk-dependent downregulation of miR-22, thereby upregulating the expression of CPEB, which in turn regulates, through functional CPE elements, the presynaptic expression of atypical PKC (aPKC), another candidate regulator of memory maintenance. Our findings support a model in which the neurotransmitter-triggered downregulation of miR-22 coordinates the regulation of genes contributing synergistically to the long-term maintenance of memory-related synaptic plasticity.

Behavioral decline and premature lethality upon pan-neuronal ferritin overexpression in Drosophila infected with a virulent form of Wolbachia.

Iron is required for organismal growth. Therefore, limiting iron availability may be a key part of the host's innate immune response to various pathogens, for example, in Drosophila infected with Zygomycetes. One way the host can transiently reduce iron bioavailability is by ferritin overexpression. To study the effects of neuronal-specific ferritin overexpression on survival and neurodegeneration we generated flies simultaneously over-expressing transgenes for both ferritin subunits in all neurons. We used two independent recombinant chromosomes bearing UAS-Fer1HCH, UAS-Fer2LCH transgenes and obtained qualitatively different levels of late-onset behavioral and lifespan declines. We subsequently discovered that one parental strain had been infected with a virulent form of the bacterial endosymbiont Wolbachia, causing widespread neuronal apoptosis and premature death. This phenotype was exacerbated by ferritin overexpression and was curable by antibiotic treatment. Neuronal ferritin overexpression in uninfected flies did not cause evident neurodegeneration but resulted in a late-onset behavioral decline, as previously reported for ferritin overexpression in glia. The results suggest that ferritin overexpression in the central nervous system of flies is tolerated well in young individuals with adverse manifestations appearing only late in life or under unrelated pathophysiological conditions.

Molecular mechanism for age-related memory loss: the histone-binding protein RbAp48.

To distinguish age-related memory loss more explicitly from Alzheimer's disease (AD), we have explored its molecular underpinning in the dentate gyrus (DG), a subregion of the hippocampal formation thought to be targeted by aging. We carried out a gene expression study in human postmortem tissue harvested from both DG and entorhinal cortex (EC), a neighboring subregion unaffected by aging and known to be the site of onset of AD. Using expression in the EC for normalization, we identified 17 genes that manifested reliable age-related changes in the DG. The most significant change was an age-related decline in RbAp48, a histone-binding protein that modifies histone acetylation. To test whether the RbAp48 decline could be responsible for age-related memory loss, we turned to mice and found that, consistent with humans, RbAp48 was less abundant in the DG of old than in young mice. We next generated a transgenic mouse that expressed a dominant-negative inhibitor of RbAp48 in the adult forebrain. Inhibition of RbAp48 in young mice caused hippocampus-dependent memory deficits similar to those associated with aging, as measured by novel object recognition and Morris water maze tests. Functional magnetic resonance imaging studies showed that within the hippocampal formation, dysfunction was selectively observed in the DG, and this corresponded to a regionally selective decrease in histone acetylation. Up-regulation of RbAp48 in the DG of aged wild-type mice ameliorated age-related hippocampus-based memory loss and age-related abnormalities in histone acetylation. Together, these findings show that the DG is a hippocampal subregion targeted by aging, and identify molecular mechanisms of cognitive aging that could serve as valid targets for therapeutic intervention.

Ferritin overexpression in Drosophila glia leads to iron deposition in the optic lobes and late-onset behavioral defects.

Cellular and organismal iron storage depends on the function of the ferritin protein complex in insects and mammals alike. In the central nervous system of insects, the distribution and relevance of ferritin remain unclear, though ferritin has been implicated in Drosophila models of Alzheimers' and Parkinsons' disease and in Aluminum-induced neurodegeneration. Here we show that transgene-derived expression of ferritin subunits in glial cells of Drosophila melanogaster causes a late-onset behavioral decline, characterized by loss of circadian rhythms in constant darkness and impairment of elicited locomotor responses. Anatomical analysis of the affected brains revealed crystalline inclusions of iron-loaded ferritin in a subpopulation of glial cells but not significant neurodegeneration. Although transgene-induced glial ferritin expression was well tolerated throughout development and in young flies, it turned disadvantageous at older age. The flies we characterize in this report contribute to the study of ferritin in the Drosophila brain and can be used to assess the contribution of glial iron metabolism in neurodegenerative models of disease.