Spatially and temporally distinct patterns of expression for VPS10P domain receptors in human cerebral organoids.

Vacuolar protein sorting 10 protein (VPS10P) domain receptors are a unique class of intracellular sorting receptors that emerge as major risk factors associated with psychiatric and neurodegenerative diseases, including bipolar disorders, autism, schizophrenia, as well as Alzheimer's disease and frontotemporal dementia. Yet, the lack of suitable experimental models to study receptor functions in the human brain has hampered elucidation of receptor actions in brain disease. Here, we have adapted protocols using human cerebral organoids to the detailed characterization of VPS10P domain receptor expression during neural development and differentiation, including single-cell RNA sequencing. Our studies uncovered spatial and temporal patterns of expression unique to individual receptor species in the human brain. While SORL1 expression is abundant in stem cells and SORCS1 peaks in neural progenitors at onset of neurogenesis, SORT1 and SORCS2 show increasing expression with maturation of neuronal and non-neuronal cell types, arguing for distinct functions in development versus the adult brain. In neurons, subcellular localization also distinguishes between types of receptor species, either mainly localized to the cell soma (SORL1 and SORT1) or also to neuronal projections (SORCS1 and SORCS2), suggesting divergent functions in protein sorting between Golgi and the endo-lysosomal system or along axonal and dendritic tracks. Taken together, our findings provide an important resource on temporal, spatial, and subcellular patterns of VPS10P domain receptor expression in cerebral organoids for further elucidation of receptor (dys) functions causative of behavioral and cognitive defects of the human brain.

An adult-stage transcriptional program for survival of serotonergic connectivity.

Neurons must function for decades of life, but how these non-dividing cells are preserved is poorly understood. Using mouse serotonin (5-HT) neurons as a model, we report an adult-stage transcriptional program specialized to ensure the preservation of neuronal connectivity. We uncover a switch in Lmx1b and Pet1 transcription factor function from controlling embryonic axonal growth to sustaining a transcriptomic signature of 5-HT connectivity comprising functionally diverse synaptic and axonal genes. Adult-stage deficiency of Lmx1b and Pet1 causes slowly progressing degeneration of 5-HT synapses and axons, increased susceptibility of 5-HT axons to neurotoxic injury, and abnormal stress responses. Axon degeneration occurs in a die back pattern and is accompanied by accumulation of α-synuclein and amyloid precursor protein in spheroids and mitochondrial fragmentation without cell body loss. Our findings suggest that neuronal connectivity is transcriptionally protected by maintenance of connectivity transcriptomes; progressive decay of such transcriptomes may contribute to age-related diseases of brain circuitry.

Reorganization of postmitotic neuronal chromatin accessibility for maturation of serotonergic identity.

Assembly of transcriptomes encoding unique neuronal identities requires selective accessibility of transcription factors to cis-regulatory sequences in nucleosome-embedded postmitotic chromatin. Yet, the mechanisms controlling postmitotic neuronal chromatin accessibility are poorly understood. Here, we show that unique distal enhancers define the Pet1 neuron lineage that generates serotonin (5-HT) neurons in mice. Heterogeneous single-cell chromatin landscapes are established early in postmitotic Pet1 neurons and reveal the putative regulatory programs driving Pet1 neuron subtype identities. Distal enhancer accessibility is highly dynamic as Pet1 neurons mature, suggesting the existence of regulatory factors that reorganize postmitotic neuronal chromatin. We find that Pet1 and Lmx1b control chromatin accessibility to select Pet1-lineage-specific enhancers for 5-HT neurotransmission. Additionally, these factors are required to maintain chromatin accessibility during early maturation suggesting that postmitotic neuronal open chromatin is unstable and requires continuous regulatory input. Together, our findings reveal postmitotic transcription factors that reorganize accessible chromatin for neuron specialization.

Postoperative cognitive dysfunction is made persistent with morphine treatment in aged rats.

Postoperative cognitive dysfunction (POCD) is the collection of cognitive impairments, lasting days to months, experienced by individuals following surgery. Persistent POCD is most commonly experienced by older individuals and is associated with a greater vulnerability to developing Alzheimer's disease, but the underlying mechanisms are not known. It is known that laparotomy (exploratory abdominal surgery) in aged rats produces memory impairments for 4 days. Here we report that postsurgical treatment with morphine extends this deficit to at least 2 months while having no effects in the absence of surgery. Indeed, hippocampal-dependent long-term memory was impaired 2, 4, and 8 weeks postsurgery only in aged, morphine-treated rats. Short-term memory remained intact. Morphine is known to have analgesic effects via µ-opioid receptor activation and neuroinflammatory effects through Toll-like receptor 4 activation. Here we demonstrate that persistent memory deficits were mediated independently of the µ-opioid receptor, suggesting that they were evoked through a neuroinflammatory mechanism and unrelated to pain modulation. In support of this, aged, laparotomized, and morphine-treated rats exhibited increased gene expression of various proinflammatory markers (IL-1ß, IL-6, TNFα, NLRP3, HMGB1, TLR2, and TLR4) in the hippocampus at the 2-week time point. Furthermore, central blockade of IL-1ß signaling with the specific IL-1 receptor antagonist (IL-1RA), at the time of surgery, completely prevented the memory impairment. Finally, synaptophysin and PSD95 gene expression were significantly dysregulated in the hippocampus of aged, laparotomized, morphine-treated rats, suggesting that impaired synaptic structure and/or function may play a key role in this persistent deficit. This instance of long-term memory impairment following surgery closely mirrors the timeline of persistent POCD in humans and may be useful for future treatment discoveries.

Lmx1b is required at multiple stages to build expansive serotonergic axon architectures.

Formation of long-range axons occurs over multiple stages of morphological maturation. However, the intrinsic transcriptional mechanisms that temporally control different stages of axon projection development are unknown. Here, we addressed this question by studying the formation of mouse serotonin (5-HT) axons, the exemplar of long-range profusely arborized axon architectures. We report that LIM homeodomain factor 1b (Lmx1b)-deficient 5-HT neurons fail to generate axonal projections to the forebrain and spinal cord. Stage-specific targeting demonstrates that Lmx1b is required at successive stages to control 5-HT axon primary outgrowth, selective routing, and terminal arborization. We show a Lmx1b→Pet1 regulatory cascade is temporally required for 5-HT arborization and upregulation of the 5-HT axon arborization gene, Protocadherin-alphac2, during postnatal development of forebrain 5-HT axons. Our findings identify a temporal regulatory mechanism in which a single continuously expressed transcription factor functions at successive stages to orchestrate the progressive development of long-range axon architectures enabling expansive neuromodulation.

Stable, long-term, spatial memory in young and aged rats achieved with a one day Morris water maze training protocol.

Here, we present data demonstrating that a 1 d Morris water maze training protocol is effective at producing stable, long-term spatial memory in both young (3 mo old) and aged (24 mo old) F344xBN rats. Four trials in each of four sessions separated by a 2.5 h ISI produced robust selective search for the platform 1 and 4 d after training, in both age groups. A 1 h ISI protocol did not produce good retention. Also, compressing the trials into just two sessions separated by a 2.5 h ISI produced limited retention in only young rats.

Diminished circadian rhythms in hippocampal microglia may contribute to age-related neuroinflammatory sensitization.

Aged animals exhibit diminished circadian rhythms, and both aging and circadian disruption sensitize neuroinflammatory responses. Microglia-the innate immune cell of the central nervous system-possess endogenous timekeeping mechanisms that regulate immune responses. Here, we explored whether aging is associated with disrupted diurnal rhythms in microglia and neuroinflammatory processes. First, hippocampal microglia isolated from young rats (4 months F344XBN) rhythmically expressed circadian clock genes, whereas microglia isolated from the hippocampus of aged rats (25 months) had aberrant Per1 and Per2 rhythms. Unstimulated microglia from young rats exhibited robust rhythms of TNFα and IL-1ß mRNA expression, whereas those from aged rats had flattened and tonically elevated cytokine expression. Similarly, microglial activation markers were diurnally regulated in the hippocampus of young but not aged rats and diurnal differences in responsiveness to both ex vivo and in vivo inflammatory challenges were abolished in aged rats. Corticosterone is an entraining signal for extra-suprachiasmatic nucleus circadian rhythms. Here, corticosterone stimulation elicited similar Per1 induction in aged and young microglia. Overall, these results indicate that aging dysregulates circadian regulation of neuroinflammatory functions.

The Alarmin HMGB1 Mediates Age-Induced Neuroinflammatory Priming.

UNLABELLED: Amplified neuroinflammatory responses following an immune challenge occur with normal aging and can elicit or exacerbate neuropathology. The mechanisms mediating this sensitized or "primed" immune response in the aged brain are not fully understood. The alarmin high mobility group box 1 (HMGB1) can be released under chronic pathological conditions and initiate inflammatory cascades. This led us to investigate whether HMGB1 regulates age-related priming of the neuroinflammatory response. Here, we show that HMGB1 protein and mRNA were elevated in the hippocampus of unmanipulated aged rats (24-month-old F344XBN rats). Furthermore, aged rats had increased HMGB1 in the CSF, suggesting increased HMGB1 release. We demonstrate that blocking HMGB1 signaling with an intracisterna magna (ICM) injection of the competitive antagonist to HMGB1, Box-A, downregulates basal expression of several inflammatory pathway genes in the hippocampus of aged rats. This indicates that blocking the actions of HMGB1 might reduce age-associated inflammatory priming. To test this hypothesis, we evaluated whether HMGB1 antagonism blocks the protracted neuroinflammatory and sickness response to peripheral Escherichia coli (E. coli) infection in aged rats. ICM pretreatment of aged rats with Box-A 24 h before E. coli infection prevented the extended hippocampal cytokine response and associated cognitive and affective behavioral changes. ICM pretreatment with Box-A also inhibited aging-induced potentiation of the microglial proinflammatory response to lipopolysaccharide ex vivo Together, these results suggest that HMGB1 mediates neuroinflammatory priming in the aged brain. Blocking the actions of HMGB1 appears to "desensitize" aged microglia to an immune challenge, thereby preventing exaggerated behavioral and neuroinflammatory responses following infection. SIGNIFICANCE STATEMENT: The world's population is aging, highlighting a need to develop treatments that promote quality of life in aged individuals. Normal aging is associated with precipitous drops in cognition, typically following events that induce peripheral inflammation (e.g., infection, surgery, heart attack). Peripheral immune stimuli cause exaggerated immune responses in the aged brain, which likely underlie these behavioral deficits. Here, we investigated whether the alarmin high mobility group box 1 (HMGB1) mediates age-associated "priming" of the neuroinflammatory response. HMGB1 is elevated in aged rodent brain and CSF. Blocking HMGB1 signaling downregulated expression of inflammatory pathway genes in aged rat brain. Further, HMGB1 antagonism prevented prolonged infection-induced neuroinflammatory and sickness responses in aged rats. Overall, blocking HMGB1 "desensitized" microglia in the aged brain, thereby preventing pathological infection-elicited neuroinflammatory responses.

Stress-induced neuroinflammatory priming is time of day dependent.

Circadian rhythms are endogenous cycles of physiology and behavior that align with the daily rotation of the planet and resulting light-dark cycle. The circadian system ensures homeostatic balance and regulates many aspects of physiology, including the stress response and susceptibility to and/or severity of stress-related sequelae. Both acute and chronic stressors amplify neuroinflammatory responses to a subsequent immune challenge, however it is not known whether circadian timing of the stressor regulates the priming response. Here, we test whether stress-induced neuroinflammatory priming is regulated by the circadian system. As has been previously shown, exposure to 100 inescapable tails shocks (IS) increased hippocampal cytokines following a subsequent inflammatory challenge. However, this effect was limited to animals that experienced the stressor during the light phase. Rats exposed to stress during the dark phase did not alter inflammatory potential following lipopolysaccharide (LPS) challenge. To determine whether microglia might be involved in diurnal differences in neuroinflammatory priming, microglia were isolated 24h after stress that occurred either during the middle of the light or dark phase. Only microglia isolated from animals stressed during the light phase demonstrated an exaggerated inflammatory response when treated ex vivo with LPS. To determine possible circadian dependency of microglia responsiveness to glucocorticoids - the likely proximal mediator for stress associated neuroinflammatory priming - microglia were isolated during the middle of the light or dark phase and treated ex vivo with corticosterone. Glucocorticoids treatment downregulated CX3CR1 and CD200R, two genes involved in microglial inflammatory "off" signaling; however, there was no effect of time of day on expression of either gene. Importantly, while absolute concentrations of corticosterone were comparable following IS during the light and dark phase, the magnitude of change in corticosterone was greater during the light phase. This work highlights the importance of studying circadian rhythms to elucidate biological mechanisms of stress.

Greater glucocorticoid receptor activation in hippocampus of aged rats sensitizes microglia.

Healthy aging individuals are more likely to suffer profound memory impairments following an immune challenge than are younger adults. These challenges produce a brain inflammatory response that is exaggerated with age. Sensitized microglia found in the normal aging brain are responsible for this amplified response, which in turn interferes with processes involved in memory formation. Here, we examine factors that may lead aging to sensitize microglia. Aged rats exhibited higher corticosterone levels in the hippocampus, but not in plasma, throughout the daytime (diurnal inactive phase). These elevated hippocampal corticosterone levels were associated with increased hippocampal 11ß-hydroxysteroid dehydrogenase type 1 protein expression, the enzyme that catalyzes glucocorticoid formation and greater hippocampal glucocorticoid receptor (GR) activation. Intracisternal administration of mifepristone, a GR antagonist, effectively reduced immune-activated proinflammatory responses, specifically from hippocampal microglia and prevented Escherichia coli-induced memory impairments in aged rats. Voluntary exercise as a therapeutic intervention significantly reduced total hippocampal GR expression. These data strongly suggest that increased GR activation in the aged hippocampus plays a critical role in sensitizing microglia.

Microglia inflammatory responses are controlled by an intrinsic circadian clock.

The circadian system regulates many physiological functions including inflammatory responses. For example, mortality caused by lipopolysaccharide (LPS) injection varies depending on the time of immunostimulation in mammals. The effects of more subtle challenges on the immune system and cellular mechanisms underlying circadian differences in neuroinflammatory responses are not well understood. Here we show that adult male Sprague-Dawley rats injected with a sub-septic dose of LPS during the light phase displayed elevated sickness behaviors and hippocampal cytokine production compared to rats injected during the dark phase. Microglia are the primary central nervous system (CNS) immune cell type and may mediate diurnal differences in sickness response, thus we explored whether microglia demonstrate temporal variations in inflammatory factors. Hippocampal microglia isolated from adult rats rhythmically expressed inflammatory factors and circadian clock genes. Microglia displayed robust rhythms of TNFα, IL1ß and IL6 mRNA, with peak cytokine gene expression occurring during the middle of the light phase. Microglia isolated during the light phase were also more reactive to immune stimulation; such that, ex vivo LPS treatment induced an exaggerated cytokine response in light phase-isolated microglia. Treating microglia with corticosterone ex vivo induced expression of the circadian clock gene Per1. However, microglia isolated from adrenalectomized rats maintained temporal differences in clock and inflammatory gene expression. This suggests circadian clock gene expression in microglia is entrained by, but oscillates in the absence of, glucocorticoids. Taken together, these findings demonstrate that microglia possess a circadian clock that influences inflammatory responses. These results indicate time-of-day is an important factor to consider when planning inflammatory interventions such as surgeries or immunotherapies.