Role of Neurocellular Endoplasmic Reticulum Stress Response in Alzheimer's Disease and Related Dementias Risk.

Currently, more than 55 million people around the world suffer from dementia, and Alzheimer's Disease and Related Dementias (ADRD) accounts for nearly 60-70% of all those cases. The spread of Alzheimer's Disease (AD) pathology and progressive neurodegeneration in the hippocampus and cerebral cortex is strongly correlated with cognitive decline in AD patients; however, the molecular underpinning of ADRD's causality is still unclear. Studies of postmortem AD brains and animal models of AD suggest that elevated endoplasmic reticulum (ER) stress may have a role in ADRD pathology through altered neurocellular homeostasis in brain regions associated with learning and memory. To study the ER stress-associated neurocellular response and its effects on neurocellular homeostasis and neurogenesis, we modeled an ER stress challenge using thapsigargin (TG), a specific inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), in the induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) of two individuals from our Mexican American Family Study (MAFS). High-content screening and transcriptomic analysis of the control and ER stress-challenged NSCs showed that the NSCs' ER stress response resulted in a significant decline in NSC self-renewal and an increase in apoptosis and cellular oxidative stress. A total of 2300 genes were significantly (moderated t statistics FDR-corrected p-value ≤ 0.05 and fold change absolute ≥ 2.0) differentially expressed (DE). The pathway enrichment and gene network analysis of DE genes suggests that all three unfolded protein response (UPR) pathways, protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF-6), and inositol-requiring enzyme-1 (IRE1), were significantly activated and cooperatively regulated the NSCs' transcriptional response to ER stress. Our results show that IRE1/X-box binding protein 1 (XBP1) mediated transcriptional regulation of the E2F transcription factor 1 (E2F1) gene, and its downstream targets have a dominant role in inducing G1/S-phase cell cycle arrest in ER stress-challenged NSCs. The ER stress-challenged NSCs also showed the activation of C/EBP homologous protein (CHOP)-mediated apoptosis and the dysregulation of synaptic plasticity and neurotransmitter homeostasis-associated genes. Overall, our results suggest that the ER stress-associated attenuation of NSC self-renewal, increased apoptosis, and dysregulated synaptic plasticity and neurotransmitter homeostasis plausibly play a role in the causation of ADRD.

Pre-Infection Innate Immunity Attenuates SARS-CoV-2 Infection and Viral Load in iPSC-Derived Alveolar Epithelial Type 2 Cells.

A large portion of the heterogeneity in coronavirus disease 2019 (COVID-19) susceptibility and severity of illness (SOI) remains poorly understood. Recent evidence suggests that SARS-CoV-2 infection-associated damage to alveolar epithelial type 2 cells (AT2s) in the distal lung may directly contribute to disease severity and poor prognosis in COVID-19 patients. Our in vitro modeling of SARS-CoV-2 infection in induced pluripotent stem cell (iPSC)-derived AT2s from 10 different individuals showed interindividual variability in infection susceptibility and the postinfection cellular viral load. To understand the underlying mechanism of the AT2's capacity to regulate SARS-CoV-2 infection and cellular viral load, a genome-wide differential gene expression analysis between the mock and SARS-CoV-2 infection-challenged AT2s was performed. The 1393 genes, which were significantly (one-way ANOVA FDR-corrected p ≤ 0.05; FC abs ≥ 2.0) differentially expressed (DE), suggest significant upregulation of viral infection-related cellular innate immune response pathways (p-value ≤ 0.05; activation z-score ≥ 3.5), and significant downregulation of the cholesterol- and xenobiotic-related metabolic pathways (p-value ≤ 0.05; activation z-score ≤ -3.5). Whilst the effect of post-SARS-CoV-2 infection response on the infection susceptibility and postinfection viral load in AT2s is not clear, interestingly, pre-infection (mock-challenged) expression of 238 DE genes showed a high correlation with the postinfection SARS-CoV-2 viral load (FDR-corrected p-value ≤ 0.05 and r2-absolute ≥ 0.57). The 85 genes whose expression was negatively correlated with the viral load showed significant enrichment in viral recognition and cytokine-mediated innate immune GO biological processes (p-value range: 4.65 × 10-10 to 2.24 × 10-6). The 153 genes whose expression was positively correlated with the viral load showed significant enrichment in cholesterol homeostasis, extracellular matrix, and MAPK/ERK pathway-related GO biological processes (p-value range: 5.06 × 10-5 to 6.53 × 10-4). Overall, our results strongly suggest that AT2s' pre-infection innate immunity and metabolic state affect their susceptibility to SARS-CoV-2 infection and viral load.

Developmental stage of transplanted neural progenitor cells influences anatomical and functional outcomes after spinal cord injury in mice.

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for replacing lost neurons following spinal cord injury (SCI). However, how graft cellular composition influences regeneration and synaptogenesis of host axon populations, or recovery of motor and sensory functions after SCI, is poorly understood. We transplanted developmentally-restricted spinal cord NPCs, isolated from E11.5-E13.5 mouse embryos, into sites of adult mouse SCI and analyzed graft axon outgrowth, cellular composition, host axon regeneration, and behavior. Earlier-stage grafts exhibited greater axon outgrowth, enrichment for ventral spinal cord interneurons and Group-Z spinal interneurons, and enhanced host 5-HT+ axon regeneration. Later-stage grafts were enriched for late-born dorsal horn interneuronal subtypes and Group-N spinal interneurons, supported more extensive host CGRP+ axon ingrowth, and exacerbated thermal hypersensitivity. Locomotor function was not affected by any type of NPC graft. These findings showcase the role of spinal cord graft cellular composition in determining anatomical and functional outcomes following SCI.

Transcription Factor Hb9 Is Expressed in Glial Cell Lineages in the Developing Mouse Spinal Cord.

Hb9 (Mnx1) is a transcription factor described as a spinal cord motor neuron (MN)-specific marker and critical factor for the postmitotic specification of these cells. To date, expression of Hb9 in other cell types has not been reported. We performed a fate-mapping approach to examine distributions of Hb9-expressing cells and their progeny ("Hb9-lineage cells") within the embryonic and adult spinal cord of Hb9cre;Ai14 mice. We found that Hb9-lineage cells are distributed in a gradient of increasing abundance throughout the rostrocaudal spinal cord axis during embryonic and postnatal stages. Furthermore, although the majority of Hb9-lineage cells at cervical spinal cord levels are MNs, at more caudal levels, Hb9-lineage cells include small-diameter dorsal horn neurons, astrocytes, and oligodendrocytes. In the peripheral nervous system, we observed a similar phenomenon with more abundant Hb9-lineage Schwann cells in muscles of the lower body versus upper body muscles. We cultured spinal cord progenitors in vitro and found that gliogenesis was increased by treatment with the caudalizing factor FGF-8B, while glial tdTomato expression was increased by treatment with both FGF-8B and GDF-11. Together, these observations suggest that early and transient expression of Hb9 in spinal cord neural progenitors may be induced by caudalizing factors such as FGF and GDF signaling. Furthermore, our work raises the possibility that early Hb9 expression may influence the development of spinal cord macroglia and Schwann cells, especially at caudal regions. Together, these findings highlight the importance of using caution when designing experiments using Hb9cre mice to perform spinal cord MN-specific manipulations.

Effects of biological sex mismatch on neural progenitor cell transplantation for spinal cord injury in mice.

Despite advancement of neural progenitor cell transplantation to spinal cord injury clinical trials, there remains a lack of understanding of how biological sex of transplanted cells influences outcomes after transplantation. To address this, we transplanted GFP-expressing sex-matched, sex-mismatched, or mixed donor cells into sites of spinal cord injury in adult male and female mice. Biological sex of the donor cells does not influence graft neuron density, glial differentiation, formation of the reactive glial cell border, or graft axon outgrowth. However, male grafts in female hosts feature extensive hypervascularization accompanied by increased vascular diameter and perivascular cell density. We show greater T-cell infiltration within male-to-female grafts than other graft types. Together, these findings indicate a biological sex-specific immune response of female mice to male donor cells. Our work suggests that biological sex should be considered in the design of future clinical trials for cell transplantation in human injury.

Inflammation increases the development of depression behaviors in male rats after spinal cord injury.

Following spinal cord injury, 18-26% of patients are diagnosed with depressive disorders, compared to 8-12% in the general population. As increased inflammation strongly correlates with depression in both animal and human studies, we hypothesized that the immune activation inherent to SCI could increase depression-like behavior. Thus, we proposed that reducing immune activation with minocycline, a microglial inhibitor, would decrease depression-like behavior following injury. Male Sprague-Dawley rats were given minocycline in their drinking water for 14 days following a moderate, mid-thoracic (T12) spinal contusion. An array of depression-like behaviors (social activity, sucrose preference, forced swim, open field activity) were examined prior to injury as well as on days 9-10, 19-20, and 29-30 post-injury. Peripheral cytokine levels were analyzed in serum collected prior to injury and 10 days post-injury. Hierarchical cluster analysis divided subjects into two groups based on behavior: depressed and not-depressed. Depressed subjects displayed lower levels of open field activity and social interaction relative to their not-depressed counterparts. Depressed subjects also showed significantly greater expression of pro-inflammatory cytokines both before and after injury and displayed lower levels of hippocampal neurogenesis than not-depressed subjects. Intriguingly, subjects who later showed depressive behaviors had higher baseline levels of the pro-inflammatory cytokine IL-6, which persisted throughout the duration of the experiment. Minocycline, however, did not affect serum cytokine levels and did not block the development of depression; equal numbers of minocycline versus vehicle-treated subjects appeared in both phenotypic groups. Despite this, these data overall suggest that molecular correlates of inflammation prior to injury could predict the development of depression after a physical stressor.

Variability in Open-Field Locomotor Scoring Following Force-Defined Spinal Cord Injury in Rats: Quantification and Implications.

Spinal cord injury research in experimental animals aims to define mechanisms of tissue damage and identify interventions that can be translated into effective clinical therapies. Highly reliable models of injury and outcome measurement are essential to achieve these aims and avoid problems with reproducibility. Functional scoring is a critical component of outcome assessment and is currently commonly focused on open field locomotion (the "BBB score"). Here we analyze variability of observed locomotor outcome after a highly regulated spinal cord contusion in a large group of rats that had not received any therapeutic intervention. Our data indicate that, despite tight regulation of the injury severity, there is considerable variability in open-field score of individual rats at 21 days after injury, when the group as a whole reaches a functional plateau. The bootstrapped reference interval (that defines boundaries that contain 95% scores in the population without regard for data distributional character) for the score at 21 days was calculated to range from 2.3 to 15.9 on the 22-point scale. Further analysis indicated that the mean day 21 score of random groups of 10 individuals drawn by bootstrap sampling from the whole study population varies between 9.5 and 13.5. Wide variability between individuals implies that detection of small magnitude group-level treatment effects will likely be unreliable, especially if using small experimental group sizes. To minimize this problem in intervention studies, consideration should be given to assessing treatment effects by comparing proportions of animals in comparator groups that attain pre-specified criterion scores.

An Analysis of Variability in "CatWalk" Locomotor Measurements to Aid Experimental Design and Interpretation.

Preclinical studies in models of neurologic injury and disease rely on behavioral outcomes to measure intervention efficacy. For spinal cord injury, the CatWalk system provides unbiased quantitative assessment of subtle aspects of locomotor function in rodents and so can powerfully detect significant differences between experimental and control groups. Although clearly of key importance, summary group-level data can obscure the variability within and between individual subjects and therefore make it difficult to understand the magnitude of effect in individual animals and the proportion of a group that may show benefit. Here, we calculate reference change intervals (RCIs) that define boundaries of normal variability for measures of rat locomotion on the CatWalk. Our results indicate that many commonly-used outcome measures are highly variable, such that differences of up to 70% from baseline value must be considered normal variation. Many CatWalk outcome variables are also highly correlated and dependent on run speed. Application of calculated RCIs to open access data (https://scicrunch.org/odc-sci) on hindlimb stride length in spinal cord-injured rats illustrates the complementarity between group-level (16 mm change; p = 0.0009) and individual-level (5/32 animals show change outside RCI boundaries) analysis between week 3 and week 6 after injury. We also conclude that interdependence among CatWalk variables implies that test "batteries" require careful composition to ensure that different aspects of defective gait are analyzed. Calculation of RCIs aids in experimental design by quantifying variability and enriches overall data analysis by providing details of change at an individual level that complement group-level analysis.

Depression-like behavior corresponds with cardiac changes in a rodent model of spinal cord injury.

In previous studies we have shown that approximately 1/3 of male Sprague Dawley rats develop symptoms of depression following a spinal cord injury (SCI). Using established behavioral tests to measure depression in rodents, we found that after SCI, subjects characterized as depressed had decreased sucrose preference, open field activity, social exploration, and burrowing behavior. As some of these tests of depression could be affected by the compromised motor function inherent to the SCI condition, the current study examined whether non-subjective, physiological differences in heart rate and heart rate variability were also associated with depression, as seen in humans. Male Sprague Dawley rats were implanted with radiotelemetry devices and either received a moderate contusion injury or remained intact. The implanted telemetry devices recorded home cage activity, body temperature, heart rate, and heart rate variability for 5 min/h throughout a 30-day post-injury assessment period. Depression behavior was evaluated using a battery of tests conducted on days 9-10 and 19-20 post-injury. Locomotor recovery and pain reactivity were also examined. Hierarchical clustering, based on the behavioral scores collected on the tests of depression, revealed that 28% of the SCI subjects displayed symptoms of depression, relative to the remaining 72% of SCI subjects. The subjects characterized as depressed had significantly lower social interaction and burrowing activity than the group that was not depressed. Interestingly, the subjects behaviorally characterized as depressed also had significantly lower heart rate variability than the not-depressed intact group. There was no difference between not-depressed SCI and intact rats on this measure. Therefore, in addition to behavior, depressed and not-depressed rats differ on measures of physiological function that are associated with depression in humans. These physiological differences further validate the rodent model of depression after SCI.

Morphine increases macrophages at the lesion site following spinal cord injury: Protective effects of minocycline.

Opioids are among the most effective and widely prescribed medications for the treatment of pain following spinal cord injury (SCI). Spinally-injured patients receive opioids within hours of arrival at the emergency room, and prolonged opioid regimens are often employed for the management of post-SCI chronic pain. However, previous studies in our laboratory suggest that the effects of opioids such as morphine may be altered in the pathophysiological context of neurotrauma. Specifically, we have shown that morphine administration in a rodent model of SCI increases mortality and tissue loss at the injury site, and decreases recovery of motor and sensory function, and overall health, even weeks after treatment. The literature suggests that opioids may produce these adverse effects by acting as endotoxins and increasing glial activation and inflammation. To better understand the effects of morphine following SCI, in this study we used flow cytometry to assess immune-competent cells at the lesion site. We observed a morphine-induced increase in the overall number of CD11b+ cells, with marked effects on microglia, in SCI subjects. Next, to investigate whether this increase in the inflammatory profile is necessary to produce morphine's effects, we challenged morphine treatment with minocycline. We found that pre-treatment with minocycline reduced the morphine-induced increase in microglia at the lesion site. More importantly, minocycline also blocked the adverse effects of morphine on recovery of function without disrupting the analgesic efficacy of this opioid. Together, our findings suggest that following SCI, morphine may exacerbate the inflammatory response, increasing cell death at the lesion site and negatively affecting functional recovery.

Osteocytes reflect a pro-inflammatory state following spinal cord injury in a rodent model.

Profound bone loss occurs following spinal cord injury (SCI) resulting in a high incidence of fractures. While likely caused in part by loss of weight-bearing, there is greater bone loss following SCI when compared to that observed in other disuse animal models. Patients with SCI have a protracted inflammatory response, with elevated circulating levels of pro-inflammatory markers. This chronic inflammation could compound the bone loss attributed to disuse and the loss of neural signaling. To assess this, we examined inflammatory markers and bone turnover regulators in osteocytes from rats with a moderate spinal contusion injury (SCI) and intact controls (CON). We counted osteocytes positive for cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-17 (IL-17), and interleukin-10 (IL-10)], osteoclastogenesis regulators RANKL and OPG, and the bone formation inhibitor sclerostin, 32 days after the spinal contusion. By day 9 post-injury, the majority of SCI rats had recovered significant locomotor function and were bearing weight on their hindlimbs. However, despite weight-bearing, peripheral QCT scans demonstrated lower bone mass due to SCI in the proximal tibia metaphysis compared to CON. SCI animals also had lower cancellous bone volume, lower bone formation rate (BFR), lower osteoid surface (OS), and higher osteoclast surface (Oc.S). Tibial mid-shaft periosteal BFR was also lower after SCI. Immunohistochemical staining of the distal femur bone revealed cancellous osteocytes positive for TNF-α, IL-6, IL-17, and IL-10 were elevated in SCI animals relative to intact controls. Protein expression of RANKL+, OPG+, and sclerostin+ osteocytes was also higher in SCI rats. At the cortical midshaft, osteocyte TNF-α, IL-6, and sclerostin were statistically higher in SCI vs. CON. With regression analysis, inflammatory factors were associated with changes in bone turnover. In conclusion, inflammatory factors as well as altered mechanical loading influence bone turnover following a moderate SCI. Treatments aimed at minimizing fracture risk after SCI may need to target both the chronically altered inflammatory state as well as disuse-induced bone loss.

Pain Input Impairs Recovery after Spinal Cord Injury: Treatment with Lidocaine.

More than 90% of spinal cord injuries are caused by traumatic accidents and are often associated with other tissue damage (polytrauma) that can provide a source of continued pain input during recovery. In a clinically relevant spinal cord contusion injury model, prior work has shown that noxious stimulation at an intensity that engages pain (C) fibers soon after injury augments secondary injury and impairs functional recovery. Noxious input increases the expression of pro-inflammatory cytokines (interleukin 1ß and 18), cellular signals associated with cell death (caspase 3 and 8), and physiological signs of hemorrhage. Here, it is shown that reducing neural excitability after spinal cord injury (SCI) with the local anesthetic lidocaine (micro-injected by means of a lumbar puncture) blocks these adverse cellular effects. In contrast, treatment with an analgesic dose of morphine had no effect. Contused rats that received nociceptive stimulation soon after injury exhibited poor locomotor recovery, less weight gain, and greater tissue loss at the site of injury. Prophylactic application of lidocaine blocked the adverse effect of nociceptive stimulation on behavioral recovery and reduced tissue loss from secondary injury. The results suggest that quieting neural excitability using lidocaine can reduce the adverse effect of pain input (from polytrauma or surgery) after SCI.

Neurobiological Effects of Morphine after Spinal Cord Injury.

Opioids and non-steroidal anti-inflammatory drugs are used commonly to manage pain in the early phase of spinal cord injury (SCI). Despite its analgesic efficacy, however, our studies suggest that intrathecal morphine undermines locomotor recovery and increases lesion size in a rodent model of SCI. Similarly, intravenous (IV) morphine attenuates locomotor recovery. The current study explores whether IV morphine also increases lesion size after a spinal contusion (T12) injury and quantifies the cell types that are affected by early opioid administration. Using an experimenter-administered escalating dose of IV morphine across the first seven days post-injury, we quantified the expression of neuron, astrocyte, and microglial markers at the injury site. SCI decreased NeuN expression relative to shams. In subjects with SCI treated with IV morphine, virtually no NeuN+ cells remained across the rostral-caudal extent of the lesion. Further, whereas SCI per se increased the expression of astrocyte and microglial markers (glial fibrillary acidic protein and OX-42, respectively), morphine treatment decreased the expression of these markers. These cellular changes were accompanied by attenuation of locomotor recovery (Basso, Beattie, Bresnahan scores), decreased weight gain, and the development of opioid-induced hyperalgesia (increased tactile reactivity) in morphine-treated subjects. These data suggest that morphine use is contraindicated in the acute phase of a spinal injury. Faced with a lifetime of intractable pain, however, simply removing any effective analgesic for the management of SCI pain is not an ideal option. Instead, these data underscore the critical need for further understanding of the molecular pathways engaged by conventional medications within the pathophysiological context of an injury.

Nor-Binaltorphimine Blocks the Adverse Effects of Morphine after Spinal Cord Injury.

Opioids are frequently used for the treatment of pain following spinal cord injury (SCI). Unfortunately, we have shown that morphine administered in the acute phase of SCI results in significant, adverse secondary consequences including compromised locomotor and sensory recovery. Similarly, we showed that selective activation of the κ-opioid receptor (KOR), even at a dose 32-fold lower than morphine, is sufficient to attenuate recovery of locomotor function. In the current study, we tested whether activation of the KOR is necessary to produce morphine's adverse effects using nor-Binaltorphimine (norBNI), a selective KOR antagonist. Rats received a moderate spinal contusion (T12) and 24 h later, baseline locomotor function and nociceptive reactivity were assessed. Rats were then administered norBNI (0, 0.02, 0.08, or 0.32 µmol) followed by morphine (0 or 0.32 µmol). Nociception was reassessed 30 min after drug treatment, and recovery was evaluated for 21 days. The effects of norBNI on morphine-induced attenuation of recovery were dose dependent. At higher doses, norBNI blocked the adverse effects of morphine on locomotor recovery, but analgesia was also significantly decreased. Conversely, at low doses, analgesia was maintained, but the adverse effects on recovery persisted. A moderate dose of norBNI, however, adequately protected against morphine's adverse effects without eliminating its analgesic efficacy. This suggests that activation of the KOR system plays a significant role in the morphine-induced attenuation of recovery. Our research suggests that morphine, and other opioid analgesics, may be contraindicated for the SCI population. Blocking KOR activity may be a viable strategy for improving the safety of clinical opioid use.

Morphine self-administration following spinal cord injury.

Neuropathic pain develops in up to two-thirds of people following spinal cord injury (SCI). Opioids are among the most effective treatments for this pain and are commonly prescribed. There is concern surrounding the use of these analgesics, however, because use is often associated with the development of addiction. Previous data suggests that this concern may not be relevant in the presence of neuropathic pain. Yet, despite the common prescription of opioids for the treatment of SCI-related pain, there has been only one previous study examining the addictive potential of morphine following spinal injury. To address this, the present study used a self-administration paradigm to examine the addictive potential of morphine in a rodent model of SCI. Animals were placed into self-administration chambers 24 h, 14 d, or 35 d following a moderate spinal contusion injury. They were placed into the chambers for seven 12-hour sessions with access to 1.5 mg morphine/lever depression (up to 30 mg/d). In the acute phase of SCI, contused animals self-administered significantly less morphine than their sham counterparts, as previously shown. However, contused animals showing signs of neuropathic pain did not self-administer less morphine than their sham counterparts when administration began 14 or 35 d after injury. Instead, these animals administered nearly the full amount of morphine available each session. This amount of morphine did not affect recovery of locomotor function but did cause significant weight loss. We suggest caution is warranted when prescribing opioids for the treatment of neuropathic pain resulting from SCI, as the addictive potential is not reduced in this model.

Assessment of depression in a rodent model of spinal cord injury.

Despite an increased incidence of depression in patients after spinal cord injury (SCI), there is no animal model of depression after SCI. To address this, we used a battery of established tests to assess depression after a rodent contusion injury. Subjects were acclimated to the tasks, and baseline scores were collected before SCI. Testing was conducted on days 9-10 (acute) and 19-20 (chronic) postinjury. To categorize depression, subjects' scores on each behavioral measure were averaged across the acute and chronic stages of injury and subjected to a principal component analysis. This analysis revealed a two-component structure, which explained 72.2% of between-subjects variance. The data were then analyzed with a hierarchical cluster analysis, identifying two clusters that differed significantly on the sucrose preference, open field, social exploration, and burrowing tasks. One cluster (9 of 26 subjects) displayed characteristics of depression. Using these data, a discriminant function analysis was conducted to derive an equation that could classify subjects as "depressed" on days 9-10. The discriminant function was used in a second experiment examining whether the depression-like symptoms could be reversed with the antidepressant, fluoxetine. Fluoxetine significantly decreased immobility in the forced swim test (FST) in depressed subjects identified with the equation. Subjects that were depressed and treated with saline displayed significantly increased immobility on the FST, relative to not depressed, saline-treated controls. These initial experiments validate our tests of depression, generating a powerful model system for further understanding the relationships between molecular changes induced by SCI and the development of depression.