Mu opioid receptor in microglia contributes to morphine analgesic tolerance, hyperalgesia, and withdrawal in mice.

A major challenge in medicine is developing potent pain therapies without the adverse effects of opiates. Neuroinflammation and in particular microglial activation have been shown to contribute to these effects. However, the implication of the microglial mu opioid receptor (MOR) is not known. We developed a novel conditional knockout (cKO) mouse line, wherein MOR is deleted in microglia. Morphine analgesic tolerance was delayed in both sexes in cKO mice in the hot plate assay. Opioid-induced hyperalgesia (OIH) as measured in the tail immersion assay was abolished in male cKO mice, and physical dependence to morphine as assessed by naloxone-induced withdrawal was attenuated in female cKO mice. Our results show a sex-dependent contribution of microglial MOR in morphine analgesic tolerance, OIH, and physical dependence. In conclusion, our data suggest that blockade of microglial MOR could represent a therapeutic target for opiate analgesia without the opiate adverse effects.

Regional vulnerability and spreading of hyperphosphorylated tau in seeded mouse brain.

We have exploited whole brain microscopy to map the progressive deposition of hyperphosphorylated tau in intact, cleared mouse brain. We found that the three-dimensional spreading pattern of hyperphosphorylated tau in the brain of an aging Tau.P301L mouse model did not resemble that observed in AD patients. Injection of synthetic or patient-derived tau fibrils in the CA1 region resulted in a more faithful spreading pattern. Atlas-guided volumetric analysis showed a connectome-dependent spreading from the injection site and also revealed hyperphosphorylated tau deposits beyond the direct anatomical connections. In fibril-injected brains, we also detected a persistent subpopulation of rod-like and swollen microglia. Furthermore, we showed that the hyperphosphorylated tau load could be reduced by intracranial co-administration of, and to a lesser extent, by repeated systemic dosing with an antibody targeting the microtubule-binding domain of tau. Thus, the combination of targeted seeding and in toto staging of tau pathology allowed assessing regional vulnerability in a comprehensive manner, and holds potential as a preclinical drug validation tool.

Phosphorylation of protein Tau by GSK3ß prolongs survival of bigenic Tau.P301L×GSK3ß mice by delaying brainstem tauopathy.

Tau.P301L transgenic mice suffer precocious mortality between ages 8 and 11 months, resulting from upper airway defects caused by tauopathy in autonomic brainstem circuits that control breathing (Dutschmann et al., 2010). In individual mice, the clinical phenotype evolves progressively and rapidly (3-6weeks) from clasping, over general motor impairment to severe reduction in body-weight into the terminal phase that announces imminent death (<3days). Surprisingly, co-expression of GSK3ß with Tau.P301L significantly prolonged survival of bigenic biGT mice (Terwel et al., 2008), which we here assign to delayed development of brainstem tauopathy. Eventually, brainstem tauopathy became as prominent in old biGT mice in the specified brainstem nuclei as in the parental Tau.P301L mice, resulting in similar clinical deterioration and terminal phase preceding death, although at later age. Biochemically, in both genotypes the pathway to neurofibrillary tangles and neuropil threads was similar: phosphorylation of protein Tau and formation of soluble oligomers and insoluble aggregates, ending in the typical tangles and threads of tauopathy. The extra GSK3ß activity led to expected increased phosphorylation of protein Tau, particularly at residues S262 and S396, which we must conclude to delay the aggregation of protein Tau in the brainstem of aging biGT mice. The unexpected, paradoxical alleviation of the brainstem problems in biGT mice allowed them to grow older and thereby develop more severe tauopathy in forebrain than Tau.P301L mice, which succumb at younger age.

Terminal hypothermic Tau.P301L mice have increased Tau phosphorylation independently of glycogen synthase kinase 3α/ß.

The microtubule-associated protein Tau is responsible for a large group of neurodegenerative disorders, known as tauopathies, including Alzheimer's disease. Tauopathy result from augmented and/or aberrant phosphorylation of Tau. Besides aging and various genetic and epigenetic defects that remain largely unknown, an important non-genetic agent that contributes is hypothermia, eventually caused by anesthesia. Remarkably, tauopathy in brains of hibernating mammals is not pathogenic, and, because it is fully reversible, is even considered to be neuroprotective. Here, we assessed the terminal phase of Tau.P301L mice and bigenic crosses with mice lacking glycogen synthase kinase 3 (GSK3)α completely, or GSK3ß specifically in neurons. We also analysed biGT bigenic mice that co-express Tau.P301L with GSK3ß.S9A and develop severe forebrain tauopathy with age. We found that the precocious mortality of Tau.P301L mice was typified by hypothermia that aggravated Tau phosphorylation, but, surprisingly, independently of GSK3α/ß. The important contribution of hypothermia at the time of death of mice with tauopathy suggests that body temperature should be included as a parameter in the analysis of pre-clinical models, and, by extension, in patients suffering from tauopathy.

Inflammasome signaling is dispensable for ß-amyloid-induced neuropathology in preclinical models of Alzheimer's disease.

Background: Alzheimer's disease (AD) is the most common neurodegenerative disorder affecting memory and cognition. The disease is accompanied by an abnormal deposition of ß-amyloid plaques in the brain that contributes to neurodegeneration and is known to induce glial inflammation. Studies in the APP/PS1 mouse model of ß-amyloid-induced neuropathology have suggested a role for inflammasome activation in ß-amyloid-induced neuroinflammation and neuropathology. Methods: Here, we evaluated the in vivo role of microglia-selective and full body inflammasome signalling in several mouse models of ß-amyloid-induced AD neuropathology. Results: Microglia-specific deletion of the inflammasome regulator A20 and inflammasome effector protease caspase-1 in the AppNL-G-F and APP/PS1 models failed to identify a prominent role for microglial inflammasome signalling in ß-amyloid-induced neuropathology. Moreover, global inflammasome inactivation through respectively full body deletion of caspases 1 and 11 in AppNL-G-F mice and Nlrp3 deletion in APP/PS1 mice also failed to modulate amyloid pathology and disease progression. In agreement, single-cell RNA sequencing did not reveal an important role for Nlrp3 signalling in driving microglial activation and the transition into disease-associated states, both during homeostasis and upon amyloid pathology. Conclusion: Collectively, these results question a generalizable role for inflammasome activation in preclinical amyloid-only models of neuroinflammation.

Early improved and late defective cognition is reflected by dendritic spines in Tau.P301L mice.

Cognitive demise correlates with progressive brain tauopathy in dementing patients. Improved cognition of young Tau.P301L mice contrasts with dysfunction later in life and remains unexplained (Boekhoorn et al., 2006). To unravel early mechanisms, we composed a correlative time line of clinical symptoms, cognitive defects, and biochemical and pathological traits, including comprehensive analysis of dendritic spines in specified regions of the cortex and hippocampus of young and adult Tau.P301L mice. Remarkably, young Tau.P301L mice have not more, but more mature spines than wild-type mice, revealing the anatomical substrate for their improved cognition and LTP. Spine maturation remained high in the hippocampus of adult Tau.P301L mice. However, spines regressed in length paralleling impaired cognition and increased Tau phosphorylation (Terwel et al., 2005). Conversely, spine maturation was unaffected in adult Tau.4R mice, while spine density was increased and length reduced similar to Tau.P301L mice. To explain how protein Tau promoted spinogenesis, we analyzed hippocampal synaptosomes and dendritic spines for mouse and human Tau. While synaptosomes were positive for both mouse and human Tau, weak variable reaction in spines was observed only after fixation according to Bouin. Mouse Tau was absent from spines in wild-type mice, dissociating the pathological actions of Tau in transgenic mice by relocalization into dendrites and spines from the physiological actions of protein Tau in axons only. We conclude that mutant protein Tau modulates cognition and morphology of spines similarly and in both directions, with pathology later in life coinciding with increased phosphorylation and relocalization of Tau from axons to soma and processes.

Delta Opioid Receptor in Astrocytes Contributes to Neuropathic Cold Pain and Analgesic Tolerance in Female Mice.

Background: The delta opioid receptor (DOR) contributes to pain control, and a major challenge is the identification of DOR populations that control pain, analgesia, and tolerance. Astrocytes are known as important cells in the pathophysiology of chronic pain, and many studies report an increased prevalence of pain in women. However, the implication of astrocytic DOR in neuropathic pain and analgesia, as well as the influence of sex in this receptor activity, remains unknown. Experimental Approach: We developed a novel conditional knockout (cKO) mouse line wherein DOR is deleted in astrocytes (named GFAP-DOR-KO), and investigated neuropathic mechanical allodynia as well as analgesia and analgesic tolerance in mutant male and female mice. Neuropathic cold allodynia was also characterized in mice of both sexes lacking DOR either in astrocytes or constitutively. Results: Neuropathic mechanical allodynia was similar in GFAP-DOR-KO and floxed DOR control mice, and the DOR agonist SNC80 produced analgesia in mutant mice of both sexes. Interestingly, analgesic tolerance developed in cKO males and was abolished in cKO females. Cold neuropathic allodynia was reduced in mice with decreased DOR in astrocytes. By contrast, cold allodynia was exacerbated in full DOR KO females. Conclusions: These findings show that astrocytic DOR has a prominent role in promoting cold allodynia and analgesic tolerance in females, while overall DOR activity was protective. Altogether this suggests that endogenous- and exogenous-mediated DOR activity in astrocytes worsens neuropathic allodynia while DOR activity in other cells attenuates this form of pain. In conclusion, our results show a sex-specific implication of astrocytic DOR in neuropathic pain and analgesic tolerance. These findings open new avenues for developing tailored DOR-mediated analgesic strategies.

Discovery and Functional Characterization of hPT3, a Humanized Anti-Phospho Tau Selective Monoclonal Antibody.

BACKGROUND: As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer's disease. OBJECTIVE: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. METHODS: By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. RESULTS AND CONCLUSION: Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer's disease.

Morphine-induced hyperalgesia involves mu opioid receptors and the metabolite morphine-3-glucuronide.

Opiates are potent analgesics but their clinical use is limited by side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). The Opiates produce analgesia and other adverse effects through activation of the mu opioid receptor (MOR) encoded by the Oprm1 gene. However, MOR and morphine metabolism involvement in OIH have been little explored. Hence, we examined MOR contribution to OIH by comparing morphine-induced hyperalgesia in wild type (WT) and MOR knockout (KO) mice. We found that repeated morphine administration led to analgesic tolerance and hyperalgesia in WT mice but not in MOR KO mice. The absence of OIH in MOR KO mice was found in both sexes, in two KO global mutant lines, and for mechanical, heat and cold pain modalities. In addition, the morphine metabolite morphine-3beta-D-glucuronide (M3G) elicited hyperalgesia in WT but not in MOR KO animals, as well as in both MOR flox and MOR-Nav1.8 sensory neuron conditional KO mice. M3G displayed significant binding to MOR and G-protein activation when using membranes from MOR-transfected cells or WT mice but not from MOR KO mice. Collectively our results show that MOR is involved in hyperalgesia induced by chronic morphine and its metabolite M3G.

Positive and negative early life experiences differentially modulate long term survival and amyloid protein levels in a mouse model of Alzheimer's disease.

Stress has been implicated as a risk factor for the severity and progression of sporadic Alzheimer's disease (AD). Early life experiences determine stress responsivity in later life, and modulate age-dependent cognitive decline. Therefore, we examined whether early life experiences influence AD outcome in a bigenic mouse model which progressively develops combined tau and amyloid pathology (biAT mice).Mice were subjected to either early life stress (ELS) or to 'positive' early handling (EH) postnatally (from day 2 to 9). In biAT mice, ELS significantly compromised long term survival, in contrast to EH which increased life expectancy. In 4 month old mice, ELS-reared biAT mice displayed increased hippocampal Aß levels, while these levels were reduced in EH-reared biAT mice. No effects of ELS or EH were observed on the brain levels of APP, protein tau, or PSD-95. Dendritic morphology was moderately affected after ELS and EH in the amygdala and medial prefrontal cortex, while object recognition memory and open field performance were not affected. We conclude that despite the strong transgenic background, early life experiences significantly modulate the life expectancy of biAT mice. Parallel changes in hippocampal Aß levels were evident, without affecting cognition of young adult biAT mice.