Glycyl-l-histidyl-l-lysine prevents copper- and zinc-induced protein aggregation and central nervous system cell death in vitro.

Common features of neurodegenerative diseases are oxidative and inflammatory imbalances as well as the misfolding of proteins. An excess of free metal ions can be pathological and contribute to cell death, but only copper and zinc strongly promote protein aggregation. Herein we demonstrate that the endogenous copper-binding tripeptide glycyl-l-histidyl-l-lysine (GHK) has the ability to bind to and reduce copper redox activity and to prevent copper- and zinc-induced cell death in vitro. In addition, GHK prevents copper- and zinc-induced bovine serum albumin aggregation and reverses aggregation through resolubilizing the protein. We further demonstrate the enhanced toxicity of copper during inflammation and the ability of GHK to attenuate this toxicity. Finally, we investigated the effects of copper on enhancing paraquat toxicity and report a protective effect of GHK. We therefore conclude that GHK has potential as a cytoprotective compound with regard to copper and zinc toxicity, with positive effects on protein solubility and aggregation that warrant further investigation in the treatment of neurodegenerative diseases.

Activation of TRPV1 receptor facilitates myelin repair following demyelination via the regulation of microglial function.

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel that is activated by capsaicin (CAP), the main component of chili pepper. Despite studies in several neurological diseases, the role of TRPV1 in demyelinating diseases remains unknown. Herein, we reported that TRPV1 expression was increased within the corpus callosum during demyelination in a cuprizone (CPZ)-induced demyelination mouse model. TRPV1 deficiency exacerbated motor coordinative dysfunction and demyelination in CPZ-treated mice, whereas the TRPV1 agonist CAP improved the behavioral performance and facilitated remyelination. TRPV1 was predominantly expressed in Iba1+ microglia/macrophages in human brain sections of multiple sclerosis patients and mouse corpus callosum under demyelinating conditions. TRPV1 deficiency decreased microglial recruitment to the corpus callosum, with an associated increase in the accumulation of myelin debris. Conversely, the activation of TRPV1 by CAP enhanced the recruitment of microglia to the corpus callosum and potentiated myelin debris clearance. Using real-time live imaging we confirmed an increased phagocytic function of microglia following CAP treatment. In addition, the expression of the scavenger receptor CD36 was increased, and that of the glycolysis regulators Hif1a and Hk2 was decreased. We conclude that TRPV1 is an important regulator of microglial function in the context of demyelination and may serve as a promising therapeutic target for demyelinating diseases such as multiple sclerosis.

Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation.

Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Our in silico drug screening reveals topoisomerase 1 (TOP1) inhibitors as promising drug candidates for microglial modulation. We show that TOP1 is highly expressed in neuroinflammatory conditions, and TOP1 inhibition using camptothecin (CPT) and its FDA-approved analog topotecan (TPT) reduces inflammatory responses in microglia/macrophages and ameliorates neuroinflammation in vivo. Transcriptomic analyses of sorted microglia from LPS-challenged mice reveal an altered transcriptional phenotype following TPT treatment. To target myeloid cells, we design a nanosystem using ß-glucan-coated DNA origami (MyloGami) loaded with TPT (TopoGami). MyloGami shows enhanced specificity to myeloid cells while preventing the degradation of the DNA origami scaffold. Myeloid-specific TOP1 inhibition using TopoGami significantly suppresses the inflammatory response in microglia and mitigates MS-like disease progression. Our findings suggest that TOP1 inhibition in myeloid cells represents a therapeutic strategy for neuroinflammatory diseases and that the myeloid-specific nanosystems we designed may also benefit the treatment of other diseases with dysfunctional myeloid cells.

Interpenetrating gallol functionalized tissue adhesive hyaluronic acid hydrogel polarizes macrophages to an immunosuppressive phenotype.

Innovative scaffold designs that modulate the local inflammatory microenvironment through favorable macrophage polarization and suppressing oxidative stress are needed for successful clinical translation of regenerative cell therapies and graft integration. We herein report derivation of a hydrazone-crosslinked gallol functionalized hyaluronic acid (HA-GA)-based hydrogel that displayed outstanding viscoelastic properties and immunomodulatory characteristics. Grafting of 6% gallol (GA) to a HA-backbone formed an interpenetrative network by promoting an additional crosslink between the gallol groups in addition to hydrazone crosslinking. This significantly enhanced the mechanical stability and displayed shear-thinning/self-healing characteristics, facilitated tissue adhesive properties to porcine tissue and also displayed radical scavenging properties, protecting encapsulated fibroblasts from peroxide challenge. The THP-1 human macrophage cell line or primary bone-marrow-derived murine macrophages cultured within HA-GA gels displayed selective polarization to a predominantly anti-inflammatory phenotype by upregulating IL4ra, IL-10, TGF-ß, and TGF-ßR1 expression when compared with HA-HA gels. Conversely, culturing of pro-inflammatory activated primary murine macrophages in HA-GA gels resulted in a significant reduction of pro-inflammatory TNF-α, IL-1ß, SOCS3 and IL-6 marker expression, and upregulated expression of anti-inflammatory cytokines including TGF-ß. Finally, when the gels were implanted subcutaneously into healthy mice, we observed infiltration of pro-inflammatory myeloid cells in HA-HA gels, while immunosuppressive phenotypes were observed within the HA-GA gels. Taken together these data suggest that HA-GA gels are an ideal injectable scaffold for viable immunotherapeutic interventions. STATEMENT OF SIGNIFICANCE: Host immune response against the implanted scaffolds that are designed to deliver stem cells or therapeutic proteins in vivo significantly limits the functional outcome. For this reason, we have designed immunomodulatory injectable scaffolds that can favorably polarize the recruited macrophages and impart antioxidant properties to suppress oxidative stress. Specifically, we have tailored a hyaluronic acid-based extracellular matrix mimetic injectable scaffold that is grafted with immunomodulatory gallol moiety. Gallol functionalization of hydrogel not only enhanced the mechanical properties of the scaffold by forming an interpenetrating network but also induced antioxidant properties, tissue adhesive properties, and polarized primary murine macrophages to immunosuppressive phenotype. We believe such immunoresponsive implants will pave the way for developing the next-generation of biomaterials for regenerative medicine applications.

Differential effects of noise exposure between substrains of CBA mice.

Noise trauma involves a plethora of mechanisms including reactive oxygen species, apoptosis, tissue damage, and inflammation. Recently, circadian mechanisms were also found to contribute to the vulnerability to noise trauma in mice, with greater damage occurring during their active phase (nighttime), when compared to similar noise exposures during their inactive phase (daytime). These effects seem to be regulated by mechanisms involving Bdnf responses to noise trauma and circulating levels of corticosterone (CORT). However, recent studies using different noise paradigms show contradicting results and it remains unclear how universal these findings are. Here we show that these findings differ even between substrains of mice and are restricted to a narrow window of noise intensity. We found that CBA/Sca mice exposed to 103 dB SPL display differential day/night noise sensitivity as measured by auditory brainstem responses (ABRs), but not at 100 (where full recovery is observed in day or night exposed mice) or 105 dB SPL (where permanent damage is found in both groups). In contrast, neither CBA/CaJ or CBA/JRj displayed such differences in day/night noise sensitivity, whatever noise intensity used. These effects appeared to be independent from outer hair cell function, as distortion product otoacoustic emissions appeared equally affected by day or night noise exposure, in all strains and in all noise conditions. Minor differences in ribbon counts or synaptic pairing were found in CBA/Sca mice, which were inconsistent with ABR wave 1 amplitude changes. Interestingly, CORT levels peaked in CBA/Sca mice at the onset of darkness at zeitgeber time 12 reaching levels of 43.8 ng/ml, while in the CBA/CaJ and the CBA/JRj, levels were 11.9 and 15.6 ng/ml respectively and peaking 4 h earlier (zeitgeber time 8). These findings were consistent with higher period of daily rhythm in CBA/Sca mice when measured in complete darkness using running wheels (23.7 h), than in CBA/CaJ (23.45 h) or CBA/JRj (23.13 h). In conclusion, our study suggests that the differential vulnerability to noise trauma between inactive and active phase is not universal and is as sensitive as substrain differences that might be governed by the circadian amplitude of the circulating CORT profiles.

Microglial PD-1 stimulation by astrocytic PD-L1 suppresses neuroinflammation and Alzheimer's disease pathology.

Chronic neuroinflammation is a pathogenic component of Alzheimer's disease (AD) that may limit the ability of the brain to clear amyloid deposits and cellular debris. Tight control of the immune system is therefore key to sustain the ability of the brain to repair itself during homeostasis and disease. The immune-cell checkpoint receptor/ligand pair PD-1/PD-L1, known for their inhibitory immune function, is expressed also in the brain. Here, we report upregulated expression of PD-L1 and PD-1 in astrocytes and microglia, respectively, surrounding amyloid plaques in AD patients and in the APP/PS1 AD mouse model. We observed juxtamembrane shedding of PD-L1 from astrocytes, which may mediate ectodomain signaling to PD-1-expressing microglia. Deletion of microglial PD-1 evoked an inflammatory response and compromised amyloid-ß peptide (Aß) uptake. APP/PS1 mice deficient for PD-1 exhibited increased deposition of Aß, reduced microglial Aß uptake, and decreased expression of the Aß receptor CD36 on microglia. Therefore, ineffective immune regulation by the PD-1/PD-L1 axis contributes to Aß plaque deposition during chronic neuroinflammation in AD.

A cell-type-specific atlas of the inner ear transcriptional response to acoustic trauma.

Noise-induced hearing loss (NIHL) results from a complex interplay of damage to the sensory cells of the inner ear, dysfunction of its lateral wall, axonal retraction of type 1C spiral ganglion neurons, and activation of the immune response. We use RiboTag and single-cell RNA sequencing to survey the cell-type-specific molecular landscape of the mouse inner ear before and after noise trauma. We identify induction of the transcription factors STAT3 and IRF7 and immune-related genes across all cell-types. Yet, cell-type-specific transcriptomic changes dominate the response. The ATF3/ATF4 stress-response pathway is robustly induced in the type 1A noise-resilient neurons, potassium transport genes are downregulated in the lateral wall, mRNA metabolism genes are downregulated in outer hair cells, and deafness-associated genes are downregulated in most cell types. This transcriptomic resource is available via the Gene Expression Analysis Resource (gEAR; https://umgear.org/NIHL) and provides a blueprint for the rational development of drugs to prevent and treat NIHL.

Microglial replacement therapy: a potential therapeutic strategy for incurable CSF1R-related leukoencephalopathy.

CSF1R-related leukoencephalopathy is an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia caused by colony stimulating factor 1 receptor (CSF1R) gene mutations. The disease has a global distribution and currently has no cure. Individuals with CSF1R-related leukoencephalopathy variably present clinical symptoms including cognitive impairment, progressive neuropsychiatric and motor symptoms. CSF1R is predominantly expressed on microglia within the central nervous system (CNS), and thus CSF1R-related leukoencephalopathy is now classified as a CNS primary microgliopathy. This urgent unmet medical need could potentially be addressed by using microglia-based immunotherapies. With the rapid recent progress in the experimental microglial research field, the replacement of an empty microglial niche following microglial depletion through either conditional genetic approaches or pharmacological therapies (CSF1R inhibitors) is being studied. Furthermore, hematopoietic stem cell transplantation offers an emerging means of exchanging dysfunctional microglia with the aim of reducing brain lesions, relieving clinical symptoms and prolonging the life of patients with CSF1R-related leukoencephalopathy. This review article introduces recent advances in microglial biology and CSF1R-related leukoencephalopathy. Potential therapeutic strategies by replacing microglia in order to improve the quality of life of CSF1R-related leukoencephalopathy patients will be presented.

Chronic Airway Allergy Induces Pro-Inflammatory Responses in the Brain of Wildtype Mice but Not 3xTgAD Mice.

The effects of systemic inflammation on the pathogenesis of Alzheimer's disease (AD) are not clarified, both beneficial and deleterious effects being reported. Allergy is accompanied by a systemic inflammatory response and some epidemiological studies have reported a positive association between a history of allergy/asthma and dementia. To investigate whether chronic airway allergy influences the inflammatory status in the brain, AD-like pathology, and behaviour in relation to AD, we induced chronic airway allergy in triple transgenic AD (3xTgAD) and wildtype (WT) mice by repeated exposure to ovalbumin (OVA) as allergen. Behavioural tests relevant for hippocampus-dependent behaviour were performed. We found that allergy significantly increased the brain levels of immunoglobulin (Ig) G, IgE. In 3xTgAD mice, allergy increased the levels of decay accelerating factor and decreased the phosphorylation of p38. In contrast, allergy increased the levels of interleukin (IL)-1ß and complement component 1q (C1q) in WT mice. Bronchoalveolar lavage fluid analysis confirmed eosinophilia in both genotypes, but the basal levels of eosinophils were lower in 3xTgAD mice. In summary, allergy induced predominantly anti-inflammatory effects in 3xTgAD mice, and pro-inflammatory effects in WT mice, thus being another potential factor to be considered when studying AD pathogenesis.

Circadian vulnerability of cisplatin-induced ototoxicity in the cochlea.

The chemotherapeutic agent cisplatin is renowned for its ototoxic effects. While hair cells in the cochlea are established targets of cisplatin, less is known regarding the afferent synapse, which is an essential component in the faithful temporal transmission of sound. The glutamate aspartate transporter (GLAST) shields the auditory synapse from excessive glutamate release, and its loss of function increases the vulnerability to noise, salicylate, and aminoglycosides. Until now, the involvement of GLAST in cisplatin-mediated ototoxicity remains unknown. Here, we test in mice lacking GLAST the effects of a low-dose cisplatin known not to cause any detectable change in hearing thresholds. When administered at nighttime, a mild hearing loss in GLAST KO mice was found but not at daytime, revealing a potential circadian regulation of the vulnerability to cisplatin-mediated ototoxicity. We show that the auditory synapse of GLAST KO mice is more vulnerable to cisplatin administration during the active phase (nighttime) when compared to WT mice and treatment during the inactive phase (daytime). This effect was not related to the abundance of platinum compounds in the cochlea, rather cisplatin had a dose-dependent impact on cochlear clock rhythms only after treatment at nighttime suggesting that cisplatin can modulate the molecular clock. Our findings suggest that the current protocols of cisplatin administration in humans during daytime may cause a yet undetectable damage to the auditory synapse, more so in already damaged ears, and severely impact auditory sensitivity in cancer survivors.

Impact of noise exposure on the circadian clock in the auditory system.

Circadian rhythms control the timing of all bodily functions, and misalignment in the rhythms can cause various diseases. Moreover, circadian rhythms are highly conserved and are regulated by a transcriptional-translational feedback loop of circadian genes that has a periodicity of approximately 24 h. The cochlea and the inferior colliculus (IC) have been shown to possess an autonomous and self-sustained circadian system as demonstrated by recording, in real time, the bioluminescence from PERIOD2::LUCIFERASE (PER2::LUC) mice. The cochlea and IC both express the core clock genes, Per1, Per2, Bmal1, and Rev-Erbα, where RNA abundance is rhythmically distributed with a 24 h cycle. Noise exposure alters clock gene expression in the cochlea and the IC after noise stimulation, although in different ways. These findings highlight the importance of circadian responses in the cochlea and the IC and emphasize the importance of circadian mechanisms for understanding the differences in central and peripheral auditory function and the subsequent molecular changes that occur after daytime (inactive phase) or nighttime (active phase) noise trauma.

Circadian Regulation of Cochlear Sensitivity to Noise by Circulating Glucocorticoids.

The cochlea possesses a robust circadian clock machinery that regulates auditory function. How the cochlear clock is influenced by the circadian system remains unknown. Here, we show that cochlear rhythms are system driven and require local Bmal1 as well as central input from the suprachiasmatic nuclei (SCN). SCN ablations disrupted the circadian expression of the core clock genes in the cochlea. Because the circadian secretion of glucocorticoids (GCs) is controlled by the SCN and GCs are known to modulate auditory function, we assessed their influence on circadian gene expression. Removal of circulating GCs by adrenalectomy (ADX) did not have a major impact on core clock gene expression in the cochlea. Rather it abolished the transcription of clock-controlled genes involved in inflammation. ADX abolished the known differential auditory sensitivity to day and night noise trauma and prevented the induction of GABA-ergic and glutamate receptors mRNA transcripts. However, these improvements were unrelated to changes at the synaptic level, suggesting other cochlear functions may be involved. Due to this circadian regulation of noise sensitivity by GCs, we evaluated the actions of the synthetic glucocorticoid dexamethasone (DEX) at different times of the day. DEX was effective in protecting from acute noise trauma only when administered during daytime, when circulating glucocorticoids are low, indicating that chronopharmacological approaches are important for obtaining optimal treatment strategies for hearing loss. GCs appear as a major regulator of the differential sensitivity to day or night noise trauma, a mechanism likely involving the circadian control of inflammatory responses.

Circadian integration of inflammation and glucocorticoid actions: Implications for the cochlea.

Auditory function has been shown to be influenced by the circadian system. Increasing evidence point towards the regulation of inflammation and glucocorticoid actions by circadian rhythms in the cochlea. Yet, how these three systems (circadian, immune and endocrine) converge to control auditory function remains to be established. Here we review the knowledge on immune and glucocorticoid actions, and how they interact with the circadian and the auditory system, with a particular emphasis on cochlear responses to noise trauma. We propose a multimodal approach to understand the mechanisms of noise-induced hearing loss by integrating the circadian, immune and endocrine systems into the bearings of the cochlea. Considering the well-established positive impact of chronotherapeutic approaches in the treatment of cardiovascular, asthma and cancer, an increased knowledge on the mechanisms where circadian, immune and glucocorticoids meet in the cochlea may improve current treatments against hearing disorders.

Microglia in Alzheimer's disease.

Microglia are brain-resident myeloid cells that mediate key functions to support the CNS. Microglia express a wide range of receptors that act as molecular sensors, which recognize exogenous or endogenous CNS insults and initiate an immune response. In addition to their classical immune cell function, microglia act as guardians of the brain by promoting phagocytic clearance and providing trophic support to ensure tissue repair and maintain cerebral homeostasis. Conditions associated with loss of homeostasis or tissue changes induce several dynamic microglial processes, including changes of cellular morphology, surface phenotype, secretory mediators, and proliferative responses (referred to as an "activated state"). Activated microglia represent a common pathological feature of several neurodegenerative diseases, including Alzheimer's disease (AD). Cumulative evidence suggests that microglial inflammatory activity in AD is increased while microglial-mediated clearance mechanisms are compromised. Microglia are perpetually engaged in a mutual interaction with the surrounding environment in CNS; thus, diverse microglial reactions at different disease stages may open new avenues for therapeutic intervention and modification of inflammatory activities. In this Review, the role of microglia in the pathogenesis of AD and the modulation of microglia activity as a therapeutic modality will be discussed.

Influence of Allergy on Immunoglobulins and Amyloid-ß in the Cerebrospinal Fluid of Patients with Alzheimer's Disease.

BACKGROUND: Peripheral inflammation has been suggested to influence the development of Alzheimer's disease (AD). Elevated levels of pro-inflammatory markers in the plasma of patients with AD indicate that a systemic pro-inflammatory status occurs concomitantly with inflammatory changes in the brain. OBJECTIVE: To investigate whether allergy influences the levels of immunoglobulins (Ig) and of pro- and anti-inflammatory cytokines in the serum and cerebrospinal fluid (CSF) from patients with AD, mild cognitive impairment (MCI), and subjective cognitive impairment (SCI). METHODS: IgA, IgG, and its subclasses, IgM, and cytokines were analyzed in CSF and serum from patients with SCI, MCI, and AD, with or without allergy. The relation between allergy and Mini-Mental State Examination (MMSE) scores, and between allergy and CSF biomarkers for AD (phosphorylated (p)-tau, total (t)-tau, amyloid-ß 42 (Aß42), were analyzed. RESULTS: In MCI, the CSF levels of IgG2 were lower in allergic patients, and in AD, the levels of IgA and the IgG1/total IgG ratio were lower in allergic patients, compared to patients without allergy. MCI subjects with allergy had higher serum IgM levels compared to those without allergy. CSF levels of Aß42 were lower and MMSE scores were higher in AD patients with allergy than in those without allergy. CONCLUSIONS: The presence of allergy was associated with seemingly beneficial effects on AD as suggested by higher Aß42 levels in CSF, and higher MMSE scores. Higher IgM levels and lower other Ig classes suggest that allergy may influence senescence of the immune response.

Chronic airway-induced allergy in mice modifies gene expression in the brain toward insulin resistance and inflammatory responses.

BACKGROUND: Chronic systemic inflammation affects brain functionality and may negatively influence the progression of neurodegenerative disorders. Allergy is a chronic inflammatory disease affecting more than 20% of the Western population. Little is known regarding the influence of allergy on brain functions. The aim of the present study was to obtain a global overview of the genes that drive the effects of peripheral inflammation associated with chronic airway-induced allergy in the brain. METHODS: Airway allergy was induced in C57B/6J mice using ovalbumin as the allergen. Microarray analysis was performed in the hippocampus and frontal cortex in association with Affymetrix. For the data analysis, principal component analysis and orthogonal to latent structures discriminant analysis followed by pathway analysis were used. Quantitative polymerase chain reaction (qPCR) and protein analysis by Western blotting were performed for the validation of microarray results. RESULTS: Microarray analysis showed low-grade changes in gene expression in the brain induced by airway-associated allergy. Changes in expression were observed for genes involved in antigen processing and presentation, cytokine-cytokine interaction, Toll-like receptor and mitogen-activated protein kinase signaling, as determined by pathway analysis. We confirmed a reduction of insulin-degrading enzyme at the protein level and a decrease in insulin receptor phosphorylation in the brains of allergic mice. Other allergy-induced gene expression changes were confirmed by qPCR, including increased levels of tumor necrosis factor receptor superfamily member 23 and lipopolysaccharide-binding protein. CONCLUSION: Airway-associated allergy induces changes in brain gene expression toward induction of insulin resistance and inflammatory responses with potential implications for neurodegenerative disorders.

Allergy influences the inflammatory status of the brain and enhances tau-phosphorylation.

Despite the existing knowledge regarding the neuropathology of Alzheimer's disease (AD), the cause of sporadic forms of the disease is unknown. It has been suggested that systemic inflammation may have a role, but the exact mechanisms through which inflammatory processes influence the pathogenesis and progress of AD are not obvious. Allergy is a chronic inflammatory disease affecting more than 20% of the Western population, but the effects of allergic conditions on brain functions are largely unknown. The aim of this study was to investigate whether or not chronic peripheral inflammation associated with allergy affects the expression of AD-related proteins and inflammatory markers in the brain. On the basis of previously described models for allergy in mice we developed a model of chronic airway allergy in mouse, with ovalbumin as allergen. The validity of the chronic allergy model was confirmed by a consistent and reproducible eosinophilia in the bronchoalveolar lavage (BAL) fluid of allergic animals. Allergic mice were shown to have increased brain levels of both immunoglobulin (Ig) G and IgE with a widespread distribution. Allergy was also found to increase phosphorylation of tau protein in the brain. The present data support the notion that allergy-dependent chronic peripheral inflammation modifies the brain inflammatory status, and influences phosphorylation of an AD-related protein, indicating that allergy may be yet another factor to be considered for the development and/or progression of neurodegenerative diseases such as AD.