Attenuating inflammatory signaling is a preemptive strategy to avoid downstream neurodegenerative processes such as inflammation-induced neuronal damage and fibrosis. One such route is the PI3K/Akt/mTOR inflammatory signaling pathway44. We have previously shown that TNF-α treatment to HMC3 microglia induces a pro-inflammatory phenotype and that cromolyn and F-cromolyn are able to significantly reduce secretion of an array of important cytokines and chemokines, including IL-1β, IL-6, IL-8, IFN-γ, CCL2, CCL3, CCL4, and CXCL1019. IL-2 is another pro-inflammatory cytokine that can activate NF-κB to promote expression of IL-6 and MCP-1, and disrupt the integrity of the blood-brain barrier by comprising components of the adherens junctions50. In contrast, IL-4 has been associated with protective mechanisms of brain ischemia and can induce expression of anti-inflammatory M2 microglial phenotype in primary rat cortical glia cultures, and encourage PPARγ-dependent microglial phagocytosis51. Increased expression of GRO-α (also known as CXCL1) in monocytes from APP/PS1 transgenic mice and AD patients have been implicated in increased monocyte infiltration into the brain52 as well as massive neutrophil recruitment in other models of brain and tissue damage53,54. The inflammatory cytokine Eotaxin is found in many cell types, including microglia and astrocytes, with roles in neurogenesis suppression and is expressed in heightened levels in aged human plasma and CSF55. Additionally, increased levels of VEGF have also been found in CSF and serum of AD patients relative to controls56,57. LPS-activated microglia from SD rats have been shown to upregulate VEGF-A expression, increase angiogenesis and promote the migration and proliferation of retinal microvascular endothelial cells while impairing tight junction protein expression58. In the context of prolonged inflammation, weakening of the BBB and increasing proliferating cells into the brain that respond to inflammatory stimuli could allow for increased microglial inflammatory activation. In this report, we further show that cromolyn and F-cromolyn promote suppression of TNF-α induced secretion of the three pro-inflammatory mediators IL-2, GRO-α, Eotaxin, and VEGF-A, and are able to increase the expression of anti-inflammatory IL-4. This finding indicates that cromolyn and F-cromolyn are able to modulate key mediators of neuroinflammation in HMC3 microglia that are related to downstream neurodegenerative disease progression.
Unbiased proteomic profiling of HMC3 microglia found that cromolyn and F-cromolyn also modulate other inflammatory proteins. Proteolipid protein 1 (PLP1) is the major form of myelin protein in the CNS with roles in myelin sheath structure, oligodendrocyte proliferation, and axonal survival59. Plp1tg mouse models exhibited heightened levels of PLP1 and significant microglial inflammation activation throughout brain parenchyma before myelinated fibers were present, leading to 24-fold TNF-α and 7-fold IL-6 increases compared to wildtype mice60. Therefore, decreases in PLP1 gene expression due to cromolyn (-14.62 fold change decrease after TNF-α exposure, Supplementary Figure S2a) may be responsible, in part, for its anti-inflammatory capabilities. We also found that cromolyn and F-cromolyn decrease Proline, Glutamate And Leucine Rich Protein 1 (PELP1) secretion following TNF-α administration. PELP1 is widely expressed in the brain, particularly in the cell membrane, dendritic shafts and synaptic terminals of neurons in the forebrain61. PELP1 has been associated with pro-survival mechanisms after ischemic injury by activating ERK and Akt pathways, as well as E2 inhibition of GSK3β, a key mediator of cell death in AD61. PELP1 can promote NF-κB gene expression to activate macrophages62. PELP1 knockdown in medulloblastoma cells results in downregulation of NF-κB, including TRAF1 and IL-8, as well as expression ECM-related genes MMP7, MMP9, and MMP1463, the latter of which is upregulated by microglia in late-stage NDD development64. Interestingly, PELP1 is a substrate of GSK3β, interacts with PI3K, and its expression may be determined by the E2-ER-GSK3β signaling loop65. Dose-dependent decreases in PELP1 secretion by both cromolyn and F-cromolyn may contribute to the ameliorating anti-inflammatory activation of HMC3 microglia observed in our experiments through modulation of downstream PI3K, GSK3β, and NF-κB signaling pathways.
The heat shock protein HSP90 is a diverse chaperone protein with regulatory roles in GSK-3β stabilization and tau phosphorylation66; as well as downstream phosphorylation of Akt and PI3K/Akt/mTOR signaling67,68. HSP90 is also associated with pro-inflammatory release of cytokines such as IL-6, IL-1β, and TNF-α, and the inhibition of the constitutive cytoplasmic HSP90β isoform69 decreases pro-inflammatory cytokine signaling, ERK phosphorylation, and STAT3 in the cytosol in N9 microglia70. Studies with TNFΔARE mice with high circulating levels of TNF-α have shown that inhibition of the C-terminal ATPase of HSP90 with novobiocin prevents Akt stabilization, increasing its degradation, and subsequent decrease in production of TNF-α71. Others found that HSP90β inhibition by novobiocin in the Hs578T cell line lowers soluble and insoluble fibronectin levels suggesting that, in addition to HSP90β binding directly to fibronectin, HSP90β is also involved in ECM assembly72. Interestingly, affinity chromatography was previously utilized to find that cromolyn binds to the N-terminus of wildtype HSP90 to inhibit its N-terminal chaperone capabilities, but not to its C-terminus73. In our experiments, treatment with cromolyn and F-cromolyn results in significant decrease in HSP90 after addition of TNF-α. The modulation of HSP90 secretion by HMC3 microglia may be partly responsible for the upstream anti-inflammatory capabilities of cromolyn and F-cromolyn, in addition to downstream PI3K/Akt/mTOR and neurodegenerative GSK-3β signaling.
Collagens are the main structural proteins of the extracellular matrix and there are many isoforms located within the body. Expression of collagen IV, a major component of the basal laminae, and fibronectin was found to be increased in frontal and temporal cortex cerebral microvessels in early Alzheimer’s Disease and was positively correlated with amyloid deposition compared to controls26. Collagen XVIII associates with vascular deposits of amyloid beta and senile plaques, but not tau, and is deposited at higher rates in the AD brain compared to age-matched controls74. Interestingly, the C-terminal fragment of collagen XVIII is endostatin, an anti-angiogenic protein75. Endostatin has strong associations with neurological disease and can dose-dependently bind and sequester nerve growth factor (NGF) to prevent neurite outgrowth and migration in PC12 cells75. Increased collagen XVIII secretion has also been associated with increased VEGF expression in experiments with hepatocellular carcinoma76. We also observe increased concomitant secretion of collagen XVIII and VEGF (Fig. 2c and Fig. 3c, respectively), but also subsequent decreases in both proteins after cromolyn and F-cromolyn treatment.
Collagen production and fibrosis progression are intimately linked and PI3K/Akt signaling has been associated with both77–79. COL6A3 has been implicated as the main isoform of collagen VI that promotes downstream pro-fibrotic regulation of myofibroblasts80. COL6A3 depletion results in reduced migratory ability of myofibroblasts and reduced recruitment of THP-1 myeloid cells to myofibroblast-conditioned media80. We observed significant downregulation of COL6A3 gene expression by TNF-α induced HMC3 microglia by 30 µM cromolyn and 30 µM F-cromolyn (-2.46 and − 2.03 fold change from TNF-α treatment alone, respectively) (Supplementary Figure S2a and S3a). Additionally, gene expression of other collagens significantly decreased by cromolyn after TNF-α administration include COL6A3, COL1A2, COL9A3, and COL8A1 (Supplementary Figure S2a). F-cromolyn also significantly reduced gene expression of COL15A1, COL6A3, COL7A1, COL9A3, COL16A1, and COL8A1 relative to treatment with TNF-α alone (Supplementary Figure S3a). Chronic immobilization stress of rats found extensive remodeling of the hippocampus with increased ECM production and reduced ECM degradation, linking the increased capacity of collagen synthesis with chronic inflammation and immunological dysregulation of the hippocampus81. It is possible that downregulation of collagen gene expression and secretion by cromolyn and F-cromolyn in HMC3 microglia in the presence of pro-inflammatory TNF-α affects Akt/PI3K signaling and may attenuate microglia-mediated fibrotic mechanisms initiated during chronic neuroinflammation.
Fibronectin is another essential glycoprotein component of the extracellular matrix in all tissues of the body and its deposition is upregulated during fibrosis82. Fibronectin functions in a regulatory role for healthy deposition of ECM as use as a structural scaffold, in addition to modulating cell function during tissue repair82. This function continues into the CNS, as fibronectin binds collagens, proteoglycans, and other ECM proteins to reinforce the endothelial basement membrane of the BBB83. Aberrant amounts of aggregate fibronectin deposition, as seen with multiple sclerosis (MS), lead to abnormal ECM structure and impaired remyelination84. Normally these aggregates activate cellular repair mechanisms that then activate microglia and macrophages to a phagocytic phenotype, but in the presence of IFN-γ it was shown to perpetuate inflammation, particularly by increased macrophage and microglia integrin-dependent production of nitric oxides84. Interestingly, cromolyn has been previously shown to inhibit mast cell-derived histamine to decrease hepatic fibrosis in Mdr2−/− mice85. Inhibition of mast cell-derived histamine with administration of cromolyn decreases hepatic fibrosis in Mdr2−/− mice85. Mdr2−/− mice have significantly higher expression of fibronectin, collagen type-1a, and α-SMA than wildtype mice and cromolyn administration significantly reduced expression of all three85. Further experiments were pursued with cholestatic rats and cromolyn decreased expression of collagen type-1a, fibronectin, and α-SMA in cultured HSCs treated with mast cell supernatants from BDL rats86. We observe in our study that TNF-α induced HMC3 microglia secretion of fibronectin was significantly abated by F-cromolyn treatment, suggesting that inflammation-induced expression of fibronectin and potential abnormalities in ECM structure may be stemmed upstream by F-cromolyn.
Epithelial cytoskeletal proteins such as actin and keratin allow for directed integrin signaling that results in fibronectin ECM assembly87, with integrins serving as nucleation points for the cytoskeleton of epithelial cells and ECM proteins of the basement membrane88. Intriguingly, keratins 5 and 8 are known to be expressed in the choroid plexus, the region of the brain that produces CSF, forms the CSF-blood barrier, and the only region of the brain with significant amounts of epithelial cells89. Keratin-1 (KRT1) is found to be excreted in extracellular vesicles by BV2 microglia90. It currently remains unclear to what extent these keratins play in the CNS or in neurodegenerative disease, but keratin-9 (KRT9) has been proposed to be a diagnostic biomarker of AD as it has been found exclusively in AD patients’ CSF compared to controls91. In our experiments, it is interesting that control HMC3 microglia passively secrete KRT1, KRT5, and KRT9 and that keratin secretion is not significantly affected by TNF-α-induced inflammation (Fig. 2c). We observe that only 3 µM cromolyn treatment significantly decreases secretion of each keratin below their respective control levels (Fig. 2c) but further studies will be necessary to more fully understand the impact secreted keratin proteins have within the CNS.
Tenascin-c (TNC) is an extracellular matrix glycoprotein that promotes tissue fibrosis in many disease models including liver, lung, MS, and systemic sclerosis; and has strong associations with other inflammatory diseases including cancer, asthma, and Alzheimer’s disease92. TNC is readily expressed after injury or during inflammation and is important for efficient tissue repair, but has also been associated with pathologic Aβ plaque deposition with concomitant reactive glia93. HDAC1 expression is induced by TNC to promote IL-6 and TNF-α secretion by microglia; these effects were strongly associated with TLR4 signaling, with additional effects on microglia phagocytosis and migration94. TNC activation of TLR4 also promotes collagen synthesis and fibroblast activation and is strongly associated with the pathogenesis of systemic sclerosis in mice33. In our experiments, TNC secretion was modestly reduced by cromolyn and significantly decreased by F-cromolyn treatment after TNF-α administration to HMC3 microglia. In the context of activation of microglia, others have shown that pretreatment of cromolyn before induced degranulation of brain mast cells reduced TLR4 signaling by microglia and subsequent inhibition of MAPK and Akt inflammatory pathways in Sprague Dawley rats95. As TNF-α is able to induce expression of TNC in HepG2 hepatoma cells96, it is possible that the reduction of TNF-α-induced TNC secretion by F-cromolyn may also reduce microglia TLR4 and Akt signaling to prevent downstream inflammatory cytokine production and fibrosis-related signaling.
Tyrosination of microtubules is essential for normal brain development and is readily found in the dendrites and growth cones of neurons97,98. α-tubulin in microtubules undergoes a tyrosine removal and addition cycle, the latter of which is catalyzed by tubulin-tyrosine ligase (TTL)99. Studies with TTL-null mice found that TTL was essential to maintain timely neurite outgrowth and prevent early axon differentiation, emphasizing that TTL is important for the formal structure and differentiation of microtubules in neurons99. This stabilization during microtubule development by TTL may also be important for axon repair post injury, as TTL regulation of tyrosinated α-tubulin has been associated with maintenance of injury signals necessary to activate axon regeneration100. Cromolyn and F-cromolyn both induce secretion of TTL by TNF-α activated microglia in our experiments, a factor that may contribute to the stabilization of axonal repair mechanisms during injury and subsequent regeneration of neurites.
Cold shock domain-containing E1 (CSDE1) is an RNA-binding protein that can directly interrupt transcription and translation of proteins and has been shown to prevent neurogenesis in human embryonic stem cells and iPSCs, with dozens of known neurogenesis-related transcripts significantly altered101. Gene variants of CSDE1 with heightened number of binding targets, particularly with FMRP, have been associated with autism spectrum disorder102. Interestingly, knockdown of CSDE1 in primary mouse cortical neurons resulted in increased neurite and axon length but with fewer and thinner dendritic spines compared to controls, suggesting that CSDE1 is important for successful, morphological outgrowth of neurons102. CSDE1 has also been found essential to the formation of stress granules in HeLa cells103. Stress granules form within many cell types during extended periods of cellular stress, including oxidative stress, heat shock, TNF-α exposure, and aging, and have been postulated to function as scaffolds for aggregation-prone proteins in Alzheimer’s disease and other neurodegenerative disorders104,105. Our results confirm that TNF-α administration strongly induces CSDE1 secretion in HMC3 microglia (Fig. 4d). We interpret the significant decreases in CSDE1 secretion following cromolyn and F-cromolyn treatment as having potential to reduce damaging cellular stress responses that may lead to downstream neurodegeneration, as well as to promote neurite outgrowth as observed in coordination with NGF in our PC12 cellular experiments.
Prospero homeobox protein 1 (PROX1) activates NFAT signaling pathway to promote IL-2 transcription in T cells and is associated with terminal differentiation of neural progenitor cells (NPCs) into mature neurons by inhibiting Notch 1 signaling106. This pathway is associated with increases in inflammatory gene expression via NF-κB, thereby increasing expression of pro-inflammatory cytokines IL-1β, TNF-α and toll-like receptor agonists107. Interestingly, oligodendrocyte progenitor NG2 + cells proliferation and differentiation is PROX1-dependent, with PROX1 playing a protective role against destructive glia response of macrophages by upregulating NG2 + cells during the regenerative response to demyelination108. PROX1 also plays an important role in suppressing Ca2+ signaling and subsequent neurite outgrowth in mouse and human neuroblastoma cell lines, suggesting that transient PROX1 expression allows newly-formed neurons time to migrate into place before extension of neurites and axons109. Although cromolyn and F-cromolyn significantly increase the expression of PROX1 in TNF-α induced HMC3 microglia, we do not observe deleterious effects by cromolyn or F-cromolyn in terms of neurite length of PC12 neuronal cells in our experiments. It is possible that cromolyn and F-cromolyn may reduce inflammation by increasing microglial expression of PROX1 to enhance NG2 + cell proliferation in vivo to help curb microglia-related neurodegeneration108, but further experiments will be required to confirm if this is an operational mechanism of cromolyn and F-cromolyn.
Ras-related protein 35 (Rab35) is a GTPase activated upstream of PI3K-Akt signaling110 that regulates neurite outgrowth in response to NGF by recruiting and complexing with MICAL-L1 and centaurin-β2/ACAP2 to form a site on Arf6 + endosomes for EHD1 association111. It was later found that Rab35 serves as a master regulator of other downstream Rab proteins to bind with MICAL-L1 to modulate neurite outgrowth after NGF administration to PC12 cells112. Experiments with neuronal CAD cells show Rab35 is necessary for the formation of tunneling nanotubes113, protruding F-actin and tubulin cellular structures involved in vesicle trafficking and cell-to-cell communication, and macrophage inflammatory activation by LPS/IFN-γ can impair their formation114. Rab35 plays multiple roles in vesicle trafficking that modulate the function and growth of neuronal protrusions and cromolyn significantly increases Rab35 secretion by TNF-α-induced HMC3 microglia. In conjunction with our neurite outgrowth experiments with PC12 cells, it is possible that the increased neurite length observed with cromolyn treatment beyond NGF treatment alone could be related to cromolyn promotion of Rab35 upstream of PI3K/Akt signaling.
Encouraging the phagocytic potential of microglia is crucial to maintaining anti-inflammatory clearance of damage associated molecular patterns, such as cellular debris of dead or dying cells, misfolded or aggregate proteins, and reduction of cell surface marker expression that signal inflammation from the local and peripheral environment115. In Alzheimer’s disease, microglia colocalize with amyloid plaques primarily consisting of amyloid β-protein, a key activator of the inflammatory microglia phenotype, that is recognized by microglia via Fc receptors, Toll-like receptors, CD36, RAGE and scavenger receptors116. Amyloid plaques are known neurotoxic entities of AD. Amyloid beta is associated with increased reactive oxygen species (ROS) production117 and its aggregate states pair with synaptoxicity and neuronal loss of function with underlying inflammation118. The ability of CNS microglia to identify and remove aggregated Aβ is a necessary component of an effective treatment strategy for AD. Cromolyn has been previously shown to upregulate the phagocytosis of Aβ42 in BV2 microglial cells37. We found in the present study that the ability of cromolyn and F-cromolyn to promote iPSC-derived microglia to phagocytize Aβ42 is upregulated relative to controls, further indicating that the cromolyn platform is able to influence microglia towards an anti-inflammatory phenotype. Figure 6 summarizes the proposed waypoints along the path to Alzheimer’s disease that cromolyn and F-cromolyn are able to measurably affect.