In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1^GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.     

Enhancing survival of photoreceptor cells in vivo using the synthetic progestin Norgestrel

Retinal degenerations such as Retinitis Pigmentosa remain difficult to treat given the diverse array of genes responsible for their aetiology. Rather than concentrate on specific genes, our focus is on identifying therapeutic avenues for the treatment of retinal disease that target general survival mechanisms or pathways. Norgestrel is a synthetic progestin commonly used in hormonal contraception. Here, we report a novel anti-apoptotic role for Norgestrel in diseased mouse retinas in vivo. Dosing with Norgestrel protects photoreceptor cells from undergoing apoptosis in two distinct models of retinal degeneration; the light damage model and the Pde6b(rd10) model. Photoreceptor rescue was assessed by analysis of cell number, structural integrity and function. Improvements in cell survival of up to 70% were achieved in both disease models, indicating that apoptosis had been halted or at least delayed. A speculative mechanism of action for Norgestrel involves activation of survival pathways in the retina. Indeed, Norgestrel increases the expression of basic fibroblast growth factor which is known to both promote cell survival and inhibit apoptosis. In summary, our results demonstrate significant protection of photoreceptor cells which may be attributed to Norgestrel mediated activation of endogenous survival pathways within the retina.     

Microglial Activation Promotes Cell Survival in Organotypic Cultures of Postnatal Mouse Retinal Explants

The role of microglia during neurodegeneration remains controversial. We investigated whether microglial cells have a neurotoxic or neuroprotective function in the retina. Retinal explants from 10-day-old mice were treated in vitro with minocycline to inhibit microglial activation, with LPS to increase microglial activation, or with liposomes loaded with clodronate (Lip-Clo) to deplete microglial cells. Flow cytometry was used to assess the viability of retinal cells in the explants and the TUNEL method to show the distribution of dead cells. The immunophenotypic and morphological features of microglia and their distribution were analyzed with flow cytometry and immunocytochemistry. Treatment of retinal explants with minocycline reduced microglial activation and simultaneously significantly decreased cell viability and increased the presence of TUNEL-labeled cell profiles. This treatment also prevented the migration of microglial cells towards the outer nuclear layer, where cell death was most abundant. The LPS treatment increased microglial activation but had no effect on cell viability or microglial distribution. Finally, partial microglial removal with Lip-Clo diminished the cell viability in the retinal explants, showing a similar effect to that of minocycline. Hence, cell viability is diminished in retinal explants cultured in vitro when microglial cells are removed or their activation is inhibited, indicating a neurotrophic role for microglia in this system.     

A Spirulina-Enhanced Diet Provides Neuroprotection in an α-Synuclein Model of Parkinson's Disease

Inflammation in the brain plays a major role in neurodegenerative diseases. In particular, microglial cell activation is believed to be associated with the pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD). An increase in microglia activation has been shown in the substantia nigra pars compacta (SNpc) of PD models when there has been a decrease in tyrosine hydroxylase (TH) positive cells. This may be a sign of neurotoxicity due to prolonged activation of microglia in both early and late stages of disease progression. Natural products, such as spirulina, derived from blue green algae, are believed to help reverse this effect due to its anti-inflammatory/anti-oxidant properties. An adeno-associated virus vector (AAV9) for α-synuclein was injected in the substantia nigra of rats to model Parkinson''s disease and to study the effects of spirulina on the inflammatory response. One month prior to surgeries, rats were fed either a diet enhanced with spirulina or a control diet. Immunohistochemistry was analyzed with unbiased stereological methods to quantify lesion size and microglial activation. As hypothesized, spirulina was neuroprotective in this α-synuclein model of PD as more TH+ and NeuN+ cells were observed; spirulina concomitantly decreased the numbers of activated microglial cells as determined by MHCII expression. This decrease in microglia activation may have been due, in part, to the effect of spirulina to increase expression of the fractalkine receptor (CX3CR1) on microglia. With this study we hypothesize that α-synuclein neurotoxicity is mediated, at least in part, via an interaction with microglia. We observed a decrease in activated microglia in the rats that received a spirulina- enhanced diet concomitant to neuroprotection. The increase in CX3CR1 in the groups that received spirulina, suggests a potential mechanism of action.     

An open-source pipeline for analysing changes in microglial morphology

Changes in microglial morphology are powerful indicators of the inflammatory state of the brain. Here, we provide an open-source microglia morphology analysis pipeline that first cleans and registers images of microglia, before extracting 62 parameters describing microglial morphology. It then compares control and ‘inflammation’ training data and uses dimensionality reduction to generate a single metric of morphological change (an ‘inflammation index’). This index can then be calculated for test data to assess inflammation, as we demonstrate by investigating the effect of short-term high-fat diet consumption in heterozygous Cx3CR1-GFP mice, finding no significant effects of diet. Our pipeline represents the first open-source microglia morphology pipeline combining semi-automated image processing and dimensionality reduction. It uses free software (ImageJ and R) and can be applied to a wide variety of experimental paradigms. We anticipate it will enable others to more easily take advantage of the powerful insights microglial morphology analysis provides.     

Neuron–glia crosstalk in health and disease: fractalkine and CX3CR1 take centre stage

An essential aspect of normal brain function is the bidirectional interaction and communication between neurons and neighbouring glial cells. To this end, the brain has evolved ligand–receptor partnerships that facilitate crosstalk between different cell types. The chemokine, fractalkine (FKN), is expressed on neuronal cells, and its receptor, CX₃CR1, is predominantly expressed on microglia. This review focuses on several important functional roles for FKN/CX₃CR1 in both health and disease of the central nervous system. It has been posited that FKN is involved in microglial infiltration of the brain during development. Microglia, in turn, are implicated in the developmental synaptic pruning that occurs during brain maturation. The abundance of FKN on mature hippocampal neurons suggests a homeostatic non-inflammatory role in mechanisms of learning and memory. There is substantial evidence describing a role for FKN in hippocampal synaptic plasticity. FKN, on the one hand, appears to prevent excess microglial activation in the absence of injury while promoting activation of microglia and astrocytes during inflammatory episodes. Thus, FKN appears to be neuroprotective in some settings, whereas it contributes to neuronal damage in others. Many progressive neuroinflammatory disorders that are associated with increased microglial activation, such as Alzheimer''s disease, show disruption of the FKN/CX₃CR1 communication system. Thus, targeting CX₃CR1 receptor hyperactivation with specific antagonists in such neuroinflammatory conditions may eventually lead to novel neurotherapeutics.     

Inosine diphosphatase as a histochemical marker of retinal microvasculature,with special reference to transformation of microglia

Summary Nucleoside diphosphatase (IDPase), localized using inosine diphosphate as substrate, allows the selective staining of blood vessels and cells of vascular origin, such as macrophages and microglia, whereas the neuroglial, the neuronal and the pigment epithelial cells remain unstained. The staining pattern observed in the retina of mouse, rat, cat and monkey are similar; some apparent quantitative differences reflect species differences in the distribution of retinal microvasculature. At the electron-microscopic level, most of the enzyme activity in the blood vessels appears to be located along the outer wall. The cell membrane, parts of the smooth endoplasmic reticulum and the nuclear membrane in the microglial perikarya appear positive; profiles of microglial processes are intensely stained.In the developing eyes of rats and mice, the blood vessels are stainable from the earliest stage of their appearance. An array of amoeboid cells precede the growing blood vessels and spread out over the future vascularized part of the retina. These cells eventually develop characteristic microglial features, and extend many elongated and branched processes between the neuroepithelial cells while remaining in contact with, or in close proximity to, the blood vessels. Intense IDPase activity in the microglial cells, in contrast to the absence of the enzyme in the neuroglial Müller cells, suggests that microglia are involved in phosphate metabolism and indicates functional compartmentalization within the glial tissue lying between the blood retinal barrier and the retinal neurons.     

Crocin,a plant-derived carotenoid,modulates microglial reactivity

Microglia activation plays an important role in immune responses in the CNS including the retina. Crocin, a plant-derived carotenoid, has been reported to possess anti-inflammatory, anti-apoptotic and anti-oxidative capacity in models of retinal damage and degeneration. If these neuroprotective effects could be mediated by direct modulation of microglial cells is unclear. Here, we examined the direct effects of crocin on key functions and pro-inflammatory gene expression in lipopolysaccharide (LPS)-activated BV-2 microglia. We found that crocin stimulation strongly promoted filopodia formation and markedly increased microglial phagocytosis, two important parameters relevant for physiological microglia functions. Moreover, crocin significantly reduced gene expression of the pro-inflammatory markers IL6, CCL2, and iNOS in LPS-challenged BV-2 cells and potently blocked NO production in these microglia. The observed immunomodulatory effects of crocin were not mediated by general inhibition of NFkB nuclear translocation. Our findings indicate that many of the anti-inflammatory effects of crocin demonstrated in animal models of neuronal degeneration could be mediated by its direct effects on microglia homeostasis.     

Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

VDAC1 regulates neuronal cell loss after retinal trauma injury by a mitochondria-independent pathway

The voltage-dependent anion channel 1 (VDAC1) was first described as a mitochondrial porin that mediates the flux of metabolites and ions, thereby integrating both cell survival and death signals. In the nervous system, the functional roles of VDAC1 remain poorly understood. Herein, the rat retina was employed to study VDAC1. First, it was observed that even subtle changes in VDAC1 levels affect neuronal survival, inducing severe alterations in the retinal morphology. We next examined the regulation of VDAC1 after traumatic retinal injury. After mechanical trauma, SOD1 translocates towards the nucleus, which is insufficient to contain the consequences of oxidative stress, as determined by the evaluation of protein carbonylation. Using in vitro models of oxidative stress and mechanical injury in primary retinal cell cultures, it was possible to determine that inhibition of VDAC1 oligomerization by 4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) rescues cell viability, impacting microglial cell activation. We next focused on the regulation of VDAC1 after retinal mechanical injury. VDAC1 was promptly upregulated 2 h after lesion in the plasma membrane and endoplasmic reticulum rather than in the mitochondria, and multimers of VDAC1 were assembled after lesion. DIDS intraocular application decreased apoptosis and prevented microglial polarization, which confirmed in vitro observations. Considering the role of microglia in neuroinflammation, multiplex evaluation of cytokines showed that DIDS application disorganized the inflammatory response 2 h after the lesion, matching the fast regulation of VDAC1. Taken together, data disclosed that fine regulation of VDAC1 influences neuronal survival, and pharmacological inhibition after trauma injury has neuroprotective effects. This protection may be attributed to the effects on VDAC1 abnormal accumulation in the plasma membrane, thereby controlling the activation of microglial cells. We concluded that VDAC1 is a putative therapeutic target in neuronal disorders since it integrates both death and survival cellular signaling.Subject terms: Cell death in the nervous system, Cellular neuroscience     

Functional Impairment of Microglia Coincides with Beta-Amyloid Deposition in Mice with Alzheimer-Like Pathology

Microglial cells closely interact with senile plaques in Alzheimer’s disease and acquire the morphological appearance of an activated phenotype. The significance of this microglial phenotype and the impact of microglia for disease progression have remained controversial. To uncover and characterize putative changes in the functionality of microglia during Alzheimer’s disease, we directly assessed microglial behavior in two mouse models of Alzheimer’s disease. Using in vivo two-photon microscopy and acute brain slice preparations, we found that important microglial functions - directed process motility and phagocytic activity - were strongly impaired in mice with Alzheimer’s disease-like pathology compared to age-matched non-transgenic animals. Notably, impairment of microglial function temporally and spatially correlated with Aβ plaque deposition, and phagocytic capacity of microglia could be restored by interventionally decreasing amyloid burden by Aβ vaccination. These data suggest that major microglial functions progressively decline in Alzheimer’s disease with the appearance of Aβ plaques, and that this functional impairment is reversible by lowering Aβ burden, e.g. by means of Aβ vaccination.     

Microglial activation mediates host neuronal survival induced by neural stem cells

The rational of neural stem cells (NSCs) in the therapy of neurological disease is either to replace dead neurons or to improve host neuronal survival, the latter of which has got less attention and the underlying mechanism is as yet little known. Using a transwell co‐culture system, we reported that, in organotypic brain slice cultures, NSCs significantly improved host neuronal viability. Interestingly, this beneficial effect of NSCs was abrogated by a microglial inhibitor minocycline, while it was mimicked by a microglial agonist, Toll‐like receptor 9 (TLR9) ligand CpG‐ODN, which supports the pro‐vital mediation by microglia on this NSCs‐improved neuronal survival. Moreover, we showed that NSCs significantly induced host microglial movement and higher expression of a microglial marker IBA‐1, the latter of which was positively correlated with TLR9 or extracellular‐regulated protein kinases 1/2 (ERK1/2) activation. Real‐time PCR revealed that NSCs inhibited the expression of pro‐inflammatory molecules, but significantly increased the expression of molecules associated with a neuroprotective phenotype such as CX3CR1, triggering receptor expressed on myeloid cells‐2 (TREM2) and insulin growth factor 1 (IGF‐1). Similarly, in the microglia cells, NSCs induced the same microglial response as that in the slices. Further treatment with TLR9 ligand CpG‐ODN, TLR9 inhibitor chloroquine (CQ) or ERK1/2 inhibitor U0126 demonstrated that TLR9‐ERK1/2 pathway was involved in the NSCs‐induced microglial activation. Collectively, this study indicated that NSCs improve host neuronal survival by switching microglia from a detrimental to a neuroprotective phenotype in adult mouse brain, and the microglial TLR9‐ERK1/2 pathway seems to participate in this NSCs‐mediated rescue action.     

CX3CL1 (Fractalkine) Protein Expression in Normal and Degenerating Mouse Retina: In Vivo Studies

We aimed to investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa. Forms of retinal protein CX3CL1, total protein and mRNA levels of CX3CL1 were analyzed at postnatal days (P) 5, 10, 14, 22, 30, 45, and 60 by Western blotting and real-time PCR. Cellular sources of CX3CL1 were investigated by in situ hybridization histochemistry (ISH) and using transgenic (CX3CL1cherry) mice. The immunoblots revealed that in both, wt and rd10 retinas, a membrane integrated ∼100 kDa CX3CL1 form and a cleaved ∼85 kDa CX3CL1 form were present at P5. At P10, accumulation of another presumably intra-neuronal ∼95 kDa form and a decrease in the ∼85-kDa form were observed. From P14, a ∼95 kDa form became principal in wt retina, while in rd10 retinas a soluble ∼85 kDa form increased at P45 and P60. In comparison, retinas of rd10 mice had significantly lower levels of total CX3CL1 protein (from P10 onwards) and lower CX3CL1 mRNA levels (from P14), even before the onset of primary rod degeneration. ISH and mCherry reporter fluorescence showed neurons in the inner retina layers as principal sites of CX3CL1 synthesis both in wt and rd10 retinas. In conclusion, our results demonstrate that CX3CL1 has a distinctive course of expression and functional regulation in rd10 retina starting at P10. The biological activity of CX3CL1 is regulated by conversion of a membrane integrated to a soluble form during neurogenesis and in response to pathologic changes in the adult retinal milieu. Viable mature neurons in the inner retina likely exhibit a dynamic intracellular storage depot of CX3CL1.     

PKG activity causes photoreceptor cell death in two retinitis pigmentosa models

Photoreceptor degeneration in retinitis pigmentosa is one of the leading causes of hereditary blindness in the developed world. Although causative genetic mutations have been elucidated in many cases, the underlying neuronal degeneration mechanisms are still unknown. Here, we show that activation of cGMP-dependent protein kinase (PKG) hallmarks photoreceptor degeneration in rd1 and rd2 human homologous mouse models. When induced in wild-type retinae, PKG activity was both necessary and sufficient to trigger cGMP-mediated photoreceptor cell death. Target-specific, pharmacological inhibition of PKG activity in both rd1 and rd2 retinae strongly reduced photoreceptor cell death in organotypic retinal explants. Likewise, inhibition of PKG in vivo, using three different application paradigms, resulted in robust photoreceptor protection in the rd1 retina. These findings suggest a pivotal role for PKG activity in cGMP-mediated photoreceptor degeneration mechanisms and highlight the importance of PKG as a novel target for the pharmacological intervention in RP.     

Prion protein expression differences in microglia and astroglia influence scrapie-induced neurodegeneration in the retina and brain of transgenic mice

Activated microglia and astroglia are known to be involved in a variety of neurodegenerative diseases, including prion diseases. In the present experiments, we studied activation of astroglia and microglia after intraocular scrapie infection in transgenic mice expressing prion protein (PrP) in multiple cell types (tg7 mice) or in neurons only (tgNSE mice). In this model, scrapie infection and protease-resistant PrP deposition occurs in the retinas of both strains of mice, but retinal degeneration is observed only in tg7 mice. Our results showed that the retinas of tg7 and tgNSE mice both had astroglial activation with increased chemokine expression during the course of infection. However, only tg7 retinas exhibited strong microglial activation compared to tgNSE retinas, which showed little microglial activation by biochemical or morphological criteria. Therefore, microglial PrP expression might be required for scrapie-induced retinal microglial activation and damage. Furthermore, microglial activation preceded retinal neurodegeneration in tg7 mice, suggesting that activated microglia might contribute to the degenerative process, rather than being a response to the damage. Surprisingly, brain differed from retina in that an altered profile of microglial activation markers was upregulated, and the profiles in the two mouse strains were indistinguishable. Microglial activation in the brain was associated with severe brain vacuolation and neurodegeneration, leading to death. Thus, retinal and brain microglia appeared to differ in their requirements for activation, suggesting that different activation pathways occur in the two tissues.     

ADPβS evokes microglia activation in the rabbit retina <Emphasis Type="Italic">in vivo</Emphasis>

To investigate whether stimulation of purinergic P2Y(1) receptors modulates the activation of microglial and Müller glial cells in the rabbit retina in vivo, adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; 2 mM in 100 mul saline), a non-hydrolyzable ADP analogue, was intravitreadly applied into control eyes or onto retinas that were experimentally detached from the pigment epithelium. Both retinal detachment and application of ADPssS onto control retinas induced phenotype alterations of the microglial cells (decrease of soma size, retraction of cell processes) and had no influence on microglial cell density. ADPssS application onto detached retinas accelerated the process retraction and resulted in a strongly decreased density of microglial cells. The effects of ADPssS on microglia density and phenotype in detached retinas were partially reversed by co-application of the selective inhibitor of P2Y(1) receptors, MRS-2317 (3 mM in 100 mul saline). ADPssS apparently did not influence Müller cell gliosis, as determined by electrophysiological and calcium imaging records. It is concluded that rabbit retinal microglial cells express functional P2Y(1) receptors in vivo, and that activation of these receptors stimulates phenotype alterations that are characteristical for microglia activation.     

Microglial MT1 activation inhibits LPS‐induced neuroinflammation via regulation of metabolic reprogramming

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. Although its pathogenesis remains unclear, a number of studies indicate that microglia‐mediated neuroinflammation makes a great contribution to the pathogenesis of PD. Melatonin receptor 1 (MT1) is widely expressed in glia cells and neurons in substantia nigra (SN). Neuronal MT1 is a neuroprotective factor, but it remains largely unknown whether dysfunction of microglial MT1 is involved in the PD pathogenesis. Here, we found that MT1 was reduced in microglia of SN in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced PD mouse model. Microglial MT1 activation dramatically inhibited lipopolysaccharide (LPS)‐induced neuroinflammation, whereas loss of microglial MT1 aggravated it. Metabolic reprogramming of microglia was found to contribute to the anti‐inflammatory effects of MT1 activation. LPS‐induced excessive aerobic glycolysis and impaired oxidative phosphorylation (OXPHOS) could be reversed by microglial MT1 activation. MT1 positively regulated pyruvate dehydrogenase alpha 1 (PDHA1) expression to enhance OXPHOS and suppress aerobic glycolysis. Furthermore, in LPS‐treated microglia, MT1 activation decreased the toxicity of conditioned media to the dopaminergic (DA) cell line MES23.5. Most importantly, the anti‐inflammatory effects of MT1 activation were observed in LPS‐stimulated mouse model. In general, our study demonstrates that MT1 activation inhibits LPS‐induced microglial activation through regulating its metabolic reprogramming, which provides a mechanistic insight for microglial MT1 in anti‐inflammation.     

Pigment Epithelium-Derived Factor (PEDF) Peptide Eye Drops Reduce Inflammation,Cell Death and Vascular Leakage in Diabetic Retinopathy in Ins2Akita Mice

Inflammation, neurodegeneration and microvascular irregularities are included in the spectrum of defects associated with diabetic retinopathy. Here, we evaluated intraocular deliverability features of two pigment epithelium-derived factor (PEDF) derivatives given as eye drops and their efficacy in modulating diabetes-induced retinal complications. The antiangiogenic PEDF60–77 (P60) and neuroprotective PEDF78–121 (P78) derivatives were applied to Ins2^Akita mouse eyes once a week for 15 wks at the onset of hyperglycemia. Peptides, labeled with Alexa Fluor 488, were observed penetrating the cornea by 1–4 h and gained access to the ciliary body, retinal pigment epithelium (RPE)-choroid complex, retina microvasculature and vitreous. Peak vitreous levels were 0.2 μg/mL for P60 and 0.9 μg/mL for P78 after 0.5 and 4 h, respectively. Both peptides reduced vascular leakage by ~60% and increased zona occludens 1 (ZO1) and occludin expression in the microvasculature to nondiabetic levels. P60 induced pERK1/2 and P78 promoted pAKT in Muller glia, two signals that were dampened in diabetic conditions. Pharmacologically inhibiting AKT signaling in the retina blocked effects of the peptides on ZO1 and occludin expression. P78 reduced levels of 9/20 cytokines in diabetic vitreous including interferon (IFN)-γ, interleukin (IL)-6, IL-3 and tumor necrosis factor (TNF)-α. P60 lowered levels of 6/20 cytokines but was less effective than P78. Neuroprotective P78 prevented diabetes-induced microglia activation by ~60%, retinal ganglion cell (RGC) death by ~22% and inner plexiform layer thinning by ~13%. In summary, we provide evidence that PEDF bioactive derivatives gained access to the retina by topical delivery and validated their efficacy in reducing diabetic retinopathy complications. Our findings argue for glia regulation of microvascular leakage and an early root cause for RGC degeneration embedded in microglia activation.     

Phosphoproteomics identifies microglial Siglec‐F inflammatory response during neurodegeneration

Alzheimer’s disease (AD) is characterized by the appearance of amyloid‐β plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Using mass spectrometry, we have quantified the phosphoproteome of the CK‐p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. We identified a shared response involving Siglec‐F which was upregulated on a subset of reactive microglia. The human paralog Siglec‐8 was also upregulated on microglia in AD. Siglec‐F and Siglec‐8 were upregulated following microglial activation with interferon gamma (IFNγ) in BV‐2 cell line and human stem cell‐derived microglia models. Siglec‐F overexpression activates an endocytic and pyroptotic inflammatory response in BV‐2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine‐based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV‐2 cells. Collectively, our results point to an important role for mouse Siglec‐F and human Siglec‐8 in regulating microglial activation during neurodegeneration.