Sporadic Alzheimer's disease (AD) is characterised by the deposition and accumulation of specific protein aggregates. Failure of clearance could underlie this process, and recent genetic association studies point towards involvement of the phagocytosis and autophagy pathways. We developed a real-time tri-color flow cytometry method to quantitate the phagocytic function of human peripheral blood monocyte subsets including non-classic CD14(dim)CD16(+), intermediate CD14(+)CD16(+) and classic CD14(+)CD16(-) monocytes. Using this method, we have measured the phagocytic ability of fresh monocytes in a study of preclinical, prodromal and clinical AD, matched with cognitively normal healthy control subjects. Basal levels of phagocytosis in all three subsets of monocytes were similar between healthy controls and AD patients, while a significant increase of basal phagocytosis was found in subjects with high Aβ-amyloid burden as assessed by PET scans. Pre-treating cells with Copaxone (CPX, to stimulate phagocytosis) or ATP (an inhibitor of P2X7-mediated phagocytosis) showed a differential response depending on clinical or Aβ-burden status, indicating a relative functional deficit. Overall the results are consistent with a perturbation of basal and stimulated innate phagocytosis in sporadic AD.

Multiple sclerosis (MS) is a chronic relapsing-remitting inflammatory disease of the central nervous system characterized by oligodendrocyte damage, demyelination and neuronal death. Genetic association studies have shown a 2-fold or greater prevalence of the HLA-DRB1*1501 allele in the MS population compared with normal Caucasians. In discovery cohorts of Australasian patients with MS (total 2941 patients and 3008 controls), we examined the associations of 12 functional polymorphisms of P2X7, a microglial/macrophage receptor with proinflammatory effects when activated by extracellular adenosine triphosphate (ATP). In discovery cohorts, rs28360457, coding for Arg307Gln was associated with MS and combined analysis showed a 2-fold lower minor allele frequency compared with controls (1.11% for MS and 2.15% for controls, P = 0.0000071). Replication analysis of four independent European MS case-control cohorts (total 2140 cases and 2634 controls) confirmed this association [odds ratio (OR) = 0.69, P = 0.026]. A meta-analysis of all Australasian and European cohorts indicated that Arg307Gln confers a 1.8-fold protective effect on MS risk (OR = 0.57, P = 0.0000024). Fresh human monocytes heterozygous for Arg307Gln have >85% loss of 'pore' function of the P2X7 receptor measured by ATP-induced ethidium uptake. Analysis shows Arg307Gln always occurred with 270His suggesting a single 307Gln-270His haplotype that confers dominant negative effects on P2X7 function and protection against MS. Modeling based on the homologous zP2X4 receptor showed Arg307 is located in a region rich in basic residues located only 12 Å from the ligand binding site. Our data show the protective effect against MS of a rare genetic variant of P2RX7 with heterozygotes showing near absent proinflammatory 'pore' function.

Genetic variants in the purinergic receptors P2RX4 and P2RX7 have been shown to affect susceptibility to multiple sclerosis (MS). In this study we set out to evaluate whether rare coding variants of major effect could also be identified in these purinergic receptors. Sequencing analysis of P2RX4 and P2RX7 in 193 MS patients and 100 controls led to the identification of a rare three variant haplotype (P2RX7 rs140915863:C>T (p.T205M), P2RX7 rs201921967:A>G (p.N361S) and P2RX4 rs765866317:G>A (p.G135S)) segregating with disease in a multi-incident family with six family members diagnosed with MS (LOD=3.07). Functional analysis of this haplotype in HEK293 cells revealed impaired P2X7 surface expression (p<0.01), resulting in over 95% inhibition of ATP-induced pore function (p<0.001) and a marked reduction in phagocytic ability (p<0.05). In addition, transfected cells showed 40% increased peak ATP-induced inward current (p<0.01), and a greater Ca 2+ response to the P2X4 135S variant compared to wild type (p<0.0001). Our study nominates rare genetic variants in P2RX4 and P2RX7 as major genetic contributors to disease, further supporting a role for these purinergic receptors in MS and suggesting the disruption of transmembrane cation channels leading to impairment of phagocytosis as the pathological mechanisms of disease.

Age-related macular degeneration (AMD) is characterized by the accumulation of debris in the posterior eye. In this study we evaluated peripheral blood monocyte phagocytic function at various stages of AMD and in aged matched control participants. Real-time tri-color flow cytometry was used to quantify phagocytic function of peripheral blood monocyte subsets (non-classic, intermediate and classic) isolated from subjects with intermediate or late AMD and compared with age matched healthy controls. Assessment of phagocytic function of monocytes isolated from those with and without reticular pseudodrusen was also made, and the effect of glatiramer acetate on phagocytic function assessed. Phagocytic function was reduced in all subjects with AMD, irrespective of stage of disease. However, there was no correlation between phagocytic function and drusen load, nor any difference between the level of phagocytosis in those with or without reticular pseudodrusen. Treatment with glatiramer acetate increased phagocytosis of classical and non-classical monocytes, normalizing the reduction in phagocytosis observed in those with AMD. These findings suggest that defective systemic phagocytosis is associated with both intermediate and late stages of AMD, highlighting a potential role in the accumulation of debris that occurs early in the disease process. Assessing peripheral monocyte phagocytic function provides further insights into the etiology of this disease and offer a novel therapeutic target.

Activation of P2X7 receptors is widely recognised to initiate proinflammatory responses. However P2X7 also has a dual function as a scavenger receptor which is active in the absence of ATP and plasma proteins and may be important in central nervous system (CNS) diseases. Here, we investigated both P2X7 pore formation and its phagocytic function in fresh human monocytes (as a model of microglia) by measuring ATP-induced ethidium dye uptake and fluorescent bead uptake respectively. This was studied in monocytes expressing various polymorphic variants as well as in the presence of different P2X7 antagonists and ionic media. P2X7-mediated phagocytosis was found to account for about half of Latrunculin (or Cytochalasin D)-sensitive bead engulfment by fresh human monocytes. Monocytes harbouring P2X7 Ala348Thr or Glu496Ala polymorphic variants showed increase or loss of ethidium uptake respectively, but these changes in pore formation did not always correspond to the changes in phagocytosis of YG beads. Unlike pore function, P2X7-mediated phagocytosis was not affected by three potent selective P2X7 antagonists and remained identical in Na and K media. Taken together, our results show that P2X7 is a scavenger receptor with important function in the CNS but its phagocytic function has features distinct from its pore function. Both P2X7 pore formation and P2X7-mediated phagocytosis should be considered in the design of new P2X7 antagonists for the treatment of CNS diseases.

Background: 7, 8-Dihydroxyflavone (7, 8-DHF) has recently been identified as a high affinity tropomyosin receptor kinase B (TrkB) agonist, which mimic BDNF's neurotrophic signaling and overcome the pharmacokinetic and side effect barriers. Our recent work suggest that 7, 8-DHF is able to bind and activate TrkB, and rescue BDNF deficits in Alzheimer's disease (AD), the most common form of dementia in elderly adults, and Fragile X syndrome (FXS), the most common form of inherited intellectual disability. Methods: We synthesized pharmacologic, behavioral, and biochemical approaches to examine the effects of 7,8-DHF on spatial and fear memory functions, and morphological spine abnormalities in fragile X mental retardation 1 (Fmr1) gene knock-out mice and Tg2576 AD mice. Results: We found a large positive effect from 7, 8-DHF on learning and memory in fragile X mental retardation 1 (Fmr1) gene knock-out mice and the Tg2576 AD mouse model. A long term treatment with 7, 8-DHF improves spatial and fear learning and memory that depend on hippocampus and amygdala. 7, 8-DHF also ameliorates morphological spine abnormalities. Further mechanism analysis reveals that 7, 8-DHF increases the phosphorylation of TrkB and enhances synapse expression of GluA1 of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. Potentially related to drug-induced changes in AMPA receptor subunits, 7, 8-DHF at the synapses leads to the phosphorylation of (i) specific serine sites including Ser818 and Ser813 of GluA1, and Ser880 of GluA2, and (ii) calcium/calmodulin-dependent protein kinase II and protein kinase C, two kinases that target these sites. 7, 8-DHF activated the phosphorylation of TrkB receptors and its downstream signals including CaMKII, Akt, Erk1/2, and cAMP-response elementbinding protein. Conclusions: Collectively, our results demonstrated that 7, 8-DHF acted on TrkB and resolved learning and memory impairments in Fmr1 gene knock-out mice and amyloid precursor protein transgenic mice partially through improving synaptic structure and enhanced synaptic AMPARs. Thus our works provide a potential therapeutic tool for FXS and AD treatment. Background: PACAP is reduced the brains and cerebrospinal fluids of Alzheimer's disease (AD) patients (Han et al 2014 Neurology. 82(19):1724-8; Han et al 2015 JAMA Neurol. 72(3):333-9). PACAP reduced Ab-induced toxicity in primary cultured mouse neurons and the neuroprotective effects could be attributed to the mitochondrial function enhancement (Han et al 2014 Neurobiol Aging. 35 (9):2064-71). Furthermore in mouse brain, PACAP enhanced the production of sAPPa while decreased Ab species, suggesting a shift of the APP metabolism from beta-secretase predominant pathway to alpha-secretase predominant pathway (Rat et al 2011.FASEB J.25 (9):3208-18.). However, the effect of PACAP on beta-secretase activity was not measured; the effects of PACAP on other key factors in AD pathogenesis had never been elucidated. In this study, we tested