A Comparison of PKD2L1-Expressing Cerebrospinal Fluid Contacting Neurons in Spinal Cords of Rodents, Carnivores, and Primates.

Cerebrospinal fluid contacting neurons (CSF-cNs) are a specific type of neurons located around the ventricles in the brain and the central canal in the spinal cord and have been demonstrated to be intrinsic sensory neurons in the central nervous system. One of the important channels responsible for the sensory function is the polycystic kidney disease 2-like 1 (PKD2L1) channel. Most of the studies concerning the distribution and function of the PKD2L1-expressing CSF-cNs in the spinal cord have previously been performed in non-mammalian vertebrates. In the present study immunohistochemistry was performed to determine the distribution of PKD2L1-immunoreactive (IR) CSF-cNs in the spinal cords of four mammalian species: mouse, rat, cat, and macaque monkey. Here, we found that PKD2L1-expressing CSF-cNs were present at all levels of the spinal cord in these animal species. Although the distribution pattern was similar across these species, differences existed. Mice and rats presented a clear PKD2L1-IR cell body labeling, whereas in cats and macaques the PKD2L1-IR cell bodies were more weakly labeled. Ectopic PKD2L1-IR neurons away from the ependymal layer were observed in all the animal species although the abundance and the detailed locations varied. The apical dendritic protrusions with ciliated fibers were clearly seen in the lumen of the central canal in all the animal species, but the sizes of protrusion bulbs were different among the species. PKD2L1-IR cell bodies/dendrites were co-expressed with doublecortin, MAP2 (microtubule-associated protein 2), and aromatic L-amino acid decarboxylase, but not with NeuN (neuronal nuclear protein), indicating their immature properties and ability to synthesize monoamine transmitters. In addition, in situ hybridization performed in rats revealed PKD2L1 mRNA expression in the cells around the central canal. Our results indicate that the intrinsic sensory neurons are conserved across non-mammalian and mammalian vertebrates. The similar morphology of the dendritic bulbs with ciliated fibers (probably representing stereocilia and kinocilia) protruding into the central canal across different animal species supports the notion that PKD2L1 is a chemo- and mechanical sensory channel that responds to mechanical stimulations and maintains homeostasis of the spinal cord. However, the differences of PKD2L1 distribution and expression between the species suggest that PKD2L1-expressing neurons may receive and process sensory signals differently in different animal species.

Synaptic vesicle glycoprotein 2A (SV2A) levels in the cerebral cortex in patients with Alzheimer's disease: a radioligand binding study in postmortem brains.

Histological and biochemical analyses in postmortem tissues have demonstrated neurodegenerative changes in the cerebral cortex in patients with Alzheimer's disease (AD), and it has been suggested that this represents a loss of synapses. PET imaging of the (pre)synaptic vesicular glycoprotein 2A (SV2A) has demonstrated a reduction in synapse density in AD in the hippocampus but not consistently in the neocortex. This investigation examines the level of [3H]UCB-J binding in postmortem cortical tissue from patients with AD and matched healthy controls using autoradiography. Among the neocortical areas examined, the binding was significantly lower only in the middle frontal gyrus in AD compared to matched controls. No differences were observed in the parietal, temporal, or occipital cortex. The binding levels in the frontal cortex in the AD cohort displayed large variability among subjects, and this revealed a highly significant negative association with the age of the patient. These results demonstrate low UCB-J binding in the frontal cortex of patients with AD, and this biomarker correlates negatively with age, which may further indicate that SV2A could be an important biomarker in AD patients.

Characterization of the Novel P2X7 Receptor Radioligand [3H]JNJ-64413739 in Human Brain Tissue.

Radioligands targeting microglia cells have been developed to identify and determine neuroinflammation in the living brain. One recently discovered ligand is JNJ-64413739 that binds selectively to the purinergic receptor P2X7R. The expression of P2X7R is increased under inflammation; hence, the ligand is considered useful in the detection of neuroinflammation in the brain. [18F]JNJ-64413739 has been evaluated in healthy subjects with positron emission tomography; however, the in vitro binding properties of the ligand in human brain tissue have not been investigated. Therefore, the purpose of this study was to measure Bmax and Kd of [3H]JNJ-64413739 using autoradiography on human cortical tissue sections resected from a total of 48 patients with treatment-resistant epilepsy. Correlations between the specific binding of [3H]JNJ-64413739 with age, sex, and duration of disease were explored. Finally, to examine the relationship between P2X7R and TSPO availability, specific binding of [3H]JNJ-64413739 and [123I]CLINDE was examined in the same tissue. The binding was measured in both cortical gray and subcortical white matter. Saturation revealed a Kd (5 nM) value similar between gray and white matter but a larger Bmax in the white than in the gray matter. The binding was completely displaced by the cold ligand and structurally different P2X7R ligands. The variability in saturable binding among the samples was found to be 38% in gray and white matter but was not correlated to either age, sex, or the duration of the disease. Interestingly, there was no significant correlation between [3H]JNJ-64413739 and [123I]CLINDE binding. These data demonstrate that [3H]JNJ-64413739 is a suitable radioligand for evaluating the distribution and expression of the P2X7R in the human brain.

Ceruloplasmin-deficient mice show changes in PTM profiles of proteins involved in messenger RNA processing and neuronal projections and synaptic processes.

Ceruloplasmin (Cp) is a multicopper oxidase with ferroxidase properties being of importance to the mobilisation and export of iron from cells and its ability to bind copper. In ageing humans, Cp deficiency is known to result in aceruloplasminemia, which among other is characterised by neurological symptoms. To obtain novel information about the functions of Cp in the central nervous system (CNS) we compared the brain proteome in forebrains from asymptomatic 4-6-month-old Cp-deficient (B6N(Cg)-Cptm1b(KOMP)Wtsi /J) and wild-type mice. Of more than 5600 quantified proteins, 23 proteins, were regulated, whereas more than 1200 proteins had regulated post-translational modifications (PTMs). The genes of the regulated proteins, glycoproteins and phosphoproteins appeared mostly to be located to neurons and oligodendrocyte precursor cells. Cp deficiency especially affected the function of proteins involved in the extension of neuronal projections, synaptic signalling and cellular mRNA processing and affected the expression of proteins involved in neurodegenerative disease and diabetes. Iron concentration and transferrin saturation were reduced in the blood of even younger, 3- to 5-month-old, Cp-deficient mice. Iron act as cofactor in many enzymatic processes and reactions. Changes in iron availability and oxidation as consequence of Cp deficiency could therefore affect the synthesis of proteins and lipids. This proteomic characterisation is to our knowledge the first to document the changes taking place in the CNS-proteome and its phosphorylation and glycosylation state in Cp-deficient mice.

Efficacy of Chronic Paroxetine Treatment in Mitigating Amyloid Pathology and Microgliosis in APPSWE/PS1ΔE9 Transgenic Mice.

BACKGROUND: Modulation of serotonergic signaling by treatment with selective serotonin reuptake inhibitors (SSRIs) has been suggested to mitigate amyloid-ß (Aß) pathology in Alzheimer's disease, in addition to exerting an anti-depressant action. OBJECTIVE: To investigate the efficacy of chronic treatment with the SSRI paroxetine, in mitigating Aß pathology and Aß plaque-induced microgliosis in the hippocampus of 18-month-old APPswe/PS1ΔE9 mice. METHODS: Plaque-bearing APPswe/PS1ΔE9 and wildtype mice were treated with paroxetine per os at a dose of 5 mg/kg/day, from 9 to 18 months of age. The per os treatment was monitored by recording of the body weights and serum paroxetine concentrations, and by assessment of the serotonin transporter occupancy by [3H]DASB-binding in wildtype mice. Additionally, 5,7-dihydroxytryptamine was administered to 9-month-old APPswe/PS1ΔE9 mice, to examine the effect of serotonin depletion on Aß pathology. Aß pathology was evaluated by Aß plaque load estimation and the Aß42/Aß40 ratio by ELISA. RESULTS: Paroxetine treatment led to > 80% serotonin transporter occupancy. The treatment increased the body weight of wildtype mice, but not of APPswe/PS1ΔE9 mice. The treatment had no effect on the Aß plaque load (p = 0.39), the number and size of plaques, or the Aß plaque-induced increases in microglial numbers in the dentate gyrus. Three months of serotonin depletion did not significantly impact the Aß plaque load or Aß42/Aß40 ratio in APPswe/PS1ΔE9 mice at 12 months. CONCLUSION: Our results show that chronic treatment with the SSRI paroxetine does not mitigate Aß pathology and Aß plaque-induced microgliosis in the hippocampus of APPswe/PS1ΔE9 mice.

The Combined Effects of Amyloidosis and Serotonin Deficiency by Tryptophan Hydroxylase-2 Knockout Impacts Viability of the APP/PS1 Mouse Model of Alzheimer's Disease.

BACKGROUND: A decline of brain serotonin (5-HT) is held responsible for the changes in mood that can be observed in Alzheimer's disease (AD). However, 5-HT'ergic signaling is also suggested to reduce the production of pathogenic amyloid-ß (Aß). OBJECTIVE: To investigate the effect of targeted inactivation of tryptophan hydroxylase-2 (Tph2), which is essential for neuronal 5-HT synthesis, on amyloidosis in amyloid precursor protein (APP)swe/presenilin 1 (PS1) ΔE9 transgenic mice. METHODS: Triple-transgenic (3xTg) APP/PS1 mice with partial (+/-) or complete Tph2 knockout (-/-) were allowed to survive until 6 months old with APP/PS1, Tph2-/-, and wildtype mice. Survival and weight were recorded. Levels of Aß42/40/38, soluble APPα (sAßPPα) and sAßPPß, and cytokines were analyzed by mesoscale, neurotransmitters by mass spectrometry, and gene expression by quantitative PCR. Tph2, microglia, and Aß were visualized histologically. RESULTS: Tph2 inactivation in APP/PS1 mice significantly reduced viability, without impacting soluble and insoluble Aß42 and Aß40 in neocortex and hippocampus, and with only mild changes of soluble Aß42/Aß40. However, sAßPPα and sAßPPß in hippocampus and Aß38 and Aß40 in cerebrospinal fluid were reduced. 3xTg-/-mice were devoid of Tph2 immunopositive fibers and 5-HT. Cytokines were unaffected by genotype, as were neocortical TNF, HTR2a and HTR2b mRNA levels in Tph2-/- mice. Microglia clustered around Aß plaques regardless of genotype. CONCLUSION: The results suggest that Tph2 inactivation influences AßPP processing, at least in the hippocampus, although levels of Aß are unchanged. The reduced viability of 3xTg-/-mice could indicate that 5-HT protects against the seizures that can impact the viability of APP/PS1 mice.

Mild Microglial Responses in the Cortex and Perivascular Macrophage Infiltration in Subcortical White Matter in Dogs with Age-Related Dementia Modelling Prodromal Alzheimer's Disease.

BACKGROUND: Microglia contribute to Alzheimer's disease (AD) pathogenesis by clearing amyloid-ß (Aß) and driving neuroinflammation. Domestic dogs with age-related dementia (canine cognitive dysfunction (CCD)) develop cerebral amyloidosis like humans developing AD, and studying such dogs can provide novel information about microglial response in prodromal AD. OBJECTIVE: The aim was to investigate the microglial response in the cortical grey and the subcortical white matter in dogs with CCD versus age-matched cognitively normal dogs. METHODS: Brains from aged dogs with CCD and age-matched controls without dementia were studied. Cases were defined by dementia rating score. Brain sections were stained for Aß, thioflavin S, hyperphosphorylated tau, and the microglial-macrophage ionized calcium binding adaptor molecule 1 (Iba1). Results were correlated to dementia rating score and tissue levels of Aß. RESULTS: Microglial numbers were higher in the Aß plaque-loaded deep cortical layers in CCD versus control dogs, while the coverage by microglial processes were comparable. Aß plaques were of the diffuse type and without microglial aggregation. However, a correlation was found between the %Iba1 area and insoluble Aß 42 and N-terminal pyroglutamate modified Aß(N3pE)-42. The %Iba1 area was higher in white matter, showing phosphorylation of S396 tau, versus grey matter. Perivascular macrophage infiltrates were abundant in the white matter particularly in CDD dogs. CONCLUSION: The results from this study of the microglial-macrophage response in dogs with CCD are suggestive of relatively mild microglial responses in the Aß plaque-loaded deep cortical layers and perivascular macrophage infiltrates in the subcortical white matter, in prodromal AD.

Tumor Necrosis Factor (TNF) Is Required for Spatial Learning and Memory in Male Mice under Physiological, but Not Immune-Challenged Conditions.

Increasing evidence demonstrates that inflammatory cytokines-such as tumor necrosis factor (TNF)-are produced at low levels in the brain under physiological conditions and may be crucial for synaptic plasticity, neurogenesis, learning and memory. Here, we examined the effects of developmental TNF deletion on spatial learning and memory using 11-13-month-old TNF knockout (KO) and C57BL6/J wild-type (WT) mice. The animals were tested in the Barnes maze (BM) arena under baseline conditions and 48 h following an injection of the endotoxin lipopolysaccharide (LPS), which was administered at a dose of 0.5 mg/kg. Vehicle-treated KO mice were impaired compared to WT mice during the acquisition and memory-probing phases of the BM test. No behavioral differences were observed between WT and TNF-KO mice after LPS treatment. Moreover, there were no differences in the hippocampal content of glutamate and noradrenaline between groups. The effects of TNF deletion on spatial learning and memory were observed in male, but not female mice, which were not different compared to WT mice under baseline conditions. These results indicate that TNF is required for spatial learning and memory in male mice under physiological, non-inflammatory conditions, however not following the administration of LPS. Inflammatory signalling can thereby modulate spatial cognition in male subjects, highlighting the importance of sex- and probably age-stratified analysis when examining the role of TNF in the brain.

Bone Marrow-Derived IL-1Ra Increases TNF Levels Poststroke.

Tumor necrosis factor (TNF) and interleukin-1 receptor antagonist (IL-1Ra) are key players in stroke, a disease in which cell-based therapies have shown great potential. Having shown an infarct-reducing effect of bone marrow (BM) cells, especially cells with high IL-1Ra expression, we here investigated the effect of BM cells on TNF and other stroke-related mediators in mice after transient middle cerebral artery occlusion (tMCAo) and in vitro using adult microglial cultures. We analyzed stroke-related genes and inflammatory mediators using qPCR stroke Tier panels, electrochemiluminescence, or enzyme-linked immunosorbent assays. We found a significant correlation and cellular colocalization between microglial-derived TNF and IL-1Ra, though IL-1Ra production was TNF independent. BM treatment significantly increased TNF, interleukin (IL)-10, and IL-4 levels, while C-X-C motif ligand 1 (CXCL1), IL-12p70, and Toll-like receptor 2 (TLR2) decreased, suggesting that BM treatment favors an anti-inflammatory environment. Hierarchical clustering identified Tnf and IL-1rn within the same gene cluster, and subsequent STRING analysis identified TLR2 as a shared receptor. Although IL-1Ra producing BM cells specifically modulated TNF levels, this was TLR2 independent. These results demonstrate BM cells as modulators of poststroke inflammation with beneficial effects on poststroke outcomes and place TNF and IL-1Ra as key players of the defense response after tMCAo.

Characterization of the TNF and IL-1 systems in human brain and blood after ischemic stroke.

Preclinical and clinical proof-of-concept studies have suggested the effectiveness of pharmacological modulation of inflammatory cytokines in ischemic stroke. Experimental evidence shows that targeting tumor necrosis factor (TNF) and interleukin (IL)-1 holds promise, and these cytokines are considered prime targets in the development of new stroke therapies. So far, however, information on the cellular expression of TNF and IL-1 in the human ischemic brain is sparse.We studied 14 cases of human post-mortem ischemic stroke, representing 21 specimens of infarcts aged 1 to > 8 days. We characterized glial and leukocyte reactions in the infarct/peri-infarct (I/PI) and normal-appearing tissue (NAT) and the cellular location of TNF, TNF receptor (TNFR)1 and TNFR2, IL-1α, IL-1ß, and IL-1 receptor antagonist (IL-1Ra). The immunohistochemically stained tissue sections received a score reflecting the number of immunoreactive cells and the intensity of the immunoreactivity (IR) in individual cells where 0 = no immunoreactive cells, 1 = many intermediately to strongly immunoreactive cells, and 2 = numerous and intensively immunoreactive cells. Additionally, we measured blood TNF, TNFR, and IL-1 levels in surviving ischemic stroke patients within the first 8 h and again at 72 h after symptom onset and compared levels to healthy controls.We observed IL-1α and IL-1ß IR in neurons, glia, and macrophages in all specimens. IL-1Ra IR was found in glia, in addition to macrophages. TNF IR was initially found in neurons located in I/PI and NAT but increased in glia in older infarcts. TNF IR increased in macrophages in all specimens. TNFR1 IR was found in neurons and glia and macrophages, while TNFR2 was expressed only by glia in I/PI and NAT, and by macrophages in I/PI. Our results suggest that TNF and IL-1 are expressed by subsets of cells and that TNFR2 is expressed in areas with increased astrocytic reactivity. In ischemic stroke patients, we demonstrate that plasma TNFR1 and TNFR2 levels increased in the acute phase after symptom onset compared to healthy controls, whereas TNF, IL-1α, IL-1ß, and IL-1Ra did not change.Our findings of increased brain cytokines and plasma TNFR1 and TNFR2 support the hypothesis that targeting post-stroke inflammation could be a promising add-on therapy in ischemic stroke patients.

Increased Inflammation and Unchanged Density of Synaptic Vesicle Glycoprotein 2A (SV2A) in the Postmortem Frontal Cortex of Alzheimer's Disease Patients.

Sections from the middle frontal gyrus (Brodmann area 46) of autopsy-confirmed Alzheimer's disease (AD) patients and non-demented subjects were examined for the prevalence of hallmark AD pathology, including amyloid-ß (Aß) plaques, phosphorylated tau (pTau) tangles, neuroinflammation and synaptic loss (n = 7 subjects/group). Dense-core deposits of Aß were present in all AD patients (7/7) and some non-demented subjects (3/7), as evidenced by 6E10 immunohistochemistry. Levels of Aß immunoreactivity were higher in AD vs. non-AD cases. For pTau, AT8-positive neurofibrillary tangles and threads were exclusively observed in AD patient tissue. Levels of [3H]PK11195 binding to the translocator protein (TSPO), a marker of inflammatory processes, were elevated in the gray matter of AD patients compared to non-demented subjects. Levels of [3H]UCB-J binding to synaptic vesicle glycoprotein 2A (SV2A), a marker of synaptic density, were not different between groups. In AD patients, pTau immunoreactivity was positively correlated with [3H]PK11195, and negatively correlated with [3H]UCB-J binding levels. No correlation was observed between Aß immunoreactivity and markers of neuroinflammation or synaptic density. These data demonstrate a close interplay between tau pathology, inflammation and SV2A density in AD, and provide useful information on the ability of neuroimaging biomarkers to diagnose AD dementia.

Ageing and amyloidosis underlie the molecular and pathological alterations of tau in a mouse model of familial Alzheimer's disease.

Despite compelling evidence that the accumulation of amyloid-beta (Aß) promotes neocortical MAPT (tau) aggregation in familial and idiopathic Alzheimer's disease (AD), murine models of cerebral amyloidosis are not considered to develop tau-associated pathology. In the present study, we show that tau can accumulate spontaneously in aged transgenic APPswe/PS1ΔE9 mice. Tau pathology is abundant around Aß deposits, and further characterized by accumulation of Gallyas and thioflavin-S-positive inclusions, which were detected in the APPswe/PS1ΔE9 brain at 18 months of age. Age-dependent increases in argyrophilia correlated positively with binding levels of the paired helical filament (PHF) tracer [18F]Flortaucipir, in all brain areas examined. Sarkosyl-insoluble PHFs were visualized by electron microscopy. Quantitative proteomics identified sequences of hyperphosphorylated and three-repeat tau in transgenic mice, along with signs of RNA missplicing, ribosomal dysregulation and disturbed energy metabolism. Tissue from the frontal gyrus of human subjects was used to validate these findings, revealing primarily quantitative differences between the tau pathology observed in AD patient vs. transgenic mouse tissue. As physiological levels of endogenous, 'wild-type' tau aggregate secondarily to Aß in APPswe/PS1ΔE9 mice, this study suggests that amyloidosis is both necessary and sufficient to drive tauopathy in experimental models of familial AD.

TNF deficiency causes alterations in the spatial organization of neurogenic zones and alters the number of microglia and neurons in the cerebral cortex.

BACKGROUND: Although tumor necrosis factor (TNF) inhibitors are used to treat chronic inflammatory diseases, there is little information about how long-term inhibition of TNF affects the homeostatic functions that TNF maintains in the intact CNS. MATERIALS AND METHODS: To assess whether developmental TNF deficiency causes alterations in the naïve CNS, we estimated the number of proliferating cells, microglia, and neurons in the developing neocortex of E13.5, P7 and adult TNF knock out (TNF-/-) mice and wildtype (WT) littermates. We also measured changes in gene and protein expression and monoamine levels in adult WT and TNF-/- mice. To evaluate long-term effects of TNF inhibitors, we treated healthy adult C57BL/6 mice with either saline, the selective soluble TNF inhibitor XPro1595, or the nonselective TNF inhibitor etanercept. We estimated changes in cell number and protein expression after two months of treatment. We assessed the effects of TNF deficiency on cognition by testing adult WT and TNF-/- mice and mice treated with saline, XPro1595, or etanercept with specific behavioral tasks. RESULTS: TNF deficiency decreased the number of proliferating cells and microglia and increased the number of neurons. At the same time, TNF deficiency decreased the expression of WNT signaling-related proteins, specifically Collagen Triple Helix Repeat Containing 1 (CTHRC1) and Frizzled receptor 6 (FZD6). In contrast to XPro1595, long-term inhibition of TNF with etanercept in adult C57BL/6 mice decreased the number of BrdU+ cells in the granule cell layer of the dentate gyrus. Etanercept, but not XPro1595, also impaired spatial learning and memory in the Barnes maze memory test. CONCLUSION: TNF deficiency impacts the organization of neurogenic zones and alters the cell composition in brain. Long-term inhibition of TNF with the nonselective TNF inhibitor etanercept, but not the soluble TNF inhibitor XPro1595, decreases neurogenesis in the adult mouse hippocampus and impairs learning and memory after two months of treatment.

Protective effect of ibuprofen in a rat model of chronic oxaliplatin-induced peripheral neuropathy.

Despite extensive preclinical and clinical investigations, a clinically relevant neuroprotective agent against oxaliplatin-induced peripheral neuropathy, which affects the quality of life following chemotherapy, has not been identified. Epidemiological data suggest that ibuprofen may reduce the risk of neuropathy. Male rats were treated with oxaliplatin (n = 6), oxaliplatin and ibuprofen (n = 5) or vehicle (n = 5) every second day for 15 days. Neuropathy was evaluated using mechanical detection thresholds (MDT) at the hind paw and sensory nerve conduction velocity (SNCV) in the tail nerve at baseline, right after and 3 weeks after the end of treatment. Intraepidermal nerve fibre density (IENFD) was evaluated in the hind paw and inflammation in the dorsal root ganglia 3 weeks after treatment. Inflammation in the dorsal root ganglia was assessed using quantitative real-time RT-PCR (qPCR) of the mRNA levels for the pro-inflammatory cytokines, TNF-α and IL-1ß, and by immunohistochemical staining for Iba1+ macrophages. SNCV was reduced in rats treated with oxaliplatin and with oxaliplatin and ibuprofen compared to control rats 3 weeks after treatment. No differences were found for MDT 3 weeks after treatment. IENFD was reduced in rats treated with oxaliplatin. There was a trend towards up-regulation of TNF-α mRNA levels in rats treated with oxaliplatin and with oxaliplatin and ibuprofen. Morphological changes of Iba1+ macrophages suggested activation, but no differences were found in area fraction or size of macrophage cell bodies. The results did not support a neuroprotective effect of ibuprofen but indicated that inflammation may play a role in oxaliplatin-induced peripheral neuropathy.

Microglia Express Insulin-Like Growth Factor-1 in the Hippocampus of Aged APPswe/PS1ΔE9 Transgenic Mice.

Insulin-like growth factor-1 (IGF-1) is a pleiotropic molecule with neurotrophic and immunomodulatory functions. Knowing the capacity of chronically activated microglia to produce IGF-1 may therefore show essential to promote beneficial microglial functions in Alzheimer's disease (AD). Here, we investigated the expression of IGF-1 mRNA and IGF-1 along with the expression of tumor necrosis factor (TNF) mRNA, and the amyloid-ß (Aß) plaque load in the hippocampus of 3- to 24-month-old APPswe/PS1ΔE9 transgenic (Tg) and wild-type (WT) mice. As IGF-1, in particular, is implicated in neurogenesis we also monitored the proliferation of cells in the subgranular zone (sgz) of the dentate gyrus. We found that the Aß plaque load reached its maximum in aged 21- and 24-month-old APPswe/PS1ΔE9 Tg mice, and that microglial reactivity and hippocampal IGF-1 and TNF mRNA levels were significantly elevated in aged APPswe/PS1ΔE9 Tg mice. The sgz cell proliferation decreased with age, regardless of genotype and increased IGF-1/TNF mRNA levels. Interestingly, IGF-1 mRNA was expressed in subsets of sgz cells, likely neuroblasts, and neurons in both genotypes, regardless of age, as well as in glial-like cells. By double in situ hybridization these were shown to be IGF1 mRNA+ CD11b mRNA+ cells, i.e., IGF-1 mRNA-expressing microglia. Quantification showed a 2-fold increase in the number of microglia and IGF-1 mRNA-expressing microglia in the molecular layer of the dentate gyrus in aged APPswe/PS1ΔE9 Tg mice. Double-immunofluorescence showed that IGF-1 was expressed in a subset of Aß plaque-associated CD11b+ microglia and in several subsets of neurons. Exposure of primary murine microglia and BV2 cells to Aß42 did not affect IGF-1 mRNA expression. IGF-1 mRNA levels remained constant in WT mice with aging, unlike TNF mRNA levels which increased with aging. In conclusion, our results suggest that the increased IGF-1 mRNA levels can be ascribed to a larger number of IGF-1 mRNA-expressing microglia in the aged APPswe/PS1ΔE9 Tg mice. The finding that subsets of microglia retain the capacity to express IGF-1 mRNA and IGF-1 in the aged APPswe/PS1ΔE9 Tg mice is encouraging, considering the beneficial therapeutic potential of modulating microglial production of IGF-1 in AD.

Proteomic signatures of neuroinflammation in Alzheimer's disease, multiple sclerosis and ischemic stroke.

Introduction: Inflammation is integral in the neuropathology of both chronic and acute neurological disorders. Knowing the inflammatory profile is important for clarification of disease mechanisms, diagnostic purposes, and ultimately treatment options. Areas covered: A systematic review was performed on literature from PubMed using the search terms 'Alzheimer's disease' (AD) or "multiple sclerosis" (MS) or "ischemic stroke" and 'proteomics'. Inflammatory proteins were assessed in blood, cerebrospinal fluid (CSF), and post-mortem brain tissue. Regulated inflammatory proteins across compartments and disorders mainly consisted of innate immune proteins, acute phase proteins and oxidative stress response proteins. In addition, immunoglobulin chains were signature proteins of MS, reflecting additional involvement of adaptive immunity. The Chitinase 3-like protein 1 was increased in ten original articles on MS and in three on AD supporting its implication in these diseases. Furthermore, CNS/CSF AD inflammatory proteins were matched to a CNS myeloid cell proteome implicating Alpha-2-Macroglobulin and Annexin A1 in AD pathogenesis. Expert opinion: Proteomics is an excellent technique for profiling inflammatory proteins in tissues and cells, but still targeted approaches are required for profiling of very low abundance proteins and peptides. Knowing the inflammatory signature of brain tissue, CSF, blood, and CNS myeloid cells holds the potential to point to novel mechanistic aspects of neurological diseases.

Neuroinflammation and amyloid-beta 40 are associated with reduced serotonin transporter (SERT) activity in a transgenic model of familial Alzheimer's disease.

BACKGROUND: Discrepant and often contradictory results have accumulated regarding the antidepressant and pro-cognitive effects of serotonin transporter (SERT) antagonists in Alzheimer's disease. METHODS: To address the discrepancy, we measured the activity and density of SERT in the neocortex of 3-24-month-old APPswe/PS1dE9 and wild-type littermate mice, by using [3H]DASB autoradiography and the [3H]5-HT uptake assay. Levels of soluble amyloid-ß (Aß), and pro-inflammatory cytokines that can regulate SERT function, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor (TNF), were measured in parallel. Neuroinflammation in aging APPswe/PS1dE9 mice was further evaluated by [3H]PK11195 autoradiography. RESULTS: Decreased SERT density was observed in the parietal and frontal cortex of 18-24-month-old APPswe/PS1dE9 mice, compared to age-matched, wild-type animals. The maximal velocity uptake rate (Vmax) of [3H]5-HT was reduced in neocortical preparations from 20-month-old transgenic vs. wild-type mice. The reduction was observed when the proportion of soluble Aß40 in the Aß40/42 ratio increased in the aged transgenic brain. At concentrations compatible with those measured in 20-month-old APPswe/PS1dE9 mice, synthetic human Aß40, but not Aß42, reduced the baseline Vmax of [3H]5-HT by ~ 20%. Neuroinflammation in APPswe/PS1dE9 vs. wild-type mice was evidenced by elevated [3H]PK11195 binding levels and increased concentration of IL-1ß protein, which preceded the reductions in neocortical SERT density and activity. Age-induced increases in the levels of IL-1ß, IL-6, and TNF were observed in both transgenic and wild-type animals. CONCLUSIONS: The progression of cerebral amyloidosis is associated with neuroinflammation and decreased presynaptic markers of serotonergic integrity and activity. The Aß40-induced reduction in the uptake kinetics of [3H]5-HT suggests that the activity of SERT, and potentially the effects of SERT antagonism, depend on the levels of interstitial Aß40.

Antibody-mediated clearance of tau in primary mouse microglial cultures requires Fcγ-receptor binding and functional lysosomes.

Neurodegenerative diseases such as Alzheimer's disease are characterized by the progressive spreading and accumulation of hyper-phosphorylated tau protein in the brain. Anti-tau antibodies have been shown to reduce tau pathology in in vivo models and antibody-mediated clearance of tau exerted by microglia has been proposed as a contributing factor. By subjecting primary microglia cultured in vitro to anti-phospho-tau antibodies in complex with pathological tau, we show that microglia internalise and degrade tau in a manner that is dependent on FcγR interaction and functional lysosomes. It has recently been discussed if anti-tau antibody effector-functions are required for induction of tau clearance. Using antibodies with compromised FcγR binding and non-compromised control antibodies we show that antibody effector functions are required for induction of microglial clearance of tau. Understanding the inflammatory consequences of targeting microglia using therapeutic antibodies is important when developing these molecules for clinical use. Using RNA sequencing, we show that treatment with anti-tau antibodies increases transcription of mRNA encoding pro-inflammatory markers, but that the mRNA expression profile of antibody-treated cells differ from the profile of LPS activated microglia. We further demonstrate that microglia activation alone is not sufficient to induce significant tau clearance.

Post-stroke inflammation-target or tool for therapy?

Inflammation is currently considered a prime target for the development of new stroke therapies. In the acute phase of ischemic stroke, microglia are activated and then circulating immune cells invade the peri-infarct and infarct core. Resident and infiltrating cells together orchestrate the post-stroke inflammatory response, communicating with each other and the ischemic neurons, through soluble and membrane-bound signaling molecules, including cytokines. Inflammation can be both detrimental and beneficial at particular stages after a stroke. While it can contribute to expansion of the infarct, it is also responsible for infarct resolution, and influences remodeling and repair. Several pre-clinical and clinical proof-of-concept studies have suggested the effectiveness of pharmacological interventions that target inflammation post-stroke. Experimental evidence shows that targeting certain inflammatory cytokines, such as tumor necrosis factor, interleukin (IL)-1, IL-6, and IL-10, holds promise. However, as these cytokines possess non-redundant protective and immunoregulatory functions, their neutralization or augmentation carries a risk of unwanted side effects, and clinical translation is, therefore, challenging. This review summarizes the cell biology of the post-stroke inflammatory response and discusses pharmacological interventions targeting inflammation in the acute phase after a stroke that may be used alone or in combination with recanalization therapies. Development of next-generation immune therapies should ideally aim at selectively neutralizing pathogenic immune signaling, enhancing tissue preservation, promoting neurological recovery and leaving normal function intact.