Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by synaptic dysfunction, protein aggregation, and widespread molecular alterations in the brain. In this study, we applied quantitative mass spectrometry-based proteomics and phosphoproteomics to characterize synaptosomes and sarkosyl-insoluble protein inclusions from the post-mortem frontal lobes of AD and control cases. We identified >3700 proteins across both fractions, revealing AD-associated changes in synaptic composition and phosphorylation patterns. Proteomic analyses indicated mitochondrial deficits and disruptions in vesicle trafficking within synapses, whereas insoluble protein inclusions showed an accumulation of spliceosomal components and glial activation markers as well as an enrichment of N-terminally truncated amyloid beta peptides in AD cases, suggesting involvement of postfibrillar processing events mediated by specific proteases in amyloid plaque pathology. Phosphoproteomic analysis revealed extensive alterations in pathways regulating vesicle trafficking, Golgi homeostasis, and synaptic function. We observed increased tau phosphorylation at AD-associated sites in insoluble inclusions and distinct phosphorylation changes in synaptic tau, particularly at S285 and S305, suggesting altered tau function and aggregation properties. These findings provide new molecular insights into AD-related nerve terminal composition and protein aggregation, advancing our understanding of disease-associated changes at the subcellular level.

Abstract Colony stimulating factor 1 receptor (CSF1R) is a tyrosine kinase receptor that is expressed exclusively in microglia within the CNS. Its endogenous ligands, colony stimulating factor-1 (CSF1) and interleukin-34 (IL-34), are released from neurons, positioning CSF1R as a key mediator receptor of neuron-glia communication. CSF1R is considered not only a potential drug target, but also a biomarker of neuroinflammation. From that perspective, selective radioligands for neuroimaging are of great interest for imaging neuroinflammation and determining drug occupancy. In this study, we have validated the binding characteristics of a CSF1R inhibitor, 4-((5-MethOxy-6-((5-methoxypyridin-2-yl)methoxy)pyridin-3-yl)methyl)-2-(1-methyl-1H-pyrazol-4-yl)pyrimidine (5-MOP) as a novel CSF1R radioligand, by performing in vitro saturation binding experiments in human and murine tissues. 5-MOP was found to be selective for CSF1R among a broad range of kinases. Autoradiography revealed that [ 3 H]5-MOP binds with high affinity (K D = 9.8 nM) to a single saturable binding site in human meningioma tissues, and this binding was displaced with known CSF1R inhibitors, including CPPC, sCSF1 inh and GW-2580. In contrast, CPPC, which has been extensively used as a CSF1R radioligand showed substantial cross-reactivity to other brain kinases, including Trk A/B/C, and [ 3 H]CPPC could only be displaced with CPPC itself, not by other ligands, including 5-MOP. These results identify [ 3 H]5-MOP as the most selective radioligand currently available, enabling accurate detection of drug occupancy and activated microglia. Significance of the study This study identifies and validates a novel selective radioligand that binds CSF1R with high selectivity and low nanomolar affinity. Because CSF1R is selectively expressed in activated microglia, this radioligand could be useful for detecting neuroinflammatory activity.

Background:Chronic microglial activation is a key characteristic of Alzheimer's disease (AD). In mouse models of AD, microglial activation is considered associated with microglial cell population expansion. Objective:To elucidate species- and gene-dosage-dependent differences and similarities in microglial cell population expansion in response to amyloid-β (Aβ) plaque pathology. Methods:The total number of Iba1+ microglia in the neocortex of hemizygous APPswe/PS1ΔE9 (APP/PS1) mice and hemi- and homozygous TgF344-AD rats, carrying the same human mutations, was estimated by use of stereological techniques. Furthermore, microglial cluster formation was assessed. Proliferation was assessed in the mouse model. Results:A significant two-fold increase in microglia was observed in the neocortex of hemizygous APP/PS1 mice at 18 months and of homozygous TgF344-AD rats at 17 months. In comparison, the number of microglia in hemizygous TgF344-AD rats, remained constant from 4 to 17 months. Microglial clusters formed prior to the increase in microglial numbers in both species. The clusters were typically small, surrounding the smaller-sized Aβ plaques, occasionally also containing recently proliferated microglia. The Aβ plaque loads were comparable in hemizygous TgF344-AD rats and APP/PS1 mice, and two-three-fold higher in homozygous TgF344-AD rats. The microglial population remained constant across ages in wild types in both species. Conclusions:Transgenic mouse and rat AD models show significant differences in microglial population expansion, with a more restrained expansion in the rat. However, in both species and regardless of gene-dosage, population expansion is preceded by microglial clustering around Aβ plaques, indicating that cluster-formation is a key event in AD neuropathology.

Background: Amyloid-beta (Ab) and tau pathology are key molecular hallmarks of Alzheimer's disease (AD), yet how their interaction contributes to cognitive decline remains unclear. We investigated the relationship between Ab burden, tau pathology, presynaptic density, and spatial learning and memory in the APPswe/PSEN1dE9 (APP/PS1) transgenic (TG) mouse model of amyloidosis. Methods: Spatial learning and memory were assessed with the Barnes maze test in male TG and wild-type (WT) littermate mice, aged 6, 12, and 18 months. Gross visual function was assessed indirectly in 18-month-old animals using the light/dark exploration test. Brains were collected for autoradiography of tau pathology and presynaptic density with [18F]Flortaucipir and [3H]UCB-J, respectively, while Ab plaque load was measured by immunohistochemistry. Correlation and linear mixed-effects regression analyses were used to assess relationships between behavioral and pathological measures. Results: APP/PS1 mice showed normal cognitive performance at 6 months, a selective long-term memory deficit at 12 months, and severe impairments in learning and retention at 18 months, independent of visual confounds. Age-dependent increases in [18F]Flortaucipir binding and Ab plaque load were observed in all brain regions of TG compared to WT mice, whereas [3H]UCB-J binding was increased in a region-dependent manner in 18-month-old TG vs. WT animals. Barnes maze performance during the final day of testing correlated negatively with both Ab and tau pathology across all areas examined. Linear regression revealed a significant association between tau and age and between tau and Ab in the cortex, indicating that memory decline in ageing TG mice was driven by the combined effects of these pathologies. Conclusions: Deficits in memory retention precede impairments in task learning performance in APP/PS1 mice. Spontaneous tau accumulation contributes to the progressive cognitive decline, capturing key aspects of the Ab-tau interaction observed in human AD. ### Competing Interest Statement The authors have declared no competing interest. SDU2020, CoPING AD: Collaborative Project on the Interaction between Neurons and Glia in AD.

Epidemiological and pre-clinical data propose that infections can accelerate the cognitive decline in Alzheimer's disease (AD) and other dementias. The implication of infectious agents, and especially the role of E.coli and other amyloid-peptide producing bacteria, on the development and progression of cerebral amyloidosis and neuroinflammation, both key neuropathological characteristics of AD, has only been studied to a limited extent. In this study, recombinant bacterial amyloid surface protein CsgA was injected intracisternally in pre-plaque 811-week-old APPSWE/PS1aE9 mice and age-matched wild type (WT) mice. Although less potent than bacterial lipopolysaccharide, CsgA significantly increased the gene expression of inflammatory cytokines, such as tumor necrosis factor, in the neocortex of both APPSWE/PS1aE9 and WT mice, and in cultured microglia. CsgA exposure also induced transient changes in neocortical amyloid-beta (A(3) peptide levels, increasing the highly fibrillogenic A(342 in the guanidine-fraction in APPSWE/PS1aE9 mice and decreasing A(340 in the PBS-fraction in WT mice. The changes in A(3 levels had dissipated 24 h post-injection. In line with the only transient changes in A(3 levels and inflammatory gene expression, CsgA did not impact on long term spatial memory in pre-plaque APPSWE/PS1aE9 mice. Our findings highlight a contribution of bacterial amyloid proteins on neuroinflammation and a possible contribution in influencing A(3-homeostasis during infections. However, findings need to be further elaborated in older APPSWE/PS1aE9 mice in which A(3 plaques are abundant and an inflammatory response already established. Also, the impact of CsgA and other bacterial amyloids should be examined after repeated and/or continuous administration and at different concentrations.

Background Chronic microglial activation is a key characteristic of Alzheimer's disease (AD). In mouse models of AD, microglial activation is considered associated with microglial cell population expansion. Objective To elucidate species- and gene-dosage-dependent differences and similarities in microglial cell population expansion in response to amyloid-β (Aβ) plaque pathology. Methods The total number of Iba1 + microglia in the neocortex of hemizygous APP swe /PS1 ΔE9 ( APP/PS1) mice and hemi- and homozygous TgF344-AD rats, carrying the same human mutations, was estimated by use of stereological techniques. Furthermore, microglial cluster formation was assessed. Proliferation was assessed in the mouse model. Results A significant two-fold increase in microglia was observed in the neocortex of hemizygous APP/PS1 mice at 18 months and of homozygous TgF344-AD rats at 17 months. In comparison, the number of microglia in hemizygous TgF344-AD rats, remained constant from 4 to 17 months. Microglial clusters formed prior to the increase in microglial numbers in both species. The clusters were typically small, surrounding the smaller-sized Aβ plaques, occasionally also containing recently proliferated microglia. The Aβ plaque loads were comparable in hemizygous TgF344-AD rats and APP/PS1 mice, and two-three-fold higher in homozygous TgF344-AD rats. The microglial population remained constant across ages in wild types in both species. Conclusions Transgenic mouse and rat AD models show significant differences in microglial population expansion, with a more restrained expansion in the rat. However, in both species and regardless of gene-dosage, population expansion is preceded by microglial clustering around Aβ plaques, indicating that cluster-formation is a key event in AD neuropathology.

Epidemiological and pre-clinical data propose that infections can accelerate the cognitive decline in Alzheimer's disease (AD) and other dementias. The implication of infectious agents, and especially the role of E.coli and other amyloid-peptide producing bacteria, on the development and progression of cerebral amyloidosis and neuroinflammation, both key neuropathological characteristics of AD, has only been studied to a limited extent. In this study, recombinant bacterial amyloid surface protein CsgA was injected intracisternally in pre-plaque 8-11-week-old APP SWE /PS1 ΔE9 mice and age-matched wild type (WT) mice. Although less potent than bacterial lipopolysaccharide, CsgA significantly increased the gene expression of inflammatory cytokines, such as tumor necrosis factor, in the neocortex of both APP SWE /PS1 ΔE9 and WT mice, and in cultured microglia. CsgA exposure also induced transient changes in neocortical amyloid-beta (Aβ) peptide levels, increasing the highly fibrillogenic Aβ42 in the guanidine-fraction in APP SWE /PS1 ΔE9 mice and decreasing Aβ40 in the PBS-fraction in WT mice. The changes in Aβ levels had dissipated 24 h post-injection. In line with the only transient changes in Aβ levels and inflammatory gene expression, CsgA did not impact on long term spatial memory in pre-plaque APP SWE /PS1 ΔE9 mice. Our findings highlight a contribution of bacterial amyloid proteins on neuroinflammation and a possible contribution in influencing Aβ-homeostasis during infections. However, findings need to be further elaborated in older APP SWE /PS1 ΔE9 mice in which Aβ plaques are abundant and an inflammatory response already established. Also, the impact of CsgA and other bacterial amyloids should be examined after repeated and/or continuous administration and at different concentrations.

Despite well-documented dysregulation in central serotonergic signaling in Alzheimer’s disease (AD), knowledge about the potential involvement of the serotonin-2B receptor (5-HT2BR) subtype remains sparse. Here, we assessed the levels of 5-HT2BRs in brain tissue from APPswe/PS1dE9 transgenic (TG) mice, AD patients, and adult microglial cells. 5-HT2BR mRNA was measured by RT-qPCR in ageing TG and wild-type (WT) mice, in samples from the middle frontal gyrus of female, AD and control subjects, and in microglia from the cerebral cortex of WT mice. The density of 5-HT2BRs was measured by autoradiography using [3H]RS 127445. Both mouse and human brains had low levels of 5-HT2BR mRNA. In whole-brain mouse samples, mRNA expression was significantly lower in TG mice compared to WT at > 18 months of age. In the Aβ-plaque-burdened neocortex and hippocampus of old TG mice, however, levels of 5-HT2BR mRNA were two-fold higher over control, with similar elevations observed in the Aβ-plaque-burdened frontal cortex of human AD patients. 5-HT2BR mRNA expression varied widely in adult microglia and was higher compared to other cortical cell subtypes. In mice, specific [3H]RS-127445 binding in the cortex was first detected after 3 months of age. The density of 5-HT2BRs was low and overall reduced in TG, compared to WT mice. Binding was detectable but too low to be reliably quantified in the human cortex. Our results document Aβ-associated increases in 5-HT2BR mRNA expression and suggest reduced receptor binding in the context of AD. Studies investigating the functional involvement of microglial 5-HT2BRs in AD are considered relevant.

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.

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.

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 (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.

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-β (Aβ). 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 Aβ42/40/38, soluble APPα (sAβPPα) and sAβPPβ, and cytokines were analyzed by mesoscale, neurotransmitters by mass spectrometry, and gene expression by quantitative PCR. Tph2, microglia, and Aβ were visualized histologically. Results: Tph2 inactivation in APP/PS1 mice significantly reduced viability, without impacting soluble and insoluble Aβ42 and Aβ40 in neocortex and hippocampus, and with only mild changes of soluble Aβ42/Aβ40. However, sAβPPα and sAβPPβ in hippocampus and Aβ38 and Aβ40 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 Aβ plaques regardless of genotype. Conclusion: The results suggest that Tph2 inactivation influences AβPP processing, at least in the hippocampus, although levels of Aβ 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.

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BACKGROUND:Modulation of serotonergic signaling by treatment with selective serotonin reuptake inhibitors (SSRIs) has been suggested to mitigate amyloid-β (Aβ) 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 Aβ pathology and Aβ 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 Aβ pathology. Aβ pathology was evaluated by Aβ plaque load estimation and the Aβ42/Aβ40 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 Aβ plaque load (p = 0.39), the number and size of plaques, or the Aβ plaque-induced increases in microglial numbers in the dentate gyrus. Three months of serotonin depletion did not significantly impact the Aβ plaque load or Aβ42/Aβ40 ratio in APPswe/PS1ΔE9 mice at 12 months.CONCLUSION:Our results show that chronic treatment with the SSRI paroxetine does not mitigate Aβ pathology and Aβ plaque-induced microgliosis in the hippocampus of APPswe/PS1ΔE9 mice.