We developed a high content screening to investigate how Alzheimer disease (AD) genetic risk factors may affect synaptic mechanisms in rat primary neuronal cultures. Out of the target genes identified, we found that Plcg2 downregulation in mouse dentate gyrus neurons consistently disrupted dendritic morphology and synaptic function. In human neuronal cultures (hNCs), PLCG2 downregulation also impaired synaptic function and increased Aβ levels and Tau phosphorylation. Very rare PLCG2 loss-of-function (LoF) variants were associated with a 10-fold increased AD risk. PLCG2 LoF carriers exhibit low mRNA/protein PLCG2/PLCγ2 levels and the R953* LoF mutation compromised synaptic function and increased AD hallmarks in hNCs. Single nuclei RNAseq analyses confirmed that the downregulation of PLCG2 impacted pathways related to synaptic and neuronal functions, potentially through neurexin in neurons. In conclusion, PLCγ2 downregulation could increase AD risk by impairing synaptic functions and increasing the Aβ levels and Tau phosphorylation in neurons.

C9orf72 hexanucleotide repeat expansion (C9-HRE) is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (FTD). However, approximately half of the FTD patients are sporadic without a clear genetic background. To compare characteristics of microglia from different FTD subtypes, we generated induced pluripotent stem cell-derived microglia (iMG) from sporadic and C9-HRE-carrying behavioral variant FTD (bvFTD) patients and healthy controls. C9-HRE iMG displayed C9-HRE-associated RNA foci and dipeptide repeat proteins. All bvFTD iMG had fewer LAMP2-A-positive vesicles compared to control iMG. Additionally, C9-HRE iMG showed significantly increased LC3BII/I conversion after bafilomycin A1 treatment and altered phagocytic activity. The gene expression profile of C9-HRE iMG only modestly differed from the control iMG, but was greatly different from the sporadic bvFTD patient iMG. Our data show alterations in phagocytic and autophagosomal/lysosomal pathways and gene expression profiles between C9-HRE and sporadic bvFTD iMG for the first time.

We developed a high-content screening to investigate how Alzheimer's disease (AD) genetic risk factors may affect synaptic mechanisms in rat primary neuronal cultures. Of the target genes identified, we found that Plcg2 downregulation in mouse dentate gyrus neurons consistently disrupted dendritic morphology and synaptic function. In human neuronal cultures (hNCs), PLCG2 downregulation also impaired synaptic function and increased amyloid-β (Aβ) levels and Tau phosphorylation. Very rare PLCG2 loss-of-function (LoF) variants were associated with a tenfold increased AD risk. PLCG2 LoF carriers show low mRNA/protein PLCG2/PLCγ2 levels and the R953* LoF mutation compromised synaptic function and increased AD hallmarks in hNCs. Single-nucleus RNA sequencing analyses confirmed that the downregulation of PLCG2 impacted pathways related to synaptic and neuronal functions, potentially through neurexins in neurons. In conclusion, PLCγ2 downregulation could increase AD risk by impairing synaptic functions and by increasing Aβ levels and Tau phosphorylation in neurons.

Phospholipase C gamma 2, proline 522 to arginine (PLCγ2-P522R) is a protective variant that reduces the risk of Alzheimer’s disease (AD). Recently, it was shown to mitigate β-amyloid pathology in a 5XFAD mouse model of AD. Here, we investigated the protective functions of the PLCγ2-P522R variant in a less aggressive APP/PS1 mouse model of AD and assessed the underlying cellular mechanisms using mouse and human microglial models. The effects of the protective PLCγ2-P522R variant on microglial activation, AD-associated β-amyloid and neuronal pathologies, and behavioral changes were investigated in PLCγ2-P522R knock-in variant mice crossbred with APP/PS1 mice. Transcriptomic, proteomic, and functional studies were carried out using microglia isolated from mice carrying the PLCγ2-P522R variant. Finally, microglia-like cell models generated from human blood and skin biopsy samples of PLCγ2-P522R variant carriers were employed. The PLCγ2-P522R variant decreased β-amyloid plaque count and coverage in female APP/PS1 mice. Moreover, the PLCγ2-P522R variant promoted anxiety in these mice. The area of the microglia around β-amyloid plaques was also increased in mice carrying the PLCγ2-P522R variant, while β-amyloid plaque-associated neuronal dystrophy and the levels of certain cytokines, including IL-6 and IL-1β, were reduced. These alterations were revealed through [18F]FEPPA PET imaging and behavioral studies, as well as various cytokine immunoassays, transcriptomic and proteomic analyses, and immunohistochemical analyses using mouse brain tissues. In cultured mouse primary microglia, the PLCγ2-P522R variant reduced the size of lipid droplets. Furthermore, transcriptomic and proteomic analyses revealed that the PLCγ2-P522R variant regulated key targets and pathways involved in lipid metabolism, mitochondrial fatty acid oxidation, and inflammatory/interferon signaling in acutely isolated adult mouse microglia and human monocyte-derived microglia-like cells. Finally, the PLCγ2-P522R variant also increased mitochondrial respiration in human iPSC-derived microglia. These findings suggest that the PLCγ2-P522R variant exerts protective effects against β-amyloid and neuronal pathologies by increasing microglial responsiveness to β-amyloid plaques in APP/PS1 mice. The changes observed in lipid/fatty acid and mitochondrial metabolism revealed by the omics and metabolic assessments of mouse and human microglial models suggest that the protective effects of the PLCγ2-P522R variant are potentially associated with increased metabolic capacity of microglia.

We developed a high content screening to investigate how Alzheimer disease (AD) genetic risk factors may impair synaptic mechanisms in rat primary neuronal cultures. Out of the gene targets identified, we found that shRNA-mediated downregulation of Plcg2 in mouse dentate gyrus neurons consistently impaired dendritic morphology and synaptic function. In human neuronal cultures (hNCs), PLCG2 downregulation also impaired synaptic function and was associated with increased levels of Aβ and Tau phosphorylation, potentially via the AKT/GSK3β axis. Very rare PLCG2 loss-of-functon(LoF) variants were associated with a 10-fold increased AD risk. PLCG2 LoF carriers exhibit low mRNA/protein PLCG2/PLCγ2 levels, consistent with nonsense-mediated mRNA decay mechanisms. Restoring PLCγ2 levels in shPLCG2-hNCs fully reversed the disease-related phenotypes. Our findings indicate that the downregulation of PLCγ2 increases the risk of AD by impairing synaptic function and increasing the levels of Aβ and Tau phosphorylation in neurons.

In Alzheimer’s disease (AD), amyloid β (Aβ)-triggered cleavage of TrkB-FL impairs Brain-derived neurotrophic factor (BDNF) signaling, thereby compromising neuronal survival, differentiation, as well as synaptic transmission and plasticity. Using cerebrospinal fluid and post-mortem human brain samples, we show that TrkB-FL cleavage occurs from the early stages of the disease and increases as function of pathology severity. To explore the therapeutic potential of this disease mechanism, we designed small TAT-fused peptides and screened their ability to prevent TrkB-FL receptor cleavage. Among these, a TAT-TrkB peptide with a lysine-lysine linker prevented TrkB-FL cleavage both in vitro and in vivo and rescued synaptic deficits induced by oligomeric Aβ in hippocampal slices. Furthermore, this TAT-TrkB peptide improved the cognitive performance, ameliorated synaptic plasticity deficits and prevented Tau pathology progression in vivo in the 5XFAD mouse model of AD. No evidence of liver or kidney toxicity was found. We provide proof-of-concept evidence for the efficacy and safety of this therapeutic strategy and anticipate that this TAT-TrkB peptide has the potential of a disease-modifying drug that can prevent and/or reverse cognitive deficits in patients with AD.

C9orf72 hexanucleotide repeat expansion (HRE) is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The role of microglia in these C9orf72 HRE-associated diseases is understudied. To elucidate effects of C9orf72 HRE on microglia, we have characterized human induced pluripotent stem cell-derived microglia (iMG) from behavioral variant frontotemporal dementia (bvFTD) patients carrying the C9orf72 HRE. C9orf72 HRE iMG were compared to iMG from healthy controls and sporadic bvFTD patients. The phenotypes of iMG were analyzed using bulk RNA sequencing, biochemical and immunofluorescence analyses, and live cell imaging. C9orf72 HRE-carrying iMG showed nuclear RNA foci and poly-GP dipeptide repeat proteins but no decreased C9orf72 mRNA or protein expression. TDP-43 pathology was absent from all bvFTD iMG. As compared to healthy control iMG, quantitative immunofluorescence analyses indicated that all bvFTD iMG had reduced number, size, and intensity of LAMP2-A-positive vesicles. C9orf72 HRE-carrying iMG additionally showed decreased number, size, and intensity of p62/SQSTM1-positive vesicles. These changes were accompanied by increased phagocytic activity of the C9orf72 HRE-carrying iMG. Serum starvation increased phagocytic activity also in the iMG of sporadic bvFTD patients. RNA sequencing revealed that iMG of C9orf72 HRE-carrying bvFTD patients as compared to the iMG of sporadic bvFTD patients showed differential gene expression in pathways related to RNA and protein regulation and mitochondrial metabolism. Our data suggest potential alterations in the autophagosomal/lysosomal pathways in bvFTD patient iMG, which are further reinforced by the C9orf72 HRE and functionally manifest as increased phagocytic activity. ### Competing Interest Statement The authors have declared no competing interest.

Background PLCγ2-P522R (phospholipase C gamma 2, proline 522 to arginine) is a protective variant that reduces the risk for late onset Alzheimer’s disease. Recently, it was shown to decrease β-amyloid pathology in 5XFAD mouse model of AD. In this study, our goal was to investigate the protective functions of PLCγ2-P522R variant in a less aggressive mouse model of AD as well as to assess the underlying mechanisms at the molecular and cellular level using mouse and human microglia models. Methods The effects of the protective PLCγ2-P522R variant on microglia activation, AD-related β-amyloid and neuronal pathologies, as well as behavioral changes were investigated in PLCγ2-P522R knock-in mice crossbred with APP/PS1 AD model mice. Transcriptomic, proteomic, and functional studies were carried out in cultured and acutely isolated adult PLCγ2-P522R mouse microglia to study molecular mechanisms. Finally, microglia-like cell models generated from blood and skin biopsy samples of the PLCγ2-P522R variant carriers were employed to translate the key findings to human cells. Results Our results demonstrate that the PLCγ2-P522R variant reduced brain β-amyloid plaque burden of APP/PS1 mice. Simultaneously, PLCγ2-P522R variant increased non-proinflammatory microglia activation and microglia clustering around β-amyloid plaques, leading to reduced β-amyloid plaque-associated neuronal dystrophy. In cultured mouse primary microglia, PLCγ2-P522R variant decreased accumulation of large lipid droplets, reduced cell stress, and increased acute response to strong inflammatory stimuli. Transcriptomic and proteomic analyses in acutely isolated adult mouse microglia as well as in human monocyte-derived microglial cells showed that PLCγ2-P522R upregulates mitochondrial fatty acid oxidation and downregulates inflammatory/interferon signaling pathways. Accordingly, PLCγ2-P522R increased mitochondrial respiration in iPSC-derived microglial cells. Conclusions Together, these findings suggest that PLCγ2-P522R variant exerts protection against AD-associated β-amyloid and neuronal pathologies via enhancing microglial barrier formation around β-amyloid plaques and suppressing pro-inflammatory activation. Observed changes in fatty acid metabolism and mitochondrial flexibility as well as the downregulation of genes involved in inflammatory signaling pathways suggest that these protective effects of the PLCγ2-P522R variant are mediated through an anti-ageing mechanism. ### Competing Interest Statement Professor Christian Haass collaborates with Denali Therapeutics and is a member of the advisory boards of AviadoBio and Cure Ventures.

Organotypic slice culture models surpass conventional in vitro methods in many aspects. They retain all tissue-resident cell types and tissue hierarchy. For studying multifactorial neurodegenerative diseases such as tauopathies, it is crucial to maintain cellular crosstalk in an accessible model system. Organotypic slice cultures from postnatal tissue are an established research tool, but adult tissue-originating systems are missing, yet necessary, as young tissue-originating systems cannot fully model adult or senescent brains. To establish an adult-originating slice culture system for tauopathy studies, we made hippocampal slice cultures from transgenic 5-month-old hTau.P301S mice. In addition to the comprehensive characterization, we set out to test a novel antibody for hyperphosphorylated TAU (pTAU, B6), with and without a nanomaterial conjugate. Adult hippocampal slices retained intact hippocampal layers, astrocytes, and functional microglia during culturing. The P301S-slice neurons expressed pTAU throughout the granular cell layer and secreted pTAU to the culture medium, whereas the wildtype slices did not. Additionally, cytotoxicity and inflammation-related determinants were increased in the P301S slices. Using fluorescence microscopy, we showed target engagement of the B6 antibody to pTAU-expressing neurons and a subtle but consistent decrease in intracellular pTAU with the B6 treatment. Collectively, this tauopathy slice culture model enables measuring the extracellular and intracellular effects of different mechanistic or therapeutic manipulations on TAU pathology in adult tissue without the hindrance of the blood–brain barrier.

Alzheimer's disease (AD) is the most common form of dementia, which is neuropathologically characterized by extracellular senile plaques containing amyloid-β and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein. Previous studies have suggested a role for septin (SEPTIN) protein family members in AD-associated cellular processes. Here, we elucidated the potential role of presynaptic SEPTIN5 protein and its post-translational modifications in the molecular pathogenesis of AD. RNA and protein levels of SEPTIN5 showed a significant decrease in human temporal cortex in relation to the increasing degree of AD-related neurofibrillary pathology. Conversely, an increase in the phosphorylation of the functionally relevant SEPTIN5 phosphorylation site S327 was observed already in the early phases of AD-related neurofibrillary pathology, but not in the cerebrospinal fluid of individuals fulfilling the criteria for mild cognitive impairment due to AD. According to the mechanistic assessments, a link between SEPTIN5 S327 phosphorylation status and the effects of SEPTIN5 on amyloid precursor protein processing and markers of autophagy was discovered in mouse primary cortical neurons transduced with lentiviral constructs encoding wild type SEPTIN5 or SEPTIN5 phosphomutants (S327A and S327D). C57BL/6 J mice intrahippocampally injected with lentiviral wild type SEPTIN5 or phosphomutant constructs did not show changes in cognitive performance after five to six weeks from the start of injections. However, SEPTIN5 S327 phosphorylation status was linked to changes in short-term synaptic plasticity ex vivo at the CA3-CA1 synapse. Collectively, these data suggest that SEPTIN5 and its S327 phosphorylation status play a pivotal role in several cellular processes relevant for AD.

Methyl-CpG-binding protein 2 (MECP2) is a critical transcriptional regulator for synaptic function. Dysfunction of synapses, as well as microglia-mediated neuroinflammation, represent the earliest pathological events in Alzheimer’s disease (AD). Here, expression, protein levels, and activity-related phosphorylation changes of MECP2 were analyzed in post-mortem human temporal cortex. The effects of wild type and phosphorylation-deficient MECP2 variants at serine 423 (S423) or S80 on microglial and neuronal function were assessed utilizing BV2 microglial monocultures and co-cultures with mouse cortical neurons under inflammatory stress conditions. MECP2 phosphorylation at the functionally relevant S423 site nominally decreased in the early stages of AD-related neurofibrillary pathology in the human temporal cortex. Overexpression of wild type MECP2 enhanced the pro-inflammatory response in BV2 cells upon treatment with lipopolysaccharide (LPS) and interferon-γ (IFNγ) and decreased BV2 cell phagocytic activity. The expression of the phosphorylation-deficient MECP2-S423A variant, but not S80A, further increased the pro-inflammatory response of BV2 cells. In neurons co-cultured with BV2 cells, the MECP2-S423A variant increased the expression of several genes, which are important for the maintenance and protection of neurons and synapses upon inflammatory stress. Collectively, functional analyses in different cellular models suggest that MECP2 may influence the inflammatory response in microglia independently of S423 and S80 phosphorylation, while the S423 phosphorylation might play a role in the activation of neuronal gene expression, which conveys neuroprotection under neuroinflammation-related stress.

Isoflurane, the most commonly used preclinical anesthetic, induces brain plasticity and long-term cellular and molecular changes leading to behavioral and/or cognitive consequences. These changes are most likely associated with network-level changes in brain function. To elucidate the mechanisms underlying long-term effects of isoflurane, we investigated the influence of a single isoflurane exposure on functional connectivity, brain electrical activity, and gene expression. Male Wistar rats (n = 22) were exposed to 1.8% isoflurane for 3 h. Control rats (n = 22) spent 3 h in the same room without exposure to anesthesia. After 1 month, functional connectivity was evaluated with resting-state functional magnetic resonance imaging (fMRI; n = 6 + 6) and local field potential measurements (n = 6 + 6) in anesthetized animals. A whole genome expression analysis (n = 10+10) was also conducted with mRNA-sequencing from cortical and hippocampal tissue samples. Isoflurane treatment strengthened thalamo-cortical and hippocampal-cortical functional connectivity. Cortical low-frequency fMRI power was also significantly increased in response to the isoflurane treatment. The local field potential results indicating strengthened hippocampal-cortical alpha and beta coherence were in good agreement with the fMRI findings. Furthermore, altered expression was found in 20 cortical genes, several of which are involved in neuronal signal transmission, but no gene expression changes were noted in the hippocampus. Isoflurane induced prolonged changes in thalamo-cortical and hippocampal-cortical function and expression of genes contributing to signal transmission in the cortex. Further studies are required to investigate whether these changes are associated with the postoperative behavioral and cognitive symptoms commonly observed in patients and animals.

Leucine-rich-alpha-2-glykoprotein (LRG) is suggested as a potential biomarker for idiopathic normal pressure hydrocephalus (iNPH). Our goal was to compare the cerebrospinal fluid (CSF) LRG levels between 119 iNPH patients and 33 age-matched controls and with the shunt responses and the brain biopsy Alzheimer’s disease (AD) pathology among the iNPH patients. CSF LRG, Aβ1-42, P-tau181, and T-tau were measured by using commercial ELISAs. The LRG levels in the CSF were significantly increased in the iNPH patients (p < 0.001) as compared to the controls, regardless of the AD pathology. However, CSF LRG did not correlate with the shunt response in contrast to the previous findings. The CSF AD biomarkers, i.e., Aβ1-42, T-tau, and P-tau correlated with the brain biopsy AD pathology as expected but were systematically lower in the iNPH patients when compared to the controls (<0.001). Our findings support that the LRG levels in the CSF are potentially useful for the diagnostics of iNPH, independent of the brain AD pathology, but contrary to previous findings, not for predicting the shunt response. Our findings also suggest a need for specific reference values of the CSF AD biomarkers for the diagnostics of comorbid AD pathology in the iNPH patients.

Microglia-specific genetic variants are enriched in several neurodegenerative diseases, including Alzheimer’s disease (AD), implicating a central role for alterations of the innate immune system in the disease etiology. A rare coding variant in the PLCG2 gene (rs72824905, p.P522R) expressed in myeloid lineage cells was recently identified and shown to reduce the risk for AD. To assess the role of the protective variant in the context of immune cell functions, we generated a Plcγ2-P522R knock-in (KI) mouse model using CRISPR/Cas9 gene editing. Functional analyses of macrophages derived from homozygous KI mice and wild type (WT) littermates revealed that the P522R variant potentiates the primary function of Plcγ2 as a Pip2-metabolizing enzyme. This was associated with improved survival and increased acute inflammatory response of the KI macrophages. Enhanced phagocytosis was observed in mouse BV2 microglia-like cells overexpressing human PLCγ2-P522R, but not in PLCγ2-WT expressing cells. Immunohistochemical analyses did not reveal changes in the number or morphology of microglia in the cortex of Plcγ2-P522R KI mice. However, the brain mRNA signature together with microglia-related PET imaging suggested enhanced microglial functions in Plcγ2-P522R KI mice. The AD-associated protective Plcγ2-P522R variant promotes protective functions associated with TREM2 signaling. Our findings provide further support for the idea that pharmacological modulation of microglia via TREM2-PLCγ2 pathway-dependent stimulation may be a novel therapeutic option for the treatment of AD.

BACKGROUND AND PURPOSE:Protein tyrosine phosphatase receptor type Q (PTPRQ) was extracted from the cerebrospinal fluid (CSF) of patients with probable idiopathic normal-pressure hydrocephalus (iNPH) by proteome analysis. We aimed to assess the feasibility of using CSF PTPRQ concentrations for the additional diagnostic criterion of iNPH in Japanese and Finnish populations. METHODS:We compared PTPRQ concentrations among patients with probable iNPH and neurologically healthy individuals (normal control [NC] group), patients with normal-pressure hydrocephalus (NPH) of acquired and congenital/developmental aetiologies, patients with Alzheimer's disease and patients with Parkinson's disease in a Japanese analysis cohort. A corresponding iNPH group and NC group in a Finnish cohort was used for validation. Patients in the Finnish cohort who underwent biopsy were classified into two groups based on amyloid and/or tau deposition. We measured PTPRQ expression levels in autopsied brain specimens of iNPH patients and the NC group. RESULTS:Cerebrospinal fluid PTPRQ concentrations in the patients with NPH of idiopathic, acquired and congenital/developmental aetiologies were significantly higher than those in the NC group and those with Parkinson's disease, but iNPH showed no significant differences when compared with those in the Alzheimer's disease group. For the patients with iNPH, the area under the receiver-operating characteristic curve was 0.860 in the Japanese iNPH and 0.849 in the Finnish iNPH cohorts. Immunostaining and in situ hybridization revealed PTPRQ expression in the ependymal cells and choroid plexus. It is highly possible that the elevated PTPRQ levels in the CSF are related to ependymal dysfunction from ventricular expansion. CONCLUSIONS:Cerebrospinal fluid PTPRQ levels indicated the validity of this assay for auxiliary diagnosis of adult chronic hydrocephalus.

Type 2 diabetes (T2D) increases the risk of Alzheimer’s disease (AD). Even though these two diseases share common molecular pathways, the mechanisms remain elusive. To shed light into these mechanisms, mice with different AD- and/or tauopathy-linked genetic backgrounds were utilized; APPswe/PS1dE9 (A+Tw), Tau P301L (AwT+), and APPswe/PS1dE9/Tau P301L (A+T+). Feeding these mice with typical Western diet (TWD) led to obesity and diabetic phenotype as compared to respective mice with a standard diet. TWD also exacerbated memory and learning impairment in A+Tw and AwT+, but not in A+T+ mice. Furthermore, RNA sequencing of mouse hippocampal samples revealed altered responses to AD-related pathologies in A+Tw and A+T+ mice upon TWD, pointing specifically towards aberrant microglial functionality and PI3K-Akt signaling. Accordingly, fewer microglia alongside an increased number of dystrophic neurites around β-amyloid plaques, and impaired PI3K-Akt signaling, were discovered in the hippocampus of TWD mice. Mechanistic elucidation revealed that disruption of the PI3K-Akt signaling pathway by pharmacological or genetic approaches significantly decreased the phagocytic uptake and proinflammatory response as well as increased the activity of Syk-kinase upon ligand-induced activation of Trem2/Dap12 signaling in mouse microglia. Finally, characterization of microglial pathology in cortical biopsies of idiopathic normal pressure hydrocephalus (iNPH) patients harboring β-amyloid plaques revealed a significant decrease in the number of microglia per β-amyloid plaque in obese iNPH patients with T2D as compared to both normal weight and obese iNPH patients without T2D. Collectively, these results suggest that the peripheral diabetic phenotype in mice and humans associates with reduced microglial response to β-amyloid pathology.

Hexanucleotide repeat expansion (HRE) in the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause underpinning frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). It leads to the accumulation of toxic RNA foci and various dipeptide repeat (DPR) proteins into cells. These C9orf72 HRE-specific hallmarks are abundant in neurons. So far, the role of microglia, the immune cells of the brain, in C9orf72 HRE-associated FTLD/ALS is unclear. In this study, we overexpressed C9orf72 HRE of a pathological length in the BV-2 microglial cell line and used biochemical methods and fluorescence imaging to investigate its effects on their phenotype, viability, and functionality. We found that BV-2 cells expressing the C9orf72 HRE presented strong expression of specific DPR proteins but no sense RNA foci. Transiently increased levels of cytoplasmic TAR DNA-binding protein 43 (TDP-43), slightly altered levels of p62 and lysosome-associated membrane protein (LAMP) 2A, and reduced levels of polyubiquitinylated proteins, but no signs of cell death were detected in HRE overexpressing cells. Overexpression of the C9orf72 HRE did not affect BV-2 cell phagocytic activity or response to an inflammatory stimulus, nor did it shift their RNA profile toward disease-associated microglia. These findings suggest that DPR proteins do not affect microglial cell viability or functionality in BV-2 cells. However, additional studies in other models are required to further elucidate the role of C9orf72 HRE in microglia.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by aberrant amyloid-β (Aβ) and hyperphosphorylated tau aggregation. We have previously investigated the involvement of SEPTIN family members in AD-related cellular processes and discovered a role for SEPTIN8 in the sorting and accumulation of β-secretase. Here, we elucidated the potential role of SEPTIN5, an interaction partner of SEPTIN8, in the cellular processes relevant for AD, including amyloid precursor protein (APP) processing and the generation of Aβ. The in vitro and in vivo studies both revealed that the downregulation of SEPTIN5 reduced the levels of APP C-terminal fragments (APP CTFs) and Aβ in neuronal cells and in the cortex of Septin5 knockout mice. Mechanistic elucidation revealed that the downregulation of SEPTIN5 increased the degradation of APP CTFs, without affecting the secretory pathway-related trafficking or the endocytosis of APP. Furthermore, we found that the APP CTFs were degraded, to a large extent, via the autophagosomal pathway and that the downregulation of SEPTIN5 enhanced autophagosomal activity in neuronal cells as indicated by altered levels of key autophagosomal markers. Collectively, our data suggest that the downregulation of SEPTIN5 increases the autophagy-mediated degradation of APP CTFs, leading to reduced levels of Aβ in neuronal cells.

The bridging integrator 1 gene (BIN1) is a major genetic risk factor for Alzheimer's disease (AD). In this report, we investigated how BIN1-dependent pathophysiological processes might be associated with Tau. We first generated a cohort of control and transgenic mice either overexpressing human MAPT (TgMAPT) or both human MAPT and BIN1 (TgMAPT;TgBIN1), which we followed-up from 3 to 15 months. In TgMAPT;TgBIN1 mice short-term memory deficits appeared earlier than in TgMAPT mice; however-unlike TgMAPT mice-TgMAPT;TgBIN1 mice did not exhibit any long-term or spatial memory deficits for at least 15 months. After killing the cohort at 18 months, immunohistochemistry revealed that BIN1 overexpression prevents both Tau mislocalization and somatic inclusion in the hippocampus, where an increase in BIN1-Tau interaction was also observed. We then sought mechanisms controlling the BIN1-Tau interaction. We developed a high-content screening approach to characterize modulators of the BIN1-Tau interaction in an agnostic way (1,126 compounds targeting multiple pathways), and we identified-among others-an inhibitor of calcineurin, a Ser/Thr phosphatase. We determined that calcineurin dephosphorylates BIN1 on a cyclin-dependent kinase phosphorylation site at T348, promoting the open conformation of the neuronal BIN1 isoform. Phosphorylation of this site increases the availability of the BIN1 SH3 domain for Tau interaction, as demonstrated by nuclear magnetic resonance experiments and in primary neurons. Finally, we observed that although the levels of the neuronal BIN1 isoform were unchanged in AD brains, phospho-BIN1(T348):BIN1 ratio was increased, suggesting a compensatory mechanism. In conclusion, our data support the idea that BIN1 modulates the AD risk through an intricate regulation of its interaction with Tau. Alteration in BIN1 expression or activity may disrupt this regulatory balance with Tau and have direct effects on learning and memory.

No single-omic approach completely elucidates the multitude of alterations taking place in Alzheimer's disease (AD). Here, we coupled transcriptomic and phosphoproteomic approaches to determine the temporal sequence of changes in mRNA, protein, and phosphopeptide expression levels from human temporal cortical samples, with varying degree of AD-related pathology. This approach highlighted fluctuation in synaptic and mitochondrial function as the earliest pathological events in brain samples with AD-related pathology. Subsequently, increased expression of inflammation and extracellular matrix-associated gene products was observed. Interaction network assembly for the associated gene products, emphasized the complex interplay between these processes and the role of addressing post-translational modifications in the identification of key regulators. Additionally, we evaluate the use of decision trees and random forests in identifying potential biomarkers differentiating individuals with different degree of AD-related pathology. This multiomic and temporal sequence-based approach provides a better understanding of the sequence of events leading to AD.