Dynamic microglia alterations associate with hippocampal network impairments: A turning point in amyloid pathology progression.

Alzheimer's disease is a progressive neurological disorder causing memory loss and cognitive decline. The underlying causes of cognitive deterioration and neurodegeneration remain unclear, leading to a lack of effective strategies to prevent dementia. Recent evidence highlights the role of neuroinflammation, particularly involving microglia, in Alzheimer's disease onset and progression. Characterizing the initial phase of Alzheimer's disease can lead to the discovery of new biomarkers and therapeutic targets, facilitating timely interventions for effective treatments. We used the AppNL-G-F knock-in mouse model, which resembles the amyloid pathology and neuroinflammatory characteristics of Alzheimer's disease, to investigate the transition from a pre-plaque to an early plaque stage with a combined functional and molecular approach. Our experiments show a progressive decrease in the power of cognition-relevant hippocampal gamma oscillations during the early stage of amyloid pathology, together with a modification of fast-spiking interneuron intrinsic properties and postsynaptic input. Consistently, transcriptomic analyses revealed that these effects are accompanied by changes in synaptic function-associated pathways. Concurrently, homeostasis- and inflammatory-related microglia signature genes were downregulated. Moreover, we found a decrease in Iba1-positive microglia in the hippocampus that correlates with plaque aggregation and neuronal dysfunction. Collectively, these findings support the hypothesis that microglia play a protective role during the early stages of amyloid pathology by preventing plaque aggregation, supporting neuronal homeostasis, and overall preserving the oscillatory network's functionality. These results suggest that the early alteration of microglia dynamics could be a pivotal event in the progression of Alzheimer's disease, potentially triggering plaque deposition, impairment of fast-spiking interneurons, and the breakdown of the oscillatory circuitry in the hippocampus.

A cell autonomous regulator of neuronal excitability modulates tau in Alzheimer's disease vulnerable neurons.

Neurons from layer II of the entorhinal cortex (ECII) are the first to accumulate tau protein aggregates and degenerate during prodromal Alzheimer's disease. Gaining insight into the molecular mechanisms underlying this vulnerability will help reveal genes and pathways at play during incipient stages of the disease. Here, we use a data-driven functional genomics approach to model ECII neurons in silico and identify the proto-oncogene DEK as a regulator of tau pathology. We show that epigenetic changes caused by Dek silencing alter activity-induced transcription, with major effects on neuronal excitability. This is accompanied by the gradual accumulation of tau in the somatodendritic compartment of mouse ECII neurons in vivo, reactivity of surrounding microglia, and microglia-mediated neuron loss. These features are all characteristic of early Alzheimer's disease. The existence of a cell-autonomous mechanism linking Alzheimer's disease pathogenic mechanisms in the precise neuron type where the disease starts provides unique evidence that synaptic homeostasis dysregulation is of central importance in the onset of tau pathology in Alzheimer's disease.

CYP46A1-mediated cholesterol turnover induces sex-specific changes in cognition and counteracts memory loss in ovariectomized mice.

The brain-specific enzyme CYP46A1 controls cholesterol turnover by converting cholesterol into 24S-hydroxycholesterol (24OH). Dysregulation of brain cholesterol turnover and reduced CYP46A1 levels are observed in Alzheimer's disease (AD). In this study, we report that CYP46A1 overexpression in aged female mice leads to enhanced estrogen signaling in the hippocampus and improved cognitive functions. In contrast, age-matched CYP46A1 overexpressing males show anxiety-like behavior, worsened memory, and elevated levels of 5α-dihydrotestosterone in the hippocampus. We report that, in neurons, 24OH contributes to these divergent effects by activating sex hormone signaling, including estrogen receptors. CYP46A1 overexpression in female mice protects from memory impairments induced by ovariectomy while having no effects in gonadectomized males. Last, we measured cerebrospinal fluid levels of 24OH in a clinical cohort of patients with AD and found that 24OH negatively correlates with neurodegeneration markers only in women. We suggest that CYP46A1 activation is a valuable pharmacological target for enhancing estrogen signaling in women at risk of developing neurodegenerative diseases.

Alzheimer's disease biomarker profiling in a memory clinic cohort without common comorbidities.

Alzheimer's disease is a multifactorial disorder with large heterogeneity. Comorbidities such as hypertension, hypercholesterolaemia and diabetes are known contributors to disease progression. However, less is known about their mechanistic contribution to Alzheimer's pathology and neurodegeneration. The aim of this study was to investigate the relationship of several biomarkers related to risk mechanisms in Alzheimer's disease with the well-established Alzheimer's disease markers in a memory clinic population without common comorbidities. We investigated 13 molecular markers representing key mechanisms underlying Alzheimer's disease pathogenesis in CSF from memory clinic patients without diagnosed hypertension, hypercholesterolaemia or diabetes nor other neurodegenerative disorders. An analysis of covariance was used to compare biomarker levels between clinical groups. Associations were analysed by linear regression. Two-step cluster analysis was used to determine patient clusters. Two key markers were analysed by immunofluorescence staining in the hippocampus of non-demented control and Alzheimer's disease individuals. CSF samples from a total of 90 participants were included in this study: 30 from patients with subjective cognitive decline (age 62.4 ± 4.38, female 60%), 30 with mild cognitive impairment (age 65.6 ± 7.48, female 50%) and 30 with Alzheimer's disease (age 68.2 ± 7.86, female 50%). Angiotensinogen, thioredoxin-1 and interleukin-15 had the most prominent associations with Alzheimer's disease pathology, synaptic and axonal damage markers. Synaptosomal-associated protein 25 kDa and neurofilament light chain were increased in mild cognitive impairment and Alzheimer's disease patients. Grouping biomarkers by biological function showed that inflammatory and survival components were associated with Alzheimer's disease pathology, synaptic dysfunction and axonal damage. Moreover, a vascular/metabolic component was associated with synaptic dysfunction. In the data-driven analysis, two patient clusters were identified: Cluster 1 had increased CSF markers of oxidative stress, vascular pathology and neuroinflammation and was characterized by elevated synaptic and axonal damage, compared with Cluster 2. Clinical groups were evenly distributed between the clusters. An analysis of post-mortem hippocampal tissue showed that compared with non-demented controls, angiotensinogen staining was higher in Alzheimer's disease and co-localized with phosphorylated-tau. The identification of biomarker-driven endophenotypes in cognitive disorder patients further highlights the biological heterogeneity of Alzheimer's disease and the importance of tailored prevention and treatment strategies.

Different effects of CYP27A1 and CYP7B1 on cognitive function: Two mouse models in comparison.

The oxysterol 27-hydroxycholesterol (27OHC) is produced by the enzyme sterol 27-hydroxylase (Cyp27A1) and is mainly catabolized to 7α-Hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) by the enzyme cytochrome P-450 oxysterol 7α-hydroxylase (Cyp7B1). 27OHC is mostly produced in the liver and can reach the brain by crossing the blood-brain barrier. A large body of evidence shows that CYP27A1 overexpression and high levels of 27OHC have a detrimental effect on the brain, causing cognitive and synaptic dysfunction together with a decrease in glucose uptake in mice. In this work, we analyzed two mouse models with high levels of 27OHC: Cyp7B1 knock-out mice and CYP27A1 overexpressing mice. Despite the accumulation of 27OHC in both models, Cyp7B1 knock-out mice maintained intact learning and memory capacities, neuronal morphology, and brain glucose uptake over time. Neurons treated with the Cyp7B1 metabolite 7-HOCA did not show changes in synaptic genes and 27OHC-treated Cyp7B1 knock-out neurons could not counteract 27OHC detrimental effects. This suggests that 7-HOCA and Cyp7B1 deletion in neurons do not mediate the neuroprotective effects observed in Cyp7B1 knock-out animals. RNA-seq of neuronal nuclei sorted from Cyp7B1 knock-out brains revealed upregulation of genes likely to confer neuroprotection to these animals. Differently from Cyp7B1 knock-out mice, transcriptomic data from CYP27A1 overexpressing neurons showed significant downregulation of genes associated with synaptic function and several metabolic processes. Our results suggest that the differences observed in the two models may be mediated by the higher levels of Cyp7B1 substrates such as 25-hydroxycholesterol and 3ß-Adiol in the knock-out mice and that CYP27A1 overexpressing mice may be a more suitable model for studying 27-OHC-specific signaling. We believe that future studies on Cyp7B1 and Cyp27A1 will contribute to a better understanding of the pathogenic mechanisms of neurodegenerative diseases like Alzheimer's disease and may lead to potential new therapeutic approaches.

Sustained Shugoshin 1 downregulation reduces tumor growth and metastasis in a mouse xenograft tumor model of triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TBNC) is an aggressive breast cancer subtype with a poor prognosis. Shugoshin-1 (SGO1) protects chromatids from early separation. Previous studies from our group have demonstrated that transient SGO1 downregulation suppresses early stages of metastasis (the epithelial-to-mesenchymal transition, or EMT, cell invasion, and cell migration) in TNBC cells. Thus, the inhibition of SGO1 activity may represent a potential therapeutic intervention against cancers that progress to metastasis. Therefore, we aimed to investigate the effects of sustained shRNA-mediated SGO1 downregulation on tumor growth and metastasis in TBNC. To that end, female NOD-SCID Gamma (NSG) mice were injected with 2.5 × 106 shRNA Control (n = 10) or shRNA SGO1 (n = 10) MDA-MB-231 cells. After eight weeks, the number of mice with metastasis to the lymph nodes was calculated. Primary and metastatic tumors, as well as lung and liver tissue, were harvested, measured, sectioned, and stained with hematoxylin and eosin (H&E) stain. RESULTS: Tumor growth and metastasis to the lymph nodes and lungs were significantly reduced in the shRNA SGO1-treated mice group, while metastasis to the liver tends to be lower in cells with downregulated SGO1, but it did not reach statistical significance. Furthermore, sustained SGO1 downregulation significantly reduced cell proliferation, cell migration, and invasion which correlated with lower levels of Snail, Slug, MMP2, MMP3, and MMP9. CONCLUSION: The supression of SGO1 activity in TNBC harboring dysregulated expression of SGO1 may be a potential target for preventing breast cancer growth and metastasis.

Defects of Nutrient Signaling and Autophagy in Neurodegeneration.

Neurons are post-mitotic cells that allocate huge amounts of energy to the synthesis of new organelles and molecules, neurotransmission and to the maintenance of redox homeostasis. In neurons, autophagy is not only crucial to ensure organelle renewal but it is also essential to balance nutritional needs through the mobilization of internal energy stores. A delicate crosstalk between the pathways that sense nutritional status of the cell and the autophagic processes to recycle organelles and macronutrients is fundamental to guarantee the proper functioning of the neuron in times of energy scarcity. This review provides a detailed overview of the pathways and processes involved in the balance of cellular energy mediated by autophagy, which when defective, precipitate the neurodegenerative cascade of Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or Alzheimer's disease.

Deciphering cell-type specific signal transduction in the brain: Challenges and promises.

Signal transduction designates the set of molecular events that take place within a cell upon extracellular stimulation to mediate a functional outcome. Decades after the discovery that dopamine triggers opposing signaling pathways in D1- and D2-expressing medium spiny neurons, it is now clear that there are as many different flavors of signaling pathways in the brain as there are neuron types. One of the biggest challenges in molecular neuroscience is to elucidate cell-type specific signaling, in order to understand neurological diseases with regional vulnerability, but also to identify targets for precision drugs devoid of off-target effects. Here, we make a case for the importance of the study of neuron-type specific molecular characteristics. We then review the technologies that exist to study neurons in their full diversity and highlight their disease-relevant idiosyncrasies.

Sex difference in flux of 27-hydroxycholesterol into the brain.

BACKGROUND AND PURPOSE: The cerebrospinal fluid (CSF)/plasma albumin ratio (QAlb) is believed to reflect the integrity of the blood-brain barrier (BBB). Recently, we reported that QAlb is lower in females. This may be important for uptake of neurotoxic 27-hydroxycholesterol (27OH) by the brain in particular because plasma levels of 27OH are higher in males. We studied sex differences in the relation between CSF and plasma levels of 27OH and its major metabolite 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) with QAlb. We tested the possibility of sex differences in the brain metabolism of 27OH and if its flux into the brain disrupted integrity of the BBB. EXPERIMENTAL APPROACH: We have examined our earlier studies looking for sex differences in CSF levels of oxysterols and their relation to QAlb. We utilized an in vitro model for the BBB with primary cultured brain endothelial cells to test if 27OH has a disruptive effect on this barrier. We measured mRNA and protein levels of CYP7B1 in autopsy brain samples. KEY RESULTS: The correlation between CSF levels of 27OH and QAlb was higher in males whereas, with 7HOCA, the correlation was higher in females. No significant sex difference in the expression of CYP7B1 mRNA in brain autopsy samples. A correlation was found between plasma levels of 27OH and QAlb. No support was obtained for the hypothesis that plasma levels of 27OH have a disruptive effect on the BBB. CONCLUSIONS AND IMPLICATIONS: The sex differences are discussed in relation to negative effects of 27OH on different brain functions. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Selective Neuronal Vulnerability in Alzheimer's Disease: A Network-Based Analysis.

A major obstacle to treating Alzheimer's disease (AD) is our lack of understanding of the molecular mechanisms underlying selective neuronal vulnerability, a key characteristic of the disease. Here, we present a framework integrating high-quality neuron-type-specific molecular profiles across the lifetime of the healthy mouse, which we generated using bacTRAP, with postmortem human functional genomics and quantitative genetics data. We demonstrate human-mouse conservation of cellular taxonomy at the molecular level for neurons vulnerable and resistant in AD, identify specific genes and pathways associated with AD neuropathology, and pinpoint a specific functional gene module underlying selective vulnerability, enriched in processes associated with axonal remodeling, and affected by amyloid accumulation and aging. We have made all cell-type-specific profiles and functional networks available at http://alz.princeton.edu. Overall, our study provides a molecular framework for understanding the complex interplay between Aß, aging, and neurodegeneration within the most vulnerable neurons in AD.

27-Hydroxycholesterol Induces Aberrant Morphology and Synaptic Dysfunction in Hippocampal Neurons.

Hypercholesterolemia is a risk factor for neurodegenerative diseases, but how high blood cholesterol levels are linked to neurodegeneration is still unknown. Here, we show that an excess of the blood-brain barrier permeable cholesterol metabolite 27-hydroxycholesterol (27-OH) impairs neuronal morphology and reduces hippocampal spine density and the levels of the postsynaptic protein PSD95. Dendritic spines are the main postsynaptic elements of excitatory synapses and are crucial structures for memory and cognition. Furthermore, PSD95 has an essential function for synaptic maintenance and plasticity. PSD95 synthesis is controlled by the REST-miR124a-PTBP1 axis. Here, we report that high levels of 27-OH induce REST-miR124a-PTBP1 axis dysregulation in a possible RxRγ-dependent manner, suggesting that 27-OH reduces PSD95 levels through this mechanism. Our results reveal a possible molecular link between hypercholesterolemia and neurodegeneration. We discuss the possibility that reduction of 27-OH levels could be a useful strategy for preventing memory and cognitive decline in neurodegenerative disorders.

Tau hyperphosphorylation induces oligomeric insulin accumulation and insulin resistance in neurons.

Insulin signalling deficiencies and insulin resistance have been directly linked to the progression of neurodegenerative disorders like Alzheimer's disease. However, to date little is known about the underlying molecular mechanisms or insulin state and distribution in the brain under pathological conditions. Here, we report that insulin is accumulated and retained as oligomers in hyperphosphorylated tau-bearing neurons in Alzheimer's disease and in several of the most prevalent human tauopathies. The intraneuronal accumulation of insulin is directly dependent on tau hyperphosphorylation, and follows the tauopathy progression. Furthermore, cells accumulating insulin show signs of insulin resistance and decreased insulin receptor levels. These results suggest that insulin retention in hyperphosphorylated tau-bearing neurons is a causative factor for the insulin resistance observed in tauopathies, and describe a novel neuropathological concept with important therapeutic implications.

Aggregation of the Inflammatory S100A8 Precedes Aß Plaque Formation in Transgenic APP Mice: Positive Feedback for S100A8 and Aß Productions.

Inflammation plays an important role in Alzheimer's disease (AD) and other neurodegenerative disorders. Although chronic inflammation in later stages of AD is well described, little is known about the inflammatory processes in preclinical or early stages of the disease prior to plaque deposition. In this study, we report that the inflammatory mediator S100A8 is increased with aging in the mouse brain. It is observed as extracellular aggregates, which do not correspond to corpora amylacea. S100A8 aggregation is enhanced in the hippocampi of two different mouse models for amyloid-ß (Aß) overproduction (Tg2576 and TgAPParctic mice). S100A8 aggregates are seen prior the formation of Aß plaques and do not colocalize. In vitro treatment of glial cells from primary cultures with Aß42 resulted in an increased production of S100A8. In parallel, treatment of a neuronal cell line with recombinant S100A8 protein resulted in enhanced Aß42 and decreased Aß40 production. Our results suggest that important inflammatory processes are occurring prior to Aß deposition and the existence of a positive feedback between S100A8 and Aß productions. The possible relevance of aging- or AD-dependent formation of S100A8 aggregates in the hippocampus thus affecting learning and memory processes is discussed.

Anthocyanins protect from complex I inhibition and APPswe mutation through modulation of the mitochondrial fission/fusion pathways.

Anthocyanins are a distinguished class of flavonoids with powerful free radical-scavenging activity that have been suggested as chemotherapeutic agents for the prevention of Alzheimer disease (AD). In this study, we examined the ability of nutraceutical Medox rich in purified cyanidin 3-O-glucoside (C3G), 3-O-b-glucosides and delphinidin 3-O-glucoside (D3G) to counteract mitochondrial deficiency induced by complex I inhibition and/or amyloid-ß peptide (Aß) induced toxicity. SH-SY5Y neuroblastoma cells were stably transfected with APP Swedish K670N/M671L double mutation (APPswe) or with the empty vector and treated with rotenone. We report that Medox treatment improves the metabolic activity and maintains cell integrity in both cell lines. At the mitochondrial level, APPswe and rotenone induced mitochondrial fragmentation, an effect that was counteracted by Medox through the modulation of fission and fusion proteins, resulting in a reshaped mitochondrial network. Although Medox was unable to fully neutralise the effects of rotenone on ATP levels and mitochondrial membrane potential, it was able to prevent rotenone-induced cytotoxicity. Our findings suggest that Medox anthocyanins, on top of their antioxidant capacity, ameliorate mitochondrial dysfunction generated by Aß overproduction or by chemical inhibition of mitochondrial complex I via stabilization of the fusion/fission processes. Modulation of the mitochondrial network has been suggested as a novel therapeutic approach in diseases involving mitochondrial dysfunction and oxidative stress. Hence, increasing the understanding of how anthocyanins influence mitochondrial dynamics in a neurodegenerative context, could be of future therapeutic value.

Underestimation of the pentose-phosphate pathway in intact primary neurons as revealed by metabolic flux analysis.

The rates of glucose oxidized at glycolysis and pentose-phosphate pathway (PPP) in neurons are controversial. Using [3-(3)H]-, [1-(14)C]-, and [6-(14)C]glucose to estimate fluxes through these pathways in resting, intact rat cortical primary neurons, we found that the rate of glucose oxidized through PPP was, apparently, ∼14% of total glucose metabolized. However, inhibition of PPP rate-limiting step, glucose-6-phosphate (G6P) dehydrogenase, increased approximately twofold the glycolytic rate; and, knockdown of phosphoglucose isomerase increased ∼1.8-fold the PPP rate. Thus, in neurons, a considerable fraction of fructose-6-phosphate returning from the PPP contributes to the G6P pool that re-enters PPP, largely underestimating its flux.

Brain energy metabolism in glutamate-receptor activation and excitotoxicity: role for APC/C-Cdh1 in the balance glycolysis/pentose phosphate pathway.

Recent advances in the field of brain energy metabolism strongly suggest that glutamate receptor-mediated neurotransmission is coupled with molecular signals that switch-on glucose utilization pathways to meet the high energetic requirements of neurons. Failure to adequately coordinate energy supply for neurotransmission ultimately results in a positive amplifying loop of receptor over-activation leading to neuronal death, a process known as excitotoxicity. In this review, we revisited current concepts in excitotoxic mechanisms, their involvement in energy substrate utilization, and the signaling pathways that coordinate both processes. In particular, we have focused on the novel role played by the E3 ubiquitin ligase, anaphase-promoting complex/cyclosome (APC/C)-Cdh1, in cell metabolism. Our laboratory identified 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) -a key glycolytic-promoting enzyme- as an APC/C-Cdh1 substrate. Interestingly, APC/C-Cdh1 activity is inhibited by over-activation of glutamate receptors through a Ca(2+)-mediated mechanism. Furthermore, by inhibiting APC/C-Cdh1 activity, glutamate-receptors activation promotes PFKFB3 stabilization, leading to increased glycolysis and decreased pentose-phosphate pathway activity. This causes a loss in neuronal ability to regenerate glutathione, triggering oxidative stress and delayed excitotoxicity. Further investigation is critical to identify novel molecules responsible for the coupling of energy metabolism with glutamatergic neurotransmission and excitotoxicity, as well as to help developing new therapeutic strategies against neurodegeneration.