RESUMO
BACKGROUND: Hepatic lipoprotein receptor-related protein 1 (LRP-1) plays a central role in peripheral amyloid beta (Aß) clearance, but its importance in Alzheimer's disease (AD) pathology is understudied. Our previous work showed that intragastric alcohol feeding to C57BL/6 J mice reduced hepatic LRP-1 expression which correlated with significant AD-relevant brain changes. Herein, we examined the role of hepatic LRP-1 in AD pathogenesis in APP/PS1 AD mice using two approaches to modulate hepatic LRP-1, intragastric alcohol feeding to model chronic heavy drinking shown by us to reduce hepatic LRP-1, and hepato-specific LRP-1 silencing. METHODS: Eight-month-old male APP/PS1 mice were fed ethanol or control diet intragastrically for 5 weeks (n = 7-11/group). Brain and liver Aß were assessed using immunoassays. Three important mechanisms of brain amyloidosis were investigated: hepatic LRP-1 (major peripheral Aß regulator), blood-brain barrier (BBB) function (vascular Aß regulator), and microglia (major brain Aß regulator) using immunoassays. Spatial LRP-1 gene expression in the periportal versus pericentral hepatic regions was confirmed using NanoString GeoMx Digital Spatial Profiler. Further, hepatic LRP-1 was silenced by injecting LRP-1 microRNA delivered by the adeno-associated virus 8 (AAV8) and the hepato-specific thyroxine-binding globulin (TBG) promoter to 4-month-old male APP/PS1 mice (n = 6). Control male APP/PS1 mice received control AAV8 (n = 6). Spatial memory and locomotion were assessed 12 weeks after LRP-1 silencing using Y-maze and open-field test, respectively, and brain and liver Aß were measured. RESULTS: Alcohol feeding reduced plaque-associated microglia in APP/PS1 mice brains and increased aggregated Aß (p < 0.05) by ELISA and 6E10-positive Aß load by immunostaining (p < 0.05). Increased brain Aß corresponded with a significant downregulation of hepatic LRP-1 (p < 0.01) at the protein and transcript level, primarily in pericentral hepatocytes (zone 3) where alcohol-induced injury occurs. Hepato-specific LRP-1 silencing significantly increased brain Aß and locomotion hyperactivity (p < 0.05) in APP/PS1 mice. CONCLUSION: Chronic heavy alcohol intake reduced hepatic LRP-1 expression and increased brain Aß. The hepato-specific LRP-1 silencing similarly increased brain Aß which was associated with behavioral deficits in APP/PS1 mice. Collectively, our results suggest that hepatic LRP-1 is a key regulator of brain amyloidosis in alcohol-dependent AD.
Assuntos
Doença de Alzheimer , Fígado , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Animais , Doença de Alzheimer/metabolismo , Masculino , Camundongos , Fígado/metabolismo , Amiloidose/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Etanol/administração & dosagem , Modelos Animais de Doenças , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Biologic TNF-α inhibitors (bTNFIs) can block cerebral TNF-α in Alzheimer's disease (AD) if these macromolecules can cross the blood-brain barrier (BBB). Thus, a model bTNFI, the extracellular domain of type II TNF-α receptor (TNFR), which can bind to and sequester TNF-α, was fused with a mouse transferrin receptor antibody (TfRMAb) to enable brain delivery via BBB TfR-mediated transcytosis. Previously, we found TfRMAb-TNFR to be protective in a mouse model of amyloidosis (APP/PS1) and tauopathy (PS19), and herein we investigated its effects in mice that combine both amyloidosis and tauopathy (3xTg-AD). METHODS: Eight-month-old female 3xTg-AD mice were injected intraperitoneally with saline (n = 11) or TfRMAb-TNFR (3 mg/kg; n = 11) three days per week for 12 weeks. Age-matched wild-type (WT) mice (n = 9) were treated similarly with saline. Brains were processed for immunostaining and high-resolution multiplex NanoString GeoMx spatial proteomics. RESULTS: We observed regional differences in proteins relevant to Aß, tau, and neuroinflammation in the hippocampus of 3xTg-AD mice compared with WT mice. From 64 target proteins studied using spatial proteomics, a comparison of the Aß-plaque bearing vs. plaque-free regions in the 3xTg-AD mice yielded 39 differentially expressed proteins (DEP) largely related to neuroinflammation (39% of DEP) and Aß and tau pathology combined (31% of DEP). Hippocampal spatial proteomics revealed that the majority of the proteins modulated by TfRMAb-TNFR in the 3xTg-AD mice were relevant to microglial function (â 33%). TfRMAb-TNFR significantly reduced mature Aß plaques and increased Aß-associated microglia around larger Aß deposits in the 3xTg-AD mice. Further, TfRMAb-TNFR increased mature Aß plaque-associated microglial TREM2 in 3xTg-AD mice. CONCLUSION: Overall, despite the low visual Aß load in the 11-month-old female 3xTg-AD mice, our results highlight region-specific AD-relevant DEP in the hippocampus of these mice. Chronic TfRMAb-TNFR dosing modulated several DEP involved in AD pathology and showed a largely microglia-centric mechanism of action in the 3xTg-AD mice.
Assuntos
Doença de Alzheimer , Amiloidose , Produtos Biológicos , Camundongos , Feminino , Animais , Doença de Alzheimer/tratamento farmacológico , Fator de Necrose Tumoral alfa/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Doenças Neuroinflamatórias , Camundongos Transgênicos , Encéfalo/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Amiloidose/metabolismo , Amiloidose/patologia , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Anticorpos/metabolismo , Produtos Biológicos/metabolismo , Modelos Animais de DoençasRESUMO
BACKGROUND: Tumor necrosis factor-α (TNF-α) plays a central role in Alzheimer's disease (AD) pathology, making biologic TNF-α inhibitors (TNFIs), including etanercept, viable therapeutics for AD. The protective effects of biologic TNFIs on AD hallmark pathology (Aß deposition and tau pathology) have been demonstrated. However, the effects of biologic TNFIs on Aß-independent tau pathology have not been reported. Existing biologic TNFIs do not cross the blood-brain barrier (BBB), therefore we engineered a BBB-penetrating biologic TNFI by fusing the extracellular domain of the type-II human TNF-α receptor (TNFR) to a transferrin receptor antibody (TfRMAb) that ferries the TNFR into the brain via receptor-mediated transcytosis. The present study aimed to investigate the effects of TfRMAb-TNFR (BBB-penetrating TNFI) and etanercept (non-BBB-penetrating TNFI) in the PS19 transgenic mouse model of tauopathy. METHODS: Six-month-old male and female PS19 mice were injected intraperitoneally with saline (n = 12), TfRMAb-TNFR (1.75 mg/kg, n = 10) or etanercept (0.875 mg/kg, equimolar dose of TNFR, n = 10) 3 days/week for 8 weeks. Age-matched littermate wild-type mice served as additional controls. Blood was collected at baseline and 8 weeks for a complete blood count. Locomotion hyperactivity was assessed by the open-field paradigm. Brains were examined for phosphorylated tau lesions (Ser202, Thr205), microgliosis, and neuronal health. The plasma pharmacokinetics were evaluated following a single intraperitoneal injection of 0.875 mg/kg etanercept or 1.75 mg/kg TfRMAb-TNFR or 1.75 mg/kg chronic TfRMAb-TNFR dosing for 4 weeks. RESULTS: Etanercept significantly reduced phosphorylated tau and microgliosis in the PS19 mouse brains of both sexes, while TfRMAb-TNFR significantly reduced these parameters in the female PS19 mice. Both TfRMAb-TNFR and etanercept treatment improved neuronal health by significantly increasing PSD95 expression and attenuating hippocampal neuron loss in the PS19 mice. The locomotion hyperactivity in the male PS19 mice was suppressed by chronic etanercept treatment. Equimolar dosing resulted in eightfold lower plasma exposure of the TfRMAb-TNFR compared with etanercept. The hematological profiles remained largely stable following chronic biologic TNFI dosing except for a significant increase in platelets with etanercept. CONCLUSION: Both TfRMAb-TNFR (BBB-penetrating) and non-BBB-penetrating (etanercept) biologic TNFIs showed therapeutic effects in the PS19 mouse model of tauopathy.
Assuntos
Gliose/prevenção & controle , Neurônios/patologia , Tauopatias/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas tau/antagonistas & inibidores , Animais , Proteína 4 Homóloga a Disks-Large/biossíntese , Proteína 4 Homóloga a Disks-Large/genética , Etanercepte/farmacocinética , Etanercepte/farmacologia , Feminino , Hipocampo/patologia , Humanos , Hipercinese , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação , Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Tauopatias/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Erythropoietin (EPO), a hematopoietic neurotrophin, is a potential therapeutic for Alzheimer's disease (AD) but has limited blood-brain barrier (BBB) permeability. EPO fused to a chimeric transferrin receptor monoclonal antibody (cTfRMAb) enters the brain via TfR-mediated transcytosis across the BBB. We previously showed that cTfRMAb-EPO is protective in a mouse model of amyloidosis, but its effects on tauopathy are not known. Given that amyloid and tau pathology are characteristics of AD, the effects of cTfRMAb-EPO were studied in a tauopathy mouse model (PS19). Six-month-old PS19 mice were injected intraperitoneally with either saline (PS19-Saline; n = 9) or cTfRMAb-EPO (PS19-cTfRMAb-EPO, 10 mg/kg; n = 10); every two or three days on alternate weeks for 8 weeks. Age-matched, saline-treated, wildtype littermates (WT-Saline; n = 12) were injected using the same protocol. After 8 weeks, locomotion, hyperactivity, and anxiety were assessed via the open-field test, and brains were harvested and sectioned. Cerebral cortex, hippocampus, amygdala, and entorhinal cortex sections were analyzed for phospho-tau (AT8) and microgliosis (Iba1). Hippocampal cellular density (H&E) was also assessed. PS19-Saline mice were hyperactive and less anxious compared to WT-Saline mice, and these behavioral phenotypes were significantly reduced in the PS19-cTfRMAb-EPO mice compared to the PS19-Saline mice. cTfRMAb-EPO significantly reduced AT8 load by ≥50% in all of the brain regions analyzed and microgliosis in the entorhinal cortex and amygdala compared to the PS19-Saline mice. Hippocampal pyramidal and granule cell layer density did not differ significantly between the PS19-cTfRMAb-EPO and PS19-Saline mice. This proof-of-concept study demonstrates the therapeutic effects of the BBB-penetrating cTfRMAb-EPO in PS19 mice.
RESUMO
Tumor necrosis factor alpha (TNF-α) plays a vital role in Alzheimer's disease (AD) pathology, and TNF-α inhibitors (TNFIs) modulate AD pathology. We fused the TNF-α receptor (TNFR), a biologic TNFI that sequesters TNF-α, to a transferrin receptor antibody (TfRMAb) to deliver the TNFI into the brain across the blood-brain barrier (BBB). TfRMAb-TNFR was protective in 6-month-old transgenic APP/PS1 mice in our previous work. However, the effects and safety following delayed chronic TfRMAb-TNFR treatment are unknown. Herein, we initiated the treatment when the male APP/PS1 mice were 10.7 months old (delayed treatment). Mice were injected intraperitoneally with saline, TfRMAb-TNFR, etanercept (non-BBB-penetrating TNFI), or TfRMAb for ten weeks. Biologic TNFIs did not alter hematology indices or tissue iron homeostasis; however, TfRMAb altered hematology indices, increased splenic iron transporter expression, and increased spleen and liver iron. TfRMAb-TNFR and etanercept reduced brain insoluble-amyloid beta (Aß) 1-42, soluble-oligomeric Aß, and microgliosis; however, only TfRMAb-TNFR reduced Aß peptides, Thioflavin-S-positive Aß plaques, and insoluble-oligomeric Aß and increased plaque-associated phagocytic microglia. Accordingly, TfRMAb-TNFR improved spatial reference memory and increased BBB-tight junction protein expression, whereas etanercept did not. Overall, despite delayed treatment, TfRMAb-TNFR resulted in a better therapeutic response than etanercept without any TfRMAb-related hematology- or iron-dysregulation in aged APP/PS1 mice.
RESUMO
The role of glycosaminoglycan sulfation patterns, particularly in regard to scar formation and inhibition of neuritogenesis, has been mainly studied in cell culture with a focus on chondroitin 4-sulfate. In this study, we investigated chondroitin 6-sulfate (C6S) and found that it also inhibits neurite outgrowth of mouse cerebellar granule neurons in vitro. To examine whether the inhibitory activity of C6S could be neutralized, seven previously characterized high-affinity C6S-binding peptides were tested, among which three peptides neutralized the inhibitory functions of C6S. We further investigated these peptides in a mouse model of spinal cord injury, since upregulation of C6S expression in the glial scar following injury has been associated with reduced axonal regrowth and functional recovery. We here subjected mice to severe compression injury at thoracic levels T7-T9, immediately followed by inserting gelfoam patches soaked in C6S-binding peptides or in a control peptide. Application of C6S-binding peptides led to functional recovery after injury and prevented fibrotic glial scar formation, as seen by decreased activation of astrocytes and microglia/macrophages. Decreased expression of several lecticans and deposition of fibronectin at the site of injury were also observed. Application of C6S-binding peptides led to axonal regrowth and inhibited the C6S-mediated activation of RhoA/ROCK and decrease of PI3K-Akt-mTOR signaling pathways. Taken together, these results indicate that treatment with C6S-binding peptides improves functional recovery in a mouse model of spinal cord injury.
Assuntos
Sulfatos de Condroitina/metabolismo , Sulfatos de Condroitina/farmacologia , Peptídeos/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Axônios/efeitos dos fármacos , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Sulfatos de Condroitina/uso terapêutico , Cicatriz/tratamento farmacológico , Modelos Animais de Doenças , Gliose/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Locomoção/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Peptídeos/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Recuperação de Função Fisiológica/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Traumatismos da Medula Espinal/etiologia , Traumatismos da Medula Espinal/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Besides several endogenous elements, exogenous factors, including exposure to pesticides, have been recognized as putative factors contributing to the onset and development of neurodegenerative diseases, including Parkinson's disease (PD). Considering the availability, success rate, and limitations associated with the current arsenals to fight PD, there is an unmet need for novel therapeutic interventions. Therefore, based on the previously reported beneficial functions of the L1 cell adhesion molecule, we hypothesized that L1 mimetic compounds may serve to neutralize neurotoxicity triggered by the pesticide paraquat (PQ). In this study, we attempt to use PQ for inducing PD-like pathology and the L1 mimetic compounds phenelzine sulfate (PS) and tacrine (TC) as potential candidates for the amelioration of PD symptoms using zebrafish as a model system. Administration of PQ together with the L1 mimetic compounds PS or TC (250 nM) improved survival of zebrafish larvae, protected them from locomotor deficits, and increased their sensorimotor reflexes. Moreover, application of PQ together with PS (500 nM) or TC (1000 nM) in adult zebrafish counteracted PQ-induced toxicity, maintaining normal locomotor functions and spatial memory in an open field and T-maze task, respectively. Both L1 mimetic compounds prevented reduction in tyrosine hydroxylase and dopamine levels, reduced reactive oxygen species (ROS) generation, protected against impairment of mitochondrial viability, improved the antioxidant enzyme system, and prevented a decrease in ATP levels. Altogether, our findings highlight the beneficial functions of the agonistic L1 mimetics PS and TC by improving several vital cell functions against PQ-triggered neurotoxicity.