RESUMO
Recent advances in immunotherapeutic approaches to the treatment of Alzheimer's disease (AD) have increased the importance of understanding the exact binding preference of each amyloid-beta (Aß) antibody employed, since this determines both efficacy and risk for potentially serious adverse events known as amyloid-related imaging abnormalities. Lecanemab is a humanized IgG1 antibody that was developed to target the soluble Aß protofibril conformation. The present study prepared extracts of post mortem brain samples from AD patients and non-demented elderly controls, characterized the forms of Aß present, and investigated their interactions with lecanemab. Brain tissue samples were homogenized and extracted using tris-buffered saline. Aß levels and aggregation states in soluble and insoluble extracts, and in fractions prepared using size-exclusion chromatography or density gradient ultracentrifugation, were analyzed using combinations of immunoassay, immunoprecipitation (IP), and mass spectrometry. Lecanemab immunohistochemistry was also conducted in temporal cortex. The majority of temporal cortex Aß (98 %) was in the insoluble extract. Aß42 was the most abundant form present, particularly in AD subjects, and most soluble Aß42 was in soluble aggregated protofibrillar structures. Aß protofibril levels were much higher in AD subjects than in controls. Protofibrils captured by lecanemab-IP contained high levels of Aß42 and lecanemab bound to large, medium, and small Aß42 protofibrils in a concentration-dependent manner. Competitive IP showed that neither Aß40 monomers nor Aß40-enriched fibrils isolated from cerebral amyloid angiopathy reduced lecanemab's binding to Aß42 protofibrils. Immunohistochemistry showed that lecanemab bound readily to Aß plaques (diffuse and compact) and to intraneuronal Aß in AD temporal cortex. Taken together, these findings indicate that while lecanemab binds to Aß plaques, it preferentially targets soluble aggregated Aß protofibrils. These are largely composed of Aß42, and lecanemab binds less readily to the Aß40-enriched fibrils found in the cerebral vasculature. This is a promising binding profile because Aß42 protofibrils represent a key therapeutic target in AD, while a lack of binding to monomeric Aß and cerebral amyloid deposits should reduce peripheral antibody sequestration and minimize risk for adverse events.
RESUMO
Amyloid beta (Aß) peptides, which aggregate to form neocortical plaques in Alzheimer's disease, exist in states that range from soluble monomers and oligomers/protofibrils to insoluble fibrillar amyloid. The present study evaluated the effects of mAb158, a mouse monoclonal antibody version of lecanemab that preferentially binds to soluble Aß protofibrils, in aged transgenic mice (Tg2576) with Aß pathology. Female Tg2576 mice (12 months old) received weekly intraperitoneal mAb158 (35 mg/kg) or vehicle for 4 weeks or for 18 weeks, with or without a subsequent 12-week off-treatment period. Aß protofibril levels were significantly lower in mAb158-treated animals at both 4 and 18 weeks, while longer treatment duration (18 weeks) was required to observe significantly lower Aß42 levels in insoluble brain fractions and lower Aß plaque load. Following the off-treatment period, comparison of the vehicle- and mAb158-treated mice demonstrated that the Aß protofibril levels, insoluble Aß42 levels and Aß plaque load remained significantly lower in mAb158-treated animals, as compared with age-matched controls. However, there was a significant increase of brain accumulation of both the Aß protofibril levels, insoluble Aß42 levels and Aß plaque load after treatment cessation. Thus, repeated mAb158 treatment of aged Tg2576 mice first reduced Aß protofibril levels within 4 weeks of treatment, which then was followed by a reduction of amyloid plaque pathology within 18 weeks of treatment. These effects were maintained 12 weeks after the final dose, indicating that mAb158 had a disease-modifying effect on the Aß pathology in this mouse model. In addition, brain accumulation of both Aß protofibril levels and amyloid pathology progressed after discontinuation of the treatment which supports the importance of continued treatment with mAb158 to maintain the effects on Aß pathology.
RESUMO
Down syndrome (DS) is caused by trisomy of chromosome 21, which leads to a propensity to develop amyloid ß (Aß) brain pathology in early adulthood followed later by cognitive and behavioral deterioration. Characterization of the Aß pathology is important to better understand the clinical deterioration of DS individuals and to identify interventive strategies. Brain samples from people with DS and Alzheimer's disease (AD), as well as non-demented controls (NDC), were analyzed with respect to different Aß species. Immunohistochemical staining using antibodies towards Aß was also performed. Elevated levels of soluble Aß protofibrils and insoluble Aßx-40 and Aßx-42 in formic acid brain extracts, and elevated immunohistochemical staining of Aß deposits were demonstrated with the antibody BAN2401 (lecanemab) in DS and AD compared with NDC. These data and the promising data in a large phase 2 CE clinical trial with lecanemab suggest that lecanemab may have the potential to preserve cognitive capacity in DS. Lecanemab is currently in a phase 3 CE clinical trial.
Assuntos
Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Síndrome de Down/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Encéfalo/patologia , Síndrome de Down/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
A growing body of evidence suggests that aggregated α-synuclein, the major constituent of Lewy bodies, plays a key role in the pathogenesis of Parkinson's disease and related α-synucleinopathies. Immunotherapies, both active and passive, against α-synuclein have been developed and are promising novel treatment strategies for such disorders. Here, we report on the humanization and pharmacological characteristics of ABBV-0805, a monoclonal antibody that exhibits a high selectivity for human aggregated α-synuclein and very low affinity for monomers. ABBV-0805 binds to a broad spectrum of soluble aggregated α-synuclein, including small and large aggregates of different conformations. Binding of ABBV-0805 to pathological α-synuclein was demonstrated in Lewy body-positive post mortem brains of Parkinson's disease patients. The functional potency of ABBV-0805 was demonstrated in several cellular assays, including Fcγ-receptor mediated uptake of soluble aggregated α-synuclein in microglia and inhibition of neurotoxicity in primary neurons. In vivo, the murine version of ABBV-0805 (mAb47) displayed significant dose-dependent decrease of α-synuclein aggregates in brain in several mouse models, both in prophylactic and therapeutic settings. In addition, mAb47 treatment of α-synuclein transgenic mice resulted in a significantly prolonged survival. ABBV-0805 selectively targets soluble toxic α-synuclein aggregates with a picomolar affinity and demonstrates excellent in vivo efficacy. Based on the strong preclinical findings described herein, ABBV-0805 has been progressed into clinical development as a potential disease-modifying treatment for Parkinson's disease.
Assuntos
Anticorpos Monoclonais , Doença de Parkinson , Sinucleinopatias , Animais , Anticorpos Monoclonais/uso terapêutico , Humanos , Longevidade , Camundongos , Camundongos Transgênicos , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Sinucleinopatias/terapia , alfa-Sinucleína/metabolismoRESUMO
Immunotherapy targeting aggregated α-synuclein has emerged as a potential treatment strategy against Parkinson's disease and other α-synucleinopathies. We have developed α-synuclein oligomer/protofibril selective antibodies that reduce toxic α-synuclein in a human cell line and, upon intraperitoneal administration, in spinal cord of transgenic mice. Here, we investigated under which conditions and by which mechanisms such antibodies can be internalized by cells. For this purpose, human neuroglioma H4 cells were treated with either monoclonal oligomer/protofibril selective α-synuclein antibodies, linear epitope monoclonal α-synuclein antibodies, or with a control antibody. The oligomer/protofibril selective antibody mAb47 displayed the highest cellular uptake and was therefore chosen for additional analyses. Next, α-synuclein overexpressing cells were incubated with mAb47, which resulted in increased antibody internalization as compared to non-transfected cells. Similarly, regular cells exposed to mAb47 together with media containing α-synuclein displayed a higher uptake as compared to cells incubated with regular media. Finally, different Fcγ receptors were targeted and we then found that blockage of FcγRI and FcγRIIB/C resulted in reduced antibody internalization. Our data thus indicate that the robust uptake of the oligomer/protofibril selective antibody mAb47 by human CNS-derived cells is enhanced by extracellular α-synuclein and mediated via Fcγ receptors. Altogether, our finding lend further support to the belief that α-synuclein pathology can be modified by monoclonal antibodies and that these can target toxic α-synuclein species in the extracellular milieu. In the context of immunotherapy, antibody binding of α-synuclein would then not only block further aggregation but also mediate internalization and subsequent degradation of antigen-antibody complexes.
Assuntos
Anticorpos Monoclonais/metabolismo , Espaço Extracelular/metabolismo , Receptores de IgG/fisiologia , alfa-Sinucleína/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Camundongos , Camundongos TransgênicosRESUMO
Several lines of evidence suggest that accumulation of aggregated alpha-synuclein (α-synuclein) in the central nervous system (CNS) is an early pathogenic event in Parkinson's disease and other Lewy body disorders. In recent years, animal studies have indicated immunotherapy with antibodies directed against α-synuclein as a promising novel treatment strategy. Since large α-synuclein oligomers, or protofibrils, have been demonstrated to possess pronounced cytotoxic properties, such species should be particularly attractive as therapeutic targets. In support of this, (Thy-1)-h[A30P] α-synuclein transgenic mice with motor dysfunction symptoms were found to display increased levels of α-synuclein protofibrils in the CNS. An α-synuclein protofibril-selective monoclonal antibody (mAb47) was evaluated in this α-synuclein transgenic mouse model. As measured by ELISA, 14month old mice treated for 14weeks with weekly intraperitoneal injections of mAb47 displayed significantly lower levels of both soluble and membrane-associated protofibrils in the spinal cord. Besides the lower levels of pathogenic α-synuclein demonstrated, a reduction of motor dysfunction in transgenic mice upon peripheral administration of mAb47 was indicated. Thus, immunotherapy with antibodies targeting toxic α-synuclein species holds promise as a future disease-modifying treatment in Parkinson's disease and related disorders.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunização Passiva , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/terapia , alfa-Sinucleína/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Encéfalo/imunologia , Encéfalo/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Injeções Intraperitoneais , Masculino , Camundongos Transgênicos , Atividade Motora/fisiologia , Mutação , Transtornos Parkinsonianos/imunologia , Índice de Gravidade de Doença , Medula Espinal/imunologia , Medula Espinal/patologia , alfa-Sinucleína/genéticaRESUMO
Therapeutic antibodies have been developed to target amyloid-beta (Aß), and some of these slow the progression of Alzheimer's disease (AD). However, they can also cause adverse events known as amyloid-related imaging abnormalities with edema (ARIA-E). We investigated therapeutic Aß antibody binding to cerebral amyloid angiopathy (CAA) fibrils isolated from human leptomeningeal tissue to study whether this related to the ARIA-E frequencies previously reported by clinical trials. The binding of Aß antibodies to CAA Aß fibrils was evaluated in vitro using immunoprecipitation, surface plasmon resonance, and direct binding assay. Marked differences in Aß antibody binding to CAA fibrils were observed. Solanezumab and crenezumab showed negligible CAA fibril binding and these antibodies have no reported ARIA-E cases. Lecanemab showed a low binding to CAA fibrils, consistent with its relatively low ARIA-E frequency of 12.6%, while aducanumab, bapineuzumab, and gantenerumab all showed higher binding to CAA fibrils and substantially higher ARIA-E frequencies (25-35%). An ARIA-E frequency of 24% was reported for donanemab, and its binding to CAA fibrils correlated with the amount of pyroglutamate-modified Aß present. The findings of this study support the proposal that Aß antibody-CAA interactions may relate to the ARIA-E frequency observed in patients treated with Aß-based immunotherapies.
Assuntos
Peptídeos beta-Amiloides , Angiopatia Amiloide Cerebral , Humanos , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Amiloide/metabolismo , Amiloide/imunologia , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Angiopatia Amiloide Cerebral/diagnóstico por imagem , Angiopatia Amiloide Cerebral/imunologia , Angiopatia Amiloide Cerebral/patologia , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Inclusions of intraneuronal alpha-synuclein (α-synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α-synuclein is a central feature of the disease pathogenesis. Among the different α-synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α-synuclein oligomers were generated. These antibodies, which do not bind amyloid-beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α-synuclein transgenic mice than linear epitope antibodies. An oligomer-selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α-synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α-synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer-selective α-synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.
Assuntos
Anticorpos Monoclonais/farmacologia , Encéfalo/patologia , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/patologia , alfa-Sinucleína/genética , alfa-Sinucleína/imunologia , Animais , Western Blotting , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática , Epitopos , Formiatos/química , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Mutação/genética , Mutação/fisiologia , Frações Subcelulares/metabolismoRESUMO
Immunotherapy against amyloid-beta (Aß) is a promising option for the treatment of Alzheimer's disease (AD). Aß exists as various species, including monomers, oligomers, protofibrils, and insoluble fibrils in plaques. Oligomers and protofibrils have been shown to be toxic, and removal of these aggregates might represent an effective treatment for AD. We have characterized the binding properties of lecanemab, aducanumab, and gantenerumab to different Aß species with inhibition ELISA, immunodepletion, and surface plasmon resonance. All three antibodies bound monomers with low affinity. However, lecanemab and aducanumab had very weak binding to monomers, and gantenerumab somewhat stronger binding. Lecanemab was distinctive as it had tenfold stronger binding to protofibrils compared to fibrils. Aducanumab and gantenerumab preferred binding to fibrils over protofibrils. Our results show different binding profiles of lecanemab, aducanumab, and gantenerumab that may explain clinical results observed for these antibodies regarding both efficacy and side effects.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismoRESUMO
BACKGROUND: Standard neuropathologic analysis of Alzheimer's brain relies on traditional fluorescence microscopy, which suffers from limited spatial resolution due to light diffraction. As a result, it fails to reveal intricate details of amyloid plaques. While electron microscopy (EM) offers higher resolution, its extensive sample preparation, involving fixation, dehydration, embedding, and sectioning, can introduce artifacts and distortions in the complex brain tissue. Moreover, EM lacks molecular specificity and has limited field of view and imaging depth. RESULTS: In our study, we employed super-resolution Stimulated Emission Depletion (STED) microscopy in conjunction with the anti-human APP recombinant antibody 1C3 fluorescently labelled with DyLightTM633 (1C3-DyLight633). This combination allowed us to visualize amyloidogenic aggregates in vitro and in brain sections from a 17-month-old 3×Tg-AD mouse with sub-diffraction limited spatial resolution. Remarkably, we achieved a spatial resolution of 29 nm in vitro and 62 nm in brain tissue sections, surpassing the capabilities of conventional confocal microscopy by 5-10 times. Consequently, we could discern individual fibrils within plaques, an achievement previously only possible with EM. CONCLUSIONS: The utilization of STED microscopy represents a groundbreaking advancement in the field, enabling researchers to delve into the characterization of local mechanisms that underlie Amyloid (Aß) deposition into plaques and their subsequent clearance. This unprecedented level of detail is especially crucial for comprehending the etiology of Alzheimer's disease and developing the next generation of anti-amyloid treatments. By facilitating the evaluation of drug candidates and non-pharmacological interventions aiming to reduce amyloid burden, STED microscopy emerges as an indispensable tool for driving scientific progress in Alzheimer's research.
RESUMO
The major characteristics of Alzheimer's disease (AD) are amyloid plaques, consisting of aggregated beta amyloid (Aß) peptides, together with tau pathology (tangles, neuropil treads and dystrophic neurites surrounding the plaques), in the brain. Down's syndrome (DS) individuals are at increased risk to develop AD-type pathology; most DS individuals have developed substantial pathology already at the age of 40. DS individuals have an extra copy of chromosome 21, harbouring the amyloid precursor protein gene (APP). Our aim was to investigate the Aß peptide pattern in DS and AD brains to investigate differences in their amyloid deposition and aggregation, respectively. Cortical tissue from patients with DS (with amyloid pathology), sporadic AD and controls were homogenized and fractionated into TBS (water soluble) and formic acid (water insoluble) fractions. Immunoprecipitation (IP) was performed using a variety of antibodies targeting different Aß species including oligomeric Aß. Mass spectrometry was then used to evaluate the presence of Aß species in the different patient groups. A large number of Aß peptides were identified including Aß1-X, 2-X, 3-X, 4-X, 5-X, 11-X, and Aß peptides extended N terminally of the BACE1 cleavage site and ending at amino 15 in the Aß sequence APP/Aß(-X to 15), as well as peptides post-translationally modified by pyroglutamate formation. Most Aß peptides had higher abundance in AD and DS compared to controls, except the APP/Aß(-X to 15) peptides which were most abundant in DS followed by controls and AD. Furthermore, the abundancies of AßX-40 and AßX-34 were increased in DS compared with AD. Aß1-40, Aß1-42, and Aß4-42 were identified as the main constitutes of protofibrils (IP'd using mAb158) and higher relative Aß1-42 signals were obtained compared with samples IP'd with 6E10 + 4G8, indicating that the protofibrils/oligomers were enriched with peptides ending at amino acid 42. All Aß peptides found in AD were also present in DS indicating similar pathways of Aß peptide production, degradation and accumulation, except for APP/Aß(-X to 15). Likewise, the Aß peptides forming protofibrils/oligomers in both AD and DS were similar, implying the possibility that treatment with clinical benefit in sporadic AD might also be beneficial for subjects with DS.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/patologia , Síndrome de Down/patologia , Fragmentos de Peptídeos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/análise , Ácido Aspártico Endopeptidases/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Fragmentos de Peptídeos/análise , Agregados ProteicosRESUMO
BACKGROUND: Currently, there is no established biomarker for Parkinson's disease (PD) and easily accessible biomarkers are crucial for developing disease-modifying treatments. OBJECTIVE: To develop a novel method to quantify cerebrospinal fluid (CSF) levels of α-synuclein protofibrils (α-syn PF) and apply it to clinical cohorts of patients with PD and atypical parkinsonian disorders. METHODS: A cohort composed of 49 patients with PD, 12 with corticobasal degeneration (CBD), 22 with progressive supranuclear palsy, and 33 controls, that visited the memory clinic but had no biomarker signs of Alzheimer's disease (AD, tau<350âpg/mL, amyloid-beta 42 (Aß42)>530âpg/mL, and phosphorylated tau (p-tau)<60âpg/mL) was used in this study. The CSF samples were analyzed with the Single molecule array (Simoa) technology. Total α-synuclein (α-syn) levels were analyzed with a commercial ELISA-kit. RESULTS: The assay is specific to α-syn PF, with no cross-reactivity to monomeric α-syn, or the ß- and γ-synuclein variants. CSF α-syn PF levels were increased in PD compared with controls (62.1 and 40.4âpg/mL, respectively, pâ=â0.03), and CBD (62.1 and 34.2âpg/mL, respectively, pâ=â0.02). The accuracy of predicting PD using α-syn PF is significantly different from controls (area under the curve 0.68, pâ=â0.0097) with a sensitivity of 62.8% and specificity of 67.7%. Levels of total α-syn were significantly different between the PD and CBD groups (pâ=â0.04). CONCLUSION: The developed method specifically quantifies α-syn PF in human CSF with increased concentrations in PD, but with an overlap with asymptomatic elderly controls.
Assuntos
Doença de Parkinson/líquido cefalorraquidiano , Transtornos Parkinsonianos/líquido cefalorraquidiano , alfa-Sinucleína/líquido cefalorraquidiano , Idoso , Biomarcadores/líquido cefalorraquidiano , Feminino , Humanos , Imunoensaio , Masculino , Pessoa de Meia-Idade , Paralisia Supranuclear Progressiva/líquido cefalorraquidianoRESUMO
Human genetics link Alzheimer's disease pathogenesis to excessive accumulation of amyloid-beta (Abeta) in brain, but the symptoms do not correlate with senile plaque burden. Since soluble Abeta aggregates can cause synaptic dysfunctions and memory deficits, these species could contribute to neuronal dysfunction and dementia. Here we explored selective targeting of large soluble aggregates, Abeta protofibrils, as a new immunotherapeutic strategy. The highly protofibril-selective monoclonal antibody mAb158 inhibited in vitro fibril formation and protected cells from Abeta protofibril-induced toxicity. When the mAb158 antibody was administered for 4 months to plaque-bearing transgenic mice with both the Arctic and Swedish mutations (tg-ArcSwe), Abeta protofibril levels were lowered while measures of insoluble Abeta were unaffected. In contrast, when treatment began before the appearance of senile plaques, amyloid deposition was prevented and Abeta protofibril levels diminished. Therapeutic intervention with mAb158 was however not proven functionally beneficial, since place learning depended neither on treatment nor transgenicity. Our findings suggest that Abeta protofibrils can be selectively cleared with immunotherapy in an animal model that display highly insoluble Abeta deposits, similar to those of Alzheimer's disease brain.
Assuntos
Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/imunologia , Amiloide/imunologia , Amiloide/metabolismo , Anticorpos Monoclonais/uso terapêutico , Envelhecimento , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Humanos , Imunização Passiva , Cinética , Aprendizagem , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Placa Amiloide/imunologia , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Multimerização Proteica , Percepção EspacialRESUMO
Deposition of amyloid-ß (Aß) is central to Alzheimer's disease (AD) pathogenesis and associated with progressive neurodegeneration in traumatic brain injury (TBI). We analyzed predisposing factors for Aß deposition including monomeric Aß40, Aß42 and Aß oligomers/protofibrils, Aß species with pronounced neurotoxic properties, following human TBI. Highly selective ELISAs were used to analyze N-terminally intact and truncated Aß40 and Aß42, as well as Aß oligomers/protofibrils, in human brain tissue, surgically resected from severe TBI patients (n = 12; mean age 49.5 ± 19 years) due to life-threatening brain swelling/hemorrhage within one week post-injury. The TBI tissues were compared to post-mortem AD brains (n = 5), to post-mortem tissue of neurologically intact (NI) subjects (n = 4) and to cortical biopsies obtained at surgery for idiopathic normal pressure hydrocephalus patients (iNPH; n = 4). The levels of Aß40 and Aß42 were not elevated by TBI. The levels of Aß oligomers/protofibrils in TBI were similar to those in the significantly older AD patients and increased compared to NI and iNPH controls (P < 0.05). Moreover, TBI patients carrying the AD risk genotype Apolipoprotein E epsilon3/4 (APOE ε3/4; n = 4) had increased levels of Aß oligomers/protofibrils (P < 0.05) and of both N-terminally intact and truncated Aß42 (P < 0.05) compared to APOE ε3/4-negative TBI patients (n = 8). Neuropathological analysis showed insoluble Aß aggregates (commonly referred to as Aß plaques) in three TBI patients, all of whom were APOE ε3/4 carriers. We conclude that soluble intermediary Aß aggregates form rapidly after TBI, especially among APOE ε3/4 carriers. Further research is needed to determine whether these aggregates aggravate the clinical short- and long-term outcome in TBI.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Amiloide/metabolismo , Apolipoproteínas E/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Amyloid-ß (Aß) immunotherapy for Alzheimer's disease (AD) has good preclinical support from transgenic mouse models and clinical data suggesting that a long-term treatment effect is possible. Soluble Aß protofibrils have been shown to exhibit neurotoxicity in vitro and in vivo, and constitute an attractive target for immunotherapy. Here, we demonstrate that the humanized antibody BAN2401 and its murine version mAb158 exhibit a strong binding preference for Aß protofibrils over Aß monomers. Further, we confirm the presence of the target by showing that both antibodies efficiently immunoprecipitate soluble Aß aggregates in human AD brain extracts. mAb158 reached the brain and reduced the brain protofibril levels by 42% in an exposure-dependent manner both after long-term and short-term treatment in tg-ArcSwe mice. Notably, a 53% reduction of protofibrils/oligomers in cerebrospinal fluid (CSF) that correlated with reduced brain protofibril levels was observed after long-term treatment, suggesting that CSF protofibrils/oligomers could be used as a potential biomarker. No change in native monomeric Aß42 could be observed in brain TBS extracts after mAb158-treatment in tg-ArcSwe mice. By confirming the specific ability of mAb158 to selectively bind and reduce soluble Aß protofibrils, with minimal binding to Aß monomers, we provide further support in favor of its position as an attractive new candidate for AD immunotherapy. BAN2401 has undergone full phase 1 development, and available data indicate a favorable safety profile in AD patients.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais/uso terapêutico , Encéfalo/metabolismo , Fatores Imunológicos/uso terapêutico , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Anticorpos Monoclonais/farmacologia , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Fatores Imunológicos/farmacologia , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Placa Amiloide , Presenilina-1/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genéticaRESUMO
Glucagon has previously been reported to increase serum levels of insulin-like growth factor binding protein-1 (IGFBP-1) in humans. The in vitro effect of glucagon and glucagon-like peptide-1 (7-36) amide (GLP-1) was investigated in Hep G2 human hepatoma cells. The expression of IGFBP-1 mRNA was determined by solution hybridization assay and IGFBP-1 secretion was measured by radioimmunoassay. In contrast to forskolin the peptides glucagon and GLP-1 had no effect on IGFBP-1 mRNA at 3, 6 and 24 h incubation or any detectable effect on the apparent half-life of IGFBP-1 mRNA. However, the exposure to glucagon (10 microg/mL, 2.87 microM) and GLP-1 (1 microM) caused a two-fold stimulation in protein levels of IGFBP-1 after 6 h incubation, declining to control levels after 24 h. This transient effect was dose dependent, remained when transcription was inhibited and required protein synthesis. The regulation of IGFBP-1 secretion by glucagon and GLP-1 appeared to be cAMP independent. In conclusion, glucagon and GPL-1 were shown to have a post-transcriptional stimulatory effect on IGFBP-1 release.
Assuntos
Proteínas de Caenorhabditis elegans , Glucagon/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Glicoproteínas de Membrana/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Humanos , RNA Mensageiro/metabolismo , Radioimunoensaio , Receptores Notch , Fatores de Tempo , Transcrição GênicaRESUMO
Immunotherapy targeting α-synuclein has evolved as a potential therapeutic strategy for neurodegenerative diseases, such as Parkinson's disease, and initial studies on cellular and animal models have shown promising results. α-synuclein vaccination of transgenic mice reduced the number of brain inclusions, whereas passive immunization studies demonstrated that antibodies against the C-terminus of α-synuclein can pass the blood-brain barrier and affect the pathology. In addition, preliminary evidence suggests that transgenic mice treated with an antibody directed against α-synuclein oligomers/protofibrils resulted in reduced levels of such species in the CNS. The underlying mechanisms of immunotherapy are not yet fully understood, but may include antibody-mediated clearance of pre-existing aggregates, prevention of protein propagation between cells and microglia-dependent protein clearance. Thus, immunotherapy targeting α-synuclein holds promise, but needs to be further developed as a future disease-modifying treatment in Parkinson's disease and other α-synucleinopathies.
Assuntos
Imunoterapia , Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/terapia , Doença de Parkinson/terapia , alfa-Sinucleína/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Humanos , Imunoterapia/tendências , Doença por Corpos de Lewy/imunologia , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular , Doença de Parkinson/imunologia , Resposta a Proteínas não Dobradas/imunologia , alfa-Sinucleína/genéticaRESUMO
The symptomatic drugs currently on the market for Alzheimer's disease (AD) have no effect on disease progression, and this creates a large unmet medical need. The type of drug that has developed most rapidly in the last decade is immunotherapy: vaccines and, especially, passive vaccination with monoclonal antibodies. Antibodies are attractive drugs as they can be made highly specific for their target and often with few side effects. Data from recent clinical AD trials indicate that a treatment effect by immunotherapy is possible, providing hope for a new generation of drugs. The first anti-amyloid-beta (anti-Aß) vaccine developed by Elan, AN1792, was halted in phase 2 because of aseptic meningoencephalitis. However, in a follow-up study, patients with antibody response to the vaccine demonstrated reduced cognitive decline, supporting the hypothesis that Aß immunotherapy may have clinically relevant effects. Bapineuzumab (Elan/Pfizer Inc./Johnson & Johnson), a monoclonal antibody targeting fibrillar Aß, was stopped because the desired clinical effect was not seen. Solanezumab (Eli Lilly and Company) was developed to target soluble, monomeric Aß. In two phase 3 studies, Solanezumab did not meet primary endpoints. When data from the two studies were pooled, a positive pattern emerged, revealing a significant slowing of cognitive decline in the subgroup of mild AD. The Arctic mutation has been shown to specifically increase the formation of soluble Aß protofibrils, an Aß species shown to be toxic to neurons and likely to be present in all cases of AD. A monoclonal antibody, mAb158, was developed to target Aß protofibrils with high selectivity. It has at least a 1,000-fold higher selectivity for protofibrils as compared with monomers of Aß, thus targeting the toxic species of the peptide. A humanized version of mAb158, BAN2401, has now entered a clinical phase 2b trial in a collaboration between BioArctic Neuroscience and Eisai without the safety concerns seen in previous phase 1 and 2a trials. Experiences from the field indicate the importance of initiating treatment early in the course of the disease and of enriching the trial population by improving the diagnostic accuracy. BAN2401 is a promising candidate for Aß immunotherapy in early AD. Other encouraging efforts in immunotherapy as well as in the small-molecule field offer hope for new innovative therapies for AD in the future.
RESUMO
BACKGROUND: The heme precursors porphobilinogen (PBG) and 5-aminolevulinic acid (ALA) accumulate during overt crises of acute intermittent porphyria (AIP), and high excretion of these metabolites often continues in the asymptomatic phase. METHODS: We measured concentrations of PBG and ALA and investigated the correlation between these metabolites in plasma and urine in 10 asymptomatic AIP carriers with high excretion and in 5 healthy individuals. We quantified plasma concentrations with an HPLC-mass spectrometric method and urine concentrations with ion-exchange chromatography. RESULTS: The mean (SD) plasma concentrations of PBG and ALA in the AIP carriers were 3.1 (1.0) and 1.7 (0.7) micromol/L, respectively. The mean 8-h urinary excretion amounts of PBG and ALA in the AIP carriers were 102 (25) and 56 (18) micromol, respectively, whereas the corresponding values for healthy individuals were 2.9 (0.7) and 9.3 (1.2) micromol. The correlations between PBG and ALA values in plasma and urine of the AIP carriers were 0.678 and 0.856, respectively. The mean PBG/ALA ratio was approximately 2.0 in both plasma and urine for the AIP carriers and 0.3 in urine for the healthy individuals. The renal clearance rates for PBG and ALA were 71 (15) and 70 (13) mL/min, respectively. CONCLUSIONS: The described HPLC-mass spectrometric method enabled characterization of variations in plasma PBG and ALA in AIP carriers during an 8-h period. The renal clearances were similar for both metabolites. This method could be used to monitor AIP patients during treatment.
Assuntos
Ácido Aminolevulínico/sangue , Ácido Aminolevulínico/urina , Heterozigoto , Porfobilinogênio/sangue , Porfobilinogênio/urina , Porfiria Aguda Intermitente/genética , Adulto , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-IdadeRESUMO
A deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy, which is characterized by accumulation of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide). Sphingolipid storage results in progressive demyelination and severe neurologic symptoms. The disease is lethal, and curative therapy is not available. To assess the therapeutic potential of enzyme replacement therapy (ERT), ASA knockout mice were treated by intravenous injection of recombinant human ASA. Plasma levels of ASA declined with a half-time of approximately 40 min, and enzyme was detectable in tissues within minutes after injection. The uptake of injected enzyme was high into liver, moderate into peripheral nervous system (PNS) and kidney and very low into brain. The apparent half-life of endocytosed enzyme was approximately 4 days. A single injection led to a time- and dose-dependent decline of the excess sulfatide in PNS and kidney by up to 70%, but no reduction was seen in brain. Four weekly injections with 20 mg/kg body weight not only reduced storage in peripheral tissues progressively, but also were surprisingly effective in reducing sulfatide storage in brain and spinal cord. The histopathology of kidney and central nervous system was ameliorated. Improved neuromotor coordination capabilities and normalized peripheral compound motor action potential demonstrate the benefits of ERT on the nervous system function. Enzyme replacement may therefore be a promising therapeutic option in this devastating disease.