Yona Levites

Neuroprotection and neurorescue against Aβ toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate

Green tea extract and its main polyphenol constituent (‐)‐epigallocatechin‐3‐gallate (EGCG) possess potent neuroprotective activity in cell culture and mice model of Parkinsons disease. The central hypothesis guiding this study is that EGCG may play an important role in amyloid precursor protein (APP) secretion and protection against toxicity induced by β‐amyloid (Aβ). The present study shows that EGCG enhances (~6‐fold) the release of the non‐amyloidogenic soluble form of the amyloid precursor protein (sAPPα) into the conditioned media of human SH‐SY5Y neuroblastoma and rat pheochromocytoma PC12 cells. sAPPα release was blocked by the hydroxamic acid‐based metalloprotease inhibitor Ro31–9790, which indicated mediation via α‐secretase activity. Inhibition of protein kinase C (PKC) with the inhibitor GF109203X, or by down‐regulation of PKC, blocked the EGCG‐induced sAPPα secretion, suggesting the involvement of PKC. Indeed, EGCG induced the phosphorylation of PKC, thus identifying a novel PKC‐dependent mechanism of EGCG action by activation of the non‐amyloidogenic pathway. EGCG is not only able to protect, but it can rescue PC12 cells against the β‐amyloid (Aβ) toxicity in a dose‐dependent manner. In addition, administration of EGCG (2 mg/kg) to mice for 7 or 14 days significantly decreased membrane‐bound holoprotein APP levels, with a concomitant increase in sAPPα levels in the hippocampus. Consistently, EGCG markedly increased PKCα and PKCε in the membrane and the cytosolic fractions of mice hippocampus. Thus, EGCG has protective effects against Aβ‐induced neurotoxicity and regulates secretory processing of non‐amyloidogenic APP via PKC pathway.

Anti-Aβ42– and anti-Aβ40–specific mAbs attenuate amyloid deposition in an Alzheimer disease mouse model

Accumulation and aggregation of amyloid β peptide 1–42 (Aβ42) in the brain has been hypothesized as triggering a pathological cascade that causes Alzheimer disease (AD). To determine whether selective targeting of Aβ42 versus Aβ40 or total Aβ is an effective way to prevent or treat AD, we compared the effects of passive immunization with an anti-Aβ42 mAb, an anti-Aβ40 mAb, and multiple Aβ1–16 mAbs. We established in vivo binding selectivity of the anti-Aβ42 and anti-Aβ40 mAbs using novel TgBRI-Aβ mice. We then conducted a prevention study in which the anti-Aβ mAbs were administered to young Tg2576 mice, which have no significant Aβ deposition, and therapeutic studies in which mAbs were administered to Tg2576 or CRND8 mice with modest levels of preexisting Aβ deposits. Anti-Aβ42, anti-Aβ40, and anti-Aβ1–16 mAbs attenuated plaque deposition in the prevention study. In contrast, anti-Aβ42 and anti-Aβ40 mAbs were less effective in attenuating Aβ deposition in the therapeutic studies and were not effective in clearing diffuse plaques following direct injection into the cortex. These data suggest that selective targeting of Aβ42 or Aβ40 may be an effective strategy to prevent amyloid deposition, but may have limited benefit in a therapeutic setting.

Massive gliosis induced by interleukin-6 suppresses Aβ deposition in vivo: evidence against inflammation as a driving force for amyloid deposition

Proinflammatory stimuli, after amyloid β (Aβ) deposition, have been hypothesized to create a self‐reinforcing positive feedback loop that increases amyloidogenic processing of the Aβ precursor protein (APP), promoting further Aβ accumulation and neuroinflammation in Alzheimers disease (AD). Interleukin‐6 (IL‐6), a proinflammatory cytokine, has been shown to be increased in AD patients implying a pathological interaction. To assess the effects of IL‐6 on Aβ deposition and APP processing in vivo,we overexpressed murine IL‐6 (mIL‐6) in the brains of APP transgenic TgCRND8 and TG2576 mice. mIL‐6 expression resulted in extensive gliosis and concurrently attenuated Aβ deposition in TgCRND8 mouse brains. This was accompanied by up‐regulation of glial phagocytic markers in vivo and resulted in enhanced microglia‐mediated phagocytosis of Aβ aggregates in vitro. Further, mIL‐6‐induced neuroinflammation had no effect on APP processing in TgCRND8 and had no effect on APP processing or steady‐state levels of Aβ in young Tg2576 mice. These results indicate that mIL‐6‐mediated reactive gliosis may be beneficial early in the disease process by potentially enhancing Aβ plaque clearance rather than mediating a neurotoxic feedback loop that exacerbates amyloid pathology. This is the first study that methodically dissects the contribution of mIL‐6 with regard to its potential role in modulating Aβ deposition in vivo.—Chakrabarty, P., Jansen‐West, K., Beccard, A., Ceballos‐Diaz, C., Levites, Y., Verbeeck, C., Zubair, A. C., Dickson, D., Golde, T. E., Das, P. Massive gliosis induced by interleukin‐6 suppresses Aβ deposition in vivo: evidence against inflammation as a driving force for amyloid deposition. FASEB J. 24, 548–559 (2010). www.fasebj.org

Intracranial Adeno-Associated Virus-Mediated Delivery of Anti-Pan Amyloid β, Amyloid β40, and Amyloid β42 Single-Chain Variable Fragments Attenuates Plaque Pathology in Amyloid Precursor Protein Mice

Accumulation of amyloid β protein (Aβ) aggregates is hypothesized to trigger a pathological cascade that causes Alzheimers disease (AD). Active or passive immunizations targeting Aβ are therefore of great interest as potential therapeutic strategies. We have evaluated the use of recombinant anti-Aβ single-chain variable fragments (scFvs) as a potentially safer form of anti-Aβ immunotherapy. We have generated and characterized three anti-Aβ scFvs that recognize Aβ1–16, Aβx-40, or Aβx-42. To achieve widespread brain delivery, constructs expressing these anti-Aβ scFvs were packaged into adeno-associated virus (AAV) vectors and injected into the ventricles of postnatal day 0 (P0) amyloid precursor protein CRND8-transgenic mice. Intracranial delivery of AAV to neonatal mice resulted in widespread neuronal delivery. In situ expression of each of the anti-Aβ scFvs after intracerebroventricular AAV serotype 1 delivery to P0 pups decreased Aβ deposition by 25–50%. These data suggest that intracranial anti-Aβ scFv expression is an effective strategy to attenuate amyloid deposition. As opposed to transgenic approaches, these studies also establish a “somatic brain transgenic” paradigm to rapidly and cost-effectively evaluate potential modifiers of AD-like pathology in AD mouse models.

Insights into the mechanisms of action of anti-abeta antibodies in Alzheimer's disease mouse models

A number of hypotheses regarding how anti‐Aβ antibodies alter amyloid deposition have been postulated, yet there is no consensus as to how Aβ immunotherapy works. We have examined the in vivo binding properties, pharmacokinetics, brain penetrance, and alterations in Aβ levels after a single peripheral dose of anti‐Aβ antibodies to both wild‐type (WT) and young non‐Aβ depositing APP and BRI‐Aβ42 mice. The rapid rise in plasma Aβ observed after antibody (Ab) administration is attributable to prolongation of the half‐life of Aβ bound to the Ab. Only a miniscule fraction of Ab enters the brain, and despite dramatic increases in plasma Aβ, we find no evidence that total brain Aβ levels are significantly altered. Surprisingly, cerebral spinal fluid Aβ levels transiently rise, and when Ab:Aβ complex is directly injected into the lateral ventricles of mice, it is rapidly cleared from the brain into the plasma where it remains stable. When viewed in context of daily turnover of Aβ, these data provide a framework to evaluate proposed mechanisms of Aβ attenuation mediated by peripheral administration of an anti‐Aβ monoclonal antibody (mAb) effective in passive immunization paradigm. Such quantitative data suggest that the mAbs are either indirectly enhancing clearance of Aβ or targeting a low abundance aggregation intermediate.—Levites, Y., Smithson, L. A., Price, R. W., Dakin. R. S., Yuan, B., Sierks, M. R., Kim, J., McGowan, E., Reed, D. K., Rosenberry, T. L., Das, P., Golde, T. E. Insights into the mechanisms of action of anti‐Aβ antibodies in Alzheimers disease mouse models. FASEB J. 20, E2002–E2014 (2006)

BRI2 (ITM2b) Inhibits Aβ Deposition In Vivo

Analyses of the biologic effects of mutations in the BRI2 (ITM2b) and the amyloid β precursor protein (APP) genes support the hypothesis that cerebral accumulation of amyloidogenic peptides in familial British and familial Danish dementias and Alzheimers disease (AD) is associated with neurodegeneration. We have used somatic brain transgenic technology to express the BRI2 and BRI2-Aβ1–40 transgenes in APP mouse models. Expression of BRI2-Aβ1–40 mimics the suppressive effect previously observed using conventional transgenic methods, further validating the somatic brain transgenic methodology. Unexpectedly, we also find that expression of wild-type human BRI2 reduces cerebral Aβ deposition in an AD mouse model. Additional data indicate that the 23 aa peptide, Bri23, released from BRI2 by normal processing, is present in human CSF, inhibits Aβ aggregation in vitro and mediates its anti-amyloidogenic effect in vivo. These studies demonstrate that BRI2 is a novel mediator of Aβ deposition in vivo.

Overlapping profiles of Aβ peptides in the Alzheimer's disease and pathological aging brains

IntroductionA hallmark of Alzheimers disease (AD) is the presence of senile plaques composed of aggregated amyloid β (Aβ) peptides. Pathological aging (PA) is a postmortem classification that has been used to describe brains with plaque pathology similar in extent to AD, minimal cortical tau pathology, and no accompanying history of cognitive decline in the brain donor prior to death. PA may represent either a prodromal phase of AD, a benign form of Aβ accumulation, or inherent individual resistance to the toxic effects of Aβ accumulation. To attempt to distinguish between these possibilities we have systematically characterized Aβ peptides in a postmortem series of PA, AD and non-demented control (NDC) brains.MethodsAβ was sequentially extracted with tris buffered saline (TBS), radioimmunoprecipitation buffer (RIPA), 2% sodium dodecyl sulfate (SDS) and 70% formic acid (FA) from the pre-frontal cortex of 16 AD, eight PA, and six NDC patients. These extracts were analyzed by 1) a panel of Aβ sandwich ELISAs, 2) immunoprecipitation followed by mass spectrometry (IP/MS) and 3) western blotting. These studies enabled us to asses Aβ levels and solubility, peptide profiles and oligomeric assemblies.ResultsIn almost all extracts (TBS, RIPA, 2% SDS and 70% FA) the average levels of Aβ1-40, Aβ1-42, Aβ total, and Aβx-42 were greatest in AD. On average, levels were slightly lower in PA, and there was extensive overlap between Aβ levels in individual PA and AD cases. The profiles of Aβ peptides detected using IP/MS techniques also showed extensive similarity between the PA and AD brain extracts. In select AD brain extracts, we detected more amino-terminally truncated Aβ peptides compared to PA patients, but these peptides represented a minor portion of the Aβ observed. No consistent differences in the Aβ assemblies were observed by western blotting in the PA and AD groups.ConclusionsWe found extensive overlap with only subtle quantitative differences between Aβ levels, peptide profiles, solubility, and SDS-stable oligomeric assemblies in the PA and AD brains. These cross-sectional data indicate that Aβ accumulation in PA and AD is remarkably similar. Such data would be consistent with PA representing a prodromal stage of AD or a resistance to the toxic effects of Aβ.

Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain.

Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24–84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.

Induction of CNS α-synuclein pathology by fibrillar and non-amyloidogenic recombinant α-synuclein.

Backgroundα-Synuclein (αS) is the major component of several types of brain inclusions including Lewy bodies, a hallmark of Parkinson’s disease. Aberrant aggregation of αS also is associated with cellular demise in multiple neurologic disorders collectively referred to as synucleinopathies. Recent studies demonstrate the induction of αS pathology by a single intracerebral injection of exogenous amyloidogenic αS in adult non-transgenic and transgenic mice expressing human αS. To further investigate the mechanism of pathology induction and evaluate an experimental paradigm with potential for higher throughput, we performed similar studies in neonatal mice injected with αS.ResultsIn non-transgenic mice, we observed limited induction of neuronal αS inclusions predominantly 8 months after brain injection of aggregated, amyloidogenic human αS. More robust inclusion pathology was induced in transgenic mice expressing wild-type human αS (line M20), and inclusion pathology was observed at earlier time points. Injection of a non-amyloidogenic (Δ71-82) deletion protein of αS was also able to induce similar pathology in a subset of M20 transgenic mice. M20 transgenic mice injected with amyloidogenic or non-amyloidogenic αS demonstrated a delayed and robust induction of brain neuroinflammation that occurs in mice with or without αS pathological inclusions implicating this mechanism in aggregate formation.ConclusionsThe finding that a non-amyloidogenic Δ71-82 αS can induce pathology calls into question the simple interpretation that exogenous αS catalyzes aggregation and spread of intracellular αS pathology solely through a nucleation dependent conformational templating mechanism. These results indicate that several mechanisms may act synergistically or independently to promote the spread of αS pathology.

Rab5 Mediates an Amyloid Precursor Protein Signaling Pathway That Leads to Apoptosis

Alzheimers disease (AD) involves activation of apoptotic pathways that may be regulated through signaling cascades initiated by the amyloid precursor protein (APP). Enlarged endosomes have been observed in postmortem AD brains at very early stages of the disease. We show here that exogenous expression of a familial AD (FAD) mutant of APP or of the APP binding protein APP–BP1 in neurons causes enlargement of early endosomes, increased receptor-mediated endocytosis via a pathway dependent on APP–BP1 binding to APP, and apoptosis. Levels of both APP–BP1 and Rab5 are elevated in early endosomes in cortical embryonic neurons expressing APP(V642I) or APP–BP1, in cultured skin fibroblast cells from Down syndrome subjects, and in postmortem hippocampal tissue of individuals with AD. Indeed, Rab5 was found to bind specifically to APP–BP1, between amino acids 443 and 479. Inhibition of Rab5 or dynamin activity, but not of Eps15 (epidermal growth factor receptor pathway substrate 15) activity, rescued neurons from apoptosis induced by either APP(V642I) or APP–BP1, without affecting levels of intracellular or secreted amyloid-β (Aβ). Induction of Rab5 activity via expression of a constitutively active mutant led to an increase in neuronal apoptosis more than twice that attributable to induction of endosome enlargement via a Rab5-independent mechanism, regardless of Aβ production. Together, these findings suggest that Rab5 activation via an APP/APP–BP1-initiated signaling pathway mediates neuronal apoptosis caused by FAD mutants of APP and that, within this pathway, Rab5 has a specific role in signaling that is distinct from, although not independent of, its role in trafficking.