Timothy J. Seabrook

Alzheimer's Disease Aβ Vaccine Reduces Central Nervous System Aβ Levels in a Non-Human Primate, the Caribbean Vervet

Amyloid β (Aβ) protein immunotherapy lowers cerebral Aβ and improves cognition in mouse models of Alzheimers disease (AD). Here we show that Caribbean vervet monkeys ( Chlorocebus aethiops, SK ) develop cerebral Aβ plaques with aging and that these deposits are associated with gliosis and neuritic dystrophy. Five aged vervets were immunized with Aβ peptide over 10 months. Plasma and cerebral spinal fluid (CSF) samples were collected periodically from the immunized vervets and five aged controls; one monkey per group expired during the study. By Day 42, immunized animals generated plasma Aβ antibodies that labeled Aβ plaques in human, AD transgenic mouse and vervet brains; bound Aβ1–7; and recognized monomeric and oligomeric Aβ but not full-length amyloid precursor protein nor its C-terminal fragments. Low anti-Aβ titers were detected in CSF. Aβx-40 levels were elevated ∼2- to 5-fold in plasma and decreased up to 64% in CSF in immunized vervets. Insoluble Aβx-42 was decreased by 66% in brain homogenates of the four immunized animals compared to archival tissues from 13 age-matched control vervets. Aβ42-immunoreactive plaques were detected in frontal cortex in 11 of the 13 control animals, but not in six brain regions examined in each of the four immunized vervets. No T cell response or inflammation was observed. Our study is the first to demonstrate age-related Aβ deposition in the vervet monkey as well as the lowering of cerebral Aβ by Aβ vaccination in a non-human primate. The findings further support Aβ immunotherapy as a potential prevention and treatment of AD.

Complement C3 Deficiency Leads to Accelerated Amyloid β Plaque Deposition and Neurodegeneration and Modulation of the Microglia/Macrophage Phenotype in Amyloid Precursor Protein Transgenic Mice

Complement factor C3 is the central component of the complement system and a key inflammatory protein activated in Alzheimers disease (AD). Previous studies demonstrated that inhibition of C3 by overexpression of soluble complement receptor-related protein y in an AD mouse model led to reduced microgliosis, increased amyloid β (Aβ) plaque burden, and neurodegeneration. To further address the role of C3 in AD pathology, we generated a complement C3-deficient amyloid precursor protein (APP) transgenic AD mouse model (APP;C3−/−). Brains were analyzed at 8, 12, and 17 months of age by immunohistochemical and biochemical methods and compared with age-matched APP transgenic mice. At younger ages (8–12 months), no significant neuropathological differences were observed between the two transgenic lines. In contrast, at 17 months of age, APP;C3−/− mice showed significant changes of up to twofold increased total Aβ and fibrillar amyloid plaque burden in midfrontal cortex and hippocampus, which correlated with (1) significantly increased Tris-buffered saline (TBS)-insoluble Aβ42 levels and reduced TBS-soluble Aβ42 and Aβ40 levels in brain homogenates, (2) a trend for increased Aβ levels in the plasma, (3) a significant loss of neuronal-specific nuclear protein-positive neurons in the hippocampus, and (4) differential activation of microglia toward a more alternative phenotype (e.g., significantly increased CD45-positive microglia, increased brain levels of interleukins 4 and 10, and reduced levels of CD68, F4/80, inducible nitric oxide synthase, and tumor necrosis factor). Our results suggest a beneficial role for complement C3 in plaque clearance and neuronal health as well as in modulation of the microglia phenotype.

Minocycline affects microglia activation, Aβ deposition, and behavior in APP-tg mice

Activated microglia and reactive astrocytes invade and surround cerebral β amyloid (Aβ) plaques in Alzheimers disease (AD), but the role of microglia in plaque development is still unclear. In this study, minocycline was administered for 3 months, prior to and early in Aβ plaque formation in amyloid precursor protein transgenic mice (APP‐tg). When minocycline was given to younger mice, there was a small but significant increase in Aβ deposition in the hippocampus, concurrent with improved cognitive performance relative to vehicle treated mice. If APP‐tg mice received minocycline after Aβ deposition had begun, microglial activation was suppressed but this did not affect Aβ deposition or improve cognitive performance. In vitro studies demonstrated that minocycline suppressed microglial production of IL‐1β, IL‐6, TNF, and NGF. Thus, minocycline has different effects on Aβ plaque deposition and microglia activation depending on the age of administration. Our data suggest that this may be due to the effects of minocycline on microglial function. Therefore, anti‐inflammatory therapies to suppress microglial activation or function may reduce cytokine production but enhance Aβ plaque formation early in AD.

Dendrimeric Aβ1–15 is an effective immunogen in wildtype and APP-tg mice

Immunization of humans and APP-tg mice with full-length beta-amyloid (Abeta) results in reduced cerebral Abeta levels. However, due to adverse events in the AN1792 trial, alternative vaccines are required. We investigated dendrimeric Abeta1-15 (dAbeta1-15), which is composed of 16 copies of Abeta1-15 peptide on a branched lysine core and thus, includes an Abeta-specific B cell epitope but lacks the reported T cell epitope. Immunization by subcutaneous, transcutaneous, and intranasal routes of B6D2F1 wildtype mice led to anti-Abeta antibody production. Antibody isotypes were mainly IgG1 for subcutaneous or transcutaneous immunization and IgG2b for intranasal immunization, suggestive of a Th2-biased response. All Abeta antibodies preferentially recognized an epitope in Abeta1-7. Intranasal immunization of J20 APP-tg mice resulted in a robust humoral immune response with a corresponding significant reduction in cerebral plaque burden. Splenocyte proliferation against Abeta peptide was minimal indicating the lack of an Abeta-specific cellular immune response. Anti-Abeta antibodies bound monomeric, oligomeric, and fibrillar Abeta. Our data suggest that dAbeta1-15 may be an effective and potentially safer immunogen for Alzheimers disease (AD) vaccination.

Novel Aβ Immunogens: Is Shorter Better?

Active and passive Aβ immunotherapy in Alzheimers disease (AD)-like mouse models lowers cerebral amyloid- β protein (Aβ) levels, especially if given early in the disease process, and improves cognitive deficits. In 2002, a Phase IIa clinical trial was halted due to meningoencephalitis in ∼6% of the AD patients. It is hypothesized that the immunogen, full-length Aβ1-42, may have led to an autoimmune response. Currently, we are developing novel Aβ peptide immunogens for active immunization in amyloid precursor protein transgenic mice (APP Tg) to target Aβ B cell epitopes (within Aβ1-15) and avoid Aβ-specific T cell epitopes (Aβ16-42) so as to generate a safe and effective AD vaccine. Intranasal immunization with dendrimeric Aβ1-15 (16 copies of Aβ1-15 on a lysine core) or a tandem repeat of Aβ1-15 joined by 2 lysines and conjugated to an RGD motif with a mutated form of an E. coli-derived adjuvant generated robust Aβ titers in both wildtype and APP Tg mice. The Aβ antibodies recognized a B cell epitope within Aβ1-7, were mostly T-helper 2 associated immunoglobulin isotypes, bound human AD and APP Tg plaques, and detected Aβ oligomers. Splenic T cells reacted to the immunogens but not full-length Aβ. Six months of intranasal immunization (from 6-to-12 months of age) of J20 mice with each immunogen lowered insoluble Aβ42 by 50%, reduced plaque burden and gliosis, and increased Aβ in plasma. Interestingly, Aβ antibody generation was influenced by route of immunization. Transcutaneous immunization with dβ1-15, but not full-length Abeta, led to high Aβ titers. In summary, our short Aβ immunogens induced robust titers of predominantly Th2 antibodies that were able to clear cerebral Aβ in the absence of Aβ-specific T cell reactivity, indicating the potential for a safer vaccine. We remain optimistic about the potential of such a vaccine for prevention and treatment of AD.

Complement C3 Deficiency Leads to Accelerated Amyloid Plaque Deposition and Neurodegeneration and Modulation of the Microglia/Macrophage Phenotype in Amyloid Precursor Protein Transgenic Mice

Complement factor C3 is the central component of the complement system and a key inflammatory protein activated in Alzheimers disease (AD). Previous studies demonstrated that inhibition of C3 by overexpression of soluble complement receptor-related protein y in an AD mouse model led to reduced microgliosis, increased amyloid β (Aβ) plaque burden, and neurodegeneration. To further address the role of C3 in AD pathology, we generated a complement C3-deficient amyloid precursor protein (APP) transgenic AD mouse model (APP;C3−/−). Brains were analyzed at 8, 12, and 17 months of age by immunohistochemical and biochemical methods and compared with age-matched APP transgenic mice. At younger ages (8–12 months), no significant neuropathological differences were observed between the two transgenic lines. In contrast, at 17 months of age, APP;C3−/− mice showed significant changes of up to twofold increased total Aβ and fibrillar amyloid plaque burden in midfrontal cortex and hippocampus, which correlated with (1) significantly increased Tris-buffered saline (TBS)-insoluble Aβ42 levels and reduced TBS-soluble Aβ42 and Aβ40 levels in brain homogenates, (2) a trend for increased Aβ levels in the plasma, (3) a significant loss of neuronal-specific nuclear protein-positive neurons in the hippocampus, and (4) differential activation of microglia toward a more alternative phenotype (e.g., significantly increased CD45-positive microglia, increased brain levels of interleukins 4 and 10, and reduced levels of CD68, F4/80, inducible nitric oxide synthase, and tumor necrosis factor). Our results suggest a beneficial role for complement C3 in plaque clearance and neuronal health as well as in modulation of the microglia phenotype.

Osteopetrotic (op/op) mice have reduced microglia, no Aβ deposition, and no changes in dopaminergic neurons

BackgroundActivation of microglia is a part of the inflammatory response in neurodegenerative diseases but its role in the pathophysiology of these diseases is still unclear. The osteopetrotic (op/op) mouse lacks colony-stimulating factor-1 (CSF-1) and thus has a deficiency in microglia and macrophages. Prior reports have demonstrated that op/op mice deposit amyloid β (Aβ) plaques, similar to those found in Alzheimers disease. The purpose of these studies was to confirm this and to determine if the lack of CSF-1 affects the development of dopaminergic neurons and the expression of CD200, a known microglial inhibitory protein.MethodWe examined the central nervous system of op/op mice at 30 days, 60 days and 7 months of age and wildtype littermates at 30 days using immunohistochemistry and histochemistry.ResultsWe found a decrease in the number of microglia in 1 month-old op/op mice compared to wildtype (WT) littermates as measured by CD11b, CD45, CD32/16, CD68, CD204 and F4/80 immunoreactivity. Aβ plaques were not detected, while the number of dopaminergic neurons appeared normal. The expression of CD200 appeared to be normal, but there appeared to be a lower expression in the substantia nigra.ConclusionIn contrast to a prior report we did not detect Aβ deposition in the central nervous system of op/op mice at 30 days, 60 days or 7 months of age and there was a normal number of dopaminergic neurons. This indicates that op/op mice may be useful to examine the effects of microglia on neurodegenerative disease progression by breeding them to different transgenic mouse models. In addition, the lack of CSF-1 does not appear to affect CD200 expression by neurons but we did note a decrease in the substantia nigra of op/op and WT mice, suggesting that this may be a mechanism by which microglia control may be attenuated in this specific area during Parkinsons disease.

Developing Novel Immunogens for an Effective, Safe Alzheimer’s Disease Vaccine

Active amyloid β (Aβ) vaccination has been shown to be effective in clearing cerebral Aβ and improving cognitive function in mouse models of Alzheimer’s disease. However, an Aβ vaccine clinical trial was suspended after meningoencephalitis was detected in a subset of subjects. Passive immunization has been suggested to be a safer alternative to active Aβ immunization but there are reports of increased risk of microhemorrhages associated with its administration in aged β-amyloid precursor protein transgenic mice bearing abundant vascular amyloid deposition. In addition, the cost may be prohibitive for large-scale clinical use. Therefore, we are designing novel Aβ immunogens that encompass the B cell epitope of Aβ but lack the T cell-reactive sites. These immunogens induced the production of Aβ-specific antibodies in the absence of an Aβ-specific cellular immune response in wild-type mice and are being tested in β-amyloid precursor protein transgenic mice. These data together with published reports from several other groups suggest that a safe, active Aβ vaccine is a tenable goal.

Boosting with Intranasal Dendrimeric Aβ1–15 but Not Aβ1–15 Peptide Leads to an Effective Immune Response Following a Single Injection of Aβ1–40/42 in APP-tg Mice

BackgroundImmunotherapy for Alzheimers disease (AD) is emerging as a potential treatment. However, a clinical trial (AN1792) was halted after adverse effects occurred in a small subset of subjects, which may have been caused by a T cell-mediated immunological response. In general, aging limits the humoral immune response, therefore, immunogens and vaccination regimes are required that induce a strong antibody response with less potential for an adverse immune response.MethodIn the current study, we immunized both wildtype and J20 APP-tg mice with a priming injection of Aβ1–40/42, followed by multiple intranasal boosts with the novel immunogen dAβ1–15 (16 copies of Aβ1–15 on a lysine tree), Aβ1–15 peptide or Aβ1–40/42 full length peptide.ResultsJ20 APP-tg mice primed with Aβ1–40/42 subcutaneously and subsequently boosted intranasally with Aβ1–15 peptide did not generate a cellular or humoral immune response. In contrast, J20 APP-tg mice boosted intranasally with dAβ1–15 or full length Aβ1–40/42 produced high levels of anti-Aβ antibodies. Splenocyte proliferation was minimal in mice immunized with dAβ1–15. Wildtype littermates of the J20 APP-tg mice produced higher amounts of anti-Aβ antibodies compared to APP-tg mice but also had low T cell proliferation. The anti-Aβ antibodies were mainly composed of IgG2b and directed to an epitope within the Aβ1–7 region, regardless of the immunogen. Examination of the brain showed a significant reduction in Aβ plaque burden in the J20 APP-tg mice producing antibodies compared to controls. Biochemically, Aβ40 or Aβ42 were also reduced in brain homogenates and elevated in plasma but the changes did not reach significance.ConclusionOur results demonstrate that priming with full length Aβ40/42 followed by boosting with dAβ1–15 but not Aβ1–15 peptide led to a robust humoral immune response with a minimal T cell response in J20 APP-tg mice. In addition, Aβ plaque burden was reduced in mice producing anti-Aβ antibodies. Interestingly, wildtype mice produced higher levels of anti-Aβ antibodies, indicating that immune tolerance may be present in J20 APP-tg mice. Together, these data suggest that dAβ1–15 but not Aβ1–15 peptide may be useful as a boosting immunogen in an AD vaccination regime.

Species-specific immune response to immunization with human versus rodent Aβ peptide

Amyloid beta (A beta) immunization of amyloid precursor protein (APP)-transgenic (tg) mice with human A beta induces humoral immunity, however, the immune response to endogenous rodent A beta is unknown. Fourteen-month J20 APP-tg mice and non-tg littermates were immunized subcutaneously followed by chronic intranasal boosting with human or rodent A beta peptide and adjuvant LT(R192G). Rodent A beta-immunized APP-tg mice had anti-rodent A beta antibody levels of 257.8 micrograms/ml and those immunized with human A beta had anti-human A beta antibodies of 120.8 micrograms/ml. Non-tg littermates had anti-rodent and anti-human A beta antibody concentrations of 98.8 and 231.1 microgram/ml, respectively. Inter-species cross-reactivity was minimal. Anti-human A beta antibodies were predominately IgG1 and IgG2b, while anti-rodent A beta antibodies were equally IgG1, IgG2a, and IgG2b. Anti-human A beta antibodies recognized an epitope within human A beta1-9. Anti-rodent A beta antibodies did not stain Alzheimers disease (AD) plaques but bound some plaques in APP-tg mice. Splenocytes proliferated modestly to their respective antigen and secreted low levels of IL-2 and IFN-gamma. Therefore, immunizing APP-tg and non-tg mice with rodent A beta resulted in a species-specific humoral response with modest T cell reactivity.