Christopher Janus

Accumulation of Pathological Tau Species and Memory Loss in a Conditional Model of Tauopathy

Neurofibrillary tangles (NFTs) are a pathological hallmark of Alzheimers disease and other tauopathies, but recent studies in a conditional mouse model of tauopathy (rTg4510) have suggested that NFT formation can be dissociated from memory loss and neurodegeneration. This suggests that NFTs are not the major neurotoxic tau species, at least during the early stages of pathogenesis. To identify other neurotoxic tau protein species, we performed biochemical analyses on brain tissues from the rTg4510 mouse model and then correlated the levels of these tau proteins with memory loss. We describe the identification and characterization of two forms of tau multimers (140 and 170 kDa), whose molecular weight suggests an oligomeric aggregate, that accumulate early in the pathogenic cascade in this mouse model. Similar tau multimers were detected in a second mouse model of tauopathy (JNPL3) and in tissue from patients with Alzheimers disease and FTDP-17 (frontotemporal dementia and parkinsonism linked to chromosome 17). Moreover, levels of the tau multimers correlated consistently with memory loss at various ages in the rTg4510 mouse model. Our findings suggest that accumulation of early-stage aggregated tau species, before the formation of NFT, is associated with the development of functional deficits during the pathogenic progression of tauopathy.

Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice.

Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene, first described in 2004 have now emerged as the most important genetic finding in both autosomal dominant and sporadic Parkinsons disease (PD). While a formidable research effort has ensued since the initial gene discovery, little is known of either the normal or the pathological role of LRRK2. We have created lines of mice that express human wild-type (hWT) or G2019S Lrrk2 via bacterial artificial chromosome (BAC) transgenesis. In vivo analysis of the dopaminergic system revealed abnormal dopamine neurotransmission in both hWT and G2019S transgenic mice evidenced by a decrease in extra-cellular dopamine levels, which was detected without pharmacological manipulation. Immunopathological analysis revealed changes in localization and increased phosphorylation of microtubule binding protein tau in G2019S mice. Quantitative biochemical analysis confirmed the presence of differential phospho-tau species in G2019S mice but surprisingly, upon dephosphorylation the tau isoform banding pattern in G2019S mice remained altered. This suggests that other post-translational modifications of tau occur in G2019S mice. We hypothesize that Lrrk2 may impact on tau processing which subsequently leads to increased phosphorylation. Our models will be useful for further understanding of the mechanistic actions of LRRK2 and future therapeutic screening.

Abnormal Long-Lasting Synaptic Plasticity and Cognition in Mice Lacking the Mental Retardation Gene Pak3

Mutations in the Pak3 gene lead to nonsyndromic mental retardation characterized by selective deficits in cognition. However, the underlying mechanisms are yet to be elucidated. We report here that the knock-out mice deficient in the expression of p21-activated kinase 3 (PAK3) exhibit significant abnormalities in synaptic plasticity, specifically hippocampal late-phase long-term potentiation, and deficiencies in learning and memory. A dramatic reduction in the active form of transcription factor cAMP-responsive element-binding protein in the knock-out mice implicates a novel signaling mechanism by which PAK3 and Rho signaling regulate synaptic function and cognition.

Short Amyloid-β (Aβ) Immunogens Reduce Cerebral Aβ Load and Learning Deficits in an Alzheimer's Disease Mouse Model in the Absence of an Aβ-Specific Cellular Immune Response

Amyloid-β (Aβ) immunotherapy lowers cerebral Aβ and improves cognition in mouse models of Alzheimers disease (AD). A clinical trial using active immunization with Aβ1–42 was suspended after ∼6% of patients developed meningoencephalitis, possibly because of a T-cell reaction against Aβ. Nevertheless, beneficial effects were reported in antibody responders. Consequently, alternatives are required for a safer vaccine. The Aβ1–15 sequence contains the antibody epitope(s) but lacks the T-cell reactive sites of full-length Aβ1–42. Therefore, we tested four alternative peptide immunogens encompassing either a tandem repeat of two lysine-linked Aβ1–15 sequences (2×Aβ1–15) or the Aβ1–15 sequence synthesized to a cross-species active T1 T-helper-cell epitope (T1-Aβ1–15) and each with the addition of a three-amino-acid RGD (Arg-Gly-Asp) motif (R-2×Aβ1–15; T1-R-Aβ1–15). High anti-Aβ antibody titers were observed in wild-type mice after intranasal immunization with R-2×Aβ1–15 or 2×Aβ1–15 plus mutant Escherichia coli heat-labile enterotoxin LT(R192G) adjuvant. Moderate antibody levels were induced after immunization with T1-R-Aβ1–15 or T1-Aβ1–15 plus LT(R192G). Restimulation of splenocytes with the corresponding immunogens resulted in moderate proliferative responses, whereas proliferation was absent after restimulation with full-length Aβ or Aβ1–15. Immunization of human amyloid precursor protein, familial AD (hAPPFAD) mice with R-2×Aβ1–15 or 2×Aβ1–15 resulted in high anti-Aβ titers of noninflammatory T-helper 2 isotypes (IgG1 and IgG2b), a lack of splenocyte proliferation against full-length Aβ, significantly reduced Aβ plaque load, and lower cerebral Aβ levels. In addition, 2×Aβ1–15-immunized hAPPFAD animals showed improved acquisition of memory compared with vehicle controls in a reference-memory Morris water-maze behavior test that approximately correlated with anti-Aβ titers. Thus, our novel immunogens show promise for future AD vaccines.

IL-10 Alters Immunoproteostasis in APP Mice, Increasing Plaque Burden and Worsening Cognitive Behavior

Anti-inflammatory strategies are proposed to have beneficial effects in Alzheimers disease. To explore how anti-inflammatory cytokine signaling affects Aβ pathology, we investigated the effects of adeno-associated virus (AAV2/1)-mediated expression of Interleukin (IL)-10 in the brains of APP transgenic mouse models. IL-10 expression resulted in increased Aβ accumulation and impaired memory in APP mice. A focused transcriptome analysis revealed changes consistent with enhanced IL-10 signaling and increased ApoE expression in IL-10-expressing APP mice. ApoE protein was selectively increased in the plaque-associated insoluble cellular fraction, likely because of direct interaction with aggregated Aβ in the IL-10-expressing APP mice. Ex vivo studies also show that IL-10 and ApoE can individually impair glial Aβ phagocytosis. Our observations that IL-10 has an unexpected negative effect on Aβ proteostasis and cognition in APP mouse models demonstrate the complex interplay between innate immunity and proteostasis in neurodegenerative diseases, an interaction we call immunoproteostasis.

Hyperdynamic Microtubules, Cognitive Deficits, and Pathology Are Improved in Tau Transgenic Mice with Low Doses of the Microtubule-Stabilizing Agent BMS-241027

Tau is a microtubule (MT)-stabilizing protein that is altered in Alzheimers disease (AD) and other tauopathies. It is hypothesized that the hyperphosphorylated, conformationally altered, and multimeric forms of tau lead to a disruption of MT stability; however, direct evidence is lacking in vivo. In this study, an in vivo stable isotope-mass spectrometric technique was used to measure the turnover, or dynamicity, of MTs in brains of living animals. We demonstrated an age-dependent increase in MT dynamics in two different tau transgenic mouse models, 3xTg and rTg4510. MT hyperdynamicity was dependent on tau expression, since a reduction of transgene expression with doxycycline reversed the MT changes. Treatment of rTg4510 mice with the epothilone, BMS-241027, also restored MT dynamics to baseline levels. In addition, MT stabilization with BMS-241027 had beneficial effects on Morris water maze deficits, tau pathology, and neurodegeneration. Interestingly, pathological and functional benefits of BMS-241027 were observed at doses that only partially reversed MT hyperdynamicity. Together, these data suggest that tau-mediated loss of MT stability may contribute to disease progression and that very low doses of BMS-241027 may be useful in the treatment of AD and other tauopathies.

IFN-γ Promotes Complement Expression and Attenuates Amyloid Plaque Deposition in Amyloid β Precursor Protein Transgenic Mice

Reactive gliosis surrounding amyloid β (Aβ) plaques is an early feature of Alzheimer’s disease pathogenesis and has been postulated to represent activation of the innate immune system in an apparently ineffective attempt to clear or neutralize Aβ aggregates. To evaluate the role of IFN-γ–mediated neuroinflammation on the evolution of Aβ pathology in transgenic (Tg) mice, we have expressed murine IFN-γ (mIFN-γ) in the brains of Aβ precursor protein (APP) Tg mice using recombinant adeno-associated virus serotype 1. Expression of mIFN-γ in brains of APP TgCRND8 mice results in robust noncell autonomous activation of microglia and astrocytes, and a concomitant significant suppression of Aβ deposition. In these mice, mIFN-γ expression upregulated multiple glial activation markers, early components of the complement cascade as well as led to infiltration of Ly-6c positive peripheral monocytes but no significant effects on APP levels, APP processing or steady-state Aβ levels were noticed in vivo. Taken together, these results suggest that mIFN-γ expression in the brain suppresses Aβ accumulation through synergistic effects of activated glia and components of the innate immune system that enhance Aβ aggregate phagocytosis.

LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors

Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation.We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis.Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.

The effects of prenatal stress on learning in adult offspring is dependent on the timing of the stressor

Impaired fetal development has been linked with deficits in behavioural and emotional development during postnatal life. In order to investigate the mechanisms underlying this relationship, we studied the effect of acute stress at two different critical phases of pregnancy on cognitive function in adult guinea pig offspring. Pregnant guinea pigs were exposed to a psychological stressor (2h/day) on gestational days (gd) 50, 51, and 52 (PS50) or 60, 61, and 62 (PS60). Male offspring were grown to adulthood and tested in the Morris water maze (MWM) to assess spatial learning and memory. Latency, path length, swim speed and the strategy used to find the platform in each session of the MWM were measured. A reverse learning trial was performed where the platform was moved to a different area of the pool and the ability of the guinea pigs to learn a new platform position was assessed. There was no effect of stress at gd50 on latency to find the platform during any of the sessions in the MWM. PS60 male offspring exhibited enhanced development of a spatial strategy during sessions 3 and 4 of the MWM, but this was not associated with decreased latency. In the reversal task PS50 male offspring demonstrated use of non-spatial strategies to find the platform during the reversal task. This would suggest decreased retention of spatial memory in these animals. In contrast, control and PS60 male offspring demonstrated no bias to a particular strategy type. In conclusion, there are subtle effects of prenatal stress on spatial learning. PS60 offspring appear to exhibit enhanced spatial learning, while PS50 male offspring exhibit impaired spatial learning. These findings are consistent with those in humans, which indicate a strong effect of maternal anxiety during pregnancy on cognition in children, and that the timing of the maternal stress is critical to determining outcome. This model will allow us to determine the mechanisms that underlie the association between prenatal stress and altered learning strategy and ability.

Interferon-γ induces progressive nigrostriatal degeneration and basal ganglia calcification

We found that CNS-directed expression of interferon-γ (IFN-γ) resulted in basal ganglia calcification, reminiscent of human idiopathic basal ganglia calcification (IBGC), and nigrostriatal degeneration. Our results indicate that IFN-γ mediates age-progressive nigrostriatal degeneration in the absence of exogenous stressors. Further study of this model may provide insight into selective nigrostriatal degeneration in human IBGC and other Parkinson syndromes.