Ayodeji A. Asuni

Immunotherapy Targeting Pathological Tau Conformers in a Tangle Mouse Model Reduces Brain Pathology with Associated Functional Improvements

Immunotherapies for various neurodegenerative diseases have recently emerged as a promising approach for clearing pathological protein conformers in these disorders. This type of treatment has not been assessed in models that develop neuronal tau aggregates as observed in frontotemporal dementia and Alzheimers disease. Here, we present that active immunization with a phosphorylated tau epitope, in P301L tangle model mice, reduces aggregated tau in the brain and slows progression of the tangle-related behavioral phenotype. Females had more tau pathology than males but were also more receptive to the immunotherapy. The tau antibodies generated in these animals recognized pathological tau on brain sections. Performance on behavioral assays that require extensive motor coordination correlated with tau pathology in corresponding brain areas, and antibody levels against the immunogen correlated inversely with tau pathology. Interestingly, age-dependent autoantibodies that recognized recombinant tau protein but not the immunogen were detected in the P301L mice. To confirm that anti-tau antibodies could enter the brain and bind to pathological tau, FITC-tagged antibodies purified from a P301L mouse, with a high antibody titer against the immunogen, were injected into the carotid artery of P301L mice. These antibodies were subsequently detected within the brain and colocalized with PHF1 and MC1 antibodies that recognize pathological tau. Currently, no treatment is available for clearing tau aggregates. Our present findings may lead to a novel therapy targeting one of the major hallmarks of Alzheimers disease and frontotemporal dementia.

GSK-3beta inhibition reverses axonal transport defects and behavioural phenotypes in Drosophila

The tauopathies are a group of disorders characterised by aggregation of the microtubule-associated protein tau and include Alzheimers disease (AD) and the fronto-temporal dementias (FTD). We have used Drosophila to analyse how tau abnormalities cause neurodegeneration. By selectively co-expressing wild-type human tau (0N3R isoform) and a GFP vesicle marker in motorneurons, we examined the consequences of tau overexpression on axonal transport in vivo. The results show that overexpression of tau disrupts axonal transport causing vesicle aggregation and this is associated with loss of locomotor function. All these effects occur without neuron death. Co-expression of constitutively active glycogen-synthase kinase-3β (GSK-3β) enhances and two GSK-3β inhibitors, lithium and AR-A014418, reverse both the axon transport and locomotor phenotypes, suggesting that the pathological effects of tau are phosphorylation dependent. These data show that tau abnormalities significantly disrupt neuronal function, in a phosphorylation-dependent manner, before the classical pathological hallmarks are evident and also suggest that the inhibition of GSK-3β might have potential therapeutic benefits in tauopathies.

An Attenuated Immune Response Is Sufficient to Enhance Cognition in an Alzheimer's Disease Mouse Model Immunized with Amyloid- Derivatives

Immunization with amyloid-β (Aβ) 1-42 has been shown to reduce amyloid burden and improve cognition in Alzheimers disease (AD) model mice. In a human trial, possible cognitive benefit was found but in association with significant toxicity in a minority of patients. We proposed that immunization with nonfibrillogenic Aβ derivatives is much less likely to produce toxicity and have previously shown that one such derivative (K6Aβ1-30) can reduce amyloid burden in mice to a similar extent as Aβ1-42. Here, we immunized AD model mice (Tg2576) with Aβ1-30[E18E19] or with K6Aβ1-30[E18E19]. These peptides were designed to be nontoxic and to produce less T-cell response, which has been linked to toxicity. K6Aβ1-30[E18E19] induced primarily an IgM response, whereas Aβ1-30[E18E19] induced an IgG titer that was lower than previously seen with K6Aβ1-30 or Aβ1-42. However, both treated animal groups performed better than Tg controls in the radial arm maze. Amyloid burden was similar in Aβ1-30[E18E19]-vaccinated mice and their Tg controls, whereas the number of medium and small sized plaques was reduced (29-34%) in K6Aβ1-30[E18E19]-immunized mice compared with Tg controls. Amyloid burden in these mice correlated inversely with plasma IgM levels. The cognitive benefit and amyloid reduction in the K6Aβ1-30[E18E19]-vaccinated mice are likely to be related to peripheral clearance of Aβ, because IgM does not cross the blood-brain barrier because of its large size. Our results indicate that these nontoxic Aβ derivatives produce an attenuated antibody response, which is less likely to be associated with negative side effects while having cognitive benefits.

Parkinson's disease alpha-synuclein mutations exhibit defective axonal transport in cultured neurons

α-Synuclein is a major protein constituent of Lewy bodies and mutations in α-synuclein cause familial autosomal dominant Parkinsons disease. One explanation for the formation of perikaryal and neuritic aggregates of α-synuclein, which is a presynaptic protein, is that the mutations disrupt α-synuclein transport and lead to its proximal accumulation. We found that mutant forms of α-synuclein, either associated with Parkinsons disease (A30P or A53T) or mimicking defined serine, but not tyrosine, phosphorylation states exhibit reduced axonal transport following transfection into cultured neurons. Furthermore, transfection of A30P, but not wild-type, α-synuclein results in accumulation of the protein proximal to the cell body. We propose that the reduced axonal transport exhibited by the Parkinsons disease-associated α-synuclein mutants examined in this study might contribute to perikaryal accumulation of α-synuclein and hence Lewy body formation and neuritic abnormalities in diseased brain.

Vaccination of Alzheimer’s model mice with Aβ derivative in alum adjuvant reduces Aβ burden without microhemorrhages

Immunotherapy holds great promise for Alzheimers disease (AD) and other conformational disorders but certain adverse reactions need to be overcome. The meningoencephalitis observed in the first AD vaccination trial was likely related to excessive cell‐mediated immunity caused by the immunogen, amyloid‐β (Aβ) 1–42, and the adjuvant, QS−21. To avoid this toxicity, we have been using Aβ derivatives in alum adjuvant that promotes humoral immunity. Other potential side effects of immunotherapy are increased vascular amyloid and associated microhemorrhages that may be related to rapid clearance of parenchymal amyloid. Here, we determined if our immunization strategy was associated with this form of toxicity, and if the therapeutic effect was age‐dependent. Tg2576 mice and wild‐type littermates were immunized from 11 or 19 months and their behaviour evaluated prior to killing at 24 months. Subsequently, plaque‐ and vascular‐Aβ burden, Aβ levels and associated pathology was assessed. The therapy started at the cusp of amyloidosis reduced cortical Aβ deposit burden by 31% and Aβ levels by 30–37%, which was associated with cognitive improvements. In contrast, treatment from 19 months, when pathology is well established, was not immunogenic and therefore did not reduce Aβ burden or improve cognition. Significantly, the immunotherapy in the 11–24 months treatment group, that reduced Aβ burden, did not increase cerebral bleeding or vascular Aβ deposits in contrast to several Aβ antibody studies. These findings indicate that our approach age‐dependently improves cognition and reduces Aβ burden when used with an adjuvant suitable for humans, without increasing vascular Aβ deposits or microhemorrhages.

Morphological and Functional Abnormalities in Mitochondria Associated with Synaptic Degeneration in Prion Disease

Synaptic and dendritic pathology is a well-documented component of prion disease. In common with other neurodegenerative diseases that contain an element of protein misfolding, little is known about the underlying mechanisms of synaptic degeneration. In particular, in prion disease the relationship between synaptic malfunction, degeneration, and mitochondria has been neglected. We investigated a wide range of mitochondrial parameters, including changes in mitochondrial density, inner membrane ultrastructure, functional properties and nature of mitochondrial DNA from hippocampal tissue of mice with prion disease, which have ongoing synaptic pathology. Our results indicate that despite a lack of detectable changes in either mitochondrial density or expression of the mitochondrial proteins, mitochondrial function was impaired when compared with age-matched control animals. We observed changes in mitochondrial inner membrane morphology and a reduction in the cytochrome c oxidase activity relative to a sustained level of mitochondrial proteins such as porin and individual, functionally important subunits of complex II and complex IV. These data support the idea that mitochondrial dysfunction appears to occur due to inhibition or modification of respiratory complex rather than deletions of mitochondrial DNA. Indeed, these changes were seen in the stratum radiatum where synaptic pathology is readily detected, indicating that mitochondrial function is impaired and could potentially contribute to or even initiate the synaptic pathology in prion disease.

Expression of the small heat shock protein family in the mouse CNS: Differential anatomical and biochemical compartmentalization

The small heat shock proteins (sHsps) are a family of molecular chaperones defined by an alpha-crystallin domain that is important for sHsps oligomerization and chaperone activity. sHsps perform many physiological functions including the maintenance of the cellular cytoskeleton, the regulation of protein aggregation and modulate cell survival in a number of cell types including glial and neuronal cells. Many of these functions have been implicated in disease processes in the CNS and indeed sHsps are considered targets for disease therapy. Despite this, there is no study that systematically and comparatively characterized sHsps expression in the CNS. In the present study we have analyzed the expression of this gene family in the mouse brain by reverse-transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and Western blotting. Gene expression analysis of the 10 known members of mammalian sHsps confirms the presence of 5 sHsps in the CNS. A distinct white matter specific expression pattern for HspB5 and overlapping expression of HspB1 and HspB8 in the lateral and dorsal ventricles of the brain is observed. We confirm protein expression of HspB1, HspB5, HspB6 and HspB8 in the brain. Further subcellular fractionation of brain and synaptosomes details a distinct subcompartment-specific association and detergent solubility of sHsps. This biochemical signature is indicative of an association with synaptic and other neural specializations. This observation will help one understand the functional role played by sHsps during physiology and pathology in the CNS.

A non-toxic ligand for voxel-based MRI analysis of plaques in AD transgenic mice

Amyloid plaques are a characteristic feature in Alzheimers disease (AD). A novel non-toxic contrast agent is presented, Gd-DTPA-K6Abeta1-30, which is homologous to Abeta, and allows plaque detection in vivo. microMRI was performed on AD model mice and controls prior to and following intracarotid injection with Gd-DTPA-K6Abeta1-30 in mannitol solution, to transiently open the blood-brain barrier. A gradient echo T2(*)-weighted sequence was used to provide 100 microm isotropic resolution with imaging times of 115 min. The scans were examined with voxel-based analysis (VBA) using statistical parametric mapping, for un-biased quantitative comparison of ligand-injected mice and controls. The results indicate that: (1) Gd-DTPA-K6Abeta1-30 is an effective, non-toxic, ligand for plaque detection when combined with VBA (p< or =0.01-0.001), comparing pre and post-ligand injection scans. (2) Large plaques can be detected without the use of a contrast agent and this detection co-localizes with iron deposition. (3) Smaller, earlier plaques require contrast ligand for MRI visualization. Our ligand when combined with VBA may be useful for following therapeutic approaches targeting amyloid in transgenic mouse models.

GSK3α exhibits β‐catenin and tau directed kinase activities that are modulated by Wnt

In the presence of a Wnt signal β‐catenin is spared from proteasomal degradation through a complex mechanism involving GSK3β, resulting in the transcription of Wnt target genes. In this study we have explored whether GSK3α, a related isoform, can also regulate nuclear β‐catenin levels and whether this and the tau‐directed kinase activity of GSK3α are modulated by Wnt. GSK3α or GSK3β and their substrates, β‐catenin and tau, were transiently expressed in mammalian cells. Immunoblotting revealed that GSK3α reduces nuclear levels of β‐catenin, whilst reporter gene assays demonstrated that GSK3α inhibits β‐catenin‐directed Tcf/Lef‐dependent transcription. Moreover, activation of the Wnt pathway was found to attenuate both the β‐catenin‐ and the tau‐directed kinase activities of GSK3α and GSK3β. By immunoprecipitation we also found that axin‐1, the β‐catenin destruction complex scaffold protein, binds GSK3α. In the light of these findings GSK3α warrants further investigation regarding its involvement in Wnt signalling and tauopathies such as Alzheimers disease.

Presenilin 1 independently regulates beta-catenin stability and transcriptional activity.

Presenilin 1 (PS1) regulates β-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous β-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of β-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of β-catenin. PS1 also inhibited the transcriptional activity of endogenous β-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type β-catenin and two mutants, ΔN89β-catenin and ΔSTβ-catenin, were also inhibited by wild-type and mutant PS1. We conclude that PS1 negatively regulates the stability and transcriptional activity of β-catenin at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3β mediated degradation of β-catenin, and that mutations in PS1 differentially affect the stability and transcriptional activity of β-catenin.