Advancing 89Zr-immuno-PET in neuroscience with a bispecific anti-amyloid-beta monoclonal antibody - The choice of chelator is essential.

The accelerated approval of the monoclonal antibody (mAb) aducanumab as a treatment option for Alzheimer's Disease and the continued discussions about its efficacy have shown that a better understanding of immunotherapy for the treatment of neurodegenerative diseases is needed. 89Zr-immuno-PET could be a suitable tool to open new avenues for the diagnosis of CNS disorders, monitoring disease progression, and assessment of novel therapeutics. Herein, three different 89Zr-labeling strategies and direct radioiodination with 125I of a bispecific anti-amyloid-beta aducanumab derivate, consisting of aducanumab with a C-terminal fused anti-transferrin receptor binding single chain Fab fragment derived from 8D3 (Adu-8D3), were compared ex vivo and in vivo with regard to brain uptake and target engagement in an APP/PS1 Alzheimer's disease mouse model and wild type animals. Methods: Adu-8D3 and a negative control antibody, based on the HIV specific B12 antibody also carrying C-terminal fused 8D3 scFab (B12-8D3), were each conjugated with NCS-DFO, NCS-DFO*, or TFP-N-suc-DFO-Fe-ester, followed by radiolabeling with 89Zr. 125I was used as a substitute for 124I for labeling of both antibodies. 30 µg of radiolabeled mAb, corresponding to approximately 6 MBq 89Zr or 2.5 MBq 125I, were injected per mouse. PET imaging was performed 1, 3 and 7 days post injection (p.i.). All mice were sacrificed on day 7 p.i. and subjected to ex vivo biodistribution and brain autoradiography. Immunostaining on brain tissue was performed after autoradiography for further validation. Results: Ex vivo biodistribution revealed that the brain uptake of [89Zr]Zr-DFO*-NCS-Adu-8D3 (2.19 ±0.12 %ID/g) was as high as for its 125I-analog (2.21 ±0.15 %ID/g). [89Zr]Zr-DFO-NCS-Adu-8D3 and [89Zr]Zr-DFO-N-suc-Adu-8D3 showed significantly lower uptake (< 0.65 %ID/g), being in the same range as for the 89Zr-labeled controls (B12-8D3). Autoradiography of [89Zr]Zr-DFO*-NCS-Adu-8D3 and [125I]I-Adu-8D3 showed an amyloid-beta related granular uptake pattern of radioactivity. In contrast, the [89Zr]Zr-DFO-conjugates and the control antibody groups did not show any amyloid-beta related uptake pattern, indicating that DFO is inferior for 89Zr-immuno-PET imaging of the brain in comparison to DFO* for Adu-8D3. This was confirmed by day 7 PET images showing only amyloid-beta related brain uptake for [89Zr]Zr-DFO*-NCS-Adu-8D3. In wild type animals, such an uptake was not observed. Immunostaining showed a co-localization of all administered Adu-8D3 conjugates with amyloid-beta plaques. Conclusion: We successfully demonstrated that 89Zr-immuno-PET is suitable for imaging and quantifying amyloid-beta specific brain uptake using a bispecific aducanumab brain shuttling antibody, Adu-8D3, but only when using the novel chelator DFO*, and not DFO, for labeling with 89Zr.

Chronic Stress Induces Hippocampal Mitochondrial Damage in APPPS1 Model Mice and Wildtype Littermates.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia worldwide. Despite decades of investigation, the etiology of AD is not fully understood, although emerging evidence suggest that chronic environmental and psychological stress plays a role in the mechanisms and contributes to the risk of developing AD. Thus, dissecting the impact of stress on the brain could improve our understanding of the pathological mechanisms. OBJECTIVE: We aimed to study the effect of chronic stress on the hippocampal proteome in male APPPS1 transgenic mice and wildtype (WT) littermates. METHODS: APPPS1 and WT mice were subjected to 4 weeks of chronic stress followed by 3 weeks of continued diurnal disruption. Hippocampal tissue was used for proteomics analysis using label-free quantitative DIA based LC-MS/MS analysis. RESULTS: We identified significantly up- and downregulated proteins in both APPPS1 and WT mice exposed to chronic stress compared to the control groups. Via interaction network mapping, significant proteins could be annotated to specific pathways of mitochondrial function (oxidative phosphorylation and TCA cycle), metabolic pathways, AD pathway and synaptic functions (long term potentiation). In WT mice, chronic stress showed the highest impact on complex I of the oxidative phosphorylation pathway, while in APPPS1 mice this pathway was compromised broadly by chronic stress. CONCLUSION: Our data shows that chronic stress and amyloidosis additively contribute to mitochondrial damage in hippocampus. Although these results do not explain all effects of chronic stress in AD, they add to the scientific knowledge on the topic.

Chronic stress induces NPD-like behavior in APPPS1 and WT mice with subtle differences in gene expression.

Neuropsychiatric disturbances (NPDs) are considered hallmarks of Alzheimer's disease (AD). Nevertheless, treatment of these symptoms has proven difficult and development of safe and effective treatment options is hampered by the limited understanding of the underlying pathophysiology. Thus, robust preclinical models are needed to increase knowledge of NPDs in AD and develop testable hypotheses and novel treatment options. Abnormal activity of the hypothalamic-pituitary-adrenal (HPA) axis is implicated in many psychiatric symptoms and might contribute to both AD and NPDs development and progression. We aimed to establish a mechanistic preclinical model of NPD-like behavior in the APPPS1 mouse model of AD and wildtype (WT) littermates. In APPPS1 and WT mice, we found that chronic stress increased anxiety-like behavior and altered diurnal locomotor activity suggestive of sleep disturbances. Also, chronic stress activated the HPA axis, which, in WT mice, remained heightened for additional 3 weeks. Chronic stress caused irregular expression of circadian regulatory clock genes (BMAL1, PER2, CRY1 and CRY2) in both APPPS1 and WT mice. Interestingly, APPPS1 and WT mice responded differently to chronic stress in terms of expression of serotonergic markers (5-HT1A receptor and MAOA) and inflammatory genes (IL-6, STAT3 and ADMA17). These findings indicate that, although the behavioral response to chronic stress might be similar, the neurobiochemical response was different in APPPS1 mice, which is an important insight in the efforts to develop safe and effective treatments options for NPDs in AD patients. Further work is needed to substantiate these findings.

Anti-Aß Antibody Aducanumab Regulates the Proteome of Senile Plaques and Closely Surrounding Tissue in a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by accumulation of amyloid-ß (Aß) species and deposition of senile plaques (SPs). Clinical trials with the anti-Aß antibody aducanumab have been completed recently. OBJECTIVE: To characterize the proteomic profile of SPs and surrounding tissue in a mouse model of AD in 10-month-old tgAPPPS1-21 mice after chronic treatment with aducanumab for four months with weekly dosing (10 mg/kg). METHODS: After observing significant reduction of SP numbers in hippocampi of aducanumab-treated mice, we applied a localized proteomic analysis by combining laser microdissection and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the remaining SPs in hippocampi. We microdissected three subregions, containing SPs, SP penumbra level 1, and an additional penumbra level 2 to follow the proteomic profile as gradient. RESULTS: In the aducanumab-treated mice, we identified 17 significantly regulated proteins that were associated with 1) mitochondria and metabolism (ACAT2, ATP5J, ETFA, EXOG, HK1, NDUFA4, NDUFS7, PLCB1, PPP2R4), 2) cytoskeleton and axons (ADD1, CAPZB, DPYSL3, MAG), 3) stress response (HIST1H1C/HIST1H1D, HSPA12A), and 4) AßPP trafficking/processing (CD81, GDI2). These pathways and some of the identified proteins are implicated in AD pathogenesis. Proteins associated with mitochondria and metabolism were mainly upregulated while proteins associated with AßPP trafficking/processing and stress response pathways were mainly downregulated, suggesting that aducanumab could lead to a beneficial proteomic profile around SPs in tgAPPPS1-21 mice. CONCLUSION: We identified novel proteomic patterns of SPs and surrounding tissue indicating that chronic treatment with aducanumab could inhibit Aß toxicity and increase phagocytosis and cell viability.

Steps Towards Developing Effective Treatments for Neuropsychiatric Disturbances in Alzheimer's Disease: Insights From Preclinical Models, Clinical Data, and Future Directions.

Alzheimer's disease (AD) is the most common form of dementia worldwide. It is mostly known for its devastating effect on memory and learning but behavioral alterations commonly known as neuropsychiatric disturbances (NPDs) are also characteristics of the disease. These include apathy, depression-like behavior, and sleep disturbances, and they all contribute to an increased caregiver burden and earlier institutionalization. The interaction between NPDs and AD pathology is not well understood, but the consensus is that they contribute to disease progression and faster decline. Consequently, recognizing and treating NPDs might improve AD pathology and increase the quality of life for both patients and caregivers. In this review article, we examine previous and current literature on apathy, depressive symptoms, and sleep disturbances in AD patients and preclinical AD mechanistic models. We hypothesize that tau accumulation, beta-amyloid (Aß) aggregation, neuroinflammation, mitochondrial damage, and loss of the locus coeruleus (LC)-norepinephrine (NE) system all collectively impact the development of NPDs and contribute synergistically to AD pathology. Targeting more than one of these processes might provide the most optimal strategy for treating NPDs and AD. The development of such clinical approaches would be preceded by preclinical studies, for which robust and reliable mechanistic models of NPD-like behavior are needed. Thus, developing effective preclinical research models represents an important step towards a better understanding of NPDs in AD.

Proteomic and Unbiased Post-Translational Modification Profiling of Amyloid Plaques and Surrounding Tissue in a Transgenic Mouse Model of Alzheimer's Disease.

Amyloid plaques are one of the hallmarks of Alzheimer's disease (AD). The main constituent of amyloid plaques is amyloid-ß peptides, but a complex interplay of other infiltrating proteins also co-localizes. We hypothesized that proteomic analysis could reveal differences between amyloid plaques and adjacent control tissue in the transgenic mouse model of AD (APPPS1-21) and in similar regions from non-transgenic littermates. Our microproteomic strategy included isolation of regions of interest by laser capture microdissection and analysis by liquid chromatography mass spectrometry-based label-free relative quantification. We consistently identified 183, 224, and 307 proteins from amyloid plaques, adjacent control and non-tg samples, respectively. Pathway analysis revealed 27 proteins that were significantly regulated when comparing amyloid plaques and corresponding adjacent control regions. We further elucidated that co-localized proteins were subjected to post-translational modifications and are the first to report 193 and 117 unique modifications associated to amyloid plaques and adjacent control extracts, respectively. The three most common modifications detected in proteins from the amyloid plaques were oxidation, deamidation, and pyroglutamylation. Together, our data provide novel information about the biological processes occurring within and around amyloid plaques in the APPPS1-21 mouse model of AD.

Dual strategy for reduced signal-suppression effects in matrix-assisted laser desorption/ionization mass spectrometry imaging.

RATIONALE: The molecular complexity of tissue features several signal-suppression effects which reduce the ionization of analytes significantly and thereby weakens the quality of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) imaging (MALDI imaging). We report a novel approach in MALDI imaging by reducing signal-suppression effects for the analysis of beta-amyloid (Aß) plaques, one pathological hallmark of Alzheimer's disease (AD). METHODS: We analyzed Aß proteoforms from postmortem AD brains and brains from transgenic mice (APPPS1-21) overexpressing familial AD mutations by combining two techniques: (1) laser capture microdissection (LCM) to accumulate Aß plaques and (2) phosphoric acid (PA) as additive to the super-2,5-dihydroxybenzoic acid matrix. RESULTS: LCM and MALDI-MS enabled tandem mass spectrometric fragmentation of stained Aß plaques. PA improved the signal-to-noise (S/N) ratio, especially of the Aß1-42 peptide, by three-fold compared with the standard matrix additive trifluoroacetic acid. The beneficial effect of the PA matrix additive in MALDI imaging was particularly important for AD brain tissue. We identified several significant differences in Aß plaque composition from AD compared with APPPS1-21, underlining the value of reducing signal-suppressing effects in MALDI imaging. CONCLUSIONS: We present a novel strategy for overcoming signal-suppression effects in MALDI imaging of Aß proteoforms.

Pathogenic tau does not drive activation of the unfolded protein response.

The unfolded protein response (UPR) is commonly associated with a range of neurodegenerative diseases, and targeting UPR components has been suggested as a therapeutic strategy. The UPR surveys protein folding within the endoplasmic reticulum. However, many of the misfolded proteins that accumulate in neurodegeneration are localized so that they do not directly cause endoplasmic reticulum triggers that activate this pathway. Here, using a transgenic mouse model and primary cell cultures along with quantitative PCR, immunoblotting, and immunohistochemistry, we tested whether the UPR is induced in in vivo and in vitro murine models of tauopathy that are based on expression of mutant tauP301L We found no evidence for the UPR in the rTg4510 mouse model, in which mutant tau is transgenically expressed under the control of tetracycline-controlled transactivator protein. This observation was supported by results from acute experiments in which neuronal cultures expressed mutant tau and accumulated misfolded cytoplasmic tau aggregates but exhibited no UPR activation. These results suggest that the UPR is not induced as a response to tau misfolding and aggregation despite clear evidence for progressive cellular dysfunction and degeneration. We propose that caution is needed when evaluating the implied significance of the UPR as a critical determinant across major neurodegenerative diseases.

Highly specific and selective anti-pS396-tau antibody C10.2 targets seeding-competent tau.

INTRODUCTION: The abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and other tauopathies. METHODS: Highly specific and selective anti-pS396-tau antibodies have been generated using peptide immunization with screening against pathologic hyperphosphorylated tau from rTg4510 mouse and AD brains and selection in in vitro and in vivo tau seeding assays. RESULTS: The antibody C10.2 bound specifically to pS396-tau with an IC50 of 104 pM and detected preferentially hyperphosphorylated tau aggregates from AD brain with an IC50 of 1.2 nM. C10.2 significantly reduced tau seeding of P301L human tau in HEK293 cells, murine cortical neurons, and mice. AD brain extracts depleted with C10.2 were not able to seed tau in vitro and in vivo, demonstrating that C10.2 specifically recognized pathologic seeding-competent tau. DISCUSSION: Targeting pS396-tau with an antibody like C10.2 may provide therapeutic benefit in AD and other tauopathies.

Antibody Engineering for Optimized Immunotherapy in Alzheimer's Disease.

There are nearly 50 million people with Alzheimer's disease (AD) worldwide and currently no disease modifying treatment is available. AD is characterized by deposits of Amyloid-ß (Aß), neurofibrillary tangles, and neuroinflammation, and several drug discovery programmes studies have focussed on Aß as therapeutic target. Active immunization and passive immunization against Aß leads to the clearance of deposits in humans and transgenic mice expressing human Aß but have failed to improve memory loss. This review will discuss the possible explanations for the lack of efficacy of Aß immunotherapy, including the role of a pro-inflammatory response and subsequent vascular side effects, the binding site of therapeutic antibodies and the timing of the treatment. We further discuss how antibodies can be engineered for improved efficacy.

Cerebrospinal fluid neurofilament light chain as a biomarker of neurodegeneration in the Tg4510 and MitoPark mouse models.

A challenge in working with preclinical models of neurodegeneration has been how to non-invasively monitor disease progression. Neurofilament proteins are established axonal damage markers and have been found to be elevated in cerebrospinal fluid (CSF) and blood from patients with neurodegenerative disorders like Alzheimer's disease (AD), Parkinson's disease (PD) and tauopathies. We hypothesized that CSF neurofilament light (NF-L) can be used to track progression of neurodegeneration and potentially monitor the efficacy of novel therapeutic agents in preclinical development. To substantiate this, we examined whether changes in NF-L levels in brain, plasma, and CSF reflect the changing disease status of preclinical models of neurodegeneration. Using Western Blot and ELISA we characterized NF-L and disease-related proteins in brain, CSF and plasma samples from Tg4510 mice (tauopathy/AD), MitoPark mice (PD), and their age-matched control littermates. We found that CSF NF-L clearly discriminates Tg4510 from control littermates, which was not observed for the MitoPark model. However, both Tg4510 and MitoPark showed altered expression and solubilization of NFs compared to control littermates. We found a significant correlation between CSF NF-L and plasma NF-L in Tg4510, suggesting a similar biomarker potential of plasma NF-L. Also, CSF NF-L correlated significantly with tau in Tg4510 brains, suggesting a surrogate biomarker potential of CSF NF-L. Overall, our findings provide further evidence that NF-L correlates with disease severity and our results suggests, that CSF NF-L has utility as a surrogate or adjunct biomarker for neurodegeneration in the Tg4510 model, but independent validation is warranted.

Antioxidant peroxiredoxin 6 protein rescues toxicity due to oxidative stress and cellular hypoxia in vitro, and attenuates prion-related pathology in vivo.

Protein misfolding, mitochondrial dysfunction and oxidative stress are common pathomechanisms that underlie neurodegenerative diseases. In prion disease, central to these processes is the post-translational transformation of cellular prion protein (PrP(c)) to the aberrant conformationally altered isoform; PrP(Sc). This can trigger oxidative reactions and impair mitochondrial function by increasing levels of peroxynitrite, causing damage through formation of hydroxyl radicals or via nitration of tyrosine residues on proteins. The 6 member Peroxiredoxin (Prdx) family of redox proteins are thought to be critical protectors against oxidative stress via reduction of H2O2, hydroperoxides and peroxynitrite. In our in vitro studies cellular metabolism of SK-N-SH human neuroblastoma cells was significantly decreased in the presence of H2O2 (oxidative stressor) or CoCl2 (cellular hypoxia), but was rescued by treatment with exogenous Prdx6, suggesting that its protective action is in part mediated through a direct action. We also show that CoCl2-induced apoptosis was significantly decreased by treatment with exogenous Prdx6. We proposed a redox regulator role for Prdx6 in regulating and maintaining cellular homeostasis via its ability to control ROS levels that could otherwise accelerate the emergence of prion-related neuropathology. To confirm this, we established prion disease in mice with and without astrocyte-specific antioxidant protein Prdx6, and demonstrated that expression of Prdx6 protein in Prdx6 Tg ME7-animals reduced severity of the behavioural deficit, decreased neuropathology and increased survival time compared to Prdx6 KO ME7-animals. We conclude that antioxidant Prdx6 attenuates prion-related neuropathology, and propose that augmentation of endogenous Prdx6 protein represents an attractive adjunct therapeutic approach for neurodegenerative diseases.

Effect of amyloid-ß (Aß) immunization on hyperphosphorylated tau: a potential role for glycogen synthase kinase (GSK)-3ß.

AIMS: Active amyloid-ß (Aß) immunotherapy in Alzheimer's disease (AD) induces removal of Aß and phosphorylated tau (ptau). Glycogen synthase kinase (GSK)-3ß is a kinase, responsible for phosphorylation of tau, activation of which can be induced by phosphorylated double-stranded RNA-dependent protein kinase (pPKR). Using a post-mortem cohort of immunized AD cases, we investigated the effect of Aß immunization on GSK-3ß expression and pPKR. METHODS: We immunostained 11 immunized AD cases and 28 unimmunized AD cases for active, inactive and total GSK-3ß, and for pPKR. Quantification of protein load was performed in the hippocampal region including CA1, subiculum and entorhinal cortex. RESULTS: All three areas showed a significant decrease in the three forms of GSK-3ß (P < 0.05) and a nonsignificant trend towards lower pPKR load in the immunized AD cases compared with the unimmunized AD cases. CONCLUSION: The lower GSK-3ß expression generated by Aß immunotherapy shows evidence of a modification of the signalling pathway induced by GSK-3ß leading to the overall reduction of tau, supporting the contention that in humans, GSK-3ß unifies Aß and tau-related neuropathology.

Blocking the apoE/Aß interaction ameliorates Aß-related pathology in APOE ε2 and ε4 targeted replacement Alzheimer model mice.

Accumulation of ß-amyloid (Aß) in the brain is essential to Alzheimer's disease (AD) pathogenesis. Carriers of the apolipoprotein E (APOE) ε4 allele demonstrate greatly increased AD risk and enhanced brain Aß deposition. In contrast, APOE ε2 allele carries show reduced AD risk, later age of disease onset, and lesser Aß accumulation. However, it remains elusive whether the apoE2 isoform exerts truly protective effect against Aß pathology or apoE2 plays deleterious role albeit less pronounced than the apoE4 isoform. Here, we characterized APPSW/PS1dE9/APOE ε2-TR (APP/E2) and APPSW/PS1dE9/APOE ε4-TR (APP/E4) mice, with targeted replacement (TR) of the murine Apoe for human ε2 or ε4 alleles, and used these models to investigate effects of pharmacological inhibition of the apoE/Aß interaction on Aß deposition and neuritic degeneration. APP/E2 and APP/E4 mice replicate differential effect of human apoE isoforms on Aß pathology with APP/E4 mice showing a several-fold greater load of Aß plaques, insoluble brain Aß levels, Aß oligomers, and density of neuritic plaques than APP/E2 mice. Furthermore, APP/E4 mice, but not APP/E2 mice, exhibit memory impairment on object recognition and radial arm maze tests. Between the age of 6 and 10 months APP/E2 and APP/E4 mice received treatment with Aß12-28P, a non-toxic, synthetic peptide homologous to the apoE binding motif within the Aß sequence, which competitively blocks the apoE/Aß interaction. In both lines, the treatment significantly reduced brain Aß accumulation, co-accumulation of apoE within Aß plaques, and neuritic degeneration, and prevented memory deficit in APP/E4 mice. These results indicate that both apoE2 and apoE4 isoforms contribute to Aß deposition and future therapies targeting the apoE/Aß interaction could produce favorable outcome in APOE ε2 and ε4 allele carriers.

Modulation of amyloid precursor protein expression reduces ß-amyloid deposition in a mouse model.

OBJECTIVE: Proteolytic cleavage of the amyloid precursor protein (APP) generates ß-amyloid (Aß) peptides. Prolonged accumulation of Aß in the brain underlies the pathogenesis of Alzheimer disease (AD) and is regarded as a principal target for development of disease-modifying therapeutics. METHODS: Using Chinese hamster ovary (CHO) APP751SW cells, we identified and characterized effects of 2-([pyridine-2-ylmethyl]-amino)-phenol (2-PMAP) on APP steady-state level and Aß production. Outcomes of 2-PMAP treatment on Aß accumulation and associated memory deficit were studied in APPSW /PS1dE9 AD transgenic model mice. RESULTS: In CHO APP751SW cells, 2-PMAP lowered the steady-state APP level and inhibited Aßx-40 and Aßx-42 production in a dose-response manner with a minimum effective concentration ≤ 0.5µM. The inhibitory effect of 2-PMAP on translational efficiency of APP mRNA into protein was directly confirmed using a 35S-methionine/cysteine metabolic labeling technique, whereas APP mRNA level remained unaltered. Administration of 2-PMAP to APPSW /PS1dE9 mice reduced brain levels of full-length APP and its C-terminal fragments and lowered levels of soluble Aßx-40 and Aßx-42 . Four-month chronic treatment of APPSW /PS1dE9 mice revealed no observable toxicity and improved animals' memory performance. 2-PMAP treatment also caused significant reduction in brain Aß deposition determined by both unbiased quantification of Aß plaque load and biochemical analysis of formic acid-extracted Aßx-40 and Aßx-42 levels and the level of oligomeric Aß. INTERPRETATION: We demonstrate the potential of modulating APP steady-state expression level as a safe and effective approach for reducing Aß deposition in AD transgenic model mice.

Analysis of the hippocampal proteome in ME7 prion disease reveals a predominant astrocytic signature and highlights the brain-restricted production of clusterin in chronic neurodegeneration.

Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ(TM)/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.

Blocking the interaction between apolipoprotein E and Aß reduces intraneuronal accumulation of Aß and inhibits synaptic degeneration.

Accumulation of ß-amyloid (Aß) in the brain is a key event in Alzheimer disease pathogenesis. Apolipoprotein (Apo) E is a lipid carrier protein secreted by astrocytes, which shows inherent affinity for Aß and has been implicated in the receptor-mediated Aß uptake by neurons. To characterize ApoE involvement in the intraneuronal Aß accumulation and to investigate whether blocking the ApoE/Aß interaction could reduce intraneuronal Aß buildup, we used a noncontact neuronal-astrocytic co-culture system, where synthetic Aß peptides were added into the media without or with cotreatment with Aß12-28P, which is a nontoxic peptide antagonist of ApoE/Aß binding. Compared with neurons cultured alone, intraneuronal Aß content was significantly increased in neurons co-cultured with wild-type but not with ApoE knockout (KO) astrocytes. Neurons co-cultured with astrocytes also showed impaired intraneuronal degradation of Aß, increased level of intraneuronal Aß oligomers, and marked down-regulation of several synaptic proteins. Aß12-28P treatment significantly reduced intraneuronal Aß accumulation, including Aß oligomer level, and inhibited loss of synaptic proteins. Furthermore, we showed significantly reduced intraneuronal Aß accumulation in APPSW/PS1dE9/ApoE KO mice compared with APPSW/PS1dE9/ApoE targeted replacement mice that expressed various human ApoE isoforms. Data from our co-culture and in vivo experiments indicate an essential role of ApoE in the mechanism of intraneuronal Aß accumulation and provide evidence that ApoE/Aß binding antagonists can effectively prevent this process.

Differential molecular chaperone response associated with various mouse adapted scrapie strains.

Prionoses are a group of neurodegenerative diseases characterized by misfolding of cellular prion protein (PrP(C)) and accumulation of its diseases specific conformer PrP(Sc) in the brain and neuropathologically, they can be associated with presence or absence of PrP amyloid deposits. Functional molecular chaperones (MCs) that constitute the unfolded protein response include heat shock proteins and glucose-regulated protein families. They protect intracellular milieu against various stress conditions including accumulation of misfolded proteins and oxidative stress, typical of neurodegenerative diseases. Little is known about the role of MCs in pathogenesis of prionoses in mammalian prion model systems. In this study we characterized MCs response pattern in mice infected with various mouse adapted scrapie strains. Rather than uniform upregulation of MCs, we encountered two distinctly different patterns of MCs response distinguishing ME7 and 87V strains from 22L and 139A strains. ME7 and 87V strains are known for the induction of amyloid deposition in infected animals, while in mice infected with 22L and 139A strains amyloid deposits are absent. MCs response pattern similar to that associated with amyloidogenic ME7 and 87V strains was also observed in APPPS1-21 Alzheimer's transgenic mice, which represent an aggressive model of cerebral amyloidosis caused by ß-amyloid deposition. Our results highlight the probability that different mechanisms of MCs regulation exist driven by amyloidogenic and non-amyloidogenic nature of prion strains.

Change in tau phosphorylation associated with neurodegeneration in the ME7 model of prion disease.

Hyperphosphorylation of the microtubule-associated protein tau is a significant determinant in AD (Alzheimer's disease), where it is associated with disrupted axonal transport and probably causes synaptic dysfunction. Although less well studied, hyperphosphorylation has been observed in prion disease. We have investigated the expression of hyperphosphorylated tau in the hippocampus of mice infected with the ME7 prion agent. In ME7-infected animals, there is a selective loss of CA1 synapse, first discernable at 13 weeks of disease. There is a potential that dysfunctional axonal transport contributes to this synaptopathy. Thus investigating hyperphosphorylated tau that is dysfunctional in AD could illuminate whether and how they are significant in prion disease. We observed no differences in the levels of phosphorylated tau (using MC1, PHF-1 and CP13 antibodies) in detergent-soluble and detergent-insoluble fractions extracted from ME7- and NBH- (normal brain homogenate) treated animals across disease. In contrast, we observed an increase in phospho-tau staining for several epitopes using immunohistochemistry in ME7-infected hippocampal sections. Although the changes were not of the magnitude seen in AD tissue, clear differences for several phospho-tau species were seen in the CA1 and CA3 of ME7-treated animals (pSer(199-202)>pSer(214)>PHF-1 antibody). Temporally, these changes were restricted to animals at 20 weeks and none of the disease-related staining was associated with the axons or dendrites that hold CA1 synapses. These findings suggest that phosphorylation of tau at the epitopes examined does not underpin the early synaptic dysfunction. These data suggest that the changes in tau phosphorylation recorded here and observed by others relate to end-stage prion pathology when early dysfunctions have progressed to overt neuronal loss.