Tina Fiorelli

LDLR Expression and Localization Are Altered in Mouse and Human Cell Culture Models of Alzheimer's Disease

Background Alzheimers disease (AD) is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the ε-4 allele of apolipoprotein E (apoE), the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR) has the highest affinity for apoE and plays an important role in brain cholesterol metabolism. Methodology/Principal Findings Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Aβ-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of γ- and α-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network. Conclusions/Significance These data suggest that increased APP expression and Aβ exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression.

Altered Processing of Amyloid Precursor Protein in Cells Undergoing Apoptosis

Altered proteolysis of amyloid precursor protein is an important determinant of pathology development in Alzheimers disease. Here, we describe the detection of two novel fragments of amyloid precursor protein in H4 neuroglioma cells undergoing apoptosis. Immunoreactivity of these 25–35 kDa fragments to two different amyloid precursor protein antibodies suggests that they contain the amyloid-β region and an epitope near the C-terminus of amyloid precursor protein. Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases. Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results. Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism. Molecular weight prediction and immunoreactivity of the fragments generated suggested that such fragments could not be generated by cleavage at any previously identified caspase, secretase, or calpain site on amyloid precursor protein. Bioinformatic analysis of the amino acid sequence of amyloid precursor protein revealed that fragments fitting the observed size and immunoreactivity could be generated by either cleavage at a novel, hitherto unidentified, caspase site or at a previously identified matrix metalloproteinase site in the extracellular domain. Proteolytic cleavage at any of these sites leads to a decrease in the generation of α-secretase cleaved secreted APP, which has both anti-apoptotic and neuroprotective properties, and thus may contribute to neurodegeneration in Alzheimers disease.

Alpha 1-Antichymotrypsin, an Inflammatory Protein Overexpressed in the Brains of Patients with Alzheimer’s Disease, Induces Tau Hyperphosphorylation through c-Jun N-Terminal Kinase Activation

The association of inflammatory proteins with neuritic plaques in the brains of Alzheimers disease (AD) patients has led to the hypothesis that inflammation plays a pivotal role in the development of pathology in AD. Earlier studies have shown that alpha 1-antichymotrypsin (ACT) enhances amyloid beta fibrillization and accelerated plaque formation in APP transgenic mice. Later studies from our laboratory have shown that purified ACT induces tau hyperphosphorylation and degeneration in neurons. In order to understand the mechanisms by which inflammatory proteins enhance tau hyperphosphorylation, we injected interleukin-1β (IL-1β) intracerebroventricularly into mice expressing human ACT, human tau, or both transgenes. It was found that the hyperphosphorylation of tau in ACT and ACT/htau mice after IL-1β injection correlated with increased phosphorylation of c-Jun N-terminal kinase (JNK). We verified the involvement of JNK in ACT-induced tau phosphorylation by utilizing JNK inhibitors in cultured primary neurons treated with ACT, and we found that the inhibitor showed complete prevention of ACT-induced tau phosphorylation. These results indicate that JNK is one of the major kinases involved in the ACT-mediated tau hyperphosphorylation and suggest that inhibitors of this kinase may protect against inflammation-induced tau hyperphosphorylation and neurodegeneration associated with AD.

The Deanna protocol supplement complex supports mitochondrial energy metabolism and prolongs lifespan in preclinical models of amyotrophic lateral sclerosis (ALS)

IntroductionAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of motor neurons causing progressive muscle weakness, paralysis, and eventual death from respiratory failure. There is currently no cure or effective treatment for ALS. The Deanna protocol (DP) is a comprehensive treatment approach that includes a metabolic therapy in the form of a supplement complex that improved neurological function, increased motor function and survival in SOD1-G93A mice and has been reported to alleviate symptoms in patients with ALS; therefore, it has been proposed as a treatment for the disease.ObjectivesWe hypothesized that the major components of the DP, including arginine alpha-ketoglutarate, gamma amino butyric acid (GABA), medium chain triglycerides (MCT), and soluble coenzyme Q10 (ubiquinol) supports energy metabolism by increasing energy intermediates of the tricarboxylic acid cycle in a mouse model of ALS (SOD1-G93A).MethodsWe explored the potential therapeutic use of DP by testing the effects of DP supplementation on the metabolomics profile of SOD1-G93A mice. In addition, we assessed time to paralysis in a Caenorhabditis elegans model of ALS (TDP-43) given DP supplementation. SOD1-G93A mice were fed a standard rodent diet (SD) or SD with low dose (LOW) or high dose of DP (HIGH). Global metabolomics analysis was performed on blood plasma from treated and untreated animals. Additionally, the time to paralysis of TDP-43 ALS C. elegans treated with and without the individual and combination DP supplements was measured.Results30 and 49 biochemicals were significantly altered in the plasma of LOW and HIGH groups, respectively. Metabolites associated with mitochondrial energy metabolism, arginine metabolism, as well as long- and medium-chain fatty acids, GABA and related intermediates were elevated in response to DP. Elements of DP, arginine and alpha-ketoglutarate, GABA, and MCTs prolonged the rate of final paralysis of C. elegans TDP-43 disease models.ConclusionTargeting energy metabolism with the DP supplement as a metabolic therapy produces a change in the global metabolic profile of ALS mice that support the role of the DP for enhanced mitochondrial energy metabolism and prolongs time to paralysis of ALS C. elegans.

Microtubule destabilization leads to APP phosphorylation and processing: Implications for Alzheimer's disease pathogenesis

However, also prion proteins produced by neurons, can form amyloid plaques in the brain. In variant Creutzfeldt Jakob disease (vCJD), sporadic CJD forms with a longer duration, hereditary CJD and Gerstmann Straussler Scheinker’s disease (GSS) prion amyloid plaques can be found. In the present immunohistochemical study we investigated the presence of amyloid associated proteins such as complement and alpha1-antichymotrypsin (ACT), microglia (HLADR), ubiquitin and tau in 4 different forms of prion diseases with prion amyloid plaques with or without prion cerebral amyloid angiopathy (CAA) and compared the results with the findings in classical AD. Methods: Cortical tissue obtained at autopsy was used from 3 vCJD (16, 37 and 49 y old), 2 sporadic CJD patients with longer duration (MM2, VV2 forms; resp. 55 y and 58 y) and 3 different GSS patients (57y, 57y, 45y) including 2 novel prion mutations, i.e. Y226X and Q227X stop codon mutations, and 5 AD cases (29y 76y). Immunohistochemistry was performed on formalin fixed tissue using antibodies against Abeta 1-17, prion proteins (3F4), complement C3b, HLADR, ubiquitin and Tau (AT8) and some sections were counterstained with Congo red. Cryostat sections were used for staining with antibodies against complement C3c and ACT. Results: Complement proteins were found in amyloid deposits both in AD and in prion diseases without Abeta. In a case with extensive prion CAA, complement was also detected in the amyloidotic vessel wall. Clustering of microglia was seen both in AD plaques and around prion plaques but also around prion CAA. Extensive neurofibrillary degeneration with numerous tangles throughout the cortex was found in the stopcodon Q227X patient (aged 45y) mimicking the AD cases. In the MM2 prion case (55 y) tau and ubiquitin was found especially near prion plaques. Prion CAA showed a mild tau and ubiquitin positivity around the vessel walls. No tau was found in the vCJD cases. Conclusions: Inflammatory changes and neurofibrillary degeneration seems to be related to cerebral amyloidosis irrespective the type of amyloid.