Space-Like Irradiation Exacerbated Cognitive Deficits and Amyloid Pathology in CRND8 Mouse Model of Alzheimer's Disease.

Background: Space radiation was linked to neurological damage and behavioral deficits which raised concerns of increased degenerative risk on the brain and development of Alzheimer's disease following space travel. Objective: In this study, we investigated the effects of irradiation by 56Fe and 28Si in CRND8 mice, an Alzheimer's disease mouse model. Methods: Six-month-old CRND8 mice were exposed to whole body irradiation by 56Fe and 28Si at 0.5 Gy and 2 Gy doses. Behavior tests were administered 1-month to 3-months post-irradiation. Amyloid deposition and other pathological changes were analyzed 3-months and/or 6-months post-irradiation. Results: The Novel Object Recognition test showed some decline in 8-month-old mice compared to non-irradiated CRND8 mice. Male mice also showed a loss of freezing behavior in the fear conditioning contextual test following irradiation. Golgi staining revealed a loss of spines in hippocampal neurons after irradiation. Total amyloid immunohistochemistry showed a robust increase in 3-months post-irradiation 56Fe groups which became normalized to non-irradiated group by 6-months post-irradiation. However, 2 Gy 28Si caused a trend towards increased plaque load at 3-months post-irradiation which became significant at 6-months post irradiation only in male CRND8 mice. While 0.5 Gy Fe did not induce obvious changes in the total number of iba-1 positive microglia, more hippocampal microglia were found to express PCNA after 0.5 Gy Fe treatment, suggesting potential involvement of microglial dysfunction. Conclusions: Overall, our study provides new evidence of gender-specific and ion-dependent effects of space radiation on cognition and amyloid pathology in AD models.

Glioma stem cells activate platelets by plasma-independent thrombin production to promote glioblastoma tumorigenesis.

Background: The interaction between platelets and cancer cells has been underexplored in solid tumor models that do not metastasize, for example, glioblastoma (GBM) where metastasis is rare. Histologically, it is known that glioma stem cells (GSCs) are found in perivascular and pseudsopalisading regions of GBM, which are also areas of platelet localization. High platelet counts have been associated with poor clinical outcomes in many cancers. While platelets are known to promote the progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Here, we aimed to understand how the bidirectional interaction between platelets and GSCs drives GBM oncogenesis. Methods: Male and female NSG mice were transplanted with GSC lines and treated with antiplatelet and anti-thrombin inhibitors. Immunofluorescence, qPCR, and Western blots were used to determine expression of coagulation mechanism in GBM tissue and subsequent GSC lines. Results: We show that GSCs activate platelets by endogenous production of all the factors of the intrinsic and extrinsic coagulation cascades in a plasma-independent manner. Therefore, GSCs produce thrombin resulting in platelet activation. We further demonstrate that the endogenous coagulation cascades of these cancer stem cells are tumorigenic: they activate platelets to promote stemness and proliferation in vitro and pharmacological inhibition delays tumor growth in vivo. Conclusions: Our findings uncover a specific preferential relationship between platelets and GSCs that drive GBM malignancies and identify a therapeutically targetable novel interaction.

Proteins inform survival-based differences in patients with glioblastoma.

BACKGROUND: Improving the care of patients with glioblastoma (GB) requires accurate and reliable predictors of patient prognosis. Unfortunately, while protein markers are an effective readout of cellular function, proteomics has been underutilized in GB prognostic marker discovery. METHODS: For this study, GB patients were prospectively recruited and proteomics discovery using liquid chromatography-mass spectrometry analysis (LC-MS/MS) was performed for 27 patients including 13 short-term survivors (STS) (≤10 months) and 14 long-term survivors (LTS) (≥18 months). RESULTS: Proteomics discovery identified 11 941 peptides in 2495 unique proteins, with 469 proteins exhibiting significant dysregulation when comparing STS to LTS. We verified the differential abundance of 67 out of these 469 proteins in a small previously published independent dataset. Proteins involved in axon guidance were upregulated in STS compared to LTS, while those involved in p53 signaling were upregulated in LTS. We also assessed the correlation between LS MS/MS data with RNAseq data from the same discovery patients and found a low correlation between protein abundance and mRNA expression. Finally, using LC-MS/MS on a set of 18 samples from 6 patients, we quantified the intratumoral heterogeneity of more than 2256 proteins in the multisample dataset. CONCLUSIONS: These proteomic datasets and noted protein variations present a beneficial resource for better predicting patient outcome and investigating potential therapeutic targets.

Generation of Glioblastoma Patient-Derived Intracranial Xenografts for Preclinical Studies.

Glioblastoma multiforme (GBM) is the most malignant primary brain cancer affecting adults. Therapeutic options for GBM have remained the same for over a decade with no significant improvement. Many therapies that are successful in culture have failed in patients, likely due to the complex microenvironment in the brain, which has yet to be reproduced in any culture model. Furthermore, the high passage number of cultured cells and clonal selection fail to recapitulate the molecular and genomic signatures of GBM. We have established orthotopic patient-derived xenografts (PDX) from 37 GBM patients with human GBM. Of the 69 patient samples analyzed, we were successful in passaging 37 lines three or more generations (53.6%). After phenotypic characterization of the xenografted tumor tissue, two different growth patterns emerged highly invasive or localized. The phenotype was dependent on malignancy and previous treatment of the patient from which the xenograft was derived. Physiologically, mice exhibited symptoms more quickly with each subsequent passage, particularly in the localized tumors. Study of these physiologically relevant human xenografts in mice will enable therapeutic screenings in a microenvironment that more closely resembles GBM and may allow development of individualized patient models which may eventually be used for simulating treatment.

Plant Virus-Like Particle In Situ Vaccine for Intracranial Glioma Immunotherapy.

Despite aggressive multi-modality treatment with surgery, radiation and chemotherapies, malignant glioma inevitably recurs and has dismal survival rates. Recent progress in immunotherapy has led to a resurgence of interest, and immunotherapies are being investigated for treatment of glioma. However, the unique brain anatomy and a highly immunosuppressive glioma microenvironment pose significant challenges to achieving efficacy. Thus, there is a critical need for assessment of next-generation immunotherapies for glioma. In this study, we have investigated the efficacy of the nanoparticle platform technology based on plant-derived Cowpea mosaic virus like particles (empty CPMV or eCPMV) to instigate a potent immune response against intracranial glioma. CPMV immunotherapy has been shown to efficiently reverse the immunosuppressive tumor microenvironments in pre-clinical murine models of dermal melanoma and metastatic melanoma, metastatic breast cancer, intraperitoneal ovarian cancer and in canine patients with oral melanoma. In the present study, we demonstrate that in situ administration of CPMV immunotherapy in the setting of glioma can effectively recruit unique subset of effector innate and adaptive immune cells to the brain parenchyma while reducing immune suppressive cellular population, leading to regression of intracranial glioma. The in situ CPMV nanoparticle vaccine offers a potent yet safe and localized immunotherapy for intracranial glioma.

Hydroxynonenal-generated crosslinking fluorophore accumulation in Alzheimer disease reveals a dichotomy of protein turnover.

Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently bind amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated in intimate association with the pathological lesions of Alzheimer disease (AD), suggesting that oxidative stress is a major component of AD pathogenesis. Some HNE-protein products result in protein crosslinking through a fluorescent compound similar to lipofuscin, linking lipid peroxidation and the lipofuscin accumulation that commonly occurs in post-mitotic cells such as neurons. In this study, brain tissue from AD and control patients was examined by immunocytochemistry and immunoelectron microscopy for evidence of HNE-crosslinking modifications of the type that should accumulate in the lipofuscin pathway. Strong labeling of granulovacuolar degeneration (GVD) and Hirano bodies was noted but lipofuscin did not contain this specific HNE-fluorophore. These findings directly implicate lipid crosslinking peroxidation products as accumulating not in the lesions or the lipofuscin pathways, but instead in a distinct pathway, GVD, that accumulates cytosolic proteins.

CD3 in Lewy pathology: does the abnormal recall of neurodevelopmental processes underlie Parkinson's disease.

CD3ζ is a subunit of the CD3 molecule that, until recently, appeared restricted to T cells and natural killer cells. However, experimental studies have demonstrated a role of CD3ζ in dendritic outgrowth in the visual system as well as in synaptic plasticity. Given the increasing evidence for uncharacteristic recapitulation of neurodevelopmental processes in neurodegenerative diseases, in this study, we evaluated brains from subjects with Parkinson's disease and Lewy body dementia for evidence of aberrant CD3 expression. Our data shows marked CD3ζ in association with the α-synuclein containing pathological lesions, i.e., Lewy bodies and Lewy neurites, in the brains of subjects with Parkinson's disease and Lewy body dementia. This finding raises the novel concept of CD3 dysregulation in these disorders as a pathogenic factor and also furthers the increasing evidence that the recall of aberrant neurodevelopmental processes underlies the pathogenesis of neurodegenerative diseases.

Expression of CD74 is increased in neurofibrillary tangles in Alzheimer's disease.

Alzheimer disease (AD) is a chronic neurodegenerative disease that is characterized by progressive memory loss. Pathological markers of AD include neurofibrillary tangles, accumulation of amyloid-beta plaques, neuronal loss, and inflammation. The exact events that lead to the neuronal dysfunction and loss are not completely understood. However, pro-inflammatory cytokines, such as interleukin-1beta, interleukin-6, and tumor necrosis factor alpha, are increased in AD, along with gene expression of major histocompatibility complex (MHC) class II molecules and macrophage migration inhibitory factor (MIF). MHC class II molecules are found in microglia of the brain, while MIF is found in both microglia and neurons of the hypothalamus, hippocampus, and cortex. MIF is not only a lymphocyte mediator but also a pituitary factor with endocrine properties and can mediate phosphorylation of the extracellular signal-regulated kinase-1/2 MAP kinases pathway. In this study, we looked at CD74, an integral membrane protein that acts as both a chaperone for MHC class II molecules as well as a receptor binding site for MIF. CD74 was recently found to be increased in microglia in AD cases compared to age-matched controls, but has not been reported in neurons. In our analysis, immunohistochemistry revealed a significant increase in CD74 primarily in neurofibrillary tangles, amyloid-beta plaques, and microglia. This is the first finding to our knowledge that CD74 is increased in neurons of AD cases compared to age-matched control cases.

Lipoic acid and N-acetyl cysteine decrease mitochondrial-related oxidative stress in Alzheimer disease patient fibroblasts.

In this study, we evaluated the effect of lipoic acid (LA) and N-acetyl cysteine (NAC) on oxidative [4-hydroxy-2-nonenal, N(epsilon)-(carboxymethyl)lysine and heme oxygenase-1] and apoptotic (caspase 9 and Bax) markers in fibroblasts from patients with Alzheimer disease (AD) and age-matched and young controls. AD fibroblasts showed the highest levels of oxidative stress, and the antioxidants, lipoic acid (1 mM) and/or N-acetyl cysteine (100 microM) exerted a protective effect as evidenced by decreases in oxidative stress and apoptotic markers. Furthermore, we observed that the protective effect of LA and NAC was more pronounced when both agents were present simultaneously. AD-type changes could be generated in control fibroblasts using N-methylprotoporphyrin to inhibit cytochrome oxidase assembly indicating that the the oxidative damage observed was associated with mitochondrial dysfunction. The effects of N-methylprotoporphyrine were reversed or attenuated by both lipoic acid and N-acetyl cysteine. These data suggest mitochondria are important in oxidative damage that occurs in AD. As such, antioxidant therapies based on lipoic acid and N-acetyl cysteine supplementation may be promising.

Cerebrotendinous xanthomatosis: case report with evidence of oxidative stress.

Cerebrotendinous xanthomatosis is an autosomal recessive disorder of bile acid synthesis, characterized by mutation in the mitochondrial enzyme 27-hydroxylase that leads to an accumulation of cholestanol and cholesterol. Characterized clinically by premature bilateral cataracts, slowly progressive neurological deterioration with dementia, cerebellar and brainstem signs, peripheral neuropathy, and seizures, the disease presents pathologically with lipid granulomata with foamy histiocytes and cholesterol clefts. Replacement therapy with chenodeoxycholic acid slows progression of the disease but does not reverse neurological deficits. Here, we present the case of a 49-year-old woman diagnosed at autopsy with cerebrotendinous xanthomatosis, on the basis of bilateral Achilles tendon granulomas, and typical foamy histiocytic infiltration of the brain, most severe in the dentate nucleus, and a typical clinical presentation. To investigate the pathological manifestations of this disease further, we performed immunohistochemistry for N(epsilon)-(carboxymethyl)-lysine, an indicator of oxidative damage, and found strong labeling of cytoplasmic material within histiocytes. In summary, this case of undiagnosed cerebrotendinous xanthomatosis during life emphasizes the need for a greater awareness of the disease, and early diagnosis and treatment. Further, the involvement of oxidative stress in cerebrotendinous xanthomatosis indicates that combined therapy with chenodeoxycholic acid and antioxidants may improve clinical outcome.

Vascular oxidative stress in Alzheimer disease.

Alzheimer disease and cerebrovascular dementia are two common causes of dementia and, by present diagnostic criteria, are mutually exclusive using vascular pathology as an arbitrary demarcation in differential diagnosis. However, evidence from epidemiological, neuropathological, clinical, pharmacological, and functional studies suggest considerable overlap in risk factors and pathological changes suggesting shared common pathogenic mechanisms between these two diseases such that vascular factors play a vital role in the pathogenesis of Alzheimer disease. A high energy demand and lack of an endogenous fuel reserve make the brain highly dependent upon a continuous blood supply where disruption of cerebral blood vessels and blood flow can have serious consequences on neural activities. Indeed, many studies implicate metabolic defects in Alzheimer disease, such a reduced brain metabolism is one of the best documented abnormalities in the disease. Notably, since endothelial reactive oxygen species such as nitric oxide act as vasodilators at low concentrations, increased production coupled with elevated reactive oxygen species scavenging of nitric oxide, can lead to reduced bioavailability of nitric oxide and increased oxidative stress that damage sensitive vascular cells. In this respect, we and others have demonstrated that oxidative stress is one of the earliest pathological changes in the brain of Alzheimer disease patients and plays a critical role in the vascular abnormalities underlying metabolic defects in Alzheimer disease. Here, we discuss vascular factors in relation to Alzheimer disease and review hypoperfusion as a potential cause by triggering mitochondrial dysfunction and increased oxidative stress initiating the pathogenic process.

Increased expression of the remodeling- and tumorigenic-associated factor osteopontin in pyramidal neurons of the Alzheimer's disease brain.

Osteopontin (OPN) is a glycophosphoprotein expressed by several cell types and has pro-adhesive, chemotactic, and cytokine-like properties. OPN is involved in a number of physiologic and pathologic events including angiogenesis, apoptosis, inflammation, oxidative stress, remyelination, wound healing, bone remodeling, cell migration and tumorigenesis. Since these functions of OPN, and the events that it regulates, are involved with neurodegeneration, we examined whether OPN was differentially expressed in the hippocampus of the Alzheimer's disease (AD) compared with age-matched (59-93 years) control brain. We report for the first time the immunocytochemical localization of OPN in the cytoplasm of pyramidal neurons. In AD brains, there was a significant 41 % increase in the expression of neuron OPN compared with age-matched control brain. No staining of other neuronal cell types was observed. Additionally, there was a significant positive correlation between OPN staining intensity and both amyloid-beta load (r(2) = 0.25; P < 0.05; n = 20) and aging (r(2) = 0.32; P < 0.01; n = 20) among all control and AD subjects. Controlling for age indicated that OPN expression was significantly influenced by amyloid-beta load, but not age. While the functional consequences of this amyloid-beta associated increase in OPN expression are unclear, it is notable that OPN is primarily localized to those neurons that are known to be vulnerable to AD-related neurite loss, degeneration and death. Given that the induction of OPN expression (and amyloid-beta generation) is associated with remodeling and tumorigenesis, our results suggest that OPN may play a role in the aberrant re-entry of neurons into the cell cycle and/or neuronal remyelination in AD.

Steroidogenic acute regulatory protein (StAR): evidence of gonadotropin-induced steroidogenesis in Alzheimer disease.

BACKGROUND: Alzheimer disease (AD) is clinically characterized by progressive memory loss, impairments in behavior, language and visual-spatial skills and ultimately, death. Epidemiological data reporting the predisposition of women to AD has led to a number of lines of evidence suggesting that age-related changes in hormones of the hypothalamic-pituitary-gonadal (HPG) axis following reproductive senescence, may contribute to the etiology of AD. Recent studies from our group and others have reported not only increases in circulating gonadotropins, namely luteinizing hormone (LH) in individuals with AD compared with control individuals, but also significant elevations of LH in vulnerable neuronal populations in individuals with AD compared to control cases as well as the highest density of gonadotropin receptors in the brain are found within the hippocampus, a region devastated in AD. However, while LH is higher in AD patients, the downstream consequences of this are incompletely understood. To begin to examine this issue, here, we examined the expression levels of steroidogenic acute regulatory (StAR) protein, which regulates the first key event in steroidogenesis, namely, the transport of cholesterol into the mitochondria, and is regulated by LH through the cyclic AMP second messenger pathway, in AD and control brain tissue. RESULTS: Our data revealed that StAR protein was markedly increased in both the cytoplasm of hippocampal pyramidal neurons as well as in the cytoplasm of other non-neuronal cell types from AD brains when compared with age-matched controls. Importantly, and suggestive of a direct mechanistic link, StAR protein expression in AD brains colocalized with LH receptor expression. CONCLUSION: Therefore, our findings suggest that LH is not only able to bind to its receptor and induce potentially pathogenic signaling in AD, but also that steroidogenic pathways regulated by LH may play a role in AD.

Nanoparticle iron chelators: a new therapeutic approach in Alzheimer disease and other neurologic disorders associated with trace metal imbalance.

Accumulating evidence suggests that oxidative stress may be a major etiologic factor in initiating and promoting neurodegeneration in Alzheimer disease. Contributing to this, there is a dyshomeostasis of metal ions in Alzheimer disease with abnormally high levels of redox-active metals, particularly iron, in affected areas of the brain. Although it is unclear whether metal excesses are the sole cause of oxidative stress and neurodegeneration or a by-product of neuronal loss, the finding that metal chelators can partially solubilize amyloid-beta deposits in Alzheimer disease suggests a promising therapeutic role for chelating agents. However, the blood-brain barrier and toxicity of known chelators limit their utility. In this study, we suggest that covalent conjugation of iron chelators with nanoparticles may help overcome the limitations in blood-brain barrier permeability of existing chelation therapy. Using in vitro studies, we have shown that a chelator-nanoparticle system and the chelator-nanoparticle system complexed with iron, when incubated with human plasma, preferentially adsorb apolipoprotein E and apolipoprotein A-I, that would facilitate transport into and out of the brain via mechanisms used for transporting low-density lipoprotein. Our studies suggest a unique approach, utilizing nanoparticles, to transport chelators and chelator-metal complexes in both directions across the blood-brain barrier, thus providing safer and more effective chelation treatment in Alzheimer disease and other neurodegenerative diseases.

Compensatory responses induced by oxidative stress in Alzheimer disease.

Oxidative stress occurs early in the progression of Alzheimer disease, significantly before the development of the pathologic hallmarks, neurofibrillary tangles and senile plaques. In the first stage of development of the disease, amyloid-beta deposition and hyperphosphorylated tau function as compensatory responses and downstream adaptations to ensure that neuronal cells do not succumb to oxidative damage. These findings suggest that Alzheimer disease is associated with a novel balance in oxidant homeostasis.

Sequestration of p27 within the cytoplasm of cardiac myocytes in chronic ischemic heart disease: pathogenic implications for ischemic cardiomyopathy.

A number of recent studies have suggested that cardiac myocytes, previously considered post-mitotic, re-enter the cell cycle and possess the ability to proliferate with certain pathogenic stimuli. To study this further, we examined cellular proliferation in myocardial tissue from subjects with chronic ischemic heart disease-associated myocardial infarction and subsequent congestive heart failure. We found striking increases in cytoplasmic phospho-p27, a well-known mitotic regulator, compared to controls by both immunocytochemical and immunoblot analyses. However, we found no evidence for cardiac myocyte proliferation in either disease or control subjects using both mitotic counting (no mitotic figures were observed) and Ki-67 immunocytochemistry, which demonstrated a 0% proliferation index. That increased cytoplasmic phospho-p27 is not accompanied by division prompts us to speculate that ectopic cell cycle activation occurs in the face of minimal to absent myocyte proliferation per se. Based on these findings, and the parallel findings in post-mitotic neurons in neurodegenerative disease, we suggest that cell-cycle activation in ischemic heart disease is a deleterious event that perpetuates disease pathogenesis culminating in myocardial failure.

Telomeres and telomerase in Alzheimer's disease: epiphenomena or a new focus for therapeutic strategy?

New approaches to the elucidation of Alzheimer disease, even those with solid data, are often ignored or dismissed as epiphenomenal when they differ from the mainstream or theoretical expectations. Here we present a new piece to the puzzle, decreases in telomere length, and telomerase expression in neuronal populations known to be vulnerable to degeneration and death in Alzheimer's disease. We can present the answers to the question "what," but the "why," "when," and "how" are not so easily answered. The goal of this report is to prompt discussion and more intensive study of this finding toward a new focus of therapeutic strategy.

Compensatory responses induced by oxidative stress in Alzheimer disease

Oxidative stress occurs early in the progression of Alzheimer disease, significantly before the development of the pathologic hallmarks, neurofibrillary tangles and senile plaques. In the first stage of development of the disease, amyloid-â deposition and hyperphosphorylated tau function as compensatory responses and downstream adaptations to ensure that neuronal cells do not succumb to oxidative damage. These findings suggest that Alzheimer disease is associated with a novel balance in oxidant homeostasis.

Ribosomal RNA in Alzheimer disease is oxidized by bound redox-active iron.

Oxidative modification of cytoplasmic RNA in vulnerable neurons is an important, well documented feature of the pathophysiology of Alzheimer disease. Here we report that RNA-bound iron plays a pivotal role for RNA oxidation in vulnerable neurons in Alzheimer disease brain. The cytoplasm of hippocampal neurons showed significantly higher redox activity and iron(II) staining than age-matched controls. Notably, both were susceptible to RNase, suggesting a physical association of iron(II) with RNA. Ultrastructural analysis further suggested an endoplasmic reticulum association. Both rRNA and mRNA showed twice the iron binding as tRNA. rRNA, extremely abundant in neurons, was considered to provide the greatest number of iron binding sites among cytoplasmic RNA species. Interestingly, the difference of iron binding capacity disappeared after denaturation of RNA, suggesting that the higher order structure may contribute to the greater iron binding of rRNA. Reflecting the difference of iron binding capacity, oxidation of rRNA by the Fenton reaction formed 13 times more 8-hydroxyguanosine than tRNA. Consistent with in situ findings, ribosomes purified from Alzheimer hippocampus contained significantly higher levels of RNase-sensitive iron(II) and redox activity than control. Furthermore, only Alzheimer rRNA contains 8-hydroxyguanosine in reverse transcriptase-PCR. Addressing the biological significance of ribosome oxidation by redox-active iron, in vitro translation with oxidized ribosomes from rabbit reticulocyte showed a significant reduction of protein synthesis. In conclusion these results suggest that rRNA provides a binding site for redox-active iron and serves as a redox center within the cytoplasm of vulnerable neurons in Alzheimer disease in advance of the appearance of morphological change indicating neurodegeneration.

Alzheimer-specific epitopes of tau represent lipid peroxidation-induced conformations.

Several recent studies support a link between tau protein phosphorylation and adduction of tau by reactive carbonyls. Indeed, the phosphorylation-dependent adduction of tau by carbonyl products resulting from lipid peroxidation creates the neurofibrillary tangle-related antigen, Alz50. To determine whether epitopes of carbonyl-modified tau are major conformational changes associated with neurofibrillary tangle formation, we examined seven distinct antibodies raised against neurofibrillary tangles that recognize unique epitopes of tau in Alzheimer disease. Consistently, all seven antibodies recognize tau more strongly (4- to 34-fold) after treatment of normal tau with the reactive carbonyl, 4-hydroxy-2-nonenal (HNE), but only when tau is in the phosphorylated state. These findings not only support the idea that oxidative stress is involved in neurofibrillary tangle formation occurring in brains of Alzheimer disease patients, but also show, for the first time, that HNE modifications of tau promote and contribute to the generation of the major conformational properties defining neurofibrillary tangles.