Allopregnanolone promotes regeneration and reduces β-amyloid burden in a preclinical model of Alzheimer's disease

Previously, we demonstrated that allopregnanolone (APα) promoted proliferation of rodent and human neural progenitor cells in vitro. Further, we demonstrated that APα promoted neurogenesis in the hippocampal subgranular zone (SGZ) and reversed learning and memory deficits in the male triple transgenic mouse model of Alzheimer's (3xTgAD). In the current study, we determined the efficacy of APα to promote the survival of newly generated neural cells while simultaneously reducing Alzheimer's disease (AD) pathology in the 3xTgAD male mouse model. Comparative analyses between three different APα treatment regimens indicated that APα administered 1/week for 6 months was maximally efficacious for simultaneous promotion of neurogenesis and survival of newly generated cells and reduction of AD pathology. We further investigated the efficacy of APα to impact Aβ burden. Treatment was initiated either prior to or post intraneuronal Aβ accumulation. Results indicated that APα administered 1/week for 6 months significantly increased survival of newly generated neurons and simultaneously reduced Aβ pathology with greatest efficacy in the pre-pathology treatment group. APα significantly reduced Aβ generation in hippocampus, cortex, and amygdala, which was paralleled by decreased expression of Aβ-binding-alcohol-dehydrogenase. In addition, APα significantly reduced microglia activation as indicated by reduced expression of OX42 while increasing CNPase, an oligodendrocyte myelin marker. Mechanistic analyses indicated that pre-pathology treatment with APα increased expression of liver-X-receptor, pregnane-X-receptor, and 3-hydroxy-3-methyl-glutaryl-CoA-reductase (HMG-CoA-R), three proteins that regulate cholesterol homeostasis and clearance from brain. Together these findings provide preclinical evidence for the optimal treatment regimen of APα to achieve efficacy as a disease modifying therapeutic to promote regeneration while simultaneously decreasing the pathology associated with Alzheimer's disease.     

IGF-1 promotes β-amyloid production by a secretase-independent mechanism

β-Amyloid peptide (Aβ) is generated via the sequential proteolysis of β-amyloid precursor protein (APP) by β- and γ-secretases, and plays a crucial role in the pathogenesis of Alzheimer’s disease (AD). Here, we sought to clarify the role of insulin-like growth factor-1 (IGF-1), implicated in the AD pathomechanism, in the generation of Aβ. Treatment of neuroblastoma SH-SY5Y cells expressing AD-associated Swedish mutant APP with IGF-1 did not alter cellular levels of APP, but significantly increased those of β-C-terminal fragment (β-CTF) and secreted Aβ. IGF-1 also enhanced APP phosphorylation at Thr668. Treatment of β-CTF-expressing cells with IGF-1 increased the levels of β-CTF and secreted Aβ. The IGF-1-induced augmentation of β-CTF was observed in the presence of γ-secretase inhibitors, but not in cells expressing β-CTF with a Thr668 to alanine substitution. These results suggest that IGF-1 promotes Aβ production through a secretase-independent mechanism involving APP phosphorylation.     

Effect of Melatonin and Melatonylvalpromide on β-amyloid and Neurofilaments in N2a Cells

In the present study, we have studied the effect of melatonin (Mt) and melatonin derivative, i.e., melatonylvalpromide (Mtv), on cell viability, β-amyloid (Aβ) production, cell morphology, and expression and phosphorylation of neurofilament proteins in wild-type murine neuroblastoma N2a (N2a/wt) and N2a stably transfected with amyloid precursor protein (N2a/APP) cell lines. The study used MTT assay, Sandwich ELISA, immunocytochemistry and Western blots techniques. The results showed that both Mt and Mtv could increase cell viability, but Mtv did so more effectively. The N2a/APP showed shorter and less amount of cell processes than N2a/wt, and Mtv but not Mt slightly improved the morphological changes in N2A/APP. Both Mt and Mtv suppressed the Aβ level in cell lysates, but the effect of Mtv was stronger than Mt. The immunoreaction to the non-phosphorylated neurofilament proteins probed by SMI32 and SMI33 were remarkably weaker in N2a/APP than N2a/wt, while the immunoreaction to the phosphorylated neurofilament proteins at SMI34 epitopes was slightly stronger in N2a/APP than N2a/wt, suggesting higher phosphorylation level of neurofilament proteins in N2a/APP. Treatment of the cells with Mt and Mtv increased the immunoreaction at SMI32 and SMI33 epitopes, while only Mtv but not Mt decreased the staining at SMI34 epitope, suggesting both Mt and Mtv promote dephosphorylation of neurofilament at SMI32 and SMI33 epitopes, while Mtv stimulates dephosphorylation of neurofilament at SMI34 epitope. These results suggest that Mtv may be a better candidate in arresting the intracellular accumulation of Aβ and protecting the cells from Aβ-related toxicity. Xiao-Chuan Wang and Yin-Chun Zhang equally contributed to the work.     

Rapamycin promotes β-amyloid production via ADAM-10 inhibition

Rapamycin is a well known immunosuppressant drug for rejection prevention in organ transplantation. Numerous clinical trials using rapamycin analogs, involving both children and adults with various disorders are currently ongoing worldwide. Most recently, rapamycin gained much attention for what appears to be life-span extending properties when administered to mice. The risk for Alzheimer disease (AD) is strongly and positively correlated with advancing age and is characterized by deposition of β-amyloid peptides (Aβ) as senile plaques in the brain. We report that rapamycin (2.5 μM), significantly increases Aβ generation in murine neuron-like cells (N2a) transfected with the human “Swedish” mutant amyloid precursor protein (APP). In concert with these observations, we found rapamycin significantly decreases the neuroprotective amino-terminal APP (amyloid precursor protein) cleavage product, soluble APP-α (sAPP-α) while increasing production of the β-carboxyl-terminal fragment of APP (β-CTF). These cleavage events are associated with decreased activation of a disintegrin and metallopeptidase domain-10 (ADAM-10), an important candidate α-secretase which opposes Aβ generation. To validate these findings in vivo, we intraperitoneal (i.p.) injected Tg2576 Aβ-overproducing transgenic mice with rapamycin (3 mg/kg/day) for 2 weeks. We found increased Aβ levels associated with decreased sAPP-α at an average rapamycin plasma concentration of 169.7 ± 23.5 ng/mL by high performance liquid chromatography (HPLC). These data suggest that although rapamycin may increase the lifespan in some mouse models, it may not decrease the risk for age-associated neurodegenerative disorders such as AD.     

Mitochondrial β-amyloid in Alzheimer's disease

It is well established that the intracellular accumulation of Aβ (amyloid β-peptide) is associated with AD (Alzheimer's disease) and that this accumulation is toxic to neurons. The precise mechanism by which this toxicity occurs is not well understood; however, identifying the causes of this toxicity is an essential step towards developing treatments for AD. One intracellular location where the accumulation of Aβ can have a major effect is within mitochondria, where mitochondrial proteins have been identified that act as binding sites for Aβ, and when binding occurs, a toxic response results. At one of these identified sites, an enzyme known as ABAD (amyloid-binding alcohol dehydrogenase), we have identified changes in gene expression in the brain cortex, following Aβ accumulation within mitochondria. Specifically, we have identified two proteins that are up-regulated not only in the brains of transgenic animal models of AD but also in those of human sufferers. The increased expression of these proteins demonstrates the complex and counteracting pathways that are activated in AD. Previous studies have identified approximate contact sites between ABAD and Aβ; on basis of these observations, we have shown that by using a modified peptide approach it is possible to reverse the expression of these two proteins in living transgenic animals and also to recover mitochondrial and behavioural deficits. This indicates that the ABAD-Aβ interaction is potentially an interesting target for therapeutic intervention. To explore this further we used a fluorescing substrate mimic to measure the activity of ABAD within living cells, and in addition we have identified chemical fragments that bind to ABAD, using a thermal shift assay.     

Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants

We have generated transgenic maize seed containing -glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant -glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant -glucuronidase (GUS) was compared to native -glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a K_m of 0.20 mM and a V_max of 0.29 mM MUG per hour, and the native enzyme had a K_m and V_max of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of -glucuronidase, the K_i of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor.     

MEK inhibitors suppress β-amyloid production by altering the level of a β-C-terminal fragment of amyloid precursor protein in neuronal cells

β-Amyloid peptide (Aβ) is generated via sequential proteolysis of amyloid precursor protein (APP) by β- and γ-secretases. Cell-based screening experiments disclosed that the MEK (MAP kinase kinase) inhibitors, U0126 and PD184352, suppress Aβ secretion from human neuronal SH-SY5Y cells expressing Swedish mutant APP. These inhibitors did not affect the cellular levels of APP but significantly reduced those of the APP β-C-terminal fragment (β-CTF). Additionally, β-CTF levels were markedly reduced by these inhibitors in cells expressing the fragment in a γ-secretase-independent and proteasome-dependent manner. Our results suggest that MEK inhibitors reduce Aβ generation via secretase-independent alteration of β-CTF levels.     

Potential role of α-synuclein in neurodegeneration: studies in a rat animal model

Neuronal protein α-synuclein (α-syn) is an essential player in the development of neurodegenerative diseases called synucleinopathies. A spontaneous autosomal recessive rat model for neurodegeneration was developed in our laboratory. These rats demonstrate progressive increases in α-syn in the brain mesencephalon followed by loss of dopaminergic terminals in the basal ganglia (BG) and motor impairments. The severity of pathology is directly related to the overexpression of α-syn and parallel decrease in dopamine (DA) level in the striatum (ST) of affected rats. The neurodegeneration in this model is characterized by the presence of perikarya and neurites Lewis bodies (LB) and diffuse marked accumulation of perikaryal α-syn in the substantia nigra (SN), brain stem (BS), and striatum (ST) along with neuronal loss. Light and ultrastructural analyses revealed that the process of neuronal degeneration is a 'dying back' type. The disease process is accompanied by gliosis and release of inflammatory cytokines. This neurodegeneration is a multisystemic disease and implicate α-syn as a major factor in the pathogenesis of this inherited autosomal recessive animal model. Decrease dopamine (DA) and overexpression of α-syn in the brain mesencephalon may provide a naturally occurring animal model for Parkinson's disease (PD) and other synucleinopathies that reproduces significant pathological, neurochemical, and behavioral features of the human disease.     

Promotion of β-amyloid production by C-reactive protein and its implications in the early pathogenesis of Alzheimer's disease

C-reactive protein (CRP) and β-amyloid protein (Aβ) are involved in the development of Alzheimer's disease (AD). However, the relationship between CRP and Aβ production is unclear. In vitro and in vivo experiments were performed to investigate the association of CRP with Aβ production. Using the rat adrenal pheochromocytoma cell line (PC12 cells) to mimic neurons, cytotoxicity was evaluated by cell viability and supernatant lactate dehydrogenase (LDH) activity. The levels of amyloid precursor protein (APP), beta-site APP cleaving enzyme (BACE-1), and presenilins (PS-1 and PS-2) were investigated using real-time polymerase chain reaction and Western blotting analysis. Aβ1-42 was measured by enzyme-linked immunosorbent assay. The relevance of CRP and Aβ as well as potential mechanisms were studied using APP/PS1 transgenic (Tg) mice. Treatment with 0.5-4.0 μM CRP for 48 h decreased cell viability and increased LDH leakage in PC12 cells. Incubation with CRP at a sub-toxic concentration of 0.2 μM increased the mRNA levels of APP, BACE-1, PS-1, and PS-2, as well as Aβ1-42 production. CRP inhibitor reversed the CRP-induced upregulations of the mRNA levels of APP, BACE-1, PS-1, and PS-2, and the protein levels of APP, BACE-1, PS-1, and Aβ1-42, but did not reversed Aβ1-42 cytotoxicity. The cerebral levels of CRP and Aβ1-42 in APP/PS1 Tg mice were positively correlated, accompanied with the elevated mRNA expressions of serum amyloid P component (SAP), complement component 1q (C1q), and tumor necrosis factor-α (TNF-α). These results suggest that CRP cytotoxicity is associated with Aβ formation and Aβ-related markers expressions; CRP and Aβ were relevant in early-stage AD; CRP may be an important trigger in AD pathogenesis.     

Physiological cholesterol concentration is a neuroprotective factor against β-amyloid and β-amyloid-metal complexes toxicity

Alzheimer's disease is one of the most common forms of dementia in the elderly. One of its hallmarks is the abnormal aggregation and deposition of β-amyloid (Aβ). Endogenous and exogenous metal ions seem to influence β-amyloid folding process, aggregation and deposition. Besides these variables other elements appear to affect β-amyloid behavior, such as cholesterol. The physiological concentration of cholesterol in the cerebrospinal fluid (CSF) was used in order to determine the extent in which Aβ and Aβ-metal complexes in vitro aggregation and their toxicity on human neuroblastoma cell cultures is affected. Cholesterol did not appear to influence Aβ and Aβ-metal complexes aggregation, but it was effective in protecting neuroblastoma cells against Aβ complexes' toxicity. The Aβ-Al complex seemed to be the most effective in disrupting and damaging membrane external layer, and simultaneously it appears to increase its toxicity on cell cultures; both of these effects are preventable by cholesterol. The presence in physiological concentrations of cholesterol seemed to compensate membrane damage that occurred to neuroblastoma cells. These findings appear to contradict some data reported in literature. We believe that our results might shed some light on the role played by cholesterol at physiological concentrations in both cellular balance and membrane protection.     

Recent progress in understanding Alzheimer's β-amyloid structures

The formation of amyloid fibrils, protofibrils and oligomers from the β-amyloid (Aβ) peptide represents a hallmark of Alzheimer's disease. Aβ-peptide-derived assemblies might be crucial for disease onset, but determining their atomic structures has proven to be a major challenge. Progress over the past 5 years has yielded substantial new data obtained with improved methodologies including electron cryo-microscopy and NMR. It is now possible to resolve the global fibril topology and the cross-β sheet organization within protofilaments, and to identify residues that are crucial for stabilizing secondary structural elements and peptide conformations within specific assemblies. These data have significantly enhanced our understanding of the mechanism of Aβ aggregation and have illuminated the possible relevance of specific conformers for neurodegenerative pathologies.     

Renin–angiotensin system regulates neurodegeneration in a mouse model of normal tension glaucoma

Glaucoma, one of the leading causes of irreversible blindness, is characterized by progressive degeneration of optic nerves and retinal ganglion cells (RGCs). In the mammalian retina, excitatory amino acid carrier 1 (EAAC1) is expressed in neural cells, including RGCs, and the loss of EAAC1 leads to RGC degeneration without elevated intraocular pressure (IOP). In the present study, we found that expressions of angiotensin II type 1 receptor (AT1-R) and Toll-like receptor 4 (TLR4) are increased in RGCs and retinal Müller glia in EAAC1-deficient (KO) mice. The orally active AT1-R antagonist candesartan suppressed TLR4 and lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expressions in the EAAC1 KO mouse retina. Sequential in vivo retinal imaging and electrophysiological analysis revealed that treatment with candesartan was effective for RGC protection in EAAC1 KO mice without affecting IOP. In cultured Müller glia, candesartan suppressed LPS-induced iNOS production by inhibiting the TLR4-apoptosis signal-regulating kinase 1 pathway. These results suggest that the renin–angiotensin system is involved in the innate immune responses in both neural and glial cells, which accelerate neural cell death. Our findings raise intriguing possibilities for the management of glaucoma by utilizing widely prescribed drugs for the treatment of high blood pressure, in combination with conventional treatments to lower IOP.Glaucoma is one of the leading causes of vision loss in the world. It is estimated that glaucoma affects nearly 70 million individuals worldwide, including at least 6.8 million who are bilaterally blind.^{1, 2} The disease is characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, together with visual field loss, which are usually associated with elevated intraocular pressure (IOP). Glaucoma is affected by multiple genes and environmental factors, and there are several inherited and experimentally induced animal models of high IOP glaucoma.³ On the other hand, normal tension glaucoma (NTG) is a subtype of glaucoma that presents with statistically normal IOP. The prevalence of NTG is reported to be higher among the Japanese than among Caucasians.^{4, 5} This raises a major problem that is faced by medical and public health sectors in Japan because simple screening programs based on detection of elevated IOP are not effective in a population where NTG is highly prevalent. In addition, these findings suggest a possibility that non-IOP-dependent factors may contribute to disease progression and that elucidating such factors is necessary to better understand the pathogenesis of glaucoma, especially in the context of NTG.^{5, 6}It is well known that an immoderate release of excitatory amino acids, such as glutamate, can cause neuronal cell death. An excessively high extracellular concentration of glutamate chronically activates glutamate receptors and allows calcium entry into the cell causing an uncontrolled elevation of intracellular calcium levels.^{7, 8} The glutamate transporter is the only mechanism for removal of glutamate from the extracellular fluid in the retina.⁹ In the inner plexiform layer where synapses exist across RGCs, at least three transporters are involved in this task: glutamate transporter 1 (GLT-1) located in the bipolar cell terminals; excitatory amino acid carrier 1 (EAAC1) in RGCs; and glutamate/aspartate transporter (GLAST) in Müller glial cells.^{10, 11} We previously reported that GLAST and EAAC1 knockout (KO) mice show progressive RGC loss and optic nerve degeneration without elevated IOP, and not only glutamate neurotoxicity but also oxidative stress is involved in its mechanism.⁶ Glutamate neurotoxicity and oxidative stress have been proposed to contribute to retinal damage in various eye diseases including glaucoma.¹² Together with the downregulation of glutamate transporters and glutathione levels observed in glaucoma patients,¹³ these mice seem to be useful as the animal models of NTG.^{6, 7, 11, 14}Apoptosis signal-regulating kinase 1 (ASK1) has key roles in human diseases closely related to the dysfunction of cellular responses to oxidative stress and endoplasmic reticulum stressors, including neurodegenerative diseases.¹⁵ We previously reported that ASK1 KO mice are less susceptible to ischemic injury and optic nerve injury.^{16, 17} In addition, RGC degeneration was partly suppressed in GLAST/ASK1 double KO mice.¹⁴ Thus, interruption of ASK1 pathways could be beneficial for RGC protection during retinal degeneration including glaucoma. Furthermore, ASK1 directly binds to Toll-like receptor 4 (TLR4) and regulates the innate immune responses during neuroinflammation.¹⁸The renin–angiotensin system (RAS) has a major role in the cardiovascular system.¹⁹ Renin, a proteolytic enzyme primarily released by the kidneys, cleaves angiotensinogen to angiotensin I (AngI). AngI is further processed by angiotensin-converting enzyme and angiotensin-converting enzyme 2 (ACE/ACE2) to different angiotensin cleavage products. Among them, angiotensin II (AngII) is the principal effector molecule of the RAS, acting on its target cells mainly via AngII type 1 receptor (AT1-R).²⁰ In addition, AngII induces TLR4 expression in various cell types.^{21, 22} Modulators of the RAS, such as ACE inhibitors or AT1-R antagonists, are utilized as prescribed drugs to treat high blood pressure.²³ A recent study showed that candesartan, an AT1-R antagonist, protected rat retinal neurons from ischemia-reperfusion injury,²⁴ but the detailed mechanisms are still unknown. Interestingly, polymorphisms in AngII receptors are reported in glaucoma patients,²⁵ and ACE inhibitors may suppress the progression of visual field defects in NTG patients.²⁶ These results suggest a possibility that the RAS is implicated in RGC death by an IOP-independent mechanism. In the present study, we show neuroprotective effects of candesartan in EAAC1 KO mice, an animal model of NTG, and we report a novel pathway for neuroprotection that involves innate immune responses in retinal glial cells.     

Ethylene production and β-cyanoalanine synthase activity in carnation flowers

The relationship between ethylene production and the CN^--assimilating enzyme -cyanoalanine synthase (CAS; EC 4.4.1.9) was examined in the carnation (Dianthus caryophyllus L.) flower. In petals from cut flowers aged naturally or treated with ethylene to accelerate senescence the several hundred-fold increase in ethylene production which occurred during irreversible wilting was accompanied by a one- to twofold increase in CAS activity. The basal parts of the petal, which produced the most ethylene, had the highest CAS activity. Studies of flower parts (styles, ovaries, receptacles, petals) showed that the styles had a high level of CAS together with the ethylene-forming enzyme (EFE) system for converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. The close association between CAS and EFE found in styles could also be observed in detached petals after induction by ACC or ethylene. Treatment of the cut flowers with cycloheximide reduced synthesis of CAS and EFE. The data indicate that CAS and ethylene production are associated, and are discussed in relation to the hypothesis that CN^- is formed during the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglyoine - CAS -cyanoalanine synthase - CHI cycloheximide - EFE ethylene-forming enzyme     

Synthesis and evaluation of a 18F-curcumin derivate for β-amyloid plaque imaging

IntroductionCurcumin is a neuroprotective compound that inhibits the formation of amyloid oligomers and fibrils and binds to β-amyloid plaques in Alzheimer’s disease (AD). We aimed to synthesize an ¹⁸F-labeled curcumin derivate ([¹⁸F]4) and to characterize its positron emission tomography (PET) tracer-binding properties to β-amyloid plaques in a transgenic APP23 mouse model of AD.MethodsWe utilized facile one-pot synthesis of [¹⁸F]4 using nucleophilic ¹⁸F-fluorination and click chemistry. Binding of [¹⁸F]4 to β-amyloid plaques in the transgenic APP23 mouse brain cryosections was studied in vitro using heterologous competitive binding against PIB. [¹⁸F]4 uptake was studied ex vivo in rodents and in vivo using PET/computed tomography of transgenic APP23 and wild-type control mice.ResultsThe radiochemical yield of [¹⁸F]4 was 21 ± 11%, the specific activity exceeded 1 TBq/μmol, and the radiochemical purity exceeded 99.3% at the end of synthesis. In vitro studies of [¹⁸F]4 with the transgenic APP23 mouse revealed high β-amyloid plaque binding. In vivo and ex vivo studies demonstrated that [¹⁸F]4 has fast clearance from the blood, moderate metabolism but low blood–brain barrier (BBB) penetration.Conclusions[¹⁸F]4 was synthesized in high yield and excellent quality. In vitro studies, metabolite profile, and fast clearance from the blood indicated a promising tracer for Aβ imaging. However, [¹⁸F]4 has low in vivo BBB penetration and thus further studies are needed to reveal the reason for this and to possibly overcome this issue.     

Exogenous seeding of cerebral β-amyloid deposition in βAPP-transgenic rats

Deposition of the amyloid-β (Aβ) peptide in senile plaques and cerebral Aβ angiopathy (CAA) can be stimulated in Aβ-precursor protein (APP)-transgenic mice by the intracerebral injection of dilute brain extracts containing aggregated Aβ seeds. Growing evidence implicates a prion-like mechanism of corruptive protein templating in this phenomenon, in which aggregated Aβ itself is the seed. Unlike prion disease, which can be induced de novo in animals that are unlikely to spontaneously develop the disease, previous experiments with Aβ seeding have employed animal models that, as they age, eventually will generate Aβ lesions in the absence of seeding. In the present study, we first established that a transgenic rat model expressing human APP (APP21 line) does not manifest endogenous deposits of Aβ within the course of its median lifespan (30?months). Next, we injected 3-month-old APP21 rats intrahippocampally with dilute Alzheimer brain extracts containing aggregated Aβ. After a 9-month incubation period, these rats had developed senile plaques and CAA in the injected hippocampus, whereas control rats remained free of such lesions. These findings underscore the co-dependence of agent and host in governing seeded protein aggregation, and show that cerebral Aβ-amyloidosis can be induced even in animals that are relatively refractory to the spontaneous origination of parenchymal and vascular deposits of Aβ.     

Alteration of β-secretase traffic by the receptor tyrosine kinase signaling pathway- a new mechanism for regulating Alzheimer's β-amyloid production

The receptor tyrosine kinases (RTKs) are a family of cellsurface proteins with diverse functions in proliferation, dif-ferentiation or cell-cell communication. When a specific li-gand binds to its cognate receptor, a conformational changeof this receptor due to the ligand-receptor interaction willlead to activation of the intrinsic tyrosine kinase residing inthe intracellular domain of the receptor. The activation ofthis tyrosine kinase is essential for transducing the signals toa cascade of its downstream molecules that eventually causerelated physiological responses [1]. For example, binding ofnerve growth factor (NGF) to its receptor TrkA is essentialfor the proper development, patterning, and maintenanceof the mammalian nervous system. This ligand and recep-tor interaction will lead to the formation of a crab-shapedhomodimeric TrkA structure [2], and the subsequent activa-tion of its intrinsic RTK will cause auto-phosphorylationof its own intracellular tyrosine residues. PhosphorylatedTrkA receptors recruit and increase the phosphorylationof PLC-γ and Shc, which leads to activation of either thePI3K/Akt pathway or Ras/raf/ERK pathway. In the brainOf Alzheimer's disease (AD) patients, alterations of nervegrowth factor (NGF) and its receptor TrkA have beenreported to associate with AD pathogenesis [3]. However,the underlying mechanisms remain elusive.     

Enzymatic production and in?situ separation of natural β-ionone from β-carotene

A biotechnological process concept for generation and in?situ separation of natural β-ionone from β-carotene is presented. The process employs carotenoid cleavage dioxygenases (CCDs), a plant-derived iron-containing nonheme enzyme family requiring only dissolved oxygen as cosubstrate and no additional cofactors. Organophilic pervaporation was found to be very well suited for continuous in?situ separation of β-ionone. Its application led to a highly pure product despite the complexity of the reaction solution containing cell homogenates. Among three different pervaporation membrane types tested, a polyoctylmethylsiloxane active layer on a porous polyetherimide support led to the best results. A laboratory-scale demonstration plant was set up, and a highly pure aqueous–ethanolic solution of β-ionone was produced from β-carotene. The described process permits generation of high-value flavor and fragrance compounds bearing the desired label “natural” according to US and European food and safety regulations and demonstrates the potential of CCD enzymes for selective oxidative cleavage of carotenoids.