Intraneuronal amyloid-beta1-42 production triggered by sustained increase of cytosolic calcium concentration induces neuronal death

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence in the brain of senile plaques which contain an amyloid core made of beta-amyloid peptide (Abeta). Abeta is produced by the cleavage of the amyloid precursor protein (APP). Since impairment of neuronal calcium signalling has been causally implicated in ageing and AD, we have investigated the influence of an influx of extracellular calcium on the metabolism of human APP in rat cortical neurones. We report that a high cytosolic calcium concentration, induced by neuronal depolarization, inhibits the alpha-secretase cleavage of APP and triggers the accumulation of intraneuronal C-terminal fragments produced by the beta-cleavage of the protein (CTFbeta). Increase in cytosolic calcium concentration specifically induces the production of large amounts of intraneuronal Abeta1-42, which is inhibited by nimodipine, a specific antagonist of l-type calcium channels. Moreover, calcium release from endoplasmic reticulum is not sufficient to induce the production of intraneuronal Abeta, which requires influx of extracellular calcium mediated by the capacitative calcium entry mechanism. Therefore, a sustained high concentration of cytosolic calcium is needed to induce the production of intraneuronal Abeta1-42 from human APP. Our results show that this accumulation of intraneuronal Abeta1-42 induces neuronal death, which is prevented by a functional gamma-secretase inhibitor.     

Intraneuronal Abeta causes the onset of early Alzheimer's disease-related cognitive deficits in transgenic mice

Progressive memory loss and cognitive dysfunction are the hallmark clinical features of Alzheimer's disease (AD). Identifying the molecular triggers for the onset of AD-related cognitive decline presently requires the use of suitable animal models, such as the 3xTg-AD mice, which develop both amyloid and tangle pathology. Here, we characterize the onset of learning and memory deficits in this model. We report that 2-month-old, prepathologic mice are cognitively unimpaired. The earliest cognitive impairment manifests at 4 months as a deficit in long-term retention and correlates with the accumulation of intraneuronal Abeta in the hippocampus and amygdala. Plaque or tangle pathology is not apparent at this age, suggesting that they contribute to cognitive dysfunction at later time points. Clearance of the intraneuronal Abeta pathology by immunotherapy rescues the early cognitive deficits on a hippocampal-dependent task. Reemergence of the Abeta pathology again leads to cognitive deficits. This study strongly implicates intraneuronal Abeta in the onset of cognitive dysfunction.     

Is there a "molecular Nirvana Principle"? Towards a unified resolutional model of the biological symbol-matter dichotomy

In the present paper, the metapsychological "Nirvana Principle" is investigated evolutionarily at the earliest forms of life in a highly tentative way. A corresponding "molecular Nirvana Principle" is proposed, where the recent suggestions of the "internal measurement" biophysical quantum-molecular research programme of modern quantum biology are introduced, in relation to the former metapsychological theory, conceived to be valid in the entire realm of living systems (just as it was intended by the original author). By an appropriate introduction of a special primordeal dynamical time inversion symmetry breaking, originating in a premeval self-measurement in a composite nucleic acid-protein system, a special internal symmetry restoration time series is defined. In this way, a strictly physically defined self-identity ("molecular Nirvana," special physical symmetry restored) is derived, which is put equal to the quantum physical equivalent and root of the goals of evolutionarily higher level fundamental drives (the "Nirvana Principle"). It is shown that it is a natural requirement that the following internal regressive time (-reversal) physical molecular relations (and so the ultimate time symmetry) is mapped onto space, as is also suggested by some symbol-theoretical propositions.     

Homing in on intracellular Abeta?

In this issue of Neuron, a study by Billings et al. points to intracellular Abeta as a possible cause of neuronal dysfunction. In a mouse model of Alzheimer's disease, Billings et al. link appearance of intraneuronal Abeta to cognitive impairments and then show that "clearance" of intraneuronal Abeta by anti-Abeta antibodies restores cognitive deficits.     

Input and output channels of quantum biocomputers]

It is proposed that "Quantum Molecular" computer of a neuron consists of the cell cytoskeleton serving as calculating media and input ionic channel sending a hypersound signal to observe these media. The sound spreads through the media travelling along microtubules and microfilaments and switching between those via molecular bridges which serve as elementary switches. The whole system works like a wave guiding net connecting input ionic channels (which generate different sound signals) and output ionic channels (which are controlled by the processed sound signals). Thus the output of such systems depends on the input (controlled by synaptic activity) and on the construction and state of these calculating media. We think that the sound waves spreading through different calculating media solve different physical problems. The construction of the calculating part of the cytoskeleton, according to the hypothesis, is different in different neurons. It is defined by special protein which is produced by DNA, RNA and protein molecular word processor (during brain development and, may be, education). We comment on how the existence of an extremal computer produces an impact on physics and mathematics exemplified by the optimality principle as substitution of physical relativity principle for a complex problem.     

Age-related intraneuronal elevation of αII-spectrin breakdown product SBDP120 in rodent forebrain accelerates in 3×Tg-AD mice

Spectrins line the intracellular surface of plasmalemma and play a critical role in supporting cytoskeletal stability and flexibility. Spectrins can be proteolytically degraded by calpains and caspases, yielding breakdown products (SBDPs) of various molecular sizes, with SBDP120 being largely derived from caspase-3 cleavage. SBDPs are putative biomarkers for traumatic brain injury. The levels of SBDPs also elevate in the brain during aging and perhaps in Alzheimer's disease (AD), although the cellular basis for this change is currently unclear. Here we examined age-related SBDP120 alteration in forebrain neurons in rats and in the triple transgenic model of AD (3×Tg-AD) relative to non-transgenic controls. SBDP120 immunoreactivity (IR) was found in cortical neuronal somata in aged rats, and was prominent in the proximal dendrites of the olfactory bulb mitral cells. Western blot and densitometric analyses in wild-type mice revealed an age-related elevation of intraneuronal SBDP120 in the forebrain which was more robust in their 3×Tg-AD counterparts. The intraneuronal SBDP120 occurrence was not spatiotemporally correlated with transgenic amyloid precursor protein (APP) expression, β-amyloid plaque development, or phosphorylated tau expression over various forebrain regions or lamina. No microscopically detectable in situ activated caspase-3 was found in the nuclei of SBDP120-containing neurons. The present study demonstrates the age-dependent intraneuronal presence of an αII-spectrin cleavage fragment in mammalian forebrain which is exacerbated in a transgenic model of AD. This novel neuronal alteration indicates that impairments in membrane protein metabolism, possibly due to neuronal calcium mishandling and/or enhancement of calcium sensitive proteolysis, occur during aging and in transgenic AD mice.     

Tau phosphorylation by GSK-3β promotes tangle-like filament morphology

Background

Neurofibrillary tangles (NFTs) are intraneuronal aggregates associated with several neurodegenerative diseases including Alzheimer's disease. These abnormal accumulations are primarily comprised of fibrils of the microtubule-associated protein tau. During the progression of NFT formation, disperse and non-interacting tau fibrils become stable aggregates of tightly packed and intertwined filaments. Although the molecular mechanisms responsible for the conversion of disperse tau filaments into tangles of filaments are not known, it is believed that some of the associated changes in tau observed in Alzheimer's disease, such as phosphorylation, truncation, ubiquitination, glycosylation or nitration, may play a role.

Results

We have investigated the effects of tau phosphorylation by glycogen synthase kinase-3β (GSK-3β) on tau filaments in an in vitro model system. We have found that phosphorylation by GSK-3β is sufficient to cause tau filaments to coalesce into tangle-like aggregates similar to those isolated from Alzheimer's disease brain.

Conclusion

These results suggest that phosphorylation of tau by GSK-3β promotes formation of tangle-like filament morphology. The in vitro cell-free experiments described here provide a new model system to study mechanisms of NFT development. Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved. This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.     

Experimental testing of the role of cytoskeleton in the solution by neurons of problems facing the brain

Investigation of the influence of cAMP on neuronal electric activity suggests that nerve cells can solve problems using an intraneuronal calculating medium based on the cytoskeleton. When a new problem is posed, this structure has to be disassembled and assembled by the neuronal molecular computer according to the program recorded in DNA. If DNA lacks an appropriate program, the cytoskeleton will not be assembled. In our experiments, fishes which were rotated simultaneously around two mutually perpendicular axes lost their swimming ability, and some dramatic changes were observed in the cytoskeleton of their Mauthner neurons. These changes disappeared after a long-term rest: the cytoskeleton was restored simultaneously with the ability for normal swimming.     

Fast spectrophotometric detection system for coupled physical and chemical electric field effects in solution

Summary A fast spectrophotometric detection system is described which extends the electric field jump relaxation techniques to complex systems of biochemical and molecular biological interest. In these systems chemical relaxation, which yields a concentration change, is accompanied by physical relaxation which is mainly due to orientation processes. The simple versatile set-up allows the measurement of the different effects using only one piece of apparatus with a transmission path and two emission channels, which can all be equipped with polarizing elements. The high intensity optical system and the low noise optoelectronic circuits result in an attainable signal-to-noise ratio of 10001 for transmission measurements with a rise time of the order of 10 nsec and 1001 for fluorescence measurements with a rise time of 50 nsec.     

Detection of biogenic monoamines in the developing nervous system of Lymnaea stagnalis mollusk embryos]

Formaldehyde-induced fluorescence of intraneuronal monoamines can be demonstrated in the Lymnaea embryos from the "late veliger" stage on. Green specific fluorescence indicating the presence of a primary catecholamine occurs in two paired formations which contain a mass of fibres and varicosities. The formations are supposed to correspond to cerebral and pedal ganglia. Single fibres of the same type can be seen in the foot and other organs of the embryo.     

Interaction between neurofilaments and mitochondria in cultured cells of the rat hippocampus

Interaction between neurofilaments and mitochondria was studied on the model of cultured hippocampal cells of newborn rats. A treatment by specific toxins -iminodipropionitril or hexanedione resulted in a disintegration and translocation of neurofilaments in the cultured neurons. These effects were accompanied by a considerable decrease in dimensions of mitochondria, an increase in their elongation coefficient, a noticeable increase in spatial density of these organelles, and their translocation within the perinuclear layer of cytoplasm. The role of neurofilaments in the intraneuronal distribution of mitochondria and modifications of their functional state is discussed. The neurofilament system is supposed to considerably influence the processes of division, growth, and translocation of the mitochondria.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 3–10, January–February, 1995.     

Cleavage of neuronal synaptosomal-associated protein of 25 kDa by exogenous matrix metalloproteinase-7

Matrix metalloproteinases (MMPs) belong to a family of zinc dependent enzymes best studied for their role in cancer and inflammation. Though MMPs typically target extracellular proteins, here we show that MMP-7, an MMP family member which lacks a C-terminal hemopexin-like domain, can cleave an intraneuronal protein that is critical to vesicular fusion and neurotransmitter release, synaptosomal-associated protein of 25 kDa (SNAP-25). Western blot analysis using an N-terminal specific antibody on extracts from cultured neurons suggests that cleavage occurs towards the C-terminal portion of SNAP 25. Additional studies with recombinant SNAP-25 demonstrate that cleavage occurs at amino acid 129. The ability of MMP-7 to cleave SNAP-25 is diminished by chlorpromazine and phenylarsine oxide, inhibitors of clathrin dependent endocytosis. Together, these results imply that exogenous MMP-7 can access an intraneuronal substrate and suggest that additional studies may be warranted to determine if SNAP function is impaired with brain inflammation.     

Determination of the folding of globular protein chain by the self-consistent field method

It is shown that and how it is possible to single out the chain fold which is thermo-dynamically most stable. The suggested approach is based on two physical ideas: A "molecular field" approximation permits to examine all protein structures which belong to the same "folding pattern". Only a limited set of the "potentially stable" folding patterns have to be examined. The general approach is illustrated by calculations of the stable folds for two beta-domains.     

Down-regulation of striatin, a neuronal calmodulin-binding protein, impairs rat locomotor activity.

Striatin, an intraneuronal, calmodulin-binding protein addressed to dendrites and spines, is expressed in the motor system, particularly the striatum and motoneurons. Striatin contains a high number of domains mediating protein-protein interactions, suggesting a role within a dendritic Ca(2+)-signaling pathway. Here, we explored the hypothesis of a direct role of striatin in the motor control of behaving rats, by using an antisense strategy based on oligodeoxynucleotides (ODN). Rats were treated by intracerebroventricular infusion of a striatin antisense ODN (A-ODN) or mismatch ODN (M-ODN) delivered by osmotic pumps over 6 days. A significant decrease in the nocturnal locomotor activity of A-ODN-treated rats was observed after 5 days of treatment. Hypomotricity was correlated with a 60% decrease in striatin content of the striata of A-ODN-treated rats sacrificed on day 6. Striatin thus plays a role in the control of motor function. To approach the cellular mechanisms in which striatin is involved, striatin down-regulation was studied in a comparatively simpler model: purified rat spinal motoneurons which retain their polarity in culture. Treatment of cells by the striatin A-ODN resulted in the impairement of the growth of dendrites but not axon. The decrease in dendritic growth paralleled the loss of striatin. This model allows analysis of the molecular basis of striatin function in the dynamic changes occurring in growing dendrites, and offers clues to unravel its function within spines.     

Biopolymers and evolution

Biopolymers are usually studied being extracted from the whole system of a cell or of an organism. Some important features are lost during such a procedure. It is necessary to take into account the behavior of proteins and nucleic acids in metabolic networks and to investigate their evolution. The substitutions of amino-acids metabolic networks residues are biologically possible in the polypeptides and proteins if they do not influence their spatial structure and function. The correlations of the primary structure with these properties are degenerate. The protein can be treated as "an edited statistical copolymer" (Ptitsyn). In the process of "edition" an important role is played by the ions of transient metals. Nucleic acids possess similar properties. It can be shown that the deleterious mutations of proteins can be compensated by the changes of their amount, spatial and temporal characteristics of the synthesis. Not only the structure of the protein is important but also the exact answers of the questions: how much, when and where? The contemporary theory of evolution unites phylogeny and onthogeny. The directionality of evolution is determined both by natural selection and by the already existing structure of an organism. Hence many characters are not adaptive. This is valid also for the molecular level of the structure. Thus three independent groups of facts and suggestions are presented, which confirm the neutral theory of evolution (Kimura) and elucidate its physical meaning. The molecular evolution does not coincide with the biological evolution.     

Intracellular amyloid-beta 1-42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis

Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of beta-amyloid peptide (A beta), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A beta 42/A beta 40 production. A beta 42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A beta 42 is a critical event in AD, but the intraneuronal A beta 42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A beta 1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A beta 1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A beta 1-42 production by a functional gamma-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A beta 1-42 is the key event in the neurodegenerative process that we observed.     

Microinjection of cyclic nucleotides provides evidence for a diffusional mechanism of intraneuronal control

Cyclic 3′,5′-AMP and cyclic 3′,5′-GMP injected into large neurons of the snail Helix lucorum altered neuron activity. The effect of cAMP is usually depolarizing and that of cGMP hyperpolarizing. The results are specific for 3′,5′-cyclic nucleotides. The experiments support the hypothesis that reaction-diffusion processes involving cyclic nucleotides from the basis of an intraneuronal system of information processing.     

Genetic and pharmacological evidence of intraneuronal Aβ accumulation in APP transgenic mice

Intraneuronal punctate immunostaining in Alzheimer’s disease brain and amyloid-β precursor protein (APP) transgenic mice has been suggested to represent Aβ, but this is somewhat controversial. Here we show that both biochemical Aβ levels and intraneuronal immunostaining are reduced in APP transgenic mice when γ-secretase is inhibited. Moreover, BACE-1 deficient APP transgenic mice show neither Aβ production nor intraneuronal immunostaining. Our findings suggest that the punctate immunostaining with APP antibodies is due to Aβ that has accumulated inside neurons. Similar type of intraneuronal Aβ accumulation, which precedes senile plaque formation, may link Aβ to tauopathy and neurodegeneration in Alzheimer’s disease pathogenesis.     

Renovation of the egg extracellular matrix at fertilization

Extracellular matrices are essential for cell survival and function. This is especially relevant for eggs, which establish a physical barrier at fertilization to protect a new embryo from additional sperm and pathogens. Formation of an extracellular matrix is most dramatic in sea urchins, in which fertilization was first observed in animals with the "sudden appearance of a perfectly transparent envelope" (A. Derbs, 1847). The process of assembling this extracellular "envelope" has been a topic of intense study ever since. Here we integrate the cellular and molecular events necessary to form this fertilization envelope within the first few minutes of a new embryo's life.