Patterns of aberrant sprouting in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by extensive synaptic and neuronal loss and by plaque formation in the cortex, but the mechanisms responsible for synaptic plasticity in the neocortex are still not completely understood. To analyze the sprouting response in AD cortex, we compared the patterns of GAP-43 with synaptophysin immunoreactivity. In AD, GAP-43 immunohistochemistry revealed extensive sprouting in the hippocampal molecular layer, stratum polymorphous, CA1 region, and prosubiculum. These regions presented abundant anti-GAP-43-immunoreactive coiled fibers and dystrophic neurites in association with plaques. Some of these sprouting structures were colocalized with anti-synapto-physin- and anti-neurofilament-positive neurites. The AD neocortex was characterized by an overall decrease in GAP-43 immunoreactivity accompanied by sprouting neurites in the areas of synaptic pathology. We conclude that GAP-43 might be involved in the mechanisms of synaptic plasticity in the AD cortex, as well as in the process of aberrant sprouting in the neuritic plaques.     

Analysis of N-glycans of pathological tau: possible occurrence of aberrant processing of tau in Alzheimer's disease

In a previous study [Wang et al. (1996) Nat. Med. 2, 871-875], Wang et al. found (i) that abnormally hyperphosphorylated tau (AD P-tau) isolated from Alzheimer's disease (AD) brain as paired helical filaments (PHF)-tau and as cytosolic AD P-tau but not tau from normal brain were stained by lectins, and (ii) that on in vitro deglycosylation the PHF untwisted into sheets of thin straight filaments, suggesting that tau only in AD brains is glycosylated. To elucidate the primary structure of N-glycans, we comparatively analyzed the N-glycan structures obtained from PHF-tau and AD P-tau. More than half of N-glycans found in PHF-tau and AD P-tau were different. High mannose-type sugar chains and truncated N-glycans were found in both taus in addition to a small amount of sialylated bi- and triantennary sugar chains. More truncated glycans were richer in PHF-tau than AD P-tau. This enrichment of more truncated glycans in PHF might be involved in promoting the assembly and or stabilizing the pathological fibrils in AD.     

Targeting alzheimer amyloid plaques in vivo

The only definitive diagnosis for Alzheimer disease (AD) at present is postmortem observation of neuritic plaques and neurofibrillary tangles in brain sections. Radiolabeled amyloid-beta peptide (Abeta), which has been shown to label neuritic plaques in vitro, therefore could provide a diagnostic tool if it also labels neuritic plaques in vivo following intravenous injection. In this study, we show that the permeability of Abeta at the blood-brain barrier can be increased by at least twofold through covalent modification with the naturally occurring polyamine, putrescine. We also show that, following intravenous injection, radiolabeled, putrescine-modified Abeta labels amyloid deposits in vivo in a transgenic mouse model of AD, as well as in vitro in human AD brain sections. This technology, when applied to humans, may be used to detect plaques in vivo, allowing early diagnosis of the disease and therapeutic intervention before cognitive decline occurs.     

Beta-amyloid from Alzheimer disease brains inhibits sprouting and survival of sympathetic neurons

The significance of the amyloid plaque core proteins (APCP) in Alzheimer's disease (AD) and its consequences for neuronal survival have been controversial. To address this problem we purified the APCP and beta A obtained from brains with AD, and assessed their biological effects in tissue culture. APCP and beta A caused severe toxicity to chick and rat sympathetic and sensory neurons whose survival is dependent upon NGF. This toxicity was dose dependent and reversible at low doses. APCP and beta A prevented sprouting of neurites in freshly plated neurons. In established cultures addition of these molecules caused vacuolation and fragmentation of neurites and disintegration of neuronal soma. We suggest that the deposition of APCP in AD may be partly responsible for the destruction of the neuritic arbor, thereby contributing to the formation of the neuritic plaque and to neuronal death.     

Chronic excitotoxin-induced axon degeneration in a compartmented neuronal culture model

Glutamate excitotoxicity is a major pathogenic process implicated in many neurodegenerative conditions, including AD (Alzheimer''s disease) and following traumatic brain injury. Occurring predominantly from over-stimulation of ionotropic glutamate receptors located along dendrites, excitotoxic axonal degeneration may also occur in white matter tracts. Recent identification of axonal glutamate receptor subunits within axonal nanocomplexes raises the possibility of direct excitotoxic effects on axons. Individual neuronal responses to excitotoxicity are highly dependent on the complement of glutamate receptors expressed by the cell, and the localization of the functional receptors. To enable isolation of distal axons and targeted excitotoxicity, murine cortical neuron cultures were prepared in compartmented microfluidic devices, such that distal axons were isolated from neuronal cell bodies. Within the compartmented culture system, cortical neurons developed to relative maturity at 11 DIV (days in vitro) as demonstrated by the formation of dendritic spines and clustering of the presynaptic protein synaptophysin. The isolated distal axons retained growth cone structures in the absence of synaptic targets, and expressed glutamate receptor subunits. Glutamate treatment (100 μM) to the cell body chamber resulted in widespread degeneration within this chamber and degeneration of distal axons in the other chamber. Glutamate application to the distal axon chamber triggered a lesser degree of axonal degeneration without degenerative changes in the untreated somal chamber. These data indicate that in addition to current mechanisms of indirect axonal excitotoxicity, the distal axon may be a primary target for excitotoxicity in neurodegenerative conditions.     

Neural cell adhesion molecule (NCAM) as a quantitative marker in synaptic remodeling

The neural cell adhesion molecule (NCAM) participates in adhesion and neuritic outgrowth during nervous system development. In the adult brain, NCAM is considered to be involved in neuronal sprouting and synaptic remodeling. the NCAM concentration of brain tissue has proved to be a useful marker of these processes, especially when viewed in comparison with the concentration of a marker of mature synapses, e.g. D3-protein (SNAP-25) or synaptophysin. The present review focusses on studies of adult brain in which NCAM concentration estimates and NCAM/D3 ratios have been used to evaluate the rate of synaptic remodeling in brain damage and degenerative diseases.Special issue dedicated to Dr. Robert Balázs.     

Pyroglutamate-Aβ: Role in the natural history of Alzheimer's disease

The accumulation of amyloid-beta (Aβ) peptides is believed to be a central contributor to the neurodegeneration typically seen in Alzheimer's disease (AD) brain. Aβ extracted from AD brains invariably possesses extensive truncations, yielding peptides of differing N- and C-terminal composition. Whilst Aβ is often abundant in the brains of cognitively normal elderly people, the brains of AD patients are highly enriched for N-terminally truncated Aβ bearing the pyroglutamate modification. Pyroglutamate-Aβ (pE-Aβ) has a higher propensity for oligomerisation and aggregation than full-length Aβ, potentially seeding the accumulation of neurotoxic Aβ oligomers and amyloid deposits. In addition, pE-Aβ has increased resistance to clearance by peptidases, causing these peptides to persist in biological fluids and tissues. The extensive deposition of pE-Aβ in human AD brain is under-represented in many transgenic mouse models of AD, reflecting major differences in the production and processing of Aβ peptides in these models compared to the human disease state.     

A(beta) generation in autophagic vacuoles

Alzheimer's disease (AD) is the most common form of dementia among older people. It is characterized by the extracellular accumulation of beta-amyloid (Abeta) deposits called senile or neuritic plaques. Abeta is generated by the proteolytic cleavage of Abeta precursor protein (APP) by beta and gamma-secretases localized in the secretory and endocytic compartments. In this issue, Yu et al. (on p. 87) report a novel mechanism for the generation of Abeta peptides, which takes place in autophagic vacuoles (AVs) that accumulate in AD brains.     

Induction of NADPH cytochrome P450 reductase by the Alzheimer β-protein. Amyloid as a 'foreign body'

A large body of data suggests that the Alzheimer's amyloid peptide (Abeta) causes degeneration and death of neurons by mechanisms that involve reactive oxygen species. The pathways involved in Abeta-mediated oxidative injury are only partially understood. We theorized that abnormal microaggregates and/or pathological conformations of Abeta peptides may behave as xenobiotics and trigger the induction of NADPH cytochrome P450 reductase (CP450r), an enzyme which, if induced by non-physiological substrates (such as xenobiotics like drugs or other 'foreign molecules'), is known to cause oxidative stress. In order to test this hypothesis, i.e. that Abeta can increase the expression of CP450r, SK-N-SH human neuroblastoma cells were exposed to Abeta25-35 and Abeta1-42 and then examined for induction of this enzyme in immunoblots, using specific antibodies. Following exposure to Abeta peptides, neuroblastoma cells showed a clear-cut induction of CP450r. To determine whether this mechanism is operational in vivo, we investigated the expression of CP450r in a transgenic mouse model of Alzheimer's disease (AD) and in brains from patients afflicted with AD, using an immunocytochemical approach. Tissue sections from brains of transgenic mice exhibited strong immunoreactivity for CP450r, surrounding amyloid deposits. The pattern of expression of CP450r was similar to that exhibited by neuritic and oxidative stress markers. Sections from non-transgenic mice showed no detectable immunoreactivity. Immunostaining of sections from four brains with neuropathologically confirmed AD showed a pattern of abnormality different from transgenic mice that was characterized by abnormal immunoreactivity for CP450r within the cytoplasm of cortical neurons. No labeling was seen in sections from aged-matched control brains. The data showed that CP450r is induced by Alzheimer amyloid peptide and that such a response must be considered as one possible mechanism whereby Abeta causes oxidative stress.     

Changes in acetylcholinesterase and butyrylcholinesterase in Alzheimer's disease resemble embryonic development--a study of molecular forms.

The pattern of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) separated by density gradient centrifugation was investigated in the brain and cerebrospinal fluid in Alzheimer's disease (AD), in human embryonic brain and in rat brain after experimental cholinergic deafferentation of the cerebral cortex. While a selective loss of the AChE G4 form was a rather constant finding in AD, a small but significant increase of G1 for both AChE and BChE was found in the most severely affected cases. Both in normal human brain and in AD a significant relationship could be established between the AChE G4/G1 ratio in different brain regions and the activity of choline acetyltransferase (ChAT). A similar decrease of the AChE G4 form as observed in AD can be induced in rat by experimental cholinergic deafferentation of the cerebral cortex. The increase in G1 of both AChE and BChE in different brain regions in AD is quantitatively related to the local density of neuritic plaques which are histochemically reactive for both enzymes. In human embryonic brain, a high abundance of G1 and a low G4/G1 ratio for both AChE and BChE was found resembling the pattern observed in AD. Furthermore, both in embryonic brain and in AD AChE shows no substrate inhibition which is a constant feature of the enzyme in the adult human brain. It is, therefore, concluded that the degeneration of the cholinergic cortical afferentation in AD as reflected by a decrease of AChE G4 is accompanied by the process of a neuritic sprouting response involved in plaque formation which is probably associated with the expression of a developmental form of the enzyme.     

Heparan sulfate proteoglycan induces the production of NO and TNF-α by murine microglia

Background  

A common feature of Alzheimer's disease (AD) pathology is the abundance of activated microglia in neuritic plaques containing amyloid-beta protein (Aβ) and associated molecules including heparan sulfate proteoglycan (HSPG). Besides the role as pathological chaperone favouring amyloidogenesis, little is known about whether or not HSPG can induce microglial activation. Cultures of primary murine microglia were used to assess the effect of HSPG on production of proinflammatory molecules that are known to be present in neuritic plaques of AD.     

The Microtubule-Associated Protein 1A (MAP1A) is an Early Molecular Target of Soluble Aβ-Peptide

A progressive accumulation of amyloid β-protein (Aβ) is widely recognized as a pathological hallmark of Alzheimer’s disease (AD). Substantial progress has been made toward understanding the neurodegenerative cascade initiated by small soluble species of Aβ and recent evidence supports the notion that microtubule rearrangements may be proximate to neuritic degeneration and deficits in episodic declarative memory. Here, we examined primary cortical neurons for changes in markers associated with synaptic function following exposure to sublethal concentrations of non-aggregated Aβ-peptide. This data show that soluble Aβ species at a sublethal concentration induce degradation of the microtubule-associated protein 1A (MAP1A) without concurrently affecting dendritic marker MAP2 and/or the pre-synaptic marker synaptophysin. In addition, MAP1A was found to highly co-localize with the postsynaptic density-95 (PSD-95) protein, proposing that microtubule perturbations might be central for the Aβ-induced neuronal dysfunctions as PSD-95 plays a key role in synaptic plasticity. In conclusion, this study suggests that disruption of MAP1A could be a very early manifestation of Aβ-mediated synaptic dysfunction—one that presages the clinical onset of AD by years. Moreover, our data support the notion of microtubule-stabilizing agents as effective AD drugs.     

Understanding molecular mechanisms of proteolysis in Alzheimer's disease: Progress toward therapeutic interventions

Amyloid beta peptide (Aβ) is not only a major constituent of extracellular fibrillary pathologies in Alzheimer's disease (AD) brains, but is also physiologically produced and metabolized in neurons. This fact led us to the notion that an age-related decrease in Aβ catabolism may contribute to the molecular pathogenesis of AD, providing a rationale for seeking proteolytic enzymes that degrade Aβ in the brain. Our recent studies have demonstrated that neprilysin is the most potent Aβ-degrading enzyme in vivo. Deficiency of endogenous neprilysin elevates the level of Aβ in brains of neprilysin-knockout mice in a gene dose-dependent manner, and an age-associated decline of neprilysin occurs in several regions of mouse brain. Neuropathological alterations in these same regions have been implicated in cognitive impairments of AD patients at an early stage of the disease. Furthermore, the level of neprilysin mRNA has been found to be significantly and selectively reduced in the hippocampus and temporal cortex of AD patients. A clarification of the role played by decreased neprilysin activity in the pathogenesis of AD has opened up the possibility of neprilysin up-regulation as a novel preventive and therapeutic approach to AD. Since the expression level and activity of neprilysin are likely to be regulated by neuropeptides and their receptors, non-peptidic agonists for these receptors might be effective agents to maintain a sufficient level of Aβ catabolism in brains of the elderly.In addition to Aβ deposits, intraneuronal fibrillary lesions, such as neurofibrillary tangles, are also a pathological hallmark of AD, and the extent of the resultant cytoskeletal disruptions may be dependent upon the activity levels of proteolytic enzymes. Among proteases for which major cytoskeletal components are good substrates, calpains were shown to participate in excitotoxic stress-induced neuritic degeneration in our recent analysis using genetically engineered mice. Moreover, we have found that this pathology can be reduced by controlling the activity of an endogenous calpain inhibitor known as calpastatin, providing a possible approach for the treatment of diverse neurodegenerative disorders, including AD.     

Transgenic mice overexpressing reticulon 3 develop neuritic abnormalities

Dystrophic neurites are swollen dendrites or axons recognizable near amyloid plaques as a part of important pathological feature of Alzheimer's disease (AD). We report herein that reticulon 3 (RTN3) is accumulated in a distinct population of dystrophic neurites named as RTN3 immunoreactive dystrophic neurites (RIDNs). The occurrence of RIDNs is concomitant with the formation of high-molecular-weight RTN3 aggregates in brains of AD cases and mice expressing mutant APP. Ultrastructural analysis confirms accumulation of RTN3-containing aggregates in RIDNs. It appears that the protein level of RTN3 governs the formation of RIDNs because transgenic mice expressing RTN3 will develop RIDNs, initially in the hippocampal CA1 region, and later in other hippocampal and cortical regions. Importantly, we show that the presence of dystrophic neurites in Tg-RTN3 mice causes impairments in spatial learning and memory, as well as synaptic plasticity, implying that RIDNs potentially contribute to AD cognitive dysfunction. Together, we demonstrate that aggregation of RTN3 contributes to AD pathogenesis by inducing neuritic dystrophy. Inhibition of RTN3 aggregation is likely a therapeutic approach for reducing neuritic dystrophy.     

Intraneuronal amyloid-beta plays a role in mediating the synergistic pathological effects of apoE4 and environmental stimulation

The allele E4 of apolipoprotein E4 (apoE4), which is the most prevalent genetic risk factor of Alzheimer's disease (AD), inhibits synaptogenesis and neurogenesis and stimulates apoptosis in brains of apoE4 transgenic mice that have been exposed to an enriched environment. In the present study, we investigated the hypothesis that the brain activity-dependent impairments in neuronal plasticity, induced by apoE4, are mediated via the amyloid cascade. Importantly, we found that exposure of mice transgenic for either apoE4, or the Alzheimer's disease benign allele apoE3, to an enriched environment elevates similarly the hippocampal levels of amyloid-beta peptide (Abeta) and apoE of these mice, but that the degree of aggregation and spatial distribution of Abeta in these mice are markedly affected by the apoE genotype. Accordingly, environmental stimulation triggered the formation of extracellular plaque-like Abeta deposits and the accumulation of intra-neuronal oligomerized Abeta specifically in brains of apoE4 mice. Further experiments revealed that hippocampal dentate gyrus neurons are particularly susceptible to apoE4 and environmental stimulation and that these neurons are specifically enriched in both oligomerized Abeta and apoE. These findings show that the impairments in neuroplasticity which are induced by apoE4 following environmental stimulation are associated with the accumulation of intraneuronal Abeta and suggest that oligomerized Abeta mediates the synergistic pathological effects of apoE4 and environmental stimulation.     

Toluidine blue-O staining of prion protein deposits

Prion diseases or transmissible spongiform encephalopathies are a group of fatal neurodegenerative diseases caused by an abnormal form of prion protein (PrP(sc)). In this study, we developed a sensitive histochemical detection of PrP(sc) deposits in a Gertsmann-Str?ussler-Scheinker disease (GSS) patient using toluidine blue-O staining, a specific reagent to stain mucins and mucopolysaccharides. Detection of prion deposits correlated with immunohistochemistry using anti-prion antibodies. Control assays were performed using amyloid-beta (Abeta) plaques from Alzheimer's disease (AD) brains. Our results demonstrated that toluidine blue-O staining allowed to recognize 69.1+/-2.6% of the total plaques recognized by the anti-prion antibody. Furthermore, in the 15 studied brain regions from the GSS patient, toluidine blue-O revealed the same recognition pattern as anti-prion labeling. Toluidine blue-O stained specifically the prion deposits but not the Abeta plaques in AD brains. The specificity of the technique was confirmed in a Creutzfeldt-Jakob disease brain. This method opens several possibilities for postmortem diagnoses. Our results also suggest the relevance of specific post-translational modifications of PrP(sc), identified by toluidine blue-O, that might participate in the transformation of PrP(c) to PrP(sc).     

Evidence against the overexpression of APP in Down syndrome

Down syndrome (DS) is the most common genetic disorder with mental retardation and is caused by trisomy 21. By the age of 40 years, virtually all adults with DS have sufficient neuropathology for a diagnosis of Alzheimer's disease (AD), which is characterized by accumulation of amyloid-beta in senile plaques and formation of neurofibrillary tangles. Amyloid-beta derives from a longer precursor protein, APP, whose gene maps to chromosome 21. In DS, the early appearance of senile plaques is commonly associated with the presence of a third copy of the APP gene. Here we show DS brains and trisomic fibroblasts in which APP is not overexpressed, compared to euploid controls, challenging the notion that the widespread amyloid-beta deposits, consistently found in DS individuals, result from an extra copy of APP.     

Neuroprotective and neurotoxic properties of glial cells in the pathogenesis of Alzheimer's disease

Alzheimer's disease (AD) affects more than 18 million people worldwide and is characterized by progressive memory deficits, cognitive impairment and personality changes. The main cause of AD is generally attributed to the increased production and accumulation of amyloid-beta (Abeta), in association with neurofibrillary tangle (NFT) formation. Increased levels of pro-inflammatory factors such as cytokines and chemokines, and the activation of the complement cascade occurs in the brains of AD patients and contributes to the local inflammatory response triggered by senile plaque. The existence of an inflammatory component in AD is now well known on the basis of epidemiological findings showing a reduced prevalence of the disease upon long-term medication with anti-inflammatory drugs, and evidence from studies of clinical materials that shows an accumulation of activated glial cells, particularly microglia and astrocytes, in the same areas as amyloid plaques. Glial cells maintain brain plasticity and protect the brain for functional recovery from injuries. Dysfunction of glial cells may promote neurodegeneration and, eventually, the retraction of neuronal synapses, which leads to cognitive deficits. The focus of this review is on glial cells and their diversity properties in AD.