Cytochemical study of the involvement of cell organelles in formation and accumulation of fibrillar amyloid in the pancreas of NORbeta transgenic mice.

Phosphatase ultrastructural cytochemistry was used to evaluate the participation of cytoplasmic organelles in the accumulation of fibrillar amyloid beta (Abeta) in exocrine acinar cells and in macrophages of the pancreas of transgenic mice overexpressing a carboxy-terminal fragment of Abeta protein precursor (ABPP). Nucleoside diphosphatase (NDPase) and glucose-6-phosphatase (G6Pase) were used as cytochemical markers of the endoplasmic reticulum (ER), thiamine pyrophosphatase (TPPase) as a marker of the Golgi apparatus (GA), and acid phosphatase (AcPase) as a marker of lysosomes. Monoclonal antibody 4G8 raised against the 17-24 aa sequence of human Abeta protein was used for immunogold localization of fibrillar Abeta. The results of this study indicate that the formation of Abeta in acinar cells occurs directly in the vacuolar areas of the rough ER (RER) without evident participation of the elements of the GA, whereas an intimate structural relation with primary lysosomes suggests their role in modification or digestion of the deposited amyloid. In macrophages, fibrillar amyloid was present in numerous cytoplasmic vacuoles located frequently in close proximity to flattened saccules of the ER. This structural pattern revealed similarity to that observed previously in microglial cells producing fibrillar PrP amyloid in scrapie-infected mice and Abeta in brains of human elderly patients and in Alzheimer's type brain pathology.

Fibrillar amyloid-beta production, accumulation, and recycling in transgenic mice pancreatic acinar cells and macrophages.

Amyloid-beta (A beta) production, accumulation, and recycling were examined by light and electron microscopy in the pancreas of transgenic mice (from 45 days to 22 months of age) that express the gene for the carboxy-terminal fragment of the human amyloid-beta protein precursor. Ultrastructural immunocytochemistry revealed four types of cells accumulating fibrillar A beta 1-40 in cytoplasmic vacuoles: acinar pancreatic cells, macrophages infiltrating stroma, epithelial cells of pancreatic ducts, and blood monocytes/macrophages in the lumen of pancreatic vessels. The ultrastructure of amyloid deposits suggests that each of these four types of cells produces fibrillar A beta. Three basic types of amyloid deposits were distinguished: primary vacuoles in different stages of amyloid aggregation and fibrillization, secondary vacuoles that are the product of fusion of primary vacuoles, and phagosome-like vacuoles with morphologically intact fibrillar amyloid and residues of ingested cells. Amyloid production in acinar pancreatic cells starts in mice younger than 45 days, progresses in 2- to 7-month-old mice, and plateaus in the second year of life. In macrophages, amyloid appears in 60-day-old mice, and the increase in the number of macrophages and the amount of amyloid in their cytoplasm correlates with age.

Characteristics of scrapie isolates derived from hay mites.

Previous epidemiological evidence suggested that in some instances a vector and/or reservoir is involved in the occurrence and spread of transmissible spongiform encephalopathies (TSEs). In a preliminary study, hay mite preparations from five Icelandic farms with a history of scrapie were injected into mice, and some of these mice became sick after long incubation periods. To confirm that the disease was scrapie, subsequent passages in mice were performed. In addition, the characteristics of the disease process in these passages were assessed and the results compared to those findings with standard scrapie strains. As expected for scrapie, subsequent passages in the same host led to shortened incubation periods compared to those in primary isolate mice, and all mice had spongiform changes in brain. Results were similar for three of four isolates with regard to clinical manifestations, the incubation periods in mice of the three scrapie incubation-period genotypes (s7s7, s7p7, p7p7), and the PrPSc Western blot (WB) pattern. The characteristics of the fourth isolate were markedly different from the other three isolates with regard to these parameters. Comparison of the characteristics of standard mouse-adapted scrapie strains and the four isolates revealed differences; these differences were particularly pronounced for the fourth isolate.

Role of perivascular cells and myocytes in vascular amyloidosis.

Amyloidogenic processing of amyloid-beta precursor protein (APP) by cells of the brain is the major pathologic component of Alzheimer's disease. Amyloid-beta (A beta) is of heterogeneous origin. Perivascular cells of monocyte-macrophage-microglial cell lineage produce fibrillar A beta in the wall of capillaries, whereas parenchymal microglial cells produce fibrillar A beta in the parenchyma of gray matter. Fibrillar A beta deposition by perivascular cells lead to endothelial cell degeneration and death, obliteration of affected capillaries, and reduction of the length of the vascular network. These changes cause local ischemia with neuronal degeneration and death. Smooth muscle cells are the source of A beta in the tunica media of parenchymal and leptomeningeal arteries and veins. Fibrillar A beta in the tunica media of leptomeningeal and parenchymal vessels causes degeneration and necrosis of smooth muscle cells and leads to multiple cortical hemorrhages. Smooth muscle cells isolated from blood vessels with amyloid deposits secrete A beta and accumulate nonfibrillar A beta intracellularly. The amyloidogenic processing of APP can be enhanced by apolipoprotein E, reduced by transthyretin, and modulated by several cytokines.

The histological validation of post mortem magnetic resonance imaging-determined hippocampal volume in Alzheimer's disease.

For 11 AD cases and four normal elderly controls, post mortem volumes of the hippocampal subdivisions were calculated by using magnetic resonance imaging and histological sections. After at least six weeks of fixation in formalin, brains were examined on a 1.5-T Philips Gyroscan imager producing T1-weighted coronal images with a 3-mm slice thickness. Brains were then processed and embedded in paraffin. Serial coronal sections, 3 mm apart and stained with Cresyl Violet, were used for the planimetry and unbiased estimation of the total numbers of neurons in the hippocampal subdivisions. For all 15 cases, magnetic resonance imaging- and histology-based measurements were performed along the whole rostrocaudal extent of the hippocampal formation and included three subvolumes: (i) the hippocampus (CA1-CA4 and the dentate gyrus); (ii) hippocampus/subiculum; and (iii) hippocampus/parahippocampal gyrus. After controlling for shrinkage, strong correlations were found between magnetic resonance imaging and histological measurements for the hippocampus (r = 0.97, P < 0.001), hippocampus/subiculum (r = 0.95, P < 0.001) and hippocampus/parahippocampal gyrus (r = 0.89, P < 0.001). We also calculated the total number of neurons in the hippocampus and hippocampus/subiculum subvolumes. Strong correlations between the magnetic resonance imaging subvolumes and neuronal counts were found for the hippocampus (r = 0.90, P < 0.001) and the hippocampus/subiculum subvolume (r = 0.84, P < 0.001). We conclude that very accurate volumetric measurements of the whole hippocampal formation can be obtained by using a magnetic resonance imaging protocol. Moreover, the strong correlations between magnetic resonance imaging-based hippocampal volumes and neuronal numbers suggest the anatomical validity of magnetic resonance imaging volume measurements.

Regulation of phosphorylation of neuronal microtubule-associated proteins MAP1b and MAP2 by protein phosphatase-2A and -2B in rat brain.

The function of the neuronal high molecular weight microtubule-associated proteins (MAPs) MAP1b and MAP2 is regulated by the degree of their phosphorylation, which in turn is controlled by the activities of protein kinases and protein phosphatases (PP). To investigate the role of PP in the regulation of the phosphorylation of MAP1b and MAP2, we used okadaic acid and cyclosporin A to selectively inhibit PP2A and PP2B activities, respectively, in metabolically competent rat brain slices. The alteration of the phosphorylation levels of MAP1b and MAP2 was examined by Western blots using several phosphorylation-dependent antibodies to these proteins. The inhibition of PP2A, and to a lesser extent of PP2B, was found to induce an increased phosphorylation of MAP1b and inhibit its microtubule binding activity. Immunocytochemically, a marked increase in neuronal staining in inhibitor-treated tissue was observed with antibodies to the phosphorylated MAP1b. The inhibition of PP2A but not of PP2B also induced phosphorylation of MAP2 at multiple sites and impaired its microtubule binding activity. These results suggest that PP2A might be the major PP that participates in regulation of the phosphorylation of MAP1b and MAP2 and their biological activities.

Plasma and cerebrospinal fluid levels of amyloid beta proteins 1-40 and 1-42 in Alzheimer disease.

BACKGROUND: In brains with AD, Abeta is a major component of diffuse plaques. Previous reports showed that CSF Abeta42 levels were lower in patients with AD than in controls. Although studies showed higher plasma Abeta42 levels in familial AD, a recent report has indicated that plasma Abeta42 levels were similar in a sporadic AD group and controls. However, no information is published on plasma Abeta40 and Abeta42 levels in relation to Apo E genotype or severity of dementia in sporadic AD. OBJECTIVE: To examine plasma and cerebrospinal fluid (CSF) levels of amyloid beta protein 1-40 (Abeta40) and 1-42 (Abeta42) levels in patients with probable Alzheimer disease (AD) and elderly nondemented control subjects in relation to the apolipoprotein E (Apo E) genotype and dementia severity. SETTING: Two university medical centers. PATIENTS AND METHODS: Levels of Abeta40 and Abeta42 were measured in plasma from 78 patients with AD and 61 controls and in CSF from 36 patients with AD and 29 controls by means of a sandwich enzyme-linked immunosorbent assay. RESULTS: Mean plasma Abeta40 levels were higher in the AD group than in controls (P = .005), but there was substantial overlap; Abeta42 levels were similar between the groups. Levels of Abeta40 and Abeta42 showed no association with sex or Mini-Mental State Examination scores. There was a significant relationship between age and Abeta40 level in controls but not in the AD group. Levels of Abeta40 were higher in patients with AD with the Apo E epsilon4 allele than in controls (P<.01). Cerebrospinal fluid Abeta40 levels were similar in the AD group and controls. However, Abeta42 levels were lower in the AD group than in controls (P<.001). The levels showed no association with severity of dementia. CONCLUSIONS: Although mean plasma Abeta40 levels are elevated in sporadic AD and influenced by Apo E genotype, measurement of plasma Abeta40 levels is not useful to support the clinical diagnosis of AD. Lower levels of CSF Abeta42 in the AD group are consistent with previous studies.

The limbic zone of the rabbit and rat claustrum: a study of the claustrocingulate connections based on the retrograde axonal transport of fluorescent tracers.

The claustrum is a subcortical structure lying under the insular and piriform cortices, whose function is still not clear. Although data exist on connections of the claustrum and the limbic cortex, the topography of the limbic zone in the rabbit and rat claustrum has not been studied extensively. The study was performed on 17 adult Wistar rats and 12 New Zealand rabbits. Two percent water solutions of fluorescent retrograde tracers fast blue and nuclear yellow were injected into the various regions of the limbic cortex. The limbic zone is localized throughout the whole rostrocaudal extent of the claustrum, mainly in its ventromedial portion lying close to the external capsule. Although this zone of the claustrum is localized similarly in both rat and rabbit, some differences between these two species exist. In the rat, neurons projecting to all limbic areas are localized mainly in the anterior and central parts of the claustrum, whereas in the rabbit, the majority of the neurons projecting to the cingulate cortex are present in the anterior and central parts of this structure, while neurons sending axons to the retrosplenial cortex are localized in the central and posterior parts. In both species, double-labeling study showed that neurons projecting to various limbic regions are intermingled and that neurons sending axons into two different limbic regions are seen only occasionally. Our findings give support to the role of the claustrum in integrating information between different areas of the cerebral cortex and the limbic system.

The combined retrograde transport and unbiased stereological study of the claustrocortical connections in the rabbit.

The quantitative analysis of the claustrocortical connections in the rabbit, labeled with the fluorescent retrograde tracer Fluoro-Gold (FG), was conducted by means of unbiased stereology. The FG was injected into selected regions of the motor, somatosensory, auditory and visual cortices and then a comparison of the various claustrocortical projections was carried out. This was achieved by comparing (1) the numerical densities of projecting neurones for each claustral projection zone and (2) the distribution of the labeled neurones throughout the rostro-caudal extent of the claustrum. No significant differences between the numerical densities of labeled neurones in the various projection zones are reported. The motor and primary somatosensory projections dominated in the anterior and central parts of the claustrum, whereas the secondary somatosensory, auditory and visual projections--in the posterior part. The difference in the distributions was significant (p < 0.001). Summarizing, the cortical projections in the claustrum, although varying topographically, do not reveal a quantitative differentiation. This may speak in favour of the integrative and modulating function of this structure in relationship to the neocortex.

Neuronal loss and beta-amyloid removal in the amygdala of people with Down syndrome.

The decrease in the number of neurons free of neurofibrillary changes, neurons with neurofibrillary degeneration, and the total volume of beta-amyloid (A beta) deposits in the amygdala of people with Down syndrome and in late stages of Alzheimer disease were estimated by using morphometry and regression analysis. This model predicts that the duration of neurofibrillary changes from the pretangle stage to ghost tangles is approximately 4.7 years. The correlation between the decrease in the number of neurons and the decrease in the amount of A beta indicates that amyloid deposition is associated with neurons and that loss of neurons causes decrease in A beta deposition. The presence of neurons only with neurofibrillary tangles, and the absence of the amyloid deposits predicted by regression analysis suggest that neurons with tangles are not engaged in amyloid deposition. The disappearance of amyloid by approximately 2.2 years after loss of neurons free of neurofibrillary changes indicates that A beta deposits are degradable and removable and that even in severely atrophic amygdala, there are mechanisms of amyloid resolution. This study shows that in normal aging in the amygdala, extracellular A beta appears later than neurofibrillary changes.

Increased levels of tau-like protein in patients with Down syndrome.

Tau-like protein levels from 40 Down syndrome (DS) persons (31-70 years old), 40 non-DS age-matched normal controls, 18 non-DS mentally retarded (MR) persons (26-91 years old), 25 probable Alzheimer disease (AD) patients (55-99 years old) and 24 non-demented elderly controls (54-79 years old) were measured using a sandwich enzyme linked immunosorbent assay. The levels were detected in 22 of 40 DS persons and were significantly higher in DS than any other group (P < 0.0001). There was no relationship between tau-like protein levels and age, gender or apolipoprotein E phenotypes in any of the five groups.

Expression of cytokine genes and increased nuclear factor-kappa B activity in the brains of scrapie-infected mice.

A number of aspects of the pathogenesis of scrapie remain to be elucidated. The cellular and molecular aspects of the neuropathology in scrapie suggest the possibility that the proinflammatory cytokines could act as pathogenic mediators in this neurodegenerative disease. To understand this possibility, we examined the expression of proinflammatory cytokine genes in brains of IM mice-infected with 87V scrapie agent. Additionally, we also analyzed the activity of nuclear factor-kappa B (NF-kappaB), which is the major transcriptional activator for inflammatory cytokines, and formation of reactive oxygen species (ROS) as a common upstream messenger for its activation. The induction of mRNAs of the inflammatory cytokines, IL-1alpha, IL-1beta and TNF-alpha, was detected only in the brains of scrapie-infected mice. The activity of NF-kappaB was significantly increased in the nuclear extracts from brains of the scrapie-infected group and the immunoreactivity of NF-kappaB was increased in the hippocampus and thalamus in the brains of scrapie-infected mice. The NF-kappaB immunoreactivity was observed mainly in GFAP-positive astrocytes and also detected in the PrP-amyloid plaques in the brains of 87V scrapie-infected mice. Gene expression of IL-6 and iNOS, the representative target genes for NF-kappaB activation, were activated only in the infected group. The production of ROS was significantly increased in the brain mitochondrial fractions of scrapie-infected mice. These results suggest that prion accumulation in astrocytes might activate NF-kappaB through the increase of ROS generation, and thus alterations in NF-kappaB-directed gene expression may contribute to both the neurodegeneration and proinflammatory responses which occur in scrapie.

Cellular processing of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type.

An important gap in our understanding of the pathogenesis of the amyloidoses is the identification of the cellular events that lead from synthesis of an amyloid precursor protein to its conversion to the amyloid fiber subunit. We address this question by characterizing the effects of an amyloidogenic mutation on the intracellular processing of its protein product. The protein, a mutant of the cysteine protease inhibitor cystatin C, is the amyloid precursor protein in Hereditary Cerebral Hemorrhage with Amyloidosis--Icelandic type (HCHWA-I). The amyloid fibers are composed of mutant cystatin C (L68Q) that lacks the first 10 amino acids. We have previously shown that processing of wild-type cystatin C entails formation of a transient intracellular dimer that dissociates prior to secretion, such that extracellular cystatin C is monomeric. We report here that the cystatin C mutation engenders several alterations in its intracellular trafficking. It forms a stable intracellular dimer that is partially retained in the endoplasmic reticulum and degraded. The bulk of mutant cystatin C that is secreted does not dissociate and is secreted as an inactive dimer. Thus, formation of the stable mutant cystatin C dimer is an early event in the pathogenesis of this disease.

Expression and distribution of various antigens of developing microglial cells in the rat telencephalon.

The distribution of microglia during the early stages of postnatal development in the rat was studied on rat brain from day of birth to postnatal day 90 (P90), using immunohistochemical methods with a panel of monoclonal antibodies that recognized the complement type 3 receptor (OX-42), macrophage antigen of unknown function (ED1), and the major histocompatibility complex (MHC) class I (OX-18) or class II (OX-6) antigens. Starting from the day of birth, ameboid microglia can be differentiated with positive immunoreactivity to OX-42, OX-18, and ED1. Labeled cells were localized mainly in the developing white matter. After P21, only positive reaction to OX-42 was present, and those cells had the typical morphology of the resting microglial cells that were located either in the white or grey matter. The changes in the appearance of different antigens are correlated with the morphological differentiation and transformation of ameboid microglial cells that are to become ramified microglia, present in the adult animals.

Pattern of neuronal loss in the rat hippocampus following experimental cardiac arrest-induced ischemia.

The pattern of neuronal loss in the rat hippocampus following 10-min-long cardiac arrest-induced global ischemia was analyzed using the unbiased, dissector morphometric technique and hierarchical sampling. On the third day after ischemia, the pyramidal layer of sector CA1 demonstrated significant (27%) neuronal loss (P<0.05). At this time, no neuronal loss was observed in other cornu Ammonis sectors or the granular layer of the dentate gyrus. On the 14th postischemic day, further neuronal loss in the sector CA1 pyramidal layer was noticed. At this time, this sector contained 31% fewer pyramidal neurons than on the third day (P<0.05) and 58% fewer than in the control group (P<0.01). On the 14th day, neuronal loss in other hippocampal subdivisions also was observed. The pyramidal layer of sector CA3 contained 36% fewer neurons than in the control group (P<0.05), whereas the granular layer of the dentate gyrus contained 40% fewer (P<0.05). The total number of pyramidal neurons in sector CA2 remained unchanged. After the 14th day, no significant alterations in the total number of neurons were observed in any subdivision of the hippocampus until the 12th month of observation. Unbiased morphometric analysis emphasizes the exceptional susceptibility of sector CA1 pyramidal neurons to hypoxia/ischemia but also demonstrates significant neuronal loss in sector CA3 and the dentate granular layer, previously considered 'relatively resistant'. The different timing of neuronal dropout in sectors CA1 and CA3 and the dentate gyrus may implicate the existence of region-related properties, which determine earlier or later reactions to ischemia. However, the hippocampus has a unique, unidirectional system of intrinsic connections, whereby the majority of dentate granular neuron projections target the sector CA3 pyramidal neurons, which in turn project mostly to sector CA1. As a result, the early neuronal dropout in sector CA1 may result in retrograde transynaptic degeneration of neurons in other areas. The lack of neuronal loss in sector CA2 can be explained by the resistance of this sector to ischemia/hypoxia and the fact that this sector is not included in the major chain of intrahippocampal connections and hence is not affected by retrograde changes.

Plasma amyloid beta-peptide 1-42 and incipient Alzheimer's disease.

Mutations in the amyloid precursor protein and presenilin 1 and 2 genes result in elevated plasma levels of the amyloid beta-peptide species terminating at amino acid residue 42 (A beta1-42). In a longitudinal study of unrelated elderly individuals, those who subsequently developed Alzheimer's disease had higher plasma levels of A beta1-42 at entry than did those who remained free of dementia. The results indicate that elevated plasma levels of the released A beta peptide A beta1-42 may be detected several years before the onset of symptoms, supporting that extracellular A beta1-42 plays an important role in the pathogenesis of late-onset Alzheimer's disease.

Alteration of free radical metabolism in the brain of mice infected with scrapie agent.

Alteration of free radical metabolism in the mouse brain by scrapie infection was evaluated. The infection of mice with scrapie agent, 87V strain, slightly increased the activities of catalase and glutathione-S-transferase, while it had no effect on glutathione peroxidase, glutathione reductase, and Cu, Zn-superoxide dismutase. Results show that the scrapie infection decreased the activity of mitochondrial Mn-superoxide dismutase by 50% but increased that of monoamine oxidase (p < 0.05). Scrapie infection also increased the rate of mitochondrial superoxide generation (p < 0.05). Following scrapie infection, the level of free-sulfhydryl compounds in brain homogenates slightly decreased, but the content of thiobarbituric-acid-reactive substances and malondialdehyde increased significantly. Electron microscopy indicated that the ultrastructure of mitochondria was destroyed in the brain of scrapie-infected mice. These results suggest that elevated oxygen free radical generation and lowered scavenging activity in mitochondria might cause the free radical damage to the brain. Such deleterious changes in mitochondria may contribute to the development of prion disease.

Inhibition of neurite outgrowth by familial Alzheimer's disease-linked presenilin-1 mutations.

Two (P117L; M146L) familial Alzheimer's disease (FAD)-causing presenilin-1 (PS1) mutations have been tested fortheir effect in stably transfected mouse neuroblastoma (N2a) cell lines. The P117L mutation is associated with the earliest onset of AD reported so far (24 years), while the M146L is less pathogenic with the onset at about 43 years. Overexpression of wild-type (wt) PS1 gene was associated with the marked increase in the number and the length of neuritic outgrowths accompanied by accumulation of PS1 immunoreactivity in neurites. The highly pathogenic P117L mutation completely suppressed this effect and the pattern of PS1 immunolabeling resembled a cup structure with all immunoreactivity gathered at one pole of the cell. The effect of less pathogenic M146L mutation was similar, but not as pronounced. These findings suggest that one of the normal functions of PS1 may be the control of neurite outgrowth, and the inhibitory effect of two FAD-linked mutations stresses its importance in the cellular mechanism that leads to the development of Alzheimer's disease (AD).

The therapeutic effect of an herbal formula Badmaev 28 (padma 28) on experimental allergic encephalomyelitis (EAE) in SJL/J mice.

A herbal formula, Badmaev 28, was evaluated in the treatment of an induced attack in a chronic relapsing model of experimental allergic encephalomyelitis (EAE) in SJL/J mice. Chronic EAE was induced by immunization of 8 week old mice with an emulsion of syngeneic spinal cords with incomplete Freund's adjuvant supplemented with Mycobacterium tuberculosis. Therapy with Badmaev 28 was started on day 25 after the immunization, and the formula was administered in the drinking water at doses of 7, 21, 83 and 166 mg/kg/day. The treatment resulted in significantly decreased mortality compared with the untreated control animals and the therapeutic effect occurred in one experiment in a dose-dependent fashion. Based on the experimental results it is difficult to name one particular mechanism responsible for the therapeutic effectiveness of the formula in the EAE model. Rather this protective effect could be explained by a broad protective mechanism of action discussed in the literature as nonspecific resistance (NSR) to the diversified biological and psychological stressors. The increase in NSR characterizes the action of pharmacological compounds termed adaptogens or bioprotectants.

Biondi ring tangles in the choroid plexus of Alzheimer's disease and normal aging brains: a quantitative study.

The choroid plexus (CP) performs the vital function of producing up to 90% (450-1000 ml/day) of cerebrospinal fluid (CSF) to nourish and to protect the brain in the CSF suspension. The CP also acts as a selective barrier between blood and CSF to regulate ions and other essential molecules. However, the accumulation of intracellular inclusions called Biondi ring tangles (BRTs) in CP cells of Alzheimer's disease (AD)/aging brains may affect these vital functions of the CP. Statistical analysis of quantitative data on the numbers of CP cells containing BRTs from 54 brains (29 AD and 25 normal control), age range 1-100 years, indicated a significant difference (p<0.00004) between AD and control brains, using analysis of covariance (ANCOVA) with age as covariate. This study compiled the first set of archives to reveal the distribution pattern of BRTs in the CP of AD brains at various ages. Electron microscopy of negatively stained isolated BRTs revealed that these tangles are made of tightly packed bundles of long filaments with diameter around 10 nm that are morphologically distinct from the more loosely packed/shorter bundles of 6-8 nm amyloid fibrils of neuritic plaques (NPs) and from the 24 nm paired helical filaments of neurofibrillary tangles (NFTs) in AD brain. These data suggest that BRTs may represent a significant and measurable biomarker for AD in addition to NPs and NFTs.