Human stem cell-derived monocytes and microglia-like cells reveal impaired amyloid plaque clearance upon heterozygous or homozygous loss of TREM2.

INTRODUCTION: Murine microglia expressing the Alzheimer's disease-linked TREM2R47H mutation display variable decrease in phagocytosis, while impaired phagocytosis is reported following loss of TREM2. However, no data exist on TREM2+/R47H human microglia. Therefore, we created human pluripotent stem cell (hPSC) monocytes and transdifferentiated microglia-like cells (tMGs) to examine the effect of the TREM2+/R47H mutation and loss of TREM2 on phagocytosis. METHODS: We generated isogenic TREM2+/R47H, TREM2+/-, and TREM2-/- hPSCs using CRISPR/Cas9. Following differentiation to monocytes and tMGs, we studied the uptake of Escherichia coli fragments and analyzed amyloid plaque clearance from cryosections of APP/PS1+/- mouse brains. RESULTS: We demonstrated that tMGs resemble cultured human microglia. TREM2+/- and TREM2-/- hPSC monocytes and tMGs phagocytosed significantly less E. coli fragments and cleared less amyloid plaques than wild-type hPSC progeny, with no difference for TREM2+/R47H progeny. DISCUSSION: In vitro phagocytosis of hPSC monocytes and tMGs was not affected by the TREM2+/R47H mutation but was significantly impaired in TREM2+/- and TREM2-/- progeny.

Sembragiline: A Novel, Selective Monoamine Oxidase Type B Inhibitor for the Treatment of Alzheimer's Disease.

Monoamine oxidase B (MAO-B) has been implicated in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. Increased MAO-B expression in astroglia has been observed adjacent to amyloid plaques in AD patient brains. This phenomenon is hypothesized to lead to increased production of hydrogen peroxide and reactive oxygen species (ROS), thereby contributing to AD pathology. Therefore, reduction of ROS-induced oxidative stress via inhibition of MAO-B activity may delay the progression of the disease. In the present study we report the pharmacological properties of sembragiline, a novel selective MAO-B inhibitor specifically developed for the treatment of AD, and on its effect on ROS-mediated neuronal injury and astrogliosis in MAO-B transgenic animals. Sembragiline showed potent and long-lasting MAO-B-selective inhibition and did not inhibit MAO-A at doses where full inhibition of MAO-B was observed. Such selectivity should translate into a favorable clinical safety profile. Indeed, sembragiline neither induced the serotonin syndrome when administered together with the serotonin precursor l-5-hydroxytryptophan in combination with antidepressants such as fluoxetine, nor potentiated the pressor effect of tyramine. Additionally, in experiments using a transgenic animal model conditionally overexpressing MAO-B in astroglia, sembragiline protected against neuronal loss and reduced both ROS formation and reactive astrogliosis. Taken together, these findings warrant further investigation of the potential therapeutic benefit of MAO-B inhibitors in patients with AD and other neurologic disorders.

A subcutaneous cellular implant for passive immunization against amyloid-ß reduces brain amyloid and tau pathologies.

Passive immunization against misfolded toxic proteins is a promising approach to treat neurodegenerative disorders. For effective immunotherapy against Alzheimer's disease, recent clinical data indicate that monoclonal antibodies directed against the amyloid-ß peptide should be administered before the onset of symptoms associated with irreversible brain damage. It is therefore critical to develop technologies for continuous antibody delivery applicable to disease prevention. Here, we addressed this question using a bioactive cellular implant to deliver recombinant anti-amyloid-ß antibodies in the subcutaneous tissue. An encapsulating device permeable to macromolecules supports the long-term survival of myogenic cells over more than 10 months in immunocompetent allogeneic recipients. The encapsulated cells are genetically engineered to secrete high levels of anti-amyloid-ß antibodies. Peripheral implantation leads to continuous antibody delivery to reach plasma levels that exceed 50 µg/ml. In a proof-of-concept study, we show that the recombinant antibodies produced by this system penetrate the brain and bind amyloid plaques in two mouse models of the Alzheimer's pathology. When encapsulated cells are implanted before the onset of amyloid plaque deposition in TauPS2APP mice, chronic exposure to anti-amyloid-ß antibodies dramatically reduces amyloid-ß40 and amyloid-ß42 levels in the brain, decreases amyloid plaque burden, and most notably, prevents phospho-tau pathology in the hippocampus. These results support the use of encapsulated cell implants for passive immunotherapy against the misfolded proteins, which accumulate in Alzheimer's disease and other neurodegenerative disorders.

Combined treatment with a BACE inhibitor and anti-Aß antibody gantenerumab enhances amyloid reduction in APPLondon mice.

Therapeutic approaches for prevention or reduction of amyloidosis are currently a main objective in basic and clinical research on Alzheimer's disease. Among the agents explored in clinical trials are anti-Aß peptide antibodies and secretase inhibitors. Most anti-Aß antibodies are considered to act via inhibition of amyloidosis and enhanced clearance of existing amyloid, although secretase inhibitors reduce the de novo production of Aß. Limited information is currently available on the efficacy and potential advantages of combinatorial antiamyloid treatment. We performed a chronic study in APPLondon transgenic mice that received treatment with anti-Aß antibody gantenerumab and BACE inhibitor RO5508887, either as mono- or combination treatment. Treatment aimed to evaluate efficacy on amyloid progression, similar to preexisting amyloidosis as present in Alzheimer's disease patients. Mono-treatments with either compound caused a dose-dependent reduction of total brain Aß and amyloid burden. Combination treatment with both compounds significantly enhanced the antiamyloid effect. The observed combination effect was most pronounced for lowering of amyloid plaque load and plaque number, which suggests effective inhibition of de novo plaque formation. Moreover, significantly enhanced clearance of pre-existing amyloid plaques was observed when gantenerumab was coadministered with RO5508887. BACE inhibition led to a significant time- and dose-dependent decrease in CSF Aß, which was not observed for gantenerumab treatment. Our results demonstrate that combining these two antiamyloid agents enhances overall efficacy and suggests that combination treatments may be of clinical relevance.

Neuronal uptake of tau/pS422 antibody and reduced progression of tau pathology in a mouse model of Alzheimer's disease.

The severity of tau pathology in Alzheimer's disease brain correlates closely with disease progression. Tau immunotherapy has therefore been proposed as a new therapeutic approach to Alzheimer's disease and encouraging results have been obtained by active or passive immunization of tau transgenic mice. This work investigates the mechanism by which immunotherapy can impact tau pathology. We demonstrate the development of Alzheimer's disease-like tau pathology in a triple transgenic mouse model of Alzheimer's disease and show that tau/pS422 is present in membrane microdomains on the neuronal cell surface. Chronic, peripheral administration of anti-tau/pS422 antibody reduces the accumulation of tau pathology. The unequivocal presence of anti-tau/pS422 antibody inside neurons and in lysosomes is demonstrated. We propose that anti-tau/pS422 antibody binds to membrane-associated tau/pS422 and that the antigen-antibody complexes are cleared intracellularly, thereby offering one explanation for how tau immunotherapy can ameliorate neuronal tau pathology.

Novel screening cascade identifies MKK4 as key kinase regulating Tau phosphorylation at Ser422.

Phosphorylation of Tau at serine 422 promotes Tau aggregation. The kinase that is responsible for this key phosphorylation event has so far not been identified but could be a potential drug target for Alzheimer's disease. We describe here an assay strategy to identify this kinase. Using a combination of screening a library of 65'000 kinase inhibitors and in vitro inhibitor target profiling of the screening hits using the Ambit kinase platform, MKK4 was identified as playing a key role in Tau-S422 phosphorylation in human neuroblastoma cells.

Phosphorylation of Tau at S422 is enhanced by Abeta in TauPS2APP triple transgenic mice.

Amyloid beta peptides and microtubule-associated protein Tau are misfolded and form aggregates in brains of Alzheimer's disease patients. To examine their specific roles in the pathogenesis of Alzheimer's disease and their relevance in neurodegenerative processes, we have created TauPS2APP triple transgenic mice that express human mutated Amyloid Precursor Protein, presenilin 2 and Tau. We present a cross-sectional analysis of these mice at 4, 8, 12 and 16 months of age. By comparing with single transgenic Tau mice, we demonstrate that accumulation of Abeta in TauPS2APP triple transgenic mice impacts on Tau pathology by increasing the phosphorylation of Tau at serine 422, as determined by a novel immunodetection method that is able to reliably measure phospho-Tau species in transgenic mouse brains. The TauPS2APP triple transgenic mouse model will be very useful for studying the effect of new therapeutic paradigms on amyloid deposition and downstream neurofibrillary tangle development.

Cortical hypoperfusion in the B6.PS2APP mouse model for Alzheimer's disease: comprehensive phenotyping of vascular and tissular parameters by MRI.

Function and morphology of the cerebral vasculature were studied in the amyloid (Abeta) plaque-containing double-transgenic (TG) B6.PS2APP Alzheimer's disease (AD) mouse model with MRI at an age range of 10 to 17 months. Perfusion, blood volume, and average vessel geometry were assessed in the brain and compared to age-matched controls (wild-type [WT] C57Bl/6). Additionally, the MR relaxation times T(1), T(2), and T(2)* were measured to detect potential pathological changes that might be associated with Abeta plaque depositions. Both decreased perfusion and decreased blood volume were observed in the occipital cortex in B6.PS2APP mice as compared to controls. A significant decrease in T(1) and T(2) was found in the frontal cortex and in the subiculum/parasubiculum. Immunohistochemistry confirmed plaque depositions in the cortex and in the subiculum/parasubiculum. In summary, our data indicate a reduced blood supply of B6.PS2APP mice in the occipital cortex that parallels the findings in cortical regions of patients with AD.

Lewy pathology in Parkinson's disease consists of crowded organelles and lipid membranes.

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.

Efficacy of chronic BACE1 inhibition in PS2APP mice depends on the regional Aß deposition rate and plaque burden at treatment initiation.

Beta secretase (BACE) inhibitors are promising therapeutic compounds currently in clinical phase II/III trials. Preclinical [18F]-florbetaben (FBB) amyloid PET imaging facilitates longitudinal monitoring of amyloidosis in Alzheimer's disease (AD) mouse models. Therefore, we applied this theranostic concept to investigate, by serial FBB PET, the efficacy of a novel BACE1 inhibitor in the PS2APP mouse, which is characterized by early and massive amyloid deposition. Methods: PS2APP and C57BL/6 (WT) mice were assigned to treatment (PS2APP: N=13; WT: N=11) and vehicle control (PS2APP: N=13; WT: N=11) groups at the age of 9.5 months. All animals had a baseline PET scan and follow-up scans at two months and after completion of the four-month treatment period. In addition to this longitudinal analysis of cerebral amyloidosis by PET, we undertook biochemical amyloid peptide quantification and histological amyloid plaque analyses after the final PET session. Results: BACE1 inhibitor-treated transgenic mice revealed a progression of the frontal cortical amyloid signal by 8.4 ± 2.2% during the whole treatment period, which was distinctly lower when compared to vehicle-treated mice (15.3 ± 4.4%, p<0.001). A full inhibition of progression was evident in regions with <3.7% of the increase in controls, whereas regions with >10% of the increase in controls showed only 40% attenuation with BACE1 inhibition. BACE1 inhibition in mice with lower amyloidosis at treatment initiation showed a higher efficacy in attenuating progression to PET. A predominant reduction of small plaques in treated mice indicated a main effect of BACE1 on inhibition of de novo amyloidogenesis. Conclusions: This theranostic study with BACE1 treatment in a transgenic AD model together with amyloid PET monitoring indicated that progression of amyloidosis is more effectively reduced in regions with low initial plaque development and revealed the need of an early treatment initiation during amyloidogenesis.

Brain Shuttle Antibody for Alzheimer's Disease with Attenuated Peripheral Effector Function due to an Inverted Binding Mode.

Receptors show promise for the transport of monoclonal antibodies (mAbs) across the blood-brain barrier. However, safety liabilities associated with peripheral receptor binding and Fc effector function have been reported. We present the Brain Shuttle-mAb (BS-mAb) technology, and we investigate the role of Fc effector function in vitro and in an Fcγ receptor (FcγR)-humanized mouse model. Strong first infusion reactions (FIRs) were observed for a conventional mAb against transferrin receptor (TfR) with a wild-type immunoglobulin G1 (IgG1) Fc. Fc effector-dead constructs completely eliminated all FIRs. Remarkably, no FIR was observed for the BS-mAb construct with a native IgG1 Fc function. Using various BS-mAb constructs, we show that TfR binding through the C-terminal BS module attenuates Fc-FcγR interactions, primarily because of steric hindrance. Nevertheless, BS-mAbs maintain effector function activity when binding their brain target. Thus, mAbs with full effector function can be transported in a stealth mode in the periphery while fully active when engaged with their brain target.

Cerebral Corpora amylacea are dense membranous labyrinths containing structurally preserved cell organelles.

Corpora amylacea are cell-derived structures that appear physiologically in the aged human brain. While their histological identification is straightforward, their ultrastructural composition and microenvironment at the nanoscale have remained unclear so far, as has their relevance to aging and certain disease states that involve the sequestration of toxic cellular metabolites. Here, we apply correlative serial block-face scanning electron microscopy and transmission electron tomography to gain three-dimensional insight into the ultrastructure and surrounding microenvironment of cerebral Corpora amylacea in the human brainstem and hippocampal region. We find that cerebral Corpora amylacea are composed of dense labyrinth-like sheets of lipid membranes, contain vesicles as well as morphologically preserved mitochondria, and are in close proximity to blood vessels and the glymphatic system, primarily within the cytoplasm of perivascular glial cells. Our results clarify the nature of cerebral Corpora amylacea and provide first hints on how they may arise and develop in the aging brain.

HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment.

Thermodynamic studies on the interaction of antibodies with beta-amyloid peptide.

Antibodies against beta-amyloid peptides (Abetas) are considered an important therapeutic opportunity in Alzheimer's disease. Despite the vast interest in Abeta no thermodynamic data on the interaction of antibodies with Abeta are available as yet. In the present study we use isothermal titration calorimetry (ITC) and surface plasmon resonance to provide a quantitative thermodynamic analysis of the interaction between soluble monomeric Abeta(1-40) and mouse monoclonal antibodies (mAb). Using four different antibodies directed against the N-terminal, middle, and C-terminal Abeta epitopes, we measured the thermodynamic parameters for the binding to Abeta. Each antibody species was found to have two independent and equal binding sites for Abeta with binding constants in the range of 10(7) to 10(8) M(-1). The binding reaction was essentially enthalpy driven with a reaction enthalpy of DeltaH(0)(Abeta) approximately -19 to -8 kcal/mol, indicating the formation of tight complexes. The loss in conformational freedom was supported by negative values for the reaction entropy DeltaS(0)(Abeta). We also measured the heat capacity change of the 1mAb:2Abeta reaction. DeltaC(0)(p, abeta) was large and negative but could not be explained exclusively by the hydrophobic effect. The free energy of binding was found to be linearly correlated with the size of the epitope.

Reproductive and developmental toxicology studies with gantenerumab in PS2APP transgenic mice.

Gantenerumab is intended for the treatment of Alzheimer's disease. It is a fully human recombinant monoclonal IgG1 which binds aggregated forms of amyloid beta such as Aß oligomers, fibrils and neuritic amyloid plaques. A full package of developmental and reproductive toxicity (DART) studies was performed in the PS2APP double-transgenic mouse model of Alzheimer's disease. A combined fertility and embryo fetal development study and a pre-and post-natal development study were performed. The PS2APP mouse model allowed a more complete DART evaluation than would have been possible in conventional species or strains which do not express the target antigen of gantenerumab. No developmental or reproductive hazards were identified.

TREM2 deficiency reduces the efficacy of immunotherapeutic amyloid clearance.

Immunotherapeutic approaches are currently the most advanced treatments for Alzheimer's disease (AD). Antibodies against amyloid ß-peptide (Aß) bind to amyloid plaques and induce their clearance by microglia via Fc receptor-mediated phagocytosis. Dysfunctions of microglia may play a pivotal role in AD pathogenesis and could result in reduced efficacy of antibody-mediated Aß clearance. Recently, heterozygous mutations in the triggering receptor expressed on myeloid cells 2 (TREM2), a microglial gene involved in phagocytosis, were genetically linked to late onset AD Loss of TREM2 reduces the ability of microglia to engulf Aß. We have now investigated whether loss of TREM2 affects the efficacy of immunotherapeutic approaches. We show that anti-Aß antibodies stimulate Aß uptake and amyloid plaque clearance in a dose-dependent manner in the presence or absence of TREM2. However, TREM2-deficient N9 microglial cell lines, macrophages as well as primary microglia showed significantly reduced uptake of antibody-bound Aß and as a consequence reduced clearance of amyloid plaques. Titration experiments revealed that reduced efficacy of amyloid plaque clearance by Trem2 knockout cells can be compensated by elevating the concentration of therapeutic antibodies.

PS2APP transgenic mice, coexpressing hPS2mut and hAPPswe, show age-related cognitive deficits associated with discrete brain amyloid deposition and inflammation.

Transgenic mice, expressing mutant beta-amyloid precursor proteins (betaAPPs), have lead to a better understanding of the pathophysiological processes in Alzheimer's disease (AD). In many of these models, however, the temporal development of cognitive decline and the relationship to Abeta deposition and inflammation are unclear. We now report a novel transgenic mouse line, PS2APP (PS2N141I x APPswe), which develops a severe cerebral amyloidosis in discrete brain regions, and present a cross-sectional analysis of these mice at 4, 8, 12, and 16 months of age. Each age cohort was investigated for changes in behavior, electrophysiology of synapse efficacy, ELISA-determined Abeta load, histopathology, and in immunoelectron microscopy. Cognitive deficits were first observed at 8 months when Abeta deposits and inflammation were restricted to discrete brain regions, namely the subiculum and frontolateral (motor and orbital) cortex. As early as 5 months, electron microscopy revealed the presence, in these regions, of pre-plaque, immunogold-labeled extracellular fibrillar Abeta. At the same age, increased levels of insoluble Abeta were detected by ELISA, with Abeta1-40 levels exceeding those of Abeta1-42. Further cognitive decline occurred in an age-related manner, and this was accompanied by the spread of amyloidosis to ultimately affect not only neo- and limbic cortices, but also thalamic and pontine nuclei. Dentate gyrus post-tetanic potentiation was significantly attenuated at 17 months, and there were also significant differences in paired-pulse parameters. This systematic cross-sectional study of the behavioral and pathological changes in the PS2APP mouse indicates that it develops age-related cognitive decline associated with severe amyloidosis and inflammation in discrete brain regions and therefore is suitable for testing a range of potential symptomatic and disease-modifying therapies for AD.

Quantification of the A beta peptide in Alzheimer's plaques by laser dissection microscopy combined with mass spectrometry.

The accumulation and aggregation of the beta-amyloid peptide (A beta) in the brain represents a key factor in the pathogenesis of Alzheimer's disease (AD). Many of the transgenic mouse models for AD exhibit an amyloid pathology with neuritic plaques but they typically vary by the type and abundance of plaques identified in their brains and by the onset and severity of cognitive impairment. Thus, an important consideration in the characterization of AD transgenic mouse models should be the quantitative evaluation of the amyloid load in the brain together with a detailed physico-chemical analysis of A beta from the deposited plaques. Here we present an analytical procedure to collect single amyloid plaques from anatomically defined brain regions by laser dissection microscopy that can be quantitatively assessed in their A beta isoforms composition by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Quantification was achieved by stable isotope dilution using calibrated 15N-labeled A beta standards that were spiked in the sample immediately after laser dissection. Using this method, we found that the amyloid loads in brain plaques isolated from the transgenic AD mouse model PS2APP or from human were similar. Total A beta composition was estimated at approximately 50-100 fmol per excised plaque disc, as confirmed by immunoblot analysis. N-Terminal truncated A beta isoforms were identified in both transgene and human amyloid plaques but with significantly elevated levels in human samples.

Acute renal effects of intravenous bisphosphonates in the rat.

Bisphosphonates are potent osteoclast inhibitors that have been associated with renal toxicity following rapid intravenous administration of high doses, which was hypothesised to be due to precipitation of bisphosphonate aggregates or complexes in the kidney. Five studies were conducted in rats investigating the characteristics of bisphosphonate-related acute renal effects. These studies included single intravenous injections of the nitrogen-containing bisphosphonates (1) ibandronate (1-20 mg/kg), or (2) zoledronate (1-10 mg/kg); (3) a single nephrotoxic dose of the non-nitrogen-containing bisphosphonate, clodronate (2 x 200 mg/kg intraperitoneal injection); (4) a single low dose of ibandronate (1 mg/kg); (5) a single high dose of zoledronate (10 mg/kg). Clinical biochemistry and kidney histopathology were performed 1 and/or 4 days after bisphosphonate dosing. The proximal convoluted tubules were the primary target for renal injury. Tubular degeneration and single cell necrosis of the these tubules were observed with all three bisphosphonates on the fourth, but not the first day after dosing. Differences between the bisphosphonates in the type and/or localisation of the lesions were apparent. Granular deposits in the lumen of distal tubules were apparent with the highest dose of zoledronate (10 mg/kg). However, intraluminal debris was proteinaceous with no evidence of any precipitation of bisphosphonate, or formation of aggregates or complexes in the kidney. Generally, biochemical parameters of renal safety and urinary enzymes did not differ significantly from controls. In summary, bisphosphonate-related renal changes did not appear to be due to the precipitation, aggregation or complexation of bisphosphonate, and biochemical parameters of renal safety did not reliably detect this renal injury.

Atherosclerosis, vascular amyloidosis and brain hypoperfusion in the pathogenesis of sporadic Alzheimer's disease.

We postulate that severe atherosclerotic occlusion of the circle of Willis and leptomeningeal arteries is an important factor in the pathogenesis of some sporadic Alzheimer's disease (AD) cases. These arterial stenoses are complicated by an overwhelming amyloid accumulation in the walls of leptomeningeal and cortical arteries resulting in a significant decrease in perfusion pressure and consequent ischemia/hypoxia of the brain tissue. We also propose that the distal areas of the white matter (WM) will be the first affected by a lack of oxygen and nutrients. Our hypotheses are supported by the following observations: (1) the number of stenoses is more frequent in AD than in the control population (p = 0.008); (2) the average index of occlusion is greater in AD than in the control group (p < 0.00001); (3) the index of stenosis and the total number of stenoses per case are positively correlated (R = 0.67); (4) the index of stenosis correlates with the neuropathological lesions of AD and with the MMSE psychometric test; (5) the number and degree of atherosclerosis of the anterior, middle and posterior cerebral arteries is more severe in cases of AD than in the control population; (6) atherosclerosis severity is apparently associated with the severity of the vascular amyloidosis; (7) the WM rarefaction correlates with the severity of the atherosclerosis and vascular amyloidosis; (8) the total cell count and microvessel count in the areas of WM rarefaction correlate with the neuropathological lesions of AD and with the MMSE score. Our data strongly suggest that severe hemodynamic disturbances contribute to sporadic AD and support the numerous observations indicating cardiovascular system participation in the pathogenesis of these dementias.