Mary Lou Previti

Minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid.

Cerebral microvascular amyloid β protein (Aβ) deposition and associated neuroinflammation is increasingly recognized as an important component leading to cognitive impairment in Alzheimers disease and related cerebral amyloid angiopathy disorders. Transgenic mice expressing the vasculotropic Dutch/Iowa (E693Q/D694N) mutant human Aβ precursor protein in brain (Tg-SwDI) accumulate abundant cerebral microvascular fibrillar amyloid deposits and exhibit robust neuroinflammation. In the present study, we investigated the effect of the anti-inflammatory drug minocycline on Aβ accumulation, neuroinflammation, and behavioral deficits in Tg-SwDI mice. Twelve-month-old mice were treated with saline or minocycline by intraperitoneal injection every other day for a total of 4 weeks. During the final week of treatment, the mice were tested for impaired learning and memory. Brains were then harvested for biochemical and immunohistochemical analysis. Minocycline treatment did not alter the cerebral deposition of Aβ or the restriction of fibrillar amyloid to the cerebral microvasculature. Similarly, minocycline-treated Tg-SwDI mice exhibited no change in the levels of total Aβ, the ratios of Aβ40 and Aβ42, or the amounts of soluble, insoluble, or oligomeric Aβ compared with the saline-treated control Tg-SwDI mice. In contrast, the numbers of activated microglia and levels of interleukin-6 were significantly reduced in minocycline-treated Tg-SwDI mice compared with saline-treated Tg-SwDI mice. In addition, there was a significant improvement in behavioral performance of the minocycline-treated Tg-SwDI mice. These finding suggest that anti-inflammatory treatment targeted for cerebral microvascular amyloid-induced microglial activation can improve cognitive deficits without altering the accumulation and distribution of Aβ.

Progression of Amyloid Pathology to Alzheimer's Disease Pathology in an Amyloid Precursor Protein Transgenic Mouse Model by Removal of Nitric Oxide Synthase 2

Alzheimers disease (AD) is characterized by three primary pathologies in the brain: amyloid plaques, neurofibrillary tangles, and neuron loss. Mouse models have been useful for studying components of AD but are limited in their ability to fully recapitulate all pathologies. We crossed the APPSwDI transgenic mouse, which develops amyloid β (Aβ)-protein deposits only, with a nitric oxide synthase 2 (NOS2) knock-out mouse, which develops no AD-like pathology. APPSwDI/NOS2−/− mice displayed impaired spatial memory compared with the APPSwDI mice, yet they have unaltered levels of Aβ. APPSwDI mice do not show tau pathology, whereas APPSwDI/NOS2−/− mice displayed extensive tau pathology associated with regions of dense microvascular amyloid deposition. Also, APPSwDI mice do not have any neuron loss, whereas the APPSwDI/NOS2−/− mice have significant neuron loss in the hippocampus and subiculum. Neuropeptide Y neurons have been shown to be particularly vulnerable in AD. These neurons appear to be particularly vulnerable in the APPSwDI/NOS2−/− mice as we observe a dramatic reduction in the number of NPY neurons in the hippocampus and subiculum. These data show that removal of NOS2 from an APP transgenic mouse results in development of a much greater spectrum of AD-like pathology and behavioral impairments.

NO synthase 2 (NOS2) deletion promotes multiple pathologies in a mouse model of Alzheimer's disease

Alzheimers disease is characterized by two primary pathological features: amyloid plaques and neurofibrillary tangles. The interconnection between amyloid and tau aggregates is of intense interest, but mouse models have yet to reveal a direct interrelationship. We now show that NO may be a key factor that connects amyloid and tau pathologies. Genetic removal of NO synthase 2 in mice expressing mutated amyloid precursor protein results in pathological hyperphosphorylation of mouse tau, its redistribution to the somatodendritic compartment in cortical and hippocampal neurons, and aggregate formation. Lack of NO synthase 2 in the amyloid precursor protein Swedish mutant mouse increased insoluble β-amyloid peptide levels, neuronal degeneration, caspase-3 activation, and tau cleavage, suggesting that NO acts at a junction point between β-amyloid peptides, caspase activation, and tau aggregation.

Scavenger receptor CD36 is essential for the cerebrovascular oxidative stress and neurovascular dysfunction induced by amyloid-β

Increasing evidence indicates that cerebrovascular dysfunction plays a pathogenic role in Alzheimers dementia (AD). Amyloid-β (Aβ), a peptide central to the pathogenesis of AD, has profound vascular effects mediated, for the most part, by reactive oxygen species produced by the enzyme NADPH oxidase. The mechanisms linking Aβ to NADPH oxidase-dependent vascular oxidative stress have not been identified, however. We report that the scavenger receptor CD36, a membrane glycoprotein that binds Aβ, is essential for the vascular oxidative stress and neurovascular dysfunction induced by Aβ1–40. Thus, topical application of Aβ1–40 onto the somatosensory cortex attenuates the increase in cerebral blood flow elicited by neural activity or by endothelium-dependent vasodilators in WT mice but not in CD36-null mice (CD360/0). The cerebrovascular effects of infusion of Aβ1–40 into cerebral arteries are not observed in mice pretreated with CD36 blocking antibodies or in CD360/0 mice. Furthermore, CD36 deficiency prevents the neurovascular dysfunction observed in transgenic mice overexpressing the Swedish mutation of the amyloid precursor protein Tg2576 despite elevated levels of brain Aβ1–40. CD36 is also required for the vascular oxidative stress induced by exogenous Aβ1–40 or observed in Tg2576 mice. These observations establish CD36 as a key link between Aβ1–40 and the NADPH oxidase-dependent vascular oxidative stress underlying the neurovascular dysfunction and suggest that CD36 is a potential therapeutical target to counteract the cerebrovascular dysfunction associated with Aβ.

Reducing Cerebral Microvascular Amyloid-β Protein Deposition Diminishes Regional Neuroinflammation in Vasculotropic Mutant Amyloid Precursor Protein Transgenic Mice

Cerebral microvascular amyloid-β (Aβ) protein deposition is emerging as an important contributory factor to neuroinflammation and dementia in Alzheimers disease and related familial cerebral amyloid angiopathy disorders. In particular, cerebral microvascular amyloid deposition, but not parenchymal amyloid, is more often correlated with dementia. Recently, we generated transgenic mice (Tg-SwDI) expressing the vasculotropic Dutch (E693Q)/Iowa (D694N) mutant human Aβ precursor protein in brain that accumulate abundant cerebral microvascular fibrillar amyloid deposits. In the present study, our aim was to assess how the presence or absence of fibrillar Aβ deposition in the cerebral microvasculature affects neuroinflammation in Tg-SwDI mice. Using Tg-SwDI mice bred onto an apolipoprotein E gene knock-out background, we found a strong reduction of fibrillar cerebral microvascular Aβ deposition, which was accompanied by a sharp decrease in microvascular-associated neuroinflammatory cells and interleukin-1β levels. Quantitative immunochemical measurements showed that this reduction of the neuroinflammation occurred in the absence of lowering the levels of total Aβ40/Aβ42 or soluble Aβ oligomers in brain. These findings suggest that specifically reducing cerebral microvascular fibrillar Aβ deposition, in the absence of lowering either the total amount of Aβ or soluble Aβ oligomers in brain, may be sufficient to ameliorate microvascular amyloid-associated neuroinflammation.

Early-Onset Subicular Microvascular Amyloid and Neuroinflammation Correlate With Behavioral Deficits in Vasculotropic Mutant APP Transgenic Mice

Cerebral microvascular amyloid beta protein (Abeta) deposition and associated neuroinflammation are increasingly recognized as an important component leading to cognitive impairment in Alzheimers disease and related cerebral amyloid angiopathy (CAA) disorders. Transgenic mice expressing the vasculotropic Dutch/Iowa (E693Q/D694N) mutant human Abeta precursor protein in brain (Tg-SwDI) accumulate abundant cerebral microvascular fibrillar amyloid deposits exhibiting robust neuroinflammation. In the present study, we sought to determine if the unique amyloid pathology of Tg-SwDI mice was associated with deficits in behavioral performance. Behavioral performance tests that assessed a variety of psychological functions, including overall activity, motor ability, balance and strength, anxiety, impulsivity, and learning were conducted on homozygous Tg-SwDI mice and similarly aged wild-type C57Bl/6 mice. Our results indicate that Tg-SwDI mice were impaired in the performance of the Barnes maze learning and memory task at 3, 9, and 12 months of age. While more widespread cerebral microvascular Abeta pathology was evident in older animals, the evaluation of the Abeta pathology in the 3 months old transgenic animals revealed specific accumulation of microvascular amyloid and markedly elevated numbers of reactive astrocytes and activated microglia restricted to the subiculum. These findings indicate that early-onset accumulation of subicular microvascular amyloid and accompanying neuroinflammation correlates with impaired performance in the learning and memory task in Tg-SwDI mice.

Age-dependent neurovascular dysfunction and damage in a mouse model of cerebral amyloid angiopathy.

Background and Purpose— Accumulation of amyloid-&bgr; in cerebral blood vessels occurs in familial and sporadic forms of cerebral amyloid angiopathy and is a prominent feature of Alzheimer disease. However, the functional correlates of the vascular pathology induced by cerebral amyloid angiopathy and the mechanisms involved have not been fully established. Methods— We used male transgenic mice expressing the Swedish, Iowa, and Dutch mutations of the amyloid precursor protein (Tg-SwDI) to examine the effect of cerebral amyloid angiopathy on cerebrovascular structure and function. Somatosensory cortex cerebral blood flow was monitored by laser-Doppler flowmetry in anesthetized Tg-SwDI mice and wild-type littermates equipped with a cranial window. Results— Tg-SwDI mice exhibited reductions in cerebral blood flow responses to whisker stimulation, endothelium-dependent vasodilators, or hypercapnia at 3 months when compared with wild-type mice, whereas the response to adenosine was not attenuated. However, at 18 and 24 months, all cerebrovascular responses were markedly reduced. At this time, there was evidence of cerebrovascular amyloid deposition, smooth muscle fragmentation, and pericyte loss. Neocortical superfusion with the free radical scavenger manganic(I-II)meso-tetrakis(4-benzoic acid) porphyrin rescued endothelium-dependent responses and functional hyperemia completely at 3 months but only partially at 18 months. Conclusions— Tg-SwDI mice exhibit a profound age-dependent cerebrovascular dysfunction, involving multiple regulatory mechanisms. Early in the disease process, oxidative stress is responsible for most of the vascular dysfunction, but with advancing disease structural alterations of the vasomotor apparatus also play a role. Early therapeutic interventions are likely to have the best chance to counteract the deleterious vascular effects of cerebral amyloid angiopathy.

Experimental Investigation of Antibody-Mediated Clearance Mechanisms of Amyloid-β in CNS of Tg-SwDI Transgenic Mice

Novel amyloid precursor protein transgenic mice, which contain the Swedish as well as the vasculotropic Dutch and Iowa mutations (Tg-SwDI), were used to investigate the mechanisms of antibody-mediated clearance of amyloid-β (Aβ) from the brain. Export of the Aβ-DI peptide across the blood–brain barrier is severely reduced because of the vasculotropic mutations. Therefore, antibody-mediated clearance of Aβ-DI is dependent on antibodies entering the brain. In this report, we immunized Tg-SwDI mice with various peptide antigens, including Aβ40-DI, Aβ42, and an Aβ epitope vaccine. Immunization of Tg-SwDI mice with substantial cortical diffuse and vascular fibrillar deposits failed to promote clearance of parenchymal or vascular amyloid deposits. We then immunized young Tg-SwDI mice before the accumulation of Aβ and saw no evidence that anti-Aβ antibodies could diminish deposition of parenchymal or vascular amyloid deposits. However, injection of anti-Aβ antibodies, affinity-purified from immunized Tg-SwDI mice, into the hippocampus induced a rapid clearance of diffuse Aβ deposits but not vascular amyloid deposits. These results further support the “peripheral sink hypothesis” as a legitimate mechanism of antibody-mediated clearance of Aβ when the blood–brain barrier remains intact. Thus, approaches that deliver immunotherapy to the brain may be more effective at clearing Aβ than immunization strategies in which the majority of the antibodies are in the periphery.

A riboswitch-based inducible gene expression system for mycobacteria.

Research on the human pathogen Mycobacterium tuberculosis (Mtb) would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

Brain and Circulating Levels of Aβ1–40 Differentially Contribute to Vasomotor Dysfunction in the Mouse Brain

Background and Purpose— Amyloid-&bgr; (A&bgr;), a peptide that accumulates in the brain and circulates in the blood of patients with Alzheimer disease, alters the regulation of cerebral blood flow and may contribute to the brain dysfunction underlying the dementia. However, the contributions of brain and circulating A&bgr;1–40 to the vascular dysfunction have not been elucidated. Methods— We used transgenic mice overexpressing mutated forms of the amyloid precursor protein in which A&bgr;1–40 is elevated in blood and brain (Tg-2576) or only in brain (Tg-SwDI). Mice were equipped with a cranial window, and the increase in cerebral blood flow induced by neural activity (whisker stimulation), or by topical application of endothelium-dependent vasodilators, was assessed by laser-Doppler flowmetry. Results— The cerebrovascular dysfunction was observed also in Tg-SwDI mice, but despite ≈40% higher levels of brain A&bgr;1–40, the effect was less marked than in Tg-2576 mice. Intravascular administration of A&bgr;1–40 elevated plasma A&bgr;1–40 and enhanced the dysfunction in Tg-SwDI mice, but not in Tg-2576 mice. Conclusions— The results provide evidence that A&bgr;1–40 acts on distinct luminal and abluminal vascular targets, the deleterious cerebrovascular effects of which are additive. Furthermore, the findings highlight the importance of circulating A&bgr;1–40 in the cerebrovascular dysfunction and may provide insight into the cerebrovascular alterations in conditions in which elevations in plasma A&bgr;1–40 occur.