Homocysteine potentiates amyloid ß -induced death receptor 4- and 5-mediated cerebral endothelial cell apoptosis, blood brain barrier dysfunction and angiogenic impairment.

Cerebrovascular dysfunction has been implicated as a major contributor to Alzheimer's Disease (AD) pathology, with cerebral endothelial cell (cEC) stress promoting ischemia, cerebral-blood flow impairments and blood-brain barrier (BBB) permeability. Recent evidence suggests that cardiovascular (CV)/cerebrovascular risk factors, including hyperhomocysteinemia (Hhcy), exacerbate AD pathology and risk. Yet, the underlying molecular mechanisms for this interaction remain unclear. Our lab has demonstrated that amyloid beta 40 (Aß40) species, and particularly Aß40-E22Q (AßQ22; vasculotropic Dutch mutant), promote death receptor 4 and 5 (DR4/DR5)-mediated apoptosis in human cECs, barrier permeability, and angiogenic impairment. Previous studies show that Hhcy also induces EC dysfunction, but it remains unknown whether Aß and homocysteine function through common molecular mechanisms. We tested the hypotheses that Hhcy exacerbates Aß-induced cEC DR4/5-mediated apoptosis, barrier dysfunction, and angiogenesis defects. This study was the first to demonstrate that Hhcy specifically potentiates AßQ22-mediated activation of the DR4/5-mediated extrinsic apoptotic pathway in cECs, including DR4/5 expression, caspase 8/9/3 activation, cytochrome-c release and DNA fragmentation. Additionally, we revealed that Hhcy intensifies the deregulation of the same cEC junction proteins mediated by Aß, precipitating BBB permeability. Furthermore, Hhcy and AßQ22, impairing VEGF-A/VEGFR2 signaling and VEGFR2 endosomal trafficking, additively decrease cEC angiogenic capabilities. Overall, these results show that the presence of the CV risk factor Hhcy exacerbates Aß-induced cEC apoptosis, barrier dysfunction, and angiogenic impairment. This study reveals specific mechanisms through which amyloidosis and Hhcy jointly operate to produce brain EC dysfunction and death, highlighting new potential molecular targets against vascular pathology in comorbid AD/CAA and Hhcy conditions.

Amyloid ß induces cardiac dysfunction and neuro-signaling impairment in the heart of an Alzheimer's disease model.

Aims: Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by cerebral amyloid ß (Aß) deposition and tau pathology. The AD-mediated degeneration of the brain neuro-signaling pathways, together with a potential peripheral amyloid accumulation, may also result in the derangement of the peripheral nervous system, culminating in detrimental effects on other organs, including the heart. However, whether and how AD pathology modulates cardiac function, neurotrophins, innervation, and amyloidosis is still unknown. Here, we report for the first time that cardiac remodeling, amyloid deposition, and neuro-signaling dysregulation occur in the heart of Tg2576 mice, a widely used model of AD and cerebral amyloidosis. Methods ad Results: Echocardiographic analysis showed significant deterioration of left ventricle function, evidenced by a decline of both ejection fraction and fraction shortening percentage in 12-month-old Tg2576 mice compared to age-matched WT littermates. Tg2576 mice hearts exhibited an accumulation of amyloid aggregates, including Aß, an increase in interstitial fibrosis and severe cardiac nervous system dysfunction. The transgenic mice also showed a significant decrease in cardiac nerve fiber density, including both adrenergic and regenerating nerve endings. This myocardial denervation was accompanied by a robust reduction in NGF and BDNF protein expression as well as GAP-43 expression (regenerating fibers) in both the brain and heart of Tg2576 mice. Accordingly, cardiomyocytes and neuronal cells challenged with Aß oligomers showed significant downregulation of BDNF and GAP-43, indicating a causal effect of Aß on the loss of cardiac neurotrophic function. Conclusions: Overall, this study uncovers possible harmful effects of AD on the heart, revealing cardiac degeneration induced by Aß through fibrosis and neuro-signaling pathway deregulation for the first time in Tg2576 mice. Our data suggest that AD pathology can cause deleterious effects on the heart, and the peripheral neurotrophic pathway may represent a potential therapeutic target to limit these effects.

Endothelial Mitochondrial Dysfunction in Cerebral Amyloid Angiopathy and Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent form of dementia. Cerebrovascular dysfunction is one of the earliest events in the pathogenesis of AD, as well as in vascular and mixed dementias. Cerebral amyloid angiopathy (CAA), the deposition of amyloid around cerebral vessels, is observed in up to 90% of AD patients and in approximately 50% of elderly individuals over 80 years of age. CAA is a strong contributor to vascular dysfunction in AD. CAA-laden brain vessels are characterized by dysfunctional hemodynamics and leaky blood-brain barrier (BBB), contributing to clearance failure and further accumulation of amyloid-ß (Aß) in the cerebrovasculature and brain parenchyma. Mitochondrial dysfunction is increasingly recognized as an important early initiator of the pathogenesis of AD and CAA. The objective of this review is to discuss the effects of Aß on cerebral microvascular cell function, focusing on its impact on endothelial mitochondria. After introducing CAA and its etiology and genetic risk factors, we describe the pathological relationship between cerebrovascular amyloidosis and brain microvascular endothelial cell dysfunction, critically analyzing its roles in disease progression, hypoperfusion, and BBB integrity. Then, we focus on discussing the effect of Aß challenge on endothelial mitochondrial dysfunction pathways, and their contribution to the progression of neurovascular dysfunction in AD and dementia. Finally, we report potential pharmacological and non-pharmacological mitochondria-targeted therapeutic strategies which may help prevent or delay cerebrovascular failure.

Carbonic anhydrase inhibition selectively prevents amyloid ß neurovascular mitochondrial toxicity.

Mounting evidence suggests that mitochondrial dysfunction plays a causal role in the etiology and progression of Alzheimer's disease (AD). We recently showed that the carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) prevents amyloid ß (Aß)-mediated onset of apoptosis in the mouse brain. In this study, we used MTZ and, for the first time, the analog CAI acetazolamide (ATZ) in neuronal and cerebral vascular cells challenged with Aß, to clarify their protective effects and mitochondrial molecular mechanism of action. The CAIs selectively inhibited mitochondrial dysfunction pathways induced by Aß, without affecting metabolic function. ATZ was effective at concentrations 10 times lower than MTZ. Both MTZ and ATZ prevented mitochondrial membrane depolarization and H2 O2 generation, with no effects on intracellular pH or ATP production. Importantly, the drugs did not primarily affect calcium homeostasis. This work suggests a new role for carbonic anhydrases (CAs) in the Aß-induced mitochondrial toxicity associated with AD and cerebral amyloid angiopathy (CAA), and paves the way to AD clinical trials for CAIs, FDA-approved drugs with a well-known profile of brain delivery.

Mitochondrial dysfunction induced by a post-translationally modified amyloid linked to a familial mutation in an alternative model of neurodegeneration.

Familial British dementia (FBD) is an early-onset non-amyloid-ß (Aß) cerebral amyloidosis that presents with severe cognitive decline and strikingly similar neuropathological features to those present in Alzheimer's disease (AD). FBD is associated with a T to A single nucleotide transition in the stop codon of a gene encoding BRI2, leading to the production of an elongated precursor protein. Furin-like proteolytic processing at its C-terminus releases a longer-than-normal 34 amino acid peptide, ABri, exhibiting amyloidogenic properties not seen in its 23 amino acid physiologic counterpart Bri1-23. Deposited ABri exhibits abundant post-translational pyroglutamate (pE) formation at the N-terminus, a feature seen in truncated forms of Aß found in AD deposits, and co-exists with neurofibrillary tangles almost identical to those found in AD. We tested the impact of the FBD mutation alone and in conjunction with the pE post-translational modification on the structural properties and associated neurotoxicity of the ABri peptide. The presence of pE conferred to the ABri molecule enhanced hydrophobicity and accelerated aggregation/fibrillization properties. ABri pE was capable of triggering oxidative stress, loss of mitochondrial membrane potential and activation of caspase-mediated apoptotic mechanisms in neuronal cells, whereas homologous peptides lacking the elongated C-terminus and/or the N-terminal pE were unable to induce similar detrimental cellular pathways. The data indicate that the presence of N-terminal pE is not in itself sufficient to induce pathogenic changes in the physiologic Bri1-23 peptides but that its combination with the ABri mutation is critical for the molecular pathogenesis of FBD.

Amyloidosis associated with cerebral amyloid angiopathy: cell signaling pathways elicited in cerebral endothelial cells.

Substantial genetic, biochemical, and in vivo data indicate that progressive accumulation of amyloid-ß (Aß) plays a central role in the pathogenesis of Alzheimer's disease (AD). Historically centered in the importance of parenchymal plaques, the role of cerebral amyloid angiopathy (CAA)--a frequently neglected amyloid deposit present in >80% of AD cases--for the mechanism of disease pathogenesis is now starting to emerge. CAA consistently associates with microvascular modifications, ischemic lesions, micro- and macro-hemorrhages, and dementia, progressively affecting cerebral blood flow, altering blood-brain barrier permeability, interfering with brain clearance mechanisms and triggering a cascade of deleterious pro-inflammatory and metabolic events that compromise the integrity of the neurovascular unit. New evidence highlights the contribution of pre-fibrillar Aß in the induction of cerebral endothelial cell dysfunction. The recently discovered interaction of oligomeric Aß species with TRAIL DR4 and DR5 cell surface death receptors mediates the engagement of mitochondrial pathways and sequential activation of multiple caspases, eliciting a cascade of cell death mechanisms while unveiling an opportunity for exploring mechanistic-based therapeutic interventions to preserve the integrity of the neurovascular unit.

Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-beta E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells.

Patients carrying mutations within the amyloid-beta (Abeta) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Abeta synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AbetaE22Q and AbetaL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Abeta peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Abeta-(1-16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Abeta degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Abeta peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AbetaE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Abeta species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype.

Dutch and Arctic mutant peptides of beta amyloid(1-40) differentially affect the FGF-2 pathway in brain endothelium.

Single point mutations of the amyloid precursor protein generate Abeta variants bearing amino acid substitutions at positions 21-23. These mutants are associated with distinct hereditary phenotypes of cerebral amyloid angiopathy, manifesting varying degrees of tropism for brain vessels, and impaired microvessel remodeling and angiogenesis. We examined the differential effects of E22Q (Dutch), and E22G (Arctic) variants in comparison to WT Abeta on brain endothelial cell proliferation, angiogenic phenotype expression triggered by fibroblast growth factor (FGF-2), pseudo-capillary sprouting, and induction of apoptosis. E22Q exhibited a potent anti-angiogenic profile in contrast to E22G, which had a much weaker effect. Investigations on the FGF-2 signaling pathway revealed the greatest differences among the peptides: E22Q and WT peptides suppressed FGF-2 expression while E22G had barely any effect. Phosphorylation of the FGF-2 receptor, FGFR-1, and the survival signal Akt were abolished by E22Q and WT peptides, but not by E22G. The biological dissimilar effect of the mutant and WT peptides on cerebral EC cannot be assigned to a particular Abeta structure, suggesting that the toxic effect of the Abeta assemblies goes beyond mere multimerization.

Neither energy collapse nor transcription underlie in vitro neurotoxicity of poly(ADP-ribose) polymerase hyper-activation.

Poly(ADP-ribose)polymerase-1 (PARP-1) overactivation is a key event in neurodegeneration but the underlying molecular mechanisms wait to be unequivocally identified. Energy failure, transcriptional derangement and deadly nucleus-mitochondria cross-talk have been proposed as mechanisms responsible for PARP-1 neurotoxicity. In this study, we sought to determine how these mechanisms contributes to PARP-1-dependent neuronal death. We report that the PARP-1 activating agent methyl-nitrosoguanidine (MNNG) caused poly(ADP-ribosyl)ation-dependent death of pure mouse cortical neurons in culture. Upon PARP-1 hyperactivation, NAD and ATP storages only partially decreased, neurons rapidly acquired apoptotic morphology, apoptosis inducing factor and cytochrome c were released from mitochondria and caspase activation occurred. No evidence for p53 activation was found, lactate dehydrogenase release occurred only 18h later, and JNK kinase was constitutively activated and not affected by PARP-1 activation. The PARP-1 inhibitors 6-(5)H-phenanthridinone and N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide (PJ-34) prevented nucleotide depletion and cell death, whereas the transcription inhibitor actinomycin D did not affect PARP-1-dependent neurotoxicity. Together, our findings provide the first evidence that neither energy collapse nor transcriptional changes are involved in PARP-1-dependent apoptotic neuronal death, and support the existence of a poly(ADP-ribose)-mediated death signaling targeting mitochondria.

Poly(ADP-ribosyl)ation regulates heat shock factor-1 activity and the heat shock response in murine fibroblasts.

Poly(ADP-ribose) polymerase-1 (PARP-1)-dependent poly(ADP-ribose) formation is emerging as a key regulator of transcriptional regulation, even though the targets and underlying molecular mechanisms have not yet been clearly identified. In this study, we gathered information on the role of PARP-1 activity in the heat shock response of mouse fibroblasts. We show that DNA binding of heat shock factor (HSF)-1 was impaired by PARP-1 activity in cellular extracts, and was higher in PARP-1(-/-) than in PARP-1+/+ cells. No evidence for HSF-1 poly(ADP-ribosyl)ation or PARP-1 interaction was found, but a poly(ADP-ribose) binding motif was identified in the transcription factor amino acid sequence. Consistent with data on HSF-1, the expression of heat-shock protein (HSP)-70 and HSP-27 was facilitated in cells lacking PARP-1. Thermosensitivity, however, was higher in PARP-1(-/-) than in PARP-1+/+ cells. Accordingly, we report that heat-shocked PARP-1 null fibroblasts showed an increased activation of proapoptotic JNK and decreased transcriptional efficiency of prosurvival NF-kappaB compared with wild-type counterparts. The data indicate that poly(ADP-ribosyl)ation finely regulates HSF-1 activity, and emphasize the complex role of PARP-1 in the heat-shock response of mammalian cells.

Poly(ADP-ribose) accumulation and enhancement of postischemic brain damage in 110-kDa poly(ADP-ribose) glycohydrolase null mice.

Poly(ADP-ribose) (PAR) is a polymer synthesized by poly(ADP-ribose) polymerases (PARPs) and metabolized into free adenosine diphosphate (ADP)-ribose units by poly(ADP-ribose) glycohydrolase (PARG). Perturbations in PAR synthesis have been shown to play a key role in brain disorders including postischemic brain damage. A single parg gene but two PARG isoforms (110 and 60 kDa) have been detected in mouse cells. Complete suppression of parg gene causes early embryonic lethality, whereas mice selectively lacking the 110 kDa PARG isoform (PARG(110)(-/-)) develop normally. We used PARG(110)(-/-) mice to evaluate the importance of PAR catabolism to postischemic brain damage. Poly(ADP-ribose) contents were higher in the brain tissue of PARG(110)(-/-) than PARG(110)(+/+) mice, both under basal conditions and after PARP activation. Distal middle cerebral artery occlusion caused higher increase of brain PAR levels and larger infarct volumes in PARG(110)(-/-) mice than in wild-type counterparts. Of note, the brain of PARG(110)(-/-) mice showed reduced heat-shock protein (HSP)-70 and increased cyclooxygenase-2 expression under both control and ischemic conditions. No differences were detected in brain expression/activation of procaspase-3, PARP-1, Akt, HSP-25 and interleukin-1beta. Our findings show that PAR accumulation worsens ischemic brain injury, and highlight the therapeutic potential of strategies capable of maintaining PAR homeostasis.

Inhibition of poly(ADP-ribose) glycohydrolase by gallotannin selectively up-regulates expression of proinflammatory genes.

Poly(ADP-ribose)-polymerase-1 (PARP-1) and poly(ADP-ribose) (PAR) are emerging key regulators of chromatin superstructure and transcriptional activation. Accordingly, both genetic inactivation of PARP-1 and pharmacological inhibition of PAR formation impair the expression of several genes, including those of the inflammatory response. In this study, we asked whether poly(ADP-ribose) glycohydrolase (PARG), the sole depoly(ADP-ribosyl)ating enzyme identified so far, also regulates gene expression. We report the novel finding that inhibition of PARG by gallotannin triggered nuclear accumulation of PAR and concomitant PAR-dependent expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), but not of interleukin-1beta and tumor necrosis factor-alpha, in cultured RAW 264.7 macrophages. Remarkably, silencing of PARG by means of small interfering RNA selectively impaired gallotannin-induced expression of iNOS and COX-2. Consistent with a PAR-dependent transcriptional activation, increases of iNOS and COX-2 transcripts were not caused by activation of transcription factors such as nuclear factor-kappaB, activator protein-1, signal transducer and activator of transcription-1 or interferon regulatory factor-1, nor by mRNA stabilization. Overall, our data provide the first evidence that pharmacological inhibition of PARG leads to PAR-dependent alteration of gene expression profiles in macrophages.