Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis.

Estradiol protects against brain injury, neurodegeneration, and cognitive decline. Our previous work demonstrates that physiological levels of estradiol protect against stroke injury and that this protection may be mediated through receptor-dependent alterations of gene expression. In this report, we tested the hypothesis that estrogen receptors play a pivotal role in mediating neuroprotective actions of estradiol and dissected the potential biological roles of each estrogen receptor (ER) subtype, ER␣ and ER␤, in the injured brain. To investigate and delineate these mechanisms, we used ER␣-knockout (ER␣KO) and ER␤-knockout (ER␤KO) mice in an animal model of stroke. We performed our studies by using a controlled endocrine paradigm, because endogenous levels of estradiol differ dramatically among ER␣KO, ER␤KO, and wild-type mice. We ovariectomized ER␣KO, ER␤KO, and the respective wild-type mice and implanted them with capsules filled with oil (vehicle) or a dose of 17␤-estradiol that produces physiological hormone levels in serum. One week later, mice underwent ischemia. Our results demonstrate that deletion of ER␣ completely abolishes the protective actions of estradiol in all regions of the brain; whereas the ability of estradiol to protect against brain injury is totally preserved in the absence of ER␤. Thus, our results clearly establish that the ER␣ subtype is a critical mechanistic link in mediating the protective effects of physiological levels of estradiol in brain injury. Our discovery that ER␣ mediates protection of the brain carries far-reaching implications for the selective targeting of ERs in the treatment and prevention of neural dysfunction associated with normal aging or brain injury. Menopause marks the end of female reproduction and is accompanied by a dramatic and permanent decrease in estrogen levels. Although the life span of women has increased significantly in the past century, the average age of menopause has remained constant. Consequently, women may now spend more than one-third of their lives in a chronic hypoestrogenic postmenopausal state. Because estradiol is an important trophic and protective factor in the adult brain (1, 2), hypoestrogenic postmenopausal women may be more vulnerable to brain injury and dysfunction caused by neurodegenerative conditions and cognitive decline. It is, therefore, crucial that we gain a complete understanding of the mechanisms underlying the neuroprotective actions of estradiol.A growing body of evidence has begun to reveal that estrogen replacement therapy may ameliorate neural dysfunctions resulting from Alzheimer's disease (3-5) and stroke (6, 7) through multiple and complex cellular and molecular mechanisms of action. The protective role of estrogen in brain function has been examined by using a variety of in vivo and in vitro models of brain injury that mimic neurotoxic environments found in Alzheimer's disease, stroke, and other neurodegenerative conditions (8-13). These studies demonstrate that physiological and pharmacological concentrations of...

Accumulation of fibrils composed of amyloid A in tissues resulting in displacement of normal structures and cellular dysfunction is the characteristic feature of systemic amyloidoses. Here we show that RAGE, a multiligand immunoglobulin superfamily cell surface molecule, is a receptor for the amyloidogenic form of serum amyloid A. Interactions between RAGE and amyloid A induced cellular perturbation. In a mouse model, amyloid A accumulation, evidence of cell stress and expression of RAGE were closely linked. Antagonizing RAGE suppressed cell stress and amyloid deposition in mouse spleens. These data indicate that RAGE is a potential target for inhibiting accumulation of amyloid A and for limiting cellular dysfunction induced by amyloid A.

BackgroundGlioblastoma (GBM) is the deadliest of brain tumors. Standard treatment for GBM is surgery, followed by combined radiation therapy and chemotherapy. Current therapy prolongs survival but does not offer a cure. We report on a novel immunotherapy against GBM, tested in an animal model of C57BL/6 mice injected intra-cranially with a lethal dose of murine GL261 glioma cells.MethodsTen week-old C57BL/6 mice were anesthetized before injection of 2 × 104 GL261 cells in the right cerebral hemisphere and after 3 days half of the mice were administered a single subcutaneous (s.c.) injection of irradiated semi-allogeneic vaccine, while mock-vaccinated mice received a s.c. injection of phosphate-buffered saline (PBS). Tumor engraftment was monitored through bioluminescence imaging (BLI). Length of animal survival was measured by Kaplan–Meier graphs and statistics. At time of sacrifice brain tissue was processed for estimation of tumor size and immunohistochemical studies.ResultsOverall survival of vaccinated mice was significantly longer compared to mock-vaccinated mice. Five to ten percent of vaccinated mice survived more than 90 days following the engraftment of GL261 cells in the brain and appeared to be free of disease by BLI. Tumor volume in the brain of vaccinated mice was on average five to ten-fold smaller compared to mock-vaccinated mice. In vaccinated mice, conspicuous microglia infiltrates were observed in tumor tissue sections and activated microglia appeared to form a fence along the perimeter of the tumor cells. The results of these animal studies persuaded the Office of Orphan Products Development of the Food and Drug Administration (FDA) to grant Orphan Drug Designation for treatment of GBM with irradiated, semi-allogeneic vaccines.ConclusionsOur preclinical observations suggest that semi-allogeneic vaccines could be tested clinically on subjects with GBM, as an adjuvant to standard treatment.

Estradiol enhances plasticity and survival of the injured brain. Our previous work demonstrates that physiological levels of estradiol protect against cerebral ischemia in the young and aging brain through actions involving estrogen receptors (ERs) and alterations in gene expression. The major goal of this study was to establish mechanisms of neuroprotective actions induced by low levels of estradiol. We first examined effects of estradiol on the time-dependent evolution of ischemic brain injury. Because estradiol is known to influence apoptosis, we hypothesized that it acts to decrease the delayed phase of cell death observed after middle cerebral artery occlusion (MCAO). Furthermore, because ERs are pivotal to neuroprotection, we examined the temporal expression profiles of both ER subtypes, ERalpha and ERbeta, after MCAO and delineated potential roles for each receptor in estradiol-mediated neuroprotection. We quantified cell death in brains at various times after MCAO and analyzed ER expression by RT-PCR, in situ hybridization, and immunohistochemistry. We found that during the first 24 h, the mechanisms of estradiol-induced neuroprotection after MCAO are limited to attenuation of delayed cell death and do not influence immediate cell death. Furthermore, we discovered that ERs exhibit distinctly divergent profiles of expression over the evolution of injury, with ERalpha induction occurring early and ERbeta modulation occurring later. Finally, we provide evidence for a new and functional role for ERalpha in estradiol-mediated protection of the injured brain. These findings indicate that physiological levels of estradiol protect against delayed cell death after stroke-like injury through mechanisms requiring ERalpha.

Transcranial laser therapy (TLT) was tested for efficacy in a mouse model of Alzheimer's disease (AD) using a near-infrared energy laser system. TLT is thought to stimulate ATP production, increase mitochondrial activity, and help maintain neuronal function. Studies were performed to determine the effect of TLT in an amyloid-β protein precursor (AβPP) transgenic mouse model. TLT was administered 3 times/week at various doses, starting at 3 months of age, and was compared to a control group (no laser treatment). Treatment was continued for a total of six months. Animals were examined for amyloid load, inflammatory markers, brain amyloid-β (Aβ) levels, plasma Aβ levels, cerebrospinal fluid Aβ levels, soluble AβPP (sAβPP) levels, and behavioral changes. The numbers of Aβ plaques were significantly reduced in the brain with administration of TLT in a dose-dependent fashion. Administration of TLT was associated with a dose-dependent reduction in amyloid load. All TLT doses mitigated the behavioral effects seen with advanced amyloid deposition and reduce the expression of inflammatory markers in the AβPP transgenic mice. All TLT doses produced an increase in sAβPPα and a decrease in CTFβ levels consistent with inhibition of the β-secretase activity. In addition, TLT showed an increase in ATP levels, mitochondrial function, and c-fos suggesting an overall improvement in neurological function. These studies suggest that TLT is a potential candidate for treatment of AD.

A cardinal feature of brain tissue injury in stroke is mitochondrial dysfunction leading to cell death, yet remarkably little is known about the mechanisms underlying mitochondrial injury in cerebral ischemia/reperfusion (IR). Ceramide, a naturally occurring membrane sphingolipid, functions as an important second messenger in apoptosis signaling and is generated by de novo synthesis, sphingomyelin hydrolysis, or recycling of sphingolipids. In this study, cerebral IR-induced ceramide elevation resulted from ceramide biosynthesis rather than from hydrolysis of sphingomyelin. Investigation of intracellular sites of ceramide accumulation revealed the elevation of ceramide in mitochondria because of activation of mitochondrial ceramide synthase via post-translational mechanisms. Furthermore, ceramide accumulation appears to cause mitochondrial respiratory chain damage that could be mimicked by exogenously added natural ceramide to mitochondria. The effect of ceramide on mitochondria was somewhat specific; dihydroceramide, a structure closely related to ceramide, did not inflict damage. Stimulation of ceramide biosynthesis seems to be under control of JNK3 signaling: IR-induced ceramide generation and respiratory chain damage was abolished in mitochondria of JNK3-deficient mice, which exhibited reduced infarct volume after IR. These studies suggest that the hallmark of mitochondrial injury in cerebral IR, respiratory chain dysfunction, is caused by the accumulation of ceramide via stimulation of ceramide synthase activity in mitochondria, and that JNK3 has a pivotal role in regulation of ceramide biosynthesis in cerebral IR.Mitochondria are known to be involved in both necrotic and apoptotic cell death, both of which have been identified in the ischemia/reperfusion (IR) 2 -injured brain (1-3). Also, restricted respiratory chain function has been found to develop in various models of cerebral IR (4 -8); specifically, mitochondrial respiration supported either by glutamate or succinate was decreased up to 40%, but ascorbate-supported respiration was not significantly altered (9, 10). Mitochondrial changes appear to be one essential step in tissue damage in cerebral IR. Treatments that slow tissue impairment were associated with better recovery of mitochondrial function (11,12).A number of cell death regulatory molecules have been implicated in neuronal injury in IR, including c-Jun N-terminal kinase (JNK) (13). Once activated, JNK can phosphorylate serine residues of several transcription factors, such as c-Jun, or non-nuclear proteins, including pro-apoptotic Bcl-2 family proteins (14 -17). Among three JNK isoforms encoded by different genes, JNK1 and JNK2 are present in most tissues, whereas JNK3 is selectively expressed in the nervous system and in the heart. A critical role of JNK3 in cerebral ischemia has been implicated, targeted deletion of JNK3-protected mice from brain IR injury (18).Numerous reports support a role for sphingolipids as second messengers in intracellular signaling pathways (19,20), especially ce...

It is well established that the extracellular deposition of amyloid ␤ (A␤) peptide plays a central role in the development of Alzheimer's disease (AD). Therefore, either preventing the accumulation of A␤ peptide in the brain or accelerating its clearance may slow the rate of AD onset. Neprilysin (NEP) is the dominant A␤ peptide-degrading enzyme in the brain; NEP becomes inactivated and down-regulated during both the early stages of AD and aging. In this study, we investigated the effect of human (h)NEP gene transfer to the brain in a mouse model of AD before the development of amyloid plaques, and assessed how this treatment modality affected the accumulation of A␤ peptide and associated pathogenetic changes (eg, inflammation, oxidative stress, and memory impairment). Overexpression of hNEP for 4 months in young APP/⌬PS1 double-transgenic mice resulted in reduction in A␤ peptide levels, attenuation of amyloid load, oxidative stress, and inflammation, and improved spatial orientation. Moreover, the overall reduction in amyloidosis and associated pathogenetic changes in the brain resulted in decreased memory impairment by ϳ50%. These data suggest that restoring NEP levels in the brain at the early stages of AD is an effective strategy to prevent or attenuate disease progression. Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a loss of neurons in discrete regions of the brain, particularly in the cortex and hippocampus.1,2 The neuronal loss is accompanied by extracellular deposition of A␤ peptide in a form of senile plaques and intracellular accumulation of neurofibrillary tangles made of a hyperphosphorylated form of the microtubule-associated protein tau.3 Clinically, AD is characterized by a gradual decline in cognition, and changes in behavior and personality including difficulty in reasoning, disorientation, and language problems. The exact cause of AD is not yet clear, but it is widely assumed that accumulation and aggregation of A␤ peptide is the initial trigger for a complex, multistep cascade that includes gliosis, inflammatory changes, oxidative stress, neuritic/ synaptic changes, tangle formation (microtubule changes), and neurotransmitter loss, leading to dementia. 4 Therefore, lowering the A␤ peptide levels in the brain would stop or delay the onset of AD. NEP as one of the A␤ peptide-degrading enzymes, has been reported to play a key role in regulating the level of A␤ peptide in the brain. 5,6 NEP [neprilysin, previously called CD10 or common acute lymphoblastic leukemia antigen (CALLA)] is a type II membrane metalloendopeptidase composed of ϳ750 residues (ϳ110 kDa) with an active site containing a zinc-binding motif (HEXXH) at the extracellular carboxyl terminal domain.7-10 NEP is capable of degrading the monomeric and (possibly) the oligomeric forms of A␤ peptide. 11,12 In recent years several reports have indicated that the soluble (eg, oligomeric) forms of A␤ peptide play a significant role in memory impairment and AD, [13][14][15] however, it is noteworthy t...