Surita Banwait

Evidence for stroke-induced neurogenesis in the human brain

Experimental stroke in rodents stimulates neurogenesis and migration of newborn neurons from their sites of origin into ischemic brain regions. We report that in patients with stroke, cells that express markers associated with newborn neurons are present in the ischemic penumbra surrounding cerebral cortical infarcts, where these cells are preferentially localized in the vicinity of blood vessels. These findings suggest that stroke-induced compensatory neurogenesis may occur in the human brain, where it could contribute to postischemic recovery and represent a target for stroke therapy.

Vascular Endothelial Growth Factor Overexpression Delays Neurodegeneration and Prolongs Survival in Amyotrophic Lateral Sclerosis Mice

We sought genetic evidence for the involvement of neuronal vascular endothelial growth factor (VEGF) in amyotrophic lateral sclerosis (ALS). Mice expressing human ALS mutant superoxide dismutase-1 (SOD1) were crossed with mice that overexpress VEGF in neurons (VEGF+/+). We report that SOD1G93A/VEGF+/+ double-transgenic mice show delayed motor neuron loss, delayed motor impairment, and prolonged survival compared with SOD1G93A single transgenics. These findings indicate that neuronal VEGF protects against motor neuron degeneration, and may have therapeutic implications for ALS.

VEGF-overexpressing transgenic mice show enhanced post-ischemic neurogenesis and neuromigration

New neurons are generated continuously in the subventricular zone and dentate gyrus of the adult brain. Neuropathologic processes, including cerebral ischemia, can enhance neurogenesis, as can growth factors and other physiologic stimuli. Vascular endothelial growth factor (VEGF) is an angiogenic and neuroprotective growth factor that can promote neurogenesis, but it is unknown whether VEGF can enhance migration of newborn neurons toward sites of ischemic injury, where they might be able to replace neurons that undergo ischemic death. In the present study we produced permanent focal cerebral ischemia in transgenic (Tg) mice that overexpress VEGF. Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (Brdu) labeling and immunostaining for cell type‐specific markers. In VEGF‐Tg mice, brains examined 7–28 days after cerebral ischemia showed markedly increased subventricular zone (SVZ) neurogenesis, chains of neuroblasts extending from the SVZ to the peri‐infarct cortex, and an increase in the number of newly generated cortical neurons at 14–28 days after ischemia. In concert with these effects, VEGF overexpression reduced infarct volume and improved postischemic motor function. These findings provide evidence that VEGF increases SVZ neurogenesis and neuromigration, consistent with a possible role in repair. Our data suggest that in addition to its neuroprotective effects, which are associated with improved outcome in the acute phase after cerebral ischemia, VEGF enhances postischemic neurogenesis, which could provide a therapeutic target for more chronic brain repair.

Neuroglobin attenuates β-amyloid neurotoxicity in vitro and transgenic Alzheimer phenotype in vivo

Neuroglobin (Ngb), a vertebrate globin expressed primarily in neurons, is induced by and protects against neuronal hypoxia and cerebral ischemia. To investigate the spectrum and mechanism of Ngbs neuroprotective action, we studied the effect of transgenic overexpression of Ngb on NMDA and β-amyloid (Aβ) toxicity in murine cortical neuron cultures in vitro and on the phenotype of Alzheimers disease (AD) transgenic (APPSw,Ind) mice. Compared with cortical neuron cultures from wild-type mice, cultures from Ngb-overexpressing transgenic (Ngb-Tg mice) were resistant to the toxic effects of NMDA and Aβ(25–35), as measured by polarization of cell membrane lipid rafts, mitochondrial aggregation, lactate dehydrogenase release, and nuclear fragmentation. In addition, compared with APPSw,Ind mice, double-transgenic (Ngb-Tg × APPSw,Ind) mice showed reductions in thioflavin-S-stained extracellular Aβ deposits, decreased levels of Aβ(1–40) and Aβ(1–42), and improved behavioral performance in a Y-maze test of spontaneous alternations. These findings suggest that the spectrum of Ngbs neuroprotective action extends beyond hypoxic–ischemic insults. Ngb may protect neurons from NMDA and Aβ toxicity by inhibiting the formation of a death-signaling membrane complex, and interventions that increase Ngb expression could have therapeutic application in AD and other neurodegenerative disorders.

Regulation of hypoxic neuronal death signaling by neuroglobin

The signal transduction pathways in volved in neuronal death are not well understood. Neuroglobin (Ngb), a recently discovered vertebrate globin expressed predominantly in the brain, shows increased expression in neurons in response to oxygen deprivation and protects neurons from ischemic and hypoxic death. The mechanism of this neuroprotection is unclear. We examined the surface distribution of raft membrane microdomains in cortical neuron cultures during hypoxia using the raft marker cholera toxin B (CTx‐B) subunit. Mechanistically, we demonstrate that hypoxia induces rapid polarization of somal mem branes and aggregation of microdomains with the sub jacent mitochondrial network. This signaling complex is formed well before neurons commit to die, consis tent with an early role in death signal transduction. Neurons from Ngb‐overexpressing transgenic (Ngb‐Tg) mice do not undergo microdomain polarization or mitochondrial aggregation in response to, and are resistant to death from hypoxia. We link the protective actions of Ngb to inhibition of Pak1 kinase activity and Racl‐GDP‐dissociation inhibitor disassociation, and in hibition of actin assembly and death‐signaling module polarization.— Khan, A. A., Mao, X. O., Banwait, S., DerMardirossian, C. M., Bokoch, G. M., Jin, K., Greenberg, D. A. Regulation of hypoxic neuronal death signaling by neuroglobin. FASEB J. 22, 1737–1747 (2008)

A pilot proteomic study of amyloid precursor interactors in Alzheimer's disease

Several approaches have been used in an effort to identify proteins that interact with β‐amyloid precursor protein (APP). However, few studies have addressed the identification of proteins associated with APP in brain tissue from patients with Alzheimers disease. We report the results of a pilot proteomic study performed on complexes immunoprecipitated with APP in brain samples of patients with Alzheimers disease and normal control subjects. The 21 proteins identified could be grouped into five functional classes: molecular chaperones, cytoskeletal and structural proteins, proteins involved in trafficking, adaptors, and enzymes. Among the proteins identified, six had been reported previously as direct, indirect, or genetically inferred APP interactors. The other 15 proteins immunoprecipitated with APP were novel potential partners. We confirmed the APP interaction by Western blotting and coimmunolocalization in brain tissues, for 5 of the 21 interactors. In agreement with previous studies, our results are compatible with an involvement of APP in axonal transport and vesicular trafficking, and with a potential association of APP with cellular protein folding/protein degradation systems. Ann Neurol 2005;58:277–289

Signal transduction in Alzheimer disease : p21-activated kinase signaling requires C-terminal cleavage of APP at Asp664

The deficits in Alzheimer disease (AD) stem at least partly from neurotoxic β‐amyloid peptides generated from the amyloid precursor protein (APP). APP may also be cleaved intracellularly at Asp664 to yield a second neurotoxic peptide, C31. Previously, we showed that cleavage of APP at the C‐terminus is required for the impairments seen in APP transgenic mice, by comparing elements of the disease in animals modeling AD, with (platelet‐derived growth factor B‐chain promoter‐driven APP transgenic mice; PDAPP) versus without (PDAPP D664A) a functional Asp664 caspase cleavage site. However, the signaling mechanism(s) by which Asp664 contributes to these deficits remains to be elucidated. In this study, we identify a kinase protein, recently shown to bind APP at the C‐terminus and to contribute to AD, whose activity is modified in PDAPP mice, but normalized in PDAPP D664A mice. Specifically, we observed a significant increase in nuclear p21‐activated kinase (isoforms 1, 2, and or 3; PAK‐1/2/3) activation in hippocampus of 3 month old PDAPP mice compared with non‐transgenic littermates, an effect completely prevented in PDAPP D664A mice. In contrast, 13 month old PDAPP mice displayed a significant decrease in PAK‐1/2/3 activity, which was once again absent in PDAPP D664A mice. Similarly, in hippocampus of early and severe AD subjects, there was a progressive and subcellular‐specific reduction in active PAK‐1/2/3 compared with normal controls. Interestingly, total PAK‐1/2/3 protein was increased in early AD subjects, but declined in moderate AD and declined further, to significantly below that of control levels, in severe AD. These findings are compatible with previous suggestions that PAK may be involved in the pathophysiology of AD, and demonstrate that both early activation and late inactivation in the murine AD model require the cleavage of APP at Asp664.

Selective Vulnerability in Alzheimer's Disease: Amyloid Precursor Protein and p75NTR Interaction

Selective neuronal vulnerability in neurodegenerative diseases is poorly understood. In Alzheimers disease, the basal forebrain cholinergic neurons are selectively vulnerable, putatively because of their expression of the cell death mediator p75NTR (the common neurotrophin receptor), and its interaction with proapoptotic ligands pro–nerve growth factor and amyloid‐β peptide. However, the relation between amyloid precursor protein (APP) and p75NTR has not been described previously.

Influence of age on the response to fibroblast growth factor-2 treatment in a rat model of stroke.

Basic fibroblast growth factor (FGF-2) has been reported to protect against ischemic injury in the brains of young adult rodents. However, little is known about whether FGF-2 retains this capability in the aged ischemic brain. Since stroke in human is much more common in older people than among younger adults, to address this question is clinically important. In this study, aged (24-month-old) rats were treated with intracerebroventricular infusion of FGF-2 or vehicle for 3 days, beginning 48 h before (pre-ischemia), 24 h after (early post-ischemia), or 96 h after (late post-ischemia) 60 min of middle cerebral artery occlusion, and were killed 10 days after ischemia. Aged rats given FGF-2 pre-ischemia showed better symmetry of movement and forepaw outstretching, and reduced infarct volumes, compared to rats treated with vehicle, but no significant improvement was found in aged rats given FGF-2 after focal ischemia. In contrast, young adult (3-month-old) rats treated with FGF-2 for 3 days beginning 24 h post-ischemia showed significant neurobehavioral improvement and better histological outcome. In addition, we also found that newborn neurons in the rostral subventricular zone (SVZ) were increased in aged rats treated with FGF-2 prior to ischemia. However, unlike in young adult ischemic rats, only a few of newly generated cells migrated into the damaged region in aged brain after focal ischemia. These findings point to differences in the response of aged versus young adult rats to FGF-2 in cerebral ischemia, and suggest that such differences need to be considered in the development of neuroprotective agents for stroke.

C-Terminal Cleavage of the Amyloid-β Protein Precursor at Asp664: A Switch Associated with Alzheimer's Disease

In addition to the proteolytic cleavages that give rise to amyloid-beta (Abeta), the amyloid-beta protein precursor (AbetaPP) is cleaved at Asp664 intracytoplasmically. This cleavage releases a cytotoxic peptide, APP-C31, removes AbetaPP-interaction motifs required for signaling and internalization, and is required for the generation of AD-like deficits in a mouse model of the disease. Although we and others had previously shown that Asp664 cleavage of AbetaPP is increased in AD brains, the distribution of the Asp664-cleaved forms of AbetaPP in non-diseased and AD brains at different ages had not been determined. Confirming previous reports, we found that Asp664-cleaved forms of AbetaPP were increased in neuronal cytoplasm and nuclei in early-stage AD brains but were absent in age-matched, non-diseased control brains and in late-stage AD brains. Remarkably, however, Asp664-cleaved AbetaPP was prominent in neuronal somata and in processes in entorhinal cortex and hippocampus of non-diseased human brains at ages <45 years. Our observations suggest that Asp664 cleavage of AbetaPP may be part of the normal proteolytic processing of AbetaPP in young (<45 years) human brain and that this cleavage is down-regulated with normal aging, but is aberrantly increased and altered in location in early AD.