Alyson Peel

Increased hippocampal neurogenesis in Alzheimer's disease

Neurogenesis, which persists in the adult mammalian brain, may provide a basis for neuronal replacement therapy in neurodegenerative diseases like Alzheimers disease (AD). Neurogenesis is increased in certain acute neurological disorders, such as ischemia and epilepsy, but the effect of more chronic neurodegenerations is uncertain, and some animal models of AD show impaired neurogenesis. To determine how neurogenesis is affected in the brains of patients with AD, we investigated the expression of immature neuronal marker proteins that signal the birth of new neurons in the hippocampus of AD patients. Compared to controls, Alzheimers brains showed increased expression of doublecortin, polysialylated nerve cell adhesion molecule, neurogenic differentiation factor and TUC-4. Expression of doublecortin and TUC-4 was associated with neurons in the neuroproliferative (subgranular) zone of the dentate gyrus, the physiological destination of these neurons (granule cell layer), and the CA1 region of Ammons horn, which is the principal site of hippocampal pathology in AD. These findings suggest that neurogenesis is increased in AD hippocampus, where it may give rise to cells that replace neurons lost in the disease, and that stimulating hippocampal neurogenesis might provide a new treatment strategy.

Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78.

Alterations in Ca2+ homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucose‐regulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stress‐induced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome c‐mediated caspase activation in a cell‐free system, and expression of GRP78 blocks both caspase activation and caspase‐mediated cell death. GRP78 forms a complex with caspase‐7 and ‐12 and prevents release of caspase‐12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase‐12 into the cytoplasm to set in motion the cytosolic component of the ER stress‐induced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stress‐induced cell death.

Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum.

Pathological processes, including cerebral ischemia, can enhance neurogenesis in the adult brain, but the fate of the newborn neurons that are produced and their role in brain repair are obscure. To determine if ischemia-induced neuronal proliferation is associated with migration of nascent neurons toward ischemic lesions, we mapped the migration of cells labeled by cell proliferation markers and antibodies against neuronal marker proteins, for up to 2 weeks after a 90-min episode of focal cerebral ischemia caused by occlusion of the middle cerebral artery. Doublecortin-immunoreactive cells in the rostral subventricular zone, but not the dentate gyrus, migrated into the ischemic penumbra of the adjacent striatum and, via the rostral migratory stream and lateral cortical stream, into the penumbra of ischemic cortex. These results indicate that after cerebral ischemia, new neurons are directed toward sites of brain injury, where they might be in a position to participate in brain repair and functional recovery.

Neuroglobin protects the brain from experimental stroke in vivo.

Neuroglobin (Ngb) is an O2-binding protein localized to cerebral neurons of vertebrates, including humans. Its physiological role is unknown but, like hemoglobin, myoglobin, and cytoglobin/histoglobin, it may transport O2, detoxify reactive oxygen species, or serve as a hypoxia sensor. We reported recently that hypoxia stimulates transcriptional activation of Ngb in cultured cortical neurons and that antisense inhibition of Ngb expression increases hypoxic neuronal injury, whereas overexpression of Ngb confers resistance to hypoxia. These findings are consistent with a role for Ngb in promoting neuronal survival after hypoxic insults in vitro. Here we report that in rats, intracerebroventricular administration of an Ngb antisense, but not sense, oligodeoxynucleotide increases infarct volume and worsens functional neurological outcome, whereas intracerebral administration of a Ngb-expressing adeno-associated virus vector reduces infarct size and improves functional outcome, after focal cerebral ischemia induced by occlusion of the middle cerebral artery. We conclude that Ngb acts as an endogenous neuroprotective factor in focal cerebral ischemia and may therefore represent a target for the development of new treatments for stroke.

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease

AbstractHuntingtons disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

Activation of the cell stress kinase PKR in Alzheimer's disease and human amyloid precursor protein transgenic mice.

Accumulation of amyloid beta peptides (Abeta) in the brain, which is a hallmark of Alzheimers disease (AD), is associated with progressive damage to neuronal processes resulting in extensive neuritic dystrophy. This process may contribute to cognitive decline, but it is not known how Abeta elicits neuritic injury. Our analysis of AD brains and related transgenic mouse models suggests an involvement of the interferon-induced serine-threonine protein kinase, PKR, which is best known for its activation upon binding to double-stranded RNA. PKR activation is a component of stress-activated pathways that mobilize somatic cell death programs, but its roles in neurological disease largely remain to be defined. An antibody specific to the activated form of PKR (phosphorylated at T451) was used to determine the pattern of PKR activation in postmortem brain tissues from humans or from transgenic mice that express high levels of familial AD-mutant human amyloid precursor protein (hAPP) and hAPP-derived Abeta in neurons. In contrast to nondemented controls, AD cases showed prominent granular phospho-PKR immunoreactivity in association with neuritic plaques and pyramidal neurons in the hippocampus and neocortex. The distribution of phospho-PKR matched the distributions of abnormally phosphorylated tau and active p38 MAP kinase in adjacent sections. Compared with nontransgenic controls, hAPP transgenic mice also showed strong increases in phospho-PKR in the brain, primarily in association with plaques and dystrophic neurites. These findings support a role for PKR activation in the pathogenesis of AD.

Proteomic and immunochemical characterization of a role for stathmin in adult neurogenesis

Stathmin is a developmentally regulated cytosolic protein expressed at high levels in the brain. Two‐dimensional differential in‐gel electrophoresis and mass spectroscopy of proteins expressed in immature and mature cultures from embryonic rat cerebral cor¬tex identified stathmin among several differentially expressed proteins, consistent with a possible role in neurogenesis. Stathmin immunohistochemistry in adult rodent brain revealed prominent expression in neuroproliferative zones and neuronal migration pathways, a pattern that resembles the expression of doublecortin, which is implicated in neuronal migration. Stathmin immunoreactivity was also associated with neurons un¬dergoing ectopic chain migration into the ischemic striatum and cerebral cortex following focal cerebral ischemia. Reducing the expression of stathmin or doublecortin with an antisense oligonucleotide inhibited the migration of new neurons from the subventricular zone to the olfactory bulb via the rostral migratory stream. These results suggest a role for stathmin in the migration of newborn neurons in the adult brain.—Jin, K., Mao, X. O., Cottrell, B., Schilling, B., Xie, L., Row, R. H., Sun, Y., Peel, A., Childs, J., Gendeh, G., Gibson, B. W. Greenberg, D. A. Proteomic and immunochemical characterization of a role for stathmin in adult neurogenesis. FASEB J. 18, 287–299 (2004)

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

Effect of aging on neuroglobin expression in rodent brain

Neuroglobin (Ngb), a recently discovered O2-binding heme protein related to hemoglobin and myoglobin, protects neurons from hypoxic-ischemic injury in vitro and in vivo. In immunostained mouse brain sections, we found widespread expression of Ngb protein in neurons, but not astrocytes, of several brain regions that are prominently involved in age-related neurodegenerative disorders. Western blots from young adult (3 month), middle-aged (12 month), and aged (24 month) rats showed an age-related decline in Ngb expression in cerebral neocortex, hippocampus, caudate-putamen, and cerebellum. Loss of this neuroprotective protein may have a role in increasing susceptibility to age-related neurological disorders.

Tau phosphorylation in Alzheimer's disease: Potential involvement of an APP-MAP kinase complex

The two predominant pathological concomitants of Alzheimer’s disease (AD) are senile plaques and neurofibrillary tangles. Although many biochemical studies have addressed the composition and formation of these AD hallmarks, very little is known about the interrelationship between the two. Here we present evidence that the tau phosphorylation characteristic of neurofibrillary tangles may be mediated by a physical association of MKK6 (mitogen-associated protein kinase kinase 6) with tau and subsequent phosphorylation of tau by the MKK6 substrate, p38 MAPK; and that APP (β-amyloid precursor protein) may be co-immunoprecipitated both with MKK6 and its upstream MAPKKK, ASK1. Taken together with recent data demonstrating APP dimerization by β-amyloid peptide (Aβ) (Lu et al., 2003), and the possible activation of ASK1 via APP dimerization (Hashimoto et al., 2003), these results suggest a model of AD in which Aβ peptide dimerizes APP directly, leading to the activation of ASK1, MKK6, and p38, with subsequent phosphorylation of tau at sites characteristic of AD.