Wenru Zhang

Amyloid-β oligomers impair fear conditioned memory in a calcineurin-dependent fashion in mice

Soluble oligomeric aggregates of the amyloid‐β (Aβ) peptide are believed to be the most neurotoxic Aβ species affecting the brain in Alzheimer disease (AD), a terminal neurodegenerative disorder involving severe cognitive decline underscored by initial synaptic dysfunction and later extensive neuronal death in the CNS. Recent evidence indicates that Aβ oligomers are recruited at the synapse, oppose expression of long‐term potentiation (LTP), perturb intracellular calcium balance, disrupt dendritic spines, and induce memory deficits. However, the molecular mechanisms behind these outcomes are only partially understood; achieving such insight is necessary for the comprehension of Aβ‐mediated neuronal dysfunction. We have investigated the role of the phosphatase calcineurin (CaN) in these pathological processes of AD. CaN is especially abundant in the CNS, where it is involved in synaptic activity, LTP, and memory function. Here, we describe how oligomeric Aβ treatment causes memory deficits and depresses LTP expression in a CaN‐dependent fashion. Mice given a single intracerebroventricular injection of Aβ oligomers exhibited increased CaN activity and decreased pCREB, a transcription factor involved in proper synaptic function, accompanied by decreased memory in a fear conditioning task. These effects were reversed by treatment with the CaN inhibitor FK506. We further found that expression of hippocampal LTP in acutely cultured rodent brain slices was opposed by Aβ oligomers and that this effect was also reversed by FK506. Collectively, these results indicate that CaN activation may play a central role in mediating synaptic and memory disruption induced by acute oligomeric Aβ treatment in mice.

α-Synuclein oligomers oppose long-term potentiation and impair memory through a calcineurin-dependent mechanism: relevance to human synucleopathic diseases.

J. Neurochem. (2012) 120, 440–452.

A peripheral neuroimmune link: glutamate agonists upregulate NMDA NR1 receptor mRNA and protein, vimentin, TNF-α, and RANTES in cultured human synoviocytes

Human primary and clonal synovial cells were incubated with glutamate receptor agonists to assess their modulating influence on glutamate receptors N-methyl-d-aspartate (NMDA) NR1 and NR2 and inflammatory cytokines to determine potential for paracrine or autocrine (neurocrine) upregulation of glutamate receptors, as has been shown for bone and chondrocytes. Clonal SW982 synoviocytes constitutively express vimentin, smooth muscle actin (SMA), and NMDA NR1 and NR2. Coincubation (6 h) with glutamate agonists NMDA (5 microM), and the NMDA NR1 glycine site activator (+/-)1-aminocyclopentane-cis-1,3-dicarboxylic acid (5 muM), significantly increases cellular mRNA and protein levels of glutamate receptors, as well as increasing vimentin, SMA, tumor necrosis factor-alpha, and RANTES (regulated on activation, normal T-cell expressed and secreted), assessed qualitatively and quantitatively with nucleotide amplification, image analysis of immunocytochemical staining, fluorescein-activated cell sorting, Western blotting, and immunoassays. Human primary synovial cells harvested from patients with arthritic conditions also constitutively expressed NMDA NR1 with increases after agonist treatment. Glutamate receptor agonist-induced increases were blocked by the noncompetitive glutamate antagonist MK-801 (8 microg/ml) and NR1 blocking antibody. Coincubation with glutamate agonists and phorbol 12-myristate 13-acetate, a protein kinase C activator, significantly enhanced mean levels of TNF-alpha and RANTES in SW982 cell supernatants compared with incubation with either agent alone. Increases were diminished with protein kinase inhibitor and NR1 blocking antibody. The functional activation of glutamate receptors on human synoviocytes establishes a neurogenic cell signaling link between neurotransmitter glutamate released from nerve terminals and target cells in the joint capsule. The influence of glutamate on subsequent release of cellular proinflammatory mediators in non-neural tissue for activation of downstream immune events supports a peripheral neuroimmune link in arthritis.

Tumour necrosis factor‐alpha‐ vs. growth factor deprivation‐promoted cell death: distinct converging pathways

Perturbations of neuronal physiological homeostasis are likely to underscore neuronal demise/impairments that are reportedly associated with aging of the central nervous system and age‐related neurodegenerative diseases such as Alzheimers disease (AD). A number of age‐ and/or disease‐associated neurotoxic events has been described. These include abnormally modified proteins such as beta amyloid and hyper‐phosphorylated Tau, cytokines such as tumour necrosis factor‐alpha (TNFα), high levels of free radicals conducive to oxidative stress, and impaired/decreased neuronal trophic support by neurotrophic factors. Overall, it could be argued that toxic events in the aged brain are either active, such as those due to a direct action of cytokines, or passive, such as those due to lack of growth factor support. It is therefore conceivable that cellular responses to such diverse toxic stimuli are different, suggesting that interventions should be targeted accordingly. In order to begin answering this question, we determined in PC12 cells the time course of activity, in response to TNFα (active) or growth factor withdrawal (passive), of protein kinase c‐zeta (PKCζ), nuclear factor kappa B (NFκB), caspases 3 and 8, and poly (ADP‐ribose) polymerase (PARP), key signal transduction elements associated with modulation of cell death/survival in PC12 cells. We found that the overall activity of PKCζ, NFκB and caspase 8 was significantly different depending on the apoptotic initiator. The pattern of caspase 3 and PARP activity, however, was not statistically different between serum‐free‐ and TNFα‐induced cell death conditions. This suggests that two distinct cell responses are elicited that converge at caspase 3, which then induces downstream events involved in the execution of a common apoptotic programme. These results contribute to the aim of differentially targeting neuronal death in the aged brain (characterized by neurotrophic factor impairments) or in the diseased brain (e.g. AD, characterized by elevated levels of pro‐inflammatory cytokines).

Free radical-dependent nuclear localization of Bcl-2 in the central nervous system of aged rats is not associated with Bcl-2-mediated protection from apoptosis.

We have previously reported that Bcl‐2 is up‐regulated in the CNS of aged F344 rats as a consequence of oxidative stress. In addition to increased levels of expression, we now report that there is a subcellular redistribution of Bcl‐2 in the CNS of aged F344 rats. Using western blotting, we found Bcl‐2 predominantly located in the cytosol of young rats. However, in aged rats Bcl‐2 was found primarily in the nucleus. This distribution, in the hippocampus and cerebellum, was reversed by treatment with the nitrone spin trap N‐tert‐butyl‐α‐phenylnitrone (PBN). Paradoxically, PBN treatment in young rats had the opposite effect, changing Bcl‐2 from predominantly cytosolic to nuclear. We also detected an increase in Bax in aged hippocampal samples (both nuclear and cytosolic), which was reversed by treatment with PBN. The distribution of Bcl‐2 and Bax in the cytosol of aged rats dramatically decreased the Bcl‐2/Bax ratio, a probable indicator of neuronal vulnerability, which was restored upon treatment with PBN. In order to assess the effect of nuclear association of Bcl‐2 we used PC12 cells stably transfected with a Bcl‐2 construct to which we added the nuclear localization sequence of the SV40 large T antigen to the N‐terminus which resulted in nuclear targeting of Bcl‐2. Measurement of cell death using lactate dehydrogenase assays showed that, contrary to wild‐type Bcl‐2, Bcl‐2 localized to the nucleus was not effective in protecting cells from treatment with 250 µm H2O2. These results suggest that nuclear localization of Bcl‐2 observed in the aged CNS may not reflect a protective mechanism against oxidative stress, a major component of age‐associated CNS impairments.

Signaling pathways mediating a selective induction of nitric oxide synthase II by tumor necrosis factor alpha in nerve growth factor-responsive cells

BackgroundInflammation and oxidative stress play a critical role in neurodegeneration associated with acute and chronic insults of the nervous system. Notably, affected neurons are often responsive to and dependent on trophic factors such as nerve growth factor (NGF). We previously showed in NGF-responsive PC12 cells that tumor necrosis factor alpha (TNFα) and NGF synergistically induce the expression of the free-radical producing enzyme inducible nitric oxide synthase (iNOS). We proposed that NGF-responsive neurons might be selectively exposed to iNOS-mediated oxidative damage as a consequence of elevated TNFα levels. With the aim of identifying possible therapeutic targets, in the present study we investigated the signaling pathways involved in NGF/TNFα-promoted iNOS induction.MethodsWestern blotting, RT-PCR, transcription factor-specific reporter gene systems, mutant cells lacking the low affinity p75NTR NGF receptor and transfections of TNFα/NGF chimeric receptors were used to investigate signalling events associated with NGF/TNFα-promoted iNOS induction in PC12 cells.ResultsOur results show that iNOS expression resulting from NGF/TNFα combined treatment can be elicited in PC12 cells. Mutant PC12 cells lacking p75NTR did not respond, suggesting that p75NTR is required to mediate iNOS expression. Furthermore, cells transfected with chimeric TNFα/NGF receptors demonstrated that the simultaneous presence of both p75NTR and TrkA signaling is necessary to synergize with TNFα to mediate iNOS expression. Lastly, our data show that NGF/TNFα-promoted iNOS induction requires activation of the transcription factor nuclear factor kappa B (NF-κB).ConclusionCollectively, our in vitro model suggests that cells bearing both the high and low affinity NGF receptors may display increased sensitivity to TNFα in terms of iNOS expression and therefore be selectively at risk during acute (e.g. neurotrauma) or chronic (e.g. neurodegenerative diseases) conditions where high levels of pro-inflammatory cytokines in the nervous system occur pathologically. Our results also suggest that modulation of NFκB-promoted transcription of selective genes could serve as a potential therapeutic target to prevent neuroinflammation-induced neuronal damage.

Alpha-synuclein oligomers induce calcineurin-dependent neurotoxicity and memory deficits

Background: Parkinson Disease (PD) is a terminal neurodegenerative disorders characterized by motor, autonomic and cognitive deficits and second only to Alzheimer Disease (AD) for population incidence. Like the pathological significance of aggregation of amyloid beta (Ab) in AD, the misfolding and aggregation into amyloid fibrils of the protein alpha synuclein (aSyn) is a pathologic signature of the PD brain. While recent evidece suggests that, rather than fibrils, pre-fibrillar small oligomeric aSyn aggregates are the most toxic species, the mechanism of action of aSyn oligomers remains elusive. Our previous studies, however, showed that calcineurin (CaN), a protein phosphatase involved in neuronal death and memory function, mediates neurotoxicity of Ab oligomers, which are structurally similar to aSyn oligomers. On this basis, here we investigated the role of CaN in the neurodysfunctional effects elicited by aSyn oligomers. Methods: The effect of monomeric, oligomeric and fibrillar aSyn on neuronal death, long term potentiation (LTP) and memory function were determined in in vitro, ex vivo and in vivo experimental models as a function of CaN activity and CaN-dependent signaling. Results: We found that oligomeric aSyn, but not monomeric or fibrillar aSyn, induced CaN activity and CaN-dependent cell death in human neuroblastoma cells, increased CaN activity and thus suppressed expression of LTP in brain slices and promoted CaN-dependent memory deficits after acute icv injection in mice. Furthermore, we found that transgenic mice overexpressing mutant aSyn had increased CaN activity in their brain as compared to wt control. Conclusions: These results suggest that activation of CaN may be a central mechanism mediating the neurodysfunctional effects of aSyn oligomers. These results further suggest that pre-fibrillar oligomeric assemblies of Ab and aSyn may share a common neuropathological mechanism of action. Supported by grants from the NIH/ NINDS (NS053986) and from the UTMB Mitchell Center for Neurodegenerative Diseases to GT.