Mee-Sook Song

Memantine protects rat cortical cultured neurons against β-amyloid-induced toxicity by attenuating tau phosphorylation

It has been suggested that accumulation of beta‐amyloid (Aβ) peptide triggers neurodegeneration, at least in part, via glutamate‐mediated excitotoxicity in Alzheimer’s disease (AD) brain. This is supported by observations that toxicity induced by Aβ peptide in cultured neurons and in adult rat brain is known to be mediated by activation of glutamatergic N‐methyl‐d‐aspartate (NMDA) receptors. Additionally, recent clinical studies have shown that memantine, a noncompetitive NMDA receptor antagonist, can significantly improve cognitive functions in some AD patients. However, very little is currently known about the potential role of memantine against Aβ‐induced toxicity. In the present study, we have shown that Aβ1–42‐induced toxicity in rat primary cortical cultured neurons is accompanied by increased extracellular and decreased intracellular glutamate levels. We subsequently demonstrated that Aβ toxicity is induced by increased phosphorylation of tau protein and activation of tau kinases, i.e. glycogen synthase kinase‐3β and extracellular signal‐related kinase 1/2. Additionally, Aβ treatment induced cleavage of caspase‐3 and decreased phosphorylation of cyclic AMP response element binding protein, which are critical in determining survival of neurons. Memantine treatment significantly protected cultured neurons against Aβ‐induced toxicity by attenuating tau‐phosphorylation and its associated signaling mechanisms. However, this drug did not alter either conformation or internalization of Aβ1–42 and it was unable to attenuate Aβ‐induced potentiation of extracellular glutamate levels. These results, taken together, provide new insights into the possible neuroprotective action of memantine in AD pathology.

An update on amine oxidase inhibitors: Multifaceted drugs

Although not used as extensively as other antidepressants for the treatment of depression, the monoamine oxidase (MAO) inhibitors continue to hold a niche in psychiatry and to have a relatively broad spectrum with regard to treatment of psychiatric and neurological disorders. Experimental and clinical research on MAO inhibitors has been expanding in the past few years, primarily because of exciting findings indicating that these drugs have neuroprotective properties (often independently of their ability to inhibit MAO). The non-selective and irreversible MAO inhibitors tranylcypromine (TCP) and phenelzine (PLZ) have demonstrated neuroprotective properties in numerous studies targeting elements of apoptotic cascades and neurogenesis. l-Deprenyl and rasagiline, both selective MAO-B inhibitors, are used in the management of Parkinsons disease, but these drugs may be useful in the treatment of other neurodegenerative disorders given that they demonstrate neuroprotective/neurorescue properties in a wide variety of models in vitro and in vivo. Although the focus of studies on the involvement of MAO inhibitors in neuroprotection has been on MAO-B inhibitors, there is a growing body of evidence demonstrating that MAO-A inhibitors may also have neuroprotective properties. In addition to MAO inhibition, PLZ also inhibits primary amine oxidase (PrAO), an enzyme implicated in the etiology of Alzheimers disease, diabetes and cardiovascular disease. These multifaceted aspects of amine oxidase inhibitors and some of their metabolites are reviewed herein.

Role of calpain and caspase in β-amyloid-induced cell death in rat primary septal cultured neurons

The invariant characteristic features associated with Alzheimers disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta (Abeta) peptide, intracellular neurofibrillary tangles containing hyper-phosphorylated tau protein and the loss of basal forebrain cholinergic neurons. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that in vivo accumulation of Abeta(1-42) may initiate the process of neurodegeneration observed in AD brains. However, the cause of degeneration of the basal forebrain cholinergic neurons and their association to Abeta peptides or phosphorylated tau protein have not been clearly established. In the present study, using rat primary septal cultures, we have shown that Abeta(1-42), in a time (1-48 h) and concentration (0.01-20 microM)-dependent manner, induce toxicity in cultured neurons. Subsequently, we have demonstrated that Abeta toxicity is mediated via activation of cysteine proteases, i.e., calpain and caspase, and proteolytic breakdown of their downstream substrates tau, microtubule-associated protein-2 and alpha II-spectrin. Additionally, Abeta-treatment was found to induce phosphorylation of tau protein along with decreased levels of phospho-Akt and phospho-Ser(9)glycogen synthase kinase-3beta. Exposure to specific inhibitors of caspase or calpain can partially protect cultured neurons against Abeta-induced toxicity but their effects are not found to be additive. These results, taken together, suggest that Abeta peptide can induce toxicity in rat septal cultured neurons by activating multiple intracellular signaling molecules. Additionally, evidence that inhibitors of caspase and calpains can partially protect the cultured basal forebrain neurons raised the possibility that their inhibitors could be of therapeutic relevance in the treatment of AD pathology.

The antidepressant phenelzine protects neurons and astrocytes against formaldehyde-induced toxicity.

J. Neurochem. (2010) 114, 1405–1413.

Apoptosis is secondary to non-apoptotic axonal degeneration in neurons exposed to Aβ in distal axons

The goal of this study was to assess if neurons exposed to amyloid-beta peptide (Abeta) exclusively in distal axons, undergo apoptosis. This is relevant to the loss of cholinergic neurons in Alzheimers disease. Using a three-compartmented culture system for rat sympathetic neurons, we demonstrate that exposure of axons to Abeta1-42 activates an independent destruction program in axons, which leads to nuclear apoptosis. Abeta-induced axonal degeneration does not involve local caspase activation, but causes caspase activation in cell bodies. Accordingly, inhibition of caspase activation blocks Abeta-induced apoptosis but not axonal degeneration. In agreement with previous suggestions that disruption of nerve growth factor (NGF)-mediated signaling might contribute to the loss of cholinergic neurons, we found that provision of NGF to cell bodies protects sympathetic neurons from Abeta-induced apoptosis. However, our data indicate that Abeta-induced axonal degeneration follows a mechanism different than that activated by NGF withdrawal. Only Abeta-induced axonal degeneration is prevented by the calpain inhibitor calpastatin and is insensitive to the inhibitor of the ubiquitin-proteasome system MG132. Importantly, inhibition of Abeta-induced axonal degeneration by calpastatin prevents nuclear apoptosis.

Inhibition of β-amyloid1-42 internalization attenuates neuronal death by stabilizing the endosomal-lysosomal system in rat cortical cultured neurons

A number of recent studies have indicated that accumulation of β amyloid (Aβ) peptides within neurons is an early event which may trigger degeneration of neurons and subsequent development of Alzheimers disease (AD) pathology. However, very little is known about the internalization and/or subcellular sites involved in trafficking of Aβ peptides into the neurons that are vulnerable in AD pathology. To address this issue we evaluated internalization of fluoroscein conjugated Aβ1-42 (FAβ1-42) and subsequent alteration of endosomal-lysosomal (EL) markers such as cathepsin D, Rab5 and Rab7 in rat cortical cultured neurons. It is evident from our results that internalization of FAβ1-42, which occurred in a dose- and time-dependent manner, triggered degeneration of neurons along with increased levels and/or altered distribution of cathepsin D, Rab5 and Rab7. Our results further revealed that FAβ1-42 internalization was attenuated by phenylarsine oxide (a general inhibitor of endocytosis) and sucrose (an inhibitor of clathrin-mediated endocytosis) but not by antagonists of N-methyl-d-aspartate (NMDA) glutamate receptors. Additionally, inhibition of FAβ1-42 endocytosis not only protected neurons against toxicity but also reversed the altered levels/distributions of EL markers. These results, taken together, suggest that internalization of exogenous Aβ1-42, which is partly mediated via a clathrin-dependent process, can lead to degeneration of neurons, possibly by activating the EL system. Inhibition of FAβ endocytosis attenuated toxicity, thus suggesting a potential strategy for preventing loss of neurons in AD pathology.

Phenelzine: An Old Drug That May Hold Clues to The Development of New Neuroprotective Agents

5 OZET: Fenelzin: Eski bir ilac, yeni noroprotektif ajanlar›n gelifltirilmesine ipuclar› tutabilir Panik bozukluk ve sosyal anksiyete bozukluu gibi anksiye- te bozukluklar›n›n tedavisinde kullan›lan monoamin oksi- daz (MAO) inhibitoru bir antidepresan olan fenelzinin geci- ci onbeyin iskemisi olan bir hayvan modelinde noroprotek- tif etkileri olduu gosterilmifltir. Fenelzinin MAO inhibisyo- nu etkisi yan› s›ra farmakolojik ve terapotik profiline ekle- nebilir cok say›da etkisi vard›r. Bu etkiler GABA transamina- z›n inhibisyonuyla beyin GABA duzeylerini artt›rmas›, glu- tamat›n ifllevsel durumu uzerine etkileri, reaktif aldehitlerin tutulumu, primer amin oksidaz inhibisyonu ve beyin kay- nakl› norotrofik faktor (BDNF) uzerindeki etkilerin inhibe edilmesidir. 2-Feniletilidenehidrazin, fenelzinin onemli bir metabolit olup GABA beyin duzeylerini artt›rd›¤› gosteril- mifltir ve gecici onbeyin iskemisi modelinde reaktif aldehit- lerin tutulumu ve noroprotektif etkileri keflfedilmifltir. Fe- nelzin ve feniletilidenehidrazin bu etkileri nedeniyle ozel- likle norodejenerasyon iceren psikiyatrik ve norolojik bo- zukluklar›n tedavisi icin gelecekte ilac tasar›m› yonunden goz onunde tutulmal›d›r. ABSTRACT: Phenelzine: An old drug that may hold clues to the development of new neuroprotective agents The monoamine oxidase (MAO)-inhibiting antidepressant phenelzine (PLZ) is also used in the treatment of anxiety disorders such as panic disorder and social anxiety disorder and has been shown to have neuroprotective actions in an animal model of transient forebrain ischemia. Phenelzine has multiple actions in addition to inhibition of MAO that may contribute to its pharmacological and therapeutic profile. These actions include inhibition of GABA transaminase and elevation of brain levels of GABA, effects on functional availability of glutamate, sequestration of reactive aldehydes, inhibition of primary amine oxidase and effects on brain-derived neurotrophic factor (BDNF). 2- Phenylethylidenehydrazine (PEH) has been identified as a major metabolite of PLZ and has also been shown to elevate brain levels of GABA, to sequester reactive aldehydes and to exert neuroprotective effects in a transient forebrain ischemia model. The actions of PLZ and PEH should be considered when designing future drugs for the treatment of psychiatric and neurologic disorders, particularly those involving neurodegeneration.

Inhibition of p53 attenuates ischemic stress-induced activation of astrocytes.

In cerebral ischemia, studies of cell death have focused primarily on neurons, but recent work indicates that ischemia also causes damage to astrocytes. Activation of astrocytes is a typical brain response to stress stimuli and is evidenced by changes in cellular function and morphology, as well as upregulation of glial fibrillary acidic protein. The tumor-suppressor transcription factor p53 has recently been implicated as a mediator of ischemia-induced neuronal death, but very little is known about its role in the activation or the death of astrocytes. The present study investigated the role of p53 in astrocyte and neuronal toxicity using in-vitro and in-vivo ischemic stroke models. We showed that p53 is activated in ischemic brains and in oxygen–glucose deprivation (OGD)-induced cell death in neurons and astrocytes. Inhibition of p53 activity using either pifithrin-&agr; or small interference RNA interference reduced OGD-induced cell death and pifithrin-&agr; reversed OGD-induced impairment of glutamate uptake in astrocytes, suggesting that p53 might play a key role in mediating neurotoxicity and gliotoxicity in ischemic brain injury. This study shows that p53 is activated in astrocytes during ischemia and that inhibition of the activity of this molecule prevents not only OGD-induced neuronal and astrocytic death but also astrocyte activation and impaired glutamate uptake. These findings suggest that p53 may be a valuable therapeutic target in ischemic brain injury.

Expression of Alzheimer-Type Neurofibrillary Epitopes in Primary Rat Cortical Neurons Following Infection with Enterococcus faecalis

The neurofibrillary tau pathology and amyloid deposits seen in Alzheimer’s disease (AD) also have been seen in bacteria-infected brains. However, few studies have examined the role of these bacteria in the generation of tau pathology. One suggested link between infection and AD is edentulism, the complete loss of teeth. Edentulism can result from chronic periodontal disease due to infection by Enterococcus faecalis. The current study assessed the ability to generate early Alzheimer-like neurofibrillary epitopes in primary rat cortical neurons through bacterial infection by E. faecalis. Seven-day old cultured neurons were infected with E. faecalis for 24 and 48 h. An upward molecular weight shift in tau by Western blotting (WB) and increased appearance of tau reactivity in cell bodies and degenerating neurites was found in the 48 h infection group for the antibody CP13 (phospho-Serine 202). A substantial increase in reactivity of Alz-50 was seen at 24 and 48 h after infection. Furthermore, extensive microtubule-associated protein 2 (MAP2) reactivity also was seen at 24 and 48 h post-infection. Our preliminary findings suggest a potential link between E. faecalis infection and intracellular changes that may help facilitate early AD-like neurofibrillary pathology. Highlights Enterococcus faecalis used in the generation of AD neurofibrillary epitopes in rat. Infection increases Alz-50, phospho-Serine 202 tau, and MAP2 expression. Infection by Enterococcus may play a role in early Alzheimer neurofibrillary changes.

AN EXPLORATION OF THE GENDER DIFFERENCES IN EARLY BEHAVIOR AND PATHOLOGY RELATING TO HEAT SHOCK PROTEIN AND AMYLOID BETA PROTEIN EXPRESSION IN THE 5XFAD MOUSE MODEL OF ALZHEIMER’S DISEASE

P4-098 AN EXPLORATION OF THE GENDER DIFFERENCES IN EARLY BEHAVIOR AND PATHOLOGY RELATING TO HEAT SHOCK PROTEIN AND AMYLOID BETA PROTEIN EXPRESSION IN THE 5XFAD MOUSE MODEL OF ALZHEIMER’S DISEASE Tia Hall, Cameron Learman, Evan Bates, Leela Paladugu, MeeSook Song, Panchanan Maiti, Julien Rossignol, Gary Dunbar, Central Michigan University, Mount Pleasant, MI, USA; Field Neurosciences Institute Laboratory for Restorative Neurology, Saginaw,MI, USA; 3 Field Neurosciences Institute, Saginaw, MI, USA; 4 Central Michigan University College of Medicine, Mount Pleasant, MI, USA. Contact e-mail: hall2tc@cmich.edu