Nikolaos K Robakis, PhD

Genetics and function of proteins involved in neurodegeneration (APP, presenilin, tau)

A general description of Alzheimer disease (AD) and relevant projects

Alzheimer disease (AD), the most common cause of dementia in the aged, is a neurodegenerative disorder caused by severe neuronal loss and synaptic abnormalities in the hippocampus and neocortical regions of the brain. Neuropathologically, the disease is defined by brain accumulation of amyloid plaques (APs) and neurofibrillary tangles (NFTs). AD is a serious humanitarian and financial problem as it afflicts a large number of individuals and is very expensive to care for its victims. Most AD cases are classified as sporadic (SAD) because they lack obvious genetic etiologies. A small percent of all cases however, segregates within families (FAD) suggesting genetic etiologies. Most FAD mutations map on three genes encoding the Amyloid Precursor Protein (APP), presenilin1 (PS1) and presenilin2 (PS2). Close to 200 FAD mutations map on the gene encoding PS1 while about 20 FAD mutations are found in APP and 10 in PS2. PSs are functional components of the γ-secretase proteolytic complexes that process many receptor proteins and APP. PS/γ-secretase processing of APP yields Aβ peptides that aggregate to form APs used to define AD. The mechanisms by which FAD mutants promote neuronal death and AD remain unclear. A common theory suggests that AD is caused by Aβ peptides and its derivatives, but inhibitors of Aβ production or anti-Aβ antibodies show no therapeutic value (for recent review see Robakis 2011). Other evidence indicates that FAD mutants affect cellular functions including inhibition of signal transduction and gene expression. Since SAD and FAD have similar clinical and neuropathological phenotypes, understanding how genetic mutations promote neurodegeneration and AD will also help our understanding of SAD.

Role of PS and FAD mutants in neoroprotection

To examine the effects of FAD mutations on neurodegeneration, we study the genetics, molecular biology, and biological functions of wild type (WT) and mutant PSs and APP. We found that PS1 FAD mutants cause a loss of γ-secretase cleavage function at ε-sites of substrates manifested by both, decreased production of cytosolic peptides that function in cell signaling and accumulation of γ-secretase substrates. These data support a theory that PS FAD mutations promote neurotoxicity by inhibiting γ-secretase-catalyzed ε-cleavage of substrates thus reducing cell signaling while causing accumulation of membrane-bound cytotoxic peptides (Marambaud et al. 2003; Georgakopoulos et al., 2006;  Litters et al., 2007; Barthet et al., 2012a). We also reported that PS mediates brain-derived neurotrophic factor (BDNF)-dependent neuroprotection against toxic insults such as oxidative stress and glutamate toxicity (Barthet et al., 2012b). We currently examine the mechanisms of the PS-dependent neuroprotection and the effects of FAD mutants on trophic factor-mediated nueroprotection of cortical neurons using both in vitro primary neuronal cultures and in vivo transgenic mouse models.

PSs and miRNAs in glucose deprivation and AD

Emerging evidence indicates that PSs regulate expression of microRNAs (miRs), non-coding small RNAs that suppress mRNA translation. In our lab we found that PS1/γ-secretase regulates expression of miR-212 that targets the mRNA of PEA15, a protein known as a promoter of cancer cell survival under glucose deprivation. We observed that absence of PS1 sensitizes neurons to glucose deprivation and increases expression of miR-212 while decreasing PEA-15. Furthermore, we found that PS/γ-secretase regulates neuronal survival by suppressing expression of miR-212. Based on our data, we hypothesize that increased expression of miR-212 and resultant decrease in PEA15 expression contribute to increased vulnerability of PS1 null neurons under reduced glucose, a condition commonly found in AD. In our Center we currently examine the relationship between PS1, miR-212 and its target protein PEA15 and examine their effects on neuronal survival under stress conditions. In addition, we ask how PS/γ-secretase affects miR-212 and whether PS modulates the glucose deprivation-induced survival signaling of PEA15. We also ask whether expression of miR-212 and its target PEA15 changes in brains of AD patients and in PS FAD mutant knockin animal models.

Brain vascular abnormalities and AD

Evidence in the last decade implicates cerebral microvasculature abnormalities in the genesis of AD neuropathology. Additional literature shows that the EphB4/ephrinB2 system regulates development/function of vascular systems. Binding of extracellular EphB4 receptor to transmembrane ephrinB2 ligand protein on surface of endothelial cells stimulates angiogenesis/growth of new vessels from existing vasculature. Thus, treatment of endothelial cells with EphB4 stimulates cell sprouting and tube formation, processes considered crucial initial steps in angiogenesis. We found that the EphB4-induced sprouting/tube formation depends on γ-secretase activity and that EphB4 stimulates γ-secretase processing of ephrinB2 producing peptide ephrinB2/CTF2 (Georgakopoulos et al., 2006; 2011). Our data suggest that EphB4/ephrinB2 regulates angiogenesis through PS/γ-secretase. In support of this hypothesis, we found that peptide ephrinB2/CTF2 stimulates sprouting of endothelial cells in vitro. In our lab, we explore the mechanisms via which the EphB4/ephrinB2 and PS1/γ-secretase systems promote angiogenesis and ask whether these mechanisms are altered in AD. Since PS1 FAD mutants affect the ε cleavage of γ-secretase substrates thus decreasing production of ephrinB2/CTF2 (see above), we ask whether FAD mutants alter the EphB4/ephrinB2-dependent angiogenesis. In our work we use both in vtro cell systems and mouse transgenic models.

Role of Progranulin in Frontotemporal lobar degeneration (FTLD)

Recent reports show that progranulin (PGRN) gene null or missense mutations are linked to frontotemporal lobar degeneration (FTLD), a form of dementia characterized by severe neuronal loss in the frontal and temporal brain regions of adult patients. The nature of these mutations suggests that survival of certain neuronal brain populations need expression of both functional alleles of PGRN (haploinsufficiency). We found that PRGN stimulates phosphorylation and activation of neuronal MEK/ERK/p90RSK and PI3K/Akt cell survival pathways and rescues cortical neurons from cell death induced by glutamate or oxidative stresses. Our data showed that extracellular PRGN acts as a neuroptotective factor supporting the hypothesis that in FTLD reduction of PGRN results in decreased survival signaling and neuroprotection against excitotoxicity and other stresses, leading to accelerated neuronal death (Xu et al., 2011). We are currently working on the identification of receptors that mediate the cellular effects of PRGN and its mutants involved in FTLD. 

 

Current Grant Support:  This research center is supported by four NIH grants and a grant from the Alzheimer's Association.

 

Robakis Laboratory

 

Tsuchida K, Shioi J, Yamada S, Boghosian G, Wu A, Cai H, Sugahara K, Robakis NK. Appican, the Proteoglycan Form of the Amyloid Precursor Protein, Contains the Chondroitin Sulfate E Motif in the Repeating Disaccharide Region and a 4-O-Sulfated Galactose Residue in the Linkage Region. J Biol Chem 2001; 276: 37155-37160.

baki l, marambaud p, efthimiopoulos s, georgakopoulos a, wen p, cui w, shioi, j, koo e, ozawa m, friedrich jr v, robakis nk. Presenilin1 binds cytoplasmic epithelial cadherin, inhibits cadherin/p120 association, and regulates stability and function of the cadherin/catenin adhesion complex. Proc Natl Acad Sci U S A 2001 Feb 27; 98(5).

marambaud p, shioi j, serban g, sarner s, georgakopoulos a, nagy v, baki l, wen p, efthimiopoulos s, shao z, wisniewski t, robakis nk. A presenilin-1/g-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions. EMBO J 2002; 21(8).

Marambaud P, Wen P, Shioi J, Siman R, Takashima A, Robakis N. A CBP-binding transcriptional repressor produced by the PS1/e-cleavage of N-cadherin is inhibited by PS1 FAD mutations. Cell 2003; 114: 635-645.

Baki L, Neve R, Shao Z, Shioi J, Georgakopoulos A, Robakis NK. Wild-type but not FAD mutant Presenilin-1, prevents neuronal degeneration by promoting phosphatidylinositol 3-kinase survival signaling. J. Neurosci. 2008; 28: 483-490.

Xu J, Litterst C, Georgakopoulos A, Zaganas I, Robakis NK. Peptide EphB2/CTF2 generated by the y-secretase processing of EphB2 receptor, promotes tyrosine phosphorylation and cell surface localization of NMDA receptors. J. Biol. Chem. 2009; 284: 27220-27228.

Robakis NK. Mechanisms of AD neurodegeneration may be independent of AB and its derivatives. Neurobiol. Aging 2010;.

Georgakopoulos A, Xu J, Xu C, Mauger G, Barthet G, Robakis NK. Presenilin1/gamma-secretase promotes the EphB2-induced phosphorylation of ephrinB2 by regulating phosphoprotein associated with glycosphingolipid-enriched microdomains/Csk binding protein. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2011 Oct; 25(10).

Xu J, Xilouri M, Bruban J, Shioi J, Shao Z, Papazoglou I, Vekrellis K, Robakis NK. Extracellular progranulin protects cortical neurons from toxic insults by activating survival signaling. Neurobiology of aging 2011 Dec; 32(12).

Barthet G, Dunys J, Shao Z, Xuan Z, Ren Y, Xu J, Arbez N, Mauger G, Bruban J, Georgakopoulos A, Shioi J, Robakis NK. Presenilin mediates neuroprotective functions of ephrinB and brain-derived neurotrophic factor and regulates ligand-induced internalization and metabolism of EphB2 and TrkB receptors. Neurobiology of aging 2013 Feb; 34(2).

Barthet G, Georgakopoulos A, Robakis NK. Cellular mechanisms of γ-secretase substrate selection, processing and toxicity. Progress in neurobiology 2012 Aug; 98(2).

Robakis NK, Georgakopoulos A. Allelic Interference: A Mechanism for Trans -Dominant Transmission of Loss of Function in the Neurodegeneration of Familial Alzheimer's Disease. Neuro-degenerative diseases 2013 Sep;.

Xuan Z, Barthet G, Shioi J, Xu J, Georgakopoulos A, Bruban J, Robakis NK. Presenilin-1/γ-secretase controls glutamate release, tyrosine phosphorylation, and surface expression of N-methyl-D-aspartate receptor (NMDAR) subunit GluN2B. The Journal of biological chemistry 2013 Oct; 288(42).