Research Interests Page

Anxiety Reactions as a Neuroprotection Strategy

Alternative splicing of pre-mRNA is an important determinant of molecular and functional complexityAnxiety reactions involve complex, incompletely understood interactions of genomic, environmental and experience-derived factors, and anxiety disorders present a major mental health problem. Recent research in the lab focuses on the putative functional role(s) of anxiety reactions as a neuroprotection strategy, and more specifically, on anxiety-induced changes in cholinergic neurotransmission. These changes modulate the motor control over movement, facilitate the consolidation of traumatic memories, and activate brain-to-body communication through the neuron-immune interface modifying blood cell composition and platelet production. Basic molecular mechanisms known to accompany stress reactions also occur during neurodegenerative processes. For example, stress and anxiety-associated changes were found in the expression pattern of the acetylcholinesterase ACHE gene, which encodes the acetylcholine hydrolyzing enzyme AChE. AChE is not one, but a combinatorial series of proteins having indistinguishable enzymatic activity yet with variant N- and C-termini due to alternate promoter usage and 3'-alternative splicing. Differentially induced under stress, they show distinct non-hydrolytic properties, interact with variant-specific protein partners and induce inverse signaling cascades. Thus, transcriptional and post-transcriptional regulation of AChE pre-mRNA protects both blood and nerve cells from acute dangers, but may also entail long-term damages. Specifically, variant-specific causal involvement of AChE in the progression of Alzheimer's and Parkinson's disease and neuromuscular syndromes like myasthenia gravis, anticipate future therapeutic needs for drugs targeting specific AChE variant(s) or the corresponding RNA transcripts.

Current Research Projects

Alternative AChE splice variants in stress-related neuropathologies

Studying cholinergic features by transgenic manipulations of acetylcholinesterase gene expression

Neuronal interactions of RACK1 and their physiological consequences

Modulation of blood cell composition by stress-induced cholinergic signaling through both hydrolytic and non-enzymatic functions of acetylcholinesterase

RNA-targeted suppression of acetylcholinesterase gene expression: from cellular tests to therapeutics

Genomic Approach to the Treatment of Anxiety

Synthetic microRNA therapeutics for immune modulation

Human acetylcholinesterase isoforms from transgenic plants

Understanding gene networks and their role in neurodegeneration

Alternative AChE splice variants in stress-related neuropathologies
The acetylcholine hydrolyzing enzyme acetylcholinesterase (AChE) is not one, but a combinatorial series of proteins having variant N- and C-termini due to alternate promoter usage and 3'-alternative splicing. Neuronal AChE variants show indistinguishable enzymatic activity yet differ in their expression, multimeric assembly and membrane association patterns. Differentially induced under stress, they show distinct non-hydrolytic properties and interact with different protein partners. Recent findings suggest that transcriptional and post-transcriptional regulation of AChE pre-mRNA serve as a neuroprotection strategy but may entail long-term damage. Specifically, variant-specific causal involvement of AChE in the progression of both neurodegenerative (e.g. Alzheimer's, Parkinson's disease) and neuromuscular syndromes like myasthenia gravis, anticipate future therapeutic needs for drugs targeting specific AChE variant(s) or the corresponding RNA transcripts. (Meshorer, E. and Soreq, H. 2006. TINS, in press)

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Studying cholinergic features by transgenic manipulations of acetylcholinesterase gene expression
Over the past decade, genetic manipulation approaches provided means for gain and loss of function of AChE gene expression in live cells and organisms. Yet more specifically, transgenic (Tg) mouse pedigrees were developed which induce over-or under-expression of specific splice variants of human or mouse AChE and antisense and siRNA tools were created for selectively suppressing their expression. ACh is in various ways a bridging signaling transmitter, responsible for neuromuscular communication, central (CNS) to peripheral (PNS) nervous system cross-talk, interaction with other neurotransmission pathways, maintenance of a neuro-immune dialog and even the transition of our system’s physiology in need of coping with stress. Mammalian stress responses provide a case study for exploring cholinergic reactions in general, and AChE gene expression in particular, under threatened homeostasis. Stress-induced changes in the alternative splicing patterns of AChE pre-mRNA attribute to this gene and its different protein products, diverse stress responsive functions that are associated with both the enzymatic and non-catalytic properties of AChE variants. Transgenic manipulations of AChE gene expression served to uncover previously non-perceived aspects of stress responses, including brain-to-blood as well as neuronal-to-immune communication. ACh is a principal stress response-regulator, which was recently found to function as a vital route by which neurons can “talk” to immune cells. Therefore, chemical, physical or mental insults to the brain might all be traced in peripheral immune cells, which serve as key determinants in the physiological reactions to stress. A new understanding is thus achieved by using genomic manipulations of AChE gene expression as tools for approaching cholinergic research. (GIACOBINI & PEPEU: The brain Cholinergic System)

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Neuronal interactions of RACK1 and their physiological consequences
The Receptor for activated protein kinase C1 (RACK1) is an intracellular adaptor
protein. Accumulating evidence attributes to this member of the tryptophan-aspartate (WD) repeat family the role of regulating several major nervous system pathways. Structurally, RACK1 is a seven-bladed-I-propeller, interacting with diverse proteins having distinct structural folds. When bound to the IP3 receptor, RACK1 regulates intracellular Ca2+ levels, potentially contributing to processes such as learning, memory and synaptic plasticity. By binding to the NMDA receptor, it dictates neuronal excitation and sensitivity to ethanol. When bound to the stress-induced acetylcholinesterase variant AChE-R, RACK1 is implicated in stress responses and behavior, compatible with reports of RACK1 modulations in brain ageing and in various neurodegenerative diseases. (Sklan, E.H., Podoly, E. and Soreq, H. 2006. Prog. Neurobiol; in press.)

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Modulation of blood cell composition by stress-induced cholinergic signaling through both hydrolytic and non-enzymatic functions of acetylcholinesterase
Stress hormone-initiated granulocytosis, excessive proliferation of myeloid blood cells, persists after cortisol levels are lowered, suggesting the involvement of additional stress mediator(s). In post-delivery patients, AChE-R-expressing granulocyte counts increase concomitantly with serum cortisol and AChE activity levels, yet persist after cortisol had declined. Ex vivo, mononuclear cells of adult peripheral blood respond to synthetic ARP26, the cleavable, cell-penetrating C-terminal peptide of AChE-R, by overproduction of both myeloid cells and hemopoietically active proinflammatory cytokines (e.g., IL-6, IL-10, and TNF-alpha), suggesting autoregulatory prolongation of ARP effects. In vivo, TgR transgenic mice over-expressing human AChE-R, unlike matched controls, maintain a stable granulocytic state following bacterial LPS exposure, suggesting that AChE-R accumulation and the consequent inflammatory events can modulate immune responses to stress stimuli. Increased AChE hydrolytic activity in the peripheral blood of TgR mice is further associated with increased thrombopoietin levels and platelet counts, and bone marrow (BM) progenitor cells from TgR mice present an elevated capacity to produce mixed (GEMM) and megakaryocyte (MK) colonies. These show intensified labeling of AChE-R and its interacting proteins RACK1 and PKC-epsilon. When injected with bacterial lipopolysaccharide (LPS), parent strain FVB/N mice, but not TgR mice, show reduced platelet counts. To challenge the hypothesis that AChE-R and/or ARP26 are causally involved in this phenomenon, human CD34(+) cells may be primed with synthetic ARP26 prior to transplantation into sub-lethally irradiated NOD/SCID mice. Engraftment of human cells (both CD45(+) and CD41(+) Mk) significantly increases in mice that receive ARP26 primed CD34(+) human cells. Moreover, ARP26 induces polyploidization and proplatelet formation in human MEG-01 promegakaryocytic cells, and human platelet engraftment increases following ex vivo expansion of ARP26-treated CD34(+) cells as compared to cells expanded with thrombopoietin and stem cell factor. These findings implicate AChE-R in myeloid and thrombopoietic activities, suggesting new therapeutic modalities for supporting blood cell production, which is currently being investigated. (see Pick et al. 2005. Blood; Grisaru et al. 2006. J. Immunol. For further details).

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RNA-targeted suppression of acetylcholinesterase gene expression: from cellular tests to therapeutics
The acetylcholinesterase (AChE) protein serves as a terminator of cholinergic neurotransmission in neuromuscular junctions and brain synapses. As a cholinergic modulator, AChE also mediates the production of proteins controlling body-brain interactions (e.g. pro-inflammatory cytokines). We employ RNA-targeted tools to suppress AChE gene expression in cultured cells, experimental animals and patient volunteers. In transfected cells, SiRNA vectors targeted at different sites in the AChE mRNA transcript suppressed the expression of both its primary "synaptic" AChE-S mRNA variant and the stress-induced "read-through" AChE-R mRNA by ca. 60%. A similar, yet transcript-specific level of suppression, primarily of AChE-R mRNA was achieved by EN101(Monarsen), a 20-Mer antisense agent with three 3' 2-O-Methyl protected nucleotides. Monarsen was tested in tumor cell lines and primary cell cultures as well as in live mice, rats and Cynomolgus monkeys. In all of these settings, it selectively suppressed in nanomolar doses the levels of AChE-R mRNA and the AChE-R protein. In central nervous system neurons of both rats and Cynomolgus monkeys, Monarsen was further found to suppress the levels of the pro-inflammatory cytokines interleukin-1 and -6. Orally delivered Monarsen was approved for testing its efficacy in alleviating the neuromuscular malfunctioning symptoms of patients with myasthenia gravis, who produce auto-immune antibodies against their own acetylcholine receptors and over-express AChE-R in their blood. Testing is now at mid-way 5 weeks phase II trials, where Monarsen daily doses effectively replace multiple daily doses of the currently employed small molecule anti-AChE drug pyridostigmine. Furthermore, patients appear to be free of the characteristic intestinal side effects caused by the non-selective suppression of AChE-S by the current small molecule drug. Monarsen thus offers potential advantages over conventional cholinesterase inhibitors with respect to dosing, specificity, side-effect profile, duration of efficacy and treatment regimen.

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Genomic Approach to the Treatment of Anxiety
A novel approach to the treatment of anxiety disorders such as post-traumatic stress (PTSD) as manifested in Gulf War veterans.

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Synthetic microRNA therapeutics for immune modulation
RNA interference (RNAi) is the term used to describe the recently described process by which short RNAs are able to exert posttranscriptional control over gene expression. One type of such RNAs is the rapidly expanding family of microRNAs (miRNAs), 20-28 nucleotide (nt) non-coding RNAs that have recently emerged as regulators of gene expression and cellular fate determination in multiple organisms. The primary miRNA transcripts are processed by the nuclear RNase III  Drosha , and the resulting ~60-120 nt hairpin precursors are exported from the nucleus via the exportin-5 mechanism. In the cytoplasm these precursors are further cleaved by Dicer, a double-stranded RNA endoribonuclease, to yield the mature miRNAs. These are incorporated into the RNA-induced silencing complex (RISC) for subsequent targeting to specific mRNAs. miRNAs are generally believed to exert their effects through targeting mRNA stability as well as translational repression.  The  mRNA targets of these miRNAs in humans are still largely unknown, but are predicted to include a number of  regulatory genes and to play a role  in cancer and hematopoiesis.

The nervous and the immune systems communicate continuously. Recently a neuronal mechanism that inhibits macrophages activity through parasympathetic outflow was identified. This newly recognized mechanism was named the “cholinergic anti-inflammatory pathway”. ACh, a principle parasympathetic neurotransmitter, effectively inhibits macrophage production of pro-inflammatory cytokines. CNS resident microglia and blood-born-Macrophages  were found to express a 7 nicotinic acetylcholine receptors (nAChR). AChR are expressed in many peripheral tissues, including lymphoid tissues , where AChE gene expression is subject to stress-induced changes. Thus, the immune function of different immune cell subsets is influenced by cholinergic activity in the local environment, and immune cells may also contribute to the regulation of systemic cholinergic activity via the release of soluble AChE-R. In fact, activation of the immune system in human volunteers by the bacterial lipopolysaccharide (LPS endotoxin) leads to an increase of plasma AChE-R thereby reducing the systemic cholinergic activity.

Little is known however about the regulatory pathways that play a role in the regulation of these processes. Recent studies in our laboratory have shown that a specific miRNA may play a role in the regulation of the immune system. Endogenous levels of this miRNA are changed in the bone marrow of stressed mice and in activated human peripheral mononuclear cells (huPBMC).  A synthetic miRNA is able to alter differentiation fates in Meg01 cells, a human hematopoietic platelet producing cell line. This work indicates that miRNAs may be a useful tool for the manipulation of the immune system and can be developed into novel therapeutics for modulation of the immune system.

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Human acetylcholinesterase isoforms from transgenic plants
A robust system for the production and delivery of effective countermeasures against pesticides and non-conventional warfare agents.

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Understanding gene networks and their role in neurodegeneration
The Soreq laboratory is a member of the EC funded VALAPODYN consortium which seeks to advance the development of multidisciplinary functional genomics relating to complex biological processes and cellular networks. The project goal is to develop a new System Biology approach to model the dynamics of Molecular Interaction Networks (MIN) related to cell death and survival in the organism. The future dynamic model will be dedicated to the selection of drug targets for human brain pathologies such as epilepsy, ischemia, Parkinsonís disease and Alzheimerís disease.
For more information please access the Project's web site:
http://www.valapodyn.eu/

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