Shai Shoham

Iron and neurodegenerative disorders.

The brain shares with other organs the need for a constant and readily available supply of iron and has a similar array of proteins available to it for iron transport, storage, and regulation. However, unlike other organs, the brain places demands on iron availability that are regional, cellular, and age sensitive. Failure to meet these demands for iron with an adequate supply in a timely manner can result in persistent neurological and cognitive dysfunction. Consequently, the brain has developed mechanisms to maintain a continuous supply of iron. However, in a number of common neurodegenerative disorders, there appears to be an excess accumulation of iron in the brain that suggests a loss of the homeostatic mechanisms responsible for regulating iron in the brain. These systems are reviewed in this article. As a result of a loss in iron homeostasis, the brain becomes vulnerable to iron-induced oxidative stress. Oxidative stress is a confounding variable in understanding the cell death that may result directly from a specific disease and is a contributing factor to the disease process. The underlying pathogenic event in oxidative stress is cellular iron mismanagement.

Stress-induced alternative splicing of acetylcholinesterase results in enhanced fear memory and long-term potentiation

Stress insults intensify fear memory; however, the mechanism(s) facilitating this physiological response is still unclear. Here, we report the molecular, neurophysiological and behavioral findings attributing much of this effect to alternative splicing of the acetylcholinesterase (AChE) gene in hippocampal neurons. As a case study, we explored immobilization-stressed mice with intensified fear memory and enhanced long-term potentiation (LTP), in which alternative splicing was found to induce overproduction of neuronal ‘readthrough’ AChE-R (AChE-R). Selective downregulation of AChE-R mRNA and protein by antisense oligonucleotides abolished the stress-associated increase in AChE-R, the elevation of contextual fear and LTP in the hippocampal CA1 region. Reciprocally, we intrahippocampally injected a synthetic peptide representing the C-terminal sequence unique to AChE-R. The injected peptide, which has been earlier found to exhibit no enzymatic activity, was incorporated into cortical, hippocampal and basal nuclei neurons by endocytosis and retrograde transport and enhanced contextual fear. Compatible with this hypothesis, inherited AChE-R overexpression in transgenic mice resulted in perikaryal clusters enriched with PKCβII, accompanied by PKC-augmented LTP enhancement. Our findings demonstrate a primary role for stress-induced alternative splicing of the AChE gene to elevated contextual fear and synaptic plasticity, and attribute to the AChE-R splice variant a major role in this process.

Interaction of “readthrough” acetylcholinesterase with RACK1 and PKCβII correlates with intensified fear-induced conflict behavior

Behavioral reactions to stress are altered in numerous psychiatric and neurodegenerative syndromes, but the corresponding molecular processes and signal transduction pathways are yet unknown. Here, we report that, in mice, the stress-induced splice variant of acetylcholinesterase, AChE-R, interacts intraneuronally with the scaffold protein RACK1 and through it, with its target, protein kinase CβII (PKCβII), which is known to be involved in fear conditioning. In stress-responsive brain regions of normal FVB/N mice, the mild stress of i.p. injection increased AChE and PKCβII levels in a manner suppressible by antisense prevention of AChE-R accumulation. Injection stress also prolonged conflict between escape and hiding in the emergence into an open field test. Moreover, transgenic FVB/N mice overexpressing AChE-R displayed prolonged delay to emerge into another field (fear-induced behavioral inhibition), associated with chronically intensified neuronal colabeling of RACK1 and PKCβII in stress-responsive brain regions. These findings are consistent with the hypothesis that stress-associated changes in cholinergic gene expression regulate neuronal PKCβII functioning, promoting fear-induced conflict behavior after stress.

Ladostigil prevents gliosis, oxidative–nitrative stress and memory deficits induced by intracerebroventricular injection of streptozotocin in rats

Glial activation and oxidative-nitrative stress occur at an early stage in Alzheimers disease (AD). In a rat model of AD, deficits in cerebral glucose utilization and memory were seen 3-4 weeks after intracerebroventricular (icv) injection of streptozotocin (STZ). This study examined whether icv STZ induced glial activation and oxidative-nitrative stress preceded the memory deficits and whether they could be prevented by ladostigil a novel drug, a cholinesterase and monoamine oxidase inhibitor with neuroprotective activity. One week after STZ injection activated microglia and astrocytes were seen in the cortex, around the cannula penetration area, in the hippocampal CA1 region, corpus callosum, medial and lateral septum. The activated astrocytes showed a significant increase in nitrotyrosine immunoreactivity, a measure of oxidative-nitrative stress. Only 3 weeks later were deficits in episodic (object recognition test) and spatial memory (place recognition) seen in STZ-injected rats. Daily oral administrations of ladostigil (1mg/kg) for 1 week, before and after STZ prevented the glial changes, increase in nitrotyrosine immunoreactivity and memory deficits. Taken together the data support the role of glial activation and oxidative-nitrative stress in discrete brain areas in the aetiology of memory deficits and indicate a potential mechanism for their prevention by drug treatment.

Neuroprotective Effects of Novel Cholinesterase Inhibitors Derived from Rasagiline as Potential Anti-Alzheimer Drugs

Abstract: TV3326, (N‐propargyl‐(3R)‐aminoindan‐5‐yl‐ethyl, methyl carbamate) was prepared in order to combine the neuroprotective effects of rasagiline, a selective inhibitor of monoamine oxidase (MAO)‐B with the cholinesterase (ChE) inhibitory activity of rivastigmine as a potential treatment for Alzheimers disease. The study reported here examined the neuropotective effects of TV3326 against various insults in vitro and in vivo. TV3326 caused a dose related (10–500 μM) reduction in death induced in NGF differentiated rat pheochromocytoma (PC12) cells by 3–4 hour exposure to oxygen‐glucose deprivation. A single sc injection of TV3326 given five minutes after closed head injury in mice significantly reduced the cerebral edema, and accelerated the recovery of motor function and spatial memory several days later. Unilateral icv injection of streptozotocin (STZ) 1.5 mg in rats, caused specific damage to myelinated neurones in the fornix and corpus callosum accompanied by microgliosis. Three bilateral injections of STZ, 0.25 mg each, caused more widespread damage, and a marked impairment in spatial memory. Chronic oral treatment with TV3326 (75 μmols/kg) reduced the neuronal damage and microgliosis and almost completely prevented the memory impairment. The neuroprotective effect in PC12 cells may be due to a combination of ChE inhibition and antiapoptotic activity. The latter does not result from ChE inhibition. It is associated with the presence of the propargyl group, since it occurs with other propargylamines that do not inhibit MAO, but not with drugs that inhibit only ChE.

Brain trauma induces X-box protein 1 processing indicative of activation of the endoplasmic reticulum unfolded protein response

Brain trauma was induced in mice using a closed head injury (CHI) model. At 1, 6 or 24 h after trauma, brains were dissected into the cortex, striatum and hippocampus. Changes in levels of processed X‐box protein 1 (xbp1), glucose‐regulated protein 78 (grp78), growth arrest and DNA damage‐inducible gene 153 (gadd153) and heat‐shock protein 70 (hsp70) mRNA, indicating impaired endoplasmic reticulum (ER) and cytoplasmic functioning, were evaluated by quantitative PCR. In the cortex, processed xbp1 mRNA levels rose to 2000% of control 1 h after CHI, and stayed high throughout the experiments. In the hippocampus and striatum, processed xbp1 mRNA levels rose in a delayed fashion, peaking at 6 h (1000% of control) and 24 h after CHI (1500% of control) respectively. Levels of grp78 mRNA were only slightly increased in the cortex 24 h after CHI (150% of control), and were unchanged or transiently decreased in the hippocampus and striatum. Levels of gadd153 mRNA did not change significantly after trauma. A transient rise in hsp70 mRNA levels was observed only in the cortex, peaking at 1 h after CHI (600% of control). Processing of xbp1 mRNA is a sign of activation of the unfolded protein response indicative of ER dysfunction. The results suggest that brain trauma induces ER dysfunction, which spreads from the ipsilateral cortex to the hippocampus and striatum. These observations may have clinical implications and should therefore be considered for future investigations on therapeutic intervention of brain injury caused by contusion‐induced neurotrauma.

Chronic brain cytochrome oxidase inhibition selectively alters hippocampal cholinergic innervation and impairs memory: prevention by ladostigil.

A 25-35% reduction of brain cytochrome oxidase (COx) activity found in Alzheimers disease (AD) could contribute to neuronal dysfunction and cognitive impairment. The present study replicated the reduction in brain COx activity in rats by administering sodium azide (NaN(3)) for 4 weeks via Alzet minipumps at the rate of 1 mg/kg/h, and determined its effect on hippocampal cholinergic transmission, spatial and episodic memory. NaN(3) caused a selective reduction in choline acetyltransferase (ChAT) immunoreactivity in the diagonal band, a major source of cholinergic input to the hippocampus and cingulate cortex, without altering the number of cholinergic neurons. NaN(3) also induced a significant increase in vesicular acetylcholine transporter (VAChT)-immunoreactive varicosities, GAP-43 in the subgranular layer and of transferrin receptors (TfR) in the hilus of the dentate gyrus. These neurochemical changes were associated with impairment in spatial learning in the Morris water maze and in episodic memory in the object recognition test. Chronic treatment with ladostigil, a novel cholinesterase and monoamine oxidase inhibitor, prevented the decrease in ChAT in the diagonal band, the compensatory increase in synaptic plasticity and TfR and the memory deficits without restoring COx activity. Ladostigil had no significant effect on ChAT activity, synaptic plasticity or TfR in control rats. Ladostigil may have a beneficial effect on cognitive deficits in AD patients that have a reduction in cortical COx activity and cholinergic hypofunction.

Ladostigil prevents age-related glial activation and spatial memory deficits in rats.

Oxidative stress and glial activation occur in the aging brain. Ladostigil is a new monoamine oxidase (MAO) and acetylcholinesterase (AChE) inhibitor designed for the treatment of Alzheimers disease. It has neuroprotective and antioxidant activities in cellular models at much lower concentrations than those inhibiting MAO or AChE. When ladostigil (1mg/kg/day) was given for 6 months to 16-month-old rats it prevented the age-related increase in activated astrocytes and microglia in several hippocampal and white matter regions and increased proNGF immunoreactivity in the hippocampus towards the levels in young rats. Ladostigil also prevented the age-related reduction in cortical AChE activity and the increase in butyrylcholinesterase activity in the hippocampus, in association with the reduction in gliosis. The immunological and enzymatic changes in aged rats were associated with improved spatial memory. Ladostigil treatment had no effect on memory, glial or proNGF immunoreactivity in young rats. Early treatment with ladostigil could slow disease progression in conditions like Alzheimers disease in which oxidative stress and inflammatory processes are present.

Impaired hippocampal plasticity and errors in cognitive performance in mice with maladaptive AChE splice site selection

Neuronal splice site selection events control multiple brain functions. Here, we report their involvement in stress‐modulated hippocampal plasticity and errors of cognitive performance. Under stress, alternative splicing changes priority from synaptic acetylcholinesterase (AChE‐S) to the normally rare, soluble and monomeric AChE‐R variant, which facilitates hippocampal long‐term potentiation (LTP) and intensifies fear‐motivated learning. To explore the adaptive value of changes in AChE splicing, we compared hippocampal plasticity and errors of executive function in TgS and TgR transgenic mice overexpressing AChE‐S or AChE‐R, respectively. Hippocampal slices from TgS and TgR mice presented delayed and facilitated transition to LTP maintenance, respectively, compared with strain‐matched FVB/N controls. TgS slices further showed failed recruitment of both the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate and N‐methyl‐d‐aspartate components of LTP, refractory response to cholinergic enhancement and suppressed protein kinase C (PKC) levels. Stable LTP could, however, be rescued by phorbol ester priming, attributing the TgS deficits to disrupted signal transduction. In serial maze tests, TgS mice displayed more errors of conflict and executive function than did FVB/N controls, reflecting maladaptive performance under chronic AChE‐S overexpression. In contrast, TgR mice displayed enhanced serial maze performance, suggesting that chronic AChE‐R overexpression facilitates adaptive reactions. Our findings are compatible with the notion that changes in the alternative splicing of AChE pre‐mRNA and consequent alterations in PKC signalling are causally involved in modulating hippocampal plasticity and cognitive performance.

Nutritional Iron Deprivation Attenuates Kainate‐Induced Neurotoxicity in Rats: Implications for Involvement of Iron in Neurodegeneration

Abstract: There is evidence suggesting that oxidative stress contributes to kainate neurotoxicity. Since iron promotes oxidative stress, the present study explores how change in nutritional iron content modulates kainate‐induced neurotoxicity. Rats received an iron‐deficient diet (ID) from 22 days of age for 4 weeks. One control group received the same diet supplemented with iron and another control group received standard rodent diet. Cellular damage after subcutaneous kainate (10 mg/kg) was assessed by silver impregnation and gliosis by staining microglia. ID reduced cellular damage in piriform and entorhinal cortex, in thalamus, and in hippocampal layers CA1‐3. ID also attenuated gliosis, except in the hippocampal CA1 layer. Given involvement of zinc in hippocampal neurotransmission and in oxidative stress, we tested for a possible interaction of nutritional iron with nutritional zinc. Rats were made iron‐deficient and then assigned to supplementation with iron, zinc, or iron + zinc. Controls were continued on ID diet. After 2 weeks, rats were treated with kainate. Iron supplementation abolished the protective effect of ID in piriform and entorhinal cortex. In hippocampal CA1 and dorsal thalamus, neither iron nor zinc supplementation alone abolished the protective effect of ID against cellular damage. Iron + zinc supplementation abolished ID protection in dorsal thalamus, but not in reuniens nucleus. Kainate‐induced gliosis in CA1 remained unaffected by nutritional treatments. Thus, in piriform and entorhinal cortex, nutritional iron has a major impact on cellular damage and gliosis. In hippocampal CA1, gliosis may associate with synaptic plasticity not modulated by nutritional iron, while cellular damage is sensitive to nutritional iron and zinc.