Haresh S. Ved

Huperzine A, a potential therapeutic agent for dementia, reduces neuronal cell death caused by glutamate

HUPERZINE A, a potential therapeutic agent for Alzheimers disease, inhibits acetylcholinesterase in primary cultures derived from forebrain, hippocampus, cortex and cerebellum of embryonic rat brain. Glutamate induces cell death in cultures from all these brain regions. Maximum cell toxicity was observed in cerebellar cultures. Pretreatment of cell cultures with Huperzine A reduced cell toxicity, as evidenced by cytotoxicity assay and general morphology. Huperzine A pretreatment also reduced glutamate-induced calcium mobilization, but did not affect elevations in intraneuronal free Ca2+ ([Ca]i) caused by KCl or (–)Bay K 8644. The data suggest that Huperzine A could be a potent neuroprotective agent not only where cholinergic neurons are impaired, but also under conditions in which glutamatergic functions are compromised.

Neuroprotective role of c-fos antisense oligonucleotide : in vitro and in vivo studies

WE investigated the dose—response and time-course of c-fos antisense oligodeoxynucleotide (ASO) treatment against excitatory amino acid (EAA)-induced neurotoxicity in rat hippocampal neurons. Glutamate (in vitro) or NMDA (in vivo) produced significant neuronal degeneration. Neuroprotection produced by 30 min or 4 h pretreatment with c-fos ASO in cultured hippocampal neurons was dose-dependent. In vivo, bilateral intrahippocampal injections of c-fos ASO (0.025 nmol/site) was neuroprotective when administered 30 min before or after NMDA treatment. However, 4 h pretreatment was ineffective. A higher dose (0.125 nmol) of c-fos ASO was neurotoxic and failed to afford neuroprotection regardless of the treatment schedule. Collectively, these results demonstrate a neuroprotective effect of c-fos ASO against EAA-nduced neuronal injury supporting a causative role of c-fos expression in EAA neurotoxicity.

RS-100642-198, a novel sodium channel blocker, provides differential neuroprotection against hypoxia/hypoglycemia, veratridine or glutamate-mediated neurotoxicity in primary cultures of rat cerebellar neurons.

The present study investigated the effects of RS-100642-198 (a novel sodium channel blocker), and two related compounds (mexiletine and QX-314), inin vitro models of neurotoxicity. Neurotoxicity was produced in primary cerebellar cultures using hypoxia/hypoglycemia (H/H), veratridine or glutamate where, in vehicle-treated neurons, 65%, 60% and 75% neuronal injury was measured, respectively. Dose-response neuroprotection experiments were carried out using concentrations ranging from 0.1-500 μM. All the sodium channel blockers were neuroprotective against H/H-induced injury, with each exhibiting similar potency and efficacy. However, against veratridine-induced neuronal injury only RS-100642-198 and mexiletine were 100% protective, whereas QX-314 neuroprotection was limited (i.e. only 54%). In contrast, RS 100642-198 and mexiletine had no effect against glutamate-induced injury, whereas QX-314 produced a consistent, but very limited (i.e. 25%), neuroprotection. Measurements of intraneuronal calcium ([Ca2+];) mobilization revealed that glutamate caused immediate and sustained increases in [Ca2+]i which were not affected by RS-100642-198 or mexiletine. However, both drugs decreased the initial amplitude and attenuated the sustained rise in [Ca2+]i mobilization produced by veratridine or KC1 depolarization. QX-314 produced similar effects on glutamate-, veratridineor KC1-induced [Ca2+]i dynamics, effectively decreasing the amplitude and delaying the initial spike in [Ca2+]i, and attenuating the sustained increase in [Ca2+]i mobilization. By using differentin vitro models of excitotoxicity, a heterogeneous profile of neuroprotective effects resulting from sodium channel blockade has been described for RS-100642-198 and related drugs, suggesting that selective blockade of neuronal sodium channels in pathological conditions may provide therapeutic neuroprotection against depolarization/excitotoxicity via inhibition of voltage-dependent Na+ channels.

Synthesis, chiral chromatographic separation, and biological activities of the enantiomers of 10,10-dimethylhuperzine A.

(+/-)-10,10-Dimethylhuperzine A (2, DMHA) has been synthesized, and its enantiomers have been separated using chiral HPLC. (-)-DMHA inhibits AChE with a Ki value approaching that of (-)-huperzine A, whereas (+)-DMHA shows no AChE inhibitory activity. On the other hand, both enantiomers are equally potent against glutamate-induced neurotoxicity when tested in neurons.

Dodecylglycerol provides partial protection against glutamate toxicity in neuronal cultures derived from different regions of embryonic rat brain

Primary cultures enriched in neurons dissociated from embryonic rat cerebral cortex, cerebellum, or hippocampus were treated in a chemically defined serum-free media with either vehicle, dodecylglycerol (DDG, 3 microM), or glutamate (75 microM), or preincubated with DDG for 4 or 24 h, and further incubated with glutamate. Their morphological and biochemical assessments (lactate dehydrogenase [LDH] release in the culture media, neuronal viability and intracellular Ca2+ mobilization) were made. Neurotoxic effects of glutamate and glutamate-mediated increases in intracellular Ca2+ were maximal in neurons from cerebellum and minimal in neurons from cortex. Cotreatment of cells with DDG and glutamate failed to provide significant neuronal protection against glutamate in the three brain regions. Pretreatment of cells with DDG for 4 or 24 h prior to glutamate treatment provided significant neuroprotection as judged by morphological changes and a decrease in LDH activity. Neuroprotection of approximately 15-35% was observed following 4 h of DDG pretreatment, increasing to 60-85% protection after 24 h of DDG pretreatment. Although the mechanism of DDGs neuroprotective action remains to be elucidated, these results demonstrate that both glutamate and DDG have differential specificity for anatomical regions of the brain.

Comparative Inhibition of Acetylcholinesterase by Tacrine, Physostigmine and Huperzine in the Adult Rat Brain

Alzheimer’s Disease (AD) is the most common type of adult-onset dementia. It is associated with a progressive and irreversible loss of memory and cognitive functions proceeding for years. The general malfunction of the cholinergic regions of the brain invariably leads to death. There seems to be a correlation between the impairment of cognitive function with an increase in the number of neuritic plaques and neurofibrillary tangles in the hippocampus, amygdala and cerebral cortex (Coyle et al., 1983; Mullan and Crawford, 1993). Moreover, the severity of the disease parallels the reduction in levels of AChE and ChAT in the frontal and temporal cortices (Perry et al., 1978). A diminished number of cholinergic neurons in basal forebrain nuclei and decreased ACh production in the brain of AD patients are thought to cause some of the characteristic cognitive impairments. Three broad strategies have been considered in dementia therapy: (1) replacement of lost neurotransmitter, (2) trophic support to slow the degeneration of the nervous system, and (3) intervention, i. e., prevention of the local inflammatory response thought to be caused by complement activation in the amyloid plaques (Davis et al., 1993). It has been suggested that anti-cholinergic drugs impair the memory of healthy individuals in a manner parallel to that observed early in the development of AD. Therefore, the principal current AD therapeutic approach, and the most promising one in the short term, is the stimulation of the cholinergic system. The first anti-ChE drug to be approved for use in AD therapy in the USA is Cognex® (1-2-3-4-tetrahydro-9 aminoacridine, Tac, tacrine). Recently, there has been a report of a multi-center, double-blind, placebo-controlled trial of Tac therapy, which included 663 patients suffering from mild to moderate AD. Huperzine A (Hup A) was recently shown to be a potent, reversible inhibitor of AChE (Ashani et al; 1992). The fairly long half-life (T0.5 = 35 min) for AChE-Hup A complex is in marked contrast to the rapid on/off rates that characterize other reversible inhibitors of AChE with similar potency (Taylor and Radic 1994).

Comparative Effects of Cholinesterase Inhibitors on Glutamate-Induced Neuronal Cell Death

Our earlier studies have shown that glutamate-induced neuronal cell death can be reduced by pretreatment with Huperzine A (Hup-A) in enriched primary neurons. These effects seem to be caused by blockage of the NMDA receptor-mediated calcium ion influxes. Hup-A is a reversible inhibitor of cholinesterase (ChEI) with a very high affinity for acetylcholinesterase as compared to butyrylcholinesterase. In the present studies we have investigated the relative potencies of other ChEIs for protecting neurons from excitatory amino acid (EAA)-induced toxicity. The ChEIs used in these studies were Hup-A, tacrine, E2020, physostigmine and dimethyl Hup-A (DMH). The cells were pretreated with graded concentrations of ChEI and then challenged with either glutamate or NMDA. Viability of the neuron was assessed after 24 hours by MTT reduction assay and in some cases also by measuring extra-cellular LDH activity. The results have shown that all these inhibitors protected the primary neurons in a dose dependant manner. However the degree of protection varied. The highest protection offered was by Hup-A followed by DMH, physostigmine, E2020, and the least protection was offered by tacrine. The present data suggest that the neuronal protection against EAA-induced cytotoxicity offered by various ChEIs is independent of their efficacy as ChE inhibitors.

Reversal of Glycerol Ether-Stimulated Acetylcholinesterase Activity by c-fos Antisense Oligonucleotide in Primary Neuronal Cultures

We have previously reported that dodecylglycerol (DDG), an alkyl glycerol lipid similar to platelet activating factor (PAF), stimulates differentiation in primary neuronal cultures obtained from fetal rat cerebral cortex, hippocampus and cerebellum (Ved et al., 1994). The neuronal differentiation was evident both morphologically as development of axon-like extensions and biochemically as an increase in neuron specific enzyme activity (Ved et al., 1991). We also reported that DDG mediated neuronal differentiation resulted in a transient increase in c-fos mRNA levels (Ved et al., 1993). The objectives of present study were to determine if c-fos antisense oligonucleotide (ASO) would reverse DDG-stimulated acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities, and neuronal differentiation. Primary enriched neurons derived from embryonic (E-17) rat cerebral cortex, cerebellum or hippocampus were treated in a serum-free media with either vehicle, DDG (4 μM) or DDG with either c-fos antisense oligonucleotide (ASO) (5–20 μM), c-myc ASO or non-sense ASO for 24 hrs. Treatment with DDG produced a maximal stimulation in AChE and ChAT activities, and outgrowth of neuronal processes in cultures obtained from cerebellum and a minimum effects in those obtained from cerebral cortex. The percent stimulation in AChE activity following DDG treatment in these neuronal cells were approximately 200%, 150% and 65% above the control levels in cerebellum, hippocampus and cortex, respectively. The percent stimulation in ChAT activity following DDG treatment in neuronal cells were approximately 260%, 220% and 45% above the control levels in cerebellum, hippocampus and cortex, respectively. Pretreatment with c-fos ASO partially inhibited DDG-stimulated AChE and ChAT activities and outgrowth of the neuronal processes, however, c-myc ASO and nonsense ASO had no significant effect. The inhibitory effect of c-fos ASO on DDG-stimulated AChE and ChAT activities and neuronal differentiation was greatest in neurons obtained from cerebellum and hippocampus (100%–52% inhibition). These results suggest a causative role of c-fos proto-oncogene in DDG-mediated neuronal differentiation and further suggest a direct role of c-fos gene product on modulation of AChE and ChAT enzyme activities in the neuronal cells.