Analysis of neuroprotective effects of valproic acid and the analogues in motor neurons

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disease characterized by substantial loss of motor neurons in the spinal cord, brain stem and motor cortex. Synaptic over-excitation (excitotoxicity) of vulnerable motoneurons is thought to be of major impact for the pathophysiology of this selective neurodegeneration. Valproic acid (2-propylpentanoic acid, VPA) is one of the most frequently prescribed antiepileptic drugs and acts mainly by a blockade of voltage dependent Na- and K-channels and by an enhancement of GABAergic inhibition as a gamma-aminobutyric acid (GABA) transaminase inhibitor.

In the present work we are investigating VPA and three analogous 3-propylheptanoic acid (3-PHA), R (+) – and S (-) -2-n-propyl-4-pentynoic acid (R and S-4-yn-VPA) for neuroprotective effects and related electrophysiological properties within the cultured network of cells. The survival effects of all these substances are investigated on dissociated rat embryonic ventral spinal cord cells enriched by density gradient centrifugation and seeded on Poly-D,L-ornithin/Laminin or on mono-layer of highly enriched neonatal rat Schwann cells, astrocytes.

We tested VPA and analogues alone or in combination with kainate after 48 hours incubation in 7–10 days old cell-cultures depending on feeding layer.

In a first set of calcium imaging experiments was showed high intracellular calcium transient after short pulses of kainate in pure motoneuron cultures 7 DIV (days in vitro) however 3 DIV did not react to kainate, probably due to a lack of expression glutamate receptor channels.

The double-staining experiments demonstrated that kainate caused 10% neuronal cell death in cocultures after 10 DIV even in low concentration such as 30µM and was dose dependent to motoneuron vulnerability. Neuroprotective effect accomplished by applying kainate with VPA, 3-PHA and S-4-yn-VPA, respectively was no significant except R-4-yn-VPA which surprisingly was neuroprotective against kainate. A direct interaction of VPA with AMPA receptor channels could be excluded using a piezo-driven fast coapplication of glutamate and VPA or analogues.

To sum up, we suppose that effect of the tested compounds is mild and the mechanism is not known yet. Neuroprotective capacities are supposed to arise from inhibition of histone deacetylases, although adverse effects such as teratogenecity and liver toxicity are also widely known.