Thiamine Deficiency Increases Ca2+ Current and CaV1.2 L-type Ca2+ Channel Levels in Cerebellum Granular Neurons

Abstract

Thiamine (vitamin B1) is co-factor for three pivotal enzymes for glycolytic metabolism: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase. Thiamine deficiency leads to neurodegeneration of several brain regions, especially the cerebellum. In addition, several neurodegenerative diseases are associated with impairments of glycolytic metabolism, including Alzheimer's disease. Therefore, understanding the link between dysfunction of the glycolytic pathway and neuronal death will be an important step to comprehend the mechanism and progression of neuronal degeneration as well as the development of new treatment for neurodegenerative states. Here, using an in vitro model to study the effects of thiamine deficiency on cerebellum granule neurons, we show an increase in Ca²⁺ current density and Ca_V1.2 expression. These results indicate a link between alterations in glycolytic metabolism and changes to Ca²⁺ dynamics, two factors that have been implicated in neurodegeneration.

Keywords: Ca2+ current; Cerebellum; Granular neuron; Neurodegeneration; Thiamine.

Fig. 1

Thiamine deficiency increases Ca²⁺ current density and shifts the voltage dependence for activation in cerebellum granular neurons. a Representative current traces recorded in response to 0 mV depolarization in thiamine (+) (Ctrl; gray line) and thiamine (−) (black line) conditions. b I–V relationships of Ca²⁺ channel current (measured using Ba²⁺ as charge carrier). Curves represent the best fit of Eq. 1 (see “Materials and Methods” section) to the data [n = 5 for thiamine (+) and n = 7 for thiamine (−)]. c Normalized G–V relationships obtained from the data in (b). Curves represent the best fit of Eq. 2 (see “Materials and Methods” section) to the data. d Steady-state inactivation. Curves represent the best-fit to Eq. 3 (see “Materials and Methods” section), *p < 0.05

Fig. 2

Immunoblotting for L-type Ca²⁺ channel in cerebellum granular neurons (CGN). CGN cultures were maintained in thiamine (−) [n = 6] or thiamine (+) [n = 6] conditions and then lysed to quantify L-type Ca²⁺ channels using anti-Ca_V1.2 antibody (see “Materials and Methods” section). *p < 0.05

Fig. 3

Computational analysis of the effects of HVA Ca²⁺ current conductance on refractory periods and temporal summation. a Refractory periods. The model neuron was stimulated with pairs of synaptic inputs with intervals ranging from 1.5 to 160 ms. The conductance of the first synaptic input was set to 1.5 times action potential (AP) threshold, and the threshold of the second synaptic input to generate an action potential was measured as a function of paired-pulse interval. The symbol indicates the absolute refractory period. Black lines represent control thiamine (+) K⁺ currents, gray lines represent modified A-type K⁺ current as reported in Cruz et al. (2012). Solid lines represent control Thiamine (+) HVA Ca²⁺ currents, broken lines represent Thiamine (−) modified HVA Ca²⁺ currents. b Temporal summation. Threshold of a second synaptic conductance was measured when the first synaptic conductance was set to 0.9 times AP threshold. Same color code as for (a). For all cases, thresholds are normalized the control values (Black solid lines) at the longest interval