In the Rat Brain Acetyl-l-carnitine Treatment Modulates the Expression of Genes Involved in Neuronal Ceroid Lipofuscinosis

Abstract

Acetyl-L-carnitine (ALC) is a naturally occurring substance that, when administered at supraphysiological concentration, is neuroprotective. It is a molecule of considerable interest for its clinical application in various neural disorders, including Alzheimer's disease and painful neuropathies. Suppression subtractive hybridization methodology was used for the generation of subtracted cDNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after ALC treatment. The method generates an equalized representation of differentially expressed genes irrespective of their relative abundance and it is based on the construction of forward and reverse cDNA libraries that allow the identification of the genes which are regulated by ALC. We report that ALC treatment: (1) upregulates lysosomal H(+)/ATPase gene expression and (2) downregulates myelin basic protein gene expression. The expression of these genes is altered in some forms of neuronal ceroid lipofuscinosis (NCL) pathologies. In this case, ALC might rebalance the disorders underlying NCL disease represented by a disturbance in pH homeostasis affecting the acidification of vesicles transported to lysosomal compartment for degradation. This study provides evidence that ALC controls genes involved in these serious neurological pathologies and provides insights into the ways in which ALC might exert its therapeutic benefits.

Modulation of Myelin Basic Protein Gene Expression by Acetyl-l-Carnitine

“…After the construction of forward and reverse cDNA libraries, the screening of the positive clones was carried out by dot blot screening. Following comparison with the sequences in the GenBank nucleotide database, we identified various clones as true positive, corresponding to differentially expressed genes after ALC treatment [3,7,9]. In this study, we reported a downregulation in the expression of the genes coding for all the MBP isoforms.…”

“…In our previous studies, we observed that a chronic ALC treatment modulates different gene expressions in the rat brain, and that the majority of detected clones are involved in neuroprotection and neuromodulation [3,9].…”

“…The proteins extracted from rat brains of control and ALCtreated (100 mg/kg bw) animals were analyzed by SDS-PAGE, transferred onto filter membrane, and stained with Ponceau S red according to [3,9]. The membranes were then incubated overnight at 4°C using either anti-MBP (1:1,000) or anti-G3PDH (1:500) antibodies (Santa Cruz Biotechnology Inc., CA, USA), diluted in the presence of PBS and 0.05% Tween 20.…”

Cytoprotective Effect of Acetyl-l-Carnitine Evidenced by Analysis of Gene Expression in the Rat Brain

“…Reverse transcriptions were carried out with total RNA (4 μg) isolated from treated and control rat brains using the SuperScript™ II RNase H − reverse transcriptase (Invitrogen) and with the Oligo(dT) 12-18 primer (Invitrogen) according to the manufacturer's instructions as reported in Traina et al [22]. One microliter of first-strand cDNAs was used for each PCR amplification.…”

“…Two different messenger RNA (mRNA) populations are compared so as to obtain clones of genes that are differentially expressed [20]. Previously, the construction of forward and reverse cDNA libraries allows us to identify genes that are up-regulated by ALC, such as subunit γ of 14,3,3 protein, heat shock protein 72, voltagedependent anion channel, lysosomal H + /ATPase, V1 subunit D, and kinesin 1 light chain, and down-regulated genes such as ATP synthase lipid-binding protein, subunit c, and myelin basic protein (MBP) [21][22][23][24].…”