Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive death of cortical and spinal motor neurons, for which there is no effective treatment. Using a cell-based assay for compounds capable of preventing motor neuron cell death in vitro, a collection of approximately 40,000 low-molecular-weight compounds was screened to identify potential small-molecule therapeutics. We report the identification of cholest-4-en-3-one, oxime (TRO19622) as a potential drug candidate for the treatment of ALS. In vitro, TRO19622 promoted motor neuron survival in the absence of trophic support in a dose-dependent manner. In vivo, TRO19622 rescued motor neurons from axotomy-induced cell death in neonatal rats and promoted nerve regeneration following sciatic nerve crush in mice. In SOD1G93A transgenic mice, a model of familial ALS, TRO19622 treatment improved motor performance, delayed the onset of the clinical disease, and extended survival. TRO19622 bound directly to two components of the mitochondrial permeability transition pore: the voltage-dependent anion channel and the translocator protein 18 kDa (or peripheral benzodiazepine receptor), suggesting a potential mechanism for its neuroprotective activity. TRO19622 may have therapeutic potential for ALS and other motor neuron and neurodegenerative diseases.Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder that selectively affects motor neurons in the spinal cord, brainstem, and cortex. ALS affects people of all races and ethnic backgrounds with an incidence approximately 2 per 100,000 individuals (McGuire and Nelson, 2006). The onset of ALS is most common in the 55 to 75 year age range, and incidence rises with advancing age; men have a higher risk of developing the disease than women (Nelson, 1995). Common clinical features of ALS include muscle weakness and fasciculations. These occur predominantly in limbs, although bulbar onset pathology can also lead to tongue atrophy and dysphagia. Failure of the respiratory muscles and cardiac complications are generally the fatal event, occurring within an average of 3 years of disease onset, with only a 5% chance of survival 5 years after diagnosis (del Aguila et al., 2003). Although 5 to 10% of ALS This work was supported by the Association Française contre les Myopathies.1 Current affiliation: Center for Motor Neuron Biology and Disease, Columbia University, New York.Article, publication date, and citation information can be found at

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1-This paper examines in detail the activation of bovine and porcine trypsinogens and of bovine chymotrypsinogens A and B by trypsin and aspergillopeptidase A. Kinetic data have also been obtained ( K , and kcat) for the hydrolysis catalyzed by these proteases of several model peptides with sequences related to the N-terminal sequence of bovine trypsinogen.2. The N-terminal sequence of (aspartyl), residues is not necessary for the recognition of the strategic Lys-Ile bond of trypsinogen.3. We have shown previously that there are two binding sites for Ca2 i-on trypsinogen. One of these sites is constituted by the 2 aspartyl residues which are the nearest neighbours of the important Lys-Ile bond. The saturation of the site by Ca2+ improves the formation of the trypsinogen-trypsin complex; Ca2+ has no effcct on the rate of decomposition of t,his complex. I n the activations by aspergillopeptidase A, trypsinogen is a much better substrate than chymotrypsinogen. The implications of this exceptionally slow hydrolysis of the Lys-Ile bond are discussed. The problem of the formation of inert proteins in particular appears to be closely related to the very poor quality of this bond as a substrate for trypsin. A mechanism is given for the formation of inert proteins ; a similar mechanism also explains the degradative autolysis of trypsin. 4.The sequence of trypsinogen has been elucidated recently [l, 21 and the covalent changes occurring in the course of the activation of the zymogen have been known for several years [3]. However, there are still two main problems related to the mechanism of the transformation into trypsin. The first one concerns the nature of the morphological changes occurring in the course of the activation which result in the correct positioning of the different elements of the active center. This problem has been studied and is still being studied in this laboratory using both physicochemical and chemical approaches [4-71. The second problem, which is the subject of this paper, is related Unusual Abbreviations. BzArgOEt, a-N-benzoyl-xuginine ethyl ester; AcTyrOEt, N-acetyl-L-tyrosine ethyl ester, to the effect of the unusual sequence Asp-Asp-AspAsp, just preceding the Lys-Ile bond which is hydrolyzed as the first step in the activation process.This sequence has been found in bovine [S, 91, porcine [lo] and ovine [ll] trypsinogens. The peptide sequences liberated in the course of the activation are Val-(Asp),-Lys [S, 9,111 and Phe-Pro-Thr-(Asp),-Lys [10,11]. The kinetic data which relate the effect of a sequence of four aspartyl residues on the rate of hydrolysis of an adjacent trypsin sensitive bond in model peptides, to the mode of activation of bovine and porcine trypsinogens, and of bovine chymotrypsinogens A and B, constitute as far as we are aware, tfhe first detailed study of the proteolytic action of trypsin (and also of aspergillopeptidase A) on a specific bond in large peptides and proteins. Up

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