<p>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.</p>

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an incidence of 1.5-2.7/100000 people/year. Today there is no cure for the disease and only symptomatic treatments are available. ALS progresses rapidly and only 50% of the patients are alive three years after the symptom debut. In ALS, the upper and lower motor neurons undergo degeneration in a process resembling apoptosis. This leads to muscle atrophy and paralysis. The causes of neuronal death are however unknown. In this thesis we have studied transgenic mice carrying human mutant superoxide dismutase, as a model for familial ALS. These mice develop ALS-like symptoms after four months of age with degeneration of the motor neurons. Our results show an involvement of endoplasmic reticulum stress, caspase-12, -9, -3 and procaspase-7 in the ALS mice spinal cord. Overexpression of the antiapoptotic protein XIAP in spinal cord neurons inhibited the activation of caspase-12 and reduced caspase-3 and calpain activity. Calpastatin, the regulator of calpain activity, was kept intact in the ALS-XIAP mice. These mice showed a 12% increase in the mean survival suggesting a beneficial effect of XIAP in ALS. The reason for the ultimate cell death of motor neurons in the ALS-XIAP mice may be due to the activation of additional cell death pathways. Thus, we observed that lysosomal proteases particularly, cathepsinB, -D, and -L were activated in the ALS mice spinal cord together with a less marked upregulation of the inhibitors, cystatinB and -C. We also found activation of astrocytes and microglial cells in the spinal cord of ALS mice indicating their involvement in the disease. The results show that both caspase-dependent and -independent pathways are activated during neuronal degeneration in the ALS spinal cord. The results obtained may help to identify novel drug targets for future treatments of ALS.

<p>The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO₂) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO₂) and the reasons for the strikingly different toxicity were investigated. </p><p>A strong bioactivation and protein adduction of 2,6-diClPh-MeSO₂ in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO₂ was observed and the bioactivation in the liver for both chlorinated isomers was negligible. <i>In vitro</i> studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO₂. The 2,6-diClPh-MeSO₂-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated <i>in vivo</i>, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. </p><p>Based on these results, the proposed mechanism for 2,6-diClPh-MeSO₂-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO₂.</p>

The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO2) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO2) and the reasons for the strikingly different toxicity were investigated. A strong bioactivation and protein adduction of 2,6-diClPh-MeSO2 in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO2 was observed and the bioactivation in the liver for both chlorinated isomers was negligible. In vitro studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO2. The 2,6-diClPh-MeSO2-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated in vivo, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. Based on these results, the proposed mechanism for 2,6-diClPh-MeSO2-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO2.