**3.3 QUIN and oxidative stress**

One of the putative causes of ALS is the increased production and accumulation of reactive oxygen species (ROS) leading to oxidative stress and lipid peroxidation. Toxicity induced by QUIN has been related to increase ROS and oxidative stress. Intracerebral injection of QUIN shows neuronal damage and increase in ROS content occurring as early as 4 hrs after administration (Ganzella *et al.* 2006).

The lipid peroxidative effect of QUIN has also been demonstrated *in vivo* in adult rat brain (Rios and Santamaria 1991), and in rat brain synaptosomes *in vitro* (Santamaria *et al.* 2001). Similarly, in sheep foetal brain infused with QUIN, 4-hydroxynonenal (4-HNE), a toxic product of lipid peroxidation, immunoreactivity was observed in Purkinje cells and in the cytoplasm of cell bodies and dendrites, reaching into the molecular layer of the cerebellum (Yan *et al.* 2005). A sub-lethal dose of 4-HNE will also lead to the loss of spinal motor neurons in mice (Vigh *et al.* 2005). This may be a consequence of microglia activation, as 4- HNE is a potent activator of microglia, which will further contribute to neuroinflammation and oxidative stress in ALS (Hall *et al.* 1998).

In sporadic ALS patients, 4-HNE was enhanced in motor neurons and glia cells in the spinal cord (Shibata *et al.* 2001), and significantly elevated in the serum and CSF, correlating positively with the stage of disease (Simpson *et al.* 2004). CSF 4-HNE levels from sporadic ALS patients were also sufficient to cause the demise of motor neurons *in vitro* (Smith *et al.* 1998).

### **3.4 QUIN and mitochondrial dysfunction**

Mitochondrial dysfunction is a prominent feature of ALS and predisposes motor neurons to ionotropic glutamate receptor-mediated excitotoxicity (Kanki *et al.* 2004). Excitotoxicity may lead to the activation of mitochondrial permeability transition pore, resulting in mitochondrial swelling and progressive motor neuron death (Bendotti *et al.* 2001). Intracerebral injection of QUIN, in addition to being excitotoxic, also produces progressive mitochondrial dysfunction leading to time-dependent energetic dysfunction, which may be a common and critical event in the cell death cascade seen in ALS (Bordelon *et al.* 1997).

### **3.5 QUIN and the inflammatory cascade**

The presence of neuroinflammation is a pathological hallmark of ALS. Activated astrocytes and microglia are often seen in the degenerating areas surrounding injured motor neurons (McGeer and McGeer 2002). Elevated levels of chemokines and cytokines, such as monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)1-, chemokine ligand 5, interleukin (IL)-1 to IL-12, TNF- and IFN-, have been detected in both G93A SOD1 mice and ALS patients (Hensley *et al.* 2003; Wilms *et al.* 2003; Henkel *et al.* 2004). It has been demonstrated that QUIN can induce astrocyte proliferation and the production of chemokines, particularly MCP-1 (Croitoru-Lamoury *et al.* 2003; Guillemin *et al.* 2003; Ting 2008), and IL-1 messenger ribonucleic acid (mRNA) expression (Guillemin *et al.* 2003) in human astrocytes and macrophages.

contributes to excessive microenvironment glutamate concentrations and neurotoxicity via at least three mechanisms: (1) stimulation of synaptosomal glutamate release by neurons (Tavares *et al.* 2002); (2) inhibition of glutamate uptake into synaptic vesicle by astrocytes (Tavares *et al.* 2000); and (3) limiting glutamate to glutamine recycling in astrocytes by

One of the putative causes of ALS is the increased production and accumulation of reactive oxygen species (ROS) leading to oxidative stress and lipid peroxidation. Toxicity induced by QUIN has been related to increase ROS and oxidative stress. Intracerebral injection of QUIN shows neuronal damage and increase in ROS content occurring as early as 4 hrs after

The lipid peroxidative effect of QUIN has also been demonstrated *in vivo* in adult rat brain (Rios and Santamaria 1991), and in rat brain synaptosomes *in vitro* (Santamaria *et al.* 2001). Similarly, in sheep foetal brain infused with QUIN, 4-hydroxynonenal (4-HNE), a toxic product of lipid peroxidation, immunoreactivity was observed in Purkinje cells and in the cytoplasm of cell bodies and dendrites, reaching into the molecular layer of the cerebellum (Yan *et al.* 2005). A sub-lethal dose of 4-HNE will also lead to the loss of spinal motor neurons in mice (Vigh *et al.* 2005). This may be a consequence of microglia activation, as 4- HNE is a potent activator of microglia, which will further contribute to neuroinflammation

In sporadic ALS patients, 4-HNE was enhanced in motor neurons and glia cells in the spinal cord (Shibata *et al.* 2001), and significantly elevated in the serum and CSF, correlating positively with the stage of disease (Simpson *et al.* 2004). CSF 4-HNE levels from sporadic ALS patients were also sufficient to cause the demise of motor neurons *in vitro* (Smith *et al.* 1998).

Mitochondrial dysfunction is a prominent feature of ALS and predisposes motor neurons to ionotropic glutamate receptor-mediated excitotoxicity (Kanki *et al.* 2004). Excitotoxicity may lead to the activation of mitochondrial permeability transition pore, resulting in mitochondrial swelling and progressive motor neuron death (Bendotti *et al.* 2001). Intracerebral injection of QUIN, in addition to being excitotoxic, also produces progressive mitochondrial dysfunction leading to time-dependent energetic dysfunction, which may be a common and critical event in the cell death cascade seen in ALS (Bordelon *et al.* 1997).

The presence of neuroinflammation is a pathological hallmark of ALS. Activated astrocytes and microglia are often seen in the degenerating areas surrounding injured motor neurons (McGeer and McGeer 2002). Elevated levels of chemokines and cytokines, such as monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein (MIP)1-, chemokine ligand 5, interleukin (IL)-1 to IL-12, TNF- and IFN-, have been detected in both G93A SOD1 mice and ALS patients (Hensley *et al.* 2003; Wilms *et al.* 2003; Henkel *et al.* 2004). It has been demonstrated that QUIN can induce astrocyte proliferation and the production of chemokines, particularly MCP-1 (Croitoru-Lamoury *et al.* 2003; Guillemin *et al.* 2003; Ting 2008), and IL-1 messenger ribonucleic acid (mRNA) expression (Guillemin *et al.* 2003) in

decreasing glutamine synthetase activity (Baverel *et al.* 1990).

**3.3 QUIN and oxidative stress** 

administration (Ganzella *et al.* 2006).

and oxidative stress in ALS (Hall *et al.* 1998).

**3.4 QUIN and mitochondrial dysfunction** 

**3.5 QUIN and the inflammatory cascade** 

human astrocytes and macrophages.

### **3.6 QUIN and apoptosis**

In ALS, apoptosis is evident from the increased expression of pro-apoptotic protooncogenes, BCl-2 and c-jun, and caspases 1 and 3 in tissue, and from the morphological features of apoptosis displayed by dying motor neurons. QUIN has been demonstrated to induce neuronal and astrocytic apoptosis involving the activation of caspase 3 (Macaya *et al.* 1994; Jeon *et al.* 1999; Guillemin *et al.* 2005). Astrocytes are essential for the homeostasis of the CNS and so, the well-being of neurons. Hence, the loss of normal astrocytes in ALS would be detrimental to motor neurons and could exacerbate disease progression in ALS (Yamanaka *et al.* 2008).
