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FIGURE 6.18 (A) A "Trojan Horse" strategy to allow a small bifunctional molecule to bind to a chaperone and gain the steric bulk required to disrupt amyloid P aggregation. One end of the bifunctional molecules binds to the FK506 binding protein, a member of the FKBP chaperone family of peptidyl prolyl cis-trans isomerases that are highly expressed in all mammalian cells. The other end of the bifunctional molecule interacts with AP to block aggregation. (B) One such bifunctional molecule is SLF (synthetic ligand for FKBP) coupled to CR (Congo Red). CR interacts with Ap. (C) Electron micrographs of AP fibrils formed in solution when incubated with or without FKBP in the presence of CR or SLF-CR. SLF-CR + FKBP clearly inhibits the formation of Ap fibrils. Reproduced with permission from Gestwicki et al., Harnessing chaperones to generate small-molecule inhibitors of amyloid P aggregation. Science 306:865-869. Copyright 2004, AAAS.

The remainder show familial inherited forms linked to mutations in the Cu/Zn-dependent superoxide dismutase-1 (SOD-1) gene. SOD-1 is a mitochondrial enzyme that converts the highly reactive and destructive superoxide anions produced by the one-electron reduction of O2 in metabolic reactions into hydrogen peroxide and O2. This mutation may thus cause cellular destruction because it does not function properly and may also cause the protein to fold abnormally and have a cytotoxic effect on motor neurons (Cova et al., 2004).

Chronic glutamate-toxicity is a major factor in the progressive death of motor neurons (Shaw and Ince, 1997). In the spinal cord and brain, astrocytes prevent glutamate toxicity by extending processes into the vicinity where two motor neurons synapse. These processes have glutamate transporters (GLT1) that recover glutamate molecules, thus turning down the glutamate signal after it has been received. In mouse models of ALS, and in the human disease, GLT1 expression is downregulated in astrocytes and ALS patients have elevated levels of glutamate in their cerebrospinal fluid (Plaitakis et al., 1988; Rothstein et al., 1990, 1992, 1995, 2005; Lin et al., 1998). There is also a defect in the editing of the mRNA encoding the GluR2 subunit of glutamate a-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors on spinal motor neurons (Kawahara et al., 2004). These features suggest that key elements of progressive neuronal cell death in ALS are astrocyte dysfunction that results in reduction in glutamate clearance and compromised functioning of glutamate receptors on spinal motor neurons. Another feature of ALS is the accumulation of neurofilaments. Mice transgenic for the mouse neurofilament light gene and the human neurofilament heavy gene display large motor neuron degeneration associated with neurofila-

ment accumulation and altered neurofilament phosphorylation (Xu et al., 1993; Cote et al., 1993); these features have been observed in human ALS as well (Manetto et al., 1988; Hirano and Kato, 1992). Neurons die by apoptosis, in which the caspase family plays a prominent role (Namura et al., 1998).

Several mouse models of ALS are useful for developing therapeutic strategies. The pmn/pmn mouse exhibits progressive motor neuron degeneration detectable as hindlimb weakness during the third week of life, and dies at ~6 weeks of age (Schmalbruch et al., 1991). The G (lysine) 93 A(alanine) mouse is transgenic for a mutated human SOD-1 gene associated with human familial ALS (Wong et al., 2002). This mouse also exhibits a progressive motor neuron degeneration that manifests itself in locomotor deficits at 13 weeks of age, and dies at ~4-5 months of age. A third model is the organotypic culture of mouse spinal cord in the presence of the glutamate transport inhibitor, threo-hydroxyaspartate (THA), which causes gluta-mate-mediated motor neuron cell death (Corse and Rothstein, 1995). These models have been used to test the efficacy of pharmaceutical and cell therapies on ALS.

a. Neuroprotective Cell Transplants

Wild-type astrocytes delay the degeneration and significantly extend the survival of mutant SOD1-expressing motoneurons in ALS mice (Clement et al., 2003; Klein et al., 2005), suggesting that they may counter the glutamate toxicity characteristic of ALS.

Transplants of stem cells into the spinal cord have been reported to have positive effects in the G93A mutant mouse model. Human umbilical cord blood cells were reported to delay the progression of ALS in these mice (Chen and Ende, 2000; Ende et al., 2000). The use of these cells was predicated on the idea that NSCs are "migrating HSCs," based on the fact that NSCs are in close contact with capillaries and on the reports that HSCs could be induced to differentiate into neurons (Chapter 13). The cells migrated toward sites of motor neuron degeneration and expressed neural and glial markers (Garbuzova-Davis et al., 2003), suggesting that they had undergone lineage conversion and replaced host motor neurons. However, the number of injected cells expressing neural markers was low. More likely, their effect was due largely to a para-crine protective action on host motor neurons through secreted factors. Dental pulp stem cells were reported to promote the survival of sensory neurons of the trigeminal ganglion and of injured motor neurons of mouse spinal cord through their production of GDNF (Nosrat et al., 2004).

Injection of neurons from a human neuron cell line (hNT) into the ventral horn of the spinal cord in the G93A mutant mouse was reported to delay the onset of motor dysfunction and extend the average lifespan of the mice (Garbuzova-Davis et al., 2002). The mechanism of action of the injected cells was not clear, however, and could have been through replacement of dying motor neurons, protection of host motor neurons through paracrine factors, or both.

Autologous peripheral blood stem cells and bone marrow cells have been injected into the spinal cords of several ALS patients in Phase I clinical cell therapy trials (Janson et al., 2001; Mazzini et al., 2003; Cova et al., 2004; Silani et al., 2004). These trials indicated that cells could be transplanted into the spinal cord with minor and reversible side effects. In the bone marrow cell trial (Mazzini et al., 2003), the linear decline in strength was slowed somewhat in some of the muscles in four of seven patients, and two of the seven actually experienced a mild increase in strength.

Fetal OECs have also been used by Huang Hongyen to treat ALS patients in Bejing's Chaoyang Hospital (Watts, 2005; Cyranoski, 2005; Curt and Dietz, 2005). Injection of over 1-2 x 106 fetal OECs into the frontal lobes of ALS patients under local anesthetic was reported to stabilize and even improve the patient's condition in about half the cases through paracrine effects. About 100 ALS patients from around the world have been treated in this way.

b. Neuroprotective Pharmaceutical Agents

Pharmaceutical neuroprotection is an area of intense focus for the treatment of ALS. Two neurotrophins known to promote the survival of motor neurons, CNTF and BDNF, were tested in clinical trials for their efficacy in halting the progression of ALS. Both drugs were failures (ALS CNTF Study Group, 1996; Cederbaum et al., 1998). Some positive effect of IGF-1 was reported in a North American clinical trial (Lai et al., 1997), but a similar European trial did not show any effect (Borasio et al., 1998). In mice, IGF-1, GDNF, and NT-4/5 were protective of motor neurons in spinal cord cultures treated with THA, whereas BDNF, CNTF, NT-3, FGF-2, and LIF were not protective (Corse et al., 1999), consistent with the results of the human clinical trials (FIGURE 6.19).

GDNF is of particular interest as a possible therapeutic agent, given its effectiveness in protecting dopaminergic neurons and motor neurons after axotomy-induced cell death in adults (Oppenheim et al., 1995) and during the embryonic period of programmed neural cell death (Zhao et al., 2004).

FIGURE 6.19 Sections of cultured rat spinal cord slices treated with the glutamate transport inhibitor threo-hydroxyaspartate (THA). Arrow indicates motor neurons. The sections were stained with a monoclonal antibody against neurofilament-H (THA). (A) Control cord. (B) THA-treated, showing severe loss of motoneurons. (C) IGF-I protects motor neurons from TA. Reproduced with permission from Corse et al., Preclinical testing of neuroprotective neurotrophic factors in a model of chronic motor neuron degeneration. Neurobiol Dis 6:335-346. Copyright 1999, Elsevier.

Control THA IGF-I + THA

FIGURE 6.19 Sections of cultured rat spinal cord slices treated with the glutamate transport inhibitor threo-hydroxyaspartate (THA). Arrow indicates motor neurons. The sections were stained with a monoclonal antibody against neurofilament-H (THA). (A) Control cord. (B) THA-treated, showing severe loss of motoneurons. (C) IGF-I protects motor neurons from TA. Reproduced with permission from Corse et al., Preclinical testing of neuroprotective neurotrophic factors in a model of chronic motor neuron degeneration. Neurobiol Dis 6:335-346. Copyright 1999, Elsevier.

GDNF administered to pmn/pmn mice by subcutane-ously implanting encapsulated BHK fibroblasts trans-fected with the GDNF gene reduced the loss of facial motor neurons by 50%, but did not extend the lifespan of the mice because, although it protected neuron cell bodies, it did not prevent axon degeneration (Sagot et al., 1996). GDNF retroviral constructs, delivered in transfected myoblasts to the gastrocnemius muscles of G93A mutant mice, resulted in a shift in the size distribution of motor neurons toward larger neurons compared with untreated G93A controls, although the average size and number of large motor neurons were less than in wild-type mice (Mohajeri et al., 2004). Quantitative RT-PCR showed that the GDNF DNA and mRNA persisted for up to 12 weeks postgrafting. Furthermore, GDNF prolonged the onset phase of the disease, slowed muscle atrophy, and delayed the deterioration of performance in tests of motor behavior.

Klein et al. (2005) demonstrated the feasibility of using human cells expressing GDNF as therapy for ALS. Human neural progenitor cells isolated from fetal cortex were expanded in vitro and infected with a GDNF lentiviral construct. When transplanted into the lumbar spinal cord of a G93A rat model of ALS, the cells differentiated into astrocytes, survived for up to 11 weeks and were integrated into the gray and white matter of the cord without observable side effects. The transplanted cells were shown to secrete GDNF in the area of transplant and to stimulate the upregulation of cholinergic markers in nerve fibers. Although counts of ChAT positive neurons revealed no differences in motor neuron number between treated rats and controls, there was a significant difference between the size of the cell body in the treated rats versus controls. However, the transplanted cells did not improve measures of limb strength and coordination.

VEGF33 mice, in which the hypoxia-response element in the VEGF promoter is deleted, have an impaired ability to upregulate VEGF levels under conditions of stress and develop ALS-like neuropathology, suggesting that motor neurons are sensitive to lack of VEGF (Oosthuyse et al., 2001). Injection of a lentiviral VEGF construct bilaterally into the gastrocnemius, diaph-gram, intercostal, facial, and tongue muscles of G93A mutant mice prior to ALS onset slowed progression of the disease and extended life expectancy by as much as 30% (table 6.7). There was a 15% increase in life expectancy when the construct was injected after disease symptoms were manifest (Azzouz et al., 2004). Interestingly, injection of the GDNF gene in a similar construct resulted in only a 5% increase in life span.

Molecules other than neurotrophic and growth factors may hold promise in treating ALS. Overexpression of the gene for the apoptosis inhibitor, Bcl-2, delayed the onset of ALS symptoms by 20%, increased survival time by 15%, and preserved significantly more motor neurons and myelinated axons in the phrenic nerve (Kostic et al., 1997). Caspase-3 is activated in mouse and human ALS (Li et al., 2000). Inhibiting caspase activity in G93A mice with N-benzyloxycarbonyl-Val-Asp-fluoromethylketone (zVAD-fmk) delayed the onset of ALS and prolonged survival time by 22%. At 110 days of age, treated mice exhibited a significantly higher number of motor neurons in the cervical region of the spinal cord. The antibiotic minocycline, which also inhibits the activity

TABLE 6.7 Effects of equine infectious anemia virus-vascular endothelial growth factor (EIAV-VEGF) gene construct on ALS age of onset, age at death and number of surviving facial motor neurons at the end stage in G93A mice. EIAV-LacZ served as a control construct. VEGF improves all parameters. Data from Azzouz et al. (2004)

EIAV Vector

LacZ VEGF

TABLE 6.7 Effects of equine infectious anemia virus-vascular endothelial growth factor (EIAV-VEGF) gene construct on ALS age of onset, age at death and number of surviving facial motor neurons at the end stage in G93A mice. EIAV-LacZ served as a control construct. VEGF improves all parameters. Data from Azzouz et al. (2004)

EIAV Vector

LacZ VEGF

Age at onset

95 t 4

123 t 11

Age at death

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