Therapies For Neurodegenerative Diseases

Demyelinating disorders, Parkinson's disease, Huntington's disease, Alzheimer's disease, and amyo-trophic lateral sclerosis (ALS) are neurodegenerative diseases that strike humans at different ages and show variable courses of progression. Huntington's disease is clearly due to a single gene mutation, as are a small percentage of ALS and Parkinson's cases. The vast majority of ALS cases, demyelinating disorders, and Parkinson's disease, however, are multifactorial in origin. Currently, about 10% of the population suffers from neurodegenerative diseases. The over-60 population will double by 2050, greatly increasing this percentage and placing a tremendous strain on healthcare systems.

Although each neurodegenerative disorder differs in the kinds of neurons affected, etiology, symptoms, and progression, a common feature is the presence of intraneuronal neurofibrillary tangles (NFTs) and intra- or interneuronal plaques in the affected areas of the CNS. These pathological inclusions are due to protein mis-folding caused by genetic mutation or undefined causes. Current thinking is that the misfolded proteins themselves are neurotoxic and that sequestering them in plaques and NFTs is a neuroprotective mechanism (Mattson, 2004; Arrasate et al., 2004). However, some results with agents that prevent aggregation of the misfolded proteins in Alzheimer's disease suggest that the plaques and NFTs themselves may also be neurotoxic. NFTs in Alzheimer's disease are composed of the hyperphosphorylated microtubule-associated tau protein. Interestingly, transgenic mice expressing doxycycline-suppressible tau accumulated NFTs, and exhibited neuronal loss, and cognitive defects that were reversed by suppressing tau expression (SantaCruz et al., 2005). However, NFTs continued to accumulate, suggesting that it is not the NFTs themselves that are neurotoxic in Alzheimer's disease.

Animal experiments indicate that fetal neural cells and adult NSCs have a remarkable ability to migrate throughout, and integrate into, the CNS of fetal and adult hosts (Tate et al., 2001). Mouse neural precursors derived from ESCs integrate into the hippocampal circuitry and differentiate as neurons when placed on slice cultures of the hippocampal region of young rats (Benninger et al., 2003). Human fetal brain cells (53-74 days postconception), marked with a retroviral lacZ construct, were transplanted into the brain ventricles of embryonic (E17-18) or neonatal rats and tracked by several markers: P-galactosidase expression; a human-specific probe to the alu repeat element; and a human-specific antibody to glutathione-S-transferase (Flax et al., 1998; Brustle et al., 1998). The transplanted cells migrated into every region of the host brain, where they differentiated into site-specific neurons and glia in the organization characteristic of each region. Human NSCs behaved likewise when injected into adult rat brains, though their response was less marked than in host neonatal brain (Fricker et al., 1999). Human NSCs differentiated in vitro from ESCs by FGF-2 or EGF plus FGF-2, localized to subventricular areas after grafting into the cerebral ventricles of neonatal mice (Zhang et al., 2001; Reubinoff et al., 2001). Freeze damage to the adult mouse brain induces the production of stem cell factor in neurons within the injury zone and the migration of NSCs by their activation of the SCF receptor, c-kit. Furthermore, damage to brain neurons by ischemia and neurodegenerative disease results in increased NSC proliferation and differentiation into new neurons (Raber et al., 2004; Curtis et al., 2003; Jin et al., 2004), indicating that the brain initiates regeneration by NSCs in response to injury. These observations, among many others, have justified the hope that pharmaceutical agents and/or cell therapy will be potentially useful in treating neurodegenerative diseases (Zhang et al., 2003; Rice and Scolding, 2004; Goldman, 2005).

1. Demyelinating Disorders

Brustle et al. (1999) injected glial precursor cells differentiated in vitro from mouse ESCs into the spinal cord and brain of rats that have the equivalent of a human demyelinating disorder, Pelizaeus-Merzbacher disease. This disease is caused by a mutation in the X-linked gene for myelin proteolipid protein (PLP). The mouse ESCs were placed in defined medium with FGF-2 and PDGF to produce proliferating glial precursors. These precursor cells were then injected into the spinal cord and brain of seven-day-old myelin-deficient rats. Two weeks after injection, electron microscopy and staining of the tissues with a probe to mouse satellite DNA and antibody to PLP showed that the injected cells had differentiated into oligodendrocytes that formed myelin sheaths around the axons (FIGURE 6.10).

Nistor et al. (2005) developed a reliable protocol for the directed differentiation of oligodendrocyte precursors from human ESC cultures over 42 days, based on signaling molecules known to regulate the survival, proliferation, migration, and differentiation of cells at various steps of the oligodendrocyte lineage (Grinspan, 2002). The principal components restricting differentiation to the oligodendroglial lineage were insulin, tri-iodothyroidin hormone, FGF, and EGF. Commitment of the hESCs to the oligo lineage was confirmed by their expression of a panel of molecular markers including Oligl, SC10, A2B5, NG2, and PDGFRa. When these cells were transplanted into the spinal cord of shiverer mice, which suffer from dysmyelination due to homozygosity for a mutation in the myelin basic protein gene (Chernoff, 1981), they integrated into the cord tissue, differentiated into oligodendrocytes and produced compact myelin. Likewise, clonal NSCs

Shiverer Mice

FIGURE 6.10 Differentiation of ESC-derived mouse glial precursors into oligodendrocytes after injecting them into the spinal cord (dorsal columns) of myelin-deficient rats. (A) Saggital section stained with antibody to myelin proteolipid protein (PLP). The asterisk marks the injection site and the arrows indicate the directions of migration of the injected cells. Abundant PLP is evident. (B) In situ hybridization of the boxed area in (A) with a probe to mouse satellite DNA shows that the myelin is generated by donor cells. (C) Double staining with a probe to mouse satellite DNA and an antibody to GFAP shows that the ESC derivatives also differentiated into astrocytes. Toluidine blue-stained semithin cross sections show the myelin deficiency in an untreated rat (D) and (E) new myelin derived from ESC glial derivatives two weeks after injection. (F) Electron micrograph of an oligodendrocyte contacing numerous myelinated axons. Inset, high magnification shows the layered structure of the myelin. Reproduced with permission from Brustle et al., Embryonic stem cell-derived glial precursors. A source of myelinating transplants. Science 285-754-756. Copyright 1999, AAAS.

FIGURE 6.10 Differentiation of ESC-derived mouse glial precursors into oligodendrocytes after injecting them into the spinal cord (dorsal columns) of myelin-deficient rats. (A) Saggital section stained with antibody to myelin proteolipid protein (PLP). The asterisk marks the injection site and the arrows indicate the directions of migration of the injected cells. Abundant PLP is evident. (B) In situ hybridization of the boxed area in (A) with a probe to mouse satellite DNA shows that the myelin is generated by donor cells. (C) Double staining with a probe to mouse satellite DNA and an antibody to GFAP shows that the ESC derivatives also differentiated into astrocytes. Toluidine blue-stained semithin cross sections show the myelin deficiency in an untreated rat (D) and (E) new myelin derived from ESC glial derivatives two weeks after injection. (F) Electron micrograph of an oligodendrocyte contacing numerous myelinated axons. Inset, high magnification shows the layered structure of the myelin. Reproduced with permission from Brustle et al., Embryonic stem cell-derived glial precursors. A source of myelinating transplants. Science 285-754-756. Copyright 1999, AAAS.

transplanted into the cerebral ventricles of shiverer mice at birth migrated throughout the brain and remy-elinated up to 52% of axons, reducing the tremor of a number of recipient animals (Yandava et al., 1999).

Human olfactory ensheathing cells were used to remyelinate rat dorsal spinal cord axons that were focally demyelinated by X-irradiation and injection of ethidium bromide (Kato et al., 2000). The OECs, prepared from adult olfactory nerves removed during surgery for nasal melanoma, were injected into the lesioned areas. Examination of tissue sections three weeks later by electron microscopy and in situ hybridization with the COT-1 human DNA probe revealed remyelination of the axons by the human cells.

Multiple sclerosis (MS) is an autoimmune CNS disorder in which myelin is damaged and astrocytes proliferate to form scar, resulting in the blockade of electrical impulses along nerve axons, loss of sensation and coordination, and in severe cases, paralysis and blindness (Steinman, 1996). It affects ~2.5 M people worldwide and is one of the most common neurological diseases of young adults (Zamvil and Steinman, 2003). Typically, the disease follows a relapsing and remitting pattern in which acute demyelinating episodes are followed by the generation of new oligodendrocytes and remyelination, but with increasing numbers of lesions that fail to remyelinate.

Arresting the progression of the disease has been a major focus of research on MS. Currently, the inter-feron-based drugs Avonex (INFP-Ia), Betaseron (INFp-Ib), and Rebif (INFp-Ia), and the synthetic peptide co-polymer glatiramer acetate (Copaxone) are FDA-approved to treat relapsing forms of MS. Experimental work on a mouse model of MS, autoimmune encephalomyelitis (EAE), and clinical trials in humans suggest that attacks of MS can be aborted or diminished by blocking the a4 integrin subunit of a4p1 and a4 p7 integrins on the surface of the attacking immune cells (Yednock et al., 1992; Steinman, 2001; Miller et al., 2003) by the use of drugs such as Tysabri (natalizumab), interfering with immune cell interactions with anti-CD154 antibody (Couzin, 2005), or by high-dose immunosuppression and reconstitution of the immune system by autologous transplantation of HSCs (Fassas and Kazis, 2003).

Regardless of how far the disease has progressed at the point of arrest, we want to be able to repair whatever damage has occurred to that point. Such repair has been accomplished in EAE mice by the intravenous injection of neurospheres derived from NSCs of the lateral ventricles of the brain (Pluchino et al., 2003). The NSCs express the same a4 integrin expressed by attacking immune cells; they homed to sites of demye-lination where they differentiated into oligodendro-cytes and new neurons. Astrogliosis and axon damage were markedly reduced, and this was associated with a reduction in the expression of TGF-P and FGF-2, factors known to promote astrogliosis, while expression of other factors such as CNTF, NT3, NGF, GDNF, BDNF, and LIF were unchanged (FIGURE 6.11). Nearly 27% of the mice experienced remyelination with complete functional recovery from paralysis, whereas controls showed no sign of recovery. Interestingly, donor NSCs provided only 20% of the new oligodendrocyte precursors in sites of remyelination, the remainder being host cells. This observation suggests that the injected cells can induce host NSCs to differentiate into oligodendrocytes and astrocytes. The cells induce apoptosis of inflammatory T-cells that infiltrate the CNS, conferring long-lasting neuroprotection (Pluchino et al., 2005).

A chronic form of EAE can be induced by injecting wild-type mice with myelin oligodendrocyte glycopro-tein (MOG). Mice deficient in Nogo A showed a significant delay in onset of EAE and significantly milder symptoms, suggesting that Nogo A plays a role in the development of EAE. MOG-induced development of EAE is suppressed in mice vaccinated against a Nogo A peptide (Nogo 623-640) or passively immunized with anti-Nogo 623-640 IgGs. This peptide thus appears able to induce an immune response that blunts the development of EAE and may be an appropriate target for the development of therapies for MS (Karnezis et al., 2004).

Recent work suggests that the bHLH transcription factor Olig1 is required to repair demyelinated lesions. Olig1 and 2 are closely related transcription factors expressed in myelinating oligodendrocytes and their progenitors in the developing CNS. Olig2 is required for specification of both motor neurons and oligoden-drocytes (Lu et al., 2002). Olig1 is not required for this process, since the CNS of Olig1-null mice develops normally, with full myelination (Arnett et al., 2004). However, Olig1-null mice treated with cuprizone or lysolecithin, which demyelinate the brain and spinal cord, respectively, are unable to remyelinate. Remyelination in wild-type mice after these demyelin-ating treatments showed that Olig1 is located in the nucleus in oligodendrocyte precursors, but translocates to the cytoplasm in the differentiating oligodendro-cytes, suggesting that this translocation plays an essential role in the repair process (Arnett et al., 2004).

2. Parkinson's Disease

The striatum of the brain consists of several basal ganglia: the caudate nucleus; the putamen; and the globus pallidus. The electrical output of these ganglia is an important regulator of movement through a

Demyelinating Disease Luxol

FGF-II TGF-ß CNTF NT-3 NGF GDNF BDNF UF ■ i.e. ■ i.v. CD Controls

FIGURE 6.11 Intravenous or intracerebroventricular injection of NSCs into EAE mice reduces glial scarring and modulates expression of neurotrophic mRNAs. a, b, reduction of area of reactive astrogliosis within demyelinating areas (arrowheads) as evidenced by Luxol fast blue staining, compared to (c) sham controls. Arrows indicate astrocyte processes. d, injection of NSCs intravenously (grey bars) or intracere-broventricularly (black bars) caused a significant drop in factors known to drive reactive gliosis (FGF-2 and TGF-P), whereas expression level of neurotrophic factors remained essentially unchanged compared to sham controls (white bars). Reproduced with permission from Pluchino et al., Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 422:688-695. Copyright 2003, Nature Publishing Group.

pathway known as the striatopallidothalamic output pathway (SPTOP) (FIGURE 6.12). The SPTOP begins with dopaminergic neurons (DANS) in the substantia nigra that project to the basal ganglia, The DANs secrete dopamine, which stimulates electrical output by the basal ganglia to the thalamus and from there to the motor cortex.

Parkinson's disease is characterized by the apoptosis of DANs in the substantia nigra, which in turn results in lower dopamine output by the striatum, hyperactiv-ity of the SPTOP, and impaired motor function (Bjorkland and Lindvall, 2000). Parkinson's is invariably progressive, characterized by tremors at rest, akinesia and bradykinesia, muscle rigidity, postural instability, and lack of facial expression (Rosenthal, 1998). Neuronal death is thought to be the result of genetic mutations and environmental toxins acting through common mechanisms that result in mitochon-drial impairment, oxidative stress, and neurotoxic aberrant a-synuclein production (Valente et al., 2004; Greenamyre and Hastings, 2004). The genes Engrailed

FIGURE 6.12 Pathway from the substantia nigra (SN) to the motor cortex (C). Dopamine is secreted at the synapses of dopaminergic neurons in the SN with neurons in the striatum (S), which consists of the globus pallidus (GP), caudate nucleus (CN) and putamen (P) basal ganglia. Axons of neurons from the basal ganglia synapse with neurons in the thalamus (T), which then project to the cortex.

En1 Dopaminergic Neurons

1 and 2 may also be involved in the apoptosis of DANS. These genes are active in DANS shortly after their formation and throughout adulthood. In En1 and 2 null mice, however, apoptosis of these neurons occurs about two-thirds of the way through embryonic development, leading to the speculation that changes in the expression levels of En 1 and 2 during adulthood might be involved in the apoptosis of DANS in Parkinson's disease (Alberi et al., 2004).

The three primary therapies for Parkinson's are (1) to increase the dopamine output of the remaining viable DANs by administration of L-dopa, which is taken up and converted to extra dopamine that compensates for the lost output of dying cells, (2) pallidot-omy, and (3) inhibitory electrical stimulation of the subthalamic nucleus, which normally stimulates the globus pallidus (Rosenthal, 1998; Bjorklund and Lindvall, 2000). These therapies reverse the akinesia, bradykinesia, and rigidity of the disease, but not the tremors. L-dopa treatment produces severe side effects after several years and eventually has no effect on disease symptoms when the number of viable DANs becomes too low. Many symptoms recur a few years after pallidotomy or electrical stimulation of the sub-thalamic nucleus. None of the therapies slows the disease progression.

Investigators have turned to transplants of NSCs in the hope of a cure. These transplants have been performed on both human patients and a rat model of Parkinson's in which the neurotoxin 6-hydroxydopa-mine is injected unilaterally into the striatum (6-OHDA model). The rats suffer muscle rigidity on the contralateral side and exhibit ipsilateral turning movements in response to amphetamine, as well as akinesia and bradykinesia. The uninjected side serves as a control.

Fetal human (6-8 weeks old) mesencephalic cells (which include dopaminergic NSCs) were the first cells to be transplanted in an attempt to cure Parkinson's. When injected into the striatum of patients, they differentiated into DANs and made synaptic connections with host neurons, restoring the activity of the SPTOP toward normal. The results of such transplants, however, have been highly variable. In the best cases there have been dramatic clinical improvements that have lasted 5-10 years (Rosenthal, 1998; Bjorklund and Lindvall, 2000; Lindvall and Hagell, 2001; Bjorklund et al., 2003). The improvements are correlated with an increased output of dopamine, as visualized by increased uptake of 18fluoro-dopa in PET scans. In other cases, improvements have been minimal, or patients have continued to deteriorate. Autopsies of two patients who died, as well as transplant experiments on Parkinsonian rats suggest that this variation is due to the differential survival of transplanted cells (Rosenthal, 1998). It is thought that a minimum of 80,000 DANs (~20% of the normal number of DANs in the human substantia nigra) are required to obtain a beneficial effect. Clouding the picture is the fact that several doubleblind studies of patients receiving transplants of fetal neural cells have suggested a large placebo effect on Parkinson's symptoms, meaning that sham control patients also showed improvement of symptoms (Lazic and Barker, 2003). This result makes it difficult to ascertain the true efficacy of the transplants. Furthermore, transplantation does not seem to benefit older patients (Freed et al., 2001).

Assuming that Parkinson's symptoms can be ameliorated by dopamine output from the transplanted cells, practical and ethical considerations dictate that fetal tissues cannot be a reliable source of cells for transplant. Thus, investigators have turned to other potential sources of DANS. Carotid body cells, which regulate respiratory rate through the medulla by monitoring changes in blood levels of O2, CO2, and H+, are rich in dopamine and thrive in low O2. Since only one carotid body is necessary, the other could be used as a source of DANS for autotransplants. When injected into the striatum of rats with Parkinson's, over 90% of the carotid body cells survived and dopamine levels increased to 65% of unlesioned controls (Espejo et al., 1998). The rat striatum is normally innervated by ~30,000 DANs, but only 400-600 carotid body cells were needed to achieve this result. Amphetamine-induced turning movements were eliminated, but there was less improvement in akinesia and bradykinesia.

Transplantation of ESCs or their derivatives is also being explored as a treatment for Parkinson's. Undifferentiated ESCs have been injected at low concentrations into the striatum of 6-OHDA Parkinsonian rats (Bjorklund et al., 2002). Low concentrations were used to allow the cells to respond to their surroundings rather than self-generated cues. In 14 of 25 rats, the cells survived and differentiated into DANS, and this was associated with recovery from amphetamine-induced turning movements. This is an exciting result that indicates that the striatal environment contains the signals necessary for site-specific differentiation of ESCs. However, the grafts did not survive in six of the animals and developed into teratomas in five others.

Kim et al. (2002) used a five-stage protocol (Lee et al., 2000) to differentiate mouse ESCs transfected with the nuclear receptor related-1 (nurr-1) gene into DANS. Nurr-1 is a transcription factor that promotes the differentiation of mesencephalic precursor cells into tyrosine hydroxylase (TH)+ dopaminergic cells in the presence of FGF-8 and Shh (Hynes and Rosenthal, 1999; Wurst and Bally-Cuif, 2001). Grafts of 5 x 105 cells were injected into the striatum of rats lesioned unilaterally by 6-OHDA. The ESC-derived TH+ cells were functional DANS as assessed by morphological, neurochemical, electrophysiological, and behavioral criteria. They released dopamine and extended axons

TABLE 6.6 Behavioral effects of Nurrl transfected ESCs grafted into the 6OH-DA lesioned rat striatum. Amphetamine-induced rotation was evaluated for 70 min, numbers are given for 8 wks post-grafting. The other measurements were made starting 9 wks post-grafting. Spontaneous ipsilateral rotations were counted over a 5 min period. The adjusting step test measures the number of adjusting steps by the paw on the lesioned side vs the paw on the non-lesioned side when the animals were moved backward by the experimenter. For the paw-reaching test, rats were maintained on a restricted diet until they lost 10-15 % of their initial weight. The number of food pellets eaten with the lesioned vs non-lesioned paw was then determined over a 7d period. They cylinder test measures the number of contacts with the cylinder wall the rats make with the lesioned paw when rearing and landing. Minus numbers indicate a change from ipsilateral turning to contralateral turning. *P < 0.05; **P < 0.001 compared with the sham group. Data from Kim et al (2004).

% adjusting # of times lesioned

Amphetamine-induced Spontaneou ipsilateral steps taken by Fraction of pellets paw used in rotations rotations lesioned paw eaten with lesioned paw cylinder test

Sham 900 8 46 0.26 30

into the host striatum. The axons formed functional synapses as indicated by significant recovery from amphetamine-induced rotation and improvement in step-adjusting, cylinder, and paw-reaching tests table 6.6. The next step in both these sets of experiments would be to determine whether DANs derived from human ESCs would improve motor function in the rat Parkinson's model.

Barberi et al. (2003) have developed a protocol to direct the differentiation of EGFP-expressing mouse ESCs, derived from blastocysts of normally fertilized or somatic nuclear transfer eggs, into a variety of neuronal phenotypes, including DANs. Transplantation of 1 x 105 DANs derived from either source into the ipsi-lateral striatum reduced amphetamine-induced turning in 6-OHDA-treated mice by over 70%. Histological analyses showed that 2%-22% of the cells survived and extended over large areas of the striatum, with the highest density of TH + cells at the interface of host and grafted cells.

The potential of xenogeneic neural precursor cells from pig fetus to alleviate symptoms of Parkinson's has been tested in the 6-OHDA rat model after expansion in culture (Armstrong et al., 2001). When implanted into the striatum of immunosuppressed rats, many of the cells differentiated into neurons that extended axons to the normal targets of DANS and made synapses at distant sites. However, only a few cells were TH+, and these cells were unable to effect any functional recovery.

In a very interesting set of experiments, Dezawa et al. (2004) reported that transducing cultured rat and human MSCs with the portion of the Notch gene that encodes the intracellular domain (NICD) and treatment of the cells with several neurotrophic factors induced them to become neurons. When GDNF was included in the trophic factor treatment, TH+ DANS were induced with an efficiency of 41%. Transplantation of either rat or human GFP-labeled cells into unilateral 6-OHDA treated rats led to substantial recovery from apomorphine-induced rotational behavior and significant improvement in step adjustment and paw-reaching. Immunohistochemistry of brain sections indicated that grafted cells had migrated and extended beyond the injection site. Concentrations of dopamine were measured in the medium of cultured tissue slices from the lesioned and unlesioned sides of control animals and from the transplanted and unlesioned sides of experimental animals. The ratio of lesioned/ engrafted to unlesioned concentration was 0.57 in control animals and 0.67 in experimental animals. These data strongly suggest that not only can MSCs be converted in high numbers to neurons, including DANS, the DANS reconstruct new tissue that is integrated into the neural circuitry of the recipients.

There is also substantial evidence that cell transplants provide host DANS of animal Parkinson's models with neuroprotective factors, thus reducing their loss. Ourednik et al. (2002) induced symptoms of Parkinson's in aged mice via systemic administration of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) and transplanted marked NSCs unilaterally into the substantia nigra one to four weeks after treatment. The cells migrated extensively into both hemispheres. Tyrosine hydroxylase (TH) and dopamine expression was increased throughout the midstriatal system. While some of the transplanted NSCs differentiated into TH+ cells, 90% of the TH+ cells were host cells. This result strongly suggests that the transplanted cells secreted neurotrophic factors that prevented further death of host DANs or induced neural regeneration (Marconi et al., 2003).

Neuroprotective factors can be delivered directly to the brain tissue of Parkinsonian animals via gene therapy or infusion of protein. Glial-derived neuro-trophic factor (GDNF) has been shown to improve the symptoms of Parkinson's disease in experimental animals. Injection of rat brains near the substantia nigra with a replication-defective adenoviral vector encoding human GDNF reduced the loss of DANS after 6-OHDA treatment by approximately threefold when the brains were examined six weeks after injec-

FIGURE 6.13 Functional improvement in Parkinsonian monkeys after treatment with a GDNF-lentiviral construct. Monkeys receiving the construct showed statistically significant improvement over controls receiving a ß-gal-lentiviral construct in clinical ratings (A) and (B) a hand reach task (seconds required to retrieve food treats from a recessed well). Reproduced with permission from Kordower et al., Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 290:767-773. Copyright 2000, AAAS.

FIGURE 6.13 Functional improvement in Parkinsonian monkeys after treatment with a GDNF-lentiviral construct. Monkeys receiving the construct showed statistically significant improvement over controls receiving a ß-gal-lentiviral construct in clinical ratings (A) and (B) a hand reach task (seconds required to retrieve food treats from a recessed well). Reproduced with permission from Kordower et al., Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 290:767-773. Copyright 2000, AAAS.

primary astrocytes of mice transduced with a retroviral GDNF/lac-Z construct were injected into the striatum or substantia nigra of mice six days prior to unilateral 6-OHDA lesioning (Cunningham and Su, 2002). GDNF levels were elevated compared to controls and TH+ immunostaining was 89% of the unlesioned side, indicating that the GDNF conferred neuroprotection on host DANS. Dopamine depletion in the substantia nigra was completely prevented, as was the acquisition of amphetamine-induced rotational behavior. These studies indicate the feasibility of treating Parkinson's disease by gene therapy.

Cells of the subventricular zone proliferate in vitro in response to TGF-a, suggesting that this growth factor might be used to activate NSCs in the striatum or lateral ventricle walls that would then migrate to the degenerating substantia nigra and differentiate into DANs. To test this idea, a total of 50 |g of TGF-a was administered to the striatum of unilateral Parkinsonian rats via a shoulder-implanted minipump and cannula at the rate of 0.5 |l/hr for two weeks (Fallon et al., 2000). The infused rats showed a 31.5% improvement in rotational behavior over controls. Studies of BrdU incorporation and immunostaining for nestin and differentiated neuron markers revealed that NSCs proliferated, migrated to the striatum and differentiated into new neurons, some of which were presumably DANs.

Finally, Shh, which is crucial for the development of the nervous system, reduced amphetamine-induced rotation and forelimb akinesia when injected into the striatum of 6-OHDA rats (Tsuboi and Shults, 2002). This effect was associated with partial preservation of striatal dopaminergic axons, but only a modest preservation of nigral TH+ neurons, suggesting that Shh may induce behavioral improvement by mechanisms other than action on nigral DANS.

3. Huntington's Disease tion (Choi-Lundberg et al., 1997). Kordower et al. (2000) induced Parkinsonian symptoms in monkeys by intracarotid injections of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), followed by injections into the substantia nigra and striatum of lentiviral constructs encoding GDNF. Three months later, motor performance of the monkeys was vastly improved (figures 6.13, 6.14) and PET scans with 18fluorodopa showed an uptake of the tracer over 300% greater than in control animals injected with constructs containing the P-galactosidase gene. Histological studies revealed abundant GDNF+/TH+ neurons in the substantia nigra, suggesting the rescue of DANS, although the activation of stem cells in the striatum and their differentiation into DANS could not be ruled out. In another study,

Huntington's disease is a progressive disorder of movement accompanied by severe cognitive deterioration. The symptoms are associated with the death of multiple populations of striatal neurons, particularly medium spiny neurons, resulting in the loss of inhibitory connections from the striatum to the globus palli-dus. In normal cells, the huntingin protein, complexed with huntingtin-associated protein-1 (HAP-1) and a dynactin subunit, p150Glued, mediates vesicular transport of brain-derived neurotrophic factor (BDNF) along neuron microtubules (FIGURE 6.15). Huntington's disease is associated with a mutation in the N-terminal region of the huntingtin gene that results in a polyglu-tamine (CAG) repeat, creating a misfolded protein that is neurotoxic (Huntington's Disease Collaborative

FIGURE 6.14 Quantification of the trophic effects of a GDNF lentiviral construct on nigral neuronal number, neuronal volume, tyrosine hydroxylase (TH) mRNA, and striatal TH immunoreactivity (ir). Asterisks indicate statistically significant decreases relative to the intact side; ttt denotes significant increases relative to the intact side. Injection of the GDNF construct clearly rescues TH + neurons. Reproduced with permission from Kordower et al., Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 290:767-773. Copyright 2000, AAAS.

FIGURE 6.14 Quantification of the trophic effects of a GDNF lentiviral construct on nigral neuronal number, neuronal volume, tyrosine hydroxylase (TH) mRNA, and striatal TH immunoreactivity (ir). Asterisks indicate statistically significant decreases relative to the intact side; ttt denotes significant increases relative to the intact side. Injection of the GDNF construct clearly rescues TH + neurons. Reproduced with permission from Kordower et al., Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 290:767-773. Copyright 2000, AAAS.

Research Group, 1993). The mutation in the protein reduces the association of molecular motor proteins with microtubles, reducing the transport of BDNF (Gauthier et al., 2004). Huntington's disease can be mimicked in primates by injecting quinolinic acid into the striatum or by intramuscular injection of nitropro-pionic acid (NPA).

Fetal striatal tissue grafted to the striatum of marmoset or macaque monkeys with NPA-induced Huntington's reversed the symptoms of the disease (Kendall et al., 1998; Palfi et al., 1998). Immunohistochemical studies indicated good survival and differentiation of the grafted neurons with establishment of functional connections with host tissue. Preliminary clinical trials in human patients given grafts of human fetal striatal tissue have shown that the tissue survives and that the symptoms of the disease are alleviated to some extent, with persistent benefits to some patients three years postgrafting (Freeman et al., 2000; Bachoud-Levy et al., 2002; Hauser et al., 2002; Rosser et al., 2002; Liu et al., 2003).

Evidence for a possible neuroprotective effect of cell transplants on striatal neurons was obtained in experiments on the quinolinic acid model of Huntington's disease in monkeys (Emerich et al., 1997). The monkeys were first given unilateral intrastriatal implants of polymer-encapsulated baby hamster kidney (BHK) fibroblasts alone or BHK fibroblasts transgenic for the human ciliary neurotrophic factor (CNTF). A week later, the implanted region was injected with quinolinic acid. The loss of striatal neurons was significantly attenuated in the CTNF implant region, compared to control implant regions with BHK cells lacking the CTNF construct. Human umbilical cord stem cells or cultured human neural precursor cells injected into a mouse model of Huntington's also improved the condition (Cova et al., 2004). While it is possible that the transplanted cells differentiated into new neurons, it is likely that their major effect was to provide paracrine factors that stabilized host neuron survival.

A conditional model of Huntington's disease has been developed that suggests that it might be possible

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