Title: The Hedgehog Pathway and Neurological Disorders Tammy Dellovade,1 Justyna T. Romer,2 Tom Curran,2 Lee L. Rubin,1

1Curis, Inc., Cambridge, MA 021382Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis Tennessee 38105

tdellovade@curis.comstysia.romer@stjude.orgtom.curran@stjude.orglrubin@curis.com

Shortened Running Title:

*Corresponding author: lrubin@curis.com

Abstract

The hedgehog pathway is a major regulator of embryonic development and mutations

that decrease its activity are known to be associated with severe defects in nervous

system development. Recent evidence suggests that hedgehog continues to function

in adult tissue, normal as well as diseased, by regulating both cell proliferation and

the production of growth and angiogenic factors. In the adult nervous system, this

dual ability is especially important in regulating the behavior of neural stem and

progenitor cells. This article summarizes information connecting hedgehog

signaling and neural diseases, including neurodegenerative disorders and brain

tumors, particularly medulloblastoma. The discovery and utility of small molecule

agonists and antagonists of this pathway and their potential as novel types of

therapeutics are also described.

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Introduction

The hedgehog (Hh) pathway is one of a small number of pathways that control the

numbers and types of cells formed during development in species ranging from

Drosophila to human. While the most well known Hh pathway ligand, Sonic Hh (Shh),

has been shown, in ever-expanding ways, to participate in the development of the central

nervous system, another ligand, Desert Hh (Dhh), is essential for proper formation of the

peripheral nervous system. The purpose of this review is to highlight information that

has become apparent, mostly in the last few years, about ways in which the Hh pathway

is involved in neural disorders, both degenerative and cancer. Therapeutic prospects for

Hh agonists and antagonists will also be discussed.

Hh Signaling Pathway

Although the three known mammalian Hh ligands (Indian Hh, Ihh, being the third) are

expressed at different times and locations during development, they all seem to bind to

the same receptors and regulate the same signaling pathway (reviewed in McMahon et al.

2003). The ligands themselves are approximately 20 kD in molecular weight and are

unique in having the N- and C-terminal amino acids both lipid modified (by palmitoyl

and cholesterol addition, respectively) resulting in secreted proteins that are not freely

diffusible. The main Hh receptor is the 12-transmembrane transporter-like protein

patched-1 (Ptc1). There are other Hh ligand-binding proteins present either on cells that

synthesize the Hh ligand (e.g., dispatched, Burke et al. 1999) or on cells that respond to

the ligand (e.g., patched-2 and hedgehog-interacting protein, Smyth et al. 1999, Chuang

& McMahon 1999). In the off (non-liganded) state, Ptc inhibits the activity of another

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membrane protein, the 7-transmembrane, GPCR-like receptor smoothened (Smo),

although exactly how this happens is still not understood. In unstimulated cells, Ptc is

located, at least in part, in the plasma membrane, with Smo being primarily intracellular.

Following Hh binding, Ptc is internalized and degraded, while Smo translocates to the

membrane, possibly after being phosphorylated (Denef et al. 2000). This triggers a

cascade of somewhat less well-delineated events, the end result generally being an

increase in levels of the transcription factor Gli1. Ultimately, there must be increased or

decreased expression of sets of genes that control, in a context-dependent way,

proliferation and differentiation. Interestingly, Ptc1 is a transcriptional target of Hh

signaling, and its up-regulation serves to dampen the degree of Hh signaling over time.

Various aspects of the Hh pathway have been discussed in detail elsewhere (McMahon et

al. 2003).

Hedgehog Ligands and Neural Development

The participation of the Hh pathway in motor neuron differentiation in the developing

spinal cord has been studied in most detail (Tanabe & Jessell 1996, Briscoe & Ericson

2001). In this case, Shh is made first by the notochord and secondarily by cells in the

floorplate. Hh signaling has a dose-dependent mechanism of action that is critical for its

developmental functions since it is distributed in concentration gradients. Several features

contribute to gradient formation across the embryonic spinal cord including the

hydrophobic nature of Hh protein, its ability to bind receptors on cells that produce as

well respond to Hh and its association with extracellular matrix proteins. Taken together,

these features enable Hh to act as a classical morphogen that promotes motor neuron

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formation by regulating expression of families of transcription factors in cells along the

ventral-to-dorsal axis. The dependence of the formation of motor neurons on Hh activity

is not unique. Other cells of ventral origin, such as nigral dopaminergic neurons, striatal

projection neurons, basal forebrain cholinergic neurons and oligodendrocyte progenitor

cells, among others, are induced, at least in part, by sonic Hh. Much of the original

interest in a potential role for Hh in treating neurodegenerative disease was based on its

participation in controlling the number of many of the types of neurons that degenerate

during the course of various neurological disorders (for example, dopaminergic neurons

in Parkinson’s disease, Ye et al. 1998).

Dhh also plays a role in nervous system formation, but its function is restricted to

peripheral nervous system development. Dhh is secreted by Schwann cells, but Ptc1 is

present only on perineurial cells that make up the nerve sheath. Mice lacking Dhh form

abnormal peripheral nerves with a discontinuous nerve sheath, a leaky blood-nerve

barrier, low rates of action potential conduction and resulting behavioral changes

(Parmantier et al. 1999). Presumably, the effects of Dhh are indirect, in that the ligand

binds to its receptors on perineurial cells that secrete growth, neurotrophic and

angiogenic factors that cooperate to ensure appropriate nerve development and function.

Hh ligands also play a significant role in regulating proliferation as well as

differentiation. Ihh is a mitogen for chondrocytes (Long et al. 2001) while Shh is

mitogenic for several cell populations, including cerebellar granule neuron precursor cells

(Wechsler-Reya & Scott 1999; the importance of which will be discussed in the context

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of brain tumors below). Shh has also recently been shown to participate in the expansion

of the dorsal part of the developing central nervous system (Palma et al. 2005). The fact

that Hh ligands can, at times, regulate both proliferation and differentiation is central to

appreciating their importance in embryonic development and to considering the various

ways in which this developmental pathway can be usurped in adult diseases.

Although Hh signaling is clearly necessary for the generation of neural and glial cell

types, there is much less evidence that it can promote cell survival. Recombinant Hh

protein introduced into developing chick embryos, in a standard test for neurotrophic

factor-like activity, failed to decrease the number of motor neurons undergoing

programmed cell death (Oppenheim et al. 1999). However, there have been a small

number of reports indicating that Hh signaling can decrease death of cultured

dopaminergic neurons after either trophic factor withdrawal or addition of MPTP (see

below).

The Hh Pathway and Developmental Disorders

The Hh gene was originally discovered in a Drosophila mutagenesis screen, and the

profound role of the Hh family in mouse development was discovered by analyzing the

phenotype of mice deficient for Hh ligands, or various components of the Hh signaling

pathway. Numerous abnormalities, ranging from embryonic lethality with severe

nervous system deficits resembling the human birth defect holoprosencephaly in the case

of Shh (Chiang et al. 1996), to dramatically shortened limbs and late prenatal/early

postnatal death, in the case of Ihh (St-Jacques et al. 1999), are seen in mice. Somewhat

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equivalent observations have been made in developing chick embryos and in fetal sheep.

Studies in both species -- the former in ovo (Cooper et al. 1998), the latter, in the field

(Binns et al. 1963) -- were carried out using natural product Hh inhibitors, such as the

teratogens jervine and cyclopamine extracted from corn lilies. Treatment of pregnant

animals at early gestation period results in severe nervous system defects, in their

offspring that bear a strong resemblance to holoprosencephaly, with associated cyclopia

(fusion of developing eyes at the midline). For some time, it was not clear how these

compounds were acting. The jervinoids are sterols, resembling cholesterol in chemical

structure. Since Hh proteins are covalently linked to cholesterol moieties, and since

disrupted cholesterol metabolism leads to similar aberrant neural phenotypes, it was

suggested that the compounds act by blocking the addition of cholesterol to Hh.

However, this turned out not to be the case; in fact the compounds were later shown to

act by binding to Smo and inhibiting Hh signaling (Chen et al. 2002, Frank-Kamenetsky

et al. 2002).

As a result of the somewhat unusual nature of the Hh signaling pathway, with the

receptor Ptc1 serving to actively inhibit the pathway in the absence of Hh ligand,

increased signaling ensues when levels of functional Ptc1 decrease. Constitutively active

forms of Smo (which, as for many GPCRs, are known to exist) also produce enhanced

signaling in the absence of ligand. Such modifications also lead to significant disruption

of normal development and, in older organisms, promote formation of certain cancers, as

will be discussed later.

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Recently, one group has suggested that, at least during avian neural development, Ptc1, in

its unbound state, and independent of the canonical Hh signaling pathway, can actively

stimulate apoptosis, thereby becoming a member of the dependence class of receptors

(Thibert et al. 2003). These results have not yet been replicated and this mechanism is

not yet known to play a role in any adult tissue or disease. Other recent experiments

suggest the possibility that the Hh pathway may be able to regulate some aspects of

neuronal behavior such as axonal growth, rapidly and without changes in gene

transcription (Trousse et al. 2001, Charron et al. 2003, Kolpak et al. 2005). Whether this

alternative means of signaling can explain any of the effects of Hh in neural disease

models will be discussed below.

Hh is Important in Humans As Well

Not only are there developmental defects in animals with diminished Hh signaling, but

similar alterations– holoprosencephaly with varying penetrance up to and including

cyclopia -- are also observed in human fetuses with sonic Hh mutations (Roessler et al.

1996). Further, peripheral nerve developmental abnormalities with consequent

neuropathies, somewhat similar to those seen in Dhh-deficient mice, are seen in a small

number of people with Dhh mutations (Umehara et al. 2000). Confirmatory evidence for

the involvement of Hh signaling in normal human development also comes from

observations made in individuals with Nevoid Basal Cell Carcinoma Syndrome (Gorlin

Syndrome), of which there are at least 5000 in the US. Gorlin syndrome patients show a

preponderance of basal cell carcinomas (BCCs), with new tumors forming throughout

life. They are also predisposed to other tumors, most notably medulloblastoma.

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Moreover, they have an increased incidence of meningioma, ovarian sarcomas and

fibromas, and they exhibit skeletal abnormalities of various types as well as radiation

hypersensitivity (Gorlin 1987). The major cause of this syndrome is heterozygosity of

the PTCH1 locus (Hahn et al. 1996, Johnson et al. 1996). Decreasing PTCH1 activity

leads to constitutive activation of the Hh pathway in the absence of Hh ligand binding. In

short, these results confirm that normal Hh signaling is important in the regulation of

proper tissue formation in species ranging from Drosophila to human, as might be

expected with the high degree of conservation of Hh and the components of its signaling

pathway.

Hh Pathway and Adult Disease

For the most part, once development has been completed, the expression of Hh ligands,

Ptc, Smo and Gli1 declines to low levels in normal healthy tissues, at least in rodents.

More than a decade ago, there was speculation among developmental biologists that

pathways like the Hh pathway could be reactivated in adult tissue to promote recovery

from damage. Possibly, according to this line of thought, supplying additional Hh protein

could rebuild functional tissue by stimulating endogenous stem cells to proliferate and

differentiate, much as happens in the embryo. However, while a role for the Hh pathway

in normal human development could perhaps have been anticipated from Drosophila and

mouse embryo studies, it was much less clear that it had any role in regulating normal or

diseased adult tissue in human (or mouse, for that matter). Therefore, the therapeutic

potential of regulating this pathway has been unclear.

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Events in the last few years have now supported a role for Hh signaling in adult biology

and provided a foundation for the idea that modulators of this pathway – both antagonists

and agonists – may have significant potential for treating different cancers and

neurodegenerative diseases. This is no surprise given that the Hh pathway can directly

control both entry into the cell cycle and cell differentiation in many different types of

cells, at least during development. A major advance was made, following recognition of

the link between PTCH1 and Gorlin syndrome, that the Hh pathway is upregulated in a

high percentage of sporadically occurring BCCs (Gailani et al. 1996, Dahmane et al.

1997, see also Oro et al. 1997). The most frequent cause is mutational inactivation of

PTCH1 (leading to activation of Hh signaling), but activating mutations of SMOH and

loss of function of suppressor of fused (SUFU), another component of the Hh pathway,

have also been seen (Reifenberger et al. 2005). Since this is the most common cancer in

the Western world, with almost 1,000,000 new cases per year in the US alone, this

certainly attracted a great deal of attention, leading to a flurry of activity concentrating on

attempts to identify other cancers in which there might be mutations in Hh pathway

genes. Approximately one third of medulloblastoma, the most common malignant

childhood brain tumor with about 500 new cases per year in the US, was found to be

associated with activation, frequently mutation-related, of the Hh pathway (Pomeroy et

al. 2002). However, to date, no other cancers have been identified in which these

mutations occur with a significant frequency.

Another view of the way in which Hh ligands might be involved in regulating tumor

growth was suggested by studies indicating that one or more Hh ligands may be

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synthesized by human tumor cells in relatively large amounts, compared to normal adult

cells. Tumor cell-derived Hh may then activate signaling in other tumor cells, stimulating

growth directly (i.e., an autocrine loop). Evidence is mounting that this autocrine effect

is characteristic of numerous types of cancers ranging from small cell lung to pancreatic

and prostate cancers (Berman et al. 2003, Thayer et al. 2003, Watkins et al. 2003,

Karhadkar et al. 2004, Sanchez et al. 2004). In many of these cases, Hh antagonists seem

to decrease the rate of tumor cell proliferation in vitro and in mouse xenograft models.

Another general observation has been that the normally quiescent Hh signaling pathway

can be reactivated after tissue damage in the adult. For example, this has been seen in

crushed sciatic nerve in which Schwann cell production of Dhh mRNA (and of Shonic

Hh mRNA) goes up enormously, yielding a large Hh response in nearby perineurial cells

which then increase their production of growth, neurotrophic and angiogenic factors.

Making hind limbs ischemic also upregulates production of Sonic Hh, ultimately leading

to enhanced stromal cell production of angiogenic and other growth factors (Pola et al.

2001, 2003). In both cases, application of an anti-Hh antibody slowed recovery from

damage and treatment with additional Sonic Hh protein stimulated recovery (Berg et al.

2000, Pepinsky et al. 2002). The ability of the Hh pathway to regulate growth factor

production may account for some of its effectiveness in different models of

neurodegenerative disease, as discussed later in this review. However, this ability, in the

context of a growing tumor producing high levels of Hh ligand, might further contribute

to tumor growth (that is, a paracrine effect) even beyond what is seen with the direct

effects on tumor cell proliferation.

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Identification of Small Molecule Hh Antagonists

Once it became reasonably clear that the Hh pathway could be involved in adult disease,

with BCC and medulloblastoma being the first ones recognized, attempts were made to

identify small molecule Hh antagonists. Treating cancers caused by inactivating PTCH1

or activating SMOH mutations obviously would require small molecule inhibitors of Hh

signaling acting at or below the level of SMOH. A cell-based reporter gene assay, in

which a multimerized regulatory sequence from the Ptc promoter was used to control the

expression of luciferase, was employed to screen a small molecule library. This led to

the identification of numerous classes of Hh antagonists that were shown to block the

effects of Hh ligands in different assays, including motor neuron induction in developing

spinal cord explants (Williams et al. 2003). Two series of antagonists were optimized by

focused medicinal chemistry; both were shown to bind directly to SMO. These

antagonists were shown to be free of inhibitory activity when tested against other

developmentally regulated pathways. Most important was the lack of inhibition of the

Wnt pathway – since Wnt receptors, the Frizzleds, are the most highly related class of

molecules to SMO. Further, the antagonists were shown to be free of inhibitory activity

on a variety of unrelated GPCRs (since SMO is a GPCR-like receptor). The first class of

these Hh antagonists has now entered a phase I clinical trial as a topical treatment for

BCC. The potential use of these antagonists in treating certain types of brain tumor will

be discussed later.

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Identification of Small Molecule Hh Agonists

Early on, it seemed obvious that developing a drug that acts by stimulating Hh signaling

in the CNS would require the use of small molecule agonists that were blood-brain

barrier permeant. Later, it also became clear that having such small molecules could

actually facilitate the study of the participation of Hh in adult CNS function since they

have provided a convenient way of activating Hh signaling in adult experimental

organisms at any time.

It was never anticipated that small molecules that acted as pseudo-ligands for PTCH

could be identified since it seemed improbable that small molecules that bind sufficiently

well to the binding pocket used by endogenous Hh ligands could be discovered. Once

assays were configured to detect Hh antagonists, they were also employed to see if small

molecule agonist “hits” could be obtained. A class of biaryl agonists was identified and

optimized, after a significant medicinal chemistry effort, for potency, oral bioavailability

and CNS penetration. The end result was a large number of structurally related

compounds with sub-nM EC50 in cell culture assays (Frank-Kamenetsky et al. 2002).

These compounds are more potent than the actual protein ligands, and they have proven

to be effective in activating the Hh pathway in various standard Hh assays carried out in

vitro and on developing mice. For example, they can regulate, in a concentration-

dependent fashion, the appearance of particular types of neural progenitors in developing

spinal cord explants. In fact, Hh agonists act essentially as morphogens under

appropriate circumstances in that, at low concentrations, they suppress the expression of

dorsal spinal cord markers, whereas at higher concentrations, they increase the levels of

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ventral markers. After oral dosing of pregnant mice with agonist, embryonic mouse

development is substantially affected with a large increase seen, for example, in the

number of Hh-responsive spinal cord progenitors. Different types of studies, ranging

from genetic and pharmacological epistasis experiments to direct binding assays,

demonstrated that these agonists bypass Ptc and bind directly to Smo. In fact, this

agonist class and the two small molecule antagonist classes described already, as well as

the jervinoid-type natural product Hh inhibitors, compete for binding, apparently to a

single site on Smo. This raises the question as to whether there may be an endogenous

ligand for this Smo biding site. The agonists have exciting therapeutic potential, detailed

later, but also have proven to be valuable reagents. Wichterle et al. (2002) showed that a

combination of retinoic acid and the Hh agonist could be used in combination to

efficiently generate motor neurons from mouse embryonic stem cells.

Hh and brain tumors: Medulloblastoma

Medulloblastoma is the most common malignant brain tumor in children, with about 500

new cases diagnosed yearly in the United States. They are included in a group of

morphologically and genetically diverse tumors, called primitive neuroectodermal tumors

(PNET), a term frequently used to describe embryonal tumors. They arise in the

cerebellum, most likely from precursors of granule neurons (GNP) residing in the

external germinal layer (EGL) of the developing cerebellum. They have a tendency to

metastasize, most often within the CNS; however, some patients develop metastases to

the bone, bone marrow, lymph nodes, liver or lung. Medulloblastomas can be subdivided

into five histologically distinct groups: the classic, desmoplastic and large-cell anaplastic

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variants being most common and melanotic and medullomyoblastoma types being

exceedingly rare.

Medulloblastoma genetics

The existence of distinct subtypes of medulloblastoma suggests that this is a

heterogeneous disease, and, indeed, there are many different genetic factors that influence

medulloblastoma initiation, progression and morphological appearance. The most

common genetic abnormality linked to medulloblastoma, and usually associated with the

large-cell anaplastic variant, is loss or mutation of chromosome 17. Approximately 40-

60% of sporadic medulloblastomas show loss of heterozygosity of 17p (for reviews of

chromosomal abnormalities in medulloblastoma see Ellison 2002, Gilbertson 2002,

Goussia et al. 2000).

Significant insights into the causes of tumorigenesis have come from the identification of

specific mutations linked to inherited syndromes associated with higher incidences of

medulloblastoma. These include Li-Fraumeni syndrome, resulting from p53 gene

inactivation (Malkin et al. 1990), and Gorlin syndrome, linked to mutation in the PTCH1

gene (Hahn et al. 1996, Johnson et al. 1996).

Since the Gorlin syndrome connection was made, many studies have investigated PTCH1

status in medulloblastoma. It was found that 10-20% of sporadic tumors harbor mutations

in this gene (Pietsch et al. 1997, Raffel et al. 1997, Wolter et al. 1997, Xie et al. 1997,

Zurawel et al. 2000a), indicating that lesions in Hh/PTCH1 signaling may contribute to

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the etiology of medulloblastoma. These data also explained the connection between LOH

at 9q22.3-q32 and medulloblastoma, as this is linked to loss of the PTCH1 allele.

Interestingly, this chromosomal abnormality is associated with preservation of the

remaining wild type allele (Vorechovsky et al. 1997), indicating that heterozygosity at the

PTCH1 locus is sufficient to promote medulloblastoma formation. PTCH1 mutations are

not the only genetic lesions associated with disrupted Shh signaling in medulloblastoma;

activating mutations of SMOH have been detected in up to 5% of sporadic

medulloblastomas (Reifenberger et al. 1998), and in one study, germline and somatic

mutations in SUFU were found in 9% of children with medulloblastoma (Taylor et al.

2002). To date, the desmoplastic subtype of the disease is proportionally more common

among medulloblastomas with mutations in Hh pathway components, including Gorlin

syndrome tumors, sporadic medulloblastomas with mutations in PTCH1, SUFU and

SMOH genes as well as ones with chromosome 9q22.3 deletions (Nicholson et al. 1999,

Pietsch et al. 1997, Pomeroy et al. 2002, Schofield et al. 1995).

Medulloblastoma caused by disruption of the Hh pathway

The first definitive proof that disruption in the Hh pathway can cause medulloblastoma

came with the analysis of mice in which one copy of the Ptc1 gene was deleted

(Goodrich et al. 1997, Hahn et al. 1998). Homozygous Ptc1-/- mice are not viable

--embryos die at 9.5 days of gestation with severe defects in the developing nervous and

cardiovascular systems. The heterozygous mice have features of Gorlin syndrome

including increased body size, a low incidence of polydactyly, and cerebellar tumors that

resemble human medulloblastoma.

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There have been contradictory reports about whether the loss of the remaining wild type

Ptc1 allele is required for tumorigenesis. Several studies indicate that the wild type allele

continues to be expressed at low levels in primary mouse tumors. This has been shown by

northern blot analysis of total RNA prepared from tumors arising in Ptc1+/- mice

(Wetmore et al. 2000), as well as sequencing of Ptc1 cDNA isolated from tumors and in

situ hybridization on tumor sections (Zurawel et al. 2000b). An independent study used in

situ hybridization and Real-Time PCR to detect expression of wild type Ptc1 in tumors

(Romer et al. 2004). One study did suggest that the wild-type Ptc1 allele might be

silenced in tumors through DNA methylation (Berman et al. 2002); however, this

conclusion was based on studying allograft-derived cell lines and should be evaluated

further in vivo tumors, as the Hh pathway has been shown to undergo silencing when

primary tumor cells are cultured in vitro (Romer et al. 2004), and methylation could be

one mechanism of this inactivation. In another study, the pre-neoplastic tumor cells in

Ptc1+/- mice were shown to lose expression of the wild type Ptc1 allele (Oliver et al.

2005). In this study, Real-Time PCR analysis was used to assay expression of Ptc1, in the

absence of contaminating non-tumor cells. This suggests that loss of Ptc1 expression is

an early event in tumorigenesis, perhaps required for tumor progression. Even in the

situation in which low levels of Ptc1 were detected, it is clear that the pathway is

defective as the increased levels of Gli1 in these tumors should have elevated Ptc1

expression if the well-known negative feedback loop was functional. It is possible that

the discrepancies reported in Ptc1 status among the various studies stem from differences

in cell handling in the various in vivo and in vitro systems.

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Medulloblastoma formation: mechanism of action

a. Disruption of cell cycle in medulloblastoma

How does loss of one allele of Ptc1 and/or increased expression of Gli1, either through

Ptc1 loss or mutations in other upstream components of the pathway, contribute to

tumorigenesis? The mechanism by which deregulation of the Hh signaling leads to

medulloblastoma formation is still unclear, but many studies point to a close connection

between activated Hh signaling and impaired cell cycle control. In part, this was inferred

through analysis of gene expression in medulloblastoma. Tumors with mutations in Hh

pathway components, express high levels of Gli1, as well and N-Myc and C-Myc,

CyclinD1 and CyclinD2 (Lee et al. 2003, Pomeroy et al. 2002).

The genes highly expressed in medulloblastoma are often also present in the developing

cerebellum (Lee et al. 2003), suggesting that medulloblastoma cells may be similar to

immature granule neuron progenitors (GNPs) and that studying mechanisms involved in

proliferation of GNPs may provide insight into the mechanisms involved in

tumorigenesis. In normal cerebellar development, Hh signaling is necessary for

proliferation of GNPs in the external germinal layer (EGL). Shh is released from Purkinje

neurons and binds to Ptc1 on GNPs. This has been shown in several different in vitro and

in vivo studies, reviewed in Wechsler-Reya and Scott (2001). This major mitogenic role

of Shh has been linked to cell-cycle control through experiments showing that Shh could

induce expression of the G1 cyclin genes (Kenney & Rowitch 2000). Addition of Shh to

isolated GNPs resulted in substantial elevation of cyclin D1, D2 and E mRNA levels.

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Interestingly, this increase required protein synthesis, implying that production of

intermediate proteins was needed to up-regulate expression of these cyclin genes. The

elevation of D-type cyclin levels was linked to activity of the Rb pathway as measured by

increased Rb phosphorylation, possibly indicating that sustained proliferation of Shh-

treated GNPs involves release of inhibition of E2F transcription factors by Rb, permitting

S-phase entry.

As well as inducing sustained proliferation through D-type cyclin genes, Shh stimulation

has also been shown to hamper exit from cell cycle, in part through blocking cell cycle

arrest induced by the cyclin-dependent kinase inhibitor (CKI) p21CIP1 (Fan & Khavari

1999). This is most probably a consequence of a Shh-induced increase in activity of the

cyclin-dependent kinases CDK2 and CDK4. This is important as during cerebellar

development immature GCPs need to exit the cell cycle before inward migration out of

the EGL and subsequent terminal differentiation.

Another connection between disruption in cell cycle and Hh-dependent medulloblastoma

comes from the finding that the Bmi1 protein, a member of the polycomb group of

transcription factors, is over-expressed in the subset of human medulloblastomas that

express high levels of Hh-target genes. Bmi1 is expressed in the EGL of the developing

cerebellum (Leung et al. 2004) and is essential for normal cerebellar development (van

der Lugt et al. 1994). Its expression appears to be regulated by Hh signaling (Leung et al.

2004). Bmi1 affects the cell cycle through its inhibition of the CKI p16Ink4a and the tumor

suppressor p19Arf. This can potentially lead to repression of both p53 and Rb function,

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through p19Arf and p16Ink4a respectively.

b. Control of growth factor production by Hh signaling

In other circumstances – following tissue damage, for example – elevated Hh signaling is

known to upregulate growth factor production. In fact, the levels of Igf2, a known

mitogen for many cell types, are high in Ptc1+/- tumors, such as rhabdomyosarcoma and,

presumably, medulloblastoma. Further, Igf2 activity appears to be necessary for the

formation of both medulloblastoma and rhabdomyosarcoma in Ptc1 heterozygous mice

(Hahn et al. 2000). This suggests that Igf2 is one of the targets of the Hh pathway that

confers an additional growth advantage to these types of tumor cells. Notch signaling

may also be important for formation of Shh-induced tumors (Hallahan et al. 2004). Mice

engineered to constitutively express an activated form of Smo have a high incidence of

medulloblastoma, and both Shh and Notch target genes are highly expressed in these

tumors. Additionally, pharmacological inhibition of Hh and Notch signaling, especially

in concert, suppresses growth of medulloblastoma cell lines and both mouse and human

primary brain tumor cells (Hallahan et al. 2004). Thus, Hh activation may not only

directly regulate cell proliferation, but also contribute to enhancing medulloblastoma

growth by ensuring a growth factor-rich environment, as may also be true for Hh in

peripheral solid tumors.

The Therapeutic Potential Of Hh Pathway Inhibitors in Medulloblastoma

Patients are diagnosed with medulloblastoma by the location of the tumors and their

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histopathological appearance. In the US, currently employed therapy consists firstly of

surgical resection (Ellison et al. 2003, Gilbertson 2004). Adjuvant chemotherapy and

radiation therapy follow the surgery, and the doses of radiation used are adjusted

according to the patient’s risk. Radiation treatment, especially in young children, has

devastating and lasting effects on intellect and neuroendocrine function (Freeman et al.

2002, Gurney et al. 2003, Palmer et al. 2001, Ris et al. 2001). For this reason, children

under the age of 3 are treated with chemotherapy alone, and, partly as a consequence,

their prognosis is usually dismal. It seems likely that further advances in

medulloblastoma therapy will focus on developing completely novel therapies based on

targeting the specific molecular defects found in medulloblastoma. The Shh pathway may

be an appropriate molecular therapeutic target, at least for certain patients.

Experimental models of medulloblastoma

Testing targeted therapies offers new challenges. Most of the standard cytotoxic therapies

are first tested using in vitro systems -- repetitively passaged cultured tumor-derived cell

lines -- or xenografts using immuno-compromised animals into which cells, most of the

time the same cultured tumor cells, are injected subcutaneously. The shortcomings of

such pre-clinical models of cancer are beginning to be fully recognized (Romer & Curran

2005), and their inability to predict clinical outcome has been apparent for some time.

This is especially important for testing targeted therapies since this relies heavily on

having models that maintain the molecular defects of the human disease being targeted.

The cultured tumor cells often undergo selective growth pressures associated specifically

with artificial in vitro culture conditions. This can lead to molecular changes in the

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cultured cells that, as a consequence, become increasingly different from their tumors of

origin. Xenografts recapitulate neither the microenvironment nor the developmental time

frame of human disease. For example, it has been noted that in many cultured

medulloblastoma cell lines the activity of the Hh pathway is down regulated, with the

resultant absence of expression of Hh target genes, and a lack of proliferative and

molecular response to Shh stimulation (Romer et al. 2004).

Efficacy of Hh antagonists in medulloblastoma models

Inhibition of the Hh pathway has been investigated in several pre-clinical

medulloblastoma models. Cyclopamine, for instance, has been tested in both in vivo and

in vitro. In one study (Berman et al. 2002), cultured cell lines derived from allografts of

medulloblastomas that arose spontaneously in Ptc1+/-p53-/- mice were treated with

cyclopamine, resulting in decreased cell numbers. When cyclopamine was injected into

the flank of allograft carrying mice, a reduction in tumor cell proliferation was again

noted and this produced a decrease in tumor volume. The therapeutic potential of Hh

pathway inhibitors was also tested in primary cultures of freshly resected human

medulloblastomas. Cyclopamine treatment decreased PTCH1 expression and reduced

tumor cell viability in 7 out of 7 samples used. This preliminary study is encouraging

since it suggests that Hh antagonists might be more broadly active than had been

predicted from knowing the percent of tumors with Hh pathway mutations, but needs to

be explored further.

A more recent study (Romer et al. 2004) aimed to avoid the problems inherent in

22

standard cell culture and allograft systems by using Ptc1+/-p53-/- mice, which

spontaneously develop medulloblastoma. They treated the mice with an Hh antagonist,

HhAntag-691, identified in the assays described earlier. This treatment decreased tumor

cell proliferation dramatically, increased tumor apoptosis and, at the highest dose used,

led to complete elimination of tumors from the brains of treated animals. This was

associated with pathway suppression as shown by assaying the expression of endogenous

Shh pathway target genes, including Gli1, by quantitative real-time PCR and in situ

hybridization. However, the treatment did not affect Ptc1 expression, indicating that in

tumors, Ptc1 transcription is not a target of Shh signaling and may have lost its normal

role in the negative feedback loop that regulates the magnitude of Hh signaling. (Sanchez

and Ruiz i Altaba 2005) obtained similar results with the Ptc1+/-p53-/- mice using

cyclopamine to inhibit Hh signaling. These studies strongly suggest that inhibition of the

Shh pathway may be an effective targeted therapy for medulloblastoma.

Use of Hh inhibitors to treat genetically diverse medulloblastomas

The groups of patients most likely to benefit from treatment with Hh antagonists are

those with Gorlin syndrome and those with sporadic medulloblastoma in which the Hh

pathway is activated by mutation. However, it is quite likely that other patients may also

be able to be treated by inhibiting Hh signaling, as over 30% of sporadic

medulloblastomas express high level of Hh target genes, including Gli1 (Lee et al. 2003).

This has also been suggested by studies comparing expression profiles of different mouse

models of medulloblastoma. Mice that develop medulloblastoma on Lig4-/-p53-/-, Kip1-/-

p53-/-, Inc4c-/-p53-/-, Ink4d-/-Kip1+/-p53-/- and Ink4d+/-Ink4c-/-p53-/- backgrounds all up-

23

regulate the signature Hh target genes (Lee et al. 2003). Additionally, medulloblastomas

arising in PARP1-/-p53-/- mice have a high level of Gli1 (Tong et al. 2003).

It is unclear how Gli1 is induced in these genetically different tumors. One example is the

loss of RENKCTD11 gene, which leads to up-regulation of Gli1 in medulloblastoma. This

gene localizes to chromosome 17p13.2, and, as already mentioned, allelic loss of 17p is

the most frequent genetic defect in human medulloblastoma. Allelic deletion or reduced

expression of RENKCTD11 gene has been detected in a subset of sporadic human

medulloblastomas (Di Marcotullio et al. 2004). Conversely, increased RENKCTD11

expression leads to decreased proliferation of medulloblastoma-derived cell lines in tissue

culture and xenografts. It is possible that this inhibitory action is related to RENKCTD11

suppression of Gli1 activity, possibly through inhibiting translocation of Gli1 to the

nucleus (Di Marcotullio et al. 2004). Taken together, these results suggest that RENKCTD11

is a negative regulator of Shh signaling, and its loss may be one of the mechanisms in

which sustained Shh activity can be achieved in medulloblastoma. Thus, it may be that

the Hh pathway has a more general role in the induction of medulloblastoma and that Hh

inhibitors may have therapeutic potential in a substantial subset of this serious childhood

brain tumor. This is consistent with the results of Berman et al. (2002) already

mentioned.

The Hh Pathway in Other Types of Brain Tumor

It appears that medulloblastomas in which the Shh pathway is activated are derived from

cerebellar granule neuron precursor cells (GNPs) that are normally regulated by Hh

24

signaling. When the Hh pathway is inappropriately maintained in this cell population,

they fail to exit the cell cycle and continue to proliferate without control, leading to tumor

formation. It can be said therefore, that medulloblastoma results from an aberration of

normal development, involving hyperactivation of the Hh pathway and over-proliferation

of progenitor cells that are normally dependent on Hh signaling for their growth.

Extrapolation of this paradigm suggests that deregulation of the Hh pathway may be

involved in formation of tumors derived from other stem/progenitor cells that depend on

Hh signaling to proliferate during normal development. If the assumption is that tumors

arise from such stem/progenitor cells that accumulate mutations allowing for their

continual and uncontrolled proliferation, then it is possible that other Gli-positive

progenitors are capable of being transformed and giving rise to different CNS tumors. In

fact, Gli1 was first described as a gene amplified in human glioma (Kinzler et al. 1987).

Additional studies have failed to confirm the link between glioma and the Hh pathway.

However, these may have been somewhat misleading, focusing more on amplification of

the Gli1 gene rather than on inappropriate Gli1 expression.

More recently, it has been shown that several CNS tumors of different origin express

high levels of GLI1, indicating that activated Hh signaling might be important for their

genesis or maintenance. Astrocytoma, oligodendroglioma, glioblastoma multiforme and

PNETs have all been found to express targets of Hh signaling, including GLI1 (Dahmane

et al. 2001). In this study, 22 independent tumors were tested by either RT-PCR alone or

by both RT-PCR and in situ hybridization, and all of the tumors consistently expressed

25

GLI1 with the majority also expressing other components of the Hh pathway, including

SHH, PTCH1 and GLI2. As controls, an ependymoma and a hemangioma were tested,

and neither expressed GLI1.

These data raise at least the possibility that Gli-positive precursors from different

locations of the dorsal brain can contribute to formation of CNS tumors. To test this idea,

selected glioma- and glioblastoma-derived cell lines were cultured in the presence of

cyclopamine. Three out of seven lines tested responded to the treatment with decreased

proliferation, as did one of three primary cortical glioma cells. Therefore, there are

perhaps brain tumors other than medulloblastoma for which Hh antagonists may

constitute an effective therapy. However, further work is needed to explore this idea

more completely as well as to identify markers that may characterize those CNS tumors

that are most reliant on Hh pathway activity.

Hh Signaling in Neurodegenerative Disease

Overview

The importance of the Hh pathway in embryonic neural development has been

thoroughly documented (Tanabe & Jessell 1996, Briscoe & Ericson 2001), but the role of

Hh signaling in the adult brain is less well understood. In the adult rodent CNS and PNS,

Hh ligands and their receptors, Ptc and Smo, are present although the magnitude of Hh

signaling seems to be lower and more restricted spatially (Traiffort et al. 1998, 1999,

Machold et al. 2003, Coulombe et al. 2004). However, the potential for Hh ligands to

function still exists. For example, viral over-expression of Shh is able to stimulate

proliferation of neural stem cells in the hippocampus (Lai et al. 2003). In addition, as

26

already mentioned, tissue damage is able to up-regulate the production of Hh ligands and

activate Hh signaling as part of the tissue repair process (Pola et al. 2001). These

observations prompted an exploration of the potential for stimulating Hh signaling to

promote functional recovery in models of neurodegenerative disease. Generally,

experiments have provided convincing support for neuroprotective and regenerative

activities following sufficient activation of the Hh pathway in disorders ranging from PD

to stroke to peripheral neuropathy.

Hh Pathway in Parkinson’s Disease

Parkinson’s disease is a progressive neurological disorder characterized by the loss of

dopaminergic neurons in the substantia nigra. Sonic Hh is known to be involved in the

differentiation of those very dopaminergic neurons during embryonic development

(Hynes et al. 1995). There are also data suggesting that Shh can promote the survival, at

least in vitro, of fetal dopaminergic neurons once they have differentiated and can protect

them from the toxic effects of N-methyl-4-phenylpyridinium (Miao et al. 1997). In

addition, neural progenitor cells isolated from the subventricular zone (SVZ) of neonatal

mice secrete Shh and have been shown to protect developing dopaminergic cells in vitro

(Rafuse et al. 2005).

Unilateral 6-hydroxydopmaine (6-OHDA) lesions are commonly used as a rat model of

PD. When injected unilaterally into the striatum, 6-OHDA is taken up into the terminals

of dopaminergic neurons resulting in a decrease in the dopaminergic innervation of the

striatum (as judged by the density of tyrosine hydroxylase positive fibers) and a loss of

27

dopaminergic neurons in the substantia nigra. Administration of dopamine agonists, such

as apomorphine, or agents that block dopamine reuptake, such as amphetamine, results in

easily quantified rotational behavior that reflects the degree of asymmetry (and, thereby,

the loss of striatal innervation). In this model system, injections of Shh protein directly

into the affected striatum significantly reduced apomorphine-induced rotation behavior

(Tsuboi & Shults 2002). Recently, Shh-secreting neural progenitor cells were further

shown to improve motor behavior when transplanted into 6-OHDA treated rats (Rafuse et

al. 2005). Finally, systemic administration of the neurotoxin 1-methyl-4-phenyl-1,2,3, 6-

tetraydropyridine (MPTP) in marmosets results in reduced dopamine levels and impaired

motor function similar to PD patients. Administration of Shh unilaterally into the

substantia nigra of MPTP-marmosets partially reversed both the behavioral and neural

effects in a dose dependent manner (Dass et al. 2002). These data indicate that Shh can be

neuroprotective in animal models of PD.

Positive behavioral effects in rodent PD models with Shh treatment are not always

associated with sparing of dopaminergic terminals in the striatum or cell bodies in the

substantia nigra. By contrast, GDNF, when injected into the brain of 6-OHDA lesioned

rats, normalized turning behavior while consistently attenuating the loss of dopamine

resulting from the lesion (Bjorklund et al. 1997). This raises the possibility that Hh

pathway activators may act via an entirely different mechanism. One such mechanism is

based on the finding that, in vitro, Shh protein can rapidly, within minutes of application,

reduce electrical transmission of adult subthalamic neurons (STN; Bezard et al. 2003).

PD symptoms are in part induced by over-activity of the STN cells, and surgical or

28

electrical inactivation of these cells generally provides symptomatic relief for PD

patients. In 6-OHDA lesioned rats, there is a reduction in the level of Shh in the globus

pallidus, the neurons that project to the STN, and hyperactivity of the STN cells (Bezard

et al. 2003). Therefore, normalization of Shh signaling via protein or agonist treatment

might improve 6-OHDA induced rotational behavior by reducing hyperactivity of STN

neurons.

Hh Pathway and Peripheral Neuropathy

While Shh is primarily important for CNS development, the morphogenic effects of Dhh

are seen in the peripheral nervous system. Treatment with Hh protein has been shown to

be protective and/or enhance regeneration in PNS injury models. In a mouse nerve crush

model, Shh and Dhh mRNA levels were dramatically elevated following injury, and Hh

signaling increased significantly. Treating mice with anti-Hh monoclonal antibodies

increased recovery time while periodic injection with recombinant Hh protein modified

to have prolonged serum half-life accelerated recovery (Pepinsky et al. 2002).

Diabetes mellitus is a chronic metabolic disease accompanied, in a significant number of

cases, by peripheral neuropathies (pain and loss of sensation being the main symptoms)

and, ultimately, by nerve fiber degeneration. In the rat streptozotocin (STZ) model of

diabetes, pancreatic beta cells are killed, leading to hyperglycemia. Over time, there is a

reduction in the rate of action potential propagation in both motor and large sensory nerve

fibers. In this model, a decreased level of Dhh mRNA in peripheral nerves associated

with the development of diabetes was observed (Calcutt et al. 2003). Treatment with

29

exogenous recombinant Hh protein starting 5 weeks after the onset of diabetes and

continued for an additional 5 weeks resulted in complete restoration of nerve conduction

velocity to control, non-diabetic, levels (Calcutt et al. 2003). Hh treatment also

attenuated the diabetes-induced decrease in nerve growth factor (NGF) expression and

the reduction in the proportion of large fibers in the sciatic nerve. In a second study,

sensory and motor conduction velocities were monitored for 12 weeks post-induction of

diabetes by STZ. Again, treatment with Hh protein after the onset of symptoms

significantly improved nerve conduction velocity in diabetic rats (Kusano et al. 2004). In

this experiment, there was also a marked improvement in nerve blood flow after

treatment. Improved blood flow and accelerated recovery of function are also seen in

ischemic hindlimbs following treatment with Shh (Pola et al. 2001). Blood flow in the

nerve does decrease in diabetes – in rodents and in humans – leaving the nerve ischemic

and perhaps accounting, in part, for its decreased function. These data, taken together

with positive effects in the nerve crush model, indicate that Shh protein can have

significant effects on nerve recovery from injury or disease. As mentioned previously,

the apparent mechanism of action underlying these effects is the ability of Hh to stimulate

the production of neurotrophic and angiogenic factors in nerve perineurial cells.

Effects of Hh Agonist in CNS Disease Models

While Hh protein is potentially useful for the treatment of PNS disorders such as diabetic

neuropathy, this type of therapy would not be practical for treating CNS disease. To

optimize the therapeutic targeting of the Hh pathway, small molecule activators of Hh

signaling (described above) have been developed. These particular agonists bind to Smo

30

and reproduce effects of Shh on neural cells in vitro, including activation of Gli1 gene

expression and induction of neural precursor cell proliferation (Frank-Kamenetsky et al.

2002). Further, these Hh agonists are active at low nanomolar concentrations and have

good bioavailability. They have also been demonstrated to readily cross the blood brain

barrier (BBB) and activate Hh signaling (as judged by an increase in Gli1 expression) in

the CNS following systemic administration (Machold et al. 2003).

To determine if these small molecules are efficacious in animal models of disease, they

were tested first under circumstances in which Shh protein had been effective. Similar to

what was seen with intrastriatal Shh protein, treatment with agonist either by ventricular

infusion or systemic administration significantly improved apomorphine-induced rotation

behavior in the rat 6-OHDA model (Boucher et al. 2004). Agonist treatment was only

required during the first week after 6-OHDA to see behavioral effects that lasted for up to

6 weeks post-lesion.

It was also important to identify other neurological indications for which Hh agonist

might be a useful therapeutic. In the United States, approximately 700,000 people each

year suffer a stroke. Although many compounds have gone into clinical trial, efficacious

treatment options are still unavailable for stroke patients. For those that survive, sensory,

motor and/or cognitive functions can be severely affected depending on location of the

infarct. Similar to what has been seen in other injury models, including nerve crush and

hind limb ischemia, Gli1 mRNA levels increased in the brains of mice following transient

middle cerebral artery occlusion (tMCAO), a stroke model, (Dellovade et al. 2002).

31

Therefore, it was hypothesized that, similar to other types of injury in which Hh pathway

genes are up regulated, exogenous Hh treatment might accelerate recovery of function. It

also had been observed previously that recombinant Hh protein and Hh agonists were

effective in limiting neuronal cell death after malonate treatment, another excitotoxicity

model, often used to test potential therapeutics for Huntington’s disease (Dellovade et al.

2002).

To test this hypothesis, small molecule Hh agonists were used in rat and mouse models of

focal ischemia. In both species, an intraluminal suture model of tMCAO in which

occlusion is induced using a poly-lysine coated suture via the internal carotid artery was

used. In rats, the MCA was occluded for 90 minutes, and rats were examined for changes

in body weight and behavioral recovery 24 and 48 hours post-stroke. Forty-eight hours

post-occlusion, rats were euthanized and infarct size measured in brain slices. Dosing

with Hh agonist 6 hrs after the onset of occlusion reduced infarct volume by about 50%

in rats (Dellovade et al. 2004). Significant effects on infarct volume were seen in both

the cortical and subcortical regions. In addition, a single dose of agonist also reduced

weight loss and improved neurological behavior (Dellovade et al. 2004). In several series

of studies, a single dose of Hh agonist administered up to at least 6 hrs post-tMCAO

significantly decreased infarct volume in both rats and mice (Dellovade et al. 2002,

2004).

Possible Mechanism of Action for Neuroprotective Effects of Hh Agonists

The idea that Hh pathway activators might promote recovery of function after stroke was

32

consistent with what had been seen in earlier studies of nerve crush, diabetic neuropathy

and hindlimb ischemia. It was somewhat unexpected that they would be neuroprotective

in stroke (and in malonate excitotoxicity) models and the mechanism of neuroprotection

is still not completely understood. One possibility is that Hh directly activates neuronal

survival pathways (such as that regulated by PI-3-kinase). Currently, there are no direct

supporting data, but Hh signaling does appear to stimulate this survival pathway in at

least endothelial cells (Kanda et al. 2003). Another possibility is that the Hh agonist

directly regulates synaptic activity such that the excitotoxic effects initiated by ischemia

are minimized. Again, there is no experimental support for this hypothesis. However, as

discussed, there has been at least one suggestion that Hh can regulate synaptic

transmission. Further, as already mentioned, there are a few recent publications

supporting the idea that Hh can have rapid effects on growth cones, at any rate. This

possibly novel mechanism will require further exploration.

Recent data showing the cells types that express the various Hh pathway members in the

adult brain might provide some clues toward mechanism. Evidence indicates that certain

neuronal populations synthesize and secrete Shh, while astrocytes, and perhaps other

types of glia, respond to secreted Shh, as demonstrated by increases in Gli1 and Ptc

expression (Allendoerfer et al. 2004). Since astrocytes are known to respond to brain

insult by secretion of various neurotrophic factors, Hh agonists may act indirectly to

mediate neuroprotection via changes in astrocyte gene expression. In support of this

hypothesis, Shh has been shown to regulate expression of such factors in vitro. For

example, Shh protein can induce brain derived neurotrophic factor (BDNF) and insulin-

33

like growth factor 1 (IGF-1) expression in cultured fibroblasts (Allendoerfer et al. 2001).

Similarly, Shh can regulate the expression of cytokines involved in angiogenesis, such as

vascular endothelial growth factor and angiopoietins from mesenchymal cells (Pola et al.

2001). Finally, in diabetic rats, treatment with Shh protein increases NGF mRNA levels

in the sciatic nerves (Calcutt et al. 2003).

Effects of Hh on Stem Cells

The Hh pathway regulates expansion of precursor cells and their differentiation in several

regions of the developing CNS, including the cerebellum and spinal cord. In all adult

mammals, including humans, there are at least two brain regions where stem cells exist,

the hippocampal dentate gyrus (DG) and the subventricular zone of the lateral ventricles

(SVZ). In both regions, new neurons are continuously being born in normal animals, and

this steady state can be influenced by a number of factors. For example, neurogenesis is

increased when animals are placed in an enhanced environment or are allowed to exercise

(Kemperman et al. 1997, Gould et al. 1999, van Praag et al. 1999). Conversely, stress

hormones and anxiety/depression have been shown to decrease precursor cell

proliferation in the DG and treatment with anti-depressants can reverse this effect (Gould

et al. 1992, 1998, Malberg et al. 2000, Santarelli et al. 2003).

These data highlight the potential of the adult brain endogenous stem cell populations to

enhance recovery and repair following brain injury or disease. Certainly, there has been a

great deal of interest in identifying therapeutics that will increase neural stem cell

proliferation, migration, neuronal differentiation and incorporation into neuronal

34

circuitry. Agents that can do this may constitute parts of therapeutic regimes for treating

disorders ranging from stroke to PD and Alzheimer’s disease to depression. Some recent

data are supportive of this general idea. Arvidsson et al. (2002) found that, following

tMCAO in rats, an increase in proliferation was seen in the SVZ, and migration of the

newly formed neurons to the area of damage in the striatum was noted. However, several

weeks after injury very few of the new neurons had survived, suggesting that the post-

stroke environment did not support incorporation of the new cells into the region of

damage. Nakatomi et al. (2002) had generally similar findings in a rat model of global

ischemia and further observed that ventricular infusion of known stem cell mitogens

(βFGF and EGF) stimulated stem cell proliferation and culminated in an enhancement of

the other processes as well, ultimately leading to the appearance of new neurons and to

behavioral improvement. These data suggest that exogenous therapies that enhance

survival, as well as proliferation and migration of neural progenitors could facilitate the

brain’s own recovery mechanisms.

In both the SVZ and DG of adult rodents, Hh pathway genes are expressed (Lai et al.

2003, Palma et al. 2005), and systemic dosing with Hh agonist increased Gli1 mRNA

levels in both regions within 24h post-dosing (Machold et al. 2003). In addition,

precursor cell proliferation was significantly elevated in both the SVZ and DG following

systemic dosing with Hh agonist as compared to vehicle treated controls. These data are

in agreement with in vitro results demonstrating increased proliferation of both DG and

SVZ precursor cells in response to Shh protein treatment (Lai et al. 2003, Palma et al.

2005). Finally, it was recently demonstrated that administration of cyclopamine reduced

35

both Gli1 mRNA levels and precursor cell proliferation in the SVZ, and prolonged

treatment ultimately reduced the numbers of bromo-deoxyuridine (BrdU)

immunoreactive neurons in the olfactory bulb (Palma et al. 2005). These data clearly

demonstrate a vital role for Shh signaling in neurogenesis in the adult brain. Studies are

underway to test whether activation of the Hh pathway is associated with long-term

behavioral recovery via neurogenesis that may be enhanced by agonist treatment after

stroke. If this were to be true, it would suggest that Hh pathway agonists would have the

unique ability to both minimize cell death and lead to the appearance of new neurons

after CNS damage.

Conclusions

The critical role played by the Hh pathway during embryonic development implies that it

regulates essential biological processes. Therefore, its reappearance as a modulator of

adult disease is not entirely unexpected. In the context of tumor growth, Hh signaling

appears to act both directly and indirectly to stimulate and maintain the proliferation of

tumor cells. In damaged neural tissue, it is neuroprotective and offers at least the

possibility of regenerating damaged tissue. Drug-like Hh agonists and antagonists have

been identified and it will be exciting and challenging to assess their true therapeutic

potential.

36

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50

CHAPTER COMPONENTS

• Key Words (as many as 6):

• Abstract

• Glossary list: define major terms used in test (as many as 10)

• Acronyms list: spell out all important acronyms used in text

• Summary list: highlight the central points of your chapter (as many as 8)

• Unresolved Issues and /or Future Directions (As many as 40: note where research

may headed

POTENTIAL SIDE EFFECTS OF AGONISTS AND ANTAGONISTS- AGE

DEPENDENT.

• Annotated References: explain the special importance of selected references from

bibliography 9As many as 10)

SUGGESTIONS 5, 6, 23, 33, 57, 83?

• Side Bar: highlight a related topic (200 words, max)

• Related Annual Reviews Chapters (as many as 6)

51