REVIEWS

NORMAL HUNTINGTIN FUNCTION: AN ALTERNATIVE APPROACH TO HUNTINGTONS DISEASE

Elena Cattaneo, Chiara Zuccato and Marzia Tartari
Abstract | Several neurological diseases are characterized by the altered activity of one or a few ubiquitously expressed cell proteins, but it is not known how these normal proteins turn into harmful executors of selective neuronal cell death. We selected huntingtin in Huntingtons disease to explore this question because the dominant inheritance pattern of the disease seems to exclude the possibility that the wild-type protein has a role in the natural history of this condition. However, even in this extreme case, there is considerable evidence that normal huntingtin is important for neuronal function and that the activity of some of its downstream effectors, such as brain-derived neurotrophic factor, is reduced in Huntingtons disease.
RNA INTERFERENCE

(RNAi). A method by which double-stranded RNA that is encoded on an exogenous vector can be used to interfere with normal RNA processing, causing rapid degradation of the endogenous RNA and thereby precluding translation. This provides a simple way of studying the effects of the absence of a gene product in simple organisms and in cells.

Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Balzaretti 9, 20133 Milano, Italy. Correspondence to E.C. e-mail: elena. cattaneo@unimi.it
doi:10.1038/nrn1806 Published online 15 November 2005

The devastating neurodegenerative disorder Huntingtons disease (HD) is caused by a mutation in a curious protein called huntingtin. Unlike many other proteins of a similar size (348 kDa), huntingtin is completely soluble, essential for embryogenesis, and ubiquitously expressed in moderate amounts in and outside the nervous system1. The disease-causing gene mutation consists of an expanded CAG tract (>35 repeats) at the 5 end, which is translated into a corresponding polyglutamine stretch (polyQ)2 that makes medium spiny neurons in the striatum particularly vulnerable to cell death, and also leads to the dysfunction and death of neurons in other brain regions, including the cortex3. The disease manifests at a mean age of 35 years, and is fatal after 1520 years of progressive neurodegeneration. Its symptoms include motor dysfunction and cognitive abnormalities4,5. Its dominant inheritance pattern and important experimental evidence, reviewed elsewhere6,7, indicate that the mutant huntingtin protein is toxic and initiates the disease insofar as there is an inverse correlation between CAG length and disease onset, mainly in relation to the largest CAG size8,9. More recently, it has been suggested that the loss of normal huntingtin function might also contribute to the pathogenesis of HD1. The reduced physiological

activity of huntingtin in HD might also affect the development of therapeutic strategies: for example, RNA INTERFERENCE (RNAi) strategies aimed at blocking the disease-triggering mutation would have to be selective for the mutant allele10. Furthermore, the identification of the affected downstream targets of wild-type huntingtin might allow the development of drugs that can restore their activity11, in addition to the current strategies aimed at blocking the toxicity of the mutant protein12,13. Here, we first discuss the structure and evolution of huntingtin, which might be important in understanding its cellular activity. We then consider studies that have investigated the biological function of huntingtin during development, and highlight its physiological role in the brain and its known downstream targets. Finally, we discuss evidence that points to the importance of the loss of wild-type huntingtin in HD. It is known that increased wild-type huntingtin expression improves brain cell survival1418; removal of wildtype huntingtin generates some of the phenotypes observed in the presence of mutant huntingtin1923; wild-type huntingtin expression mitigates the effect of the mutant protein16,2326; and deletion of the wildtype allele in an animal model of HD causes more damage27 BOX 1.

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Box 1 | Wild-type huntingtin and neuronal pathology Various experimental approaches have been used to investigate normal huntingtin function and its possible involvement in the pathogenesis of Huntingtons disease. The data show that increased wild-type huntingtin expression leads to improved brain cell survival; removal of the wild-type protein generates some of the phenotypes observed in the presence of mutant huntingtin; wild-type huntingtin expression mitigates the effect of the mutant protein; and depletion of the wild-type protein in a Huntingtons disease background causes more damage. Wild-type huntingtin is shown in green, mutant huntingtin in orange.

Increased expression of wild-type huntingtin leads to:

Protection from apoptotic neuronal cell death in response to toxic stimuli in vitro14,66,87 Reduced apoptosis after toxic stimuli in vivo in mice17,25 Dose-dependent neuroprotection from excitotoxicity in vivo in mice18 Increased transcription of brain-derived neurotrophic factor (BDNF) and other Repressor element 1/neuron-restrictive silencer element (RE1/NRSE)-controlled neuronal genes in mice15,16

Reduced levels of wild-type huntingtin lead to:

Embryonic lethality in huntingtin-knockout (Hdh/) mice7173 Abnormal brain development with huntingtin levels of <50% in mice80,81 Neuronal cell death and neurological dysfunctions in Hdh+/ mice20,71 Apoptosis in the brain and testes, and a Huntingtons disease-like phenotype in adult conditional Hdh/ mice (in which Hdh is knocked out in both endogenous alleles only in post-mitotic neurons)20 Fewer haematopoietic and neuronal progenitors from cultured Hdh/ embryonic stem cells82,83 Abnormal distribution and morphology of cellular organelles33 Reduced vesicle and mitochondria transport in Drosophila melanogaster huntingtin (htt) RNAi (in which huntingtin mRNA is depleted by RNA interference) and in neurons from conditional Hdh/ mice21,22 Reduced BDNF vesicular transport in a mouse cell line after htt RNAi23

Increased expression of wild-type huntingtin in a mutant huntingtin background causes:

Reduced toxicity in peripheral cells overexpressing mutant huntingtin24 Inactivation of the RE1/NRSE silencer in heterozygous mutant huntingtin knock-in cells16 Reduced neuronal toxicity mediated by NMDA (N-methyl-d-aspartate) or kainate receptors in rodents26

Depletion of wild-type huntingtin in a mutant huntingtin background causes:

Increased apoptotic cell death in the testes of YAC72/ mice (in which Hdh is knocked out in both endogenous alleles, and an additional yeast-derived artificial chromosome (YAC) containing the entire human mutant huntingtin 72-CAG gene is inserted) in comparison with YAC72+/+ mice (in which Hdh is wild-type in both endogenous alleles, and an additional YAC containing the entire human mutant huntingtin gene is inserted)25 Worsening of behavioural phenotype and increased apoptosis in the testes of YAC128/ mice compared with YAC128+/+ mice27

Although it does not yet show conclusively that the loss of normal huntingtin functions has an impact on human pathology, this growing list of findings provides strong support for this proposal and offers a useful example of how investigating the biology of diseaselinked proteins can increase our knowledge of brain function and dysfunction. Examples of other diseases whose natural history might be at least partially due to the loss of physiological function of the normal protein are discussed briefly in BOX 2.
From structure to potential function

Huntingtin is a completely soluble protein of 3,144 amino acids. It has many potential domains, the boundaries and activities of which are not fully

understood. It has no sequence homology with other proteins and is expressed ubiquitously in humans and rodents, with the highest levels in CNS neurons and the testes2830. Intracellularly, huntingtin is associated with various organelles, including the nucleus, endoplasmic reticulum and Golgi complex3135, and it is also found in neurites and at synapses, where it associates with vesicular structures such as clathrin-coated vesicles, endosomal compartments or caveolae, and microtubules36. This widespread subcellular localization does not facilitate the definition of its function. The primary amino acid sequence of huntingtin reveals little, as there are only a few known sequence motifs and no structural domains with defined functions (FIG. 1). One obviously important portion of the

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Box 2 | Normal PrPC and FMRP function in human pathology Two types of disease whose natural history might be at least partially due to the loss of physiological function of the normal protein, despite the involvement of greater toxicity of the altered protein, are the transmissible spongiform encephalopathies (TSEs) and fragile X syndrome. TSEs are fatal neurodegenerative diseases, such as CreutzfeldtJakob disease, that affect humans and other mammals. They are caused by the accumulation of PrPSc, an abnormally folded form of the prion protein. PrPSc acts in a dominant-negative manner by recruiting and modifying the conformation of the normal cellular form (PrPC), therefore leading to loss of its normal function in the absence of genetic insufficiency. PrPC is conserved across species, which suggests that it has some relevance to basic physiological processes. Although its precise function is not known, accumulating evidence shows that it is involved in: copper and/or zinc ion transport or metabolism139,140; protection from oxidative stress141; cell signalling142,143; membrane excitability and synaptic transmission144,145; neuritogenesis146; and apoptosis147. Like huntingtin, these functions can be achieved by means of PrPC binding to different interactors at different cellular locations148. The co-expression of both cytosolic and membrane-bound PrPC with Bax abolishes Bax-mediated cell death, which indicates that PrPC is involved in antiapoptotic activity147. Fragile X syndrome is an X-linked dominant disorder that involves the fragile X mental retardation protein (FMRP). In the presence of the mutation, FMRP loses its physiological activity, and this has been associated with the synaptic dysfunction observed in the disease. FMRP acts as a translational repressor of specific synaptic mRNAs (brain cytoplasmic RNA 1, BC1 RNA), and its mutation impairs this normal and necessary break in translation and leads to an abnormally increased synaptic presence of active proteins149.

POLAR ZIPPER

A term used by Max Perutz to describe a three-dimensional protein motif consisting of interactions between polar residues on separate subunits or separate proteins.

mammalian protein is the polyQ region itself, which is also present in many transcription factors and aberrantly expanded in at least eight other disease-causing proteins37. Because each of these diseases is characterized by the loss of a specific subset of neurons, we have previously proposed that sequences around the glutamine tract might have a crucial role in disease pathogenesis1. The polyQ stretch in huntingtin begins at the eighteenth amino acid, and, in unaffected individuals, contains up to 34 glutamine residues2. In 1994, Perutz et al. showed that this portion forms a POLAR ZIPPER structure, and suggested that its physiological function is to bind transcription factors that contain a polyQ region38; it has now been shown that wild-type huntingtin interacts with several partners and that the polyQ tract is a key regulator of such binding3941. In higher vertebrates, the polyQ region is followed by a polyproline (polyP) stretch, which might help to keep the protein in solution42. Downstream of these regions are the so-called HEAT repeats, which are ~40-amino-acid-long sequences that occur multiple times within a given protein and are involved in protein protein interactions43,44 (FIG. 1). Bioinformatics analysis has found 37 putative HEAT repeats in huntingtin45 and, despite contradictory findings concerning the number of functionally active repeats46, three main clusters have been identified43. The repeats are well conserved in huntingtin from all vertebrates, which may indicate that it interacts with the same proteins across vertebrates, whereas 28 putative consensus repeats have been found in Drosophila melanogaster huntingtin, whose degree of conservation with respect to humans has yet to be fully defined46. Huntingtin contains a functionally active carboxy (C)-terminal nuclear export signal (NES) sequence and a less active nuclear localization signal (NLS), which might indicate that the protein (or a portion of it) is involved in transporting molecules from the nucleus to the cytoplasm47. This hypothesis is supported by

huntingtins perinuclear and nuclear distribution, and the recent demonstration that the 17 amino acids before the polyQ region interact with the nuclear pore protein TPR (translocated promoter region), which exports proteins from the nucleus. Removal of these amino acids causes huntingtin to accumulate in the nucleus48. Huntingtin also contains three well-characterized protease cleavage consensus sites4953 (FIG. 1), where cleavage generally leads to fragments of both normal and mutant huntingtin, although the latter is more susceptible to proteolysis and generates fragments that are found in the cytoplasm and nucleus5458. Caspase cleavage sites are typically conserved in vertebrate huntingtin, but not in that of D. melanogaster4951,58,59. Functionally active calpain cleavage sites have been described for mouse and human huntingtin52,53. Other sites, the exact amino acid positions of which are not well defined, are preferentially cleaved in some brain regions60 (FIG. 1). The contribution of huntingtin proteolysis to cell function is unclear. However, modifications in the activity of caspase and calpain reduce the proteolysis and toxicity of the mutant protein, and delay disease progression51,53,61. Huntingtin is subject to four types of post-translational modification: the amino (N)-terminal lysines K6, K9 and K15 compete for sumoylation and ubiquitination42,62, and phosphorylation at serines 421 and 434 influences cleavage and toxicity, and is reduced in HD6366. Huntingtin is also palmitoylated by its co-partner, huntingtin-interacting protein 14 (HIP14, a palmitoyl transferase), but the precise amino acid position involved is unknown. The palmitoylation of huntingtin is consistent with its proposed role in regulating vesicular trafficking, because palmitoylated proteins are often involved in the dynamic assembly of components that control vesicle trafficking and synaptic vesicle function31,67.

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born with no apparent defects76,77, which suggests that the CAG tract does not affect the physiological function of the protein during development, and also shows that mutant huntingtin can compensate for the loss of wild-type function in development (FIG. 2). Patients with WolfHirschhorn syndrome, a rare condition in which there is a deletion on chromosome 4 that comprises the CAG triplet repeats region (IT15), are born and do not develop HD78. In addition, a rare patient with a balanced translocation with a breakpoint between exons 40 and 41 shows no disease phenotype79. This evidence indicates that the presence of one fully functional allele (at least 50% huntingtin expression) is compatible with life in humans and that HD is not caused by a simple loss of function of the huntingtin gene. However, we now know that, in addition to its early extra-embryonic function, huntingtin is involved in other important activities that, if brought below a certain level (<50%), might render cells more vulnerable to the genetic stress imposed by the CAG mutation1. In fact, during embryogenesis in mice, an experimental reduction in huntingtin levels to below 50% of normal reveals a phenotype within the embryo itself 80 (FIG. 2). After gastrulation, huntingtin becomes important for NEUROGENESIS mice carrying a <50% dose of wild-type huntingtin show defects in the epiblast (the structure that will give rise to the neural tube), which lead to reduced neurogenesis and profound malformations of the cortex and striatum80. These findings indicate that huntingtin participates in the formation of the CNS80. Another study has shown that greatly reduced huntingtin levels are insufficient to support normal mouse development81. In vitro differentiation of Hdh/ embryonic stem cells also showed reduced production of neuronal (and haematopoietic) progenitors82,83. Analyses of CHIMAERAS created by injection of Hdh/ embryonic stem cells into the blastocyst have shown that wild-type huntingtin is also crucial for establishing and maintaining neuronal identity, especially in the cortex and striatum84. Although some adult brain regions were appropriately colonized by Hdh/ neurons (their functional activity has not been evaluated), few donor cells were found in the cerebral cortex, striatum and basal ganglia, which indicates that huntingtin might have a region-specific role in neuronal survival. Preliminary analyses of chimaeras at embryonic day 12.5 showed ongoing Hdh/ cell degeneration specific to the striatum, cortex and thalamus, which supports the view that neuroblasts in these areas need to synthesize huntingtin if they are to progress in development and differentiation85. These data indicate that huntingtin is required at different stages of development, and that its total absence or a >50% reduction in its levels generates an early phenotype in mice BOX 1; FIG. 1). By contrast, like humans, mice carrying 50% full-length wild-type huntingtin (that is, one allelic dose) reach normal adult life7173. However, in adulthood, one in three heterozygous knockout mice shows behavioural
469 536

HEAT group 1

HEAT group 2

HEAT group 3

SUMO/UBI

A
0 250 500 750

B
1,000 1,250

NES

(Q)n (P)n

Glu, Ser, Pro rich C AA

3,000

Ser rich 530

513

586

1,500 1,750 2,000 2,250 2,500 2,750

Figure 1 | Schematic diagram of huntingtin amino acid sequence. (Q)n indicates the polyglutamine tract, which is followed by the polyproline sequence, (P)n, and the red squares indicate the three main clusters of HEAT repeats. The arrows indicate the caspase cleavage sites and their amino acid positions, and the blue arrowheads the calpain cleavage sites and their amino acid position. B identifies the regions cleaved preferentially in the cerebral cortex, C indicates those cleaved mainly in the striatum, and A indicates regions cleaved in both. Green and orange arrowheads point to the approximate amino acid regions for protease cleavage. NES is the nuclear export signal. The red and blue circles indicate post-translational modifications: ubiquitination (UBI) and/or sumoylation (SUMO) (red), and phosphorylation at serine 421 and serine 434 (blue). The glutamic acid (Glu)-, serine (Ser)- and proline (Pro)-rich regions are indicated (serine-rich regions encircled in green).

EXTRAEMBRYONIC TISSUE

All tissues that do not contribute to the embryo but support its development.
VISCERAL ENDODERM

Extra-embryonic tissue in pre-gastrulation stages of the mouse embryo.

ONTOGENESIS

Taken together, these studies have identified potential activities of wild-type huntingtin, the most remarkable of which is its ability to bind several proteins in both its normal and mutated forms39,40. Huntingtin might, therefore, have a flexible or multifunctional structure capable of assuming specific conformations and activities depending on its subcellular location and time of maturation in a given cell6,39,41,45,68. Alternatively, it might be born as a natively unfolded protein. The evidence in favour of the structural flexibility of the huntingtin backbone is that different protein epitopes are differently accessible to various antibodies depending on their subcellular compartments69,70, and that the elongated polyQ region can modify the three-dimensional structure of the entire protein and so change its interactors38,39.
Huntingtin function during development

The events involved in the development of an organism, from the earliest embryonic stage to maturity.
PHYLOGENESIS

The events involved in the evolution of a species.

NEUROGENESIS

The birth of new neurons, which occurs not only in developing organisms but also throughout adult life in both vertebrates and invertebrates. Ongoing neurogenesis is thought to be an important mechanism underlying neuronal plasticity, allowing organisms to adapt to environmental changes, and influencing learning and memory throughout life.
CHIMAERA

An organism that is composed of cells derived from at least two genetically different zygotes.

Huntingtin is essential for embryonic development: its complete inactivation in huntingtin-knockout mice (Hdh/) causes embryonic death before day 8.5 (before gastrulation and the formation of the nervous system)7173. This correlates with defects in the organization of EXTRAEMBRYONIC TISSUE73, possibly as a consequence of an alteration in the nutritive function of the 74 VISCERAL ENDODERM . On the basis of Haeckels suggestion in 1899 that mammalian ONTOGENESIS recapitulates PHYLOGENESIS75, the early non-neuronal activity of huntingtin can be likened to its ancestral function in species with a poorly organized or no nervous system. The extra-embryonic function of huntingtin (that is, in tissues such as the placenta) does not depend on the CAG tract and ancestral huntingtin has no CAG domain BOX 3. Furthermore, the expression of mammalian huntingtin with a pathological polyQ expansion (even up to 128 CAG repetitions) rescues huntingtin-null mice from embryonic lethality25,27. Finally, patients with HD who are homozygous for the CAG expansion do not express wild-type huntingtin but develop normally and are

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are important for the newly formed nervous system and essential for post-mitotic neurons. This hypothesis has been tested in gene overexpression and deletion studies of cultured brain cells and in vivo, and the results suggest that wild-type huntingtin is important for the survival of mammalian neurons, in which it controls many cell functions, such as neuronal gene transcription, and axonal and vesicular transport.
Huntingtin is anti-apoptotic

Box 3 | Huntingtin during evolution: a route to its function?

Insects (Drosophila) Vertebrates (from fishes to human)

Halocynthia roretzi Nematodes Strongylocentrotus purpuratus Heliocidaris erythrogramma Deuterostoma

Protostoma

Dictyostelium

Saccharomyces cerevisiae Fungi/Myxomycetes

In the absence of information about its three-dimensional structure, comparisons of huntingtin homologues might help to define conserved or newly emergent functional domains in mammalian cells. Huntingtin homologues in the vertebrate subphylum are highly conserved (80%)150, whereas Drosophila melanogaster huntingtin, the only entirely known invertebrate sequence, is characterized by an additional region and by five 2050% conserved regions59; these may represent a residue of the ancestral huntingtin at the origin of the protostomadeuterostoma branches (yellowgreen). In support of this, huntingtin seems to be present in Dictyostelium discoideum, but not in the older Saccharomyces cerevisiae (fungi, light blue) or in previously divergent plants, which suggests that huntingtin is an evolutionarily old gene that has been lost in some later animals46,59 (for example, Caenorhabditis elegans151). Only partial sequences are available from other deuterostoma invertebrates (one tunicata, two echinodermata), in which expression of the partially identified carboxy-terminal huntingtin seems to be confined to non-neural tissues152. By contrast, in vertebrates huntingtin is expressed throughout life and in all tissues, but is particularly enriched in the brain, which suggests that it may have acquired individual activities important for the newly formed nervous system and essential for post-mitotic neurons. Although huntingtin passages linking vertebrates and D. melanogaster along the evolutionary tree have not been fully described, the available information raises the possibility that D. melanogaster huntingtin has developed some different functions from those of the mammalian protein21,22. The polyglutamine (polyQ) region seems to have appeared in huntingtin for the first time in fishes and has been maintained during vertebrate evolution, becoming highly polymorphic only in humans (D. melanogaster huntingtin has no polyQ region59). The four glutamine residues in fishes huntingtin are not followed by a polyproline (polyP) tract, which appeared later in vertebrate evolution, possibly providing structural advantages150,153,154. Because polyP helps to maintain protein solubility42, it is also possible that, during evolution, an expanded polyQ has conferred important molecular function(s) to the protein, in part because of cooperation with the emerging polyP tract.

DOMINANTNEGATIVE

Describes a mutant molecule that can form a heteromeric complex with the normal molecule, knocking out the activity of the entire complex.

abnormalities, cognitive deficits and significant neuronal loss in the subthalamic nucleus, suggesting that reduced huntingtin levels could contribute to HD pathogenesis19,71. This phenotype, which is not replicated in the other two knockout mice, might reflect a DOMINANTNEGATIVE effect of a remaining 20 kDa N-terminal fragment over the full-length wild-type protein, regardless of the CAG tract. From these data we can conclude that wild-type huntingtin has important functions that build up during mammalian development, and possibly reflect the evolutionary steps of the protein (FIG. 2; BOX 3. In higher vertebrates (late deuterostoma species, see BOX 3), huntingtin might have acquired activities that

Various data highlight the continued importance of wild-type huntingtin function in post-mitotic brain cells and adulthood BOX 1; FIG. 1). In vitro studies of conditionally immortalized striatum-derived cells overexpressing the human wild-type protein14 provided the first direct demonstration that wild-type huntingtin is neuroprotective in brain cells exposed to various apoptotic stimuli, such as serum deprivation, mitochondrial toxins or the transfection of death genes14. It has also been shown that the in vitro anti-apoptotic activity of wild-type huntingtin is contained within the 548 N-terminal amino acids of the normal protein, the first recognized domain endowed with biological function14. These data also suggest that this specific function of huntingtin is cell-autonomous. The in vivo function of the wild-type protein in neurons was tested using the Cre/loxP SITESPECIFIC RECOMBI NATION SYSTEM driven by the neuron-specific -subunit of the calcium/calmodulin-dependent protein kinase II (-CaMKII) promoter to generate a null mutation of huntingtin at late stages of mouse forebrain development20. The neuronal inactivation of huntingtin led to neurological abnormalities and progressive degeneration (apoptotic cells in the hippocampus, cortex and striatum, and a lack of axons), with death occurring by the age of 1 year20 . Earlier morphometric and ultrastructural analyses of one heterozygous knockout mouse strain yielded similar results19. The overexpression of wild-type huntingtin also protects against ischaemic injury in vivo17 and against 18 EXCITOTOXICITY , to which cells are made more susceptible by the full-length mutant protein18,86. Neuroprotection is enhanced with a progressive increase in the level of wild-type huntingtin, which indicates a gene-dosage effect18. All of these studies indicate that wild-type huntingtin can trigger molecular events that lead to increased cell survival well beyond the developmental period. Investigations of the underlying molecular mechanisms have shown that wild-type huntingtin prevents cells from dying by inhibiting the processing of pro-caspase 9 REFS 14,87, and that it also inhibits the formation of the pro-apoptotic protein interactor of huntingtin-interacting protein 1 (HIPPI)HIP1 (huntingtin-interacting protein 1) complex88. Huntingtin can also be a substrate for Akt, a serine/threonine kinase that activates pro-survival pathways63,89.
Huntingtin controls BDNF production

Wild-type huntingtin has also been linked to brainderived neurotrophic factor (BDNF), a neurotrophin that is particularly important for the survival of striatal

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In rodents, the Bdnf gene contains four 5 exons (IIV) that are linked to separate promoters to produce four different transcripts (mRNAs IIV), which are spliced to the fifth 3 exon (V) to produce the BDNF protein. The different promoter-like sequences upstream of exons IIV act independently to modulate the tissue transcription of mRNA IIV in a development- and stimulus-specific manner105,106. These different transcripts may also have differential subcellular localization and targets107. Human BDNF is even more complex and contains a larger number of exons, some of which are still under investigation (GenBank accession number NT_009237.17, nucleotide positions 2646368526530516). The use of promoter reporter assays and polymerase chain reaction (PCR) for the specific mRNAs IIV showed that enhanced transcription from Bdnf exon II accounts for the increased amount of BDNF found in the presence of wild-type huntingtin, whereas transcription from exons I, III and IV is not affected15. Conversely, reduced BDNF mRNA levels are found in samples without wild-type huntingtin (C. Z., unpublished observations), as well as in heterozygous huntingtin-knockout mice16. Huntingtin mutation causes a similar reduction in the transcription of BDNF mRNA II, and also decreases transcription from Bdnf exons III and IV REFS 15,16. These findings have led to the conclusion that wild-type huntingtin contributes to the pool of BDNF protein produced in the cerebral cortex, and that a loss of or reduction in wild-type huntingtin activity might affect the stability of the cortical afferents and decrease BDNF support to striatal targets. Other studies have concentrated on BDNF trafficking and suggest that, in addition to controlling BDNF mRNA production, wild-type huntingtin may also regulate BDNF transport along the cortico-striatal afferents, and that huntingtin mutation affects the ability of BDNF to reach its striatal targets.
Huntingtin facilitates vesicular transport

Embryo Gastrulation Nervous system

Adult Post-mitotic neuron

Wild type

Wild type

Wild type

Cre/loxP SITESPECIFIC RECOMBINATION SYSTEM

A system derived from the Escherichia coli bacteriophage P1. Two short DNA sequences (loxP sites) are engineered to flank the target DNA. Activation of the Crerecombinase enzyme catalyses recombination between the loxP sites, which leads to excision of the intervening sequence. This tool is used at late stages of maturation or in the adult to study the function of genes whose deletion causes embryonic lethality.
EXCITOTOXICITY

Mutant

Mutant

Mutant

Figure 2 | Huntingtin function over a lifetime. Wild-type huntingtin function during development and in adults may arise from the use of different protein domains (pale or dark green), and by recruiting different sets of interactors (coloured circles). During embryogenesis, huntingtin is essential for gastrulation and the development of the nervous system; during development, mutant huntingtin has the same activities as the wild-type protein (and is therefore coloured green); in adulthood, wild-type huntingtin controls the activity of mammalian neurons. In Huntingtons disease, the altered three-dimensional structure of huntingtin bearing an expanded polyglutamine region (orange) gives rise to aberrant interactions that cause toxicity and also possibly interfere with normal huntingtin function.

Cellular toxicity involving the activation of glutamate receptors in the CNS. Glutamate, an excitatory amino acid neurotransmitter, activates different types of ionotropic (ion channel-forming) and metabotropic (G-proteincoupled) receptor. Excessive activation of these receptors by high concentrations of glutamate or by neurotoxins acting at the same receptors leads to cell death.
CORTICOSTRIATAL SYNAPSE

Cortical afferents reaching the striatum are intermingled with other cellular elements (the striatal targets, dopaminergic inputs and glial cells), which may be able to influence the output of the cortex. The activity of each of these cellular elements is finely regulated through a complex interplay between the receptor systems they express.
FAST AXONAL TRAFFICKING

Several motor proteins move various cargoes on microtubule tracks such as membrane organelles, protein complexes, complexes of nucleic acids, signalling molecules, neuroprotective and repair molecules, and vesicular and cytoskeletal components to deliver them from the neuronal cell body through the long axon to their final destination.

neurons and the activity of CORTICOSTRIATAL SYNAPSES9094. BDNF is co-localized with huntingtin in cortical neurons that project to the striatum95 and, despite some reports of BDNF mRNA transcription in adult striatal neurons96, most striatal BDNF is produced in the cerebral cortex97,98. After being anterogradely transported into vesicles along the cortico-striatal afferents, cortical BDNF is released at the axon terminals and captured by striatal neurons, so giving rise to rapid intracellular signals93,99,100. At the cortico-striatal synapse, BDNF also controls glutamate release94, and its exogenous administration allows striatal neurons to survive excitotoxin-induced neurodegeneration101103. In vitro and in vivo data show that wild-type huntingtin, but not the mutant protein, stimulates cortical BDNF production by acting at the level of Bdnf gene transcription. Cultured brain cells overexpressing wild-type huntingtin produce increased BDNF mRNA and protein, an effect that is lost in cells overexpressing mutant huntingtin15. Mice carrying an additional yeast-derived artificial chromosome (YAC) that includes the full-length wild-type huntingtin gene, in which huntingtin levels are increased, also show high BDNF protein levels in the cerebral cortex, an effect that is mediated by the positive regulation of Bdnf transcription by wild-type huntingtin15,104. As a consequence, YAC mice also have higher levels of BDNF in the striatum15. A more thorough assessment of the molecular mechanism by which wild-type huntingtin affects Bdnf transcription has shown that the normal protein regulates the activity of the BDNF promoter15,16 (FIG. 3).

Wild-type huntingtin specifically enhances the vesicular transport of BDNF along microtubules23 (FIG. 4). In vitro, full-length wild-type huntingtin stimulates (but mutant huntingtin represses) BDNF vesicular trafficking in neuronal cells, and BDNF transport can be attenuated by reducing the levels of wild-type huntingtin using RNAi. The ability of wild-type huntingtin to enhance vesicular transport involves huntingtin-associated protein 1 (HAP1, the first identified huntingtin interactor) and the p150(Glued) subunit of dynactin, which is an essential component of molecular motors. Huntingtin normally interacts with the p150(Glued) subunit via HAP1, and thereby stimulates BDNF transport23. Support for this idea is provided by earlier studies in D. melanogaster in which levels of the normal protein were experimentally reduced21. The loss of huntingtin in D. melanogaster neurons causes defects in FAST AXONAL TRAFFICKING and degeneration of the eye, a phenotype that is identical to that obtained after the expression of an exogenous mutant huntingtin fragment21,108. Huntingtin is found predominantly in the

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Wild type

Huntingtin controls neuronal gene transcription

NTYAGMRCCNNRGMSAG

II

III

IV

Bdnf gene

Figure 3 | Wild-type but not mutant huntingtin facilitates cortical BDNF mRNA production. Wild-type huntingtin contributes to brain-derived neurotrophic factor (Bdnf ) transcription in the cortical neurons that project to the striatum by inhibiting the Repressor element 1/neuron-restrictive silencer element (RE1/NRSE) that is located in BDNF promoter exon II. IIV indicate BDNF promoter exons in rodent Bdnf; V indicates the coding region. The RE1/NRSE consensus sequence is shown. Inactivation of the RE1/NRSE in Bdnf leads to increased mRNA transcription and protein production in the cortex. BDNF, which is also produced through translation from exons III and IV is then made available to the striatal targets via the cortico-striatal afferents. Wild-type huntingtin might also facilitate vesicular BDNF transport from the cortex to the striatum.

cytoplasm of neurons and is enriched in compartments containing vesicle-associated proteins31, and its anteroand retrograde transport in rat sciatic nerve axons is consistent with a vesicle association109. As the same has been found for HAP1, the protein that interacts with both huntingtin and the p150 subunit of dynactin, both huntingtin and HAP1 might have vital roles as scaffolding proteins in axonal transport and (perhaps with dynactin) a role in establishing bidirectional transport109,110. Wild-type huntingtin is also involved in fast axonal trafficking in mammalian neurons22. In embryonic striatal neurons taken from mice expressing only one copy of the wild-type allele or <50% of normal huntingtin levels upon CRE-mediated recombination (knockout), mitochondria became progressively immobilized. This effect was significantly stronger in complete knockout neurons than in those with a 50% loss of huntingtin, which points to a dose-dependent effect. The expression of mutant huntingtin in otherwise normal cells also impaired mitochondria trafficking, although to a lesser extent, further indicating that there might not be a simple loss-of-function mechanism in HD22. However, no mitochondria movement defects were detected in another study using mutant huntingtin knock-in cells, probably because of differences in the experimental systems23.

Further investigations of the mechanism by which wild-type huntingtin stimulates Bdnf transcription have concentrated on BDNF promoter exon II (FIG. 4), in which a conserved 2123 base pair DNA Repressor element 1 (RE1; also known as the neuronrestrictive silencer element, NRSE) is recognized by the RE1-silencing transcription factor (REST; also known as neuronal restrictive silencing factor, NRSF) transcriptional regulator, which is proposed to act as a transcriptional silencer111115. RE1/NRSE sequences are also found in genes involved in neuronal development and normal neuronal functions, such as ion channels, neurotransmitter receptors and their synthesizing enzymes, and synaptic vesicle proteins. Recent computational analyses of human and mouse genomes have found 1,892 and 1,894 RE1/NRSE sites, respectively116, so it is possible that wild-type huntingtin stimulates the transcription of many RE1/NRSE-controlled neuronal genes, other than Bdnf, by silencing the RE1/NRSEs. We have found that wild-type huntingtin promotes Bdnf transcription because it sequesters the available REST/NRSF in the cytoplasm, thereby preventing it from forming the nuclear co-repressor complex at the RE1/NRSE nuclear site and allowing gene transcription16. Mutant huntingtin causes the pathological entry of REST/NRSF into the nucleus, so leading to repressor complex formation and reduced transcription (FIG. 4). Further data indicate that wild-type huntingtin has a broader role in regulating neuronal gene transcription cells and mice expressing increased wild-type but not mutant huntingtin levels also show higher levels of mRNAs transcribed from many other RE1/NRSEcontaining neuronal genes, whereas mutant huntingtin is associated with reduced transcription from neuronal genes with RE1/NRSEs16 (C. Z., unpublished observations). Huntingtin might therefore act in the nervous system as a general facilitator of neuronal gene transcription.
Huntingtin at synaptic terminals

Huntingtin is involved in the cell machinery that controls synaptic transmission. Normal huntingtin interacts with a number of cytoskeletal and synaptic vesicle proteins that are essential for exo- and endocytosis at synaptic terminals117. One key molecule in synaptic transmission is PSD-95 (postsynaptic density protein 95), a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that binds the NMDA (N-methyld-aspartate) and kainate receptors at the postsynaptic density118. Wild-type huntingtin directly binds the Src homology 3 (SH3) domains of PSD-95 REF. 26. The decreased interaction of mutant huntingtin with PSD-95 indicates that more PSD-95 is released in HD, which affects the activity of NMDA receptors and might lead to their overactivation or sensitization, and excitotoxicity26. By contrast, the overexpression of wild-type huntingtin seems to attenuate the neuronal toxicity induced by NMDA receptors and mutant huntingtin26.

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Wild-type huntingtin reduces mutant huntingtin toxicity. Expression of mutant huntingtin in the absence of wild-type huntingtin results in massive apoptotic cell death in the testes of male mice25. This cell death can be modulated by the expression of normal huntingtin. In fact, no evidence of apoptosis is seen in the testes of mice expressing human mutant huntingtin when wildtype huntingtin is expressed from both Hdh alleles25; the same data also indicate that wild-type huntingtin protects non-neuronal cells from death. Similar results were obtained in in vitro experiments involving nonneuronal cells, which showed that wild-type protein overexpression reduces the polyQ toxicity induced by an exogenous mutant huntingtin construct24. Another approach considers crossing mice expressing exogenous wild-type huntingtin with mice carrying the mutant gene. Restoration of at least some parameters is expected (B. Leavitt, unpublished observations). Reduced wild-type huntingtin increases HD damage. Van Raamsdonk et al. compared the phenotypic severity of YAC128+/+ mice with YAC128/ mice. YAC128+/+ animals express, over the two endogenous wild-type alleles (+/+), a dose of mutant 128-CAG huntingtin that corresponds to 75% of endogenous levels, whereas YAC128/ mice do not express endogenous wild-type huntingtin (/) but express the same amount (75% of endogenous huntingtin levels) of the mutant protein. The loss of wild-type huntingtin in the YAC128/ mice led to a slight worsening of striatal atrophy and neuronal loss, and a small but significant decrease in neuronal cross-sectional area27. YAC128/ mice performed worse than YAC128+/+ mice in the rotarod test of motor coordination and were hypoactive at 2 months of age. In addition, testicular atrophy and degeneration were markedly worsened in the absence of wild-type huntingtin. Finally, YAC128+/+ mice showed a malespecific deficit in survival at 12 months of age, which was exacerbated in YAC128/ mice. It is concluded that elimination of wild-type huntingtin expression in YAC128 mice results in the exacerbation of behavioural deficits and survival, with a mild worsening of neuropathology at 12 months. The absence of more severe striatal abnormalities suggests that the striatal phenotype is dependent primarily on mutant huntingtin toxicity. However, it might be important to characterize these mice at a molecular level and at later time points. In addition, it is possible that YAC128/ mice carry a fragment of wild-type huntingtin (not deleted in the original Hdh/ mice)71. The fact that a reduction of wild-type huntingtin worsens the behaviour of HD mice in the absence of a clear striatal pathology also suggests that a simple increase in the wild-type huntingtin allelic dose might not be sufficient to counteract all phenotypes in HD, and that more appropriate strategies should be aimed at restoring the activity of the downstream targets of wild-type huntingtin11,122. However, it should be noted that the total amount of intact huntingtin protein in this study was always 50%, whereas this might not be true in patients. Another mouse model that stably

a
REST HDAC HDAC SIN3A coREST NRSE BDNF*

b
HDAC HDAC SIN3A coREST REST Mutant NRSE BDNF*

Wild type

HA

P1 HA P1

HA

P1 HA P1

Figure 4 | Molecular mechanism of wild-type huntingtin function. Huntingtin activity on (a) the transcription of brain-derived neurotrophic factor (BDNF) and of many other neuronal genes and (b) BDNF transport. a | Wild-type huntingtin sequesters Repressor element 1 (RE1)-silencing transcription factor (REST, also known as neuronal restrictive silencing factor, NRSF) in the cytoplasm in such a way that the co-repressor complex does not form at the RE1 (also known as the neuron-restrictive silencer element, NRSE) sites and the BDNF gene is transcribed. The asterisk indicates that wild-type huntingtin facilitates the transcription of many other RE1/NRSEcontrolled neuronal genes (for example, synapsin I, pro-enkephalin, muscarinic acetylcholine receptor 4 and dynamin 1) as well as BDNF. b | In Huntingtons disease, mutant huntingtin is less capable of retaining REST/NRSF in the cytoplasm, and it enters the nucleus where it leads to the formation of the repressor complex and reduced transcription of neuronal genes, including BDNF. c | Wild-type huntingtin is also part of a motor complex that controls vesicle BDNF transport along microtubules: it binds to huntingtin-associated protein 1 (HAP1) and indirectly regulates the assembly of p150(Glued) (grey) with the dynein (blue) and dynactin complexes (pink) that ultimately control the microtubule transport of BDNF vesicles (yellow). Arrows indicate the movement of the BDNF vesicles. The transient binding of wild-type huntingtin with cytoskeletal proteins might allow the movement of the BDNF vesicles. d | Under pathological conditions, mutant huntingtin binds more tightly to HAP1, reducing the transport of BDNF vesicles along microtubules. coREST, REST co-repressor; HDAC, histone deacetylase; SIN3A, SIN3 homologue A, a transcription regulator.

Reduced huntingtin activity in HD

The above data indicate that wild-type huntingtin has beneficial functions in the mature brain, so it is possible that its loss in patients with HD reduces the ability of neurons to survive the toxic effects of the mutant protein and thereby contributes significantly to disease pathogenesis. In mouse models, homozygosity for the HD mutation leads to a more severe phenotype than heterozygosity for an equivalent CAG expansion in the HD gene119,120. Similarly, a small cohort of patients with HD who were homozygous for the CAG expansion showed more severe disease progression than those who were heterozygous for the mutation121. However, these studies were unable to determine the relative contributions of the loss of wild-type huntingtin and the increased expression of mutant huntingtin to the worsening phenotype. In an attempt to address this question, a new set of experiments were carried out to investigate the ability of increased wild-type huntingtin to mitigate the damage caused by the mutant protein; the impact of reducing wild-type huntingtin levels in mice carrying the mutant gene; and the specific consequences of reduced levels of wild-type huntingtin downstream effectors (for example, BDNF) in a mouse model of HD.

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expresses <50% of total huntingtin protein (wild-type plus mutant) shows an early phenotype at a striatal level81; although this analysis was mainly morphological and focused on the early stages compatible with life, the data seem to indicate the presence of striatal defects when huntingtin is below an acceptable level. Reduced BDNF levels in HD. The finding that normal huntingtin stimulates Bdnf gene transcription/protein production and its axonal transport has prompted analyses of BDNF levels in the cortex and striatum of transgenic mice and patients with HD. As BDNF is synthesized in the cerebral cortex but not in the striatum, mutant huntingtin should reduce mRNA and protein levels in the cortex and lead to reduced striatal BDNF levels. With the exceptions discussed below, a consistent reduction in cortical BDNF protein and its mRNA has been reported in all cases. In fact, reduced BDNF mRNA and protein levels are found in the cerebral cortex from: YAC mice expressing the full-length mutant protein15,123; R6/2 mice, which express a 63 amino acid N-terminal portion of mutant huntingtin17,122,124; mice expressing a 517 amino acid N-terminal portion of huntingtin with 82 polyQ repeats125; mutant huntingtin knock-in mice126; and post-mortem cortical tissue from a small cohort of patients with HD15. These data indicate that reduced Bdnf transcription occurs in HD. This is due to reduced wild-type huntingtin activity on Bdnf exon II. Furthermore, mutant huntingtin also reduces transcription of exons III and IV, which suggests that the lower cortical BDNF levels in HD are due to both the loss of wildtype huntingtin function and increased toxicity of the mutant protein15,122. Wild-type huntingtin also stimulates BDNF transport, and analysis of BDNF protein in a mouse model of HD shows that the mutation causes a reduction in the efficiency of BDNF vesicle transport along microtubules 23 . This study was conducted using heterozygous knock-in mice. However, another study that used the same mice, but in homozygosity, showed a 2.2-fold reduction in BDNF production in the cortex with respect to control mice, possibly affecting its transport along the cortico-striatal afferents126. The possibility that a defect in BDNF transport from cortex to striatum occurs in human HD was inferred from western blot data showing reduced BDNF levels in the striatum but not in the cortex of post-mortem brain extracts from ten patients with HD23. An earlier study also reported reduced amounts of BDNF in the striatum but not in the cortex 127 . Reduced striatal but not cortical BDNF has also been reported in the R6/1 mouse line, which shows a milder and more delayed phenotype than R6/2 mice128. However, in another study, R6/1 mice show normal levels of BDNF in the cortex and striatum129. In addition, the results of studies of many other mouse models and a limited number of human cases of HD (reviewed above)15,17,122126 have shown reduced levels of cortical BDNF protein (and mRNA), which is consistent with reduced transcriptional activity in HD. Despite these differences, which subsequent studies will clarify, the data reviewed so far suggest that the reduction in the amount of BDNF reaching striatal targets might cause the preferential vulnerability of striatal neurons in HD. The impact of reduced cortical BDNF has been studied in EmxBdnf conditional knockout mice, in which cortical BDNF is almost completely eliminated98. In addition to finding that striatal and cortical volumes were decreased and the morphology of medium-sized spiny striatal neurons was altered, the study revealed a long-term in vivo requirement for cortical BDNF to support striatal neuronal survival. Adult EmxBdnf-knockout mice also have a hindlimb clasping phenotype similar to that observed in mouse models of HD98. In another experiment, an earlier onset and enhanced severity of motor alterations was detected in mice expressing only one functional Bdnf allele on a background of an overexpressed N-terminal mutant huntingtin fragment (the R6/1 line)129. In particular, it was found that an insufficient level of BDNF in the striatum of R6/1 mice leads to degeneration of the enkephalinergic striatal projection neurons (those most affected in HD), a phenotype that could be restored by exogenous BDNF administration129. Reduced BDNF also seems to exacerbate dopaminergic neuronal dysfunction in the same mouse model130. Together, these data indicate that wild-type huntingtin contributes to the pool of BDNF found in the brain, and that reduced BDNF levels affect the development of HD in mice. Reduced huntingtin function in HD might ultimately occur as a result of a dominantnegative mechanism in the presence of the mutant protein1,131 and/or wild-type huntingtin sequestration in aggregates132,133, or because of its degradation by active caspases and calpains17,52,53,56,134,135.
Concluding remarks

Huntingtin is a large protein that interacts with numerous other proteins, the roles of many of which still await biological confirmation39,41. Huntingtin has been found in different cellular locations, where it may act as a scaffold protein for the activation and correct assembly, in time and space, of specific molecular partners and pathways45,68. It is ubiquitously expressed and has anti-apoptotic properties in many intra- and extracerebral cell types. But why is Huntingtons disease limited to neurons, and particularly to striatal neurons? Given the function of wild-type huntingtin in the brain, its reduced activity on specific downstream effectors, such as BDNF, might exacerbate the sensitivity of the striatal neurons to the expanded glutamine repeats86,136. In addition, reduced BDNF might severely affect the activity of the cortico-striatal synapse, therefore contributing to selective vulnerability. It is known that striatal neurons from HD mice are much more sensitive to excitotoxins and that changes in BDNF levels can influence the glutamatergic output of the cortex94,86,137. Other neurons besides those in the striatum are also affected in HD3,138, and reduced wild-type huntingtin activity on

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axonal transport, synaptic activity and neuronal gene transcription might contribute to this generalized neuronal dysfunction. The neuronal specificity of the RE1/NRSEREST/NRSF pathway might be related to the limited presence of REST/NRSF in the brain (where its nuclear translocation is regulated by huntingtin), compared with its level in non-neuronal tissues, where it represses neuronal genes regardless of the presence of wild-type or mutant huntingtin16. Is the loss of wild-type huntingtin involved in the disease process? If wild-type huntingtin has a function in mammalian neurons and this is inhibited by mutant huntingtin, the defects observed in mutant mice and cells should also be observed in the absence or reduced presence of wild-type huntingtin. Evidence reviewed here indicates that reducing wild-type huntingtin levels or expressing the mutant protein might have similar effects on specific phenotypes, which suggests that at least some of the molecular dysfunctions observed in HD are a consequence of reduced wild-type huntingtin activity. Discrimination of the abnormalities that depend on the activity of the mutant protein versus the reduced function of wild-type huntingtin becomes crucial. In the first case, to restore the normal phenotype one would have to interfere with the events elicited by the mutant protein. In the second case, compounds able to mimic normal huntingtin activity on its downstream targets would be ideal. The next phase of research on huntingtin should also consider how this protein has acquired such important activities; if there are specific functional domains in the protein and what their boundaries are; how the activity of these domains relates to the natural history of HD; and whether there are wild-type huntingtin effectors that can be exploited in drug-screening strategies. The RE1/NRSEREST/NRSF pathway could be a first target. Although the structure of huntingtin remains unknown, a lot of information is now available about its function in brain cells and the interactions of its 3,144 amino acids with other proteins39,41. Research on huntingtin physiology will continue to contribute to our understanding of neuronal development and function. It will also give us molecular cues as to how a protein with a range of important functions becomes detrimental for HD neurons. But the next big challenge will be to transform the study of normal huntingtin function into an additional avenue for the treatment of HD.

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Acknowledgements
The work described in this review has been supported by grants from the Huntingtons Disease Society of America (HDSA; USA), the Hereditary Disease Foundation (HDF; USA), HighQ Foundation (USA), Telethon (Italy), the Cariplo Foundation (Italy), Ministero dellIstruzione dellUniversita e della Ricerca (Italy) and the European Commission Framework VI Programme (NeuroNE). E.C. is member and coordinator of the Huntingtin Function HDSA Coalition For the Cure Team. We apologize for the omission of a number of significant papers that could not be cited because of space limitations.

Competing interests statement

The authors declare no competing financial interests.

Online links
DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene BDNF | HAP1 | HIP1 | HIP14 | HIPPI | huntingtin | PSD-95 | REST | TPR Entrez Nucleotide: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=Nucleotide BDNF OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=OMIM CreutzfeldtJakob disease | Fragile X mental retardation | Huntingtons disease | WolfHirschhorn syndrome FURTHER INFORMATION Cattaneos lab: http://users.unimi.it/~spharm/cattaneo/ Access to this interactive links box is free online.

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