Benefits of global mutant huntingtin lowering diminish over time in a Huntington's disease mouse model.

We have developed an inducible Huntington's disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt (~50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow-up revealed that the benefits, in all mHtt-lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12 months of age, and late mHtt lowering did not ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefits in humans.

Dynamics of huntingtin protein interactions in the striatum identifies candidate modifiers of Huntington disease.

Huntington disease (HD) is a monogenic neurodegenerative disorder with one causative gene, huntingtin (HTT). Yet, HD pathobiology is multifactorial, suggesting that cellular factors influence disease progression. Here, we define HTT protein-protein interactions (PPIs) perturbed by the mutant protein with expanded polyglutamine in the mouse striatum, a brain region with selective HD vulnerability. Using metabolically labeled tissues and immunoaffinity purification-mass spectrometry, we establish that polyglutamine-dependent modulation of HTT PPI abundances and relative stability starts at an early stage of pathogenesis in a Q140 HD mouse model. We identify direct and indirect PPIs that are also genetic disease modifiers using in-cell two-hybrid and behavioral assays in HD human cell and Drosophila models, respectively. Validated, disease-relevant mHTT-dependent interactions encompass mediators of synaptic neurotransmission (SNAREs and glutamate receptors) and lysosomal acidification (V-ATPase). Our study provides a resource for understanding mHTT-dependent dysfunction in cortico-striatal cellular networks, partly through impaired synaptic communication and endosomal-lysosomal system. A record of this paper's Transparent Peer Review process is included in the supplemental information.

Characterization of a Knock-In Mouse Model with a Huntingtin Exon 1 Deletion.

BACKGROUND: The Huntingtin (HTT) N-terminal domains encoded by Huntingtin's (HTT) exon 1 consist of an N17 domain, the polyglutamine (polyQ) stretch and a proline-rich region (PRR). These domains are conserved in mammals and have been hypothesized to modulate HTT's functions in the developing and adult CNS, including DNA damage repair and autophagy. OBJECTIVE: This study longitudinally characterizes the in vivo consequences of deleting the murine Htt N-terminal domains encoded by Htt exon 1. METHODS: Knock-in mice with a deletion of Htt exon 1 sequences (HttΔE1) were generated and bred into the C57BL/6J congenic genetic background. Their behavior, DNA damage response, basal autophagy, and glutamatergic synapse numbers were evaluated. RESULTS: Progeny from HttΔE1/+ intercrosses are born at the expected Mendelian frequency but with a distorted male to female ratio in both the HttΔE1/ΔE1 and Htt+/+ offspring. HttΔE1/ΔE1 adults exhibit a modest deficit in accelerating rotarod performance, and an earlier increase in cortical and striatal DNA damage with elevated neuronal pan-nuclear 53bp1 levels compared to Htt+/+ mice. However, a normal response to induced DNA damage, normal levels of basal autophagy markers, and no significant differences in corticocortical, corticostriatal, thalamocortical, or thalamostriatal synapses numbers were observed compared to controls. CONCLUSION: Our results suggest that deletion of the Htt N-terminus encoded by the Htt exon 1 does not affect Htt's critical role during embryogenesis, but instead, may have a modest effect on certain motor tasks, basal levels of DNA damage in the brain, and Htt function in the testis.

Huntington's disease alters human neurodevelopment.

Although Huntington's disease is a late-manifesting neurodegenerative disorder, both mouse studies and neuroimaging studies of presymptomatic mutation carriers suggest that Huntington's disease might affect neurodevelopment. To determine whether this is actually the case, we examined tissue from human fetuses (13 weeks gestation) that carried the Huntington's disease mutation. These tissues showed clear abnormalities in the developing cortex, including mislocalization of mutant huntingtin and junctional complex proteins, defects in neuroprogenitor cell polarity and differentiation, abnormal ciliogenesis, and changes in mitosis and cell cycle progression. We observed the same phenomena in Huntington's disease mouse embryos, where we linked these abnormalities to defects in interkinetic nuclear migration of progenitor cells. Huntington's disease thus has a neurodevelopmental component and is not solely a degenerative disease.

Columnar-Intrinsic Cues Shape Premotor Input Specificity in Locomotor Circuits.

Control of movement relies on the ability of circuits within the spinal cord to establish connections with specific subtypes of motor neuron (MN). Although the pattern of output from locomotor networks can be influenced by MN position and identity, whether MNs exert an instructive role in shaping synaptic specificity within the spinal cord is unclear. We show that Hox transcription-factor-dependent programs in MNs are essential in establishing the central pattern of connectivity within the ventral spinal cord. Transformation of axially projecting MNs to a limb-level lateral motor column (LMC) fate, through mutation of the Hoxc9 gene, causes the central afferents of limb proprioceptive sensory neurons to target MNs connected to functionally inappropriate muscles. MN columnar identity also determines the pattern and distribution of inputs from multiple classes of premotor interneurons, indicating that MNs broadly influence circuit connectivity. These findings indicate that MN-intrinsic programs contribute to the initial architecture of locomotor circuits.

Is Huntingtin Dispensable in the Adult Brain?

Huntingtin (HTT) is an essential protein during early embryogenesis and the development of the central nervous system (CNS). Conditional knock-out of mouse Huntingtin (Htt) expression in the CNS beginning during neural development, as well as reducing Htt expression only during embryonic and early postnatal stages, results in neurodegeneration in the adult brain. These findings suggest that HTT is important for the development and/or maintenance of the CNS, but they do not address the question of whether HTT is required specifically in the adult CNS for its normal functions and/or homeostasis. Recently, it was reported that although removing Htt expression in young adult mice causes lethality due to acute pancreatitis, loss of Htt expression in the adult brain is well tolerated and does not result in either motor deficits or neurodegeneration for up to 7 months after Htt inactivation. However, recent studies have also demonstrated that HTT participates in several cellular functions that are important for neuronal homeostasis and survival including sensing reactive oxygen species (ROS), DNA damage repair, and stress responses, in addition to its role in selective macroautophagy. In this review, HTT's functions in development and in the adult CNS will be discussed in the context of these recent discoveries, together with a discussion of their potential impact on the design of therapeutic strategies for Huntington's disease (HD) aimed at lowering total HTT expression.

Generation and Characterization of Knock-in Mouse Models Expressing Versions of Huntingtin with Either an N17 or a Combined PolyQ and Proline-Rich Region Deletion.

BACKGROUND: The polyglutamine (polyQ) stretch of the Huntingtin protein (HTT) in mammals is flanked by a highly conserved 17 amino acid N-terminal domain (N17), and a proline-rich region (PRR). The PRR is a binding site for many HTT-interacting proteins, and the N17 domain regulates several normal HTT functions, including HTT's ability to associate with membranes and organelles. OBJECTIVE: This study investigates the consequence of deleting mouse Huntingtin's (Htt's) N17 domain or a combination of its polyQ stretch and PRR (QP) on normal Htt function in mice. METHODS: Knock-in mice expressing versions of Htt lacking either the N17 domain (HttΔN17) or both the polyQ and PRR domains (HttΔQP) were generated, and their behavior, autophagy function, and neuropathology were evaluated. RESULTS: Homozygous and hemizygous HttΔQP/ΔQP, HttΔN17/ΔN17, HttΔQP/-, and HttΔN17/- mice were generated at the expected Mendelian frequency. HttΔQP/ΔQP mutants exhibit improvements in motor coordination compared to controls (Htt+/+). In contrast, HttΔN17/ΔN17 mutants do not exhibit any changes in motor coordination, but they do display variable changes in spatial learning that are dependent on their age at testing. Neither mutant exhibited any changes in basal autophagy in comparison to controls, but thalamostriatal synapses in the dorsal striatum of 24-month-old HttΔN17/ΔN17 mice were decreased compared to controls. CONCLUSIONS: These findings support the hypothesis that Htt's N17 and QP domains are dispensable for its critical functions during early embryonic development, but are likely more important for Htt functions in CNS development or maintenance.

Hox Proteins Coordinate Motor Neuron Differentiation and Connectivity Programs through Ret/Gfrα Genes.

The accuracy of neural circuit assembly relies on the precise spatial and temporal control of synaptic specificity determinants during development. Hox transcription factors govern key aspects of motor neuron (MN) differentiation; however, the terminal effectors of their actions are largely unknown. We show that Hox/Hox cofactor interactions coordinate MN subtype diversification and connectivity through Ret/Gfrα receptor genes. Hox and Meis proteins determine the levels of Ret in MNs and define the intrasegmental profiles of Gfrα1 and Gfrα3 expression. Loss of Ret or Gfrα3 leads to MN specification and innervation defects similar to those observed in Hox mutants, while expression of Ret and Gfrα1 can bypass the requirement for Hox genes during MN pool differentiation. These studies indicate that Hox proteins contribute to neuronal fate and muscle connectivity through controlling the levels and pattern of cell surface receptor expression, consequently gating the ability of MNs to respond to limb-derived instructive cues.

A series of N-terminal epitope tagged Hdh knock-in alleles expressing normal and mutant huntingtin: their application to understanding the effect of increasing the length of normal Huntingtin's polyglutamine stretch on CAG140 mouse model pathogenesis.

BACKGROUND: Huntington's disease (HD) is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ) stretch within Huntingtin (htt), the protein product of the HD gene. Although studies in vitro have suggested that the mutant htt can act in a potentially dominant negative fashion by sequestering wild-type htt into insoluble protein aggregates, the role of the length of the normal htt polyQ stretch, and the adjacent proline-rich region (PRR) in modulating HD mouse model pathogenesis is currently unknown. RESULTS: We describe the generation and characterization of a series of knock-in HD mouse models that express versions of the mouse HD gene (Hdh) encoding N-terminal hemaglutinin (HA) or 3xFlag epitope tagged full-length htt with different polyQ lengths (HA7Q-, 3xFlag7Q-, 3xFlag20Q-, and 3xFlag140Q-htt) and substitution of the adjacent mouse PRR with the human PRR (3xFlag20Q- and 3xFlag140Q-htt). Using co-immunoprecipitation and immunohistochemistry analyses, we detect no significant interaction between soluble full-length normal 7Q- htt and mutant (140Q) htt, but we do observe N-terminal fragments of epitope-tagged normal htt in mutant htt aggregates. When the sequences encoding normal mouse htt's polyQ stretch and PRR are replaced with non-pathogenic human sequence in mice also expressing 140Q-htt, aggregation foci within the striatum, and the mean size of htt inclusions are increased, along with an increase in striatal lipofuscin and gliosis. CONCLUSION: In mice, soluble full-length normal and mutant htt are predominantly monomeric. In heterozygous knock-in HD mouse models, substituting the normal mouse polyQ and PRR with normal human sequence can exacerbate some neuropathological phenotypes.

The long and the short of aberrant ciliogenesis in Huntington disease.

Huntington disease (HD) is a dominantly inherited neurodegenerative disorder that is caused by a mutant huntingtin (HTT) gene encoding a version of the Htt protein with an expanded polyglutamine stretch. Although the HTT gene was discovered more than 18 years ago, the functions of normal Htt and the mechanisms by which mutant Htt causes disease are not well defined. In this issue of the JCI, Keryer et al. uncovered a novel function for normal Htt in ciliogenesis and report that mutant Htt causes hypermorphic ciliogenesis and ciliary dysfunction. These observations suggest that it is now critical to understand the extent to which ciliary dysfunction contributes to the different symptoms of HD and to determine whether therapeutic strategies designed to normalize ciliary function can ameliorate the disease.

Gdf11 facilitates temporal progression of neurogenesis in the developing spinal cord.

Various types of neurons and glia are generated following a precise spatial and temporal order during neurogenesis. The mechanisms that control this sequential generation of neuronal and glial cell types from the same progenitor population are not well understood. Growth differentiation factor 11 (Gdf11) belongs to the TGF-ß family of proteins and is expressed transiently in newly born neurons adjacent to the progenitor domain in the developing spinal cord. We examined the phenotypes of Gdf11(-/-) mouse embryos and found that without Gdf11, neuronal differentiation in the spinal cord progresses at a slower rate. Higher progenitor proliferation rate, along with a delay in gliogenesis, is also observed in Gdf11(-/-) spinal cord but only after the peak of Gdf11 expression, indicating that Gdf11 can cause long-lasting changes in progenitor properties. These changes can be preserved in vitro, as neurospheres derived from Gdf11(-/-) and wild-type littermates at a stage after, but not before the onset of Gdf11 expression, exhibit differences in proliferation and differentiation potential. Moreover, these changes in progenitor properties can be induced in vitro by the addition of Gdf11. We also demonstrate that the effects of Gdf11 on progenitor cells are associated with its ability to upregulate p57(Kip2) and p27(Kip1) while downregulating Pax6 expression. These results support a model in which Gdf11 secreted by newly born neurons in the developing spinal cord facilitates the temporal progression of neurogenesis by acting as a positive feedback signal on the progenitor cells to promote cell cycle exit and decrease proliferation ability, thus changing their differentiation potential.

Generation of conditional Hoxc8 loss-of-function and Hoxc8-->Hoxc9 replacement alleles in mice.

The Hox family of transcription factors are expressed at different domains along the rostrocaudal (R-C) body axis during development. To examine the function of Hoxc8 and Hoxc9 in specific cell types and at different developmental times, we have generated and characterized loxP flanked (floxed) Hoxc8 and Hoxc8-->Hoxc9 replacement alleles of mice, with either GFP or LacZ reporters. Although all four alleles of mice behave like wild-type controls in motor behavioral testing, slight differences in endogenous Hox gene expression were observed among these alleles depending on the type of reporters used and the presence of Hoxc9 cDNA in the targeting constructs. The efficiency of Cre-mediated recombination was evaluated by crossing these mice with the Nestin-cre and Isl1-cre mice, and the loss of Hoxc8 expression with or without Hoxc9 misexpression was confirmed in embryonic spinal cord. In addition, an upregulation of reporter gene expression was observed after Cre-mediated recombination. These mice will be useful tools to analyze Hox gene function in a cell type-specific manner.

The function of growth/differentiation factor 11 (Gdf11) in rostrocaudal patterning of the developing spinal cord.

Hoxc family transcription factors are expressed in different domains along the rostrocaudal (RC) axis of the developing spinal cord and they define RC identities of spinal neurons. Our previous study using an in vitro assay system demonstrated that Fgf and Gdf11 signals located around Hensen's node of chick embryos have the ability to induce profiled Hoxc protein expression. To investigate the function of Gdf11 in RC patterning of the spinal cord in vivo, we expressed Gdf11 in chick embryonic spinal cord by in ovo electroporation and found that ectopic expression of Gdf11 in the neural tissue causes a rostral displacement of Hoxc protein expression domains, accompanied by rostral shifts in the positions of motoneuron columns and pools. Moreover, ectopic expression of follistatin (Fst), an antagonist of Gdf11, has a converse effect and causes caudal displacement of Hox protein expression domains, as well as motoneuron columns and pools. Mouse mutants lacking Gdf11 function exhibit a similar caudal displacement of Hox expression domains, but the severity of phenotype increases towards the caudal end of the spinal cord, indicating that the function of Gdf11 is more important in the caudal spinal cord. We also provide evidence that Gdf11 induces Smad2 phosphorylation and activated Smad2 is able to induce caudal Hox gene expression. These results demonstrate that Gdf11 has an important function in determining Hox gene expression domains and RC identity in the caudal spinal cord.

Motor neuron columnar fate imposed by sequential phases of Hox-c activity.

The organization of neurons into columns is a prominent feature of central nervous system structure and function. In many regions of the central nervous system the grouping of neurons into columns links cell-body position to axonal trajectory, thus contributing to the establishment of topographic neural maps. This link is prominent in the developing spinal cord, where columnar sets of motor neurons innervate distinct targets in the periphery. We show here that sequential phases of Hox-c protein expression and activity control the columnar differentiation of spinal motor neurons. Hox expression in neural progenitors is established by graded fibroblast growth factor signalling and translated into a distinct motor neuron Hox pattern. Motor neuron columnar fate then emerges through cell autonomous repressor and activator functions of Hox proteins. Hox proteins also direct the expression of genes that establish motor topographic projections, thus implicating Hox proteins as critical determinants of spinal motor neuron identity and organization.