Caspase-6 activity in a BACHD mouse modulates steady-state levels of mutant huntingtin protein but is not necessary for production of a 586 amino acid proteolytic fragment.

Huntington's disease (HD) is caused by a mutation in the huntingtin (htt) gene encoding an expansion of glutamine repeats at the N terminus of the Htt protein. Proteolysis of Htt has been identified as a critical pathological event in HD models. In particular, it has been postulated that proteolysis of Htt at the putative caspase-6 cleavage site (at amino acid Asp-586) plays a critical role in disease progression and pathogenesis. However, whether caspase-6 is indeed the essential enzyme that cleaves Htt at this site in vivo has not been determined. To evaluate, we crossed the BACHD mouse model with a caspase-6 knock-out mouse (Casp6(-/-)). Western blot and immunocytochemistry confirmed the lack of caspase-6 protein in Casp6(-/-) mice, regardless of HD genotype. We predicted the Casp6(-/-) mouse would have reduced levels of caspase-6 Htt fragments and increased levels of full-length Htt protein. In contrast, we found a significant reduction of full-length mutant Htt (mHtt) and fragments in the striatum of BACHD Casp6(-/-) mice. Importantly, we detected the presence of Htt fragments consistent with cleavage at amino acid Asp-586 of Htt in the BACHD Casp6(-/-) mouse, indicating that caspase-6 activity cannot fully account for the generation of the Htt 586 fragment in vivo. Our data are not consistent with the hypothesis that caspase-6 activity is critical in generating a potentially toxic 586 aa Htt fragment in vivo. However, our studies do suggest a role for caspase-6 activity in clearance pathways for mHtt protein.

Posttranslational modification of ataxin-7 at lysine 257 prevents autophagy-mediated turnover of an N-terminal caspase-7 cleavage fragment.

Polyglutamine (polyQ) expansion within the ataxin-7 protein, a member of the STAGA [SPT3-TAF(II)31-GCN5L acetylase] and TFTC (GCN5 and TRRAP) chromatin remodeling complexes, causes the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7). Proteolytic processing of ataxin-7 by caspase-7 generates N-terminal toxic polyQ-containing fragments that accumulate with disease progression and play an important role in SCA7 pathogenesis. To elucidate the basis for the toxicity of these fragments, we evaluated which posttranslational modifications of the N-terminal fragment of ataxin-7 modulate turnover and toxicity. Here, we show that mutating lysine 257 (K257), an amino acid adjacent to the caspase-7 cleavage site of ataxin-7 regulates turnover of the truncation product in a repeat-dependent manner. Modification of ataxin-7 K257 by acetylation promotes accumulation of the fragment, while unmodified ataxin-7 is degraded. The degradation of the caspase-7 cleavage product is mediated by macroautophagy in cell culture and primary neuron models of SCA7. Consistent with this, the fragment colocalizes with autophagic vesicle markers, and enhanced fragment accumulation increases in these lysosomal structures. We suggest that the levels of fragment accumulation within the cell is a key event in SCA7 neurodegeneration, and enhancing clearance of polyQ-containing fragments may be an effective target to reduce neurotoxicity in SCA7.

In-vitro analysis of Pitx3 in mesodiencephalic dopaminergic neuron maturation.

The transcription factor Pitx3 is expressed exclusively by mesodiencephalic dopaminergic neurons; however, ablation of Pitx3 results in selective degeneration of primarily dopaminergic neurons of the substantia nigra pars compacta, the neuronal population that is most vulnerable in Parkinson's disease. Although the exact molecular mechanisms of the action of Pitx3 are unclear, roles in both terminal maturation and/or survival of substantia nigra dopaminergic neurons have been suggested. To investigate the connection between Pitx3 and selective neurodegeneration, we generated embryonic stem cells from a Pitx3-deficient mouse (aphakia) for in-vitro differentiation to dopaminergic neurons. This 'loss of function'in-vitro system allowed us to examine characteristic features in dopaminergic neuron development and to assess the role that Pitx3 plays in the differentiation/maturation process. We found that aphakia embryonic stem cells generated 50% fewer tyrosine hydroxylase-positive/microtubule-associated protein (Map)2-positive mature neurons compared with control cultures. The expression of dopamine transport regulators and vesicle release proteins was reduced and dopamine release was unregulated in the Pitx3-deficient tyrosine hydroxylase-positive neurons generated. Treatment of aphakia embryonic stem cell cultures with retinoic acid resulted in a significant increase in mesodiencephalic tyrosine hydroxylase-positive neurons, providing further support for the role of Pitx3 in dopaminergic neuron specification through the retinoic acid pathway. Our study, using Pitx3-deficient embryonic stem cells in an in-vitro differentiation culture system, allowed us to assess the role of Pitx3 in the specification and final maturation of dopaminergic neurons.

In vitro generation of dopaminergic neurons from adult subventricular zone neural progenitor cells.

The adult subventricular zone (SVZ) supports a population of cells that display the hallmarks of stem cells: they are self-renewing and multipotent-capable of generating neurons, oligodendrocytes, and astrocytes. In vivo, these adult neural stem cells (aNSCs) are fated primarily for a gamma-amino butyric acid (GABA)-ergic lineage of olfactory bulb interneurons, a small subpopulation of which is dopaminergic. Here, we investigate the plasticity of aNSCs in vitro, in particular, their ability to generate a specific neuronal lineage, midbrain dopamine neurons. Previous work using mouse embryonic stem (ES) cells showed that introduction of early developmental inductive cues, sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF-8), directed ES cell-derived neuroepithelial cells to generate midbrain dopaminergic neurons, those lost in Parkinson's disease. Placing aNSCs under similar culture conditions, immunocytochemistry and RT-PCR analysis revealed early dopaminergic neuron specification. However, aNSC-derived neurons remained morphologically immature, exhibiting concurrent nestin and tyrosine hydroxylase (TH) expression, with cell death occurring in the final differentiation stage. High-performance liquid chromatography (HPLC) analysis revealed that while aNSC-derived neurons released dopamine, release was not significantly increased following depolarization with K+. In contrast, ES cell-generated TH+ neurons expressed the mature markers MAP2 and NeuN and showed K+-evoked release of dopamine. Reduced culture time of aNSC-derived nestin+ progenitors in FGF-2-containing medium improved survival of TH+ neurons. However, these neurons exhibited characteristics of forebrain dopamine neurons and also expressed low levels of midbrain transcription factors. Together, our data indicate that when presented with in vitro conditions that promote midbrain-specific dopamine neuron specification, aNSCs instead generate forebrain-like dopamine neurons, demonstrating their restricted and prescribed nature.

Histone deacetylase-3 interacts with ataxin-7 and is altered in a spinocerebellar ataxia type 7 mouse model.

Spinocerebellar ataxia type 7 (SCA7) is caused by a toxic polyglutamine (polyQ) expansion in the N-terminus of the protein ataxin-7. Ataxin-7 has a known function in the histone acetylase complex, Spt/Ada/Gcn5 acetylase (STAGA) chromatin-remodeling complex. We hypothesized that some histone deacetylase (HDAC) family members would impact the posttranslational modification of normal and expanded ataxin-7 and possibly modulate ataxin-7 function or neurotoxicity associated with the polyQ expansion. Interestingly, when we coexpressed each HDAC family member in the presence of ataxin-7 we found that HDAC3 increased the posttranslational modification of normal and expanded ataxin-7. Specifically, HDAC3 stabilized ataxin-7 and increased modification of the protein. Further, HDAC3 physically interacts with ataxin-7. The physical interaction of HDAC3 with normal and polyQ-expanded ataxin-7 affects the toxicity in a polyQ-dependent manner. We detect robust HDAC3 expression in neurons and glia in the cerebellum and an increase in the levels of HDAC3 in SCA7 mice. Consistent with this we found altered lysine acetylation levels and deacetylase activity in the brains of SCA7 transgenic mice. This study implicates HDAC3 and ataxin-7 interaction as a target for therapeutic intervention in SCA7, adding to a growing list of neurodegenerative diseases that may be treated by HDAC inhibitors.

Pizotifen Activates ERK and Provides Neuroprotection in vitro and in vivo in Models of Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is a dominantly inherited neurodegenerative condition characterized by dysfunction in striatal and cortical neurons. There are currently no approved drugs known to slow the progression of HD. OBJECTIVE: To facilitate the development of therapies for HD, we identified approved drugs that can ameliorate mutant huntingtin-induced toxicity in experimental models of HD. METHODS: A chemical screen was performed in a mouse Hdh(Q111/Q111) striatal cell model of HD. This screen identified a set of structurally related approved drugs (pizotifen, cyproheptadine, and loxapine) that rescued cell death in this model. Pizotifen was subsequently evaluated in the R6/2 HD mouse model. RESULTS: We found that in striatal Hdh(Q111/Q111) cells, pizotifen treatment caused transient ERK activation and inhibition of ERK activation prevented rescue of cell death in this model. In the R6/2 HD mouse model, treatment with pizotifen activated ERK in the striatum, reduced neurodegeneration and significantly enhanced motor performance. CONCLUSIONS: These results suggest that pizotifen and related approved drugs may provide a basis for developing disease modifying therapeutic interventions for HD.

Autophagy: polyQ toxic fragment turnover.

Recent studies have highlighted the importance of the lysosome in degrading proteins that misfold in neurodegenerative diseases. In this study we explore the role for autophagy in the clearance of an N-terminal caspase-7-generated fragment of ataxin-7, a protein with a pathogenic polyglutamine (polyQ) expansion in the neurodegenerative disease spinocerebellar ataxia 7 (SCA7). Using both cellular and transgenic mouse models of SCA7 we show that the stability of wild-type ataxin-7 is modified by macroautophagy, but not by proteasomal, inhibition, whereas both autophagy and proteasomal degradation have little effect on polyQ-expanded ataxin-7. We also create a post-translational modification-deficient ataxin-7 mutant that has increased protein turnover of both wild-type and polyQ-expanded ataxin-7, mediated through the autophagy pathway. Histological analysis reveals that wild-type ataxin-7 colocalizes with markers of chaperone-mediated autophagy (CMA) and macroautophagy, indicating that both of these mechanisms may play a role in the clearance of ataxin-7. Furthermore, there is an increase in LC3, a marker of autophagy initiation, in the cerebellum of SCA7 transgenic mice. Our findings indicate that the ataxin-7 fragment may be cleared via autophagy and that this process is altered in SCA7. Identification of the different types of autophagy involved in ataxin-7 turnover and the influence of post-translational modifications on these processes will be pursued in future studies.

IGF-1: elixir for motor neuron diseases.

Modulation of testosterone levels is a therapeutic approach for spinal and bulbar muscular atrophy (SBMA), a polyglutamine disorder that affects the motor neurons. The article by Palazzolo et al. in this issue of Neuron provides compelling evidence that the expression of insulin growth hormone is a potential therapeutic for SBMA.

Nuclear transfer embryonic stem cells provide an in vitro culture model for Parkinson's disease.

Somatic cell nuclear transfer enables the generation of embryonic stem cells (ESCs) that genetically match the donor and can be used to treat disease through cell replacement therapies or to recapitulate patient-specific disease via in vitro differentiation. We performed a "proof-of-principle" study using tail tip fibroblasts from a mouse model of Parkinson's disease (Aphakia) as the donor cell nuclei for nuclear transfer and derived "customized" ESCs for in vitro analysis. Aphakia mice contain deletions in the pitx3 gene and show selective loss of dopamine neurons of the substantia nigra, specifically the neuron population susceptible to degeneration in Parkinson's disease. Using electrofusion nuclear transfer, we produced cloned Aphakia oocytes at rates similar to those for control, cloned oocytes. Aphakia ESCs were isolated and live mice were generated using tetraploid embryo complementation. In vitro differentiation of Aphakia ESCs to dopaminergic neurons revealed significantly fewer TH+ neurons that expressed MAP2, DAT, synaptophysin, VMAT2, and AHD2 compared to control nuclear transfer ESC cultures, supporting a role for Pitx3 in mesodiencephalic dopamine neuron maturation. Taken together, our studies define a customized in vitro ESC culture system used to analyze gene-specific contribution to dopamine neuron generation, maturation, and susceptibility to degeneration.