Full-length huntingtin is palmitoylated at multiple sites and post-translationally myristoylated following caspase-cleavage.

Introduction: Huntington disease is an autosomal dominant neurodegenerative disorder which is caused by a CAG repeat expansion in the HTT gene that codes for an elongated polyglutamine tract in the huntingtin (HTT) protein. Huntingtin is subjected to multiple post-translational modifications which regulate its cellular functions and degradation. We have previously identified a palmitoylation site at cysteine 214 (C214), catalyzed by the enzymes ZDHHC17 and ZDHHC13. Reduced palmitoylation level of mutant huntingtin is linked to toxicity and loss of function. Moreover, we have described N-terminal myristoylation by the N-myristoyltransferases of a short fragment of huntingtin (HTT553-586) at glycine 553 (G553) following proteolysis at aspartate 552 (D552). Results: Here, we show that huntingtin is palmitoylated at numerous cysteines: C105, C433, C3134 and C3144. In addition, we confirm that full-length huntingtin is cleaved at D552 and post-translationally myristoylated at G553. Importantly, blocking caspase cleavage at the critical and pathogenic aspartate 586 (D586) significantly increases posttranslational myristoylation of huntingtin. In turn, myristoylation of huntingtin promotes the co-interaction between C-terminal and N-terminal huntingtin fragments, which is also protective. Discussion: This suggests that the protective effect of inhibiting caspase-cleavage at D586 may be mediated through post-translational myristoylation of huntingtin at G553.

Rescue of aberrant huntingtin palmitoylation ameliorates mutant huntingtin-induced toxicity.

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion in the HTT gene that codes for an elongated polyglutamine tract in the huntingtin (HTT) protein. HTT is subject to multiple post-translational modifications (PTMs) that regulate its cellular function. Mutating specific PTM sites within mutant HTT (mHTT) in HD mouse models can modulate disease phenotypes, highlighting the key role of HTT PTMs in the pathogenesis of HD. These findings have led to increased interest in developing small molecules to modulate HTT PTMs in order to decrease mHTT toxicity. However, the therapeutic efficacy of pharmacological modulation of HTT PTMs in preclinical HD models remains largely unknown. HTT is palmitoylated at cysteine 214 by the huntingtin-interacting protein 14 (HIP14 or ZDHHC17) and 14-like (HIP14L or ZDHHC13) acyltransferases. Here, we assessed if HTT palmitoylation should be regarded as a therapeutic target to treat HD by (1) investigating palmitoylation dysregulation in rodent and human HD model systems, (2) measuring the impact of mHTT-lowering therapy on brain palmitoylation, and (3) evaluating if HTT palmitoylation can be pharmacologically modulated. We show that palmitoylation of mHTT and some HIP14/HIP14L-substrates is decreased early in multiple HD mouse models, and that mHTT palmitoylation decreases further with aging. Lowering mHTT in the brain of YAC128 mice is not sufficient to rescue aberrant palmitoylation. However, we demonstrate that mHTT palmitoylation can be normalized in COS-7 cells, in YAC128 cortico-striatal primary neurons and HD patient-derived lymphoblasts using an acyl-protein thioesterase (APT) inhibitor. Moreover, we show that modulating palmitoylation reduces mHTT aggregation and mHTT-induced cytotoxicity in COS-7 cells and YAC128 neurons.

Reliable Resolution of Full-Length Huntingtin Alleles by Quantitative Immunoblotting.

BACKGROUND: Therapeutics that lower mutant huntingtin (mHTT) have shown promise in preclinical studies and are in clinical development for the treatment of Huntington disease (HD). Multiple assays have been developed that either quantify mHTT or total HTT but may not accurately measure levels of wild type HTT (wtHTT) in biological samples. OBJECTIVE: To optimize a method that can be used to resolve, quantify and directly compare levels of full length wtHTT and mHTT in HD samples. METHODS: We provide a detailed quantitative immunoblotting protocol to reproducibly resolve full length wtHTT and mHTT in multiple HD mouse and patient samples. RESULTS: We show that this assay can be modified, depending on the sample, to resolve wtHTT and mHTT with a wide range of polyglutamine length differences (ΔQs 22-179). We also demonstrate that this method can be used to quantify allele-selective lowering of mHTT using an antisense oligonucleotide in HD patient-derived cells. CONCLUSION: This quantitative immunoblotting method can be used to reliably resolve full-length HTT alleles with ΔQs≥22 and allows for direct comparison of wtHTT and mHTT levels in HD samples.

DAPK1 Promotes Extrasynaptic GluN2B Phosphorylation and Striatal Spine Instability in the YAC128 Mouse Model of Huntington Disease.

Huntington disease (HD) is a devastating neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Disrupted cortico-striatal transmission is an early event that contributes to neuronal spine and synapse dysfunction primarily in striatal medium spiny neurons, the most vulnerable cell type in the disease, but also in neurons of other brain regions including the cortex. Although striatal and cortical neurons eventually degenerate, these synaptic and circuit changes may underlie some of the earliest motor, cognitive, and psychiatric symptoms. Moreover, synaptic dysfunction and spine loss are hypothesized to be therapeutically reversible before neuronal death occurs, and restoration of normal synaptic function may delay neurodegeneration. One of the earliest synaptic alterations to occur in HD mouse models is enhanced striatal extrasynaptic NMDA receptor expression and activity. This activity is mediated primarily through GluN2B subunit-containing receptors and is associated with increased activation of cell death pathways, inhibition of survival signaling, and greater susceptibility to excitotoxicity. Death-associated protein kinase 1 (DAPK1) is a pro-apoptotic kinase highly expressed in neurons during development. In the adult brain, DAPK1 becomes re-activated and recruited to extrasynaptic NMDAR complexes during neuronal death, where it phosphorylates GluN2B at S1303, amplifying toxic receptor function. Approaches to reduce DAPK1 activity have demonstrated benefit in animal models of stroke, Alzheimer's disease, Parkinson's disease, and chronic stress, indicating that DAPK1 may be a novel target for neuroprotection. Here, we demonstrate that dysregulation of DAPK1 occurs early in the YAC128 HD mouse model, and contributes to elevated extrasynaptic GluN2B S1303 phosphorylation. Inhibition of DAPK1 normalizes extrasynaptic GluN2B phosphorylation and surface expression, and completely prevents YAC128 striatal spine loss in cortico-striatal co-culture, thus validating DAPK1 as a potential target for synaptic protection in HD and warranting further development of DAPK1-targeted therapies for neurodegeneration.