Huntington's disease (HD) is an age-dependent neurodegenerative disease. DNA repair pathways have recently been implicated as the most predominant modifiers of age of onset in HD patients. We report that endogenous huntingtin protein directly participates in oxidative DNA damage repair. Using novel chromobodies to detect endogenous human huntingtin in live cells, we show that localization of huntingtin to DNA damage sites is dependent on the kinase activity of ataxia telangiectasia mutated (ATM) protein. Super-resolution microscopy and biochemical assays revealed that huntingtin co-localizes with and scaffolds proteins of the DNA damage response pathway in response to oxidative stress. In HD patient fibroblasts bearing typical clinical HD allele lengths, we demonstrate that there is deficient oxidative DNA damage repair. We propose that DNA damage in HD is caused by dysfunction of the mutant huntingtin protein in DNA repair, and accumulation of DNA oxidative lesions due to elevated reactive oxygen species may contribute to the onset of HD.

The huntingtin N17 domain is a modulator of mutant huntingtin toxicity and is hypophosphorylated in Huntington's disease (HD). We conducted high-content analysis to find compounds that could restore N17 phosphorylation. One lead compound from this screen was N6-furfuryladenine (N6FFA). N6FFA was protective in HD model neurons, and N6FFA treatment of an HD mouse model corrects HD phenotypes and eliminates cortical mutant huntingtin inclusions. We show that N6FFA restores N17 phosphorylation levels by being salvaged to a triphosphate form by adenine phosphoribosyltransferase (APRT) and used as a phosphate donor by casein kinase 2 (CK2). N6FFA is a naturally occurring product of oxidative DNA damage. Phosphorylated huntingtin functionally redistributes and colocalizes with CK2, APRT, and N6FFA DNA adducts at sites of induced DNA damage. We present a model in which this natural product compound is salvaged to provide a triphosphate substrate to signal huntingtin phosphorylation via CK2 during low-ATP stress under conditions of DNA damage, with protective effects in HD model systems.

Recent genome-wide association studies of Huntington's disease (HD) primarily highlighted genes involved in DNA damage repair mechanisms as modifiers of age at onset and disease severity, consistent with evidence that more DNA repair genes are being implicated in late age-onset neurodegenerative diseases. This provides an exciting opportunity to advance therapeutic development in HD, as these pathways have already been under intense investigation in cancer research. Also emerging are the roles of other polyglutamine disease proteins in DNA damage repair mechanisms. A potential universal trigger of oxidative DNA damage shared in these late age-onset diseases is the increase of reactive oxygen species (ROS) in human aging, defining an agerelated mechanism that has defied other hypotheses of neurodegeneration. We discuss the potential commonality of DNA damage repair pathways in HD and other neurodegenerative diseases. Potential targets for therapy that may prove beneficial across many of these diseases are also identified, defining nodes in the ataxia telangiectasia-mutated (ATM) complex, mismatch repair, and poly ADP-ribose polymerases (PARPs).

Contactin-1 has been shown to promote cancer metastasis. However, the underlying mechanisms remain unclear. We report here that knockdown of contactin-1 in A549 lung cancer cells reduced A549 cell invasion and the cell's ability to grow in soft agar without affecting cell proliferation. Reduction of contactin-1 resulted in upregulation of E-cadherin, consistent with E-cadherin being inhibitive of cancer cell invasion. In an effort to investigate the mechanisms whereby contactin-1 reduces E-cadherin expression, we observed that contactin-1 plays a role in AKT activation, as knockdown of contactin-1 attenuated AKT activation. Additionally, inhibition of AKT activation significantly enhanced E-cadherin expression, an observation that mimics the situation observed in contactin-1 knockdown, suggesting that activation of AKT plays a role in contactin-1-mediated downregulation of E-cadherin. In addition, we were able to show that knockdown of contactin-1 did not further reduce A549 cell's invasion ability, when AKT activation was inhibited by an AKT inhibitor. To further support our findings, we overexpressed CNTN-1 in two CNTN-1 null breast cancer cell lines expressing E-cadherin. Upon overexpression, CNTN-1 reduced E-cadherin levels in one cell line and increased AKT activation in the other. Furthermore, in our study of 63 primary lung cancers, we observed 65% of primary lung cancers being contactin-1 positive and in these carcinomas, 61% were E-cadherin negative. Collectively, we provide evidence that contactin-1 plays a role in the downregulation of E-cadherin in lung cancer and that AKT activation contributes to this process. In a study of mechanisms responsible for contactin-1 to activate AKT, we demonstrated that knockdown of CNTN-1 in A549 cells did not enhance PTEN expression but upregulated PHLPP2, a phosphatase that dephosphorylates AKT. These observations thus suggest that contactin-1 enhances AKT activation in part by preventing PHLPP2-mediated AKT dephosphrorylation.

The development of neural connectivity is essential for brain function, and disruption of this process is associated with autism spectrum disorders (ASDs). DIX domain containing 1 (DIXDC1) has previously been implicated in neurodevelopmental disorders, but its role in postnatal brain function remains unknown. Using a knockout mouse model, we determined that DIXDC1 is a regulator of excitatory neuron dendrite development and synapse function in the cortex. We discovered that MARK1, previously linked to ASDs, phosphorylates DIXDC1 to regulate dendrite and spine development through modulation of the cytoskeletal network in an isoform-specific manner. Finally, rare missense variants in DIXDC1 were identified in ASD patient cohorts via genetic sequencing. Interestingly, the variants inhibit DIXDC1 isoform 1 phosphorylation, causing impairment to dendrite and spine growth. These data reveal that DIXDC1 is a regulator of cortical dendrite and synaptic development and provide mechanistic insight into morphological defects associated with neurodevelopmental disorders.

Huntington's disease (HD) is an autosomal dominant, neurodegenerative disorder that can be characterized by the presence of protein inclusions containing mutant huntingtin within a subset of neurons in the brain. Since their discovery, the relevance of inclusions to disease pathology has been controversial. We show using super-resolution fluorescence imaging and Förster resonance energy transfer (FRET) in live cells, that mutant huntingtin fragments can form two morphologically and conformationally distinct inclusion types. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that the two huntingtin inclusion types have unique dynamic properties. The ability to form one or the other type of inclusion can be influenced by the phosphorylation state of serine residues at amino acid positions 13 and 16 within the huntingtin protein. We can define two types of inclusions: fibrillar, which are tightly packed, do not exchange protein with the soluble phase, and result from phospho-modification at serines 13 and 16 of the N17 domain, and globular, which are loosely packed, can readily exchange with the soluble phase, and are not phosphorylated in N17. We hypothesize that the protective effect of N17 phosphorylation or phospho-mimicry seen in animal models, at the level of protein inclusions with elevated huntingtin levels, is to induce a conformation of the huntingtin amino-terminus that causes fragments to form tightly packed inclusions that do not exit the insoluble phase, and hence exert less toxicity. The identification of these sub-types of huntingtin inclusions could allow for drug discovery to promote protective inclusions of mutant huntingtin protein in HD.

The huntingtin protein participates in several cellular processes that are disrupted when the polyglutamine tract is expanded beyond a threshold of 37 CAG DNA repeats in Huntington’s disease (HD). Cellular biology approaches to understand these functional disruptions in HD have primarily focused on cell lines with synthetically long CAG length alleles that clinically represent outliers in this disease and a more severe form of HD that lacks age onset. Patient-derived fibroblasts are limited to a finite number of passages before succumbing to cellular senescence. We used human telomerase reverse transcriptase (hTERT) to immortalize fibroblasts taken from individuals of varying age, sex, disease onset, and CAG repeat length, which we have termed TruHD cells. TruHD cells display classic HD phenotypes of altered morphology, size and growth rate, increased sensitivity to oxidative stress, aberrant adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratios, and hypophosphorylated huntingtin protein. We additionally observed dysregulated reactive oxygen species (ROS)-dependent huntingtin localization to nuclear speckles in HD cells. We report the generation and characterization of a human, clinically relevant cellular model for investigating disease mechanisms in HD at the single-cell level, which, unlike transformed cell lines, maintains functions critical for huntingtin transcriptional regulation and genomic integrity.

The use of genome wide association studies (GWAS) in Huntington’s disease (HD) research, driven by unbiased human data analysis, has transformed the focus of new targets that could affect age at onset. While there is a significant depth of information on DNA damage repair, with many drugs and drug targets, most of this development has taken place in the context of cancer therapy. DNA damage repair in neurons does not rely on DNA replication correction mechanisms. However, there is a strong connection between DNA repair and neuronal metabolism, mediated by nucleotide salvaging and the poly ADP-ribose (PAR) response, and this connection has been implicated in other age-onset neurodegenerative diseases. Validation of leads including the mismatch repair protein MSH3, and interstrand cross-link repair protein FAN1, suggest the mechanism is driven by somatic CAG instability, which is supported by the protective effect of CAA substitutions in the CAG tract. We currently do not understand: how somatic instability is triggered; the state of DNA damage within expanding alleles in the brain; whether this damage induces mismatch repair and interstrand cross-link pathways; whether instability mediates toxicity, and how this relates to human ageing. We discuss DNA damage pathways uncovered by HD GWAS, known roles of other polyglutamine disease proteins in DNA damage repair, and a panel of hypotheses for pathogenic mechanisms.