Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene which results in progressive neurodegeneration in the striatum, cortex, and eventually most brain areas. Despite being a monogenic disorder, environmental factors influence HD characteristics. Both human and mouse studies suggest that mutant HTT (mHTT) leads to gene expression changes that harbor potential to be modulated by the environment. Yet, the underlying mechanisms integrating environmental cues into the gene regulatory program have remained largely unclear. To better understand gene-environment interactions in the context of mHTT, we employed RNA-seq to examine effects of maternal separation (MS) and environmental enrichment (EE) on striatal gene expression during development of BACHD rats. We integrated our results with striatal consensus modules defined on HTT-CAG length and age-dependent co-expression gene networks to relate the environmental factors with disease progression. While mHTT was the main determinant of expression changes, both MS and EE were capable of modulating these disturbances, resulting in distinctive and in several cases opposing effects of MS and EE on consensus modules. This bivalent response to maternal separation and environmental enrichment may aid in explaining their distinct effects observed on disease phenotypes in animal models of HD and related neurodegenerative disorders.

Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher free NADH suggesting a possible synergism between metabolic dysfunction and transcriptional regulation. Our results indicate that metabolic dysfunction in HD shifts to increased glycolysis leading to oxidative stress and cell death. This powerful label free method can be used to screen native HD tissue samples and for potential drug screening.

Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN. Using phasor approach, we characterized the fluorescence of LAURDAN that is sensitive to the polarity of the immediate environment. LAURDAN is affected by the physical order of phospholipids (lipid order) and reports the membrane fluidity. We also validated our results using a different fluorescent membrane probe, Nile Red (NR). The plasma membrane in the cells expressing expanded polyQ shows a shift toward increased membrane fluidity revealed by both LAURDAN and NR spectral phasors. This finding brings a new perspective in the understanding of the early stages of HD that can be used as a target for drug screening.

Blood-brain barrier (BBB) breakdown, due to the concomitant disruption of the tight junctions (TJs), normally required for the maintenance of BBB function, and to the altered transport of molecules between blood and brain and vice-versa, has been suggested to significantly contribute to the development and progression of different brain disorders including Huntington's disease (HD). Although the detrimental consequence the BBB breakdown may have in the clinical settings, the timing of its alteration remains elusive for many neurodegenerative diseases. In this study we demonstrate for the first time that BBB disruption in HD is not confined to established symptoms, but occurs early in the disease progression. Despite the obvious signs of impaired BBB permeability were only detectable in concomitance with the onset of the disease, signs of deranged TJs integrity occur precociously in the disease and precede the onset of overt symptoms. To our perspective this finding may add a new dimension to the horizons of pathological mechanisms underlying this devastating disease, however much remains to be elucidated for understanding how specific BBB drug targets can be approached in the future.

Post-translational modifications (PTMs) are key modulators of protein function. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin (HTT) gene. A spectrum of PTMs have been shown to modify the normal functions of HTT, including proteolysis, phosphorylation and lipidation, but the full contribution of these PTMs to the molecular pathogenesis of HD remains unclear. In this study, we examine all commonly occurring missense mutations in HTT to identify potential human modifiers of HTT PTMs relevant to HD biology. We reveal a SNP that modifies post-translational myristoylation of HTT, resulting in downstream alterations to toxic HTT proteolysis in human cells. This is the first SNP shown to functionally modify a PTM in HD and the first validated genetic modifier of post-translational myristoylation. This SNP is a high-priority candidate modifier of HD phenotypes and may illuminate HD biology in human studies.

Huntington disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Expression of the mutant protein disrupts various intracellular pathways and impairs overall cell function. In particular striatal neurons seem to be most vulnerable to mutant huntingtin-related changes. A well-known and commonly used model to study molecular aspects of Huntington disease are the striatum-derived STHdh cell lines generated from wild type and huntingtin knock-in mouse embryos. However, obvious morphological differences between wild type and mutant cell lines exist, which have rarely been described and might not have always been considered when designing experiments or interpreting results. Here, we demonstrate that STHdh cell lines display differences in cell size, proliferation rate and chromosomal content. While the chromosomal divergence is considered to be a result of the cells' tumour characteristics, differences in size and proliferation, however, were confirmed in a second non-immortalized Huntington disease cell model. Importantly, our results further suggest that the reported phenotypes can confound other study outcomes and lead to false conclusions. Thus, careful experimental design and data analysis are advised when using these cell models.

Increasing evidence supports a role for abnormal immune activation and inflammatory responses in Huntington disease (HD). In this study, we evaluated the therapeutic potential of laquinimod (1 and 10 mg/kg), a novel immunomodulatory agent shown to be protective in a number of neuroinflammatory conditions, in the YAC128 mouse model of HD. Treatment with laquinimod for 6 months rescued atrophy in the striatum, in certain cortical regions, and in the corpus callosum of YAC128 HD mice. Diffusion tensor imaging showed that white matter microstructural abnormalities in the posterior corpus callosum were improved following treatment with low dose (1 mg/kg) laquinimod, and were paralleled by reduced levels of interleukin-6 in the periphery of YAC128 HD mice. Functionally, treatment with laquinimod (1 and 10 mg/kg) led to modest improvements in motor function and in depressive-like behaviour. Taken together, these results suggest that laquinimod may improve some features of pathology in HD, and provides support for the role of immune activation in the pathogenesis of HD.

Huntington disease (HD) is a lethal neurodegenerative disorder caused by expansion of a CAG repeat within the huntingtin (HTT) gene. Disease prevention can be facilitated by preimplantation genetic testing for this monogenic disorder (PGT-M). We developed a strategy for HD PGT-M, involving whole genome amplification (WGA) followed by combined triplet-primed PCR (TP-PCR) for HTT CAG repeat expansion detection and multi-microsatellite marker genotyping for disease haplotype phasing. The strategy was validated and tested pre-clinically in a simulated PGT-M case before clinical application in five cycles of a PGT-M case. The assay reliably and correctly diagnosed all embryos, even where allele dropout (ADO) occurred at the HTT CAG repeat locus or at one or more linked markers. Ten of the 27 embryos analyzed were diagnosed as unaffected. Four embryo transfers were performed, two of which involved fresh cycle double embryo transfers and two were frozen-thawed single embryo transfers. Pregnancies were achieved from each of the frozen-thawed single embryo transfers and confirmed to be unaffected by amniocentesis, culminating in live births at term. This strategy enhances diagnostic confidence for PGT-M of HD and can also be employed in situations where disease haplotype phase cannot be established prior to the start of PGT-M.

Alzheimer's disease (AD) is the most common cause of dementia. A common finding in AD is DNA damage. Double-strand DNA breaks (DSBs) are particularly hazardous to neurons because their post-mitotic state forces neurons to rely on error-prone and potentially mutagenic mechanisms to repair DNA breaks. However, it remains unclear whether DNA damage results from increased DNA damage or failure of DNA repair. Oligomerization of the tumor suppressor protein p53 is an essential part of DSB repair, and p53 phosphorylated on S15 is an indicator of DNA damage. We report that the monomer:dimer ratio of phosphorylated (S15) p53 is increased by 2.86-fold in temporal lobes of AD patients compared to age-matched controls, indicating that p53 oligomerization is compromised in AD. In vitro oxidation of p53 with 100 nM H2O2 produced a similar shift in the monomer:dimer ratio. A COMET test showed a higher level of DNA degradation in AD consistent with double-strand DNA damage or inhibition of repair. Protein carbonylation was also elevated (190% of control), indicating elevated oxidative stress in AD patients. Levels of the DNA repair support protein 14-3-3σ, γ-H2AX, a phosphorylated histone marking double strand DNA breaks, and phosphorylated ataxia telangiectasia mutated (ATM) protein were all increased. cGAS-STING-interferon signaling was impaired in AD and was accompanied by a depletion of STING protein from Golgi and a failure to elevate interferon despite the presence of DSBs. The results suggest that oxidation of p53 by ROS could inhibit the DDR and decrease its ability to orchestrate DSB repair by altering the oligomerization state of p53. The failure of immune-stimulated DNA repair may contribute to cell loss in AD and suggests new therapeutic targets for AD.

Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington's disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine- and temperature-dependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperature- and polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients.

Periodontal disease (PD) has been suggested to be a risk factor for Alzheimer's disease (AD). We tested the impact of ligature-induced PD on 5xFAD mice and WT littermates. At baseline, 5xFAD mice presented significant alveolar bone loss compared to WT mice. After the induction of PD, both WT and 5xFAD mice experienced alveolar bone loss. PD increased the level of Iba1-immunostained microglia in WT mice. In 5xFAD mice, PD increased the level of insoluble Aβ42. The increased level in Iba1 immunostaining that parallels the accumulation of Aβ in 5xFAD mice was not affected by PD except for a decrease in the dentate gyrus. Analysis of double-label fluorescent images showed a decline in Iba1 in the proximity of Aβ plaques in 5xFAD mice with PD compared to those without PD suggesting a PD-induced decrease in plaque-associated microglia (PAM). PD reduced IL-6, MCP-1, GM-CSF, and IFN-γ in brains of WT mice and reduced IL-10 in 5xFAD mice. The data demonstrated that PD increases neuroinflammation in WT mice and disrupts the neuroinflammatory response in 5xFAD mice and suggest that microglia is central to the association between PD and AD.

Meclizine is a well-tolerated drug routinely used as an anti-histamine agent in the management of disequilibrium. Recently, meclizine has been assessed for its neuroprotective properties in ischemic stroke and Huntington disease models. We found that meclizine protected against 6-hydroxydopamine-induced apoptosis and cell death in both SH-SY5Y cells and rat primary cortical cultures. Meclizine increases the level of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which activates phosphofructokinase, a rate-determining enzyme of glycolysis. This protection is therefore mediated by meclizine's ability to enhance glycolysis and increase mitochondrial hyperpolarization. Meclizine represents an interesting candidate for further investigation to re-purpose for its potential to be neuroprotective in Parkinson disease.

Lipid metabolism is abnormal in Alzheimer's disease (AD) brain leading to ceramide and sphingosine accumulation and reduced levels of brain sphingosine-1-phosphate (S1P). We hypothesize that changes in S1P signaling are central to the inflammatory and immune-pathogenesis of AD and the therapeutic benefits of fingolimod, a structural analog of sphingosine that is FDA approved for the treatment of multiple sclerosis. We recently reported that the neuroprotective effects of fingolimod in 5xFAD transgenic AD mice treated from 1-3 months of age were greater at 1 mg/kg/day than at 5 mg/kg/day. Here we performed a dose-response study using fingolimod from 0.03 to 1 mg/kg/day in 5xFAD mice treated from 1-8 months of age. At 1 mg/kg/day, fingolimod decreased both peripheral blood lymphocyte counts and brain Aβ levels, but at the lowest dose tested (0.03 mg/kg/day), we detected improved memory, decreased activation of brain microglia and astrocytes, and restored hippocampal levels of GABA and glycerophosphocholine with no effect on circulating lymphocyte counts. These findings suggests that, unlike the case in multiple sclerosis, fingolimod may potentially have therapeutic benefits in AD at low doses that do not affect peripheral lymphocyte function.

The Hippo signaling pathway is involved in organ size regulation and tumor suppression. Although inhibition of Hippo leads to tumorigenesis, activation of Hippo may play a role in neurodegeneration. Specifically, activation of the upstream regulator, mammalian sterile 20 (STE20)-like kinase 1 (MST1), reduces activity of the transcriptional co-activator Yes-Associated Protein (YAP), thereby mediating oxidative stress-induced neuronal death. Here, we investigated the possible role of this pathway in Huntington's disease (HD) pathogenesis. Our results demonstrate a significant increase in phosphorylated MST1, the active form, in post-mortem HD cortex and in the brains of CAG knock-in HdhQ111/Q111 mice. YAP nuclear localization was also decreased in HD post-mortem cortex and in neuronal stem cells derived from HD patients. Moreover, there was a significant increase in phosphorylated YAP, the inactive form, in HD post-mortem cortex and in HdhQ111/Q111 brain. In addition, YAP was found to interact with huntingtin (Htt) and the chaperone 14-3-3, however this interaction was not altered in the presence of mutant Htt. Lastly, YAP/TEAD interactions and expression of Hippo pathway genes were altered in HD. Together, these results demonstrate that activation of MST1 together with a decrease in nuclear YAP could significantly contribute to transcriptional dysregulation in HD.

Huntington disease (HD) is a fatal neurodegenerative disorder without a cure that is caused by an aberrant expansion of CAG repeats in exon 1 of the huntingtin (HTT) gene. Although a negative correlation between the number of CAG repeats and the age of disease onset is established, additional factors may contribute to the high heterogeneity of the complex manifestation of symptoms among patients. This variability is also observed in mouse models, even under controlled genetic and environmental conditions. To better understand this phenomenon, we analysed the R6/1 strain in search of potential correlates between pathological motor/cognitive phenotypical traits and transcriptional alterations. HD-related genes (e.g., Penk, Plk5, Itpka), despite being downregulated across the examined brain areas (the prefrontal cortex, striatum, hippocampus and cerebellum), exhibited tissue-specific correlations with particular phenotypical traits that were attributable to the contribution of the brain region to that trait (e.g., striatum and rotarod performance, cerebellum and feet clasping). Focusing on the striatum, we determined that the transcriptional dysregulation associated with HD was partially exacerbated in mice that showed poor overall phenotypical scores, especially in genes with relevant roles in striatal functioning (e.g., Pde10a, Drd1, Drd2, Ppp1r1b). However, we also observed transcripts associated with relatively better outcomes, such as Nfya (CCAAT-binding transcription factor NF-Y subunit A) plus others related to neuronal development, apoptosis and differentiation. In this study, we demonstrated that altered brain transcription can be related to the manifestation of HD-like symptoms in mouse models and that this can be extrapolated to the highly heterogeneous population of HD patients.

Huntington's disease (HD) is a progressive and fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin gene. Although HD is monogenic, its molecular manifestation appears highly complex and involves multiple cellular processes. The recent application of high throughput platforms such as microarrays and mass-spectrometry has indicated multiple pathogenic routes. The massive data generated by these techniques together with the complexity of the pathogenesis, however, pose considerable challenges to researchers. Network-based methods can provide valuable tools to consolidate newly generated data with existing knowledge, and to decipher the interwoven molecular mechanisms underlying HD. To facilitate research on HD in a network-oriented manner, we have developed HDNetDB, a database that integrates molecular interactions with many HD-relevant datasets. It allows users to obtain, visualize and prioritize molecular interaction networks using HD-relevant gene expression, phenotypic and other types of data obtained from human samples or model organisms. We illustrated several HDNetDB functionalities through a case study and identified proteins that constitute potential cross-talk between HD and the unfolded protein response (UPR). HDNetDB is publicly accessible at http://hdnetdb.sysbiolab.eu .

Magnetic resonance (MR) imaging is a powerful technique for non-invasive in-vivo imaging of the human brain. We employed a recently validated method for robust cross-sectional and longitudinal segmentation of MR brain images from the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. Specifically, we segmented 5074 MR brain images into 138 anatomical regions and extracted time-point specific structural volumes and volume change during follow-up intervals of 12 or 24 months. We assessed the extracted biomarkers by determining their power to predict diagnostic classification and by comparing atrophy rates to published meta-studies. The approach enables comprehensive analysis of structural changes within the whole brain. The discriminative power of individual biomarkers (volumes/atrophy rates) is on par with results published by other groups. We publish all quality-checked brain masks, structural segmentations, and extracted biomarkers along with this article. We further share the methodology for brain extraction (pincram) and segmentation (MALPEM, MALPEM4D) as open source projects with the community. The identified biomarkers hold great potential for deeper analysis, and the validated methodology can readily be applied to other imaging cohorts.

Hypothalamic pathology, metabolic dysfunction and psychiatric symptoms are part of Huntington disease (HD), which is caused by an expanded CAG repeat in the huntingtin (HTT) gene. Inactivation of mutant HTT selectively in the hypothalamus prevents the development of metabolic dysfunction and depressive-like behavior in the BACHD mouse model. The hypothalamic paraventricular nucleus (PVN) is implicated in metabolic and emotional control, therefore we here tested whether inactivation of mutant HTT in the PVN affects metabolic and psychiatric manifestations of HD in BACHD mice. BACHD mice were crossed with mice expressing Cre-recombinase under the Sim1 promoter (Sim1-Cre) to inactivate mutant HTT in Sim1 expressing cells, i.e. the PVN of the hypothalamus. We found that inactivation of mutant HTT in Sim1 cells had a sex-specific effect on both the metabolic and the psychiatric phenotype, as these phenotypes were no longer different in male BACHD/Sim1-Cre mice compared to wild-type littermates. We also found a reduced number of GnRH neurons specifically in the anterior hypothalamus and an increased testes weight in male BACHD mice compared to wild-type littermates. Taken together, expression of mutant HTT in Sim1 cells may play a role for the development of metabolic dysfunction and depressive-like behavior in male BACHD mice.

Although β-amyloid (Aβ) may be the primary driver of Alzheimer's disease (AD) pathology, accumulation of pathological tau correlates with dementia in AD patients. Thus, the prevention/inhibition of AD may require vaccine/s targeting Aβ and tau simultaneously or sequentially. Since high antibody titers are required for AD vaccine efficacy, we have decided to generate vaccines, targeting Aβ (AV-1959R), Tau (AV-1980R) or Aβ/tau (AV-1953R) B cell epitopes, based on immunogenic MultiTEP platform and evaluate the immunogenicity of these vaccines formulated with Advax(CpG), delta inulin, Alhydrogel(®), Montanide-ISA51, Montanide-ISA720, MPLA-SM pharmaceutical grade adjuvants. Formulation of AV-1959R in Advax(CpG) induced the highest cellular and humoral immune responses in mice. The dual-epitope vaccine, AV-1953R, or the combination of AV-1959R and AV-1980R vaccines formulated with Advax(CpG) induced robust antibody responses against various forms of both, Aβ and tau pathological molecules. While anti-Aβ antibody titers after AV-1953R immunization were similar to that in mice vaccinated with AV-1959R or AV-1959R/AV-1980R combination, anti-tau titers were significantly lower after AV-1953R injection when compared to the AV-1980R or AV-1959R/AV-1980R. In silico 3D-modeling provided insight into the differences in immunogenicity of these vaccine constructs. In sum, AV-1959R and AV-1980R formulated with Advax(CpG) adjuvant were identified as promising immunogenic vaccines for ongoing pre-clinical assessment and future human clinical trials.

Recently, AAV2.retro, a new capsid variant capable of efficient retrograde transport in brain, was generated in mice using a directed evolution approach. However, it remains unclear to what degree transport will be recapitulated in the substantially larger and more complex nonhuman primate (NHP) brain. Here, we compared the biodistribution of AAV2.retro with its parent serotype, AAV2, in adult macaques following delivery into the caudate and putamen, brain regions which comprise the striatum. While AAV2 transduction was primarily limited to the injected brain regions, AAV2.retro transduced cells in the striatum and in dozens of cortical and subcortical regions with known striatal afferents. We then evaluated the capability of AAV2.retro to deliver disease-related gene cargo to biologically-relevant NHP brain circuits by packaging a fragment of human mutant HTT, the causative gene mutation in Huntington's disease. Following intra-striatal delivery, pathological mHTT-positive protein aggregates were distributed widely among cognitive, motor, and limbic cortico-basal ganglia circuits. Together, these studies demonstrate strong retrograde transport of AAV2.retro in NHP brain, highlight its utility in developing novel NHP models of brain disease and suggest its potential for querying circuit function and delivering therapeutic genes in the brain, particularly where treating dysfunctional circuits, versus single brain regions, is warranted.