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Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre‐clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non‐human or non‐neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra‐nuclear inclusions in a polyQ length‐dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction.

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene (HTT). Disease progression is characterized by the loss of vulnerable neuronal populations within the striatum. A consistent phenotype across HD models is disruption of nucleocytoplasmic transport and nuclear pore complex (NPC) function. Here we demonstrate that high content imaging is a suitable method for detecting mislocalization of lamin-B1, RAN and RANGAP1 in striatal neuronal cultures thus allowing a robust, unbiased, highly powered approach to assay nuclear pore deficits. Furthermore, nuclear pore deficits extended to the selectively vulnerable DARPP32 + subpopulation neurons, but not to astrocytes. Striatal neuron cultures are further affected by changes in gene and protein expression of RAN, RANGAP1 and lamin-B1. Lowering total HTT using HTT-targeted anti-sense oligonucleotides partially restored gene expression, as well as subtly reducing mislocalization of proteins involved in nucleocytoplasmic transport. This suggests that mislocalization of RAN, RANGAP1 and lamin-B1 cannot be normalized by simply reducing expression of CAG-expanded HTT in the absence of healthy HTT protein.

Background Neuroinflammation may contribute to the pathogenesis of Huntington’s disease, given evidence of activated microglia and elevated levels of inflammatory molecules in disease gene carriers, even those many years from symptom onset. We have shown previously that monocytes from Huntington’s disease patients are hyper-reactive to stimulation in a manner dependent on their autonomous expression of the disease-causing mutant HTT protein. To date, however, whether human microglia are similarly hyper-responsive in a cell-autonomous manner has not been determined. Methods Microglial-like cells were derived from human pluripotent stem cells (PSCs) expressing mutant HTT containing varying polyglutamine lengths. These included lines that are otherwise isogenic, such that any observed differences can be attributed with certainty to the disease mutation itself. Analyses by quantitative PCR and immunofluorescence microscopy respectively of key genes and protein markers were undertaken to determine whether Huntington’s disease PSCs differentiated normally to a microglial fate. The resultant cultures and their supernatants were then assessed by various biochemical assays and multiplex ELISAs for viability and responses to stimulation, including the release of pro-inflammatory cytokines and reactive oxygen species. Conditioned media were applied to PSC-derived striatal neurons, and vice versa, to determine the effects that the secretomes of each cell type might have on the other. Results Human PSCs generated microglia successfully irrespective of the expression of mutant HTT. These cells, however, were hyper-reactive to stimulation in the production of pro-inflammatory cytokines such as IL-6 and TNFα. They also released elevated levels of reactive oxygen species that have neurotoxic potential. Accompanying such phenotypes, human Huntington’s disease PSC-derived microglia showed increased levels of apoptosis and were more susceptible to exogenous stress. Such stress appeared to be induced by supernatants from human PSC-derived striatal neurons expressing mutant HTT with a long polyglutamine tract. Conclusions These studies show, for the first time, that human Huntington’s disease PSC-derived microglia are hyper-reactive due to their autonomous expression of mutant HTT. This provides a cellular basis for the contribution that neuroinflammation might make to Huntington’s disease pathogenesis.

Additional file 2. comprises Supplementary figures S1-S10.

Additional file 1. comprises Supplementary tables 1-4.

Huntington’s disease (HD) is an inherited neurodegenerative disease caused by an expanded CAG repeat in the huntingtin (HTT) gene. CAG repeat length explains around half of the variation in age at onset (AAO) but genetic variation elsewhere in the genome accounts for a significant proportion of the remainder. Genome-wide association studies have identified a bidirectional signal on chromosome 15, likely underlain by FANCD2- and FANCI-associated nuclease 1 (FAN1), a nuclease involved in DNA interstrand cross link repair. Here we show that increased FAN1 expression is significantly associated with delayed AAO and slower progression of HD, suggesting FAN1 is protective in the context of an expanded HTT CAG repeat. FAN1 overexpression in human cells reduces CAG repeat expansion in exogenously expressed mutant HTT exon 1, and in patient-derived stem cells and differentiated medium spiny neurons, FAN1 knockdown increases CAG repeat expansion. The stabilizing effects are FAN1 concentration and CAG repeat length-dependent. We show that FAN1 binds to the expanded HTT CAG repeat DNA and its nuclease activity is not required for protection against CAG repeat expansion. These data shed new mechanistic insights into how the genetic modifiers of HD act to alter disease progression and show that FAN1 affects somatic expansion of the CAG repeat through a nuclease-independent mechanism. This provides new avenues for therapeutic interventions in HD and potentially other triplet repeat disorders.

Huntington’s disease (HD) is an inherited neurodegenerative disease caused by an expanded CAG repeat in the HTT gene. CAG repeat length explains around half of the variation in age-at-onset, but genetic variation elsewhere in the genome accounts for a significant proportion of the remainder. Genome-wide association studies have identified a bidirectional signal on chromosome 15, likely underlain by FAN1 (FANCD2 and FANCI Associated Nuclease 1), a nuclease involved in DNA interstrand cross link repair. Here we show that increased FAN1 expression is significantly associated with delayed age-at-onset and slower progression of HD suggesting FAN1 is protective in the context of an expanded HTT CAG repeat. FAN1 overexpression in human cells reduces CAG repeat expansion in exogenously expressed mutant HTT exon 1, and in patient-derived stem cells and differentiated medium spiny neurons, FAN1 knockdown increases CAG repeat expansion. The stabilising effect is FAN1 concentration and CAG repeat length dependent. We show that FAN1 binds to the expanded HTT CAG repeat DNA and its nuclease activity is not required for protection against CAG repeat expansion. These data shed new mechanistic insights into how the genetic modifiers of HD act to alter disease progression, and show that FAN1 affects somatic expansion of the CAG repeat through a nuclease-independent mechanism. This provides new avenues for therapeutic interventions in HD and potentially other triplet repeat disorders.

Background Pluripotent stem cell technology is allowing the study of disease-relevant cell types of human origin, and is accompanied by much interest in their application for the screening of disease phenotypes and potential therapeutics. Here, we have generated and characterised induced pluripotent stem cells (iPSCs) from HD gene carriers differentiated into disease-specific striatal medium spiny neurons (MSNs) by high content imaging to investigate the effects of increasing HTT CAG repeat length on neuronal function. Aims To adapt a protocol for the directed differentiation of iPSCs to MSNs to a high throughput format, in order to carry out a broad phenotypic screen of HD-related phenotypes using high content imaging. Methods The iPSC lines we used were derived from closely related individuals, three of whom were suffering from juvenile HD and their unaffected mother. This created a unique familial allelic series with increasing HTT CAG repeat lengths of 20, 56, 67 and 73 CAGs. High content imaging was carried out at differentiation day 36 using the Opera Phenix High Content Screening System and data analysed using Columbus software (Perkin Elmer). Results We report here significant phenotypic differences in endpoint total cell number and culture composition between control and HD lines with the latter accumulating fewer cells over time and a reduction in the proliferative precursor pool. Interestingly, there were no significant differences in the proportions of CTIP2+ cells, neurons or MSNs generated. However, in terms of pathology, we find evidence of enhanced cell death, reduced DARPP-32 expression within MSNs and mHTT inclusion formation in the longest (73 CAG) length line. Conclusions We find no differences in holistic measurements of neuronal differentiation between control and HD lines suggesting that the pathway to neuronal development is unaffected at this stage. However, differences in endpoint cell number and HD-related pathologies were found which could be used as potential endpoints for high throughput drug screening. Support Takeda, Cerevance Ltd, UCLH Biomedical Research Centre, Medical Research Council

Background The corticostriatal (CS) pathway, comprising layer V cortical projection neurons (CPN) and medium spiny neurons (MSN), is one of the first brain pathways to succumb to Huntington’s disease (HD) pathology. As a result, disrupted CS connectivity is evident and contributes to the motor and cognitive symptoms experienced by HD patients. Aims The aim of this work is to investigate the CS pathway using a purely human tissue-derived in vitro system. Methods This project utilizes two familial iPSC lines; the control line, with 20/20 HTT CAG repeat lengths (20Q), and a juvenile HD line, with 20/73 CAG repeats (73Q). These lines were differentiated in parallel to either MSNs or CPNs, and co-cultured in microfluidic chambers to physically recapitulate the human CS pathway. Results High-resolution fluorescence microscopy has revealed the formation of CS synapses within MFC co-cultures, complimented by live cell imaging with calcium binding dye Fluo4, which demonstrates the successful transmission of calcium between neuronal populations within MFCs. CPN cultures show a HD phenotype in their cytoskeletal dynamics, as axon projection efficiency is drastically reduced in 73Q CPNs compared to 20Q. Furthermore, 73Q MSNs exhibit enhanced cell death after BDNF-withdrawal compared to 20Q cultures. Finally, the intrinsic membrane properties of iPSC-derived MSNs also differ with disease state, as 73Q MSNs are hyper-excitable, with an extended latency to fire and extended refractory period. Conclusion These results provide a novel insight into the human CS pathway and suggest subtle differences in both the development and function of the CS pathway in HD.

The serine/threonine kinase glycogen synthase kinase-3beta (GSK-3beta) is expressed in two, alternatively spliced, isoforms: a short form (GSK-3beta1) and a long form containing a 13 amino acid insert in the catalytic domain (GSK-3beta2). We examined the expression of these isoforms in the rat using specific antibodies and found that GSK-3beta2, in contrast to GSK-3beta1, is only expressed in the nervous system. The highest levels of GSK-3beta2 are found in the developing nervous system but expression persists into adulthood. In the adult central nervous system the highest expression of GSK-3beta2 occurs in regions with a high proportion of white matter, suggesting that GSK-3beta2 is expressed in axons. Consistent with this finding, sub-cellular fractionation of neonatal rat brain showed that GSK-3beta2 is present in fractions enriched in neurites and growth cones. Furthermore, we found that when we separated neuronal cell bodies from neurites by culturing embryonic cortical neurons in neurite outgrowth inserts, GSK-3beta2 was present in both compartments. Finally, a rabbit polyclonal antibody raised to the 13 amino acid insert of GSK-3beta2 (anti-8A) that specifically recognises GSK-3beta2, labels the cell body, including the nucleus, neurites and growth cones of embryonic neurons in culture. To compare functionally the two isoforms, we performed in vitro kinase assays. These showed that GSK-3beta1 is more efficient at phosphorylating the microtubule-associated protein MAP1B than GSK-3beta2, consistent with previous findings with the microtubule-associated protein tau. However, when co-expressed with MAP1B in COS-7 cells, both GSK-3beta isoforms equally efficiently phosphorylated MAP1B and had a similar influence on the regulation of microtubule dynamics by MAP1B in these cells. We conclude that the alternatively spliced isoform of GSK-3beta, GSK-3beta2, is neuron-specific and has overlapping activities with GSK-3beta1.

Summary Mutations in PINK1 cause autosomal recessive Parkinson's disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na+/Ca2+ exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload. We show that calcium overload stimulates reactive oxygen species (ROS) production via NADPH oxidase. ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration. We demonstrate that impaired respiration may be restored by provision of mitochondrial complex I and II substrates. Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells. Our findings propose a mechanism by which PINK1 dysfunction renders neurons vulnerable to cell death.

Parkinson's disease (PD) is a common, disabling, neurodegenerative disease. Our knowledge of the molecular events leading to PD is being greatly enhanced by the study of relatively rare familial form of the disease. Nevertheless, the pathways leading from the genetic mutations to nigral cell degeneration and the other features in PD remain poorly understood. The identification of PINK1, a mitochondrial putative protein kinase, has helped understand the pathophysiology of mitochondria and their potential role in PD. Mutations in PINK1 are associated with the PARK6 autosomal recessive, early-onset, PD-susceptibility locus. Point mutations in another mitochondrial protein, HtrA2, are a susceptibility factor for PD (PARK13 locus). We report here the results of investigations into the interactors and pathways of these two mitochondrial molecules (PINK1 and HtrA2) in a range of models and human PD tissue.

Parkinson's disease (PD) is a neurodegenerative disease that is both common and incurable. The majority of cases are sporadic and of unknown origin but several genes have been identified that, when mutated, give rise to rare, familial forms of the disease. The principal genes that have been shown to cause PD are alpha-synuclein (SNCA), parkin, leucine-rich repeat kinase 2 (LRRK2), PTEN-induced putative kinase 1 (PINK1) and DJ-1. Here, we discuss what has been learnt from the study of these genes and what has been elucidated of the molecular pathways that lead to cell degeneration. Of importance is what these molecular events and pathways tell scientists of the common sporadic form of PD. Although complete knowledge of these genes' functions remains elusive, recent work implicates abnormal protein accumulation, protein phosphorylation, mitochondrial dysfunction and oxidative stress as common pathways to PD pathogenesis.

Crystallizer design is often hindered by the lack of scaleup rules, hydrodynamic information, and predictive crystallization modeling tools. A hybrid CFD compartmentalization batch cooling crystallizer model is proposed to take into account localized mixing, heat transfer, and fluid hydrodynamics, combined with key process engineering information obtained on a laboratory scale. The compartments were identified using CFD simulations based on the crystallizer geometry and operating conditions. The population, mass, concentration, and energy balance of each compartment is modeled separately as a well-mixed MSMPR unit with input and output streams. The software gPROMS (Process Systems Enterprise Limited) is a process-modeling tool that can facilitate compartmental modeling and will be used for the prediction of the crystallization behavior upon scaleup.

Computational fluid dynamics (CFD) is a powerful simulation tool that was successfully used to investigate mixing, turbulence, and shear in a laboratory-scale MSMPR and batch cooling crystallizer for an organic fine chemical. CFD gives a qualitative engineering insight into the effects of the impeller configuration on the crystallization rates and particle size distribution. A process-modelling tool, gPROMS (Process Systems Enterprise), was used to model particle size and size distribution in both batch and continuous laboratory-scale crystallization processes with predictive simulations in good agreement with experimental results. CFD simulations of large-scale crystallizations using constant specific power input per unit mass, predict an increase in macromixing and decrease in micromixing and turbulence. This effect should improve process performance of batch cooling crystallizers on scale-up including the product quality of the final solid form in terms of the particle size and crystal habit. This is d...

The focussed beam reflectance monitoring (FBRM) instrument developed by Lasentec is a ‘powerful’ tool used as an ‘in-situ’ particle monitoring technique for in-line real-time measurement of particle size. This technique was successfully used to monitor particulate attrition and breakage of an organic fine chemical in a turbulently agitated suspension. The great advantage of using the FBRM technique is that the change in the crystal size distribution (CSD) for different particle size classes (fine, intermediate and coarse) can be monitored as a function of time. The attrition rates can be calculated to produce a model for the disruption kernel for the organic compound. The shift in the CSD that was observed with an increase in the specific power input was found to be largely due to micro-attrition effects rather than particle breakage (splitting).

Modelling, design and control of organic batch cooling crystallization processes requires accurate solute concentration and supersaturation measurement. De-supersaturation rate controls the crystallization kinetics and determines the crystal size distribution and product quality. This paper describes a quick, robust and convenient technique for the successful measurement of solute concentration of an organic fine chemical using attenuated total reflectance ultraviolet spectroscopy. The use of multivariate analysis and partial least squares using UMETRICS SIMCA-P software for the calibration and measurement of solute concentration is also described.

Recent experiments show that the microtubule-associated protein (MAP) 1B is a major phosphorylation substrate for the serine/threonine kinase glycogen synthase kinase-3beta (GSK-3beta) in differentiating neurons. GSK-3beta phosphorylation of MAP1B appears to act as a molecular switch regulating the control that MAP1B exerts on microtubule dynamics in growing axons and growth cones. Maintaining a population of dynamically unstable microtubules in growth cones is important for axon growth and growth cone pathfinding. We have mapped two GSK-3beta phosphorylation sites on mouse MAP1B to Ser1260 and Thr1265 using site-directed point mutagenesis of recombinant MAP1B proteins, in vitro kinase assays and phospho-specific antibodies. We raised phospho-specific polyclonal antibodies to these two sites and used them to show that MAP1B is phosphorylated by GSK-3beta at Ser1260 and Thr1265 in vivo. We also showed that in the developing nervous system of rat embryos, the expression of GSK-3beta phosphorylated MAP1B is spatially restricted to growing axons, in a gradient that is highest distally, despite the expression of MAP1B and GSK-3beta throughout the entire neuron. This suggests that there is a mechanism that spatially regulates the GSK-3beta phosphorylation of MAP1B in differentiating neurons. Heterologous cell transfection experiments with full-length MAP1B, in which either phosphorylation site was separately mutated to a valine or, in a double mutant, in which both sites were mutated, showed that these GSK-3beta phosphorylation sites contribute to the regulation of microtubule dynamics by MAP1B.

The crystallization kinetics of an important organic fine chemical was determined using a modified laboratory scale continuous cooling mixed suspension mixed product removal (MSMPR) crystallizer with a product recycle loop designed for use with limited quantities of available material. A convenient method of monitoring steady state in the MSMPR using lasentec focussed beam reflectance measurement (FBRM) is also described. The effect of supersaturation, suspension density of the crystallizer content, impeller velocity (turnover time) and configuration on the growth and nucleation rates was studied. An observed deviation from the McCabe DeltaL law for small crystal sizes was attributed to either size-dependent growth (SDG) or growth rate dispersion (GRD) mechanisms and hence complicated the estimation of growth and nucleation rates. SDG was successfully modelled using a three-parameter exponential SDG model. Growth rates estimated from the SDG model were compared to a GRD model using discrete growth probability distributions. (C) 2004 Elsevier B.V. All rights reserved.

The FBRM instrument is a ‘powerful’ tool developed by Lasentec as an ‘in situ’ particle monitoring technique for in-line real-time measurement of particle size. This technique was successfully used to monitor steady-state operation in a modified MSMPR crystallizer. The FBRM technique was also used to estimate crystallization kinetics. The FBRM particle size measurements were complimented by an in-line process video imaging (PVI) system developed ‘ in-house ’ (Microscopy and Microanalysis, 6 (Suppl. 2) (2000) 996–997), to visualize habit and crystal behaviour within an MSMPR crystallizer. A comparison of the steady-state crystal size distributions measured by low angle light scattering (LALLS) and FBRM was made, showing poor sensitivity of the FBRM technique to particles of less than 1 μm hence the technique was not suitable for the measurement of crystallization kinetics for this organic system.

Reaction of 4-bromo-3-methylaniline with 4-chlorobutyryl chloride/TEA and subsequent treatment of the resulting secondary amide intermediate with KOt-Bu gives 1-(4-bromo-3-methylphenyl)pyrrolidin-2-one in 65% yield. This procedure has been optimised (74−76% overall yield) and has been carried out on 41 molar scale. In a variation of this process, we have employed NaOH as the ring-closing base under phase-transfer conditions. NaOH is added to a mixture of 4-bromo-3-methylaniline, 4-chlorobutyryl chloride, and catalytic TBAC in THF/H2O. A further 2 equiv of aqueous NaOH is added, and the mixture is heated at 40−45 °C, providing access to cyclised product in an improved 86% yield. 1-(4-Bromo-3-methylphenyl)pyrrolidin-2-one is subsequently coupled with 4-carboxyphenylboronic acid under standard Suzuki coupling conditions [Pd(PPh3)4, Na2CO3, DME/H2O] to give 2‘-methyl-4‘-(2-oxo-1-pyrrolidinyl)biphenyl-4-carboxylic acid in 64% yield, contaminated with 40−80 ppm of residual Pd. In a modification of this process,...

Background Mitochondrial dysfunction is a known component of HD pathogenesis, but the precise mechanisms and temporal order of events linked to mHTT inclusion formation and neurodegeneration are unclear. For example, observations from clinical samples and in vitro models implicate respiratory impairment and enhanced glycolysis in HD cells. Aim To identify early signs of mitochondrial dysfunction and perturbed bioenergetics in a human HD neuronal model, and better understand the interaction between the proteotoxic environment of mHTT-expressing cells and their metabolic capacity. Methods We are using a human neural stem cell (NSC) line (ReNcell VM) that over-expresses huntingtin (HTT) exon 1 fragments with either normal (29Q) or pathogenic polyglutamine tracts (71Q and 129Q), and can be rapidly differentiated into mixed populations of neurons and glia in various culture formats. The pathogenic HTT exon 1 lines develop both intranuclear and cytoplasmic mHTT-containing inclusions, as well as accumulating a diffuse aggregated form of mHTT in a time and poly Q-dependent manner. We have correlated with this measures of oxygen consumption rate and extracellular acidification rates using an XF96 Seahorse Bioanalyser, mitochondrial volume and basal ΔΨm by live confocal imaging and potential impairment individual respiratory chain complexes using in vitro assays. Results Despite no overt reduction in cell viability in the mHTT exon 1 expressing lines we have uncovered sub-pathological alterations to mitochondrial function. We report differences in key respiratory parameters (e.g. basal and maximal respiration) in line with a significant decrease in basal mitochondrial ΔΨm in mHTT exon 1 cells. Here, we will present our findings correlating the inclusion phenotypes and mitochondrial dysfunction in the presence of mHTT exon 1. Conclusions We have established a robust and disease-relevant in vitro cellular model of HD that has revealed potentially important early changes to neuronal bioenergetics and mitochondrial function. Using this system we aim to not only identify novel pathways in disease pathogenesis that may be amenable to therapeutic targeting, but also to simultaneously develop a novel in vitro cellular platform for high-throughput compound screening.

Background Neuronal inclusion formation is a pathognomonic feature of Huntington’s disease (HD). Recent evidence has suggested that these inclusion bodies (IBs) may comprise a heterogeneous population of huntingtin (HTT) protein species, a subset of which may lead to impaired cell viability and ultimately cell death. Aim To investigate the formation of inclusions in human neurons derived from neural stem cells (NSCs) (ReNcell VM) stably transduced to over-express HTT exon 1 fragments with either normal (29Q) or pathogenic polyglutamine tracts (71 Q and 129Q). Methods High-content screening of neurons stained with anti-HTT antibody S830 was carried out using a Perkin Elmer Opera LX confocal microscope. Subsequent quantitative image analysis was carried out using ImageJ and R. Individual cells were analysed in more detail using super resolution imaging (N-SIM Super Resolution System). The effect of mHTT exon 1 on neuronal viability was assessed using a variety of biochemical techniques (LDH assay, MTT and Alamar Blue). Results Inclusion bodies form in a small proportion of (1%) of neurons, and are predominantly intra-nuclear, though a lower proportion of cells display smaller perinuclear inclusions. The formation of nuclear inclusions increases in a polyQ- length dependent manner and over time. Super resolution microscopy of individual cells has demonstrated this to be an all-or-nothing phenomenon. A diffuse cytosolic form of mHTT also accumulates in the cells over time. There is no overt effect on basal viability in these mHTT exon 1 expressing neurons. Conclusions We have characterised the spatio-temporal profile of mHTT inclusion formation in mHTT expressing NSCs and neurons, and have demonstrated poly-Q and time-dependent accumulation of IBs. A deeper understanding of relationship between the different forms of mHTT and neuronal vulnerability is essential for the design of targeted therapeutics.