Inhibition of 7α,26-dihydroxycholesterol biosynthesis promotes midbrain dopaminergic neuron development.

Dysregulated cholesterol metabolism has been linked to neurodegeneration. We previously found that free, non-esterified, 7α,(25R)26-dihydroxycholesterol (7α,26-diHC), was significantly elevated in the cerebrospinal fluid of patients with Parkinson's disease (PD). In this study we investigated the role of 7α,26-diHC in midbrain dopamine (mDA) neuron development and survival. We report that 7α,26-diHC induces apoptosis and reduces the number of mDA neurons in hESC-derived cultures and in mouse progenitor cultures. Voriconazole, an oxysterol 7α-hydroxylase (CYP7B1) inhibitor, increases the number of mDA neurons and prevents the loss of mDA neurons induced by 7α,26-diHC. These effects are specific since neither 7α,26-diHC nor voriconazole alter the number of Islet1+ oculomotor neurons. Furthermore, our results suggest that elevated 24(S),25-epoxycholesterol, which has been shown to promote mDA neurogenesis, may be partially responsible for the effect of voriconazole on mDA neurons. These findings suggest that voriconazole, and/or other azole CYP7B1 inhibitors may have implications in PD therapy development.

Impaired cognitive and motor function are coincident with L-DOPA-induced dyskinesia in a model of Parkinson's disease.

Dopamine transmission has been implicated in motor and cognitive function. In Parkinson's disease (PD), dopamine replacement using the precursor drug L-DOPA is the predominant treatment approach, but long-term exposure leads to the onset of dyskinesias (LIDs). Chronic L-DOPA exposure has been associated with changes in gene expression and altered cortico-striatal plasticity. The aim of this research was to assess the functional consequence of long-term L-DOPA exposure on cognitive and motor function using a rodent model of PD. Across two independent experiments, we assessed the impact of chronic L-DOPA exposure, or a control D2R agonist, on motor and cognitive function in intact and in hemi parkinsonian rats, in the absence of drug. Abnormal involuntary movements associated with LID were measured and brain tissues were subsequently harvested for immunohistochemical analysis. Exposure to chronic L-DOPA, but not the D2R agonist, impaired motor and cognitive function, when animals were tested in the absence of drug. A meta-analysis of the two experiments allowed further dissociation of L-DOPA -treated rats into those that developed LIDs (dyskinetic) and those that did not develop LIDs (non-dyskinetic). This analysis revealed impaired cognitive and motor performance were evident only in dyskinetic, but not in non-dyskinetic, rats. These data reveal a functional consequence of the altered plasticity associated with LID onset and have implications for understanding symptom progression in the clinic.

Nuclear ERK1/2 signaling potentiation enhances neuroprotection and cognition via Importinα1/KPNA2.

Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneration and cognitive decline. Here, we show that selective genetic potentiation of neuronal ERK signaling prevents cell death in vitro and in vivo in the mouse brain, while attenuation of ERK signaling does the opposite. This neuroprotective effect mediated by an enhanced nuclear ERK activity can also be induced by the novel cell penetrating peptide RB5. In vitro administration of RB5 disrupts the preferential interaction of ERK1 MAP kinase with importinα1/KPNA2 over ERK2, facilitates ERK1/2 nuclear translocation, and enhances global ERK activity. Importantly, RB5 treatment in vivo promotes neuroprotection in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) disease, and enhances ERK signaling in a human cellular model of HD. Additionally, RB5-mediated potentiation of ERK nuclear signaling facilitates synaptic plasticity, enhances cognition in healthy rodents, and rescues cognitive impairments in AD and HD models. The reported molecular mechanism shared across multiple neurodegenerative disorders reveals a potential new therapeutic target approach based on the modulation of KPNA2-ERK1/2 interactions.

Neurite Outgrowth and Gene Expression Profile Correlate with Efficacy of Human Induced Pluripotent Stem Cell-Derived Dopamine Neuron Grafts.

Transplantation of human induced pluripotent stem cell-derived dopaminergic (iPSC-DA) neurons is a promising therapeutic strategy for Parkinson's disease (PD). To assess optimal cell characteristics and reproducibility, we evaluated the efficacy of iPSC-DA neuron precursors from two individuals with sporadic PD by transplantation into a hemiparkinsonian rat model after differentiation for either 18 (d18) or 25 days (d25). We found similar graft size and dopamine (DA) neuron content in both groups, but only the d18 cells resulted in recovery of motor impairments. In contrast, we report that d25 grafts survived equally as well and produced grafts rich in tyrosine hydroxylase-positive neurons, but were incapable of alleviating any motor deficits. We identified the mechanism of action as the extent of neurite outgrowth into the host brain, with d18 grafts supporting significantly more neurite outgrowth than nonfunctional d25 grafts. RNAseq analysis of the cell preparation suggests that graft efficacy may be enhanced by repression of differentiation-associated genes by REST, defining the optimal predifferentiation state for transplantation. This study demonstrates for the first time that DA neuron grafts can survive well in vivo while completely lacking the capacity to induce recovery from motor dysfunction. In contrast to other recent studies, we demonstrate that neurite outgrowth is the key factor determining graft efficacy and our gene expression profiling revealed characteristics of the cells that may predict their efficacy. These data have implication for the generation of DA neuron grafts for clinical application.

Acyl-Ghrelin Attenuates Neurochemical and Motor Deficits in the 6-OHDA Model of Parkinson's Disease.

The feeding-related hormone, acyl-ghrelin, protects dopamine neurones in murine 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-based models of experimental Parkinson's disease (PD). However, the potential protective effect of acyl-ghrelin on substantia nigra pars compacta (SNpc) dopaminergic neurones and consequent behavioural correlates in the more widely used 6-hydroxydopamine (6-OHDA) rat medial forebrain bundle (MFB) lesion model of PD are unknown. To address this question, acyl-ghrelin levels were raised directly by mini-pump infusion for 7 days prior to unilateral injection of 6-OHDA into the MFB with assessment of amphetamine-induced rotations on days 27 and 35, and immunohistochemical analysis of dopaminergic neurone survival. Whilst acyl-ghrelin treatment was insufficient to elevate food intake or body weight, it attenuated amphetamine-induced circling behaviour and SNpc dopamine neurone loss induced by 6-OHDA. These data support the notion that elevating circulating acyl-ghrelin may be a valuable approach to slow or impair progression of neurone loss in PD.

Challenges in progressing cell therapies to the clinic for Huntington's disease: A review of the progress made with pluripotent stem cell derived medium spiny neurons.

Huntington's disease (HD) is a hereditary, neurodegenerative disorder characterized by a triad of symptoms: motor, cognitive and psychiatric. HD is caused by a genetic mutation, expansion of the CAG repeat in the huntingtin gene, which results in loss of medium spiny neurons (MSNs) of the striatum. Cell replacement therapy (CRT) has emerged as a possible therapy for HD, aiming to replace those cells lost to the disease process and alleviate its symptoms. Initial pre-clinical studies used primary fetal striatal cells to provide proof-of-principal that CRT can bring about functional recovery on some behavioral tasks following transplantation into HD models. Alternative donor cell sources are required if CRT is to become a viable therapeutic option and human pluripotent stem cell (hPSC) sources, which have undergone differentiation toward the MSNs lost to the disease process, have proved to be strong candidates. The focus of this chapter is to review work conducted on the functional assessment of animals following transplantation of hPSC-derived MSNs. We discuss different ways that graft function has been assessed, and the results that have been achieved to date. In addition, this chapter presents and discusses challenges that remain in this field.

Investigating cell therapies in animal models of Parkinson's and Huntington's disease: Current challenges and considerations.

Cell therapeutics have entered into an exciting era, with first-in-person clinical trials underway for Parkinson's disease and novel cell therapies in development for other neurodegenerative diseases. In the hope of ensuring successful translation of these novel cell products to the clinic, a significant amount of preclinical work continues to be undertaken. Rodent models of neural transplantation are required to thoroughly assess the survival, safety and efficacy of novel therapeutics. It is critical to produce robust and reliable preclinical data, in order to increase the likelihood of clinical success. As a result, significant effort has been driven into generating ever more relevant model systems, from genetically modified disease models to mice with humanized immune systems. Despite this, several challenges remain in the quest to assess human cells in the rodent brain long-term. Here, with a focus on models of Parkinson's and Huntington's disease, we discuss key considerations for choosing an appropriate rodent model for neural transplantation. We also consider the challenges associated with long-term survival and assessment of functional efficacy in these models, as well as the need to consider the clinical relevance of the model. While the choice of model will be dependent on the scientific question, by considering the caveats associated with each model, we identify opportunities to optimize the preclinical assessment and generate reliable data on our novel cell therapeutics.

Defining the unknowns for cell therapies in Parkinson's disease.

First-in-human clinical trials have commenced to test the safety and efficacy of cell therapies for people with Parkinson's disease (PD). Proof of concept that this neural repair strategy is efficacious is based on decades of preclinical studies and clinical trials using primary foetal cells, as well as a significant literature exploring more novel stem cell-derived products. Although several measures of efficacy have been explored, including the successful in vitro differentiation of stem cells to dopamine neurons and consistent alleviation of motor dysfunction in rodent models, many unknowns still remain regarding the long-term clinical implications of this treatment strategy. Here, we consider some of these outstanding questions, including our understanding of the interaction between anti-Parkinsonian medication and the neural transplant, the impact of the cell therapy on cognitive or neuropsychiatric symptoms of PD, the role of neuroinflammation in the therapeutic process and the development of graft-induced dyskinesias. We identify questions that are currently pertinent to the field that require further exploration, and pave the way for a more holistic understanding of this neural repair strategy for treatment of PD.

Can cell and gene therapies improve cognitive symptoms in Parkinson's disease?

Advanced therapeutic medicinal products (ATMPs), including cell and gene therapies, are in development for Parkinson's disease (PD). In many cases, the goal is to replace the lost dopamine (DA), which is anticipated to improve motor dysfunctions associated with DA loss. However, it is less clear the extent to which these therapeutic interventions may impact on the wide range of cognitive symptoms that manifest as the disease progresses. Although the accepted perception is that cognitive symptoms are predominately non-DAergic in origin, in this commentary, it is argued that several, specific cognitive processes, such as habit formation, working memory and reward processing, have been reported to be DA-dependent. Furthermore, there is evidence of DAergic medications modulating these behaviours in PD patients. Finally, the potential for cell and gene ATMPs to influence these symptoms is considered. It is concluded that DA replacement through ATMPs is likely to improve certain DA-dependent symptoms, but only sparse clinical data are currently available and the ability to precisely titrate DA transmission is likely to be complex.

[18F]Difluorocarbene for positron emission tomography.

The advent of total-body positron emission tomography (PET) has vastly broadened the range of research and clinical applications of this powerful molecular imaging technology1. Such possibilities have accelerated progress in fluorine-18 (18F) radiochemistry with numerous methods available to 18F-label (hetero)arenes and alkanes2. However, access to 18F-difluoromethylated molecules in high molar activity is mostly an unsolved problem, despite the indispensability of the difluoromethyl group for pharmaceutical drug discovery3. Here we report a general solution by introducing carbene chemistry to the field of nuclear imaging with a [18F]difluorocarbene reagent capable of a myriad of 18F-difluoromethylation processes. In contrast to the tens of known difluorocarbene reagents, this 18F-reagent is carefully designed for facile accessibility, high molar activity and versatility. The issue of molar activity is solved using an assay examining the likelihood of isotopic dilution on variation of the electronics of the difluorocarbene precursor. Versatility is demonstrated with multiple [18F]difluorocarbene-based reactions including O-H, S-H and N-H insertions, and cross-couplings that harness the reactivity of ubiquitous functional groups such as (thio)phenols, N-heteroarenes and aryl boronic acids that are easy to install. The impact is illustrated with the labelling of highly complex and functionalized biologically relevant molecules and radiotracers.

Spontaneous Graft-Induced Dyskinesias Are Independent of 5-HT Neurons and Levodopa Priming in a Model of Parkinson's Disease.

BACKGROUND: The risk of graft-induced dyskinesias (GIDs) presents a major challenge in progressing cell transplantation as a therapy for Parkinson's disease. Current theories implicate the presence of grafted serotonin neurons, hotspots of dopamine release, neuroinflammation and established levodopa-induced dyskinesia. OBJECTIVE: To elucidate the mechanisms of GIDs. METHODS: Neonatally desensitized, dopamine denervated rats received intrastriatal grafts of human embryonic stem cells (hESCs) differentiated into either ventral midbrain dopaminergic progenitor (vmDA) (n = 15) or ventral forebrain cells (n = 14). RESULTS: Of the eight rats with surviving grafts, two vmDA rats developed chronic spontaneous GIDs, which were observed at 30 weeks post-transplantation. GIDs were inhibited by D2 -like receptor antagonists and not affected by 5-HT1A/1B/5-HT6 agonists/antagonists. Grafts in GID rats showed more microglial activation and lacked serotonin neurons. CONCLUSIONS: These findings argue against current thinking that rats do not develop spontaneous GID and that serotonin neurons are causative, rather indicating that GID can be induced in rats by hESC-derived dopamine grafts and, critically, can occur independently of both previous levodopa exposure and grafted serotonin neurons. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Do foetal transplant studies continue to be justified in Huntington's disease?

Early CNS transplantation studies used foetal derived cell products to provide a foundation of evidence for functional recovery in preclinical studies and early clinical trials. However, it was soon recognised that the practical limitations of foetal tissue make it unsuitable for widespread clinical use. Considerable effort has since been directed towards producing target cell phenotypes from pluripotent stem cells (PSCs) instead, and there now exist several publications detailing the differentiation and characterisation of PSC-derived products relevant for transplantation in Huntington's disease (HD). In light of this progress, we ask if foetal tissue transplantation continues to be justified in HD research. We argue that (i) the extent to which accurately differentiated target cells can presently be produced from PSCs is still unclear, currently making them undesirable for studying wider CNS transplantation issues; (ii) foetal derived cells remain a valuable tool in preclinical research for advancing our understanding of which products produce functional striatal grafts and as a reference to further improve PSC-derived products; and (iii) until PSC-derived products are ready for human trials, it is important to continue using foetal cells to gather clinical evidence that transplantation is a viable option in HD and to use this opportunity to optimise practical parameters (such as trial design, clinical practices, and delivery strategies) to pave the way for future PSC-derived products.

Generating Excitotoxic Lesion Models of Huntington's Disease.

In Huntington's disease (HD), the medium spiny projection neurons of the neostriatum degenerate early in the course of the disease. While genetic mutant models of HD provide an excellent resource for studying the molecular and cellular effects of the inherited polyQ huntingtin mutation, they do not typically present with overt atrophy of the basal ganglia, despite this being a major pathophysiological hallmark of the disease. By contrast, excitotoxic lesion models, which use quinolinic acid to specifically target the striatal projection neurons, are employed to study the functional consequences of striatal atrophy and to investigate potential therapeutic interventions that target the neuronal degeneration. This chapter provides a detailed guide to the generation of excitotoxic lesion models of HD in rats.

Rethinking Functional Outcome Measures: The Development of a Novel Upper Limb Token Transfer Test to Assess Basal Ganglia Dysfunction.

The basal ganglia are implicated in a wide range of motor, cognitive and behavioral activities required for normal function. This region is predominantly affected in Huntington's disease (HD), meaning that functional ability progressively worsens. However, functional outcome measures for HD, particularly those for the upper limb, are limited meaning there is an imperative for well-defined, quantitative measures. Here we describe the development and evaluation of the Moneybox test (MBT). This novel, functional upper limb assessment was developed in accordance with translational neuroscience and physiological principles for people with a broad disease manifestation, such as HD. Participants with HD (n = 64) and healthy controls (n = 21) performed the MBT, which required subjects to transfer tokens into a container in order of size (Baseline Transfer), value (Complex Transfer) with and without reciting the alphabet (Dual Transfer). Disease specific measures of motor, cognition, behavior, and function were collected. HD patients were grouped into disease stage, from which, discriminative and convergent validity was assessed using Analysis of Variance and Pearson's correlation respectively. Manifest HD participants were slower than pre-manifest and control participants, and achieved significantly lower MBT total scores. Performance in the Complex Transfer and Dual Transfer tasks were significantly different between pre-manifest and stage 1 HD. All MBT performance variables significantly correlated with routinely used measures of motor, cognition, behavior, and function. The MBT provides a valid, sensitive, and affordable functional outcome measure. Unlike current assessments, MBT performance significantly distinguished the subtle differences between the earliest disease stages of HD, which are the populations typically targeted in clinical trials.

Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease.

Stem cell treatments for neurodegenerative diseases are expected to reach clinical trials soon. Most of the approaches currently under development involve transplantation of immature progenitors that subsequently undergo phenotypic and functional maturation in vivo, and predicting the long-term graft outcome already at the progenitor stage remains a challenge. Here, we took an unbiased approach to identify predictive markers expressed in dopamine neuron progenitors that correlate with graft outcome in an animal model of Parkinson's disease through gene expression analysis of >30 batches of grafted human embryonic stem cell (hESC)-derived progenitors. We found that many of the commonly used markers did not accurately predict in vivo subtype-specific maturation. Instead, we identified a specific set of markers associated with the caudal midbrain that correlate with high dopaminergic yield after transplantation in vivo. Using these markers, we developed a good manufacturing practice (GMP) differentiation protocol for highly efficient and reproducible production of transplantable dopamine progenitors from hESCs.

Cognitive training modifies disease symptoms in a mouse model of Huntington's disease.

Huntington's disease (HD) is an incurable neurodegenerative disorder which causes a triad of motor, cognitive and psychiatric disturbances. Cognitive disruptions are a core feature of the disease, which significantly affect daily activities and quality of life, therefore cognitive training interventions present an exciting therapeutic intervention possibility for HD. We aimed to determine if specific cognitive training, in an operant task of attention, modifies the subsequent behavioural and neuropathological phenotype of the Hdh(Q111) mouse model of HD. Three testing groups comprising both Hdh(Q111) mice and wildtype controls were used. The first group received cognitive training in an operant task of attention at 4months of age. The second group received cognitive training in a comparable non-attentional operant task at 4months of age, and the third group were control animals that did not receive cognitive training. All groups were then tested in an operant task of attention at 12months of age. Relative to naïve untrained mice, both wildtype and Hdh(Q111) mice that received cognitive training in the operant task of attention demonstrated an increased number of trials initiated, greater accuracy, and fewer 'time out' errors. A specific improvement in response time performance was observed in Hdh(Q111) mice, relative to naïve untrained Hdh(Q111) mice. Relative to the group that received comparable training in a non-attentional task, both wildtype and Hdh(Q111) mice that received attentional training demonstrated superior accuracy in the task and made fewer 'time out' errors. Despite significant behavioural change, in both wildtype and Hdh(Q111) mice that had received cognitive training, no significant changes in neuropathology were observed between any of the testing groups. These results demonstrate that attentional cognitive training implemented at a young age significantly improves attentional performance, at an older age, in both wildtype and Hdh(Q111) mice. Attentional cognitive training also improved motor performance in Hdh(Q111) mice, thus leading to the conclusion that cognitive training can improve disease symptoms in a mouse model of HD.

Direct Comparison of Rat- and Human-Derived Ganglionic Eminence Tissue Grafts on Motor Function.

Huntington's disease (HD) is a debilitating, genetically inherited neurodegenerative disorder that results in early loss of medium spiny neurons from the striatum and subsequent degeneration of cortical and other subcortical brain regions. Behavioral changes manifest as a range of motor, cognitive, and neuropsychiatric impairments. It has been established that replacement of the degenerated medium spiny neurons with rat-derived fetal whole ganglionic eminence (rWGE) tissue can alleviate motor and cognitive deficits in preclinical rodent models of HD. However, clinical application of this cell replacement therapy requires the use of human-derived (hWGE), not rWGE, tissue. Despite this, little is currently known about the functional efficacy of hWGE. The aim of this study was to directly compare the ability of the gold standard rWGE grafts, against the clinically relevant hWGE grafts, on a range of behavioral tests of motor function. Lister hooded rats either remained as unoperated controls or received unilateral excitotoxic lesions of the lateral neostriatum. Subsets of lesioned rats then received transplants of either rWGE or hWGE primary fetal tissue into the lateral striatum. All rats were tested postlesion and postgraft on the following tests of motor function: staircase test, apomorphine-induced rotation, cylinder test, adjusting steps test, and vibrissae-evoked touch test. At 21 weeks postgraft, brain tissue was taken for histological analysis. The results revealed comparable improvements in apomorphine-induced rotational bias and the vibrissae test, despite larger graft volumes in the hWGE cohort. hWGE grafts, but not rWGE grafts, stabilized behavioral performance on the adjusting steps test. These results have implications for clinical application of cell replacement therapies, as well as providing a foundation for the development of stem cell-derived cell therapy products.

Novel Application of Behavioral Assays Allows Dissociation of Joint Pathology from Systemic Extra-Articular Alterations Induced by Inflammatory Arthritis.

Introduction: Although rheumatoid arthritis (RA) is a disease of articular joints, patients often suffer from co-morbid neuropsychiatric changes, such as anxiety, that may reflect links between heightened systemic inflammation and abnormal regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we apply behavioral neuroscience methods to assess the impact of antigen-induced arthritis (AIA) on behavioral performance in wild type (WT) and interleukin-10 deficient (Il10-/-) mice. Our aim was to identify limb-specific motor impairments, as well as neuropsychological responses to inflammatory arthritis. Methods: Behavioral testing was performed longitudinally in WT and Il10-/- mice before and after the induction of arthritic joint pathology. Footprint analysis, beam walking and open field assessment determined a range of motor, exploratory and anxiety-related parameters. Specific gene changes in HPA axis tissues were analyzed using qPCR. Results: Behavioral assessment revealed transient motor and exploratory impairments in mice receiving AIA, coinciding with joint swelling. Hind limb coordination deficits were independent of joint pathology. Behavioral impairments returned to baseline by 10 days post-AIA in WT mice. Il10-/- mice demonstrated comparable levels of swelling and joint pathology as WT mice up to 15 days post-AIA, but systemic differences were evident in mRNA expression in HPA axis tissues from Il10-/- mice post-AIA. Interestingly, the behavioral profile of Il10-/- mice revealed a significantly longer time post-AIA for activity and anxiety-related behaviors to recover. Conclusions: The novel application of sensitive behavioral tasks has enabled dissociation between behaviors that occur due to transient joint-specific pathology and those generated by more subtle systemic alterations that manifest post-AIA.

ß-Amyloid pathology alters neural network activation during retrieval of contextual fear memories in a mouse model of Alzheimer's disease.

Although episodic memory deficits are the most conspicuous cognitive change in patients with Alzheimer's disease (AD), patients also display alterations in emotional expression, including anxiety and impaired conditioned fear behaviours. The neural circuitry underlying emotional learning is known to involve the amygdala and hippocampus, although the precise impact of amyloid pathology on the interaction between these brain regions remains unclear. Recent evidence suggests that Tg2576 mice, which express a human amyloid precursor protein (APP) mutation associated with early-onset AD, demonstrate normal acquisition of conditioned freezing to auditory and contextual stimuli paired with footshock. However, examination of the expression of c-Fos revealed altered neural network activity in transgenic mice. In the present study we examined the effects of the APP mutation on the expression of c-Fos following the retrieval of emotional memories. To this end, stimulus-induced cellular activity was measured by analysing expression of the immediate-early gene c-Fos after the retrieval of auditory or contextual fear memories. To characterize regional interdependencies of c-Fos expression, structural equation modelling was used to compare patterns of neural network activity. Consistent with previous findings, Tg2576 mice displayed reduced freezing elicited by the auditory stimulus but not by the conditioning context. Interestingly, the analysis of c-Fos expression revealed that the APPswe mutation disrupted dentate gyrus and amygdala function, as well as altering the influence of these regions on the neural network dynamics activated during context memory retrieval. These results provide novel insight into the influence of excess amyloid production on neural network activity during memory retrieval.