Biodistribution of Radioactively Labeled Splice Modulating Antisense Oligonucleotides After Intracerebroventricular and Intrathecal Injection in Mice.

Antisense oligonucleotides (AONs) are promising therapeutic candidates, especially for neurological diseases. Intracerebroventricular (ICV) injection is the predominant route of administration in mouse studies, while in clinical trials, intrathecal (IT) administration is mostly used. There is little knowledge on the differences in distribution of these injection methods within the same species over time. In this study, we compared the distribution of splice-switching AONs targeting exon 15 of amyloid precursor protein pre-mRNA injected via the ICV and IT route in mice. The AON was labeled with radioactive indium-111 and mice were imaged using single-photon emission computed tomography (SPECT) 0, 4, 24, 48, 72, and 96 h after injection. In vivo SPECT imaging showed 111In-AON activity diffused throughout the central nervous system (CNS) in the first hours after injection. The 111In-AON activity in the CNS persisted over the course of 4 days, while signal in the kidneys rapidly decreased. Postmortem counting in different organs and tissues showed very similar distribution of 111In-AON activity throughout the body, while the signal in the different brain regions was higher with ICV injection. Overall, IT and ICV injection have very similar distribution patterns in the mouse, but ICV injection is much more effective in reaching the brain.

Glucocorticoid receptor antagonist CORT113176 attenuates motor and neuropathological symptoms of Huntington's disease in R6/2 mice.

Huntington's Disease (HD) is a progressive neurodegenerative disease caused by a mutation in the huntingtin gene. The mutation leads to a toxic gain of function of the mutant huntingtin (mHtt) protein resulting in cellular malfunction, aberrant huntingtin aggregation and eventually neuronal cell death. Patients with HD show impaired motor functions and cognitive decline. Elevated levels of glucocorticoids have been found in HD patients and in HD mouse models, and there is a positive correlation between increased glucocorticoid levels and the progression of HD. Therefore, antagonism of the glucocorticoid receptor (GR) may be an interesting strategy for the treatment of HD. In this study, we evaluated the efficacy of the selective GR antagonist CORT113176 in the commonly used R6/2 mouse model. In male mice, CORT113176 treatment significantly delayed the loss of grip strength, the development of hindlimb clasping, gait abnormalities, and the occurrence of epileptic seizures. CORT113176 treatment delayed loss of DARPP-32 immunoreactivity in the dorsolateral striatum. It also restored HD-related parameters including astrocyte markers in both the dorsolateral striatum and the hippocampus, and microglia markers in the hippocampus. This suggests that CORT113176 has both cell-type and brain region-specific effects. CORT113176 delayed the formation of mHtt aggregates in the striatum and the hippocampus. In female mice, we did not observe major effects of CORT113176 treatment on HD-related symptoms, with the exception of the anti-epileptic effects. We conclude that CORT113176 effectively delays several key symptoms related to the HD phenotype in male R6/2 mice and believe that GR antagonism may be a possible treatment option.

GLUT-1 changes in paediatric Huntington disease brain cortex and fibroblasts: an observational case-control study.

BACKGROUND: Paediatric Huntington disease with highly expanded mutations (HE-PHD; >80 CAG repeats) presents atypically, compared to adult-onset Huntington disease (AOHD), with neurodevelopmental delay, epilepsy, abnormal brain glucose metabolism, early striatal damage, and reduced lifespan. Since genetic GLUT-1 deficiency syndrome shows a symptom spectrum similar to HE-PHD, we investigated the potential role of the two main glucose transporters, GLUT-1 and GLUT-3, in HE-PHD. METHODS: We compared GLUT-1 and GLUT-3 protein expression in HE-PHD, juvenile-onset (JOHD), and AOHD brains (n = 2; n = 3; n = 6) and periphery (n = 3; n = 2; n = 2) versus healthy adult controls (n = 6; n = 6). We also investigated mitochondrial complexes and hexokinase-II protein expression. FINDINGS: GLUT-1 and GLUT-3 expression were significantly lower in HE-PHD frontal cortex (p = 0.009, 95% [CI 13.4, 14.7]; p = 0.017, 95% [CI 14.2, 14.5]) versus controls. In fibroblasts, GLUT-1 and GLUT-3 expression were lower compared to controls (p < 0.0001, 95% [CI 0.91, 1.09]; p = 0.046, 95% [CI 0.93, 1.07]). In the frontal cortex, this occurred without evidence of extensive neuronal degeneration. Patients with HE-PHD had deregulated mitochondrial complex expression, particularly complexes II-III, levels of which were lower in frontal cortex versus controls (p = 0.027, 95% [CI 17.1, 17.6]; p = 0.002, 95% CI [16.6, 16.9]) and patients with AOHD (p = 0.052, 95% [CI 17.0, 17.6]; p = 0.002, 95% [CI 16.6, 16.7]). Hexokinase-II expression was also lower in HE-PHD frontal cortex and striatum versus controls (p = 0.010, 95% [CI 17.8, 18.2]; p = 0.045, 95% [CI 18.6, 18.7]) and in frontal cortex versus patients with AOHD (p = 0.013, 95% [CI 17.7, 18.1]). Expression JOHD levels were consistently different to those of HE-PHD but similar to those of AOHD. INTERPRETATION: Our data suggest a dysfunctional hypometabolic state occurring specifically in paediatric Huntington disease brains. FUNDING: '5 × 1000' Personal Income Tax donation to LIRH Foundation; Italian Ministry of HealthRC2301MH04 and RF-2016-02364123 to CSS.

Machine learning in Huntington's disease: exploring the Enroll-HD dataset for prognosis and driving capability prediction.

BACKGROUND: In biomedicine, machine learning (ML) has proven beneficial for the prognosis and diagnosis of different diseases, including cancer and neurodegenerative disorders. For rare diseases, however, the requirement for large datasets often prevents this approach. Huntington's disease (HD) is a rare neurodegenerative disorder caused by a CAG repeat expansion in the coding region of the huntingtin gene. The world's largest observational study for HD, Enroll-HD, describes over 21,000 participants. As such, Enroll-HD is amenable to ML methods. In this study, we pre-processed and imputed Enroll-HD with ML methods to maximise the inclusion of participants and variables. With this dataset we developed models to improve the prediction of the age at onset (AAO) and compared it to the well-established Langbehn formula. In addition, we used recurrent neural networks (RNNs) to demonstrate the utility of ML methods for longitudinal datasets, assessing driving capabilities by learning from previous participant assessments. RESULTS: Simple pre-processing imputed around 42% of missing values in Enroll-HD. Also, 167 variables were retained as a result of imputing with ML. We found that multiple ML models were able to outperform the Langbehn formula. The best ML model (light gradient boosting machine) improved the prognosis of AAO compared to the Langbehn formula by 9.2%, based on root mean squared error in the test set. In addition, our ML model provides more accurate prognosis for a wider CAG repeat range compared to the Langbehn formula. Driving capability was predicted with an accuracy of 85.2%. The resulting pre-processing workflow and code to train the ML models are available to be used for related HD predictions at: https://github.com/JasperO98/hdml/tree/main . CONCLUSIONS: Our pre-processing workflow made it possible to resolve the missing values and include most participants and variables in Enroll-HD. We show the added value of a ML approach, which improved AAO predictions and allowed for the development of an advisory model that can assist clinicians and participants in estimating future driving capability.

Spinocerebellar Ataxia Type 1 Characteristics in Patient-Derived Fibroblast and iPSC-Derived Neuronal Cultures.

BACKGROUND: Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by a polyglutamine expansion in the ataxin-1 protein resulting in neuropathology including mutant ataxin-1 protein aggregation, aberrant neurodevelopment, and mitochondrial dysfunction. OBJECTIVES: Identify SCA1-relevant phenotypes in patient-specific fibroblasts and SCA1 induced pluripotent stem cells (iPSCs) neuronal cultures. METHODS: SCA1 iPSCs were generated and differentiated into neuronal cultures. Protein aggregation and neuronal morphology were evaluated using fluorescent microscopy. Mitochondrial respiration was measured using the Seahorse Analyzer. The multi-electrode array (MEA) was used to identify network activity. Finally, gene expression changes were studied using RNA-seq to identify disease-specific mechanisms. RESULTS: Bioenergetics deficits in patient-derived fibroblasts and SCA1 neuronal cultures showed altered oxygen consumption rate, suggesting involvement of mitochondrial dysfunction in SCA1. In SCA1 hiPSC-derived neuronal cells, nuclear and cytoplasmic aggregates were identified similar in localization as aggregates in SCA1 postmortem brain tissue. SCA1 hiPSC-derived neuronal cells showed reduced dendrite length and number of branching points while MEA recordings identified delayed development in network activity in SCA1 hiPSC-derived neuronal cells. Transcriptome analysis identified 1050 differentially expressed genes in SCA1 hiPSC-derived neuronal cells associated with synapse organization and neuron projection guidance, where a subgroup of 151 genes was highly associated with SCA1 phenotypes and linked to SCA1 relevant signaling pathways. CONCLUSIONS: Patient-derived cells recapitulate key pathological features of SCA1 pathogenesis providing a valuable tool for the identification of novel disease-specific processes. This model can be used for high throughput screenings to identify compounds, which may prevent or rescue neurodegeneration in this devastating disease. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Early molecular layer interneuron hyperactivity triggers Purkinje neuron degeneration in SCA1.

Toxic proteinaceous deposits and alterations in excitability and activity levels characterize vulnerable neuronal populations in neurodegenerative diseases. Using in vivo two-photon imaging in behaving spinocerebellar ataxia type 1 (Sca1) mice, wherein Purkinje neurons (PNs) degenerate, we identify an inhibitory circuit element (molecular layer interneurons [MLINs]) that becomes prematurely hyperexcitable, compromising sensorimotor signals in the cerebellum at early stages. Mutant MLINs express abnormally elevated parvalbumin, harbor high excitatory-to-inhibitory synaptic density, and display more numerous synaptic connections on PNs, indicating an excitation/inhibition imbalance. Chemogenetic inhibition of hyperexcitable MLINs normalizes parvalbumin expression and restores calcium signaling in Sca1 PNs. Chronic inhibition of mutant MLINs delayed PN degeneration, reduced pathology, and ameliorated motor deficits in Sca1 mice. Conserved proteomic signature of Sca1 MLINs, shared with human SCA1 interneurons, involved the higher expression of FRRS1L, implicated in AMPA receptor trafficking. We thus propose that circuit-level deficits upstream of PNs are one of the main disease triggers in SCA1.

Therapeutic Strategies for Spinocerebellar Ataxia Type 1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder that affects one or two individuals per 100,000. The disease is caused by an extended CAG repeat in exon 8 of the ATXN1 gene and is characterized mostly by a profound loss of cerebellar Purkinje cells, leading to disturbances in coordination, balance, and gait. At present, no curative treatment is available for SCA1. However, increasing knowledge on the cellular and molecular mechanisms of SCA1 has led the way towards several therapeutic strategies that can potentially slow disease progression. SCA1 therapeutics can be classified as genetic, pharmacological, and cell replacement therapies. These different therapeutic strategies target either the (mutant) ATXN1 RNA or the ataxin-1 protein, pathways that play an important role in downstream SCA1 disease mechanisms or which help restore cells that are lost due to SCA1 pathology. In this review, we will provide a summary of the different therapeutic strategies that are currently being investigated for SCA1.

The potential value of disease-modifying therapy in patients with spinocerebellar ataxia type 1: an early health economic modeling study.

OBJECTIVE: There currently is no disease-modifying therapy for spinocerebellar ataxia type 1 (SCA1). Genetic interventions, such as RNA-based therapies, are being developed but those currently available are very expensive. Early evaluation of costs and benefits is, therefore, crucial. By developing a health economic model, we aimed to provide first insights into the potential cost-effectiveness of RNA-based therapies for SCA1 in the Netherlands. METHODS: We simulated disease progression of individuals with SCA1 using a patient-level state-transition model. Five hypothetical treatment strategies with different start and endpoints and level of effectiveness (5-50% reduction in disease progression) were evaluated. Consequences of each strategy were measured in terms of quality-adjusted life years (QALYs), survival, healthcare costs, and maximum costs to be cost effective. RESULTS: Most QALYs (6.68) are gained when therapy starts during the pre-ataxic stage and continues during the entire disease course. Incremental costs are lowest (- €14,048) if therapy is stopped when the severe ataxia stage is reached. The maximum costs per year to be cost-effective are €19,630 in the "stop after moderate ataxia stage" strategy at 50% effectiveness. DISCUSSION: Our model indicates that the maximum price for a hypothetical therapy to be cost-effective is considerably lower than currently available RNA-based therapies. Most value for money can be gained by slowing progression in the early and moderate stages of SCA1 and by stopping therapy upon entering the severe ataxia stage. To allow for such a strategy, it is crucial to identify individuals in early stages of disease, preferably just before symptom onset.

Potential disease-modifying therapies for Huntington's disease: lessons learned and future opportunities.

Huntington's disease is the most frequent autosomal dominant neurodegenerative disorder; however, no disease-modifying interventions are available for patients with this disease. The molecular pathogenesis of Huntington's disease is complex, with toxicity that arises from full-length expanded huntingtin and N-terminal fragments of huntingtin, which are both prone to misfolding due to proteolysis; aberrant intron-1 splicing of the HTT gene; and somatic expansion of the CAG repeat in the HTT gene. Potential interventions for Huntington's disease include therapies targeting huntingtin DNA and RNA, clearance of huntingtin protein, DNA repair pathways, and other treatment strategies targeting inflammation and cell replacement. The early termination of trials of the antisense oligonucleotide tominersen suggest that it is time to reflect on lessons learned, where the field stands now, and the challenges and opportunities for the future.

Iron accumulation induces oxidative stress, while depressing inflammatory polarization in human iPSC-derived microglia.

Iron accumulation in microglia has been observed in Alzheimer's disease and other neurodegenerative disorders and is thought to contribute to disease progression through various mechanisms, including neuroinflammation. To study this interaction, we treated human induced pluripotent stem cell-derived microglia (iPSC-MG) with iron, in combination with inflammatory stimuli such as interferon gamma (IFN-γ) and amyloid ß. Both IFN-γ and iron treatment increased labile iron levels, but only iron treatment led to a consistent increase of ferritin levels, reflecting long-term iron storage. Therefore, in iPSC-MG, ferritin appeared to be regulated by iron revels rather than inflammation. Further investigation showed that while IFN-γ induced pro-inflammatory activation, iron treatment dampened both classic pro- and anti-inflammatory activation on a transcriptomic level. Notably, iron-loaded microglia showed strong upregulation of cellular stress response pathways, the NRF2 pathway, and other oxidative stress pathways. Functionally, iPSC-MG exhibited altered phagocytosis and impaired mitochondrial metabolism following iron treatment. Collectively, these data suggest that in MG, in contrast to current hypotheses, iron treatment does not result in pro-inflammatory activation, but rather dampens it and induces oxidative stress.

Co-expression patterns of microglia markers Iba1, TMEM119 and P2RY12 in Alzheimer's disease.

Microglia have been identified as key players in Alzheimer's disease pathogenesis, and other neurodegenerative diseases. Iba1, and more specifically TMEM119 and P2RY12 are gaining ground as presumedly more specific microglia markers, but comprehensive characterization of the expression of these three markers individually as well as combined is currently missing. Here we used a multispectral immunofluorescence dataset, in which over seventy thousand microglia from both aged controls and Alzheimer patients have been analysed for expression of Iba1, TMEM119 and P2RY12 on a single-cell level. For all markers, we studied the overlap and differences in expression patterns and the effect of proximity to ß-amyloid plaques. We found no difference in absolute microglia numbers between control and Alzheimer subjects, but the prevalence of specific combinations of markers (phenotypes) differed greatly. In controls, the majority of microglia expressed all three markers. In Alzheimer patients, a significant loss of TMEM119+-phenotypes was observed, independent of the presence of ß-amyloid plaques in its proximity. Contrary, phenotypes showing loss of P2RY12, but consistent Iba1 expression were increasingly prevalent around ß-amyloid plaques. No morphological features were conclusively associated with loss or gain of any of the markers or any of the identified phenotypes. All in all, none of the three markers were expressed by all microglia, nor can be wholly regarded as a pan- or homeostatic marker, and preferential phenotypes were observed depending on the surrounding pathological or homeostatic environment. This work could help select and interpret microglia markers in previous and future studies.

Delivery of Antisense Oligonucleotides to the Mouse Brain by Intracerebroventricular Injections.

The use of antisense oligonucleotides (AONs) is a promising therapeutic strategy for central nervous system disorders. However, the delivery of AONs to the central nervous system is challenging because their size does not allow them to diffuse over the blood-brain barrier (BBB) when injected systemically. The BBB can be bypassed by administering directly into the brain. Here we describe a method to perform single and repeated intracerebroventricular injections into the lateral ventricle of the mouse brain.

Huntington Disease Gene Expression Signatures in Blood Compared to Brain of YAC128 Mice as Candidates for Monitoring of Pathology.

While the genetic cause of Huntington disease (HD) is known since 1993, still no cure exists. Therapeutic development would benefit from a method to monitor disease progression and treatment efficacy, ideally using blood biomarkers. Previously, HD-specific signatures were identified in human blood representing signatures in human brain, showing biomarker potential. Since drug candidates are generally first screened in rodent models, we aimed to identify HD signatures in blood and brain of YAC128 HD mice and compare these with previously identified human signatures. RNA sequencing was performed on blood withdrawn at two time points and four brain regions from YAC128 and control mice. Weighted gene co-expression network analysis was used to identify clusters of co-expressed genes (modules) associated with the HD genotype. These HD-associated modules were annotated via text-mining to determine the biological processes they represented. Subsequently, the processes from mouse blood were compared with mouse brain, showing substantial overlap, including protein modification, cell cycle, RNA splicing, nuclear transport, and vesicle-mediated transport. Moreover, the disease-associated processes shared between mouse blood and brain were highly comparable to those previously identified in human blood and brain. In addition, we identified HD blood-specific pathology, confirming previous findings for peripheral pathology in blood. Finally, we identified hub genes for HD-associated blood modules and proposed a strategy for gene selection for development of a disease progression monitoring panel.

Juvenile-Onset Huntington Disease Pathophysiology and Neurodevelopment: A Review.

Huntington disease is an autosomal dominant inherited brain disorder that typically becomes manifest in adulthood. Juvenile-onset Huntington disease refers to approximately 5% of patients with symptom onset before the age of 21 years. The causal factor is a pathologically expanded CAG repeat in the Huntingtin gene. Age at onset is inversely correlated with CAG repeat length. Juvenile-onset patients have distinct symptoms and signs with more severe pathology of involved brain structures in comparison with disease onset in adulthood. The aim of this review is to compare clinical and pathological features in juvenile- and adult-onset Huntington disease and to explore which processes potentially contribute to the observed differences. A specific focus is placed on molecular mechanisms of mutant huntingtin in early neurodevelopment and the interaction of a neurodegenerative disease and postnatal brain maturation. The importance of a better understanding of pathophysiological differences between juvenile- and adult-onset Huntington disease lies in development and implementation of new therapeutic strategies. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Human Induced Pluripotent Stem Cell-Based Modelling of Spinocerebellar Ataxias.

Dominant spinocerebellar ataxias (SCAs) constitute a large group of phenotypically and genetically heterogeneous disorders that mainly present with dysfunction of the cerebellum as their main hallmark. Although animal and cell models have been highly instrumental for our current insight into the underlying disease mechanisms of these neurodegenerative disorders, they do not offer the full human genetic and physiological context. The advent of human induced pluripotent stem cells (hiPSCs) and protocols to differentiate these into essentially every cell type allows us to closely model SCAs in a human context. In this review, we systematically summarize recent findings from studies using hiPSC-based modelling of SCAs, and discuss what knowledge has been gained from these studies. We conclude that hiPSC-based models are a powerful tool for modelling SCAs as they contributed to new mechanistic insights and have the potential to serve the development of genetic therapies. However, the use of standardized methods and multiple clones of isogenic lines are essential to increase validity and reproducibility of the insights gained.

Preparing n-of-1 Antisense Oligonucleotide Treatments for Rare Neurological Diseases in Europe: Genetic, Regulatory, and Ethical Perspectives.

Antisense oligonucleotide (ASO) therapies present a promising disease-modifying treatment approach for rare neurological diseases (RNDs). However, the current focus is on "more common" RNDs, leaving a large share of RND patients still without prospect of disease-modifying treatments. In response to this gap, n-of-1 ASO treatment approaches are targeting ultrarare or even private variants. While highly attractive, this emerging, academia-driven field of ultimately individualized precision medicine is in need of systematic guidance and standards, which will allow global scaling of this approach. We provide here genetic, regulatory, and ethical perspectives for preparing n-of-1 ASO treatments and research programs, with a specific focus on the European context. By example of splice modulating ASOs, we outline genetic criteria for variant prioritization, chart the regulatory field of n-of-1 ASO treatment development in Europe, and propose an ethically informed classification for n-of-1 ASO treatment strategies and level of outcome assessments. To accommodate the ethical requirements of both individual patient benefit and knowledge gain, we propose a stronger integration of patient care and clinical research when developing novel n-of-1 ASO treatments: each single trial of therapy should inherently be driven to generate generalizable knowledge, be registered in a ASO treatment registry, and include assessment of generic outcomes, which allow aggregated analysis across n-of-1 trials of therapy.

Amyloid beta accumulations and enhanced neuronal differentiation in cerebral organoids of Dutch-type cerebral amyloid angiopathy patients.

Introduction: ADutch-type cerebral amyloid angiopathy (D-CAA) is a hereditary brain disorder caused by a point mutation in the amyloid precursor protein (APP) gene. The mutation is located within the amyloid beta (Aß) domain of APP and leads to Aß peptide accumulation in and around the cerebral vasculature. There lack of disease models to study the cellular and molecular pathological mechanisms of D-CAA together with the absence of a disease phenotype in vitro in overexpression cell models, as well as the limited availability of D-CAA animal models indicates the need for a D-CAA patient-derived model. Methods: We generated cerebral organoids from four D-CAA patients and four controls, cultured them up to 110 days and performed immunofluorescent and targeted gene expression analyses at two time points (D52 and D110). Results: D-CAA cerebral organoids exhibited Aß accumulations, showed enhanced neuronal and astrocytic gene expression and TGFß pathway de-regulation. Conclusions: These results illustrate the potential of cerebral organoids as in vitro disease model of D-CAA that can be used to understand disease mechanisms of D-CAA and can serve as therapeutic intervention platform for various Aß-related disorders.

Antisense Oligonucleotide-Induced Amyloid Precursor Protein Splicing Modulation as a Therapeutic Approach for Dutch-Type Cerebral Amyloid Angiopathy.

Dutch-type cerebral amyloid angiopathy (D-CAA) is a monogenic form of cerebral amyloid angiopathy and is inherited in an autosomal dominant manner. The disease is caused by a point mutation in exon 17 of the amyloid precursor protein (APP) gene that leads to an amino acid substitution at codon 693. The mutation is located within the amyloid beta (Aß) domain of APP, and leads to accumulation of toxic Aß peptide in and around the cerebral vasculature. We have designed an antisense oligonucleotide (AON) approach that results in skipping of exon 17, generating a shorter APP isoform that lacks part of the Aß domain and the D-CAA mutation. We demonstrate efficient AON-induced skipping of exon 17 at RNA level and the occurrence of a shorter APP protein isoform in three different cell types. This resulted in a reduction of Aß40 in neuronally differentiated, patient-derived induced pluripotent stem cells. AON-treated wild-type mice showed successful exon skipping on RNA and protein levels throughout the brain. These results illustrate APP splice modulation as a promising therapeutic approach for D-CAA.