Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy.

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.

Gene Therapy for Dopamine Dyshomeostasis: From Parkinson's to Primary Neurotransmitter Diseases.

Neurological disorders encompass a broad range of neurodegenerative and neurodevelopmental diseases that are complex and almost universally without disease modifying treatments. There is, therefore, significant unmet clinical need to develop novel therapeutic strategies for these patients. Viral gene therapies are a promising approach, where gene delivery is achieved through viral vectors such as adeno-associated virus and lentivirus. The clinical efficacy of such gene therapies has already been observed in two neurological disorders of pediatric onset; for spinal muscular atrophy and aromatic L-amino acid decarboxylase (AADC) deficiency, gene therapy has significantly modified the natural history of disease in these life-limiting neurological disorders. Here, we review recent advances in gene therapy, focused on the targeted delivery of dopaminergic genes for Parkinson's disease and the primary neurotransmitter disorders, AADC deficiency and dopamine transporter deficiency syndrome (DTDS). Although recent European Medicines Agency and Medicines and Healthcare products Regulatory Agency approval of Upstaza (eladocagene exuparvovec) signifies an important landmark, numerous challenges remain. Future research will need to focus on defining the optimal therapeutic window for clinical intervention, better understanding of the duration of therapeutic efficacy, and improved brain targeting. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Aromatic l-amino acid decarboxylase deficiency: a patient-derived neuronal model for precision therapies.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a complex inherited neurological disorder of monoamine synthesis which results in dopamine and serotonin deficiency. The majority of affected individuals have variable, though often severe cognitive and motor delay, with a complex movement disorder and high risk of premature mortality. For most, standard pharmacological treatment provides only limited clinical benefit. Promising gene therapy approaches are emerging, though may not be either suitable or easily accessible for all patients. To characterize the underlying disease pathophysiology and guide precision therapies, we generated a patient-derived midbrain dopaminergic neuronal model of AADC deficiency from induced pluripotent stem cells. The neuronal model recapitulates key disease features, including absent AADC enzyme activity and dysregulated dopamine metabolism. We observed developmental defects affecting synaptic maturation and neuronal electrical properties, which were improved by lentiviral gene therapy. Bioinformatic and biochemical analyses on recombinant AADC predicted that the activity of one variant could be improved by l-3,4-dihydroxyphenylalanine (l-DOPA) administration; this hypothesis was corroborated in the patient-derived neuronal model, where l-DOPA treatment leads to amelioration of dopamine metabolites. Our study has shown that patient-derived disease modelling provides further insight into the neurodevelopmental sequelae of AADC deficiency, as well as a robust platform to investigate and develop personalized therapeutic approaches.

Phosphorylation of histone H2AX in the mouse brain from development to senescence.

Phosphorylation of the histone H2AX (γH2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases.

Aneuploid cells are differentially susceptible to caspase-mediated death during embryonic cerebral cortical development.

Neural progenitor cells, neurons, and glia of the normal vertebrate brain are diversely aneuploid, forming mosaics of intermixed aneuploid and euploid cells. The functional significance of neural mosaic aneuploidy is not known; however, the generation of aneuploidy during embryonic neurogenesis, coincident with caspase-dependent programmed cell death (PCD), suggests that a cell's karyotype could influence its survival within the CNS. To address this hypothesis, PCD in the mouse embryonic cerebral cortex was attenuated by global pharmacological inhibition of caspases or genetic removal of caspase-3 or caspase-9. The chromosomal repertoire of individual brain cells was then assessed by chromosome counting, spectral karyotyping, fluorescence in situ hybridization, and DNA content flow cytometry. Reducing PCD resulted in markedly enhanced mosaicism that was comprised of increased numbers of cells with the following: (1) numerical aneuploidy (chromosome losses or gains); (2) extreme forms of numerical aneuploidy (>5 chromosomes lost or gained); and (3) rare karyotypes, including those with coincident chromosome loss and gain, or absence of both members of a chromosome pair (nullisomy). Interestingly, mildly aneuploid (<5 chromosomes lost or gained) populations remained comparatively unchanged. These data demonstrate functional non-equivalence of distinguishable aneuploidies on neural cell survival, providing evidence that somatically generated, cell-autonomous genomic alterations have consequences for neural development and possibly other brain functions.

Histone lysine methyltransferase-related neurodevelopmental disorders: current knowledge and saRNA future therapies.

Neurodevelopmental disorders encompass a group of debilitating diseases presenting with motor and cognitive dysfunction, with variable age of onset and disease severity. Advances in genetic diagnostic tools have facilitated the identification of several monogenic chromatin remodeling diseases that cause Neurodevelopmental disorders. Chromatin remodelers play a key role in the neuro-epigenetic landscape and regulation of brain development; it is therefore not surprising that mutations, leading to loss of protein function, result in aberrant neurodevelopment. Heterozygous, usually de novo mutations in histone lysine methyltransferases have been described in patients leading to haploinsufficiency, dysregulated protein levels and impaired protein function. Studies in animal models and patient-derived cell lines, have highlighted the role of histone lysine methyltransferases in the regulation of cell self-renewal, cell fate specification and apoptosis. To date, in depth studies of histone lysine methyltransferases in oncology have provided strong evidence of histone lysine methyltransferase dysregulation as a determinant of cancer progression and drug resistance. As a result, histone lysine methyltransferases have become an important therapeutic target for the treatment of different cancer forms. Despite recent advances, we still lack knowledge about the role of histone lysine methyltransferases in neuronal development. This has hampered both the study and development of precision therapies for histone lysine methyltransferases-related Neurodevelopmental disorders. In this review, we will discuss the current knowledge of the role of histone lysine methyltransferases in neuronal development and disease progression. We will also discuss how RNA-based technologies using small-activating RNAs could potentially provide a novel therapeutic approach for the future treatment of histone lysine methyltransferase haploinsufficiency in these Neurodevelopmental disorders, and how they could be first tested in state-of-the-art patient-derived neuronal models.

Dopamine Transporter Deficiency Syndrome (DTDS): Expanding the Clinical Phenotype and Precision Medicine Approaches.

Infantile parkinsonism-dystonia due to dopamine transporter deficiency syndrome (DTDS) is an ultrarare childhood movement disorder caused by biallelic loss-of-function mutations in the SLC6A3 gene. Advances in genomic analysis have revealed an evolving spectrum of SLC6A3-related neurological and neuropsychiatric disorders. Since the initial clinical and genetic characterisation of DTDS in 2009, there have been thirty-one published cases with a variety of protein-truncating variants (nonsense variants, splice-site changes, and deletions) and missense changes. Amino acid substitutions result in mutant proteins with impaired dopamine transporter function due to reduced transporter activity, impaired dopamine binding, reduced cell-surface expression, and aberrant posttranslational protein modification with impaired glycosylation. In this review, we provide an overview of the expanding clinical phenotype of DTDS and the precision therapies in development, including pharmacochaperones and gene therapy.

Automated high-content imaging in iPSC-derived neuronal progenitors.

Induced pluripotent stem cells (iPSCs) have great potential as physiological disease models for human disorders where access to primary cells is difficult, such as neurons. In recent years, many protocols have been developed for the generation of iPSCs and the differentiation into specialised cell subtypes of interest. More recently, these models have been modified to allow large-scale phenotyping and high-content screening of small molecule compounds in iPSC-derived neuronal cells. Here, we describe the automated seeding of day 11 ventral midbrain progenitor cells into 96-well plates, administration of compounds, automated staining for immunofluorescence, the acquisition of images on a high-content screening platform and workflows for image analysis.

Cardiac glycosides restore autophagy flux in an iPSC-derived neuronal model of WDR45 deficiency.

Beta-Propeller Protein-Associated Neurodegeneration (BPAN) is one of the commonest forms of Neurodegeneration with Brain Iron Accumulation, caused by mutations in the gene encoding the autophagy-related protein, WDR45. The mechanisms linking autophagy, iron overload and neurodegeneration in BPAN are poorly understood and, as a result, there are currently no disease-modifying treatments for this progressive disorder. We have developed a patient-derived, induced pluripotent stem cell (iPSC)-based midbrain dopaminergic neuronal cell model of BPAN (3 patient, 2 age-matched controls and 2 isogenic control lines) which shows defective autophagy and aberrant gene expression in key neurodegenerative, neurodevelopmental and collagen pathways. A high content imaging-based medium-throughput blinded drug screen using the FDA-approved Prestwick library identified 5 cardiac glycosides that both corrected disease-related defective autophagosome formation and restored BPAN-specific gene expression profiles. Our findings have clear translational potential and emphasise the utility of iPSC-based modelling in elucidating disease pathophysiology and identifying targeted therapeutics for early-onset monogenic disorders.

Isolation and characterization of living primary astroglial cells using the new GLAST-specific monoclonal antibody ACSA-1.

Astrocytes show large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocyte subpopulations has been hampered by the lack of a suitable antibody for their direct detection and isolation. Here, we describe a new monoclonal antibody, ACSA-1, which was generated by immunization of GLAST1 knockout mice. The antibody specifically detects an extracellular epitope of the astrocyte-specific L-glutamate/L-aspartate transporter GLAST (EAAT1, Slc1a3). As shown by immunohistochemistry, immunocytochemistry, and flow cytometry, ACSA-1 was cross-reactive for mouse, human, and rat. It labeled virtually all astrocytes positive for GFAP, GS, BLBP, RC2, and Nestin, including protoplastic, fibrous, and reactive astrocytes as well as Bergmann glia, Müller glia, and radial glia. Oligodendrocytes, microglia, neurons, and neuronal progenitors were negative for ACSA-1. Using an immunomagnetic approach, we established a method for the isolation of GLAST-positive cells with high purity. Binding of the antibody to GLAST and subsequent sorting of GLAST-positive cells neither interfered with cellular glutamate transport nor compromised astrocyte viability in vitro. The ACSA-1 antibody is not only a valuable tool to identify and track astrocytes by immunostaining, but also provides the possibility of separation and further analysis of pure astrocytes.

Electrophysiological Properties of Human Cortical Organoids: Current State of the Art and Future Directions.

Human cortical development is an intricate process resulting in the generation of many interacting cell types and long-range connections to and from other brain regions. Human stem cell-derived cortical organoids are now becoming widely used to model human cortical development both in physiological and pathological conditions, as they offer the advantage of recapitulating human-specific aspects of corticogenesis that were previously inaccessible. Understanding the electrophysiological properties and functional maturation of neurons derived from human cortical organoids is key to ensure their physiological and pathological relevance. Here we review existing data on the electrophysiological properties of neurons in human cortical organoids, as well as recent advances in the complexity of cortical organoid modeling that have led to improvements in functional maturation at single neuron and neuronal network levels. Eventually, a more comprehensive and standardized electrophysiological characterization of these models will allow to better understand human neurophysiology, model diseases and test novel treatments.

On-demand cell-autonomous gene therapy for brain circuit disorders.

Several neurodevelopmental and neuropsychiatric disorders are characterized by intermittent episodes of pathological activity. Although genetic therapies offer the ability to modulate neuronal excitability, a limiting factor is that they do not discriminate between neurons involved in circuit pathologies and "healthy" surrounding or intermingled neurons. We describe a gene therapy strategy that down-regulates the excitability of overactive neurons in closed loop, which we tested in models of epilepsy. We used an immediate early gene promoter to drive the expression of Kv1.1 potassium channels specifically in hyperactive neurons, and only for as long as they exhibit abnormal activity. Neuronal excitability was reduced by seizure-related activity, leading to a persistent antiepileptic effect without interfering with normal behaviors. Activity-dependent gene therapy is a promising on-demand cell-autonomous treatment for brain circuit disorders.

Genome Editing in iPSC-Based Neural Systems: From Disease Models to Future Therapeutic Strategies.

Therapeutic advances for neurological disorders are challenging due to limited accessibility of the human central nervous system and incomplete understanding of disease mechanisms. Many neurological diseases lack precision treatments, leading to significant disease burden and poor outcome for affected patients. Induced pluripotent stem cell (iPSC) technology provides human neuronal cells that facilitate disease modeling and development of therapies. The use of genome editing, in particular CRISPR-Cas9 technology, has extended the potential of iPSCs, generating new models for a number of disorders, including Alzheimers and Parkinson Disease. Editing of iPSCs, in particular with CRISPR-Cas9, allows generation of isogenic pairs, which differ only in the disease-causing mutation and share the same genetic background, for assessment of phenotypic differences and downstream effects. Moreover, genome-wide CRISPR screens allow high-throughput interrogation for genetic modifiers in neuronal phenotypes, leading to discovery of novel pathways, and identification of new therapeutic targets. CRISPR-Cas9 has now evolved beyond altering gene expression. Indeed, fusion of a defective Cas9 (dCas9) nuclease with transcriptional repressors or activation domains allows down-regulation or activation of gene expression (CRISPR interference, CRISPRi; CRISPR activation, CRISPRa). These new tools will improve disease modeling and facilitate CRISPR and cell-based therapies, as seen for epilepsy and Duchenne muscular dystrophy. Genome engineering holds huge promise for the future understanding and treatment of neurological disorders, but there are numerous barriers to overcome. The synergy of iPSC-based model systems and gene editing will play a vital role in the route to precision medicine and the clinical translation of genome editing-based therapies.

The role of manganese dysregulation in neurological disease: emerging evidence.

Manganese is an essential trace metal. The dysregulation of manganese seen in a broad spectrum of neurological disorders reflects its importance in brain development and key neurophysiological processes. Historically, the observation of acquired manganism in miners and people who misuse drugs provided early evidence of brain toxicity related to manganese exposure. The identification of inherited manganese transportopathies, which cause neurodevelopmental and neurodegenerative syndromes, further corroborates the neurotoxic potential of this element. Moreover, manganese dyshomoeostasis is also implicated in Parkinson's disease and other neurodegenerative conditions, such as Alzheimer's disease and Huntington's disease. Ongoing and future research will facilitate the development of better targeted therapeutical strategies than are currently available for manganese-associated neurological disorders.

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-µ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.

A reevaluation of tetraploidy in the Alzheimer's disease brain.

Alzheimer's disease (AD) is characterized by extensive neuronal death in distinct brain regions, including the frontal cortex and hippocampus, although the specific mechanisms of neuronal degeneration in AD remain a topic of intense scientific pursuit. One model for cell death in AD postulates that abortive cell cycle events in neurons, including tetraploidy, precede neuronal death, and novel therapeutics based on suppressing cell cycle re-entry are being pursued. Using DNA content fluorescence-activated cell sorting combined with fluorescence in situ hybridization and immunostaining, we analyzed neuronal nuclei from postmortem human brain samples from the frontal cortex and hippocampus of nondiseased and AD patients for evidence of tetraploidy. Here, we show that tetraploid nuclei are similarly prevalent in AD and control brains and are exclusively non-neuronal, contrasting with an absence of tetraploid neurons. Our findings demonstrate that neuronal tetraploidy is nonexistent in the AD brain and intimate a reevaluation of neuronal cell cycle re-entry as a therapeutic target for AD.

IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP+ mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP+ cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP+ mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies.

Mutations in the histone methyltransferase gene KMT2B cause complex early-onset dystonia.

Histone lysine methylation, mediated by mixed-lineage leukemia (MLL) proteins, is now known to be critical in the regulation of gene expression, genomic stability, cell cycle and nuclear architecture. Despite MLL proteins being postulated as essential for normal development, little is known about the specific functions of the different MLL lysine methyltransferases. Here we report heterozygous variants in the gene KMT2B (also known as MLL4) in 27 unrelated individuals with a complex progressive childhood-onset dystonia, often associated with a typical facial appearance and characteristic brain magnetic resonance imaging findings. Over time, the majority of affected individuals developed prominent cervical, cranial and laryngeal dystonia. Marked clinical benefit, including the restoration of independent ambulation in some cases, was observed following deep brain stimulation (DBS). These findings highlight a clinically recognizable and potentially treatable form of genetic dystonia, demonstrating the crucial role of KMT2B in the physiological control of voluntary movement.

Utility of Induced Pluripotent Stem Cells for the Study and Treatment of Genetic Diseases: Focus on Childhood Neurological Disorders.

The study of neurological disorders often presents with significant challenges due to the inaccessibility of human neuronal cells for further investigation. Advances in cellular reprogramming techniques, have however provided a new source of human cells for laboratory-based research. Patient-derived induced pluripotent stem cells (iPSCs) can now be robustly differentiated into specific neural subtypes, including dopaminergic, inhibitory GABAergic, motorneurons and cortical neurons. These neurons can then be utilized for in vitro studies to elucidate molecular causes underpinning neurological disease. Although human iPSC-derived neuronal models are increasingly regarded as a useful tool in cell biology, there are a number of limitations, including the relatively early, fetal stage of differentiated cells and the mainly two dimensional, simple nature of the in vitro system. Furthermore, clonal variation is a well-described phenomenon in iPSC lines. In order to account for this, robust baseline data from multiple control lines is necessary to determine whether a particular gene defect leads to a specific cellular phenotype. Over the last few years patient-derived neural cells have proven very useful in addressing several mechanistic questions related to central nervous system diseases, including early-onset neurological disorders of childhood. Many studies report the clinical utility of human-derived neural cells for testing known drugs with repurposing potential, novel compounds and gene therapies, which then can be translated to clinical reality. iPSCs derived neural cells, therefore provide great promise and potential to gain insight into, and treat early-onset neurological disorders.