Drosophila Orthologues to Human Disease Genes: An Update on Progress

Translation: Screening for Novel Therapeutics With Disease-Relevant Cell Types Derived from Human Stem Cell Models

Kidney organoids—a new tool for kidney therapeutic development

The cerebellum is a highly conserved brain compartment of vertebrates. Genetic diseases of the human cerebellum often lead to degeneration of the principal neuron, the Purkinje cell, resulting in locomotive deficits and socio-emotional impairments. Due to its relatively simple but highly conserved neuroanatomy and circuitry, these human diseases can be modeled well in vertebrates amenable for genetic manipulation. In the recent years, cerebellar research in zebrafish has contributed to understanding cerebellum development and function, since zebrafish larvae are not only molecularly tractable, but also accessible for high resolution in vivo imaging due to the transparency of the larvae and the ease of access to the zebrafish cerebellar cortex for microscopy approaches. Therefore, zebrafish is increasingly used for genetic modeling of human cerebellar neurodegenerative diseases and in particular of different types of Spinocerebellar Ataxias (SCAs). These models are well suited to address the underlying pathogenic mechanisms by means of in vivo cell biological studies. Furthermore, accompanying circuitry characterizations, physiological studies and behavioral analysis allow for unraveling molecular, structural and functional relationships. Moreover, unlike in mammals, zebrafish possess an astonishing ability to regenerate neuronal populations and their functional circuitry in the central nervous system including the cerebellum. Understanding the cellular and molecular processes of these regenerative processes could well serve to counteract acute and chronic loss of neurons in humans. Based on the high evolutionary conservation of the cerebellum these regeneration studies in zebrafish promise to open therapeutic avenues for counteracting cerebellar neuronal degeneration. The current review aims to provide an overview over currently existing genetic models of human cerebellar neurodegenerative diseases in zebrafish as well as neuroregeneration studies using the zebrafish cerebellum. Due to this solid foundation in cerebellar disease modeling and neuronal regeneration analysis, the zebrafish promises to become a popular model organism for both unraveling pathogenic mechanisms of human cerebellar diseases and providing entry points for therapeutic neuronal regeneration approaches.

Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons

Introduction Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease, caused by the expansion of a CAG repeat within exon 1 of IT15 gene. HD exhibits the typical phenomenon of genetic anticipation and the symptoms of the disease appear earlier and more severe in subsequent generations due to meiotic instability. The CAG repeat accounts only for approximately 56%–70% of the variation in age at onset in HD. It is likely that modifying genetic variations, which segregate independently from the primary mutation, could influence the age at onset. Aims of the study Genetic, pathological, and clinical similarities exist between HD and spino-cerebellar ataxias (SCAs). In this study, seven SCAs genes have been studied as modifiers of age at onset in a cohort of HD patients. Materials and Methods We enrolled 50 HD patients. For every HD subject, CAG repeats have been measured on the larger allele of ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, PPP2R2B, and TBP genes. Regression analysis was used to evaluate the effects of CAG repeats in SCAs genes on age at motor, cognitive and psychiatric onset in HD patients. Results We did not find extensive correlations between CAG repeats in SCA genes and age at onset of HD. The only exceptions were represented by ATXN2 and CACNA1 genes for age at motor onset, and ATXN2 for age at psychiatric onset. When a multiple regression model was tested, a small additional effect on age at motor onset was identified only for CACNA1A. CAG repeats in expanded HD gene and larger CACNA1A alleles account for 64% of age at motor onset in HD patients. Conclusions CACNA1A gene could represent a mild genetic modifier of age at onset in HD patients. Further studies, conducted on larger HD patients‘ cohorts, are needed to confirm our data.

Protein Fate in Neurodegenerative Proteinopathies: Polyglutamine Diseases Join the (Mis)Fold

BackgroundIn this article, the authors discuss how they utilized the genetic mutation data in Sri Lankan Duchenne muscular dystrophy (DMD), Spinal muscular atrophy (SMA), Spinocerebellar ataxia (SCA) and Huntington’s disease (HD) patients and compare the available literature from South Asian countries to identifying potential candidates for available gene therapy for DMD, SMA, SCA and HD patients.MethodsRare disease patients (n = 623) with the characteristic clinical findings suspected of HD, SCA, SMA and Muscular Dystrophy were genetically confirmed using Multiplex Ligation Dependent Probe Amplification (MLPA), and single plex PCR. A survey was conducted in the “Wiley database on Gene Therapy Trials Worldwide” to identify DMD, SMA, SCA, and HD gene therapy clinical trials performed worldwide up to April 2021. In order to identify candidates for gene therapy in other neighboring countries we compared our findings with available literature from India and Pakistan which has utilized the same molecular diagnostic protocol to our study.ResultsFrom the overall cohort of 623 rare disease patients with the characteristic clinical findings suspected of HD, SCA, SMA and Muscular Dystrophy, n = 343 (55%) [Muscular Dystrophy- 65%; (DMD-139, Becker Muscular Dystrophy -BMD-11), SCA type 1–3–53% (SCA1–61,SCA2- 23, SCA3- 39), HD- 52% (45) and SMA- 34% (22)] patients were positive for molecular diagnostics by MLPA and single plex PCR. A total of 147 patients in Sri Lanka amenable to available gene therapy; [DMD-83, SMA-15 and HD-49] were identified. A comparison of Sri Lankan finding with available literature from India and Pakistan identified a total of 1257 patients [DMD-1076, SMA- 57, and HD-124] from these three South Asian Countries as amenable for existing gene therapy trials. DMD, SMA, and HD gene therapy clinical trials (113 studies) performed worldwide up to April 2021 were concentrated mostly (99%) in High Income Countries (HIC) and Upper Middle-Income Countries (UMIC). However, studies on the potential use of anti-sense oligonucleotides (ASO) for treatment of SCAs have yet to reach clinical trials.ConclusionMost genetic therapies for neurodegenerative and neuromuscular disorders have been evaluated for efficacy primarily in Western populations. No multicenter gene therapy clinical trial sites for DMD, SMA and HD in the South Asian region, leading to lack of knowledge on the safety and efficacy of such personalized therapies in other populations, including South Asians. By fostering collaboration between researchers, clinicians, patient advocacy groups, government and industry in gene therapy initiatives for the inherited-diseases community in the developing world would link the Global North and Global South and breathe life into the motto “Together we can make a difference”.

Contribution of Glial Cells to Polyglutamine Diseases: Observations from Patients and Mouse Models.

Heat shock proteins are up-regulated as a physiological response to stressful stimuli and generally function as molecular chaperones for improperly folded protein substrates. The small heat shock protein HSP27 (or HSPB1) has multiple cytoplasmic roles. HSP27 also can translocate to the nucleus in response to stress, but the functional significance of this nuclear distribution has not been elucidated. We have previously implicated HSP27 as a genetic modifier of spinocerebellar ataxia 17 (SCA17), a neurological disease caused by a polyglutamine expansion in the TATA-binding protein (TBP). Altered expression of HSP27 is also found in cell models of other polyglutamine diseases, including Huntington disease as well as SCA3 and SCA7. Here, we show that Hsp27, unlike Hsp70, is not detected in mutant TBP aggregates in primary cerebellar granule neurons from transgenic SCA17 mice. Although HSP27 overexpression does not reduce the aggregation of cotransfected mutant TBP containing 105 glutamines, it potentiates activated transcription from both TATA-containing and TATA-lacking promoters. Neither HSP40 nor HSP70 elicits the same transcriptional effect. Moreover, HSP27 interacts with the transcription factor SP1, and coexpression of SP1 and nuclear localization signal-tagged HSP27 synergistically activates reporter constructs for the SP1-responsive neurotrophic receptor genes Ngfr(p75) and TRKA. Overexpression of nuclear localization signal-tagged HSP27 also rescues mutant TBP-mediated down-regulation of TrkA in a PC12 cell model of SCA17. These results indicate that nuclear HSP27 can modulate SP1-dependent transcriptional activity to promote neuronal protection.

In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS) for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS) represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers “in vitro”. In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases.

BackgroundThere are no current disease-modifying treatments for Huntington’s disease (HD). Further analysis of previously curated HD-associated datasets using updated annotations and integration of multiple datasets could highlight therapeutic targets: however,...

Recent advances in the techniques that differentiate induced pluripotent stem cells (iPSCs) into specific types of cells enabled us to establish in vitro cell-based models as a platform for drug discovery. iPSC-derived disease models are advantageous to generation of a large number of cells required for high-throughput screening. Furthermore, disease-relevant cells differentiated from patient-derived iPSCs are expected to recapitulate the disorder-specific pathogenesis and physiology in vitro. Such disease-relevant cells will be useful for developing effective therapies. We demonstrated that cerebellar tissues are generated from human PSCs (hPSCs) in 3D culture systems that recapitulate the in vivo microenvironments associated with the isthmic organizer. Recently, we have succeeded in generation of spinocerebellar ataxia (SCA) patient-derived Purkinje cells by combining the iPSC technology and the self-organizing stem cell 3D culture technology. We demonstrated that SCA6-derived Purkinje cells exhibit vulnerability to triiodothyronine depletion, which is suppressed by treatment with thyrotropin-releasing hormone and Riluzole. We further discuss applications of patient-specific iPSCs to intractable cerebellar disease.

A Peptide Fusion a Day Keeps the Aggregates Away

Huntington’s disease (HD), with its writhing dancelike movements (chorea) and cardinal loss of neurons in the striatum (1), is the result of an unstable expanded CAG trinucleotide repeat that lengthens a variable glutamine tract in a novel protein called huntingtin (HD) (2). HD shares elements of a common pathogenic mechanism with at least seven other inherited neurodegenerative diseases, including spinobulbar muscular atrophy (SBMA) (3), dentatorubral-pallidoluysian atrophy (DRPLA/Haw River syndrome) (4–6), and several spinocerebellar ataxias (SCA1, SCA2, SCA3/MJD, SCA6 and SCA7) (7–14) (Fig. 1). Expanded glutamine segments in otherwise unrelated proteins cause specific neuronal cell loss in each case, suggesting unique protein context-dependent modulation of some intrinsic toxic property of polyglutamine (15–17). In this view, some feature of huntingtin produces HD pathology by presenting the embedded toxic glutamine tract to cells in a manner that culminates in a graded loss of striatal neurons. One molecular possibility is a glutamine-induced conformational change that alters huntingtin’s association with its normal or abnormal protein partners (18,19).

In 1991, a novel mutational mechanism in human genetics was discovered: the expansion of an unstable trinucleotide repeat (Fu et al., 1991; La Spada et al., 1991). To date, trinucleotide repeat expansions have been found to be associated with 16 neurological disorders. Although the sequence of the unstable repeat and its location within the affected gene varies among these disorders, by far the largest category of disorders are those in which the neurodegenerative disease results from the expansion of a CAG repeat. Because the CAG tract is located in the coding region of each gene and encodes a polyglutamine stretch in each respective protein, these disorders are often designated as polyglutamine diseases (Ross, 1997). The eight polyglutamine repeat diseases currently include Kennedy disease or spinobulbar muscular atrophy (SBMA), Huntington disease (HD), and the spinocerebellar ataxias (SCA1, SCA2, SCA3, Machado-Joseph disease [MJD], SCA6, and SCA7), including dentatorubropallidoluysian atrophy (DRPLA). Except for Kennedy disease (SBMA), these neurodegenerative disorders are dominantly inherited. All eight polyglutamine disorders are progressive, often with an onset in mid-life with an increase in neuronal dysfunction and eventual neuronal loss 10–20 yr after onset. Other features that characterize this group of diseases are (1) an inverse relationship between the number of CAG repeats on expanded alleles and age of onset and severity of disease and (2) an intergenerational instability that leads to repeat expansions and earlier age of onset and more rapid disease progression in affected offspring of affected parents. Most interesting, despite the widespread expression of the relevant protein throughout the brain and other tissues, only a subset of neurons that is unique to each disease appears to be vulnerable to the mutation in each of these diseases. This review focuses on one of these polyglutamine disorders, spinocerebellar ataxia type 1 (SCA1). The reader is referred to other chapters for reviews on some of the other polyglutamine disorders: Huntington disease (Chapters 9–11 and 13), Kennedy disease (Chapter 14), and SCA3/MJD (Chapter 15).