Deep Intronic FGF14 GAA Repeat Expansion in Late-Onset Cerebellar Ataxia.

BACKGROUND: The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis. METHODS: We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines. RESULTS: In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P<0.001) and in the German series (odds ratio, 8.76; 95% CI, 3.45 to 20.84; P<0.001). The repeat expansion was present in 61%, 18%, 15%, and 10% of French Canadian, German, Australian, and Indian index patients, respectively. In total, we identified 128 patients with LOCA who carried an FGF14 (GAA)≥250 expansion. Postmortem cerebellum specimens and iPSC-derived motor neurons from patients showed reduced expression of FGF14 RNA and protein. CONCLUSIONS: A dominantly inherited deep intronic GAA repeat expansion in FGF14 was found to be associated with LOCA. (Funded by Fondation Groupe Monaco and others.).

SORD-deficient rats develop a motor-predominant peripheral neuropathy unveiling novel pathophysiological insights.

Biallelic SORD mutations cause one of the most frequent forms of recessive hereditary neuropathy, estimated to affect ∼10 000 patients in North America and Europe alone. Pathogenic SORD loss-of-function changes in the encoded enzyme sorbitol dehydrogenase result in abnormally high sorbitol levels in cells and serum. How sorbitol accumulation leads to peripheral neuropathy remains to be elucidated. A reproducible animal model for SORD neuropathy is essential to illuminate the pathogenesis of SORD deficiency and for preclinical studies of potential therapies. Therefore, we have generated a Sord knockout (KO), Sord-/-, Sprague Dawley rat, to model the human disease and to investigate the pathophysiology underlying SORD deficiency. We have characterized the phenotype in these rats with a battery of behavioural tests as well as biochemical, physiological and comprehensive histological examinations. Sord-/- rats had remarkably increased levels of sorbitol in serum, CSF and peripheral nerve. Moreover, serum from Sord-/- rats contained significantly increased levels of neurofilament light chain, an established biomarker for axonal degeneration. Motor performance significantly declined in Sord-/- animals starting at ∼7 months of age. Gait analysis evaluated with video motion-tracking confirmed abnormal gait patterns in the hindlimbs. Motor nerve conduction velocities of the tibial nerves were slowed. Light and electron microscopy of the peripheral nervous system revealed degenerating myelinated axons, de- and remyelinated axons, and a likely pathognomonic finding-enlarged 'ballooned' myelin sheaths. These findings mainly affected myelinated motor axons; myelinated sensory axons were largely spared. In summary, Sord-/- rats develop a motor-predominant neuropathy that closely resembles the human phenotype. Our studies revealed novel significant aspects of SORD deficiency, and this model will lead to an improved understanding of the pathophysiology and the therapeutic options for SORD neuropathy.

Customized antisense oligonucleotide-based therapy for neurofilament-associated Charcot-Marie-Tooth disease.

DNA-based therapeutics have emerged as a revolutionary approach for addressing the treatment gap in rare inherited conditions by targeting the fundamental genetic causes of disease. Charcot-Marie-Tooth (CMT) disease, a group of inherited neuropathies, represents one of the most prevalent Mendelian disease groups in neurology and is characterized by diverse genetic etiology. Axonal forms of CMT, known as CMT2, are caused by dominant mutations in over 30 different genes which lead to degeneration of lower motor neuron axons. Recent advances in antisense oligonucleotide (ASO) therapeutics have shown promise in targeting neurodegenerative disorders. Here we elucidate pathomechanistic changes contributing to variant specific molecular phenotypes in CMT2E, caused by a single nucleotide substitution (p.N98S) in the neurofilament light chain gene (NEFL). We used a patient-derived pluripotent stem cell (iPSC)-induced motor neuron model, which recapitulates several cellular and biomarker phenotypes associated with CMT2E. Using an ASO treatment strategy targeting a heterozygous gain-of-function variant, we aimed to resolve molecular phenotypic changes observed in the CMT2E p.N98S subtype. To determine ASO therapeutic potential, we employed our treatment strategy in iPSC-derived motor neurons and used established as well as novel biomarkers of peripheral nervous system axonal degeneration. Our findings have demonstrated a significant decrease in clinically relevant biomarkers of axonal degeneration, presenting the first clinically viable genetic therapeutic for CMT2E. Similar strategies could be used to develop precision medicine approaches for otherwise untreatable gain of function inherited disorders.

Somatic instability of the FGF14-SCA27B GAA•TTC repeat reveals a marked expansion bias in the cerebellum.

Spinocerebellar ataxia 27B (SCA27B) is a common autosomal dominant ataxia caused by an intronic GAA•TTC repeat expansion in FGF14. Neuropathological studies have shown that neuronal loss is largely restricted to the cerebellum. Although the repeat locus is highly unstable during intergenerational transmission, it remains unknown whether it exhibits cerebral mosaicism and progressive instability throughout life. We conducted an analysis of the FGF14 GAA•TTC repeat somatic instability across 156 serial blood samples from 69 individuals, fibroblasts, induced pluripotent stem cells, and post-mortem brain tissues from six controls and six patients with SCA27B, alongside methylation profiling using targeted long-read sequencing. Peripheral tissues exhibited minimal somatic instability, which did not significantly change over periods of more than 20 years. In post-mortem brains, the GAA•TTC repeat was remarkably stable across all regions, except in the cerebellar hemispheres and vermis. The levels of somatic expansion in the cerebellar hemispheres and vermis were, on average, 3.15 and 2.72 times greater relative to other examined brain regions, respectively. Additionally, levels of somatic expansion in the brain increased with repeat length and tissue expression of FGF14. We found no significant difference in methylation of wild-type and expanded FGF14 alleles in post-mortem cerebellar hemispheres between patients and controls. In conclusion, our study revealed that the FGF14 GAA•TTC repeat exhibits a cerebellar-specific expansion bias, which may explain the pure cerebellar involvement in SCA27B.

Mutant Huntingtin Is Cleared from the Brain via Active Mechanisms in Huntington Disease.

Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Therapeutics that lower HTT have shown preclinical promise and are being evaluated in clinical trials. However, clinical assessment of brain HTT lowering presents challenges. We have reported that mutant HTT (mHTT) in the CSF of HD patients correlates with clinical measures, including disease burden as well as motor and cognitive performance. We have also shown that lowering HTT in the brains of HD mice results in correlative reduction of mHTT in the CSF, prompting the use of this measure as an exploratory marker of target engagement in clinical trials. In this study, we investigate the mechanisms of mHTT clearance from the brain in adult mice of both sexes to elucidate the significance of therapy-induced CSF mHTT changes. We demonstrate that, although neurodegeneration increases CSF mHTT concentrations, mHTT is also present in the CSF of mice in the absence of neurodegeneration. Importantly, we show that secretion of mHTT from cells in the CNS followed by glymphatic clearance from the extracellular space contributes to mHTT in the CSF. Furthermore, we observe secretion of wild type HTT from healthy control neurons, suggesting that HTT secretion is a normal process occurring in the absence of pathogenesis. Overall, our data support both passive release and active clearance of mHTT into CSF, suggesting that its treatment-induced changes may represent a combination of target engagement and preservation of neurons.SIGNIFICANCE STATEMENT: Changes in CSF mutant huntingtin (mHTT) are being used as an exploratory endpoint in HTT lowering clinical trials for the treatment of Huntington disease (HD). Recently, it was demonstrated that intrathecal administration of a HTT lowering agent leads to dose-dependent reduction of CSF mHTT in HD patients. However, little is known about how HTT, an intracellular protein, reaches the extracellular space and ultimately the CSF. Our findings that HTT enters CSF by both passive release and active secretion followed by glymphatic clearance may have significant implications for interpretation of treatment-induced changes of CSF mHTT in clinical trials for HD.

Generation of 3 patient induced Pluripotent stem cell lines containing SORD mutations linked to a recessive neuropathy.

The SORD neuropathy has been identified as the most common autosomal recessive inherited neuropathy, occurring in thousands of patients worldwide. Fibroblast lines from 3 different patients containing the c.753delG; p.Ala253GlnfsTer27 SORD mutations were reprogrammed into induced Pluripotent Stem Cell (iPSC) lines. These iPSC lines demonstrate an apparent normal karyotype and have positive expression of pluripotency markers. These iPSC lines also stain positively for Ectoderm, Endoderm and Mesoderm markers following Embryoid body differentiation. These lines pose to serve as a valuable disease modeling resource for studying the SORD neuropathy, including studying disease phenotype and treatment efficacy.

Somatic instability of the FGF14 -SCA27B GAA•TTC repeat reveals a marked expansion bias in the cerebellum.

Spinocerebellar ataxia 27B (SCA27B) is a common autosomal dominant ataxia caused by an intronic GAA•TTC repeat expansion in FGF14 . Neuropathological studies have shown that neuronal loss is largely restricted to the cerebellum. Although the repeat locus is highly unstable during intergenerational transmission, it remains unknown whether it exhibits cerebral mosaicism and progressive instability throughout life. We conducted an analysis of the FGF14 GAA•TTC repeat somatic instability across 156 serial blood samples from 69 individuals, fibroblasts, induced pluripotent stem cells, and post-mortem brain tissues from six controls and six patients with SCA27B, alongside methylation profiling using targeted long-read sequencing. Peripheral tissues exhibited minimal somatic instability, which did not significantly change over periods of more than 20 years. In post-mortem brains, the GAA•TTC repeat was remarkably stable across all regions, except in the cerebellar hemispheres and vermis. The levels of somatic expansion in the cerebellar hemispheres and vermis were, on average, 3.15 and 2.72 times greater relative to other examined brain regions, respectively. Additionally, levels of somatic expansion in the brain increased with repeat length and tissue expression of FGF14 . We found no significant difference in methylation of wild-type and expanded FGF14 alleles in post-mortem cerebellar hemispheres between patients and controls. In conclusion, our study revealed that the FGF14 GAA•TTC repeat exhibits a cerebellar-specific expansion bias, which may explain the pure and late-onset cerebellar involvement in SCA27B.

Neuron-Schwann cell interactions in peripheral nervous system homeostasis, disease, and preclinical treatment.

Schwann cells (SCs) have a critical role in the peripheral nervous system. These cells are able to support axons during homeostasis and after injury. However, mutations in genes associated with the SCs repair program or myelination result in dysfunctional SCs. Several neuropathies such as Charcot-Marie-Tooth (CMT) disease, diabetic neuropathy and Guillain-Barré syndrome show abnormal SC functions and an impaired regeneration process. Thus, understanding SCs-axon interaction and the nerve environment in the context of homeostasis as well as post-injury and disease onset is necessary. Several neurotrophic factors, cytokines, and regulators of signaling pathways associated with proliferation, survival and regeneration are involved in this process. Preclinical studies have focused on the discovery of therapeutic targets for peripheral neuropathies and injuries. To study the effect of new therapeutic targets, modeling neuropathies and peripheral nerve injuries (PNIs) in vitro and in vivo are useful tools. Furthermore, several in vitro protocols have been designed using SCs and neuron cell lines to evaluate these targets in the regeneration process. SCs lines have been used to generate effective myelinating SCs without success. Alternative options have been investigated using direct conversion from somatic cells to SCs or SCs derived from pluripotent stem cells to generate functional SCs. This review will go over the advantages of these systems and the problems associated with them. In addition, there have been challenges in establishing adequate and reproducible protocols in vitro to recapitulate repair SC-neuron interactions observed in vivo. So, we also discuss the mechanisms of repair SCs-axon interactions in the context of peripheral neuropathies and nerve injury (PNI) in vitro and in vivo. Finally, we summarize current preclinical studies evaluating transgenes, drug, and novel compounds with translational potential into clinical studies.

Sorbitol reduction via govorestat ameliorates synaptic dysfunction and neurodegeneration in sorbitol dehydrogenase deficiency.

Sorbitol dehydrogenase (SORD) deficiency has been identified as the most frequent autosomal recessive form of hereditary neuropathy. Loss of SORD causes high sorbitol levels in tissues due to the inability to convert sorbitol to fructose in the 2-step polyol pathway, leading to degenerative neuropathy. The underlying mechanisms of sorbitol-induced degeneration have not been fully elucidated, and no current FDA-approved therapeutic options are available to reduce sorbitol levels in the nervous system. Here, in a Drosophila model of SORD deficiency, we showed synaptic degeneration in the brain, neurotransmission defect, locomotor impairment, and structural abnormalities in the neuromuscular junctions. In addition, we found reduced ATP production in the brain and ROS accumulation in the CNS and muscle, indicating mitochondrial dysfunction. Applied Therapeutics has developed a CNS-penetrant next-generation aldose reductase inhibitor (ARI), AT-007 (govorestat), which inhibits the conversion of glucose to sorbitol. AT-007 significantly reduced sorbitol levels in patient-derived fibroblasts, induced pluripotent stem cell-derived (iPSC-derived) motor neurons, and Drosophila brains. AT-007 feeding in Sord-deficient Drosophila mitigated synaptic degeneration and significantly improved synaptic transduction, locomotor activity, and mitochondrial function. Moreover, AT-007 treatment significantly reduced ROS accumulation in Drosophila CNS, muscle, and patient-derived fibroblasts. These findings uncover the molecular and cellular pathophysiology of SORD neuropathy and provide a potential treatment strategy for patients with SORD deficiency.

Sord deficient rats develop a motor-predominant peripheral neuropathy unveiling novel pathophysiological insights.

Biallelic SORD mutations cause one of the most frequent forms of recessive hereditary neuropathy, estimated to affect approximately 10,000 patients in North America and Europe alone. Pathogenic SORD loss-of-function changes in the encoded enzyme sorbitol dehydrogenase result in abnormally high sorbitol levels in cells and serum. How sorbitol accumulation leads to peripheral neuropathy remains to be elucidated. A reproducible animal model for SORD neuropathy is essential to illuminate the pathogenesis of SORD deficiency and for preclinical studies of potential therapies. Therefore, we have generated a Sord knockout (KO), Sord -/- , Sprague Dawley rat, to model the human disease and to investigate the pathophysiology underlying SORD deficiency. We have characterized the phenotype in these rats with a battery of behavioral tests as well as biochemical, physiological, and comprehensive histological examinations. Sord -/- rats had remarkably increased levels of sorbitol in serum, cerebral spinal fluid (CSF), and peripheral nerve. Moreover, serum from Sord -/- rats contained significantly increased levels of neurofilament light chain, NfL, an established biomarker for axonal degeneration. Motor performance significantly declined in Sord -/- animals starting at ∼7 months of age. Gait analysis evaluated with video motion tracking confirmed abnormal gait patterns in the hindlimbs. Motor nerve conduction velocities of the tibial nerves were slowed. Light and electron microscopy of the peripheral nervous system revealed degenerating myelinated axons, de- and remyelinated axons, and a likely pathognomonic finding - enlarged "ballooned" myelin sheaths. These findings mainly affected myelinated motor axons; myelinated sensory axons were largely spared. In summary, Sord -/- rats develop a motor-predominant neuropathy that closely resembles the human phenotype. Our studies revealed novel significant aspects of SORD deficiency, and this model will lead to an improved understanding of the pathophysiology and the therapeutic options for SORD neuropathy.

Extracellular vesicle-mediated amyloid transfer to neural progenitor cells: implications for RAGE and HIV infection.

Amyloid beta (Aß) deposition was demonstrated to be elevated in the brains of HIV-infected patients and associated with neurocognitive decline; however, the mechanisms of these processes are poorly understood. The goal of the current study was to address the hypothesis that Aß can be transferred via extracellular vesicles (ECVs) from brain endothelial cells to neural progenitor cells (NPCs) and that this process can contribute to abnormal NPC differentiation. Mechanistically, we focused on the role of the receptor for advanced glycation end products (RAGE) and activation of the inflammasome in these events. ECVs loaded with Aß (Aß-ECVs) were readily taken up by NPCs and Aß partly colocalized with the inflammasome markers ASC and NLRP3 in the nuclei of the recipient NPCs. This colocalization was affected by HIV and RAGE inhibition by a high-affinity specific inhibitor FPS-ZM1. Blocking RAGE resulted also in an increase in ECV number produced by brain endothelial cells, decreased Aß content in ECVs, and diminished Aß-ECVs transfer to NPC nuclei. Interestingly, both Aß-ECVs and RAGE inhibition altered NPC differentiation. Overall, these data indicate that RAGE inhibition affects brain endothelial ECV release and Aß-ECVs transfer to NPCs. These events may modulate ECV-mediated amyloid pathology in the HIV-infected brain and contribute to the development of HIV-associated neurocognitive disorders.