Biotin Homeostasis and Human Disorders: Recent Findings and Perspectives.

Biotin (vitamin B7, or vitamin H) is a water-soluble B-vitamin that functions as a cofactor for carboxylases, i.e., enzymes involved in the cellular metabolism of fatty acids and amino acids and in gluconeogenesis; moreover, as reported, biotin may be involved in gene regulation. Biotin is not synthesized by human cells, but it is found in food and is also produced by intestinal bacteria. Biotin status/homeostasis in human individuals depends on several factors, including efficiency/deficiency of the enzymes involved in biotin recycling within the human organism (biotinidase, holocarboxylase synthetase), and/or effectiveness of intestinal uptake, which is mainly accomplished through the sodium-dependent multivitamin transporter. In the last years, administration of biotin at high/"pharmacological" doses has been proposed to treat specific defects/deficiencies and human disorders, exhibiting mainly neurological and/or dermatological symptoms and including biotinidase deficiency, holocarboxylase synthetase deficiency, and biotin-thiamine-responsive basal ganglia disease. On the other hand, according to warnings of the Food and Drug Administration, USA, high biotin levels can affect clinical biotin-(strept)avidin assays and thus lead to false results during quantification of critical biomarkers. In this review article, recent findings/advancements that may offer new insight in the abovementioned research fields concerning biotin will be presented and briefly discussed.

4-Hydroxybenzoic acid rescues multisystemic disease and perinatal lethality in a mouse model of mitochondrial disease.

Coenzyme Q (CoQ) deficiency syndrome is conventionally treated with limited efficacy using exogenous CoQ10. Poor outcomes result from low absorption and bioavailability of CoQ10 and the clinical heterogenicity of the disease. Here, we demonstrate that supplementation with 4-hydroxybenzoic acid (4HB), the precursor of the benzoquinone ring in the CoQ biosynthetic pathway, completely rescues multisystemic disease and perinatal lethality in a mouse model of CoQ deficiency. 4HB stimulates endogenous CoQ biosynthesis in tissues of Coq2 mutant mice, normalizing mitochondrial function and rescuing cardiac insufficiency, edema, and neurodevelopmental delay. In contrast, exogenous CoQ10 supplementation falls short in fully restoring the phenotype. The treatment is translatable to human use, as proven by in vitro studies in skin fibroblasts from patients with pathogenic variants in COQ2. The therapeutic approach extends to other disorders characterized by deficiencies in the production of 4HB and early steps of CoQ biosynthesis and instances of secondary CoQ deficiency.

Mitochondrial dysfunction in brain tissues and Extracellular Vesicles Fragile X-associated tremor/ataxia syndrome.

OBJECTIVE: Mitochondrial impairments have been implicated in the pathogenesis of Fragile X-associated tremor/ataxia syndrome (FXTAS) based on analysis of mitochondria in peripheral tissues and cultured cells. We sought to assess whether mitochondrial abnormalities present in postmortem brain tissues of patients with FXTAS are also present in plasma neuron-derived extracellular vesicles (NDEVs) from living carriers of fragile X messenger ribonucleoprotein1 (FMR1) gene premutations at an early asymptomatic stage of the disease continuum. METHODS: We utilized postmortem frozen cerebellar and frontal cortex samples from a cohort of eight patients with FXTAS and nine controls and measured the quantity and activity of the mitochondrial proteins complex IV and complex V. In addition, we evaluated the same measures in isolated plasma NDEVs by selective immunoaffinity capture targeting L1CAM from a separate cohort of eight FMR1 premutation carriers and four age-matched controls. RESULTS: Lower complex IV and V quantity and activity were observed in the cerebellum of FXTAS patients compared to controls, without any differences in total mitochondrial content. No patient-control differences were observed in the frontal cortex. In NDEVs, FMR1 premutation carriers compared to controls had lower activity of Complex IV and Complex V, but higher Complex V quantity. INTERPRETATION: Quantitative and functional abnormalities in mitochondrial electron transport chain complexes IV and V seen in the cerebellum of patients with FXTAS are also manifest in plasma NDEVs of FMR1 premutation carriers. Plasma NDEVs may provide further insights into mitochondrial pathologies in this syndrome and could potentially lead to the development of biomarkers for predicting symptomatic FXTAS among premutation carriers and disease monitoring.

Role of fragile X messenger ribonucleoprotein 1 in the pathophysiology of brain disorders: a glia perspective.

Fragile X messenger ribonucleoprotein 1 (FMRP) is a widely expressed RNA binding protein involved in several steps of mRNA metabolism. Mutations in the FMR1 gene encoding FMRP are responsible for fragile X syndrome (FXS), a leading genetic cause of intellectual disability and autism spectrum disorder, and fragile X-associated tremor-ataxia syndrome (FXTAS), a neurodegenerative disorder in aging men. Although FMRP is mainly expressed in neurons, it is also present in glial cells and its deficiency or altered expression can affect functions of glial cells with implications for the pathophysiology of brain disorders. The present review focuses on recent advances on the role of glial subtypes, astrocytes, oligodendrocytes and microglia, in the pathophysiology of FXS and FXTAS, and describes how the absence or reduced expression of FMRP in these cells can impact on glial and neuronal functions. We will also briefly address the role of FMRP in radial glial cells and its effects on neural development, and gliomas and will speculate on the role of glial FMRP in other brain disorders.

Genetic and sporadic forms of tauopathies-TAU as a disease driver for the majority of patients but the minority of tauopathies.

Ageing-associated tauopathies like frontotemporal dementia (FTD), variants thereof (like progressive supranuclear palsy (PSP), pick diseases (PiD), corticobasal degeneration (CBD)), and of course the most prevalent form of dementia, Alzheimer Disease (AD), are widely recognized forms of tauopathies. The list of tauopathies is expanding. We now include: (i) tauopathies where the disease cause or trigger is clearly either physical, such as in Traumatic Brain Injury (TBI) or Chronic Traumatic Encephalopathy (CTE), and (ii) genetic diseases that result in tauopathy but have pathogenic genetic variants in genes not related to TAU. Examples of the latter are myotonic dystrophy Type 1 and Type 2 (DM1, DM2, due to pathogenic genetic variants in the genes DMPK and CNBP, respectively), Niemann-Pick Disease Type C (NPD, due to mutations in NPC1 or NPC2), Kufs Disease (CLN6), Christianson Syndrome (SLC9A6), familial forms of Parkinson Disease (PD), and many others. In terms of affected brain regions and cell types, intracellular distribution of TAU pathology/aggregates, age of disease onset, velocity of disease progression and spreading of TAU pathology, there is, however, little in common in most of these disease entities. Here, I reason that TAU/MAPT is causative for the minority of tauopathies (e.g., MAPT-related FTD/PSP and Vacuolar Tauopathy (VCP)) and a critical mediator for others, like shown by overwhelming evidence for AD. However, TAU may also be a mere bystander or even protective in other settings. Improved understanding of rare tauopathies is necessary to develop specific treatments, but also to improve our understanding of the pathomechanistic role of TAU and to identify diseases that may profit from TAU-based therapies.

Hem25p is required for mitochondrial IPP transport in fungi.

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor composed of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing and targeted uptake assays, we reveal that Hem25p-a mitochondrial glycine transporter required for haem biosynthesis-doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p failed to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enabled robust IPP uptake and incorporation into the CoQ biosynthetic pathway. HEM25 orthologues from diverse fungi, but not from metazoans, were able to rescue hem25∆ CoQ deficiency. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in fungi.

A mitochondrial-targeted antioxidant (MitoQ) improves motor coordination and reduces Purkinje cell death in a mouse model of ARSACS.

Mitochondrial deficits have been observed in animal models of Autosomal-recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) and in patient-derived fibroblasts. We investigated whether mitochondrial function could be restored in Sacs-/- mice, a mouse model of ARSACS, using the mitochondrial-targeted antioxidant ubiquinone MitoQ. After 10weeks of chronic MitoQ administration in drinking water, we partially reversed motor coordination deficits in Sacs-/- mice but did not affect litter-matched wild-type control mice. MitoQ administration led to a restoration of superoxide dismutase 2 (SOD2) in cerebellar Purkinje cell somata without altering Purkinje cell firing deficits. Purkinje cells in anterior vermis of Sacs-/- mice normally undergo cell death in ARSACS; however, Purkinje cells numbers were elevated after chronic MitoQ treatment. Furthermore, Purkinje cell innervation of target neurons in the cerebellar nuclei of Sacs-/- mice was also partially restored with MitoQ treatment. Our data suggest that MitoQ is a potential therapeutic treatment for ARSACS and that it improves motor coordination via increasing cerebellar Purkinje cell mitochondria function and reducing Purkinje cell death.

Retinoic acid supplementation ameliorates motor incoordination via RARα-CBLN2 in the cerebellum of a prenatal valproic acid-exposed rat autism model.

In addition to their core symptoms, most individuals with autism spectrum disorder (ASD) also experience motor impairments. These impairments are often linked to the cerebellum, which is the focus of the current study. Herein, we utilized a prenatal valproic acid (VPA)-induced rat model of autism and performed RNA sequencing in the cerebellum. Relative to control animals, the VPA-treated offspring demonstrated both abnormal motor coordination and impaired dendritic arborization of Purkinje cells (PCs). Concurrently, we observed a decrease in the cerebellar expression of retinoic acid (RA) synthesis enzymes (RDH10, ALDH1A1), metabolic enzyme (CYP26A2), and lower levels of RA, retinoic acid receptor α (RARα), and Cerebellin2 (CBLN2) in the VPA-treated offspring. However, RA supplementation ameliorated these deficits, restoring motor coordination, normalizing PCs dendritic arborization, and increasing the expression of RA, RARα, and CBLN2. Further, ChIP assays confirmed that RA supplementation enhanced RARα's binding capacity to CBLN2 promoters. Collectively, these findings highlight the therapeutic potential of RA for treating motor incoordination in VPA-induced autism, acting through the RARα-CBLN2 pathway.

Mitochondrial dysfunction and calcium dysregulation in COQ8A-ataxia Purkinje neurons are rescued by CoQ10 treatment.

COQ8A-ataxia is a rare form of neurodegenerative disorder due to mutations in the COQ8A gene. The encoded mitochondrial protein is involved in the regulation of coenzyme Q10 biosynthesis. Previous studies on the constitutive Coq8a-/- mice indicated specific alterations of cerebellar Purkinje neurons involving altered electrophysiological function and dark cell degeneration. In the present manuscript, we extend our understanding of the contribution of Purkinje neuron dysfunction to the pathology. By generating a Purkinje-specific conditional COQ8A knockout, we demonstrate that loss of COQ8A in Purkinje neurons is the main cause of cerebellar ataxia. Furthermore, through in vivo and in vitro approaches, we show that COQ8A-depleted Purkinje neurons have abnormal dendritic arborizations, altered mitochondria function and intracellular calcium dysregulation. Furthermore, we demonstrate that oxidative phosphorylation, in particular Complex IV, is primarily altered at presymptomatic stages of the disease. Finally, the morphology of primary Purkinje neurons as well as the mitochondrial dysfunction and calcium dysregulation could be rescued by CoQ10 treatment, suggesting that CoQ10 could be a beneficial treatment for COQ8A-ataxia.

Between Order and Chaos: Understanding the Mechanism and Pathology of RAN Translation.

Repeat-associated non-AUG (RAN) translation is a pathogenic mechanism in which repetitive sequences are translated into aggregation-prone proteins from multiple reading frames, even without a canonical AUG start codon. Since its discovery in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1), RAN translation is now known to occur in the context of 12 disease-linked repeat expansions. This review discusses recent advances in understanding the regulatory mechanisms controlling RAN translation and its contribution to the pathophysiology of repeat expansion diseases. We discuss the key findings in the context of Fragile X Tremor Ataxia Syndrome (FXTAS), a neurodegenerative disorder caused by a CGG repeat expansion in the 5' untranslated region of FMR1.

[The neuropathological mechanism on guanine-rich repeat expansion diseases].

Repeat expansion diseases are caused by the aberrant repeat expansions within specific genes. RNAs derived from aberrant repeat sequences form non-canonical secondary structures, contributing to induce cell toxicity. In particular, RNA G-quadruplexes (G4RNAs) formed in guanine-rich repeat expanded RNAs trigger neurodegeneration. We have previously shown that the expanded CGG repeat-derived G4RNAs initiate aggregation of FMRpolyG, a neuropathogenic protein generated by repeat-associated non-AUG (RAN) translation in Fragile X-associated tremor/ataxia syndrome (FXTAS). In this review, we describe the neuropathological mechanism attributed to G4RNAs in guanine-rich repeat expansion diseases, including FXTAS.

Coenzyme Q biochemistry and biosynthesis.

Coenzyme Q (CoQ) is a remarkably hydrophobic, redox-active lipid that empowers diverse cellular processes. Although most known for shuttling electrons between mitochondrial electron transport chain (ETC) complexes, the roles for CoQ are far more wide-reaching and ever-expanding. CoQ serves as a conduit for electrons from myriad pathways to enter the ETC, acts as a cofactor for biosynthetic and catabolic reactions, detoxifies damaging lipid species, and engages in cellular signaling and oxygen sensing. Many open questions remain regarding the biosynthesis, transport, and metabolism of CoQ, which hinders our ability to treat human CoQ deficiency. Here, we recount progress in filling these knowledge gaps, highlight unanswered questions, and underscore the need for novel tools to enable discoveries and improve the treatment of CoQ-related diseases.

The humanised CYP2C19 transgenic mouse exhibits cerebellar atrophy and movement impairment reminiscent of ataxia.

AIMS: CYP2C19 transgenic mouse expresses the human CYP2C19 gene in the liver and developing brain, and it exhibits altered neurodevelopment associated with impairments in emotionality and locomotion. Because the validation of new animal models is essential for the understanding of the aetiology and pathophysiology of movement disorders, the objective was to characterise motoric phenotype in CYP2C19 transgenic mice and to investigate its validity as a new animal model of ataxia. METHODS: The rotarod, paw-print and beam-walking tests were utilised to characterise the motoric phenotype. The volumes of 20 brain regions in CYP2C19 transgenic and wild-type mice were quantified by 9.4T gadolinium-enhanced post-mortem structural neuroimaging. Antioxidative enzymatic activity was quantified biochemically. Dopaminergic alterations were characterised by chromatographic quantification of concentrations of dopamine and its metabolites and by subsequent immunohistochemical analyses. The beam-walking test was repeated after the treatment with dopamine receptor antagonists ecopipam and raclopride. RESULTS: CYP2C19 transgenic mice exhibit abnormal, unilateral ataxia-like gait, clasping reflex and 5.6-fold more paw-slips in the beam-walking test; the motoric phenotype was more pronounced in youth. Transgenic mice exhibited a profound reduction of 12% in cerebellar volume and a moderate reduction of 4% in hippocampal volume; both regions exhibited an increased antioxidative enzyme activity. CYP2C19 mice were hyperdopaminergic; however, the motoric impairment was not ameliorated by dopamine receptor antagonists, and there was no alteration in the number of midbrain dopaminergic neurons in CYP2C19 mice. CONCLUSIONS: Humanised CYP2C19 transgenic mice exhibit altered gait and functional motoric impairments; this phenotype is likely caused by an aberrant cerebellar development.

Melittin ameliorates motor function and prevents autophagy-induced cell death and astrogliosis in rat models of cerebellar ataxia induced by 3-acetylpyridine.

BACKGROUND: Cerebellar ataxia (CA) is a form of ataxia that adversely affects the cerebellum. This study aims to investigate the therapeutic effects of melittin (MEL) on a 3-acetylpyridine-induced (3-AP) cerebellar ataxia (CA) rat model. METHODS: Initially, CA rat models were generated by 3-AP administration followed by the subcutaneous injection of MEL. The open-field test was used for the evaluation of locomotion and anxiety. Immunohistochemistry was also conducted for the autophagy markers of LC3 and Beclin1. In the next step, the morphology of the astrocyte, the cell responsible for maintaining homeostasis in the CNS, was evaluated by the Sholl analysis. RESULTS: The findings suggested that the administration of MEL in a 3-AP model of ataxia improved locomotion and anxiety (P < 0.001), decreased the expression of LC3 (P < 0.01) and Beclin1 (P < 0.05), increased astrocyte complexity (P < 0.05) and reduced astrocyte cell soma size (P < 0.001). CONCLUSIONS: Overall, the findings imply that the MEL attenuates the 3-AP-induced autophagy, causes cell death and improves motor function. As such, it could be used as a therapeutic procedure for CA due to its neuroprotective effects.

The ataxia-linked E1081Q mutation affects the sub-plasma membrane Ca2+-microdomains by tuning PMCA3 activity.

Calcium concentration must be finely tuned in all eukaryotic cells to ensure the correct performance of its signalling function. Neuronal activity is exquisitely dependent on the control of Ca2+ homeostasis: its alterations ultimately play a pivotal role in the origin and progression of many neurodegenerative processes. A complex toolkit of Ca2+ pumps and exchangers maintains the fluctuation of cytosolic Ca2+ concentration within the appropriate threshold. Two ubiquitous (isoforms 1 and 4) and two neuronally enriched (isoforms 2 and 3) of the plasma membrane Ca2+ATPase (PMCA pump) selectively regulate cytosolic Ca2+ transients by shaping the sub-plasma membrane (PM) microdomains. In humans, genetic mutations in ATP2B1, ATP2B2 and ATP2B3 gene have been linked with hearing loss, cerebellar ataxia and global neurodevelopmental delay: all of them were found to impair pump activity. Here we report three additional mutations in ATP2B3 gene corresponding to E1081Q, R1133Q and R696H amino acids substitution, respectively. Among them, the novel missense mutation (E1081Q) immediately upstream the C-terminal calmodulin-binding domain (CaM-BD) of the PMCA3 protein was present in two patients originating from two distinct families. Our biochemical and molecular studies on PMCA3 E1081Q mutant have revealed a splicing variant-dependent effect of the mutation in shaping the sub-PM [Ca2+]. The E1081Q substitution in the full-length b variant abolished the capacity of the pump to reduce [Ca2+] in the sub-PM microdomain (in line with the previously described ataxia-related PMCA mutations negatively affecting Ca2+ pumping activity), while, surprisingly, its introduction in the truncated a variant selectively increased Ca2+ extrusion activity in the sub-PM Ca2+ microdomains. These results highlight the importance to set a precise threshold of [Ca2+] by fine-tuning the sub-PM microdomains and the different contribution of the PMCA splice variants in this regulation.

LINE-1 activation in the cerebellum drives ataxia.

Dysregulation of long interspersed nuclear element 1 (LINE-1, L1), a dominant class of transposable elements in the human genome, has been linked to neurodegenerative diseases, but whether elevated L1 expression is sufficient to cause neurodegeneration has not been directly tested. Here, we show that the cerebellar expression of L1 is significantly elevated in ataxia telangiectasia patients and strongly anti-correlated with the expression of epigenetic silencers. To examine the role of L1 in the disease etiology, we developed an approach for direct targeting of the L1 promoter for overexpression in mice. We demonstrated that L1 activation in the cerebellum led to Purkinje cell dysfunctions and degeneration and was sufficient to cause ataxia. Treatment with a nucleoside reverse transcriptase inhibitor blunted ataxia progression by reducing DNA damage, attenuating gliosis, and reversing deficits of molecular regulators for calcium homeostasis in Purkinje cells. Our study provides the first direct evidence that L1 activation can drive neurodegeneration.

The neglected role of endocannabinoid actions at TRPC channels in ataxia.

Transient receptor potential (TRP) channels are highly expressed in cells of the cerebellum including in the dendrites and somas of Purkinje cells (PCs). Their endogenous activation promotes influx of Ca2+ and Na+, resulting in depolarization. TRP channels can be activated by endogenous endocannabinoids (eCBs) and activity of TRP channels has been shown to modulate GABA and glutamate transmission. Ataxia is caused by disruption of multiple intracellular pathways which often involve changes in Ca2+ homeostasis that can result in neural cellular dysfunction and cell death. Based on available literature, alteration of transmission of eCBs would be expected to change activity of cerebellar TRP channels. Antagonists of the endocannabinoid system (ECS) including enzymes which break eCBs down have been shown to result in reductions in postsynaptic excitatory activity mediated by TRPC channels. Further, TRPC channel antagonists could modulate both pre and postsynaptically-mediated glutamatergic and GABAergic transmission, resulting in reductions in cell death due to excitotoxicity and dysfunctions caused by abnormal inhibitory signaling. Accordingly, TRP channels, and in particular the TRPC channel, represent a potential therapeutic target for management of ataxia.

Extracellular Vesicles From Hyperammonemic Rats Induce Neuroinflammation in Cerebellum of Normal Rats: Role of Increased TNFα Content.

Hyperammonemia plays a main role in the neurological impairment in cirrhotic patients with hepatic encephalopathy. Rats with chronic hyperammonemia reproduce the motor incoordination of patients with minimal hepatic encephalopathy, which is due to enhanced GABAergic neurotransmission in cerebellum as a consequence of neuroinflammation. Extracellular vesicles (EVs) could play a key role in the transmission of peripheral alterations to the brain to induce neuroinflammation and neurological impairment in hyperammonemia and hepatic encephalopathy. EVs from plasma of hyperammonemic rats (HA-EVs) injected to normal rats induce neuroinflammation and motor incoordination, but the underlying mechanisms remain unclear. The aim of this work was to advance in the understanding of these mechanisms. To do this we used an ex vivo system. Cerebellar slices from normal rats were treated ex vivo with HA-EVs. The aims were: 1) assess if HA-EVs induce microglia and astrocytes activation and neuroinflammation in cerebellar slices of normal rats, 2) assess if this is associated with activation of the TNFR1-NF-kB-glutaminase-GAT3 pathway, 3) assess if the TNFR1-CCL2-BDNF-TrkB pathway is activated by HA-EVs and 4) assess if the increased TNFα levels in HA-EVs are responsible for the above effects and if they are prevented by blocking the action of TNFα. Our results show that ex vivo treatment of cerebellar slices from control rats with extracellular vesicles from hyperammonemic rats induce glial activation, neuroinflammation and enhance GABAergic neurotransmission, reproducing the effects induced by hyperammonemia in vivo. Moreover, we identify in detail key underlying mechanisms. HA-EVs induce the activation of both the TNFR1-CCL2-BDNF-TrkB-KCC2 pathway and the TNFR1-NF-kB-glutaminase-GAT3 pathway. Activation of these pathways enhances GABAergic neurotransmission in cerebellum, which is responsible for the induction of motor incoordination by HA-EVs. The data also show that the increased levels of TNFα in HA-EVs are responsible for the above effects and that the activation of both pathways is prevented by blocking the action of TNFα. This opens new therapeutic options to improve motor incoordination in hyperammonemia and also in cirrhotic patients with hepatic encephalopathy and likely in other pathologies in which altered cargo of extracellular vesicles contribute to the propagation of the pathology.

A step forward for stress-induced ataxia.

Patients with episodic ataxia type 2 (EA2) display attacks of severe incoordination and dystonia that can be triggered by stress. In a recent study, Snell, Vitenzon, Tara, and colleagues found a mechanistic pathway by which norepinephrine (NE) alters cerebellar Purkinje output to trigger attacks in a mouse model of EA2 and identified a pharmacological intervention that effectively reduces them.