IMPDH2 filaments protect from neurodegeneration in AMPD2 deficiency.

Metabolic dysregulation is one of the most common causes of pediatric neurodegenerative disorders. However, how the disruption of ubiquitous and essential metabolic pathways predominantly affect neural tissue remains unclear. Here we use mouse models of a childhood neurodegenerative disorder caused by AMPD2 deficiency to study cellular and molecular mechanisms that lead to selective neuronal vulnerability to purine metabolism imbalance. We show that mouse models of AMPD2 deficiency exhibit predominant degeneration of the hippocampal dentate gyrus, despite a general reduction of brain GTP levels. Neurodegeneration-resistant regions accumulate micron-sized filaments of IMPDH2, the rate limiting enzyme in GTP synthesis, while these filaments are barely detectable in the hippocampal dentate gyrus. Furthermore, we show that IMPDH2 filament disassembly reduces GTP levels and impairs growth of neural progenitor cells derived from individuals with human AMPD2 deficiency. Together, our findings suggest that IMPDH2 polymerization prevents detrimental GTP deprivation, opening the possibility of exploring the induction of IMPDH2 assembly as a therapy for neurodegeneration.

Glutamate-dependent translational control through ribosomal protein S6 phosphorylation in cultured bergmann glial cells.

Glutamate (Glu) the main excitatory neurotransmitter of the central nervous system regulates gene expression at different levels through the activation of specific membrane receptors and transporters expressed in neurons and glia cells. A membrane to nucleus signaling cascade triggered by this neurotransmitter has been described in cultured cerebellar Bergmann glia cells isolated from chick embryos. Furthermore, it has also been described that Glu receptors activation is linked to a modulation of [(35)S]-methionine incorporation into newly synthesized polypeptides. In order to gain insight into the signal transduction cascades that participate in this effect, in the present study we characterized the phosphorylation of a critical component of the translational machinery, namely the ribosomal protein S6. The phosphorylation sites in rpS6 have been mapped to five clustered residues, Ser235, Ser236, Ser240, Ser244 and Ser247. Nevertheless, Ser236 phosphorylation is the primary phosphorylation site. The kinases responsible of this modification are p70(S6K) and p90(RSK). rpS6 phosphorylation increases the affinity of 40s subunit for mRNAs and thus facilitates translational initiation. Glutamate exposure of cultured cerebellar Bergmann glia cells results in a time- and dose-dependent increase in rpS6 phosphorylation. This effect is mainly observed at cytoplasm, and involves the phosphoinositol-3 kinase/protein kinase B pathway. Our results favor the notion of a continuous neuronal signaling to glia cells that regulates the proteome of these cells not only at the transcriptional level but also at the level of protein synthesis.

IMPDH2 filaments protect from neurodegeneration in AMPD2 deficiency.

Metabolic dysregulation is one of the most common causes of pediatric neurodegenerative disorders. However, how the disruption of ubiquitous and essential metabolic pathways predominantly affect neural tissue remains unclear. Here we use mouse models of AMPD2 deficiency to study cellular and molecular mechanisms that lead to selective neuronal vulnerability to purine metabolism imbalance. We show that AMPD deficiency in mice primarily leads to hippocampal dentate gyrus degeneration despite causing a generalized reduction of brain GTP levels. Remarkably, we found that neurodegeneration resistant regions accumulate micron sized filaments of IMPDH2, the rate limiting enzyme in GTP synthesis. In contrast, IMPDH2 filaments are barely detectable in the hippocampal dentate gyrus, which shows a progressive neuroinflammation and neurodegeneration. Furthermore, using a human AMPD2 deficient neural cell culture model, we show that blocking IMPDH2 polymerization with a dominant negative IMPDH2 variant, impairs AMPD2 deficient neural progenitor growth. Together, our findings suggest that IMPDH2 polymerization prevents detrimental GTP deprivation in neurons with available GTP precursor molecules, providing resistance to neurodegeneration. Our findings open the possibility of exploring the involvement of IMPDH2 assembly as a therapeutic intervention for neurodegeneration.

Altered lipid homeostasis is associated with cerebellar neurodegeneration in SNX14 deficiency.

Dysregulated lipid homeostasis is emerging as a potential cause of neurodegenerative disorders. However, evidence of errors in lipid homeostasis as a pathogenic mechanism of neurodegeneration remains limited. Here, we show that cerebellar neurodegeneration caused by Sorting Nexin 14 (SNX14) deficiency is associated with lipid homeostasis defects. Recent studies indicate that SNX14 is an interorganelle lipid transfer protein that regulates lipid transport, lipid droplet (LD) biogenesis, and fatty acid desaturation, suggesting that human SNX14 deficiency belongs to an expanding class of cerebellar neurodegenerative disorders caused by altered cellular lipid homeostasis. To test this hypothesis, we generated a mouse model that recapitulates human SNX14 deficiency at a genetic and phenotypic level. We demonstrate that cerebellar Purkinje cells (PCs) are selectively vulnerable to SNX14 deficiency while forebrain regions preserve their neuronal content. Ultrastructure and lipidomic studies reveal widespread lipid storage and metabolism defects in SNX14-deficient mice. However, predegenerating SNX14-deficient cerebella show a unique accumulation of acylcarnitines and depletion of triglycerides. Furthermore, defects in LD content and telolysosome enlargement in predegenerating PCs suggest lipotoxicity as a pathogenic mechanism of SNX14 deficiency. Our work shows a selective cerebellar vulnerability to altered lipid homeostasis and provides a mouse model for future therapeutic studies.

Targeted therapy improves cellular dysfunction, ataxia, and seizure susceptibility in a model of a progressive myoclonus epilepsy.

The recurrent variant KCNC1-p.Arg320His causes progressive myoclonus epilepsy (EPM) type 7, defined by progressive myoclonus, epilepsy, and ataxia, and is without effective treatment. KCNC1 encodes the voltage-gated potassium channel subunit Kv3.1, specifically expressed in high-frequency-firing neurons. Variant subunits act via loss of function; hence, EPM7 pathogenesis may involve impaired excitability of Kv3.1-expressing neurons, while enhancing Kv3 activity could represent a viable therapeutic strategy. We generate a mouse model, Kcnc1-p.Arg320His/+, which recapitulates the core features of EPM7, including progressive ataxia and seizure susceptibility. Kv3.1-expressing cerebellar granule cells and neocortical parvalbumin-positive GABAergic interneurons exhibit abnormalities consistent with Kv3 channel dysfunction. A Kv3-specific positive modulator (AUT00206) selectively enhances the firing frequency of Kv3.1-expressing neurons and improves motor function and seizure susceptibility in Kcnc1-Arg320His/+ mice. This work identifies a cellular and circuit basis of dysfunction in EPM7 and demonstrates that Kv3 positive modulators such as AUT00206 have therapeutic potential for the treatment of EPM7.

GLAST/EAAT1-induced glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling.

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium-dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium-dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so-called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time-dependent Na⁺-dependent glutamate/aspartate transporter/EAAT1-induced System N-mediated glutamine release could be demonstrated. Furthermore, D-aspartate, a specific glutamate transporter ligand, was capable of enhancing the co-immunoprecipitation of Na⁺-dependent glutamate/aspartate transporter and Na⁺-dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron-derived glutamate through their contribution to the neurotransmitter turnover.

Glutamate-dependent translational control in cultured Bergmann glia cells: eIF2α phosphorylation.

Glutamate (Glu), the major excitatory amino acid, activates a wide variety of signal transduction cascades. Synaptic plasticity relies on activity-dependent differential protein expression. Glu receptors have been critically involved in long-term synaptic changes, although recent findings suggest that Na(+)-dependent Glu transporters participate in Glu-induced signalling. Within the cerebellum, Bergmann glia cells are in close proximity to glutamatergic synapses and through their receptors and transporters, sense and respond to neuronal glutamatergic activity. Translational control represents the fine-tuning stage of protein expression regulation and Glu modulates this event in glial cells. In this context, we decided to explore the involvement of Glu receptors and transporters in the regulation of the initiation phase of protein synthesis. To this end, Bergmann glia cells were exposed to glutamatergic ligands and the serine 51-phosphorylation pattern of the main regulator of the initiation phase of translation, namely the α subunit of eukaryotic initiation factor 2 (eIF2α), determined. A time and dose-dependent increase in eIF2α phosphorylation was detected. The signalling cascade included Ca(2+) influx, activation of the Ca(2+)/calmodulin-dependent protein kinase II and protein kinase C. These results provide an insight into the molecular targets of the Glu effects at the translational level and strengthen the notion of the critical involvement of glia cells in glutamatergic synaptic function.

Heterozygous Nonsense Variants in the Ferritin Heavy Chain Gene FTH1 Cause a Novel Pediatric Neuroferritinopathy.

Ferritin, the iron storage protein, is composed of light and heavy chain subunits, encoded by FTL and FTH1 , respectively. Heterozygous variants in FTL cause hereditary neuroferritinopathy, a type of neurodegeneration with brain iron accumulation (NBIA). Variants in FTH1 have not been previously associated with neurologic disease. We describe the clinical, neuroimaging, and neuropathology findings of five unrelated pediatric patients with de novo heterozygous FTH1 variants. Children presented with developmental delay, epilepsy, and progressive neurologic decline. Nonsense FTH1 variants were identified using whole exome sequencing, with a recurrent de novo variant (p.F171*) identified in three unrelated individuals. Neuroimaging revealed diffuse volume loss, features of pontocerebellar hypoplasia and iron accumulation in the basal ganglia. Neuropathology demonstrated widespread ferritin inclusions in the brain. Patient-derived fibroblasts were assayed for ferritin expression, susceptibility to iron accumulation, and oxidative stress. Variant FTH1 mRNA transcripts escape nonsense-mediated decay (NMD), and fibroblasts show elevated ferritin protein levels, markers of oxidative stress, and increased susceptibility to iron accumulation. C-terminus variants in FTH1 truncate ferritin's E-helix, altering the four-fold symmetric pores of the heteropolymer and likely diminish iron-storage capacity. FTH1 pathogenic variants appear to act by a dominant, toxic gain-of-function mechanism. The data support the conclusion that truncating variants in the last exon of FTH1 cause a novel disorder in the spectrum of NBIA. Targeted knock-down of mutant FTH1 transcript with antisense oligonucleotides rescues cellular phenotypes and suggests a potential therapeutic strategy for this novel pediatric neurodegenerative disorder.

Heterozygous nonsense variants in the ferritin heavy-chain gene FTH1 cause a neuroferritinopathy.

Ferritin, the iron-storage protein, is composed of light- and heavy-chain subunits, encoded by FTL and FTH1, respectively. Heterozygous variants in FTL cause hereditary neuroferritinopathy, a type of neurodegeneration with brain iron accumulation (NBIA). Variants in FTH1 have not been previously associated with neurologic disease. We describe the clinical, neuroimaging, and neuropathology findings of five unrelated pediatric patients with de novo heterozygous FTH1 variants. Children presented with developmental delay, epilepsy, and progressive neurologic decline. Nonsense FTH1 variants were identified using whole-exome sequencing, with a recurrent variant (p.Phe171∗) identified in four unrelated individuals. Neuroimaging revealed diffuse volume loss, features of pontocerebellar hypoplasia, and iron accumulation in the basal ganglia. Neuropathology demonstrated widespread ferritin inclusions in the brain. Patient-derived fibroblasts were assayed for ferritin expression, susceptibility to iron accumulation, and oxidative stress. Variant FTH1 mRNA transcripts escape nonsense-mediated decay (NMD), and fibroblasts show elevated ferritin protein levels, markers of oxidative stress, and increased susceptibility to iron accumulation. C-terminal variants in FTH1 truncate ferritin's E helix, altering the 4-fold symmetric pores of the heteropolymer, and likely diminish iron-storage capacity. FTH1 pathogenic variants appear to act by a dominant, toxic gain-of-function mechanism. The data support the conclusion that truncating variants in the last exon of FTH1 cause a disorder in the spectrum of NBIA. Targeted knockdown of mutant FTH1 transcript with antisense oligonucleotides rescues cellular phenotypes and suggests a potential therapeutic strategy for this pediatric neurodegenerative disorder.

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.

Effect of ß-Hydroxybutyrate on Autophagy Dynamics During Severe Hypoglycemia and the Hypoglycemic Coma.

Glucose supply from blood is mandatory for brain functioning and its interruption during acute hypoglycemia or cerebral ischemia leads to brain injury. Alternative substrates to glucose such as the ketone bodies (KB), acetoacetate (AcAc), and ß-hydroxybutyrate (BHB), can be used as energy fuels in the brain during hypoglycemia and prevent neuronal death, but the mechanisms involved are still not well understood. During glucose deprivation adaptive cell responses can be activated such as autophagy, a lysosomal-dependent degradation process, to support cell survival. However, impaired or excessive autophagy can lead to cell dysfunction. We have previously shown that impaired autophagy contributes to neuronal death induced by glucose deprivation in cortical neurons and that D isomer of BHB (D-BHB) reestablishes the autophagic flux increasing viability. Here, we aimed to investigate autophagy dynamics in the brain of rats subjected to severe hypoglycemia (SH) without glucose infusion (GI), severe hypoglycemia followed by GI (SH + GI), and a brief period of hypoglycemic coma followed by GI (Coma). The effect of D-BHB administration after the coma was also tested (Coma + BHB). The transformation of LC3-I to LC3-II and the abundance of autophagy proteins, Beclin 1 (BECN1), ATG7, and ATG12-ATG5 conjugate, were analyzed as an index of autophagosome formation, and the levels of sequestrosome1/p62 (SQSTM1/p62) were determined as a hallmark of autophagic degradation. Data suggest that autophagosomes accumulate in the cortex and the hippocampus of rats after SH, likely due to impaired autophagic degradation. In the cortex, autophagosome accumulation persisted at 6 h after GI in animals exposed to SH but recovered basal levels at 24 h, while in the hippocampus no significant effect was observed. In animals subjected to coma, autophagosome accumulation was observed at 24 h after GI in both regions. D-BHB treatment reduced LC3-II and SQSTM1/p62 content and reduced ULK1 phosphorylation by AMPK, suggesting it stimulates the autophagic flux and decreases AMPK activity reducing autophagy initiation. D-BHB also reduced the number of degenerating cells. Together, data suggest different autophagy dynamics after GI in rats subjected to SH or the hypoglycemic coma and support that D-BHB treatment can modulate autophagy dynamics favoring the autophagic flux.

Multimerization of Zika Virus-NS5 Causes Ciliopathy and Forces Premature Neurogenesis.

Zika virus (ZikV) is a flavivirus that infects neural tissues, causing congenital microcephaly. ZikV has evolved multiple mechanisms to restrict proliferation and enhance cell death, although the underlying cellular events involved remain unclear. Here we show that the ZikV-NS5 protein interacts with host proteins at the base of the primary cilia in neural progenitor cells, causing an atypical non-genetic ciliopathy and premature neuron delamination. Furthermore, in human microcephalic fetal brain tissue, ZikV-NS5 persists at the base of the motile cilia in ependymal cells, which also exhibit a severe ciliopathy. Although the enzymatic activity of ZikV-NS5 appears to be dispensable, the amino acids Y25, K28, and K29 that are involved in NS5 oligomerization are essential for localization and interaction with components of the cilium base, promoting ciliopathy and premature neurogenesis. These findings lay the foundation for therapies that target ZikV-NS5 multimerization and prevent the developmental malformations associated with congenital Zika syndrome.

Glia plasma membrane transporters: Key players in glutamatergic neurotransmission.

Glutamate, the main excitatory amino acid in the central nervous system, elicits its functions through the activation of specific membrane receptors that are expressed in neurons and glial cells. The re-cycling of this amino acid is carried out mostly through a continuous interplay between neurons and glia cells, given the fact that the removal of glutamate from the synaptic cleft depends mainly on glial glutamate transporters. Therefore, a functional and physical interaction between membrane transporters links glutamate uptake, transformation to glutamine and its release to the extra-synaptic space and its uptake to the pre-synaptic terminal. This sequence of events, best known as the glutamate/glutamine shuttle is central to glutamatergic transmission. In this sense, the uptake process triggers a complex series of biochemical cascades that modify the physiology of glial cells in the immediate, short and long term so as to be capable to take up, transform and release these amino acids in a regulated amount and in an appropriate time frame to sustain glutamatergic neurotransmission. Among the signaling cascades activated in glial cells by glutamate transporters, a sustained Na(+) and Ca(2+) influx, protein posttranslational modifications and gene expression regulation at the transcriptional and translational levels are present. Therefore, it is clear that the pivotal role of glial cells in the context of excitatory transmission has been constantly underestimated.

Fluoride exposure regulates the elongation phase of protein synthesis in cultured Bergmann glia cells.

Fluoride is an environmental pollutant present in dental products, food, pesticides and water. The latter, is the greatest source of exposure to this contaminant. Structural and functional damages to the central nervous system are present in exposed population. An established consequence of the neuronal is the release of a substantial amount of glutamate to the extracellular space, leading to an excitotoxic insult. Glutamate exerts its actions through the activation of specific plasma membrane receptors and transporters present in neurons and in glia cells and it is the over-activation of glutamate receptors and transporters, the biochemical hallmark of neuronal and oligodendrocyte cell death. In this context, taking into consideration that fluoride leads to degeneration of cerebellar cells, we took the advantage of the well-established model of cerebellar Bergmann glia cultures to gain insight into the molecular mechanisms inherent to fluoride neurotoxicity that might be triggered in glia cells. We could establish that fluoride decreases [(35)S]-methionine incorporation into newly synthesized polypeptides, in a time-dependent manner, and that this halt in protein synthesis is the result of a decrease in the elongation phase of translation, mediated by an augmentation of eukaryotic elongation factor 2 phosphorylation. These results favor the notion of glial cells as targets of fluoride toxicity and strengthen the idea of a critical involvement of glia cells in the function and dysfunction of the brain.

Glutamate regulates eEF1A phosphorylation and ribosomal transit time in Bergmann glial cells.

Glutamate, the major excitatory transmitter in the vertebrate brain, is involved in neuronal development and synaptic plasticity. Glutamatergic stimulation leads to differential gene expression patterns in neuronal and glial cells. A glutamate-dependent transcriptional control has been established for several genes. However, much less is known about the molecular events that modify the translational machinery upon exposure to this neurotransmitter. In a glial model of cerebellar cultured Bergmann cells, glutamate induces a biphasic effect on [(35)S]-methionine incorporation into proteins that suggests that the elongation phase of protein biosynthesis is the target for regulation. Indeed, after a 15 min exposure to glutamate a transient increase in elongation factor 2 phosphorylation has been reported, an effect mediated through the activation of the elongation factor 2 kinase. In this contribution, we sought to characterize the phosphorylation status of the eukaryotic elongation factor 1A (eEF1A) and the ribosomal transit time under glutamate exposure. A dose-dependent increase in eEF1A phosphorylation was found after a 60 min glutamate treatment; this phenomenon is Ca(2+)/CaM dependent, blocked with Src and phosphatidyl-inositol 3-kinase inhibitors and with rapamicyn. Concomitantly, the ribosomal transit time was increased with a 15 min glutamate exposure. After 60 more minutes, the average time used by the ribosomes to complete a polypeptide chain had almost returned to its initial level. These results strongly suggest that glutamate exerts an exquisite time-dependent translational control in glial cells, a process that might be critical for glia-neuron interactions.