S-adenosylmethionine and nicotinamide riboside therapy in Arts syndrome: A case report and literature review.

Phospho-ribosyl-pyrophosphate synthetase 1 (PRPS1) deficiency is secondary to loss of function variants in PRPS1. This enzyme generates phospho-ribosyl-pyrophosphate (PRPP), which is utilized in the synthesis of purines, nicotinamide adenine dinucleotide (NAD), and NAD phosphate (NADP), among other metabolic pathways. Arts syndrome, or severe PRPS1 deficiency, is an X-linked condition characterized by congenital sensorineural hearing loss, optic atrophy, developmental delays, ataxia, hypotonia, and recurrent infections that can cause progressive clinical decline, often resulting in death before 5 years of age. Supplementation of the purine and NAD pathways outside of PRPP-dependent reactions is a logical approach and has been reported in a handful of patients, two with S-adenosylmethionine (SAMe) and one with SAMe and nicotinamide riboside (NR). We present the clinical course of a fourth Arts syndrome patient who was started on therapy and review previously reported patients. All patients had stability or improvement of symptoms, suggesting that SAMe and NR can be a treatment option in Arts syndrome, though further studies are warranted.

Post-Translational Modifications of the DUX4 Protein Impact Toxic Function in FSHD Cell Models.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is caused by abnormal de-repression of the myotoxic transcription factor DUX4. Although the transcriptional targets of DUX4 are known, the regulation of DUX4 protein and the molecular consequences of this regulation are unclear. Here, we used in vitro models of FSHD to identify and characterize DUX4 post-translational modifications (PTMs) and their impact on the toxic function of DUX4. METHODS: We immunoprecipitated DUX4 protein and performed mass spectrometry to identify PTMs. We then characterized DUX4 PTMs and potential enzyme modifiers using mutagenesis, proteomics, and biochemical assays in HEK293 and human myoblast cell lines. RESULTS: We identified 17 DUX4 amino acids with PTMs, and generated 55 DUX4 mutants designed to prevent or mimic PTMs. Five mutants protected cells against DUX4-mediated toxicity and reduced the ability of DUX4 to transactivate FSHD biomarkers. These mutagenesis results suggested that DUX4 toxicity could be counteracted by serine/threonine phosphorylation and/or inhibition of arginine methylation. We therefore sought to identify modifying enzymes that could play a role in regulating DUX4 PTMs. We found several enzymes capable of modifying DUX4 protein in vitro, and confirmed that protein kinase A (PKA) and protein arginine methyltransferase (PRMT1) interact with DUX4. INTERPRETATION: These results support that DUX4 is regulated by PTMs and set a foundation for developing FSHD drug screens based mechanistically on DUX4 PTMs and modifying enzymes. ANN NEUROL 2023;94:398-413.

Targeted Treatments for Inherited Neuromuscular Diseases of Childhood.

In the past decade, the number of genes linked to neuromuscular diseases of childhood has expanded dramatically, and this genetic information is forming the basis for gene-specific and even mutation-specific therapies. At the forefront of these advances are the two recently approved treatments for spinal muscular atrophy: one, an antisense oligonucleotide that modifies splicing of the SMN2 gene, and, the other, a gene therapy vector that delivers the SMN1 gene to motor neurons, both of which are allowing patients to acquire developmental milestones previously unseen in this fatal disease. This review highlights these advances and emerging targeted therapies for Duchenne muscular dystrophy and centronuclear myopathy, while also covering enzyme replacement therapy and small molecule-based targeted therapies for conditions such as Pompe's disease and congenital myasthenic syndromes. With these and other newer techniques for targeted correction of genetic defects, such as CRISPR/Cas9, there is now hope that treatments for many more genetic diseases of the nervous system will follow in the near future.

Autism risk in offspring can be assessed through quantification of male sperm mosaicism.

De novo mutations arising on the paternal chromosome make the largest known contribution to autism risk, and correlate with paternal age at the time of conception. The recurrence risk for autism spectrum disorders is substantial, leading many families to decline future pregnancies, but the potential impact of assessing parental gonadal mosaicism has not been considered. We measured sperm mosaicism using deep-whole-genome sequencing, for variants both present in an offspring and evident only in father's sperm, and identified single-nucleotide, structural and short tandem-repeat variants. We found that mosaicism quantification can stratify autism spectrum disorders recurrence risk due to de novo mutations into a vast majority with near 0% recurrence and a small fraction with a substantially higher and quantifiable risk, and we identify novel mosaic variants at risk for transmission to a future offspring. This suggests, therefore, that genetic counseling would benefit from the addition of sperm mosaicism assessment.

Hypoxia-ischemia modifies postsynaptic GluN2B-containing NMDA receptor complexes in the neonatal mouse brain.

The N-methyl-d-aspartate-type glutamate receptor (NMDAR)-associated multiprotein complexes are indispensable for synaptic plasticity and cognitive functions. While purification and proteomic analyses of these signaling complexes have been performed in adult rodent and human brain, much less is known about the protein composition of NMDAR complexes in the developing brain and their modifications by neonatal hypoxic-ischemic (HI) brain injury. In this study, the postsynaptic density proteins were prepared from postnatal day 9 naïve, sham-operated and HI-injured mouse cortex. The GluN2B-containing NMDAR complexes were purified by immunoprecipitation with a mouse GluN2B antibody and subjected to mass spectrometry analysis for determination of the GluN2B binding partners. A total of 71 proteins of different functional categories were identified from the naïve animals as native GluN2B-interacting partners in the developing mouse brain. Neonatal HI reshaped the postsynaptic GluN2B interactome by recruiting new proteins, including multiple kinases, into the complexes; and modifying the existing associations within 1h of reperfusion. The early responses of postsynaptic NMDAR complexes and their related signaling networks may contribute to molecular processes leading to cell survival or death, brain damage and/or neurological disorders in term infants with neonatal encephalopathy.

Proteomic Analysis of Mouse Cortex Postsynaptic Density following Neonatal Brain Hypoxia-Ischemia.

Proteomics of the synapses and postsynaptic densities (PSDs) have provided a deep understanding of protein composition and signal networks in the adult brain, which underlie neuronal plasticity and neurodegenerative or psychiatric disorders. However, there is a paucity of knowledge about the architecture and organization of PSDs in the immature brain, and how it is modified by brain injury in an early developing stage. Mass spectrometry (MS)-based proteomic analysis was performed on PSDs prepared from cortices of postnatal day 9 naïve mice or pups which had suffered hypoxic-ischemic (HI) brain injury. 512 proteins of different functional groups were identified from PSDs collected 1 h after HI injury, among which 60 have not been reported previously. Seven newly identified proteins involved in neural development were highlighted. HI injury increased the yield of PSDs at early time points upon reperfusion, and multiple proteins were recruited into PSDs following the insult. Quantitative analysis was performed using spectral counting, and proteins whose relative expression was more than 50% up- or downregulated compared to the sham animals 1 h after HI insult were reported. Validation with Western blotting demonstrated changes in expression and phosphorylation of the N-methyl-D-aspartate receptor, activation of a series of postsynaptic protein kinases and dysregulation of scaffold and adaptor proteins in response to neonatal HI insult. This work, along with other recent studies of synaptic protein profiling in the immature brain, builds a foundation for future investigation on the molecular mechanisms underlying developing plasticity. Furthermore, it provides insights into the biochemical changes of PSDs following early brain hypoxia-ischemia, which is helpful for understanding not only the injury mechanisms, but also the process of repair or replenishment of neuronal circuits during recovery from brain damage.

Surgical management of medically refractory epilepsy in patients with polymicrogyria.

OBJECTIVE: Polymicrogyria (PMG) is a malformation of cortical development characterized by formation of an excessive number of small gyri. Sixty percent to 85% of patients with PMG have epilepsy that is refractory to medication, but surgical options are usually limited. We characterize a cohort of patient with polymicrogyria who underwent epilepsy surgery and document seizure outcomes. METHODS: A retrospective study of all patients with PMG who underwent epilepsy surgery (focal seizure foci resection and/or hemispherectomy) at our center was performed by review of all clinical data related to their treatment. RESULTS: We identified 12 patients (7 males and 5 female) with mean age of 18 (ranging from 3 months to 44 years) at time of surgery. Mean age at seizure onset was 8 years, with the majority (83%) having childhood onset. Six patients had focal, five had multifocal, and one patient had diffuse PMG. Perisylvian PMG was the most common pattern seen on magnetic resonance imaging (MRI). Eight patients had other cortical malformations including hemimegalencephaly and cortical dysplasia. Scalp electroencephalography (EEG) often showed diffuse epileptic discharges that poorly lateralized but were focal on intracranial electrocorticography (ECoG). Eight patients underwent seizure foci resection and four underwent hemispherectomy. Mean follow-up was 7 years (ranging from one to 19 years). Six patients (50%) were seizure-free at last follow-up. One patient had rare seizures (Engel class II). Three patients were Engel class III, having either decreased seizure frequency or severity, and two patients were Engel class IV. Gross total resection of the PMG cortex trended toward good seizure control. SIGNIFICANCE: Our study shows that even in patients with extensive or bilateral PMG malformations, some may still be good candidates for surgery because the epileptogenic zone may involve only a portion of the malformation. Intracranial ECoG can provide additional localizing information compared to scalp EEG in guiding resection of epileptogenic foci.

Fyn in Neurodevelopment and Ischemic Brain Injury.

The Src family kinases (SFKs) are nonreceptor protein tyrosine kinases that are implicated in many normal and pathological processes in the nervous system. The SFKs Fyn, Src, Yes, Lyn, and Lck are expressed in the brain. This review will focus on Fyn, as Fyn mutant mice have striking phenotypes in the brain and Fyn has been shown to be involved in ischemic brain injury in adult rodents and, with our work, in neonatal animals. An understanding of Fyn's role in neurodevelopment and disease will allow researchers to target pathological pathways while preserving protective ones.

NR2B phosphorylation at tyrosine 1472 contributes to brain injury in a rodent model of neonatal hypoxia-ischemia.

BACKGROUND AND PURPOSE: The NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor is phosphorylated by the Src family kinase Fyn in brain, with tyrosine (Y) 1472 as the major phosphorylation site. Although Y1472 phosphorylation is important for synaptic plasticity, it is unknown whether it is involved in NMDA receptor-mediated excitotoxicity in neonatal brain hypoxia-ischemia (HI). This study was designed to elucidate the specific role of Y1472 phosphorylation of NR2B in neonatal HI in vivo and in NMDA-mediated neuronal death in vitro. METHODS: Neonatal mice with a knockin mutation of Y1472 to phenylalanine (YF-KI) and their wild-type littermates were subjected to HI using the Vannucci model. Brains were scored 5 days later for damage using cresyl violet and iron staining. Western blotting and immunoprecipitation were performed to determine NR2B tyrosine phosphorylation. Expression of NADPH oxidase subunits and superoxide production were measured in vivo. NMDA-induced calcium response, superoxide formation, and cell death were evaluated in primary cortical neurons. RESULTS: After neonatal HI, YF-KI mice have reduced expression of NADPH oxidase subunit gp91phox and p47phox and superoxide production, lower activity of proteases implicated in necrotic and apoptotic cell death, and less brain damage when compared with the wild-type mice. In vitro, YF-KI mutation diminishes superoxide generation in response to NMDA without effect on calcium accumulation and inhibits NMDA and glutamate-induced cell death. CONCLUSIONS: Upregulation of NR2B phosphorylation at Y1472 after neonatal HI is involved in superoxide-mediated oxidative stress and contributes to brain injury.

Hypoxic preconditioning protection is eliminated in HIF-1α knockout mice subjected to neonatal hypoxia-ischemia.

BACKGROUND: Hypoxic preconditioning (HPc) protects the neonatal brain in the setting of hypoxia-ischemia (HI). The mechanisms of protection may depend on activation of hypoxia-inducible factor (HIF-1α). This study sought to clarify the role of HIF-1α after HPc and HI. METHODS: To induce HPc, HIF-1α knockout and wild-type (WT) mice were exposed to hypoxia at postnatal day 6. At day 7, the mice underwent HI. Brain injury was determined by histology. HIF-1α, downstream targets, and markers of cell death were measured by western blot. RESULTS: HPc protected the WT brain compared with WT without HPc, but did not protect the HIF-1α knockout brain. In WT, HIF-1α increased after hypoxia and after HI, but not with HPc. The HIF-1α knockout showed no change in HIF-1α after hypoxia, HI, or HPc/HI. After HI, spectrin 145/150 was higher in HIF-1α knockout, but after HPc/HI, it was higher in WT. Lysosome-associated membrane protein was higher in WT early after HI, but not later. After HPc/HI, lysosome-associated membrane protein was higher in HIF-1α knockout. CONCLUSION: These results indicate that HIF-1α is necessary for HPc protection in the neonatal brain and may affect cell death after HI. Different death and repair mechanisms depend on the timing of HPc.

Enhanced NMDA receptor tyrosine phosphorylation and increased brain injury following neonatal hypoxia-ischemia in mice with neuronal Fyn overexpression.

The Src family kinases (SFKs) Src and Fyn are implicated in hypoxic-ischemic (HI) injury in the developing brain. However, it is unclear how these particular SFKs contribute to brain injury. Using neuron-specific Fyn overexpressing (OE) mice, we investigated the role of neuronal Fyn in neonatal brain HI. Wild type (WT) and Fyn OE mice were subjected to HI using the Vannucci model at postnatal day 7. Brains were scored five days later for evaluation of damage using cresyl violet and iron staining. Western blotting with postsynaptic density (PSD)-associated synaptic membrane proteins and co-immunoprecipitation with cortical lysates were performed at various time points after HI to determine NMDA receptor tyrosine phosphorylation and Fyn kinase activity. Fyn OE mice had significantly higher mortality and brain injury compared to their WT littermates. Neuronal Fyn overexpression led to sustained NR2A and NR2B tyrosine phosphorylation and enhanced NR2B phosphorylation at tyrosine (Y) 1472 and Y1252 in synaptic membranes. These early changes correlated with higher calpain activity 24h after HI in Fyn OE mice relative to WT animals. Our findings suggest a role for Fyn kinase in neuronal death after neonatal HI, possibly via up-regulation of NMDA receptor tyrosine phosphorylation.

Developmental localization of NMDA receptors, Src and MAP kinases in mouse brain.

Activation of NMDA receptors (NMDAR) is associated with divergent downstream signaling leading to neuronal survival or death that may be regulated in part by whether the receptor is located synaptically or extrasynaptically. Distinct activation of the MAP kinases ERK and p38 by synaptic and extrasynaptic NMDAR is one of the mechanisms underlying these differences. We have recently shown that the Src family kinases (SFKs) play an important role in neonatal hypoxic-ischemic brain injury by regulating NMDAR phosphorylation. In this study, we characterized the distribution of NMDAR, SFKs and MAP kinases in synaptic and extrasynaptic membrane locations in the postnatal day 7 and adult mouse cortex. We found that the NMDAR, SFKs and phospho-NR2B were predominantly at synapses, whereas striatal-enriched protein tyrosine phosphatase (STEP) and its substrates ERK and p38 were much more concentrated extrasynaptically. NR1/NR2B was the main subunit at extrasynaptic membrane with concomitant NR2B phosphorylation at tyrosine (Y) 1336 in the immature brain. STEP expression increased, while p38 decreased with development in the extrasynaptic membrane. These results suggest that SFKs and STEP are poised to differentially regulate NMDAR-mediated signaling pathways due to their distinct subcellular localization, and thus may contribute to the age-specific differences seen in vulnerability, pathology and consequences of hypoxic-ischemic brain injury.