Protein Oxidative Modifications in Neurodegenerative Diseases: From Advances in Detection and Modelling to Their Use as Disease Biomarkers.

Oxidation-reduction post-translational modifications (redox-PTMs) are chemical alterations to amino acids of proteins. Redox-PTMs participate in the regulation of protein conformation, localization and function, acting as signalling effectors that impact many essential biochemical processes in the cells. Crucially, the dysregulation of redox-PTMs of proteins has been implicated in the pathophysiology of numerous human diseases, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. This review aims to highlight the current gaps in knowledge in the field of redox-PTMs biology and to explore new methodological advances in proteomics and computational modelling that will pave the way for a better understanding of the role and therapeutic potential of redox-PTMs of proteins in neurodegenerative diseases. Here, we summarize the main types of redox-PTMs of proteins while providing examples of their occurrence in neurodegenerative diseases and an overview of the state-of-the-art methods used for their detection. We explore the potential of novel computational modelling approaches as essential tools to obtain insights into the precise role of redox-PTMs in regulating protein structure and function. We also discuss the complex crosstalk between various PTMs that occur in living cells. Finally, we argue that redox-PTMs of proteins could be used in the future as diagnosis and prognosis biomarkers for neurodegenerative diseases.

Neuroinflammation and Lysosomal Abnormalities Characterise the Essential Role for Oxidation Resistance 1 in the Developing and Adult Cerebellum.

Loss-of-function mutations in the TLDc family of proteins cause a range of severe childhood-onset neurological disorders with common clinical features that include cerebellar neurodegeneration, ataxia and epilepsy. Of these proteins, oxidation resistance 1 (OXR1) has been implicated in multiple cellular pathways related to antioxidant function, transcriptional regulation and cellular survival; yet how this relates to the specific neuropathological features in disease remains unclear. Here, we investigate a range of loss-of-function mouse model systems and reveal that constitutive deletion of Oxr1 leads to a rapid and striking neuroinflammatory response prior to neurodegeneration that is associated with lysosomal pathology. We go on to show that neuroinflammation and cell death in Oxr1 knockouts can be completely rescued by the neuronal expression of Oxr1, suggesting that the phenotype is driven by the cell-intrinsic defects of neuronal cells lacking the gene. Next, we generate a ubiquitous, adult inducible knockout of Oxr1 that surprisingly displays rapid-onset ataxia and cerebellar neurodegeneration, establishing for the first time that the distinctive pathology associated with the loss of Oxr1 occurs irrespective of developmental stage. Finally, we describe two new homozygous human pathogenic variants in OXR1 that cause neurodevelopmental delay, including a novel stop-gain mutation. We also compare functionally two missense human pathogenic mutations in OXR1, including one newly described here, that cause different clinical phenotypes but demonstrate partially retained neuroprotective activity against oxidative stress. Together, these data highlight the essential role of Oxr1 in modulating neuroinflammatory and lysosomal pathways in the mammalian brain and support the hypothesis that OXR1 protein dosage may be critical for pathological outcomes in disease.

Enhancing research culture in academia: a spotlight on early career researchers.

This editorial highlights common challenges faced by early career researchers (ECRs) who play a crucial role in our research community. We propose that enhancing the experiences of ECRs will yield benefits to the entire scientific community and we give practical suggestions on how such improvements may be achieved.

The Ncoa7 locus regulates V-ATPase formation and function, neurodevelopment and behaviour.

Members of the Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) protein family are associated with multiple neurodevelopmental disorders, although their exact roles in disease remain unclear. For example, nuclear receptor coactivator 7 (NCOA7) has been associated with autism, although almost nothing is known regarding the mode-of-action of this TLDc protein in the nervous system. Here we investigated the molecular function of NCOA7 in neurons and generated a novel mouse model to determine the consequences of deleting this locus in vivo. We show that NCOA7 interacts with the cytoplasmic domain of the vacuolar (V)-ATPase in the brain and demonstrate that this protein is required for normal assembly and activity of this critical proton pump. Neurons lacking Ncoa7 exhibit altered development alongside defective lysosomal formation and function; accordingly, Ncoa7 deletion animals exhibited abnormal neuronal patterning defects and a reduced expression of lysosomal markers. Furthermore, behavioural assessment revealed anxiety and social defects in mice lacking Ncoa7. In summary, we demonstrate that NCOA7 is an important V-ATPase regulatory protein in the brain, modulating lysosomal function, neuronal connectivity and behaviour; thus our study reveals a molecular mechanism controlling endolysosomal homeostasis that is essential for neurodevelopment.

Detection and quantification of novel C-terminal TDP-43 fragments in ALS-TDP.

The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C-terminal fragments of the protein TDP-43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS-PRM) can generate a high-resolution map of pathological TDP-43 peptide ratios to form the basis for quantitation of abnormal C-terminal TDP-43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of p-TDP-43, p-tau, alpha-synuclein, and beta-amyloid pathology, were biochemically fractionated and analyzed by immunoblot and MS for the detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed the synthesis of heavy isotope-labeled peptides for absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson's, 8 Alzheimer's disease, and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C-terminal fragments in ALS-TDP urea fractions. Subsequent MS analysis resolved specific TDP-43 N- and C-terminal peptides, including a novel N-terminal truncation site-specific peptide. Absolute quantification of peptides by MS-PRM showed an increased C:N-terminal TDP-43 peptide ratio in ALS-TDP brain compared to normal and disease controls. A C:N-terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6-100) and specificity of 100% (CI 68-100), and from Parkinson's and Alzheimer's disease with a sensitivity of 93% (CI 70-100) and specificity of 100% (CI 68-100). N-terminal truncation site-specific peptides were increased in ALS in line with C-terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N-terminal ratio but coexistent limbic TDP-43 neuropathological changes. In conclusion this is a novel, sensitive, and specific method to quantify the enrichment of pathological TDP-43 fragments in human brain, which could form the basis for an antibody-free assay. Our methodology has the potential to help clarify if specific pathological TDP-43 peptide signatures are associated with primary or secondary TDP-43 proteinopathies.

Redox Post-translational Modifications of Protein Thiols in Brain Aging and Neurodegenerative Conditions-Focus on S-Nitrosation.

Reactive oxygen species and reactive nitrogen species (RONS) are by-products of aerobic metabolism. RONS trigger a signaling cascade that can be transduced through oxidation-reduction (redox)-based post-translational modifications (redox PTMs) of protein thiols. This redox signaling is essential for normal cellular physiology and coordinately regulates the function of redox-sensitive proteins. It plays a particularly important role in the brain, which is a major producer of RONS. Aberrant redox PTMs of protein thiols can impair protein function and are associated with several diseases. This mini review article aims to evaluate the role of redox PTMs of protein thiols, in particular S-nitrosation, in brain aging, and in neurodegenerative diseases. It also discusses the potential of using redox-based therapeutic approaches for neurodegenerative conditions.

An ALS-linked mutation in TDP-43 disrupts normal protein interactions in the motor neuron response to oxidative stress.

TDP-43 pathology is a key feature of amyotrophic lateral sclerosis (ALS), but the mechanisms linking TDP-43 to altered cellular function and neurodegeneration remain unclear. We have recently described a mouse model in which human wild-type or mutant TDP-43 are expressed at low levels and where altered stress granule formation is a robust phenotype of TDP-43M337V/- expressing cells. In the present study we use this model to investigate the functional connectivity of human TDP-43 in primary motor neurons under resting conditions and in response to oxidative stress. The interactome of human TDP-43WT or TDP-43M337V was compared by mass spectrometry, and gene ontology enrichment analysis identified pathways dysregulated by the M337V mutation. We found that under normal conditions the interactome of human TDP-43WT was enriched for proteins involved in transcription, translation and poly(A)-RNA binding. In response to oxidative stress, TDP-43WT recruits proteins of the endoplasmic reticulum and endosomal-extracellular transport pathways, interactions which are reduced in the presence of the M337V mutation. Specifically, TDP-43M337V impaired protein-protein interactions involved in stress granule formation including reduced binding to the translation initiation factors Poly(A)-binding protein and Eif4a1 and the endoplasmic reticulum chaperone Grp78. The M337V mutation also affected interactions involved in endosomal-extracellular transport and this this was associated with reduced extracellular vesicle secretion in primary motor neurons from TDP-43M337V/- mice and in human iPSCs-derived motor neurons. Taken together, our analysis highlights a TDP-43 interaction network in motor neurons and demonstrates that an ALS associated mutation may alter the interactome to drive aberrant pathways involved in the pathogenesis of ALS.

Neuronal over-expression of Oxr1 is protective against ALS-associated mutant TDP-43 mislocalisation in motor neurons and neuromuscular defects in vivo.

A common pathological hallmark of amyotrophic lateral sclerosis (ALS) and the related neurodegenerative disorder frontotemporal dementia, is the cellular mislocalization of transactive response DNA-binding protein 43 kDa (TDP-43). Additionally, multiple mutations in the TARDBP gene (encoding TDP-43) are associated with familial forms of ALS. While the exact role for TDP-43 in the onset and progression of ALS remains unclear, the identification of factors that can prevent aberrant TDP-43 localization and function could be clinically beneficial. Previously, we discovered that the oxidation resistance 1 (Oxr1) protein could alleviate cellular mislocalization phenotypes associated with TDP-43 mutations, and that over-expression of Oxr1 was able to delay neuromuscular abnormalities in the hSOD1G93A ALS mouse model. Here, to determine whether Oxr1 can protect against TDP-43-associated phenotypes in vitro and in vivo, we used the same genetic approach in a newly described transgenic mouse expressing the human TDP-43 locus harbouring an ALS disease mutation (TDP-43M337V). We show in primary motor neurons from TDP-43M337V mice that genetically-driven Oxr1 over-expression significantly alleviates cytoplasmic mislocalization of mutant TDP-43. We also further quantified newly-identified, late-onset neuromuscular phenotypes of this mutant line, and demonstrate that neuronal Oxr1 over-expression causes a significant reduction in muscle denervation and neuromuscular junction degeneration in homozygous mutants in parallel with improved motor function and a reduction in neuroinflammation. Together these data support the application of Oxr1 as a viable and safe modifier of TDP-43-associated ALS phenotypes.

Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase.

Glucose metabolism is essential for the brain: it not only provides the required energy for cellular function and communication but also participates in balancing the levels of oxidative stress in neurons. Defects in glucose metabolism have been described in neurodegenerative disease; however, it remains unclear how this fundamental process contributes to neuronal cell death in these disorders. Here, we investigated the molecular mechanisms driving the selective neurodegeneration in an ataxic mouse model lacking oxidation resistance 1 (Oxr1) and discovered an unexpected function for this protein as a regulator of the glycolytic enzyme, glucose-6-phosphate isomerase (GPI/Gpi1). Initially, we present a dysregulation of metabolites of glucose metabolism at the pre-symptomatic stage in the Oxr1 knockout cerebellum. We then demonstrate that Oxr1 and Gpi1 physically and functionally interact and that the level of Gpi1 oligomerisation is disrupted when Oxr1 is deleted in vivo. Furthermore, we show that Oxr1 modulates the additional and less well-understood roles of Gpi1 as a cytokine and neuroprotective factor. Overall, our data identify a new molecular function for Oxr1, establishing this protein as important player in neuronal survival, regulating both oxidative stress and glucose metabolism in the brain.

The epilepsy-associated protein TBC1D24 is required for normal development, survival and vesicle trafficking in mammalian neurons.

Mutations in the Tre2/Bub2/Cdc16 (TBC)1 domain family member 24 (TBC1D24) gene are associated with a range of inherited neurological disorders, from drug-refractory lethal epileptic encephalopathy and DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, seizures) to non-syndromic hearing loss. TBC1D24 has been implicated in neuronal transmission and maturation, although the molecular function of the gene and the cause of the apparently complex disease spectrum remain unclear. Importantly, heterozygous TBC1D24 mutation carriers have also been reported with seizures, suggesting that haploinsufficiency for TBC1D24 is significant clinically. Here we have systematically investigated an allelic series of disease-associated mutations in neurons alongside a new mouse model to investigate the consequences of TBC1D24 haploinsufficiency to mammalian neurodevelopment and synaptic physiology. The cellular studies reveal that disease-causing mutations that disrupt either of the conserved protein domains in TBC1D24 are implicated in neuronal development and survival and are likely acting as loss-of-function alleles. We then further investigated TBC1D24 haploinsufficiency in vivo and demonstrate that TBC1D24 is also crucial for normal presynaptic function: genetic disruption of Tbc1d24 expression in the mouse leads to an impairment of endocytosis and an enlarged endosomal compartment in neurons with a decrease in spontaneous neurotransmission. These data reveal the essential role for TBC1D24 at the mammalian synapse and help to define common synaptic mechanisms that could underlie the varied effects of TBC1D24 mutations in neurological disease.

Oxidation resistance 1 regulates post-translational modifications of peroxiredoxin 2 in the cerebellum.

Protein aggregation, oxidative and nitrosative stress are etiological factors common to all major neurodegenerative disorders. Therefore, identifying proteins that function at the crossroads of these essential pathways may provide novel targets for therapy. Oxidation resistance 1 (Oxr1) is a protein proven to be neuroprotective against oxidative stress, although the molecular mechanisms involved remain unclear. Here, we demonstrate that Oxr1 interacts with the multifunctional protein, peroxiredoxin 2 (Prdx2), a potent antioxidant enzyme highly expressed in the brain that can also act as a molecular chaperone. Using a combination of in vitro assays and two animal models, we discovered that expression levels of Oxr1 regulate the degree of oligomerization of Prdx2 and also its post-translational modifications (PTMs), specifically suggesting that Oxr1 acts as a functional switch between the antioxidant and chaperone functions of Prdx2. Furthermore, we showed in the Oxr1 knockout mouse that Prdx2 is aberrantly modified by overoxidation and S-nitrosylation in the cerebellum at the presymptomatic stage; this in-turn affected the oligomerization of Prdx2, potentially impeding its normal functions and contributing to the specific cerebellar neurodegeneration in this mouse model.

TLDc proteins: new players in the oxidative stress response and neurological disease.

Oxidative stress (OS) arises from an imbalance in the cellular redox state, which can lead to intracellular damage and ultimately cell death. OS occurs as a result of normal ageing, but it is also implicated as a common etiological factor in neurological disease; thus identifying novel proteins that modulate the OS response may facilitate the design of new therapeutic approaches applicable to many disorders. In this review, we describe the recent progress that has been made using a range of genetic approaches to understand a family of proteins that share the highly conserved TLDc domain. We highlight their shared ability to prevent OS-related cell death and their unique functional characteristics, as well as discussing their potential application as new neuroprotective factors. Furthermore, with an increasing number of pathogenic mutations leading to epilepsy and hearing loss being discovered in the TLDc protein TBC1D24, understanding the function of this family has important implications for a range of inherited neurological diseases.

Comprehensive mapping of 5-hydroxymethylcytosine epigenetic dynamics in axon regeneration.

In contrast to central nervous system neurons, dorsal root ganglia (DRG) neurons can switch to a regenerative state after peripheral axotomy. In a screen for chromatin regulators of the regenerative responses in this conditioning lesion paradigm, we identified Tet methylcytosine dioxygenase 3 (Tet3) as upregulated in DRG neurons, along with increased 5-hydroxymethylcytosine (5hmC). We generated genome-wide 5hmC maps in adult DRG, which revealed that peripheral and central axotomy (leading to no regenerative effect) triggered differential 5hmC changes that are associated with distinct signaling pathways. 5hmC was altered in a large set of regeneration-associated genes (RAGs), including well-known RAGs, such as Atf3, Bdnf, and Smad1, that regulate axon growth potential of DRG neurons, thus supporting its role for RAG regulation. Our analyses also predicted HIF-1, STAT, and IRF as potential transcription factors that may collaborate with Tet3 for 5hmC modifications. Intriguingly, central axotomy resulted in widespread 5hmC modifications that had little overlap with those of peripheral axotomy, thus potentially constituting a roadblock for regeneration. Our study revealed 5hmC dynamics as a previously unrecognized epigenetic mechanism underlying the divergent responses after axonal injury.

TBC1D24 genotype-phenotype correlation: Epilepsies and other neurologic features.

OBJECTIVE: To evaluate the phenotypic spectrum associated with mutations in TBC1D24. METHODS: We acquired new clinical, EEG, and neuroimaging data of 11 previously unreported and 37 published patients. TBC1D24 mutations, identified through various sequencing methods, can be found online (http://lovd.nl/TBC1D24). RESULTS: Forty-eight patients were included (28 men, 20 women, average age 21 years) from 30 independent families. Eighteen patients (38%) had myoclonic epilepsies. The other patients carried diagnoses of focal (25%), multifocal (2%), generalized (4%), and unclassified epilepsy (6%), and early-onset epileptic encephalopathy (25%). Most patients had drug-resistant epilepsy. We detail EEG, neuroimaging, developmental, and cognitive features, treatment responsiveness, and physical examination. In silico evaluation revealed 7 different highly conserved motifs, with the most common pathogenic mutation located in the first. Neuronal outgrowth assays showed that some TBC1D24 mutations, associated with the most severe TBC1D24-associated disorders, are not necessarily the most disruptive to this gene function. CONCLUSIONS: TBC1D24-related epilepsy syndromes show marked phenotypic pleiotropy, with multisystem involvement and severity spectrum ranging from isolated deafness (not studied here), benign myoclonic epilepsy restricted to childhood with complete seizure control and normal intellect, to early-onset epileptic encephalopathy with severe developmental delay and early death. There is no distinct correlation with mutation type or location yet, but patterns are emerging. Given the phenotypic breadth observed, TBC1D24 mutation screening is indicated in a wide variety of epilepsies. A TBC1D24 consortium was formed to develop further research on this gene and its associated phenotypes.

The Evolutionarily Conserved Tre2/Bub2/Cdc16 (TBC), Lysin Motif (LysM), Domain Catalytic (TLDc) Domain Is Neuroprotective against Oxidative Stress.

Oxidative stress is a pathological feature of many neurological disorders; therefore, utilizing proteins that are protective against such cellular insults is a potentially valuable therapeutic approach. Oxidation resistance 1 (OXR1) has been shown previously to be critical for oxidative stress resistance in neuronal cells; deletion of this gene causes neurodegeneration in mice, yet conversely, overexpression of OXR1 is protective in cellular and mouse models of amyotrophic lateral sclerosis. However, the molecular mechanisms involved are unclear. OXR1 contains the Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) domain, a motif present in a family of proteins including TBC1 domain family member 24 (TBC1D24), a protein mutated in a range of disorders characterized by seizures, hearing loss, and neurodegeneration. The TLDc domain is highly conserved across species, although the structure-function relationship is unknown. To understand the role of this domain in the stress response, we carried out systematic analysis of all mammalian TLDc domain-containing proteins, investigating their expression and neuroprotective properties in parallel. In addition, we performed a detailed structural and functional study of this domain in which we identified key residues required for its activity. Finally, we present a new mouse insertional mutant of Oxr1, confirming that specific disruption of the TLDc domain in vivo is sufficient to cause neurodegeneration. Our data demonstrate that the integrity of the TLDc domain is essential for conferring neuroprotection, an important step in understanding the functional significance of all TLDc domain-containing proteins in the cellular stress response and disease.

The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

Oxr1 improves pathogenic cellular features of ALS-associated FUS and TDP-43 mutations.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neuron-like cells. Mutations in the RNA- and DNA-binding proteins, fused in sarcoma (FUS) and transactive response DNA-binding protein 43 kDa (TDP-43), are responsible for 5-10% of familial and 1% of sporadic ALS cases. Importantly, aggregation of misfolded FUS or TDP-43 is also characteristic of several neurodegenerative disorders in addition to ALS, including frontotemporal lobar degeneration. Moreover, splicing deregulation of FUS and TDP-43 target genes as well as mitochondrial abnormalities are associated with disease-causing FUS and TDP-43 mutants. While progress has been made to understand the functions of these proteins, the exact mechanisms by which FUS and TDP-43 cause ALS remain unknown. Recently, we discovered that, in addition to being up-regulated in spinal cords of ALS patients, the novel protein oxidative resistance 1 (Oxr1) protects neurons from oxidative stress-induced apoptosis. To further understand the function of Oxr1, we present here the first interaction study of the protein. We show that Oxr1 binds to Fus and Tdp-43 and that certain ALS-associated mutations in Fus and Tdp-43 affect their Oxr1-binding properties. We further demonstrate that increasing Oxr1 levels in cells expressing specific Fus and Tdp-43 mutants improves the three main cellular features associated with ALS: cytoplasmic mis-localization and aggregation, splicing changes of a mitochondrial gene and mitochondrial defects. Taken together, these findings suggest that OXR1 may have therapeutic benefits for the treatment of ALS and related neurodegenerative disorders with TDP-43 pathology.

Neuron-specific antioxidant OXR1 extends survival of a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a devastating neurodegenerative disorder characterized by the progressive loss of spinal motor neurons. While the aetiological mechanisms underlying the disease remain poorly understood, oxidative stress is a central component of amyotrophic lateral sclerosis and contributes to motor neuron injury. Recently, oxidation resistance 1 (OXR1) has emerged as a critical regulator of neuronal survival in response to oxidative stress, and is upregulated in the spinal cord of patients with amyotrophic lateral sclerosis. Here, we tested the hypothesis that OXR1 is a key neuroprotective factor during amyotrophic lateral sclerosis pathogenesis by crossing a new transgenic mouse line that overexpresses OXR1 in neurons with the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Interestingly, we report that overexpression of OXR1 significantly extends survival, improves motor deficits, and delays pathology in the spinal cord and in muscles of SOD1(G93A) mice. Furthermore, we find that overexpression of OXR1 in neurons significantly delays non-cell-autonomous neuroinflammatory response, classic complement system activation, and STAT3 activation through transcriptomic analysis of spinal cords of SOD1(G93A) mice. Taken together, these data identify OXR1 as the first neuron-specific antioxidant modulator of pathogenesis and disease progression in SOD1-mediated amyotrophic lateral sclerosis, and suggest that OXR1 may serve as a novel target for future therapeutic strategies.

Laf4/Aff3, a gene involved in intellectual disability, is required for cellular migration in the mouse cerebral cortex.

Members of the AFF (AF4/FMR2) family of putative transcription factors are involved in infant acute leukaemia and intellectual disability (ID), although very little is known about their transcriptional targets. For example, deletion of human lymphoid nuclear protein related to AF4/AFF member 3 (LAF4/AFF3) is known to cause severe neurodevelopmental defects, and silencing of the gene is also associated with ID at the folate-sensitive fragile site (FSFS) FRA2A; yet the normal function of this gene in the nervous system is unclear. The aim of this study was to further investigate the function of Laf4 in the brain by focusing on its role in the cortex. By manipulating expression levels in organotypic slices, we demonstrate here that Laf4 is required for normal cellular migration in the developing cortex and have subsequently identified Mdga2, an important structural protein in neurodevelopment, as a target of Laf4 transcriptional activity. Furthermore, we show that the migration deficit caused by loss of Laf4 can be partially rescued by Mdga2 over-expression, revealing an important functional relationship between these genes. Our study demonstrates the key transcriptional role of Laf4 during early brain development and reveals a novel function for the gene in the process of cortical cell migration relevant to the haploinsufficiency and silencing observed in human neurodevelopmental disorders.

Epigenetic regulation of sensory axon regeneration after spinal cord injury.

Axon regeneration is hindered by a decline of intrinsic axon growth capability in mature neurons. Reversing this decline is associated with the induction of a large repertoire of regeneration-associated genes (RAGs), but the underlying regulatory mechanisms of the transcriptional changes are largely unknown. Here, we establish a correlation between diminished axon growth potential and histone 4 (H4) hypoacetylation. When neurons are triggered into a growth state, as in the conditioning lesion paradigm, H4 acetylation is restored, and RAG transcription is initiated. We have identified a set of target genes of Smad1, a proregenerative transcription factor, in conditioned DRG neurons. We also show that, during the epigenetic reprogramming process, histone-modifying enzymes work together with Smad1 to facilitate transcriptional regulation of RAGs. Importantly, targeted pharmacological modulation of the activity of histone-modifying enzymes, such as histone deacetylases, leads to induction of multiple RAGs and promotion of sensory axon regeneration in a mouse model of spinal cord injury. Our findings suggest epigenetic modulation as a potential therapeutic strategy to enhance axon regeneration.