Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.

Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment.

SNCA/α-synuclein aggregation plays a crucial role in synucleinopathies such as Parkinson disease and dementia with Lewy bodies. Aggregating and nonaggregating SNCA species are degraded by the autophagy-lysosomal pathway (ALP). Previously, we have shown that the ALP is not only responsible for SNCA degradation but is also involved in the intracellular aggregation process of SNCA. An additional role of extracellular SNCA in the pathology of synucleinopathies substantiating a prion-like propagation hypothesis has been suggested since released SNCA species and spreading of SNCA pathology throughout neural cells have been observed. However, the molecular interplay between intracellular pathways, SNCA aggregation, release, and response of the local microenvironment remains unknown. Here, we attributed SNCA-induced toxicity mainly to secreted species in a cell culture model of SNCA aggregation and in SNCA transgenic mice: We showed that ALP inhibition by bafilomycinA1 reduced intracellular SNCA aggregation but increased secretion of smaller oligomers that exacerbated microenvironmental response including uptake, inflammation, and cellular damage. Low-aggregated SNCA was predominantly released by exosomes and RAB11A-associated pathways whereas high-aggregated SNCA was secreted by membrane shedding. In summary, our study revealed a novel role of the ALP by linking protein degradation to nonclassical secretion for toxic SNCA species. Thus, impaired ALP in the diseased brain not only limits intracellular degradation of misfolded proteins, but also leads to a detrimental microenvironmental response due to enhanced SNCA secretion. These findings suggest that the major toxic role of SNCA is related to its extracellular species and further supports a protective role of intracellular SNCA aggregation.

α-Synuclein induces alterations in adult neurogenesis in Parkinson disease models via p53-mediated repression of Notch1.

Parkinson disease is characterized by the loss of dopaminergic neurons mainly in the substantia nigra. Accumulation of α-synuclein and cell loss has been also reported in many other brain regions including the hippocampus, where it might impair adult neurogenesis, contributing to nonmotor symptoms. However, the molecular mechanisms of these alterations are still unknown. In this report we show that α-synuclein-accumulating adult rat hippocampus neural progenitors present aberrant neuronal differentiation, with reduction of Notch1 expression and downstream signaling targets. We characterized a Notch1 proximal promoter that contains p53 canonical response elements. In vivo binding of p53 represses the transcription of Notch1 in neurons. Moreover, we demonstrated that α-synuclein directly binds to the DNA at Notch1 promoter vicinity and also interacts with p53 protein, facilitating or increasing Notch1 signaling repression, which interferes with maturation and survival of neural progenitors cells. This study provides a molecular basis for α-synuclein-mediated disruption of adult neurogenesis in Parkinson disease.

Heterozygous mutations in SIX3 and SHH are associated with schizencephaly and further expand the clinical spectrum of holoprosencephaly.

Schizencephaly (SCH) is a clinically and etiologically heterogeneous cerebral malformation presenting as unilateral or bilateral hemispheric cleft with direct connection between the inner and outer liquor spaces. The SCH cleft is usually lined by gray matter, which appears polymicrogyric implying an associated impairment of neuronal migration. The majority of SCH patients are sporadic, but familial SCH has been described. An initial report of heterozygous mutations in the homeobox gene EMX2 could not be confirmed in 52 patients investigated in this study in agreement with two independent SCH patient cohorts published previously. SCH frequently occurs with additional cerebral malformations like hypoplasia or aplasia of the septum pellucidum or optic nerve, suggesting the involvement of genes important for the establishment of midline forebrain structures. We therefore considered holoprosencephaly (HPE)-associated genes as potential SCH candidates and report for the first time heterozygous mutations in SIX3 and SHH in a total of three unrelated patients and one fetus with SCH; one of them without obvious associated malformations of midline forebrain structures. Three of these mutations have previously been reported in independent patients with HPE. SIX3 acts directly upstream of SHH, and the SHH pathway is a key regulator of ventral forebrain patterning. Our data indicate that in a subset of patients SCH may develop as one aspect of a more complex malformation of the ventral forebrain, directly result from mutations in the SHH pathway and hence be considered as yet another feature of the broad phenotypic spectrum of holoprosencephaly.

Human in vitro reporter model of neuronal development and early differentiation processes.

BACKGROUND: During developmental and adult neurogenesis, doublecortin is an early neuronal marker expressed when neural stem cells assume a neuronal cell fate. To understand mechanisms involved in early processes of neuronal fate decision, we investigated cell lines for their capacity to induce expression of doublecortin upon neuronal differentiation and develop in vitro reporter models using doublecortin promoter sequences. RESULTS: Among various cell lines investigated, the human teratocarcinoma cell line NTERA-2 was found to fulfill our criteria. Following induction of differentiation using retinoic acid treatment, we observed a 16-fold increase in doublecortin mRNA expression, as well as strong induction of doublecortin polypeptide expression. The acquisition of a neuronal precursor phenotype was also substantiated by the establishment of a multipolar neuronal morphology and expression of additional neuronal markers, such as Map2, betaIII-tubulin and neuron-specific enolase. Moreover, stable transfection in NTERA-2 cells of reporter constructs encoding fluorescent or luminescent genes under the control of the doublecortin promoter allowed us to directly detect induction of neuronal differentiation in cell culture, such as following retinoic acid treatment or mouse Ngn2 transient overexpression. CONCLUSION: Induction of doublecortin expression in differentiating NTERA-2 cells suggests that these cells accurately recapitulate some of the very early events of neuronal determination. Hence, the use of reporter genes under the control of the doublecortin promoter in NTERA-2 cells will help us to investigate factors involved early in the course of neuronal differentiation processes. Moreover the ease to detect the induction of a neuronal program in this model will permit to perform high throughput screening for compounds acting on the early neuronal differentiation mechanisms.

Long-term course and mutational spectrum of spatacsin-linked spastic paraplegia.

OBJECTIVE: Hereditary spastic paraplegias (HSPs) comprise a heterogeneous group of neurodegenerative disorders resulting in progressive spasticity of the lower limbs. One form of autosomal recessive hereditary spastic paraplegia (ARHSP) with thin corpus callosum (TCC) was linked to chromosomal region 15q13-21 (SPG11) and associated with mutations in the spatacsin gene. We assessed the long-term course and the mutational spectrum of spatacsin-associated ARHSP with TCC. METHODS: Neurological examination, cerebral magnetic resonance imaging (MRI), 18fluorodeoxyglucose positron emission tomography (PET), nerve biopsy, linkage and mutation analysis are presented. RESULTS: Spastic paraplegia in patients with spatacsin mutations (n = 20) developed during the second decade of life. The Spastic Paraplegia Rating Scale (SPRS) showed severely compromised walking between the second and third decades of life (mean SPRS score, >30). Impaired cognitive function was associated with severe atrophy of the frontoparietal cortex, TCC, and bilateral periventricular white matter lesions. Progressive cortical and thalamic hypometabolism in the 18fluorodeoxyglucose PET was observed. Sural nerve biopsy showed a loss of unmyelinated nerve fibers and accumulation of intraaxonal pleomorphic membranous material. Mutational analysis of spatacsin demonstrated six novel and one previously reported frameshift mutation and two novel nonsense mutations. Furthermore, we report the first two splice mutations to be associated with SPG11. INTERPRETATION: We demonstrate that not only frameshift and nonsense mutations but also splice mutations result in SPG11. Mutations are distributed throughout the spatacsin gene and emerge as major cause for ARHSP with TCC associated with severe motor and cognitive impairment. The clinical phenotype and the ultrastructural analysis suggest a disturbed axonal transport of long projecting neurons.

Relevance of correction for rotational targeting error in functional neurosurgery.

OBJECTIVE: A prospective study is presented on the amount of targeting error that is due to rotational deviations between the atlas and the stereotactic coordinate system. MATERIALS AND METHODS: We investigated 14 volunteers with a stereotactic frame fixed to their heads by tight adhesive bands. Sagittal, coronal and axial T2-weighted MRI scans, as well as MPRage sequences, were performed. The anterior and posterior commissures and one additional point on the midline (the septum pellucidum) were determined on the axial T2-weighted images. Bilateral atlas coordinates for the subthalamic nucleus (STN), globus pallidus pars interna (GPi) and nucleus ventralis intermedius (Vim) were transformed to stereotactic frame coordinates, either without correction or by 2-point or 3-point correction. A total of 896 coordinates (x, y, z for the STN, GPi and Vim in both hemispheres) were calculated. RESULTS: Although the mean differences between the two algorithms (0.24 +/- standard deviation of 0.33 mm) were within the range of system-immanent inaccuracies in MRI-guided stereotaxy, deviations of up to 2.8 mm occurred. No significant correlation was found regarding the amount of rotational angle and the differences in x-, y-, or z-coordinates when 2-point and 3-point transformations were compared. CONCLUSIONS: The reliability of meticulous trajectory planning might be compromised significantly by using only 2-point-based correction or no calculations at all.

Adult neural progenitor cell grafts survive after acute spinal cord injury and integrate along axonal pathways.

The main rationale for cell-based therapies following spinal cord injury are: (i) replacement of degenerated spinal cord parenchyma by an axon growth supporting scaffold; (ii) remyelination of regenerating axons; and (iii), local delivery of growth promoting molecules. A potential source to meet these requirements is adult neural progenitor cells, which were examined in the present study. Fibroblast growth factor 2-responsive adult spinal cord-derived syngenic neural progenitor cells were either genetically modified in vitro to express green fluorescent protein (GFP) using retroviral vectors or prelabelled with bromodeoxyuridine (BrdU). Neural progenitor cells revealed antigenic properties of neurons and glial cells in vitro confirming their multipotency. This differentiation pattern was unaffected by retroviral transduction. GFP-expressing or BrdU-prelabelled neural progenitor cells were grafted as neurospheres directly into the acutely injured rat cervical spinal cord. Animals with lesions only served as controls. Three weeks postoperatively, grafted neural progenitor cells integrated along axonal profiles surrounding the lesion site. In contrast to observations in culture, grafted neural progenitor cells differentiated only into astro- and oligodendroglial lineages, supporting the notion that the adult spinal cord provides molecular cues for glial, but not for neuronal, differentiation. This study demonstrates that adult neural progenitor cells will survive after transplantation into the acutely injured spinal cord. The observed oligodendroglial and astroglial differentiation and integration along axonal pathways represent important prerequisites for potential remyelination and support of axonal regrowth.

High efficacy of clonal growth and expansion of adult neural stem cells.

Neural stem cells (NSCs) from the adult central nervous system are currently being investigated for their potential use in autologous cell replacement strategies. High expansion rates of NSCs in culture are crucial for the generation of a sufficient amount of cells needed for transplantation. Here, we describe efficient growth of adult NSCs in Neurobasal medium containing B27 supplement under clonal and low-density conditions in the absence of serum or conditioned medium. Expansion of up to 15-fold within 1 week was achieved on low-density NSC cultures derived from the lateral ventricle wall, the hippocampal formation, and the spinal cord of adult rats. A 27% single-cell cloning efficiency in Neurobasal/B27 combination further demonstrates its growth-promoting ability. Multipotency and nontumorgenicity of NSCs were retained despite the high rate of culture expansion. In addition, increased cell survival was obtained when Accutase, instead of trypsin, was used for enzymatic dissociation of NSC cultures. This work provides an important step toward the development of standardized protocols for highly efficient in vitro expansion of NSCs from the adult central nervous system to move more closely to the clinical use of NSCs.