Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons.

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)] and compared them with neurons from an epileptic encephalopathy (EE) patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. In contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations.

EFhd2/Swiprosin-1 is a common genetic determinator for sensation-seeking/low anxiety and alcohol addiction.

In many societies, the majority of adults regularly consume alcohol. However, only a small proportion develops alcohol addiction. Individuals at risk often show a high sensation-seeking/low-anxiety behavioural phenotype. Here we asked which role EF hand domain containing 2 (EFhd2; Swiprosin-1) plays in the control of alcohol addiction-associated behaviours. EFhd2 knockout (KO) mice drink more alcohol than controls and spontaneously escalate their consumption. This coincided with a sensation-seeking and low-anxiety phenotype. A reversal of the behavioural phenotype with ß-carboline, an anxiogenic inverse benzodiazepine receptor agonist, normalized alcohol preference in EFhd2 KO mice, demonstrating an EFhd2-driven relationship between personality traits and alcohol preference. These findings were confirmed in a human sample where we observed a positive association of the EFhd2 single-nucleotide polymorphism rs112146896 with lifetime drinking and a negative association with anxiety in healthy adolescents. The lack of EFhd2 reduced extracellular dopamine levels in the brain, but enhanced responses to alcohol. In confirmation, gene expression analysis revealed reduced tyrosine hydroxylase expression and the regulation of genes involved in cortex development, Eomes and Pax6, in EFhd2 KO cortices. These findings were corroborated in Xenopus tadpoles by EFhd2 knockdown. Magnetic resonance imaging (MRI) in mice showed that a lack of EFhd2 reduces cortical volume in adults. Moreover, human MRI confirmed the negative association between lifetime alcohol drinking and superior frontal gyrus volume. We propose that EFhd2 is a conserved resilience factor against alcohol consumption and its escalation, working through Pax6/Eomes. Reduced EFhd2 function induces high-risk personality traits of sensation-seeking/low anxiety associated with enhanced alcohol consumption, which may be related to cortex function.

Adult neurogenesis and neurite outgrowth are impaired in LRRK2 G2019S mice.

The generation and maturation of adult neural stem/progenitor cells are impaired in many neurodegenerative diseases, among them is Parkinson's disease (PD). In mammals, including humans, adult neurogenesis is a lifelong feature of cellular brain plasticity in the hippocampal dentate gyrus (DG) and in the subventricular zone (SVZ)/olfactory bulb system. Hyposmia, depression, and anxiety are early non-motor symptoms in PD. There are parallels between brain regions associated with non-motor symptoms in PD and neurogenic regions. In autosomal dominant PD, mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are frequent. LRRK2 homologs in non-vertebrate systems play an important role in chemotaxis, cell polarity, and neurite arborization. We investigated adult neurogenesis and the neurite development of new neurons in the DG and SVZ/olfactory bulb system in bacterial artificial chromosome (BAC) human Lrrk2 G2019S transgenic mice. We report that mutant human Lrrk2 is highly expressed in the hippocampus in the DG and the SVZ of adult Lrrk2 G2019S mice. Proliferation of newly generated cells is significantly decreased and survival of newly generated neurons in the DG and olfactory bulb is also severely impaired. In addition, after stereotactic injection of a GFP retrovirus, newly generated neurons in the DG of Lrrk2 G2019S mice exhibited reduced dendritic arborization and fewer spines. This loss in mature, developed spines might point towards a decrease in synaptic connectivity. Interestingly, physical activity partially reverses the decrease in neuroblasts observed in Lrrk2 G2010S mice. These data further support a role for Lrrk2 in neuronal morphogenesis and provide new insights into the role of Lrrk2 in adult neurogenesis.

Frequency and phenotype of SPG11 and SPG15 in complicated hereditary spastic paraplegia.

BACKGROUND: Hereditary spastic paraplegias (HSP) are clinically and genetically highly heterogeneous. Recently, two novel genes, SPG11 (spatacsin) and SPG15 (spastizin), associated with autosomal recessive HSP, were identified. Clinically, both are characterised by complicated HSP and a rather similar phenotype consisting of early onset spastic paraplegia, cognitive deficits, thin corpus callosum (TCC), peripheral neuropathy and mild cerebellar ataxia. OBJECTIVE: To compare the frequency of SPG11 and SPG15 in patients with early onset complicated HSP and to further characterise the phenotype of SPG11 and SPG15. RESULTS: A sample of 36 index patients with early onset complicated HSP and a family history compatible with autosomal recessive inheritance was collected and screened for mutations in SPG11 and SPG15. Overall frequency of SPG11 was 14% (5/36) but was considerably higher in patients with TCC (42%). One patient with mental retardation and thinning of the corpus callosum was compound heterozygous for two novel SPG15 mutations. Additionally, several new polymorphisms and sequence variants of unknown significance have been identified in the SPG15 gene. CONCLUSIONS: TCC seems to be the best phenotypic predictor for SPG11 as well as SPG15. No clinical features could discriminate between SPG11 and SPG15. Therefore, priority of genetic testing should be driven by mutation frequency that appears to be substantially higher in SPG11 than in SPG15.

Glyc-oxidized LDL impair endothelial function more potently than oxidized LDL: role of enhanced oxidative stress.

Hypercholesterolemia is associated with impairment of endothelial function due to increased levels of LDL. In diabetic patients, however, attenuation of endothelial function occurs even under normocholesterolemic conditions. Here we assessed whether glycation of LDL potentiates their influence on endothelial function, with particular emphasis on the oxidizability of LDL and the role of O2-. Human LDL was glycated by dialyzation for 7 days against buffer containing 200 mmol/l glucose, or sham-treated without glucose, and oxidized by incubation with Cu2+. Glycation significantly enhanced the oxidizability of LDL, as detected by diene formation and by electrophoretic mobility (27.5 mm for oxidized LDL vs. 34 mm for oxidized glycated LDL at 20 h of oxidation). Isolated rings of rabbit aorta were superfused with physiological salt solution, and isometric tension was recorded. Incubation of the aortic rings with sham-treated or with glycated LDL, not oxidized, had no influence on acetylcholine-induced, endothelium-dependent relaxation. Exposure of the aortic rings to oxidized non-glycated LDL caused a significant inhibition (30% at 1 microM acetylcholine) of the endothelium-dependent relaxation only in the presence of diethyl-dithiocarbamate (DDC), an inhibitor of the endogenous superoxide dismutase (SOD). Incubation of aortic rings with oxidized glycated LDL attenuated endothelium-dependent relaxation even in the absence of DDC (by 31% at 1 microM acetylcholine). The presence of DDC potentiated the inhibition of relaxation (65% inhibition at 1 microM acetylcholine), and co-incubation with exogenous SOD and catalase prevented the inhibition of relaxation, indicating a mediator role of O2-. Endothelium-independent relaxation induced by forskolin was unaffected by any of the lipoproteins. Using a chemiluminescence assay, significantly increased O2- production of aortic rings pretreated with oxidized glycated LDL (4101 +/- 360 counts/s) in comparison to control rings (753 +/- 81 counts/s) or arteries pretreated with oxidized non-glycated LDL (2358 +/- 169 counts/s) could be detected, suggesting that enhanced NO-inactivation by O2- could be the underlying mechanism for the stronger impairment of endothelium-dependent dilations by oxidized glycated LDL. Glycation increases the oxidizability of LDL and potentiates its endothelium-damaging influence. The likely mechanism for attenuation of endothelium-dependent dilations is increased formation of O2-, resulting in inactivation of nitric oxide. This mechanism may play an important role in diabetic patients and may contribute to disturbed organ perfusion.

Workshop II: "neuroprotection"--the Lugano consensus.

Consensus could be reached that there is overwhelming evidence of preclinical neuroprotection. However, the evidence of neuroprotection/neurorescue under clinical conditions is limited. Lessons from clinical trials designed to show neuroprotection (selegiline, amantadine, dopamine agonists) demonstrate that with the drugs available neuroprotection/neurorescue has to start as early as possible. A PET-controlled clinical trial with ropinirole shows that there seems to be a good chance for neuroprotection in the early phase of Parkinson's disease in patients treated from the very beginning of the disease while there is no such benefit in patients with a late start of a neuroprotective therapeutic strategy. Also long-term neuroprotection cannot be reached. Complicating factors to demonstrate clinical neuroprotection are discussed.

Impaired olfactory bulb neurogenesis depends on the presence of human wild-type alpha-synuclein.

Synucleinopathies including Parkinson's disease (PD) are characterized by the accumulation of alpha-synuclein (α-syn) within neural cell bodies and their processes. Transgenic mice overexpressing human wild-type or mutant forms of α-syn under the control of different promoters were developed to analyse the underlying neuropathology of PD. One of the earliest clinical symptoms associated with PD is olfactory impairment. The generation of new neurons persists up to adulthood in mammals, in particular the olfactory bulb (OB). In order to assess this process in relation to α-syn accumulation, we used mice overexpressing human wild-type α-syn under the regulatable control (tet-off) of the calcium/calmodulin-dependent protein kinase IIα-promoter (CaMKII). We observed a decrease in OB neurogenesis in transgenic animals compared to controls using 5-bromo-2'-deoxyuridine (BrdU) to label newly generated cells (neuron-specific nuclear protein; NeuN). After cessation of transgene expression we detected an increase in newly generated cells both in granular (GCL) and glomerular (GLOM) layers of the OB. This led to a rescue of newly generated neurons (BrdU(+)/NeuN(+)) within the GLOM with a distinct specificity for the dopaminergic subpopulation. In contrast, we did not detect a cell-specific rescue of neuronal cells in the GCL suggesting diverse effects of alpha-synucleinopathy in both interneuronal layers of the OB. Colabelling of BrdU with glial markers showed that a differentiation into neither astroglia nor microglia attributed to the observed phenotype in the GCL. In particular, BrdU(+) particles located within microglial cells were predominantly associated close to the membrane therefore the resembling phagocytosed nuclear fragments of BrdU(+) cells. Thus, our study further contributes insights into α-syn accumulation as a causative player in the impairment of adult neurogenesis and emphasizes its diverse role in cell renewal of distinct OB cell layers.

The Spastic Paraplegia Rating Scale (SPRS): a reliable and valid measure of disease severity.

OBJECTIVE: To develop and evaluate a clinical Spastic Paraplegia Rating Scale (SPRS) to measure disease severity and progression. METHODS: A 13-item scale was designed to rate functional impairment occurring in pure forms of spastic paraplegia (SP). Additional symptoms constituting a complicated form of SP are recorded in an inventory. Two independent patient cohorts were evaluated in a two-step validation procedure. RESULTS: Application of SPRS requires less than 15 minutes and does not require any special equipment, so it is suitable for an outpatient setting. Interrater agreement of SPRS was high (intraclass correlation coefficient = 0.99). Reliability was further supported by high internal consistency (Cronbach alpha = 0.91). SPRS values were almost normally distributed without apparent floor or ceiling effect. Construct validity was shown by high correlation of SPRS to Barthel Index and the International Cooperative Ataxia Rating Scale (convergent validity) and low correlation to Mini-Mental Status Examination (discriminant validity). CONCLUSION: The Spastic Paraplegia Rating Scale is a reliable and valid measure of disease severity.

Further clinical and genetic characterization of SPG11: hereditary spastic paraplegia with thin corpus callosum.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders leading to progressive spasticity of the lower limbs. Clinically, HSPs are divided into "pure" and "complicated" forms. In pure HSP, the spasticity of the lower limbs is the sole symptom, whereas in complicated forms additional neurological and non-neurological features are observed. Genetically, HSPs are divided into autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) forms. Up to date, 30 different HSPs are linked to different chromosomal loci and 11 genes could be defined for AR-HSP, AD-HSP and XL-HSP. SPG11, an AR-HSP (synonym: HSP11), is a complicated HSP associated with a slowly progressive spastic paraparesis, mental impairment and the development of a thin corpus callosum (TCC) during the course of the disease. SPG11 has been previously linked to chromosomal region 15q13 - 15. First, we applied rigid diagnostic criteria to systematically examine 20 Turkish families with autosomal recessive HSP for characteristic features of SPG11. We detected four large Turkish families with AR-HSP and TCC consistent with SPG11. Subsequent genetic linkage analysis of those 4 families refines the SPG11 locus further down to a small region of 2.93 cM with a maximum lod score of 11.84 at marker D15S659 and will guide further candidate gene analysis.

Novel truncating and missense mutations of the KCC3 gene associated with Andermann syndrome.

BACKGROUND: Andermann syndrome (OMIM 218000) is an autosomal recessive motor-sensory neuropathy associated with developmental and neurodegenerative defects. The cerebral MRI reveals a variable degree of agenesis of the corpus callosum. Recently, truncating mutations of the KCC3 gene (also known as SLC12A6) have been associated with Andermann syndrome. METHODS: The authors assessed clinically and genetically three isolated cases from Germany and Turkey with symptoms consistent with Andermann syndrome. RESULTS: The authors detected four novel mutations within the KCC3 gene in their patients: two different truncating mutations in the first patient, a homozygous truncating mutation in the second, and a homozygous missense mutation in the third patient. In contrast to the classic phenotype of the Andermann syndrome linked to truncating KCC3 mutations the phenotype and the course of the disease linked to the missense mutation appeared to be different (i.e., showing additional features like diffuse and widespread white matter abnormalities). CONCLUSIONS: Not only truncating but also missense mutations of the KCC3 gene are associated with Andermann syndrome. Different types of KCC3 mutations may determine different clinical phenotypes.

Impairment of endothelial function induced by glyc-oxidized lipoprotein a [Lp(a)].

UNLABELLED: Diabetic patients develop endothelial dysfunction early in the course of the disease. Atherogenic lipoproteins such as LDL and Lp(a) are important risk factors for endothelial dysfunction and undergo nonenzymatic glycation in hyperglycaemia. Here we assessed whether glycation of Lp(a) potentiates its damaging influence on endothelial function. Human Lp(a) was glycated by dialyzation for 7 days against buffer containing 200 mmol/l glucose, or sham-treated without glucose and oxidized by incubation with Cu++. The degree of glycation accounted to 32 +/- 4%, and glycation rendered Lp(a) more susceptible to oxidative modification when exposed to Cu++. Isolated rings of rabbit aorta were superfused with physiological salt solution, and isometric tension was recorded. Incubation of the aortic rings with sham-treated or with 30 microg/ml glycated Lp(a), not oxidized, had no influence on acetylcholine-induced, endothelium-dependent relaxation. Exposure of the aortic rings to 30 microg/ml oxidized non-glycated (ox) Lp(a) caused a significant inhibition (19% at 1 microM acetylcholine) of the endothelium-dependent relaxation. Incubation of aortic rings with 30 microg/ml oxidized glycated (glyc-ox) Lp(a) attenuated endothelium-dependent relaxation more potently than oxLp(a) (by 34% at 1 microM acetylcholine). The presence of diethyl-dithio-carbamate (DDC), an inhibitor of the endogenous superoxide dismutase (SOD), potentiated the inhibition of relaxation induced by oxLp(a) and by glyc-oxLp(a) [38% inhibition at 1 microM acetylcholine for oxLp(a), and 49% inhibition at 1 microM acetylcholine for glyc-oxLp(a)]. Co-incubation with the O2- scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt (TIRON) prevented the inhibition of relaxation by the oxidized lipoproteins, suggesting that enhanced NO-inactivation by O2- could be the underlying mechanism for the impairment of endothelium-dependent dilations by ox- and glyc-oxLp(a). The concentration of lysophosphatidycholine, a lipoprotein oxidation product and stimulus for O2- formation, was significantly enhanced in oxLp(a) and in glyc-oxLp(a) compared to native lipoproteins. CONCLUSION: Glycation enhances the endothelium-damaging influence of oxLp(a), presumably by enhancing oxidative stress. The likely mechanism for attenuation of endothelium-dependent dilations is increased formation of O2-, resulting in inactivation of nitric oxide. This mechanism may play an important role in diabetic patients and may contribute to disturbed organ perfusion.

Long-term stabilization in patients with malignant glioma after treatment with liposomal doxorubicin.

BACKGROUND: Resistance to chemotherapeutic agents and poor blood-brain barrier penetration are major limitations in the treatment of malignant glioma. To improve drug delivery across the blood-brain barrier, the authors used doxorubicin as liposomal encapsulated formulation (Caelyx, Scheringh-Plough, Munich, Germany) in therapy of recurrent malignant glioma. METHODS: Fifteen patients with recurrent high-grade gliomas were included in the study. Of these, 13 patients could be evaluated, including 6 patients with glioblastoma, 1 patient with gliosarcoma and 6 patients with anaplastic astrocytoma. The treatment consisted of liposomal doxorubicin (20 mg/m(2)), applied intravenously every 2 weeks. RESULTS: Stabilization of the disease was observed in 54% (7 of 13) of patients. Partial response and complete response (CR) were not observed. Median time-to-progression was 11 weeks. Progression free survival at 12 months was 15%. Median overall survival (OS) after doxorubicin therapy was 40.0 weeks, whereas the median OS after diagnosis reached 20.0 months (87.0 weeks). Doxorubicin was well tolerated, with main side effects being palmoplantar erythrodysesthesia occurring in 38% and myelotoxicity (World Health Organization Grade 3-4) in 31% of the patients. CONCLUSIONS: Doxorubicin has been shown to be a safe treatment with moderate activity that may lead to long-term stabilization in recurrent high-grade glioma patients. Of note, median OS after all and after initiation of recurrence therapy was prolonged in comparison with the OS in other Phase II studies, as recently described by Wong et al. (Wong ET, Hess KR, Gleason MJ, Jaeckle KA, Kyritsis AP, Prados MD, et al. Outcomes and prognostic factors in recurrent glioma patients enrolled onto phase II clinical trials. J Clin Oncol 1999;17:2572.).