SCN1A-deficient excitatory neuronal networks display mutation-specific phenotypes

Dravet syndrome is a severe epileptic encephalopathy, characterized by (febrile) seizures, behavioural problems and developmental delay. Eighty per cent of patients with Dravet syndrome have a mutation in SCN1A, encoding Nav1.1. Milder clinical phenotypes, such as GEFS+ (generalized epilepsy with febrile seizures plus), can also arise from SCN1A mutations. Predicting the clinical phenotypic outcome based on the type of mutation remains challenging, even when the same mutation is inherited within one family. This clinical and genetic heterogeneity adds to the difficulties of predicting disease progression and tailoring the prescription of anti-seizure medication. Understanding the neuropathology of different SCN1A mutations may help to predict the expected clinical phenotypes and inform the selection of best-fit treatments. Initially, the loss of Na+-current in inhibitory neurons was recognized specifically to result in disinhibition and consequently seizure generation. However, the extent to which excitatory neurons contribute to the pathophysiology is currently debated and might depend on the patient clinical phenotype or the specific SCN1A mutation. To examine the genotype-phenotype correlations of SCN1A mutations in relation to excitatory neurons, we investigated a panel of patient-derived excitatory neuronal networks differentiated on multi-electrode arrays. We included patients with different clinical phenotypes, harbouring various SCN1A mutations, along with a family in which the same mutation led to febrile seizures, GEFS+ or Dravet syndrome. We hitherto describe a previously unidentified functional excitatory neuronal network phenotype in the context of epilepsy, which corresponds to seizurogenic network prediction patterns elicited by proconvulsive compounds. We found that excitatory neuronal networks were affected differently, depending on the type of SCN1A mutation, but did not segregate according to clinical severity. Specifically, loss-of-function mutations could be distinguished from missense mutations, and mutations in the pore domain could be distinguished from mutations in the voltage sensing domain. Furthermore, all patients showed aggravated neuronal network responses at febrile temperatures compared with controls. Finally, retrospective drug screening revealed that anti-seizure medication affected GEFS+ patient- but not Dravet patient-derived neuronal networks in a patient-specific and clinically relevant manner. In conclusion, our results indicate a mutation-specific excitatory neuronal network phenotype, which recapitulates the foremost clinically relevant features, providing future opportunities for precision therapies.</p

Mutations in Potassium Channel KCND3 Cause Spinocerebellar Ataxia Type 19

OBJECTIVE: To identify the causative gene for the neurodegenerative disorder spinocerebellar ataxia type 19 (SCA19) located on chromosomal region 1p21-q21. METHODS: Exome sequencing was used to identify the causal mutation in a large SCA19 family. We then screened 230 ataxia families for mutations located in the same gene (KCND3, also known as Kv4.3) using high-resolution melting. SCA19 brain autopsy material was evaluated, and in vitro experiments using ectopic expression of wild-type and mutant Kv4.3 were used to study protein localization, stability, and channel activity by patch-clamping. RESULTS: We detected a T352P mutation in the third extracellular loop of the voltage-gated potassium channel KCND3 that cosegregated with the disease phenotype in our original family. We identified 2 more novel missense mutations in the channel pore (M373I) and the S6 transmembrane domain (S390N) in 2 other ataxia families. T352P cerebellar autopsy material showed severe Purkinje cell degeneration, with abnormal intracellular accumulation and reduced protein levels of Kv4.3 in their soma. Ectopic expression of all mutant proteins in HeLa cells revealed retention in the endoplasmic reticulum and enhanced protein instability, in contrast to wild-type Kv4.3 that was localized on the plasma membrane. The regulatory β subunit Kv channel interacting protein 2 was able to rescue the membrane localization and the stability of 2 of the 3 mutant Kv4.3 complexes. However, this either did not restore the channel function of the membrane-located mutant Kv4.3 complexes or restored it only partially. INTERPRETATION: KCND3 mutations cause SCA19 by impaired protein maturation and/or reduced channel function

Entry of Human Papillomavirus Type 16 by Actin-Dependent, Clathrin- and Lipid Raft-Independent Endocytosis

Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV) infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants) to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus) with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na+/H+ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH

Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions

The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30–60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing

Simian virus 40 depends on ER protein folding and quality control factors for entry into host cells

SummaryCell entry of Simian Virus 40 (SV40) involves caveolar/lipid raft-mediated endocytosis, vesicular transport to the endoplasmic reticulum (ER), translocation into the cytosol, and import into the nucleus. We analyzed the effects of ER-associated processes and factors on infection and on isolated viruses and found that SV40 makes use of the thiol-disulfide oxidoreductases, ERp57 and PDI, as well as the retrotranslocation proteins Derlin-1 and Sel1L. ERp57 isomerizes specific interchain disulfides connecting the major capsid protein, VP1, to a crosslinked network of neighbors, thus uncoupling about 12 of 72 VP1 pentamers. Cryo-electron tomography indicated that loss of interchain disulfides coupled with calcium depletion induces selective dissociation of the 12 vertex pentamers, a step likely to mimic uncoating of the virus in the cytosol. Thus, the virus utilizes the protein folding machinery for initial uncoating before exploiting the ER-associated degradation machinery presumably to escape from the ER lumen into the cytosol

Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells.

Cell entry of Simian Virus 40 (SV40) involves caveolar/lipid raft-mediated endocytosis, vesicular transport to the endoplasmic reticulum (ER), translocation into the cytosol, and import into the nucleus. We analyzed the effects of ER-associated processes and factors on infection and on isolated viruses and found that SV40 makes use of the thiol-disulfide oxidoreductases, ERp57 and PDI, as well as the retrotranslocation proteins Derlin-1 and Sel1L. ERp57 isomerizes specific interchain disulfides connecting the major capsid protein, VP1, to a crosslinked network of neighbors, thus uncoupling about 12 of 72 VP1 pentamers. Cryo-electron tomography indicated that loss of interchain disulfides coupled with calcium depletion induces selective dissociation of the 12 vertex pentamers, a step likely to mimic uncoating of the virus in the cytosol. Thus, the virus utilizes the protein folding machinery for initial uncoating before exploiting the ER-associated degradation machinery presumably to escape from the ER lumen into the cytosol

Muscle ultrasonography to predict survival in amyotrophic lateral sclerosis

International audienceWe assessed the prognostic value of muscle ultrasonography in 31 patients with amyotrophic lateral sclerosis (ALS) and compared it with accepted prognostic variables like functional capacity (measured with the ALSFRS-R rating scale) and muscle strength. Each patient was examined once. The following ultrasonography parameters were determined: muscle thickness, echo intensity (EI) and the presence of fasciculations. Correlations between baseline measurements, preslope values and survival were calculated. EI, disease duration, muscle strength preslope and ALSFRS-R preslope correlated with survival. Using a stepwise multivariate analysis, the combination of EI preslope and ALSFRS-R preslope was shown to have the best predictive value for survival

Mood, anxiety, and perceived quality of life in adults with epilepsy and intellectual disability

Objective: Depression and anxiety symptoms are common among patients with epilepsy, but are relatively under-researched in patients with both epilepsy and intellectual disability (ID). The aim was to investigate whether epilepsy and ID characteristics are associated with mood, anxiety, and quality of life. Materials and Methods: Adult patients with epilepsy and ID who rely on tertiary epilepsy care were included (N = 189). Mood, anxiety, and quality of life were assessed by standardized questionnaires. Epilepsy and ID characteristics were retrieved from patient charts or determined by psychometric instruments. Results: Elevated levels of depressive and anxiety symptoms were present in 21.7% and 12.7%, respectively. Anxiety was significantly associated with a focal epilepsy type and ID domain discrepancy (substantial difference between two domains of adaptive behavior), but was negatively related to seizure frequency and drug load of mood-stabilizing antiepileptic drugs. Depressive symptoms were not significantly related to epilepsy characteristics, but a severe ID and ID domain discrepancy was associated with more depressive symptoms. Quality of life was significantly worse in those with multiple seizure types and ID domain discrepancy. Conclusion: Whereas anxiety and quality of life are associated with individual epilepsy characteristics, this could not be confirmed for depressive symptoms in patients with epilepsy and ID, despite its high prevalence

A systematic review of neuropsychiatric comorbidities in patients with both epilepsy and intellectual disability

Epilepsy is a neurological condition that is particularly common in people with intellectual disability (ID). The care for people with both epilepsy and ID is often complicated by the presence of neuropsychiatric disorders, defined as psychiatric symptoms, psychiatric disorders, and behavioral problems. The aim of this study was to investigate associations between epilepsy or epilepsy-related factors and neuropsychiatric comorbidities in patients with ID and between ID and neuropsychiatric comorbidities in patients with epilepsy. We performed a systematic review of the literature, published between January 1995 and January 2015 and retrieved from PubMed/Medline, PsycINFO, and ERIC and assessed the risk of bias using the SIGN-50 methodology. Forty-two studies were identified, fifteen of which were assessed as having a low or acceptable risk-of-bias evaluation. Neuropsychiatric comorbidities were examined in relation to epilepsy in nine studies; in relation to epilepsy-related factors, such as seizure activity, seizure type, and medication in four studies; and in relation to the presence and degree of ID in five studies. We conclude that the presence of epilepsy only was not a clear determinant of neuropsychiatric comorbidity in patients with ID, although a tendency towards negative mood symptoms was identified. Epilepsy-related factors indicating a more severe form of epilepsy were associated with neuropsychiatric comorbidity as was the presence of ID as compared to those without ID in patients with epilepsy, although this should be validated in future research. A large proportion of the studies in this area is associated with a substantial risk of bias. There is a need for high quality studies using standardized methods to enable clear conclusions to be drawn that might assist in improving the quality of care for this population