Rapidly developed, optimized, and applied wastewater surveillance system for real-time monitoring of low-incidence, high-impact MPOX outbreak.

Recent MPOX viral resurgences have mobilized public health agencies around the world. Recognizing the significant risk of MPOX outbreaks, large-scale human testing, and immunization campaigns have been initiated by local, national, and global public health authorities. Recently, traditional clinical surveillance campaigns for MPOX have been complemented with wastewater surveillance (WWS), building on the effectiveness of existing wastewater programs that were built to monitor SARS-CoV-2 and recently expanded to include influenza and respiratory syncytial virus surveillance in wastewaters. In the present study, we demonstrate and further support the finding that MPOX viral fragments agglomerate in the wastewater solids fraction. Furthermore, this study demonstrates that the current, most commonly used MPOX assays are equally effective at detecting low titers of MPOX viral signal in wastewaters. Finally, MPOX WWS is shown to be more effective at passively tracking outbreaks and/or resurgences of the disease than clinical testing alone in smaller communities with low human clinical case counts of MPOX.

Vorinostat Improves Myotonic Dystrophy Type 1 Splicing Abnormalities in DM1 Muscle Cell Lines and Skeletal Muscle from a DM1 Mouse Model.

Myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy, is caused by an abnormal expansion of CTG repeats in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The expanded repeats of the DMPK mRNA form hairpin structures in vitro, which cause misregulation and/or sequestration of proteins including the splicing regulator muscleblind-like 1 (MBNL1). In turn, misregulation and sequestration of such proteins result in the aberrant alternative splicing of diverse mRNAs and underlie, at least in part, DM1 pathogenesis. It has been previously shown that disaggregating RNA foci repletes free MBNL1, rescues DM1 spliceopathy, and alleviates associated symptoms such as myotonia. Using an FDA-approved drug library, we have screened for a reduction of CUG foci in patient muscle cells and identified the HDAC inhibitor, vorinostat, as an inhibitor of foci formation; SERCA1 (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) spliceopathy was also improved by vorinostat treatment. Vorinostat treatment in a mouse model of DM1 (human skeletal actin-long repeat; HSALR) improved several spliceopathies, reduced muscle central nucleation, and restored chloride channel levels at the sarcolemma. Our in vitro and in vivo evidence showing amelioration of several DM1 disease markers marks vorinostat as a promising novel DM1 therapy.

Timing of therapy and neurodevelopmental outcomes in 18 families with pyridoxine-dependent epilepsy.

BACKGROUND: Seventy-five percent of patients with pyridoxine-dependent epilepsy due to α-aminoadipic semialdehyde dehydrogenase deficiency (PDE-ALDH7A1) suffer intellectual developmental disability despite pyridoxine treatment. Adjunct lysine reduction therapies (LRT), aimed at lowering putative neurotoxic metabolites, are associated with improved cognitive outcomes. However, possibly due to timing of treatment, not all patients have normal intellectual function. METHODS: This retrospective, multi-center cohort study evaluated the effect of timing of pyridoxine monotherapy and pyridoxine with adjunct LRT on neurodevelopmental outcome. Patients with confirmed PDE-ALDH7A1 with at least one sibling with PDE-ALDH7A1 and a difference in age at treatment initiation were eligible and identified via the international PDE registry, resulting in thirty-seven patients of 18 families. Treatment regimen was pyridoxine monotherapy in ten families and pyridoxine with adjunct LRT in the other eight. Primary endpoints were standardized and clinically assessed neurodevelopmental outcomes. Clinical neurodevelopmental status was subjectively assessed over seven domains: overall neurodevelopment, speech/language, cognition, fine and gross motor skills, activities of daily living and behavioral/psychiatric abnormalities. RESULTS: The majority of early treated siblings on pyridoxine monotherapy performed better than their late treated siblings on the clinically assessed domain of fine motor skills. For siblings on pyridoxine and adjunct LRT, the majority of early treated siblings performed better on clinically assessed overall neurodevelopment, cognition, and behavior/psychiatry. Fourteen percent of the total cohort was assessed as normal on all domains. CONCLUSION: Early treatment with pyridoxine and adjunct LRT may be beneficial for neurodevelopmental outcome. When evaluating a more extensive neurodevelopmental assessment, the actual impairment rate may be higher than the 75% reported in literature. TAKE- HOME MESSAGE: Early initiation of lysine reduction therapies adjunct to pyridoxine treatment in patients with PDE-ALDH7A1 may result in an improved neurodevelopmental outcome.

Quantitation of phosphatidylethanols in dried blood spots to determine rates of prenatal alcohol exposure in Ontario.

BACKGROUND: Estimating rates of prenatal alcohol exposure (PAE) in a population is necessary to ensure that proper medical and social supports and interventions are in place. This study sought to estimate PAE in Ontario, Canada by quantifying phosphatidylethanol (PEth) homologues in over 2000 residual neonatal dried blood spots (DBS). METHODS: A random selection of 2011 residual DBS collected over a 1-week time period were anonymized and extracted. A targeted liquid chromatography-mass spectrometry method was used to quantify 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (PEth (16:0/18:1) or POPEth), the clinically accepted biomarker, and six additional PEth homologues. A POPEth level above the United States Drug Testing Laboratories (USDTL) cutoff up to 4 weeks predelivery was indicative of PAE. All PEth homologues were correlated to one another and logistic regression was used to determine the association between PAE status and infant characteristics. RESULTS: The estimated rate of PAE in Ontario, up to the last 4 weeks of gestation, was 15.5% (POPEth >28.5 nM). Most PEth homologues were moderately to strongly correlated to one another. A low birth weight and preterm birth were both associated with PAE, while being small for gestational age had lower odds of PAE. CONCLUSIONS: The results of this study suggest that PAE may be more prevalent in Ontario than previous estimates by self-report or meconium testing. These findings support the need to consider the effectiveness of current interventions and the design of new interventions to address this significant public health issue.

Rare-disease genetics in the era of next-generation sequencing: discovery to translation.

Work over the past 25 years has resulted in the identification of genes responsible for ~50% of the estimated 7,000 rare monogenic diseases, and it is predicted that most of the remaining disease-causing genes will be identified by the year 2020, and probably sooner. This marked acceleration is the result of dramatic improvements in DNA-sequencing technologies and the associated analyses. We examine the rapid maturation of rare-disease genetic analysis and successful strategies for gene identification. We highlight the impact of discovering rare-disease-causing genes, from clinical diagnostics to insights gained into biological mechanisms and common diseases. Last, we explore the increasing therapeutic opportunities and challenges that the resulting expansion of the 'atlas' of human genetic pathology will bring.

Sewer transport conditions and their role in the decay of endogenous SARS-CoV-2 and pepper mild mottle virus from source to collection.

This study presents a comprehensive analysis of the decay patterns of endogenous SARS-CoV-2 and Pepper mild mottle virus (PMMoV) within wastewaters spiked with stool from infected patients expressing COVID-19 symptoms, and hence explores the decay of endogenous SARS-CoV-2 and PMMoV targets in wastewaters from source to collection of the sample. Stool samples from infected patients were used as endogenous viral material to more accurately mirror real-world decay processes compared to more traditionally used lab-propagated spike-ins. As such, this study includes data on early decay stages of endogenous viral targets in wastewaters that are typically overlooked when performing decay studies on wastewaters harvested from wastewater treatment plants that contain already-degraded endogenous material. The two distinct sewer transport conditions of dynamic suspended sewer transport and bed and near-bed sewer transport were simulated in this study at temperatures of 4 °C, 12 °C and 20 °C to elucidate decay under these two dominant transport conditions within wastewater infrastructure. The dynamic suspended sewer transport was simulated over 35 h, representing typical flow conditions, whereas bed and near-bed transport extended to 60 days to reflect the prolonged settling of solids in sewer systems during reduced flow periods. In dynamic suspended sewer transport, no decay was observed for SARS-CoV-2, PMMoV, or total RNA over the 35-h period, and temperature ranging from 4 °C to 20 °C had no noticeable effect. Conversely, experiments simulating bed and near-bed transport conditions revealed significant decreases in SARS-CoV-2 and total RNA concentrations by day 2, and PMMoV concentrations by day 3. Only PMMoV exhibited a clear trend of increasing decay constant with higher temperatures, suggesting that while temperature influences decay dynamics, its impact may be less significant than previously assumed, particularly for endogenous RNA that is bound to dissolved organic matter in wastewater. First order decay models were inadequate for accurately fitting decay curves of SARS-CoV-2, PMMoV, and total RNA in bed and near-bed transport conditions. F-tests confirmed the superior fit of the two-phase decay model compared to first order decay models across temperatures of 4 °C-20 °C. Finally, and most importantly, total RNA normalization emerged as an appropriate approach for correcting the time decay of SARS-CoV-2 exposed to bed and near-bed transport conditions. These findings highlight the importance of considering decay from the point of entry in the sewers, sewer transport conditions, and normalization strategies when assessing and modelling the impact of viral decay rates in wastewater systems. This study also emphasizes the need for ongoing research into the diverse and multifaceted factors that influence these decay rates, which is crucial for accurate public health monitoring and response strategies.

Effective method to mitigate impact of rain or snowmelt sewer flushing events on wastewater-based surveillance measurements.

Wastewater-based surveillance (WBS) is increasingly used for monitoring disease targets in wastewaters around the world. This study, performed in Ottawa, Canada, identifies a decrease in SARS-CoV-2 wastewater measurements during snowmelt-induced sewer flushing events. Observations first revealed a correlation between suppressed viral measurements and periods of increased sewage flowrates, air temperatures above 0 °C during winter months, and solids mass flux increases. These correlations suggest that high sewage flowrates from snowmelt events or intense precipitation events lead to the scouring of previously settled solids in sewers and the subsequent entrainment of these solids into the transported wastewaters. Collection of WBS samples during flushing events hence contains a heterogeneous mixture of solids, including resuspended solids with varying degrees of decay. Therefore flushing events can present a challenge for accurately measuring disease target viral signals when using solids-based analytical methods. This study demonstrates that resuspended solids entrained in the wastewaters during flushing events retain PMMoV signal while the SARS-CoV-2 signal is significantly reduced due to the slower decay rate of pepper mild mottle virus (PMMoV) compared to SARS-CoV-2 within wastewaters. Hence current normalization methods using PMMoV are shown to be ineffective in correcting for flushing events and the associated resuspension of settled solids, as the PMMoV signal of settled solids within sewers does not account for the differential decay rates experiences by SARS-CoV-2 signal in settled solids. Instead, this study identifies RNA to PMMoV correction factor as an effective approach to correct for flushing events and to realign SARS-CoV-2 signal with COVID-19 hospital admission rates within communities. As such, the study highlights the key physicochemical parameters necessary to identify flushing events that affect SARS-CoV-2 WBS measurements and introduces a novel RNA to PMMoV correction factor approach for solids-based analysis of SARS-CoV-2 during flushing events, enhancing the accuracy of WBS data for public health decision-making.

Impact of coagulation on SARS-CoV-2 and PMMoV viral signal in wastewater solids.

Wastewater surveillance (WWS) of SARS-CoV-2 has become a crucial tool for monitoring COVID-19 cases and outbreaks. Previous studies have indicated that SARS-CoV-2 RNA measurement from testing solid-rich primary sludge yields better sensitivity compared to testing wastewater influent. Furthermore, measurement of pepper mild mottle virus (PMMoV) signal in wastewater allows for precise normalization of SARS-CoV-2 viral signal based on solid content, enhancing disease prevalence tracking. However, despite the widespread adoption of WWS, a knowledge gap remains regarding the impact of ferric sulfate coagulation, commonly used in enhanced primary clarification, the initial stage of wastewater treatment where solids are sedimented and removed, on SARS-CoV-2 and PMMoV quantification in wastewater-based epidemiology. This study examines the effects of ferric sulfate addition, along with the associated pH reduction, on the measurement of SARS-CoV-2 and PMMoV viral measurements in wastewater primary clarified sludge through jar testing. Results show that the addition of Fe3+ concentrations in the conventional 0 to 60 mg/L range caused no effect on SARS-CoV-2 N1 and N2 gene region measurements in wastewater solids. However, elevated Fe3+ concentrations were shown to be associated with a statistically significant increase in PMMoV viral measurements in wastewater solids, which consequently resulted in the underestimation of PMMoV-normalized SARS-CoV-2 viral signal measurements (N1 and N2 copies/copies of PMMoV). The observed pH reduction from coagulant addition did not contribute to the increased PMMoV measurements, suggesting that this phenomenon arises from the partitioning of PMMoV viral particles into wastewater solids.

Corrigendum: Wastewater-based surveillance identifies start to the pediatric respiratory syncytial virus season in two cities in Ontario, Canada.

[This corrects the article DOI: 10.3389/fpubh.2023.1261165.].

Wastewater-based surveillance identifies start to the pediatric respiratory syncytial virus season in two cities in Ontario, Canada.

Introduction: Detection of community respiratory syncytial virus (RSV) infections informs the timing of immunoprophylaxis programs and hospital preparedness for surging pediatric volumes. In many jurisdictions, this relies upon RSV clinical test positivity and hospitalization (RSVH) trends, which are lagging indicators. Wastewater-based surveillance (WBS) may be a novel strategy to accurately identify the start of the RSV season and guide immunoprophylaxis administration and hospital preparedness. Methods: We compared citywide wastewater samples and pediatric RSVH in Ottawa and Hamilton between August 1, 2022, and March 5, 2023. 24-h composite wastewater samples were collected daily and 5 days a week at the wastewater treatment facilities in Ottawa and Hamilton, Ontario, Canada, respectively. RSV WBS samples were analyzed in real-time for RSV by RT-qPCR. Results: RSV WBS measurements in both Ottawa and Hamilton showed a lead time of 12 days when comparing the WBS data set to pediatric RSVH data set (Spearman's ρ = 0.90). WBS identify early RSV community transmission and declared the start of the RSV season 36 and 12 days in advance of the provincial RSV season start (October 31) for the city of Ottawa and Hamilton, respectively. The differing RSV start dates in the two cities is likely associated with geographical and regional variation in the incidence of RSV between the cities. Discussion: Quantifying RSV in municipal wastewater forecasted a 12-day lead time of the pediatric RSVH surge and an earlier season start date compared to the provincial start date. These findings suggest an important role for RSV WBS to inform regional health system preparedness, reduce RSV burden, and understand variations in community-related illness as novel RSV vaccines and monoclonal antibodies become available.

Physical Activity, Gut Microbiota, and Genetic Background for Children and Adolescents with Autism Spectrum Disorder.

It is estimated that one in 100 children worldwide has been diagnosed with autism spectrum disorder (ASD). Children with ASD frequently suffer from gut dysbiosis and gastrointestinal issues, findings which possibly play a role in the pathogenesis and/or severity of their condition. Physical activity may have a positive effect on the composition of the intestinal microbiota of healthy adults. However, the effect of exercise both on the gastrointestinal problems and intestinal microbiota (and thus possibly on ASD) itself in affected children is unknown. In terms of understanding the physiopathology and manifestations of ASD, analysis of the gut-brain axis holds some promise. Here, we discuss the physiopathology of ASD in terms of genetics and microbiota composition, and how physical activity may be a promising non-pharmaceutical approach to improve ASD-related symptoms.

Understanding the dynamic relation between wastewater SARS-CoV-2 signal and clinical metrics throughout the pandemic.

Wastewater surveillance (WWS) of SARS-CoV-2 was proven to be a reliable and complementary tool for population-wide monitoring of COVID-19 disease incidence but was not as rigorously explored as an indicator for disease burden throughout the pandemic. Prior to global mass immunization campaigns and during the spread of the wildtype COVID-19 and the Alpha variant of concern (VOC), viral measurement of SARS-CoV-2 in wastewater was a leading indicator for both COVID-19 incidence and disease burden in communities. As the two-dose vaccination rates escalated during the spread of the Delta VOC in Jul. 2021 through Dec. 2021, relations weakened between wastewater signal and community COVID-19 disease incidence and maintained a strong relationship with clinical metrics indicative of disease burden (new hospital admissions, ICU admissions, and deaths). Further, with the onset of the vaccine-resistant Omicron BA.1 VOC in Dec. 2021 through Mar. 2022, wastewater again became a strong indicator of both disease incidence and burden during a period of limited natural immunization (no recent infection), vaccine escape, and waned vaccine effectiveness. Lastly, with the populations regaining enhanced natural and vaccination immunization shortly prior to the onset of the Omicron BA.2 VOC in mid-Mar 2022, wastewater is shown to be a strong indicator for both disease incidence and burden. Hospitalization-to-wastewater ratio is further shown to be a good indicator of VOC virulence when widespread clinical testing is limited. In the future, WWS is expected to show moderate indication of incidence and strong indication of disease burden in the community during future potential seasonal vaccination campaigns.

Municipal and neighbourhood level wastewater surveillance and subtyping of an influenza virus outbreak.

Recurrent influenza epidemics and pandemic potential are significant risks to global health. Public health authorities use clinical surveillance to locate and monitor influenza and influenza-like cases and outbreaks to mitigate hospitalizations and deaths. Currently, global integration of clinical surveillance is the only reliable method for reporting influenza types and subtypes to warn of emergent pandemic strains. The utility of wastewater surveillance (WWS) during the COVID-19 pandemic as a less resource intensive replacement or complement for clinical surveillance has been predicated on analyzing viral fragments in wastewater. We show here that influenza virus targets are stable in wastewater and partitions favorably to the solids fraction. By quantifying, typing, and subtyping the virus in municipal wastewater and primary sludge during a community outbreak, we forecasted a citywide flu outbreak with a 17-day lead time and provided population-level viral subtyping in near real-time to show the feasibility of influenza virus WWS at the municipal and neighbourhood levels in near real time using minimal resources and infrastructure.

Wastewater to clinical case (WC) ratio of COVID-19 identifies insufficient clinical testing, onset of new variants of concern and population immunity in urban communities.

Clinical testing has been the cornerstone of public health monitoring and infection control efforts in communities throughout the COVID-19 pandemic. With the anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) will have greater value as an important diagnostic tool. An in-depth analysis and understanding of the metrics derived from WWS is required to interpret and utilize WWS-acquired data effectively (McClary-Gutierrez et al., 2021; O'Keeffe, 2021). In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven cities in Canada over periods ranging from 8 to 21 months. This work demonstrates that significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing (resulting in a reduction to testing access and a reduction in the number of daily tests) in these communities, despite increases in the wastewater signal. Furthermore, the WC ratio decreased significantly in 6 of the 7 studied locations, serving as a potential signal of the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized community (40-60 % allelic proportion), while a more muted decrease in the WC ratio signaled the emergence of the Delta VOC in a relatively well-immunized community (40-60 % allelic proportion). Finally, a significant decrease in the WC ratio signaled the emergence of the Omicron VOC, likely because of the variant's greater effectiveness at evading immunity, leading to a significant number of new reported clinical cases, even when community immunity was high. The WC ratio, used as an additional monitoring metric, could complement clinical case counts and wastewater signals as individual metrics in its potential ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.

COVID-19 wastewater surveillance in rural communities: Comparison of lagoon and pumping station samples.

Wastewater-based epidemiology/wastewater surveillance has been a topic of significant interest over the last year due to its application in SARS-CoV-2 surveillance to track prevalence of COVID-19 in communities. Although SARS-CoV-2 surveillance has been applied in more than 50 countries to date, the application of this surveillance has been largely focused on relatively affluent urban and peri-urban communities. As such, there is a knowledge gap regarding the implementation of reliable wastewater surveillance in small and rural communities for the purpose of tracking rates of incidence of COVID-19 and other pathogens or biomarkers. This study examines the relationships existing between SARS-CoV-2 viral signal from wastewater samples harvested from an upstream pumping station and from an access port at a downstream wastewater treatment lagoon with the community's COVID-19 rate of incidence (measured as percent test positivity) in a small, rural community in Canada. Real-time quantitative polymerase chain reaction (RT-qPCR) targeting the N1 and N2 genes of SARS-CoV-2 demonstrate that all 24-h composite samples harvested from the pumping station over a period of 5.5 weeks had strong viral signal, while all samples 24-h composite samples harvested from the lagoon over the same period were below the limit of quantification. RNA concentrations and integrity of samples harvested from the lagoon were both lower and more variable than from samples from the upstream pumping station collected on the same date, indicating a higher overall stability of SARS-CoV-2 RNA upstream of the lagoon. Additionally, measurements of PMMoV signal in wastewater allowed normalizing SARS-CoV-2 viral signal for fecal matter content, permitting the detection of actual changes in community prevalence with a high level of granularity. As a result, in sewered small and rural communities or low-income regions operating wastewater lagoons, samples for wastewater surveillance should be harvested from pumping stations or the sewershed as opposed to lagoons.

Catching a resurgence: Increase in SARS-CoV-2 viral RNA identified in wastewater 48 h before COVID-19 clinical tests and 96 h before hospitalizations.

Curtailing the Spring 2020 COVID-19 surge required sweeping and stringent interventions by governments across the world. Wastewater-based COVID-19 epidemiology programs have been initiated in many countries to provide public health agencies with a complementary disease tracking metric and non-discriminating surveillance tool. However, their efficacy in prospectively capturing resurgences following a period of low prevalence is unclear. In this study, the SARS-CoV-2 viral signal was measured in primary clarified sludge harvested every two days at the City of Ottawa's water resource recovery facility during the summer of 2020, when clinical testing recorded daily percent positivity below 1%. In late July, increases of >400% in normalized SARS-CoV-2 RNA signal in wastewater were identified 48 h prior to reported >300% increases in positive cases that were retrospectively attributed to community-acquired infections. During this resurgence period, SARS-CoV-2 RNA signal in wastewater preceded the reported >160% increase in community hospitalizations by approximately 96 h. This study supports wastewater-based COVID-19 surveillance of populations in augmenting the efficacy of diagnostic testing, which can suffer from sampling biases or timely reporting as in the case of hospitalization census.

Quantitative analysis of SARS-CoV-2 RNA from wastewater solids in communities with low COVID-19 incidence and prevalence.

In the absence of an effective vaccine to prevent COVID-19 it is important to be able to track community infections to inform public health interventions aimed at reducing the spread and therefore reduce pressures on health-care, improve health outcomes and reduce economic uncertainty. Wastewater surveillance has rapidly emerged as a potential tool to effectively monitor community infections through measuring trends of RNA signal in wastewater systems. In this study SARS-CoV-2 viral RNA N1 and N2 gene regions are quantified in solids collected from influent post grit solids (PGS) and primary clarified sludge (PCS) in two water resource recovery facilities (WRRF) serving Canada's national capital region, i.e., the City of Ottawa, ON (pop. ≈ 1.1M) and the City of Gatineau, QC (pop. ≈ 280K). PCS samples show signal inhibition using RT-ddPCR compared to RT-qPCR, with PGS samples showing similar quantifiable concentrations of RNA using both assays. RT-qPCR shows higher frequency of detection of N1 and N2 gene regions in PCS (92.7, 90.6%, n = 6) as compared to PGS samples (79.2, 82.3%, n = 5). Sampling of PCS may therefore be an effective approach for SARS-CoV-2 viral quantification, especially during periods of declining and low COVID-19 incidence in the community. The pepper mild mottle virus (PMMoV) is determined to have a less variable RNA signal in PCS over a three month period for two WRRFs, regardless of environmental conditions, compared to Bacteroides 16S rRNA or human 18S rRNA, making PMMoV a potentially useful biomarker for normalization of SARS-CoV-2 signal. PMMoV-normalized PCS RNA signal from WRRFs of two cities correlated with the regional public health epidemiological metrics, identifying PCS normalized to a fecal indicator (PMMoV) as a potentially effective tool for monitoring trends during decreasing and low-incidence of infection of SARS-Cov-2 in communities.

Electrophysiological Alterations of Pyramidal Cells and Interneurons of the CA1 Region of the Hippocampus in a Novel Mouse Model of Dravet Syndrome.

Dravet syndrome is a developmental epileptic encephalopathy caused by pathogenic variation in SCN1A To characterize the pathogenic substitution (p.H939R) of a local individual with Dravet syndrome, fibroblast cells from the individual were reprogrammed to pluripotent stem cells and differentiated into neurons. Sodium currents of these neurons were compared with healthy control induced neurons. A novel Scn1aH939R/+ mouse model was generated with the p.H939R substitution. Immunohistochemistry and electrophysiological experiments were performed on hippocampal slices of Scn1aH939R/+ mice. We found that the sodium currents recorded in the proband-induced neurons were significantly smaller and slower compared to wild type (WT). The resting membrane potential and spike amplitude were significantly depolarized in the proband-induced neurons. Similar differences in resting membrane potential and spike amplitude were observed in the interneurons of the hippocampus of Scn1aH939R/+ mice. The Scn1aH939R/+ mice showed the characteristic features of a Dravet-like phenotype: increased mortality and both spontaneous and heat-induced seizures. Immunohistochemistry showed a reduction in amount of parvalbumin and vesicular acetylcholine transporter in the hippocampus of Scn1aH939R/+ compared to WT mice. Overall, these results underline hyper-excitability of the hippocampal CA1 circuit of this novel mouse model of Dravet syndrome which, under certain conditions, such as temperature, can trigger seizure activity. This hyper-excitability is due to the altered electrophysiological properties of pyramidal neurons and interneurons which are caused by the dysfunction of the sodium channel bearing the p.H939R substitution. This novel Dravet syndrome model also highlights the reduction in acetylcholine and the contribution of pyramidal cells, in addition to interneurons, to network hyper-excitability.

Neurodevelopmental abnormalities in neurosphere-derived neural stem cells from SMN-depleted mice.

Spinal muscular atrophy (SMA) is a genetic disorder caused by depletion of survival motor neuron (SMN) protein and characterized by degeneration of alpha-motor neurons in the spinal cord. We investigated the morphology and differentiation of neurosphere-derived neural stem cells (NSCs) generated from the brains of a hypomorphic series of SMA mice. Neurospheres from the Smn(-/-);SMN2 mice, which represent a model of very severe SMA, produced NSCs with increased proliferation during growth and differentiation. These cells produced fewer Tuj1-positive neuronal cells, which displayed morphological alterations and had fewer and shorter neurites. The decrease in the number of Tuj1-positive cells was not a result of enhanced apoptosis but was accompanied by an increase in the number of nestin-positive cells. These results provide insight into the most severe model of SMA, in which SMN is nearly completely depleted, and suggest that SMN has a role in neurodevelopment as well as in neuromaintenance. Our work raises the possibility that SMN depletion affects neurodevelopment and neuromaintenance to varying extents, leading to SMA pathogenesis.

Distribution of neuronal apoptosis inhibitory protein in human tissues.

The neuronal apoptosis inhibitory protein (NAIP) gene, also known as the baculovirus inhibitor of apoptosis repeat-containing protein 1 (BIRC1) gene, is a member of the inhibitors of apoptosis (IAP) family and was first characterized as a candidate gene for spinal muscular atrophy (SMA). The expression of NAIP has been thoroughly studied in the central nervous system and overlaps the pattern of neurodegeneration in SMA. Recent studies have pointed to a role for NAIP in non-neuronal cells. We report here the production of a specific anti-NAIP antibody and the profile of NAIP expression in human adult tissues by Western blot and immunohistochemical detection methods. NAIP was detected in a number of tissues by Western blot analysis, but immunohistochemistry revealed that NAIP's presence in certain tissues, such as liver, lung, and spleen, is most likely due to macrophage infiltration. In the small intestine, the expression of NAIP coincides with the expression of p21(WAF1). This observation, coupled with findings from other groups, suggests a role for NAIP in increasing the survival of cells undergoing terminal differentiation as well as the possibility that the protein serves as an intestinal pathogen recognition protein. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.