Identification of early gene expression changes in primary cultured neurons treated with topoisomerase I poisons.

Topoisomerase 1 (TOP1) poisons like camptothecin (CPT) are currently used in cancer chemotherapy but these compounds can have damaging, off-target effects on neurons leading to cognitive, sensory and motor deficits. To understand the molecular basis for the enhanced sensitivity of neurons to CPT, we examined the effects of compounds that inhibit TOP1-CPT, actinomycin D (ActD) and β-lapachone (β-Lap)-on primary cultured rat motor (MN) and cortical (CN) neurons as well as fibroblasts. Neuronal cells expressed higher levels of Top1 mRNA than fibroblasts but transcript levels are reduced in all cell types after treatment with CPT. Microarray analysis was performed to identify differentially regulated transcripts in MNs in response to a brief exposure to CPT. Pathway analysis of the differentially expressed transcripts revealed activation of ERK and JNK signaling cascades in CPT-treated MNs. Immediate-early genes like Fos, Egr-1 and Gadd45b were upregulated in CPT-treated MNs. Fos mRNA levels were elevated in all cell types treated with CPT; Egr-1, Gadd45b and Dyrk3 transcript levels, however, increased in CPT-treated MNs and CNs but decreased in CPT-treated fibroblasts. These transcripts may represent new targets for the development of therapeutic agents that mitigate the off-target effects of chemotherapy on the nervous system

Transcriptome profiling of spinal muscular atrophy motor neurons derived from mouse embryonic stem cells.

Proximal spinal muscular atrophy (SMA) is an early onset, autosomal recessive motor neuron disease caused by loss of or mutation in SMN1 (survival motor neuron 1). Despite understanding the genetic basis underlying this disease, it is still not known why motor neurons (MNs) are selectively affected by the loss of the ubiquitously expressed SMN protein. Using a mouse embryonic stem cell (mESC) model for severe SMA, the RNA transcript profiles (transcriptomes) between control and severe SMA (SMN2+/+;mSmn-/-) mESC-derived MNs were compared in this study using massively parallel RNA sequencing (RNA-Seq). The MN differentiation efficiencies between control and severe SMA mESCs were similar. RNA-Seq analysis identified 3,094 upregulated and 6,964 downregulated transcripts in SMA mESC-derived MNs when compared against control cells. Pathway and network analysis of the differentially expressed RNA transcripts showed that pluripotency and cell proliferation transcripts were significantly increased in SMA MNs while transcripts related to neuronal development and activity were reduced. The differential expression of selected transcripts such as Crabp1, Crabp2 and Nkx2.2 was validated in a second mESC model for SMA as well as in the spinal cords of low copy SMN2 severe SMA mice. Furthermore, the levels of these selected transcripts were restored in high copy SMN2 rescue mouse spinal cords when compared against low copy SMN2 severe SMA mice. These findings suggest that SMN deficiency affects processes critical for normal development and maintenance of MNs

Evaluation of the Orally Bioavailable 4-Phenylbutyrate-Tethered Trichostatin a Analogue AR42 in Models of Spinal Muscular Atrophy

Proximal spinal muscular atrophy (SMA) is a leading genetic cause for infant death in the world and results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of SMN protein and small molecules that can increase SMN expression are of considerable interest as potential therapeutics. Previous studies have shown that both 4-phenylbutyrate (4PBA) and trichostatin A (TSA) increase SMN expression in dermal fibroblasts derived from SMA patients. AR42 is a 4PBA-tethered TSA derivative that is a very potent histone deacetylase inhibitor. SMA patient fibroblasts were treated with either AR42, AR19 (a related analogue), 4PBA, TSA or vehicle for 5 days and then immunostained for SMN localization. AR42 as well as 4PBA and TSA increased the number of SMN-positive nuclear gems in a dose-dependent manner while AR19 did not show marked changes in gem numbers. While gem number was increased in AR42-treated SMA fibroblasts, there were no significant changes in FL-SMN mRNA or SMN protein. The neuroprotective effect of this compound was then assessed in SMNΔ7 SMA (SMN2+/+;SMNΔ7+/+;mSmn−/−) mice. Oral administration of AR42 prior to disease onset increased the average lifespan of SMNΔ7 SMA mice by ~ 27% (20.1 ± 1.6 days for AR42-treated mice vs. 15.8 ± 0.4 days for vehicle-treated mice). AR42 treatment also improved motor function in these mice. AR42 treatment inhibited histone deacetylase (HDAC) activity in treated spinal cord although it did not affect SMN protein expression in these mice. AKT and GSK3β phosphorylation were both significantly increased in SMNΔ7 SMA mouse spinal cords. In conclusion, presymptomatic administration of the HDAC inhibitor AR42 ameliorates the disease phenotype in SMNΔ7 SMA mice in a SMN-independent manner possibly by increasing AKT neuroprotective signaling

State of the climate in 2018

In 2018, the dominant greenhouse gases released into Earth’s atmosphere—carbon dioxide, methane, and nitrous oxide—continued their increase. The annual global average carbon dioxide concentration at Earth’s surface was 407.4 ± 0.1 ppm, the highest in the modern instrumental record and in ice core records dating back 800 000 years. Combined, greenhouse gases and several halogenated gases contribute just over 3 W m−2 to radiative forcing and represent a nearly 43% increase since 1990. Carbon dioxide is responsible for about 65% of this radiative forcing. With a weak La Niña in early 2018 transitioning to a weak El Niño by the year’s end, the global surface (land and ocean) temperature was the fourth highest on record, with only 2015 through 2017 being warmer. Several European countries reported record high annual temperatures. There were also more high, and fewer low, temperature extremes than in nearly all of the 68-year extremes record. Madagascar recorded a record daily temperature of 40.5°C in Morondava in March, while South Korea set its record high of 41.0°C in August in Hongcheon. Nawabshah, Pakistan, recorded its highest temperature of 50.2°C, which may be a new daily world record for April. Globally, the annual lower troposphere temperature was third to seventh highest, depending on the dataset analyzed. The lower stratospheric temperature was approximately fifth lowest. The 2018 Arctic land surface temperature was 1.2°C above the 1981–2010 average, tying for third highest in the 118-year record, following 2016 and 2017. June’s Arctic snow cover extent was almost half of what it was 35 years ago. Across Greenland, however, regional summer temperatures were generally below or near average. Additionally, a satellite survey of 47 glaciers in Greenland indicated a net increase in area for the first time since records began in 1999. Increasing permafrost temperatures were reported at most observation sites in the Arctic, with the overall increase of 0.1°–0.2°C between 2017 and 2018 being comparable to the highest rate of warming ever observed in the region. On 17 March, Arctic sea ice extent marked the second smallest annual maximum in the 38-year record, larger than only 2017. The minimum extent in 2018 was reached on 19 September and again on 23 September, tying 2008 and 2010 for the sixth lowest extent on record. The 23 September date tied 1997 as the latest sea ice minimum date on record. First-year ice now dominates the ice cover, comprising 77% of the March 2018 ice pack compared to 55% during the 1980s. Because thinner, younger ice is more vulnerable to melting out in summer, this shift in sea ice age has contributed to the decreasing trend in minimum ice extent. Regionally, Bering Sea ice extent was at record lows for almost the entire 2017/18 ice season. For the Antarctic continent as a whole, 2018 was warmer than average. On the highest points of the Antarctic Plateau, the automatic weather station Relay (74°S) broke or tied six monthly temperature records throughout the year, with August breaking its record by nearly 8°C. However, cool conditions in the western Bellingshausen Sea and Amundsen Sea sector contributed to a low melt season overall for 2017/18. High SSTs contributed to low summer sea ice extent in the Ross and Weddell Seas in 2018, underpinning the second lowest Antarctic summer minimum sea ice extent on record. Despite conducive conditions for its formation, the ozone hole at its maximum extent in September was near the 2000–18 mean, likely due to an ongoing slow decline in stratospheric chlorine monoxide concentration. Across the oceans, globally averaged SST decreased slightly since the record El Niño year of 2016 but was still far above the climatological mean. On average, SST is increasing at a rate of 0.10° ± 0.01°C decade−1 since 1950. The warming appeared largest in the tropical Indian Ocean and smallest in the North Pacific. The deeper ocean continues to warm year after year. For the seventh consecutive year, global annual mean sea level became the highest in the 26-year record, rising to 81 mm above the 1993 average. As anticipated in a warming climate, the hydrological cycle over the ocean is accelerating: dry regions are becoming drier and wet regions rainier. Closer to the equator, 95 named tropical storms were observed during 2018, well above the 1981–2010 average of 82. Eleven tropical cyclones reached Saffir–Simpson scale Category 5 intensity. North Atlantic Major Hurricane Michael’s landfall intensity of 140 kt was the fourth strongest for any continental U.S. hurricane landfall in the 168-year record. Michael caused more than 30 fatalities and $25 billion (U.S. dollars) in damages. In the western North Pacific, Super Typhoon Mangkhut led to 160 fatalities and$6 billion (U.S. dollars) in damages across the Philippines, Hong Kong, Macau, mainland China, Guam, and the Northern Mariana Islands. Tropical Storm Son-Tinh was responsible for 170 fatalities in Vietnam and Laos. Nearly all the islands of Micronesia experienced at least moderate impacts from various tropical cyclones. Across land, many areas around the globe received copious precipitation, notable at different time scales. Rodrigues and Réunion Island near southern Africa each reported their third wettest year on record. In Hawaii, 1262 mm precipitation at Waipā Gardens (Kauai) on 14–15 April set a new U.S. record for 24-h precipitation. In Brazil, the city of Belo Horizonte received nearly 75 mm of rain in just 20 minutes, nearly half its monthly average. Globally, fire activity during 2018 was the lowest since the start of the record in 1997, with a combined burned area of about 500 million hectares. This reinforced the long-term downward trend in fire emissions driven by changes in land use in frequently burning savannas. However, wildfires burned 3.5 million hectares across the United States, well above the 2000–10 average of 2.7 million hectares. Combined, U.S. wildfire damages for the 2017 and 2018 wildfire seasons exceeded $40 billion (U.S. dollars)

Broad proteomics analysis of seeding-induced aggregation of α-synuclein in M83 neurons reveals remodeling of proteostasis mechanisms that might contribute to Parkinson’s disease pathogenesis

Abstract Aggregation of misfolded α-synuclein (α-syn) is a key characteristic feature of Parkinson’s disease (PD) and related synucleinopathies. The nature of these aggregates and their contribution to cellular dysfunction is still not clearly elucidated. We employed mass spectrometry-based total and phospho-proteomics to characterize the underlying molecular and biological changes due to α-syn aggregation using the M83 mouse primary neuronal model of PD. We identified gross changes in the proteome that coincided with the formation of large Lewy body-like α-syn aggregates in these neurons. We used protein-protein interaction (PPI)-based network analysis to identify key protein clusters modulating specific biological pathways that may be dysregulated and identified several mechanisms that regulate protein homeostasis (proteostasis). The observed changes in the proteome may include both homeostatic compensation and dysregulation due to α-syn aggregation and a greater understanding of both processes and their role in α-syn-related proteostasis may lead to improved therapeutic options for patients with PD and related disorders

Validation of differentially expressed transcripts determined by RNA-Seq using qRT-PCR.

<p>The levels of <i>Crabp1</i>, <i>Crabp2</i>, <i>Isl1</i>, <i>Nkx2.2</i>, <i>Pla2g1b</i>, <i>Smn1</i> and <i>Vim</i> transcripts were measured in total RNAs from control and SMA mESC-derived MNs. (<b>A</b>) The magnitude of change (log₂(fold change)) of selected transcripts in A2 SMA MNs relative to Hb9 control MNs (n = 3/genotype) as determined by RNA-Seq (black bars) or qRT-PCR (grey bars). (<b>B</b>) The magnitude of change of selected transcripts in HB9:eGFP-labeled (black bars) or unlabeled (grey bars) SMA MNs (A2 and E2 cells, respectively) relative to control MNs (Hb9 and C4 cells, respectively; n = 3/genotype).</p

Upstream regulator analysis of differentially expressed transcripts between mESC-derived Hb9 control and A2 SMA MNs.

<p>IPA of significantly modified upstream (<b>A</b>) transcriptional regulator, (<b>B</b>) endogenous signaling and (<b>C</b>) drug pathways in upregulated or downregulated transcripts in A2 SMA MNs compared to Hb9 control MNs. Significant upstream regulators were identified as those having an activation z-score greater than or equal to 2.0 for activated regulators or less than or equal to −2.0 for inhibited regulators.</p

RNA-Seq reads mapped to NCBI mouse genome build 37 using TopHat.

<p>RNA-Seq reads mapped to NCBI mouse genome build 37 using TopHat.</p

Relationship between changes in mRNA levels measured by RNA-Seq and protein levels measured by 2D-DIGE or immunoblot (*) of selected genes in normal versus SMA mESC-derived motor neurons.

<p>The protein expression data is taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106818#pone.0106818-Wu1" target="_blank">[46]</a>.</p><p>Relationship between changes in mRNA levels measured by RNA-Seq and protein levels measured by 2D-DIGE or immunoblot (*) of selected genes in normal versus SMA mESC-derived motor neurons.</p

Expression of RNA-Seq-identified differentially expressed transcripts in SMA mouse spinal cords.

<p>The levels of <i>Crabp1</i>, <i>Crabp2</i>, <i>Isl1</i>, <i>Nkx2.2</i>, <i>Pla2g1b</i>, <i>Smn1</i> and <i>Vim</i> transcripts were measured in spinal cord total RNAs from control and SMA mice. (<b>A</b>) The magnitude of change (log₂(fold change)) of selected transcripts in low copy <i>SMN2</i> SMA (<i>SMN2(89)^+/+;mSmn^−/−</i>; black bars) or high copy <i>SMN2</i> rescue (<i>SMN2(566)^+/+;mSmn^−/−</i>; grey bars) mouse spinal cord samples (n = 3/genotype) relative to control samples. (<b>B</b>) The magnitude of change of selected transcripts in low copy SMN2 SMA mouse spinal cord samples relative to controls at embryonic day 13.5 (e13.5; black bars) and postnatal day 3 (PND03; grey bars).</p