Phosphatidylserine improves axonal transport by inhibition of HDAC and has potential in treatment of neurodegenerative diseases

Familial dysautonomia (FD) is a rare children neurodegenerative disease caused due to a point mutation in the IKBKAP gene that results in decreased IKK complex-associated protein (IKAP) protein production. The disease affects mostly the dorsal root ganglion (DRG) and the sympathetic ganglion. Recently, we found that the molecular mechanisms underlying neurodegeneration in FD patients are defects in axonal transport of nerve growth factors and microtubule stability in the DRG. Neurons are highly polarized cells with very long axons. In order to survive and maintain proper function, neurons depend on transport of proteins and other cellular components from the neuronal body along the axons. We further demonstrated that IKAP is necessary for axon maintenance and showed that phosphatidylserine acts as an HDAC6 inhibitor to rescue neuronal function in FD cells. In this review, we will highlight our latest research findings

Regulation of alternative splicing through coupling with transcription and chromatin structure

Alternative precursor messenger RNA (pre-mRNA) splicing plays a pivotal role in the flow of genetic information from DNA to proteins by expanding the coding capacity of genomes. Regulation of alternative splicing is as important as regulation of transcription to determine cell- and tissue-specific features, normal cell functioning, and responses of eukaryotic cells to external cues. Its importance is confirmed by the evolutionary conservation and diversification of alternative splicing and the fact that its deregulation causes hereditary disease and cancer. This review discusses the multiple layers of cotranscriptional regulation of alternative splicing in which chromatin structure, DNA methylation, histone marks, and nucleosome positioning play a fundamental role in providing a dynamic scaffold for interactions between the splicing and transcription machineries. We focus on evidence for how the kinetics of RNA polymerase II (RNAPII) elongation and the recruitment of splicing factors and adaptor proteins to chromatin components act in coordination to regulate alternative splicing.Fil: Naftelberg S. Sackler Medical School - Tel Aviv University; IsraelFil: Schor, Ignacio Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Ast, Gil. Sackler Medical School, Tel Aviv; IsraelFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

How Are Short Exons Flanked by Long Introns Defined and Committed to Splicing?

The splice sites (SSs) delimiting an intron are brought together in the earliest step of spliceosome assembly yet it remains obscure how SS pairing occurs, especially when introns are thousands of nucleotides long. Splicing occurs in vivo in mammals within minutes regardless of intron length, implying that SS pairing can instantly follow transcription. Also, factors required for SS pairing, such as the U1 small nuclear ribonucleoprotein (snRNP) and U2AF65, associate with RNA polymerase II (RNAPII), while nucleosomes preferentially bind exonic sequences and associate with U2 snRNP. Based on recent publications, we assume that the 5′ SS-bound U1 snRNP can remain tethered to RNAPII until complete synthesis of the downstream intron and exon. An additional U1 snRNP then binds the downstream 5′ SS, whereas the RNAPII-associated U2AF65 binds the upstream 3′ SS to facilitate SS pairing along with exon definition. Next, the nucleosome-associated U2 snRNP binds the branch site to advance splicing complex assembly. This may explain how RNAPII and chromatin are involved in spliceosome assembly and how introns lengthened during evolution with a relatively minimal compromise in splicing.Fil: Hollander, Dror. Tel Aviv University; IsraelFil: Naftelberg, Shiran. Tel Aviv University; IsraelFil: Lev Maor, Galit. Tel Aviv University; IsraelFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Ast, Gil. Tel Aviv University; Israe

Phosphatidylserine Ameliorates Neurodegenerative Symptoms and Enhances Axonal Transport in a Mouse Model of Familial Dysautonomia.

Familial Dysautonomia (FD) is a neurodegenerative disease in which aberrant tissue-specific splicing of IKBKAP exon 20 leads to reduction of IKAP protein levels in neuronal tissues. Here we generated a conditional knockout (CKO) mouse in which exon 20 of IKBKAP is deleted in the nervous system. The CKO FD mice exhibit developmental delays, sensory abnormalities, and less organized dorsal root ganglia (DRGs) with attenuated axons compared to wild-type mice. Furthermore, the CKO FD DRGs show elevated HDAC6 levels, reduced acetylated α-tubulin, unstable microtubules, and impairment of axonal retrograde transport of nerve growth factor (NGF). These abnormalities in DRG properties underlie neuronal degeneration and FD symptoms. Phosphatidylserine treatment decreased HDAC6 levels and thus increased acetylation of α-tubulin. Further PS treatment resulted in recovery of axonal outgrowth and enhanced retrograde axonal transport by decreasing histone deacetylase 6 (HDAC6) levels and thus increasing acetylation of α-tubulin levels. Thus, we have identified the molecular pathway that leads to neurodegeneration in FD and have demonstrated that phosphatidylserine treatment has the potential to slow progression of neurodegeneration

Deletion of <i>IKBKAP</i> exon 20 results in grossly reduced DRG size and interruption of peripheral projections.

<p>(<b>A-J</b>) Frozen cryo cross-sections of E13.5 CKO^<i>Tyrp2</i> FD and control littermate embryos were immunostained for DNA marker Drq5 (blue), DRG markers Brn3a (pink) and Isl-1 (green), and IKAP (red). Lumbar DRG cross-sections show decreases in DRG size as indicated by the gross morphology and decreased numbers of cells expressing Brn3a and Isl-1 in CKO^<i>Tyrp2</i> FD mice compared to controls. Scale bars 250 μm for panels A and F and 50 μm for panels B-E and G-J. (<b>K</b>) Quantification of average cell counts based on Drq5 immunostaining (n = 5 per group,*p<0.05), of total DRG size (in cm³, ***p<0.001), and of the Isl-1/Drq5 ratio for the CKO^<i>Tyrp2</i> FD DRGs compared to controls at E13.5 (***p<0.001). (<b>L-Q</b>) Embryos at E13.5 were whole-mount stained for neuronal marker Tuj-1 (blue). Scale bars: panels L and M, 750 μm; panels N and O, 250 μm; panels P and Q, 100 μm. (<b>R</b>) Quantification of Tuj-1 intensity (mean ROI intensity ± SEM,), average neurite length (mean ROI length/forelimb ROI size ± SEM), and numbers of branches (mean of total branches/neurite length ± SEM) based on ImageJ analysis of whole-mount staining (n = 40 per group, *p<0.05). (<b>S</b>) DRGs from 3 month-old CKO^<i>Tyrp2</i> FD and control mice were cultured for 24 h and stained using calcein to detect neurites and Hoechst dye to detect cell bodies. (<b>T</b>) Plots of neurite lengths and total numbers of branches normalized to cells number of DRGs cultured from 3 month-old CKO^<i>Tyrp2</i> FD and control mice (n = 300 per group, ***p<0.001 and *p<0.05, respectively). Error bars represent ± SEM.</p

A suggested model for the effect of PS on impaired axonal transport in FD.

<p>Phosphatidylserine affects axonal transport and microtubule stabilization by balancing the interplay of IKAP and HDAC6 levels. IKAP is part of the Elongator complex that contains the catalytic acetyltransferase subunit of ELP3. The Elongator complex acetylates α-tubulin, which is crucial for dynein movement and polymerization of microtubules. HDAC6 destabilizes acetylated α-tubulin, and its levels are influenced by levels of IKAP and other Elongator components. Phosphatidylserine elevates IKAP levels and downregulates HDAC6 levels and thus facilitates axonal transport and microtubule stabilization.</p

IKAP is a cytoplasmic protein that interacts with α-tubulin and HDAC6.

<p>(<b>A-D</b>) Right panels: Representative western blots for IKAP, acetylated α-tubulin, and HDAC6. Left panels: Relative levels of acetylated α-tubulin in control (set to 1) and IKAP-deficient samples from (<b>A</b>) control and CKO^<i>Tyrp2</i> FD DRG extracts, (<b>B</b>) control and CKO^<i>Tyrp2</i> FD forebrains, (<b>C</b>) extracts generated from fibroblasts from normal controls and an FD patient, and (<b>D</b>) HEK 293nt controls and shIKAP cells. HSC-70 levels were used as a protein loading control. Quantifications are of three biological replicates (*p<0.05). (<b>E-F</b>) Western blots and quantification of histone H3K9ac levels in (<b>E</b>) DRGs (*p<0.05) and (<b>F</b>) forebrain CKO^<i>Tyrp2</i> FD DRGs and control DRGs (***p<0.001). (<b>G</b>) Western blots of nuclear (Nuc) and cytoplasmic (Cyt) fractions of HEK 293nt lysates. Histone H3 was present only in the nuclear fraction and α-tubulin only in the cytoplasmic fraction. (<b>H</b>) HEK 293nt cell lysate was immunoprecipitated with anti-IKAP antibody followed by immunoblot analysis for indicated proteins. Error bars represent SEM.</p

Deletion of <i>IKBKAP</i> exon 20 results in disrupted NGF axonal transport.

<p>(<b>A</b>) Schematic for the microfluidic system to track retrograde transport in DRG neurons. (<b>B</b>) Representation of retrograde axonal transport after addition of NGF-Qdot (pink). (<b>C</b>) DRG E13.5 explants were grown in microfluidic chambers, labeled NGF was added to the distal side, and bright field and fluorescent images were taken 24 h after plating. Arrows indicate transported particles. Scale bars: horizontal, 10 μm; vertical, 50 s. (<b>D</b>) The average velocities and speeds of labeled NGF were lower in CKO^<i>Tyrp2</i> FD DRGs than control DRGs (***p<0.001). Error bars represent ± SEM.</p

Phosphatidylserine elevates acetylated α-tubulin levels and rescues axonal transport.

<p>(<b>A</b>) HEK 293nt cells were treated with 200 μg/μl PS for the indicated time. Left panel: Proteins were extracted, and IKAP, acetylated α-tubulin, and HSC-70 levels were analyzed by western blot. Right panel: Quantification of 24-h data with control levels set to 1 (n = 3, *p<0.05). (<b>B</b>) Western blot of extract of HEK 293nt shIKAP cells treated with 200 μg/μl PS for 24 h. (<b>C-G</b>) PS alters NGF transport in DRG explants culture from CKO/⁺ embryos. Labeled NGF was added to the distal side of the culture, and bright field and fluorescent images were taken 24 hours after addition of PS or vehicle. (C) NGF-Qdot transport was imaged in DRG neurons upon PS treatment. The arrowheads track representative faster Q-dots along the axon of PS treatment neurons. Below is a representative kymograph demonstrating faster NGF-Qdot transport in PS-treated compared to control cells. (<b>D</b>) Mean average velocities and speeds (***p<0.001), (<b>E</b>) displacement, and (<b>F</b>) mean square displacement plotted vs. time of labeled NGF in CKO/⁺ DRG cultures treated with PS or vehicle. Error bars represent SEM. (<b>G</b>) Comparisons of the distribution profiles for instantaneous velocities show that PS treatment induces an overall shift toward faster transport velocities.</p

Generation of <i>Tyrp2-Cre;IKBKAP</i>^{FDloxP/FDloxP} (CKO^<i>Tyrp2</i> FD) mice.

<p>(<b>A</b>) Two <i>loxP</i> sequences were inserted in the introns flanking exon 20 of the <i>IKBKAP</i> gene (<i>IKBAKP</i>^{FDloxP/FDloxP} mouse, the mouse shown on the left). <i>IKBAKP</i>^{FDloxP/FDloxP} mice were mated with <i>Tyrp2-Cre</i> mice (the mouse on the right). The lower panel shows a schematic representation of the <i>IKBKAP</i>^{<i>FDloxP/FDloxP</i>} construct. <i>Cre</i> activation leads to exon 20 deletion in targeted tissues. (<b>B-E</b>) Whole-mount immunostaining using Cre (red) and Tuj-1 (green) antibodies of (B) control and (C-E) CKO^<i>Tyrp2</i> FD mice; enlargements are shown in D and E. (<b>F</b>) DNA from the indicated organs was extracted and analyzed to examine exon 20 deletion. Green arrowhead indicates removal of <i>IKBKAP</i> exon 20. (<b>G</b>) Western blot of IKAP in the lungs, DRGs, cerebellums (Cere.), and forebrains (FB) of control and CKO^<i>Tyrp2</i> FD mice. (<b>H</b>) Left panel: Photographs of CKO^<i>Tyrp2</i> FD and control littermates 10 days after birth (P10). Middle panel: Weights of CKO^<i>Tyrp2</i> FD and control mice (n = 40 per group, ***p<0.001). Right panel: Weights of brains from CKO^<i>Tyrp2</i> FD and control littermates (n = 10 per group, **p<0.01). (<b>I</b>) CKO^<i>Tyrp2</i> FD mice have brownish and swollen intestines (indicated by arrow). (<b>J</b>) Tail hanging test of three-month old CKO^<i>Tyrp2</i> FD and control littermates and plots of hindpaw gaps during tail hanging (n = 5 per group, ***p<0.001). (<b>K</b>) Hot-plate analgesia evaluation of thermal sensation and peripheral sensory nerve function of CKO^<i>Tyrp2</i> FD and control mice (n = 20 per group, *p<0.05). (<b>L</b>) qRT-PCR analysis of genes, known to be abnormally regulated in FD patients in brains (n = 3 of each group) and DRGs (pool of four mice for each group) of CKO^<i>Tyrp2</i> FD and control mice. Error bars represent ± SEM and for DRGs error bars represent technical standard deviation of three repeats.</p