INVESTIGATING THE MECHANISMS OF p35-MEDIATED NEURODEGENERATION AND ITS EFFECTS ON AGING

Aging and adult-onset neurodegenerative diseases (NDDs) share multiple cellular phenotypes stemming from dysfunction of an overlapping set of molecular mechanisms; however, aging by itself fails to cause neurodegeneration in all individuals, indicating that NDDs are the result of more than brain aging alone. Thus, while aging is the greatest risk factor for NDDs, a complete understanding of NDD etiology and pathogenesis will require the full elucidation of the relationship between aging and degeneration. My thesis project investigates this relationship by probing the mechanisms underlying a model of adult-onset neurodegeneration in Drosophila. In humans, cyclin dependent kinase 5 (Cdk5) has been linked to multiple NDDs, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Cdk5 activity requires binding an activator subunit; in Drosophila, the sole activator is the neuron-specific p35. We develop a comprehensive metric for physiological age in Drosophila based on genome-wide expression profiling, and show that loss (mutant) or overexpression (OE) of p35 in young, presymptomatic flies accelerates the intrinsic rate of aging of brain and thorax tissue. Gene ontology analysis revealed that biological processes affected by altered p35 levels are also affected by aging. Further, modulation of p35 leads to degenerative phenotypes including impaired autophagy, loss of central brain neurons, motor defects, and shortened lifespan, all of which are observed in human ND diseases. These findings suggest that neuronal dysfunction can cause accelerated aging, which then acts as a driving mechanism to induce neurodegeneration. In parallel work, we also investigated the regulatory role of p35 in regards to the axon initial segment (AIS) and its potential link to neurodegeneration. p35 activity modulates the size of the AIS of mushroom body (MB) neurons, and deletion of p35 results in swelling of the proximal axon in the vicinity of the AIS; furthermore, p35-mutant flies exhibited an accelerated loss of MB neurons. Thus, we sought to investigate whether perturbation of the AIS contributed to p35-mediated neurodegeneration. We identified a novel regulator that robustly modulates the AIS independently of p35, and showed that shortening of the AIS may be sufficient to induce MB neurodegeneration. Taken together, our results suggest p35-mediated neurodegeneration results from accelerated aging effects in combination with cell-autonomous neuronal insults. These data fundamentally recast our picture of the relationship between neurodegenerative processes and their most prominent risk factor, natural aging, and have profound implications for identifying which aspects of NDDs could be productive targets for therapy

Ontogeny-Driven rDNA Rearrangement, Methylation, and Transcription, and Paternal Influence

Gene rearrangement occurs during development in some cell types and this genome dynamics is modulated by intrinsic and extrinsic factors, including growth stimulants and nutrients. This raises a possibility that such structural change in the genome and its subsequent epigenetic modifications may also take place during mammalian ontogeny, a process undergoing finely orchestrated cell division and differentiation. We tested this hypothesis by comparing single nucleotide polymorphism-defined haplotype frequencies and DNA methylation of the rDNA multicopy gene between two mouse ontogenic stages and among three adult tissues of individual mice. Possible influences to the genetic and epigenetic dynamics by paternal exposures were also examined for Cr(III) and acid saline extrinsic factors. Variables derived from litters, individuals, and duplicate assays in large mouse populations were examined using linear mixed-effects model. We report here that active rDNA rearrangement, represented by changes of haplotype frequencies, arises during ontogenic progression from day 8 embryos to 6-week adult mice as well as in different tissue lineages and is modifiable by paternal exposures. The rDNA methylation levels were also altered in concordance with this ontogenic progression and were associated with rDNA haplotypes. Sperm showed highest level of methylation, followed by lungs and livers, and preferentially selected haplotypes that are positively associated with methylation. Livers, maintaining lower levels of rDNA methylation compared with lungs, expressed more rRNA transcript. In vitro transcription demonstrated haplotype-dependent rRNA expression. Thus, the genome is also dynamic during mammalian ontogeny and its rearrangement may trigger epigenetic changes and subsequent transcriptional controls, that are further influenced by paternal exposures

Altered expression of the Cdk5 activator-like protein, Cdk5α, causes neurodegeneration, in part by accelerating the rate of aging

Aging is the greatest risk factor for neurodegeneration, but the connection between the two processes remains opaque. This is in part for want of a rigorous way to define physiological age, as opposed to chronological age. Here, we develop a comprehensive metric for physiological age in Drosophila, based on genome-wide expression profiling. We applied this metric to a model of adult-onset neurodegeneration, increased or decreased expression of the activating subunit of the Cdk5 protein kinase, encoded by the gene Cdk5α, the ortholog of mammalian p35. Cdk5α-mediated degeneration was associated with a 27-150% acceleration of the intrinsic rate of aging, depending on the tissue and genetic manipulation. Gene ontology analysis and direct experimental tests revealed that affected age-associated processes included numerous core phenotypes of neurodegeneration, including enhanced oxidative stress and impaired proteostasis. Taken together, our results suggest that Cdk5α-mediated neurodegeneration results from accelerated aging, in combination with cell-autonomous neuronal insults. These data fundamentally recast our picture of the relationship between neurodegeneration and its most prominent risk factor, natural aging

Sex differences in vocal communication of freely interacting adult mice depend upon behavioral context.

Ultrasonic vocalizations (USVs) are believed to play a critical role in mouse communication. Although mice produce USVs in multiple contexts, signals emitted in reproductive contexts are typically attributed solely to the male mouse. Only recently has evidence emerged showing that female mice are also vocally active during mixed-sex interactions. Therefore, this study aimed to systematically quantify and compare vocalizations emitted by female and male mice as the animals freely interacted. Using an eight-channel microphone array to determine which mouse emitted specific vocalizations during unrestrained social interaction, we recorded 13 mixed-sex pairs of mice. We report here that females vocalized significantly less often than males during dyadic interactions, with females accounting for approximately one sixth of all emitted signals. Moreover, the acoustic features of female and male signals differed. We found that the bandwidths (i.e., the range of frequencies that a signal spanned) of female-emitted signals were smaller than signals produced by males. When examining how the frequency of each signal changed over time, the slopes of male-emitted signals decreased more rapidly than female signals. Further, we revealed notable differences between male and female vocal signals when the animals were performing the same behaviors. Our study provides evidence that a female mouse does in fact vocalize during interactions with a male and that the acoustic features of female and male vocalizations differ during specific behavioral contexts

HIV-1 Nef Down-Modulates C-C and C-X-C Chemokine Receptors via Ubiquitin and Ubiquitin-Independent Mechanism

<div><p>Human and Simian Immunodeficiency virus (HIV-1, HIV-2, and SIV) encode an accessory protein, Nef, which is a pathogenesis and virulence factor. Nef is a multivalent adapter that dysregulates the trafficking of many immune cell receptors, including chemokine receptors (CKRs). Physiological endocytic itinerary of agonist occupied CXCR4 involves ubiquitinylation of the phosphorylated receptor at three critical lysine residues and dynamin-dependent trafficking through the ESCRT pathway into lysosomes for degradation. Likewise, Nef induced CXCR4 degradation was critically dependent on the three lysines in the C-terminal -SSLKILSKGK- motif. Nef directly recruits the HECT domain E3 ligases AIP4 or NEDD4 to CXCR4 in the resting state. This mechanism was confirmed by ternary interactions of Nef, CXCR4 and AIP4 or NEDD4; by reversal of Nef effect by expression of catalytically inactive AIP4-C830A mutant; and siRNA knockdown of AIP4, NEDD4 or some ESCRT-0 adapters. However, ubiquitinylation dependent lysosomal degradation was not the only mechanism by which Nef downregulated CKRs. Agonist and Nef mediated CXCR2 (and CXCR1) degradation was ubiquitinylation independent. Nef also profoundly downregulated the naturally truncated CXCR4 associated with WHIM syndrome and engineered variants of CXCR4 that resist CXCL12 induced internalization via an ubiquitinylation independent mechanism.</p></div

Subcellular distribution of CXCR4-YFP in HeLa transfectants co-expressing Nef-Cer or Cer. Host endo-lysosomal markers, E3 ubiquitin ligases AIP4 and NEDD4 and HRS were identified by primary antibody staining (as described under Methods) followed by Alex-647 conjugated secondary antibodies [47].

<p>Confocal images corresponding to Nef-Cer and Cer transfectants co-expressing CXCR4-YFP are shown pairwise with co-staining for clathrin, EEA1, AP2, LAMP and CD63 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086998#pone-0086998-g006" target="_blank">Figure <b>6A</b></a>. Similar results for costaining AIP4, NEDD4 and HRS are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086998#pone-0086998-g006" target="_blank">Figure <b>6B</b></a>. Individual channels corresponding to the respective cellular proteins (R), CXCR4-YFP (G) and Nef-CerFP or Cer (B) fluorescence are shown below the composite RGB images. 4-X cropped images of Nef-Cer transfectants are shown in the top row of each figure, with the arrows denoting colocalization of the respective indicated proteins. 7.5 or 10 µm scale bars are shown. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086998#s3" target="_blank">Results</a> are representative of three independent experiments.</p

HIV Nef inhibits agonist mediated chemokine receptor internalization.

<p>Agonist dose response of wt or WM (WHIM syndrome) CXCR4 (<b>A1</b>), CCR2B or CCR5 (<b>A2</b>) clearance from the plasma membranes of Jurkat (CXCR4), CEM (CXCR4 or CCR5), K562 (wt or WM CXCR4) cells, fresh PBMCs (CXCR4), or monocytes (CCR2B or CXCR4) in the context of Nef expression. Cells were nucleofected (Amaxa Subdivision, Lonza Corp.) or not with a mixture of plasmids for GFP (for PBMCs) or CD8 (CEM, Jurkat and monocytes) and wt, some Nef mutants or null plasmids. In each case, ∼1×10⁶ transfected cells were treated in duplicate for 20 min with the indicated concentrations of CXCL12, CCL2 or CCL5. MFVs of CXCR4, CCR2B or CCR5 were determined by FACS analysis. Data are shown as relative mean fluorescent values (MFV) (%) of untreated sample(s) as a function of agonist concentration. The relative % downregulation was calculated after assigning receptor MFV in the absence of agonist to 100% for Nef (−) or Nef (+) cells. MFV data analysis was limited to GFP or CD8 gated cells. K562 cells were nucleofected with wt or WM CXCR4 and a bicistronic IRES plasmid encoding wt Nef or a null mutant and GFP. Data for Nef (−) & GFP (+) and Nef (+) & GFP (+) K562 cells expressing wt (top) or WM CXCR4 (bottom) were separately analyzed. For each transfection, data (in duplicate) for each cell population were used to fit a polynomial regression curve with standard deviation (n = 4). In A1 & A2, *** represents p<0.001, **p<0.01 when mean is compared with plasmid transfected cells. For experiments with Nef (−) K562 cells, ** represents p<0.01 when GFP positive cells were compared to GFP negative cells. Nef did not significantly enhance the intrinsic (non-agonist driven) internalization rates of CXCR4 (<b>B1</b>) or CCR5 (<b>B2</b>). CEM-NKR cell line expressing CCR5 and CXCR4 was nucleofected with GFP and Nef or null plasmid. At 16 h post-nucleofection, cells were stained (at 1×10⁷cells/ml) in RPMI with 2% FBS and containing unlabeled CCR5 (2D7) or CXCR4 (12G5) mAb at 4°C for 15 min. They were then shifted to 37°C, and left untreated or treated with 100 nM CCL5 or CXCL12 (vector and GFP co-transfectants only) at 37°C. At each indicated time point, aliquots were shifted to 4°C, washed thrice with 10× volumes of RPMI and the amount of bound antibody at the cell surface visualized and quantified in a flow cytometer after staining with Alexa 647 conjugated goat anti-mouse antibody (Invitrogen Corp). Each point (for GFP gated cells) is the mean of duplicate MFVs, expressed relative to MFV at time zero, which was arbitrarily set in each case to 100%. The MFV plots of GFP gated cells represent averaged results of three experiments. (*** indicates p<0.05 compared to vector transfected cells for all time points after 15 min). All data in this figure are presented as mean ± standard deviation.</p

Nef downregulated cell surface expression of wt and C-terminally truncated CCR2B, CXCR1 and CXCR2 receptors in T cell line and epithelial cells.

<p>A) C-terminal sequence coordinates of CCR2B, CXCR1 and CXCR2 are shown with the arrows denoting the C-termini of the respective truncation mutants. <b>B1</b>) Nef effect on wt and C-terminally truncated derivatives of CCR2B, CXCR1 and CXCR2 was evaluated in Jurkat, CHO and K562 cells. Cells were co-transfected (nucleofection of Jurkat and K562 cells and lipofection of CHO cells) with expression plasmids for the indicated receptors and IRES plasmids encoding Nef and GFP or a null Nef mutant and GFP (vector). For all the comparisons of receptor levels between the Nef and plasmid transfected cells, the p value was less than 0.05 (**). <b>B2</b>) CD4 expression plasmid was introduced in CHO and K562 transfectants to monitor Nef effect (***p<0.01). Cell surface expression of receptors was analyzed and data presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086998#pone-0086998-g003" target="_blank">Figure <b>3 B</b></a> (n = 4).</p

Cell surface expression of C-terminally truncated CXCR4 mutants and CXCR4/CCR5 chimeras were downregulated by Nef almost as well if not better than their wild type counterparts.

<p>A) C-terminal sequence coordinates of the chemokine receptors (i) C-terminal sequence of wt CXCR4 and selected C-terminally truncated mutants, including the natural WHIM mutant, numbers within parentheses denote the position of respective deletion; (ii) C-terminal sequence of wt CCR5 and CCR5/CXCR4 chimeras swapping the respective C-terminal sequence. The swap position(s) are denoted on the left. In each case, CCR5 sequence is underlined. <b>B</b>) Nef effect on genetically engineered CXCR4 mutants, the naturally occurring WHIM mutant (WM CXCR4) and chimeras (X4R5 and R5X4) was evaluated in CHO cells or CXCR4 negative K562 cell line. CD4 was cotransfected in each case to monitor Nef effect (<b>B2</b>). Cells were transfected with an IRES plasmid encoding GFP and Nef or the null mutant. Expression of CXCR4 or CCR5 (in the case R5X4 chimera) and CD4 in GFP gated cells was evaluated by flow cytometry. Average MFVs for CXCR4 (and CCR5 for R5X4 transfection) and CD4 on null and Nef (+) cells are plotted in the histograms (with standard deviation) for CXCR4 (and CCR5) expression in the left panel and for CD4 in the right panel. MFVs for Nef (−) cells were set to 100 (n = 4). *** represents p<0.01) when Nef transfected cells are compared to plasmid transfected controls. Black bars indicate relative % downregulation in Nef (wt or mutant) expressers relative to control cells transfected with empty vector.</p

Figure 7

<p>siRNA knockdown of AP2, E3 ubiquitin ligases, AIP4 and NEDD4 and Hrs/Vps27, a candidate ESCRT-0 protein reversed Nef induced downregulation of CXCR4 or CCR5. <b>A</b>) Histograms of relative (%) MFVs (with standard deviation) of native CXCR4 (left) in Jurkat cells or CCR5 in CEM cell line (right) expressing Nef and GFP are shown in the context of siRNA knockdown of AIP4, AP2, NEDD4 and HRS (*p<0.05 compared with Nef and mock siRNA transfected cells). <b>B</b>) Nef induced CXCR4 downregulation was not reversed by siRNA knockdown of AP1, clathrin, β-arrestin, deubiquitinases, AMSH, STAM, and USP14 and a candidate ESCRT adapter, TSG101/Vps23P. CD4 downregulation by Nef was resistant to all siRNA knockdowns except for clathrin (<b>B</b>, right, n = 5; <b>*</b>p<0.01). Expression levels of proteins targeted by cognate siRNAs were monitored by immuno-blots shown underneath panels <b>A</b> and <b>B</b>.</p