No abstract

Promoter-based genetic recombination (via, e.g., Cre-lox) is most useful when all cells of interest express a particular gene. The discovery that the actin-binding protein advillin is expressed in all somatic sensory neurons has been exploited repeatedly to drive DNA recombination therein, yet specificity of expression has not been well demonstrated. Here, we characterize advillin expression amongst sensory neurons and in several other neural and non-neural tissues. We first validate an advillin antibody against advillin knock-out tissue, advillin promoter-driven EGFP, and advillin mRNA expression. In the dorsal root ganglion (DRG), advillin is enriched in non-peptidergic nociceptors. We also show that advillin expression, and advillin promotor-driven EGFP and Cre-recombinase expression, occurs in multiple tissues including the dorsal habenula of the epithalamus, endocrine cells of the gut, Merkel cells in the skin, and most strikingly, throughout the autonomic nervous system (sympathetic, parasympathetic, and enteric neurons) in mice, rats, and non-human primates. In the mouse pelvic ganglion, advillin immunoreactivity is most intense in pairs of small neurons, and concentrated in spine-like structures on the axon initial segment contacted by sympathetic preganglionic axons. In autonomic targets (iris and blood vessels), advillin is distributed along cholinergic parasympathetic axons and in sympathetic varicosities. Developmentally, advillin expression is absent from sympathetics at postnatal day 4 but begins to emerge by day 7, accounting for previous reports (based on embryonic expression) of advillin's specificity to sensory neurons. These results indicate that caution is warranted in interpreting previous studies in which advillin-driven genomic editing is either constitutive or performed after postnatal day 4.

Type 1 long-interspersed nuclear elements (L1s) are autonomous retrotransposable elements that retain the potential for activity in the human genome but are suppressed by host factors. Retrotransposition of L1s into chromosomal DNA can lead to genomic instability, whereas reverse transcription of L1 in the cytosol has the potential to activate innate immune sensors. We hypothesized that HIV-1 infection would compromise cellular control of L1 elements, resulting in the induction of retrotransposition events. Here, we show that HIV-1 infection enhances L1 retrotransposition in Jurkat cells in a Vif-and Vpr-dependent manner. In primary CD4؉ cells, HIV-1 infection results in the accumulation of L1 DNA, at least the majority of which is extrachromosomal. These data expose an unrecognized interaction between HIV-1 and endogenous retrotransposable elements, which may have implications for the innate immune response to HIV-1 infection, as well as for HIV-1-induced genomic instability and cytopathicity. L 1 element DNA sequences comprise approximately 17% of the human genome (1, 2). Although the bulk of these sequences are in the form of short 5= truncated insertions, an estimated 100 full-length intact elements are present (3, 4). These intact L1 elements represent the only retrotransposons encoded by the human genome known to be capable of autonomous replication (4-7). Full-length L1 elements are ϳ6 kb in length, comprising a 5=-untranslated region (5=UTR) two open reading frames (ORF1 and ORF2) and a 3=UTR ending in a poly(A) tail (8). ORF1 encodes a 40-kDa protein with RNA chaperone activity, while ORF2 encodes a 150-kDa protein which possesses the reverse transcriptase (RT) and endonuclease functions required for retrotransposition (6,(9)(10)(11)(12)(13)(14)(15)(16)(17). Productive retrotransposition is thought to occur by a mechanism termed target-primed reverse transcription (TPRT), where reverse transcription is primed against genomic DNA at the insertion site and thus occurs in concert with integration (18)(19)(20).Several cases of genetic disease have been traced to gene disruptions caused by L1 retrotransposition events in germ line cells, and L1 retrotransposition in somatic cells has been implicated in oncogenesis and cancer progression (21-26). L1 retrotransposition may also play a role in normal physiology. Previous studies have demonstrated the ability for tagged, engineered L1 elements to retrotranspose in neural progenitor cells, and this, supported by quantitative PCR (qPCR) data showing elevated copy numbers of L1 elements in the adult human brain, has led to the suggestion that L1 retrotransposition may play a role in the generation of neuronal somatic mosaicism (27, 28). The vast amount of L1 element sequence fixed in the human genome has, however, presented a technical challenge to the isolation of novel endogenous L1 genomic insertions in somatic cells.Although TPRT appears to be the primary mechanism by which novel genomic L1 insertions are generated, there is considerable evidence that cytosolic...

SummaryThe secreted growth factor progranulin (PGRN) has been shown to be important for regulating neuronal survival and outgrowth, as well as synapse formation and function. Mutations in the PGRN gene that result in PGRN haploinsufficiency have been identified as a major cause of frontotemporal dementia (FTD). Here we demonstrate that PGRN is colocalized with dense-core vesicle markers and is cotransported with brain-derived neurotrophic factor (BDNF) within axons and dendrites of cultured hippocampal neurons in both anterograde and retrograde directions. We also show that PGRN is secreted in an activity-dependent manner from synaptic and extrasynaptic sites, and that the temporal profiles of secretion are distinct in axons and dendrites. Neuronal activity is also shown to increase the recruitment of PGRN to synapses and to enhance the density of PGRN clusters along axons. Finally, treatment of neurons with recombinant PGRN is shown to increase synapse density, while decreasing the size of the presynaptic compartment and specifically the number of synaptic vesicles per synapse. Together, this indicates that activity-dependent secretion of PGRN can regulate synapse number and structure.

The presence of interleukin-2 (IL-2)-producing human immunodeficiency virus type 1 (HIV-1)-specific CD4؉ T-cell responses has been associated with the immunological control of HIV-1 replication; however, the causal relationship between these factors remains unclear. Here we show that IL-2-producing HIV-1-specific CD4 ؉ T cells can be cloned from acutely HIV-1-infected individuals. Despite the early presence of these cells, each of the individuals in the present study exhibited progressive disease, with one individual showing rapid progression. In this rapid progressor, three IL-2-producing HIV-1 Gag-specific CD4؉ T-cell responses were identified and mapped to the following optimal epitopes: HIVWASRELER, REPRGSDIAGT, and FRDYVDRFYKT. Responses to these epitopes in peripheral blood mononuclear cells were monitored longitudinally to >1 year postinfection, and contemporaneous circulating plasma viruses were sequenced. A variant of the FRDYVDR FYKT epitope sequence, FRDYVDQFYKT, was observed in 1/21 plasma viruses sequenced at 5 months postinfection and 1/10 viruses at 7 months postinfection. This variant failed to stimulate the corresponding CD4 ؉ T-cell clone and thus constitutes an escape mutant. Responses to each of the three Gag epitopes were rapidly lost, and this loss was accompanied by a loss of antigen-specific cells in the periphery as measured by using an FRDYVDRFYKT-presenting major histocompatibility complex class II tetramer. Highly active antiretroviral therapy was associated with the reemergence of FRDYVDRFYKT-specific cells by tetramer. Thus, our data support that IL-2-producing HIV-1-specific CD4 ؉ T-cell responses can exert immune pressure during early HIV-1 infection but that the inability of these responses to enforce enduring control of viral replication is related to the deletion and/or dysfunction of HIV-1-specific CD4 ؉ T cells rather than to the fixation of escape mutations at high frequencies.