VTechWorks Repository :: Browsing by Author "Valdez, Gregorio"

Browsing by Author "Valdez, Gregorio"

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Agrin and Synaptic Laminin Are Required to Maintain Adult Neuromuscular Junctions
Samuel, Melanie; Valdez, Gregorio; Tapia, Juan C.; Lichtman, Jeff W.; Sanes, Joshua R. (PLOS, 2012-10-03)
As synapses form and mature the synaptic partners produce organizing molecules that regulate each other’s differentiation and ensure precise apposition of pre- and post-synaptic specializations. At the skeletal neuromuscular junction (NMJ), these molecules include agrin, a nerve-derived organizer of postsynaptic differentiation, and synaptic laminins, muscle-derived organizers of presynaptic differentiation. Both become concentrated in the synaptic cleft as the NMJ develops and are retained in adulthood. Here, we used mutant mice to ask whether these organizers are also required for synaptic maintenance. Deletion of agrin from a subset of adult motor neurons resulted in the loss of acetylcholine receptors and other components of the postsynaptic apparatus and synaptic cleft. Nerve terminals also atrophied and eventually withdrew from muscle fibers. On the other hand, mice lacking the presynaptic organizer laminin-a4 retained most of the synaptic cleft components but exhibited synaptic alterations reminiscent of those observed in aged animals. Although we detected no marked decrease in laminin or agrin levels at aged NMJs, we observed alterations in the distribution and organization of these synaptic cleft components suggesting that such changes could contribute to age-related synaptic disassembly. Together, these results demonstrate that pre- and post-synaptic organizers actively function to maintain the structure and function of adult NMJs.
The ALS-inducing factors, TDP43A315T and SOD1G93A, directly affect and sensitize sensory neurons to stress
Vaughan, Sydney K.; Sutherland, Natalia M.; Zhang, Sihui; Hatzipetros, Theo; Vieira, Fernando; Valdez, Gregorio (Nature, 2018-11-08)
There is increased recognition that sensory neurons located in dorsal root ganglia (DRG) are affected in amyotrophic lateral sclerosis (ALS). However, it remains unknown whether ALS-inducing factors, other than mutant superoxide dismutase 1 (SOD1G93A), directly affect sensory neurons. Here, we examined the effect of mutant TAR DNA-binding protein 1 (TDP43A315T) on sensory neurons in culture and in vivo. In parallel, we reevaluated sensory neurons expressing SOD1G93A. We found that cultured sensory neurons harboring either TDP43A315T or SOD1G93A grow neurites at a slower rate and elaborate fewer neuritic branches compared to control neurons. The presence of either ALS-causing mutant gene also sensitizes sensory neurons to vincristine, a microtubule inhibitor that causes axonal degeneration. Interestingly, these experiments revealed that cultured sensory neurons harboring TDP43A315T elaborate shorter and less complex neurites, and are more sensitive to vincristine compared to controls and to SOD1G93A expressing sensory neurons. Additionally, levels of two molecules involved in stress responses, ATF3 and PERK are significantly different between sensory neurons harboring TDP43A315T to those with SOD1G93A in vitro and in vivo. These findings demonstrate that sensory neurons are directly affected by two ALS-inducing factors, suggesting important roles for this neuronal subpopulation in ALS-related pathogenesis.
Attenuating Cholinergic Transmission Increases the Number of Satellite Cells and Preserves Muscle Mass in Old Age
Vaughan, Sydney K.; Sutherland, Natalia M.; Valdez, Gregorio (2019-09-24)
In addition to driving contraction of skeletal muscles, acetylcholine (ACh) acts as an anti-synaptogenic agent at neuromuscular junctions (NMJs). Previous studies suggest that aging is accompanied by increases in cholinergic activity at the NMJ, which may play a role in neuromuscular degeneration. In this study, we hypothesized that moderately and chronically reducing ACh could attenuate the deleterious effects of aging on NMJs and skeletal muscles. To test this hypothesis, we analyzed NMJs and muscle fibers from heterozygous transgenic mice with reduced expression of the vesicular ACh transporter (VAChT; VKDHet), which present with approximately 30% less synaptic ACh compared to control mice. Because ACh is constitutively decreased in VKDHet, we first analyzed developing NMJs and muscle fibers. We found no obvious morphological or molecular differences between NMJs and muscle fibers of VKDHet and control mice during development. In contrast, we found that moderately reducing ACh has various effects on adult NMJs and muscle fibers. VKDHet mice have significantly larger NMJs and muscle fibers compared to age-matched control mice. They also present with reduced expression of the pro-atrophy gene, Foxo1, and have more satellite cells in skeletal muscles. These molecular and cellular features may partially explain the increased size of NMJs and muscle fibers. Thus, moderately reducing ACh may be a therapeutic strategy to prevent the loss of skeletal muscle mass that occurs with advancing age.
Cell-specific roles for CASK in the pathology of Optic Nerve Hypoplasia
Kerr, Alicia Marie (Virginia Tech, 2019-06-25)
Optic Nerve Hypoplasia (ONH) is the leading cause of childhood blindness in developed nations and its prevalence has been rising. Yet, we know little about the genetic, molecular, or cellular mechanisms underlying ONH. A previous study described ONH in a cohort of patients with mutations in CASK, an X-linked gene with established roles in neural development and synaptic function. I have demonstrated that heterozygous deletion of CASK in mice (Cask+/-) recapitulates many of the phenotypes observed in patients with CASK mutations, including ONH. This includes reduced optic nerve size, reduced numbers of retinal ganglion cells (RGCs), reduced RGC axonal diameter, and deficits in vision-related tasks. Further analysis on a homozygous partial loss of function variant (Caskfl/fl) also displayed ONH with reduced numbers of RGCs. In order to understand the mechanisms underlying CASK-associated ONH, I explored whether RGCs, the projection neurons of the retina and the cells whose axons comprise the optic nerve, generate CASK. Indeed, mRNA analysis revealed expression of CASK by a large cohort of RGCs. In order to assess whether loss of CASK from a majority of RGCs leads to ONH, I crossed a conditional allele of CASK (CASKfl/fl) with transgenic mice that express Cre Recombinase (Cre) in RGCs. Deletion of CASK from RGCs did not further alter ONH size nor RGC survival. These results demonstrate that loss of CASK signaling in this discrete neuronal populations is not sufficient to lead to further disruption in the assembly of the subcortical visual circuit, suggesting a non-cell autonomous mechanism for loss of CASK in ONH.
Dynamic UTR Usage Regulates Alternative Translation to Modulate Gap Junction Formation during Stress and Aging
Zeitz, Michael J.; Calhoun, Patrick J.; James, Carissa C.; Taetzsch, Thomas; George, Kijana K.; Robel, Stefanie; Valdez, Gregorio; Smyth, James W. (Elsevier, 2019-05-28)
Connexin43 (Cx43; gene name GJA1) is the most ubiquitously expressed gap junction protein, and understanding of its regulation largely falls under transcription and post-translational modification. In addition to Cx43, Gja1 mRNA encodes internally translated isoforms regulating gap junction formation, whose expression is modulated by TGF-b. Here, using RLM-RACE, we identify distinct Gja1 transcripts differing only in 50 UTR length, of which two are upregulated during TGF-b exposure and hypoxia. Introduction of these transcripts into Gja1/ cells phenocopies the response of Gja1 to TGF-b with reduced internal translation initiation. Inhibiting pathways downstream of TGF-b selectively regulates levels of Gja1 transcript isoforms and translation products. Reporter assays reveal enhanced translation of fulllength Cx43 from shorter Gja1 50 UTR isoforms. We also observe a correlation among UTR selection, translation, and reduced gap junction formation in aged heart tissue. These data elucidate a relationship between transcript isoform expression and translation initiation regulating intercellular communication.
EvoCor: a platform for predicting functionally related genes using phylogenetic and expression profiles
Dittmar, W. James; McIver, Lauren; Michalak, Pawel; Garner, Harold R.; Valdez, Gregorio (2014-07-01)
The wealth of publicly available gene expression and genomic data provides unique opportunities for computational inference to discover groups of genes that function to control specific cellular processes. Such genes are likely to have co-evolved and be expressed in the same tissues and cells. Unfortunately, the expertise and computational resources required to compare tens of genomes and gene expression data sets make this type of analysis difficult for the average end-user. Here, we describe the implementation of a web server that predicts genes involved in affecting specific cellular processes together with a gene of interest. We termed the server 'EvoCor', to denote that it detects functional relationships among genes through evolutionary analysis and gene expression correlation. This web server integrates profiles of sequence divergence derived by a Hidden Markov Model (HMM) and tissue-wide gene expression patterns to determine putative functional linkages between pairs of genes.
Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction
Nishimune, Hiroshi; Valdez, Gregorio; Jarad, George; Moulson, Casey L.; Müller, Ulrich; Miner, Jeffrey H.; Sanes, Joshua R. (The Rockefeller University Press, 2008-09-15)
A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the α4, α5, and β2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin 5 and arrested in mutants lacking both α4 and α5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.
Mechanism of CASK-linked ophthalmological disorders
Liang, Chen (Virginia Tech, 2018-09-21)
Calcium/calmodulin-dependent serine protein kinase (CASK) is a membrane-associated guanylate kinase (MAGUK) family protein, which is encoded by a gene of identical name present on the X chromosome. CASK may participate in presynaptic scaffolding, gene expression regulation, and cell junction formation. CASK is essential for survival in mammals. Heterozygous mutations in the CASK gene (in females) produce X-linked intellectual disability (XLID) and mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH, OMIM# 300749). CASK mutations are also frequently associated with optic nerve hypoplasia (ONH) which is the most common cause of childhood blindness in developed countries. Some patients with mutations in CASK have been also diagnosed with optic nerve atrophy (ONA) and glaucoma. We have used floxed CASK (CASKfloxed), CASK heterozygous knockout (CASK(+/-)), CASK neuronal knockout (CASKNKO) and tamoxifen inducible CASK knockout (CASKiKO) mouse models to investigate the mechanism and pathology of CASK-linked ONH. Our observations indicate that ONH occurs with 100% penetrance in CASK(+/-) mice, which also displayed microcephaly and disproportionate cerebellar hypoplasia. Further, we found that CASK-linked ONH is a complex developmental neuropathology with some degenerative components. Cellular pathologies include loss of retinal ganglion cells (RGC), astrogliosis, axonopathy, and synaptopathy. The onset of ONH is late in development, observed only around the early postnatal stage in mice reaching the plateau phase by three weeks of birth. The developmental nature of the disorder is confirmed by deleting CASK after maturity since CASKiKO mice did not produce any obvious optic nerve pathology. Strikingly the CASKfloxed mice expressing ~49% level of CASK did not manifest ONH despite displaying a slightly smaller brain and cerebellar hypoplasia indicating that ONH may not simply be an extension of microcephaly. We discovered that deleting CASK in neurons produced lethality before the onset of adulthood. The CASKNKO mice exhibited delayed myelination of the optic nerve. Overall this work suggests that CASK is critical for neuronal maturation and CASK-linked ONH is a pervasive developmental disorder of the subcortical visual pathway. Finally, in a side project, I also described a new methodology of targeting neurons using receptor-mediated endocytosis which would help target retinal neurons for therapeutic purposes in the future.
Mechanisms underlying neural circuit remodeling in Toxoplasma gondii infection
Carrillo, Gabriela Lizana (Virginia Tech, 2022-09-20)
The central nervous system (CNS) is protected by a vascular blood-brain barrier that prevents many types of pathogens from entering the brain. Still, some pathogens have evolved mechanisms to traverse this barrier and establish a long-term infection. The apicomplexan parasite, Toxoplasma gondii, is one such pathogen with the ability to infect the CNS in virtually all warm-blooded animals, including humans. Across the globe, an estimated 30% of the human population is infected with Toxoplasma, an infection for which mounting evidence suggests increases the risk for developing neurological and neuropsychiatric disorders, like seizures and schizophrenia. In my dissertation, I investigate the telencephalic neural circuit changes induced by long-term Toxoplasma infection in the mouse brain and the neuroimmune signaling role of the complement system in mediating microglial remodeling of neural circuits following parasitic infection. While there has been keen interest in investigating neural circuit changes in the amygdala – a region of the brain involved in fear response and which Toxoplasma infection alters in many species of infected hosts – the hippocampus and cortex have been less explored. These are brain regions for which Toxoplasma also has tropism, and moreover, are rich with fast-spiking parvalbumin perisomatic synapses, a type of GABAergic synapse whose dysfunction has been implicated in epilepsy and schizophrenia. By employing a range of visualization techniques to assess cell-to-cell connectivity and neuron-glia interactions (including immunohistochemistry, ultrastructural microscopy, and microglia-specific reporter mouse lines), I discovered that longterm Toxoplasma infection causes microglia to target and ensheath neuronal somata in these regions and subsequently phagocytose their perisomatic inhibitory synapses. These findings provide a novel model by which Toxoplasma infection within the brain can lead to seizure susceptibility and a wider range of behavioral and cognitive changes unrelated to fear response. In the Toxoplasma infected brain, microglia, along with monocytes recruited to the brain from the periphery, coordinate a neuroinflammatory response against pathogenic invasion. This is characterized by a widespread activation of these cells and their increased interaction with neurons and their synaptic inputs. Yet, whether T. gondii infection triggers microglia and monocytes (i.e. phagocytes) to target, ensheath, and remove perisomatic inhibitory synapses on neuronal somata indiscriminately, or whether specificity exists in this type of circuit remodeling, remained unclear. Through a comprehensive assessment of phagocyte interactions with cortical neuron subtypes, I demonstrate that phagocytes selectively target and ensheath excitatory pyramidal cells in long-term infection. Moreover, coupling of in situ hybridization with transgenic reporter lines and immunolabeling revealed that in addition to phagocytes, excitatory neurons also express complement component C3 following infection (while inhibitory interneurons do not). Lastly, by employing targeted deletion of complement components, C1q and C3, I show that complement is required for phagocyte ensheathment of excitatory cells and the subsequent removal of perisomatic inhibitory synapses on these cells (albeit not through the classical pathway). Together, these studies highlight a novel role for complement in mediating synapse-type and cell-type specific circuit remodeling in the Toxoplasma infected brain.
Mechanisms underlying retinogeniculate synapse formation in mouse visual thalamus
Monavarfeshani, Aboozar (Virginia Tech, 2018-01-22)
Retinogeniculate (RG) synapses connect retinal ganglion cells to the thalamic relay cells of the dorsal lateral geniculate nucleus (dLGN). They are critical for regulating the flow of visual information from retina to primary visual cortex (V1). RG synapses in dLGN are uniquely larger and stronger than their counterparts in other retinorecipient regions. Moreover, in dLGN, RG synapses can be classified into two groups: simple RG synapses, which contain glia-encapsulated single RTs synapsing onto relay cell dendrites, and complex RG synapses, which contain numerous RTs that converge onto the shared regions of relay cell dendrites. To identify target-derived molecules that direct the transformation of RTs into unique RG synapses in dLGN, I used RNAseq to obtain the whole transcriptome of dLGN and its adjacent retinorecipient nucleus, vLGN, at different time points during RG synapses development. Leucine-Rich Repeat Transmembrane Neuronal 1 (LRRTM1), a synaptogenic adhesion molecule, was the candidate I selected based on its expression pattern. Here, I discovered that LRRTM1 regulates the development of complex RG synapses. Mice lacking LRRTM1 (lrrtm1-/-) not only show a significant reduction in the number of complex RG synapses but they exhibit abnormal visual behaviors. This work reveals, for the first time, a high level of retinal convergence onto dLGN relay cells in thalamus and the functional significance of this convergence for vision.
𝛼-Motor neurons are spared from aging while their synaptic inputs degenerate in monkeys and mice
Maxwell, Nicholas; Castro, Ryan W.; Sutherland, Natalia M.; Vaughan, Kelli L.; Szarowicz, Mark D.; de Cabo, Rafael; Mattison, Julie A.; Valdez, Gregorio (Wiley, 2017)
Motor function deteriorates with advancing age, increasing the risk of adverse health outcomes. While it is well established that skeletal muscles and neuromuscular junctions (NMJs) degenerate with increasing age, the effect of aging on 𝛼-motor neurons and their innervating synaptic inputs remains largely unknown. In this study, we examined the soma of 𝛼-motor neurons and innervating synaptic inputs in the spinal cord of aged rhesus monkeys and mice, two species with vastly different lifespans. We found that, in both species, 𝛼-motor neurons retain their soma size despite an accumulation of large amounts of cellular waste or lipofuscin. Interestingly, the lipofuscin profile varied considerably, indicating that 𝛼-motor neurons age at different rates. Although the rate of aging varies, 𝛼-motor neurons do not atrophy in old age. In fact, there is no difference in the number of motor axons populating ventral roots in old mice compared to adult mice. Moreover, the transcripts and proteins associated with 𝛼-motor neurons do not decrease in the spinal cord of old mice. However, in aged rhesus monkeys and mice, there were fewer cholinergic and glutamatergic synaptic inputs directly abutting 𝛼-motor neurons, evidence that aging causes 𝛼-motor neurons to shed synaptic inputs. Thus, the loss of synaptic inputs may contribute to age-related dysfunction of 𝛼-motor neurons. These findings broaden our understanding of the degeneration of the somatic motor system that precipitates motor dysfunction with advancing age.
The role of autonomic neurons in the pathegenesis of herpes simplex virus infection
Lee, Sung Seok (Virginia Tech, 2016-01-27)
Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are major human pathogens. HSV establishes latency in the nervous system and reactivates to cause recurrent disease, resulting in transmission of progeny virions to naïve individuals. Though HSV-1 and HSV-2 share similar structure and genes, they have distinctive recurrence profiles. Generally, HSV-1 reactivation is associated with disease 'above the waist' and HSV-2 reactivation is associated with disease 'below the waist'. This phenomenon was described decades ago but still remains unexplained. The mechanism of HSV latent infection in the peripheral nervous system (PNS) has been extensively investigated, especially with in sensory neurons. Another component of the peripheral nervous system (PNS), autonomic neurons, were also known to be infected with HSV productively and latently, but largely ignored because of the assumption that there is no difference in the pathogenesis of HSV in the neurons and that both HSV-1 and HSV-2 behave in the same way in different types of neurons. However, autonomic neurons differ in physiological function compared to sensory neurons. Activation factors of autonomic neurons, such as emotional stress, trauma and hormonal fluctuation, are also known HSV reactivation triggering factors. Therefore, I hypothesized that autonomic neurons innervating the site of HSV infection are responsible the different reactivation frequencies of HSV-1 and HSV-2 after peripheral invasion. In this report, the role of autonomic neurons in HSV pathogenesis were examined using the female guinea pig reactivation model. Major findings of this report are that 1) parasympathetic ganglia innervating the ocular region support latent infection of HSV-1 selectively, thus contributing the more frequent HSV-1 reactivation, 2) mixed autonomic ganglia in the genital area support HSV-2 latent infection selectively, and 3) sympathetic neurons in the genital region supported productive and latent infection of HSV-1 and HSV-2 differently. All of the results in this report indicate that autonomic neurons play a distinctive role in HSV pathogenesis compared to the sensory neurons and are responsible for the different reactivation frequencies of HSV-1 and HSV-2. This report raises the importance of autonomic neurons in HSV pathogenesis and challenges the paradigm of HSV pathogenesis.
The role of Lynx1, an endogenous modulator of cholinergic transmission, in NMJ development, maintenance, and repair
Vaughan, Sydney Katherine (Virginia Tech, 2019-05-08)
The cholinergic system drives muscle contraction and plays a central role in the formation, maintenance, and repair of mammalian neuromuscular junctions (NMJs) and skeletal muscles. Because of these essential actions, much effort has been devoted to identifying primary and auxiliary modulatory components of the cholinergic system at NMJs and throughout skeletal muscles. Here, I asked if Lynx1, a GPI-anchored protein shown to modulate nAChRs in the brain, is present and affects the activity of nAChRs at NMJs. Molecular and cellular analysis revealed that Lynx1 levels increase in skeletal muscles, specifically at NMJs, during development. Its expression pattern also closely mirrors changes in cholinergic transmission in vivo and in vitro. As expected, I found by co-immunoprecipitation that Lynx1 interacts with muscle nAChRs and using electrophysiology, I show that Lynx1 desensitizes nAChRs to ACh at NMJs. These findings demonstrate that Lynx1 regulates the cholinergic system at NMJs, suggesting roles for this gene in developing and adult NMJs. To determine the role of Lynx1 at NMJs, I examined Lynx1 knockout mice at different ages. While deletion of Lynx1 has no discernable effect on developing NMJs, its absence increases the incidence of NMJs with age-related morphological features, such as fragmentation and denervation, in young adult and middle-aged mice. Loss of Lynx1 also increases the number of slow-type muscle fibers in young and middle-aged mice, another hallmark of aging. Along with these morphological changes, deletion of Lynx1 affects expression of genes associated with NMJ stability, myogenesis, and muscle atrophy in young adult and middle-aged mice. Not surprisingly, the loss of Lynx1 reduces the density and stability of nAChRs at NMJs. Because of these findings, I surmised that loss of Lynx1 would adversely affect NMJs under other physiological stressors. However, I found the opposite as the loss of Lynx1 augments the capacity of NMJs to repair damages during exercise, following injury to motor axons, and during the initial symptomatic stage of amyotrophic lateral sclerosis (ALS). Since Lynx1 modulates the activity of nAChRs, these contrasting findings likely represent the positive and negative effects of heightened cholinergic transmission on aging compared to injury and disease-afflicted NMJs.
The Role of Muscle microRNAs in Repairing the Neuromuscular Junction
Valdez, Gregorio; Heyer, Mary P.; Feng, Guoping; Sanes, Joshua R. (PLOS, 2014-03-24)
microRNAs have been implicated in mediating key aspects of skeletal muscle development and responses to diseases and injury. Recently, we demonstrated that a synaptically enriched microRNA, miR-206, functions to promote maintenance and repair of the neuromuscular junction (NMJ); in mutant mice lacking miR-206, reinnervation is impaired following nerve injury and loss of NMJs is accelerated in a mouse model of amyotrophic lateral sclerosis (ALS). Here, we asked whether other microRNAs play similar roles. One attractive candidate is miR-133b because it is in the same transcript that encodes miR-206. Like miR-206, miR-133b is concentrated near NMJs and induced after denervation. In miR-133b null mice, however, NMJ development is unaltered, reinnervation proceeds normally following nerve injury, and disease progression is unaffected in the SOD1(G93A) mouse model of ALS. To determine if miR-206 compensates for the loss of miR-133b, we generated mice lacking both microRNAs. The phenotype of these double mutants resembled that of miR-206 single mutants. Finally, we used conditional mutants of Dicer, an enzyme required for the maturation of most microRNAs, to generate mice in which microRNAs were depleted from skeletal muscle fibers postnatally, thus circumventing a requirement for microRNAs in embryonic muscle development. Reinnervation of muscle fibers following injury was impaired in these mice, but the defect was similar in magnitude to that observed in miR-206 mutants. Together, these results suggest that miR-206 is the major microRNA that regulates repair of the NMJ following nerve injury.
Role of retinal inputs and astrocytes for the development of visual thalamus
Somaiya, Rachana Deven (Virginia Tech, 2022-06-01)
Axons of retinal ganglion cells (RGCs) send visual information to a number of retinorecipient regions in the brain. In rodents, visual thalamus receives dense innervations from RGC axons and is important for both image-forming and nonimage-forming visual functions. Retinal inputs invade visual thalamus during embryonic development, before the arrival of non-retinal inputs (such as local interneurons and axonal inputs from other brain regions). In this dissertation, I explore how early innervation of RGC axons affects circuitry in visual thalamus and the role of visual experience, neural activity, and molecular cues in the development. While the development of astrocytes in cortex has been well-described, they have been largely overlooked in visual thalamus. Using immunohistochemical, functional, and ultrastructural analysis, I show that astrocytes in visual thalamus reach adult-like morphological properties and functionality at retinogeniculate synapses early in development, by eye-opening and before visual experience. These studies reveal that while experience-dependent visual activity from RGC axons is critical for many aspects of visual thalamus development, astrocytic maturation occurs independent of that information about our visual environment. As with astrocytes, little progress has been made in understanding the development of interneurons in the visual thalamus. Here, I show that retinal inputs interact with thalamic astrocytes to influence the recruitment of GABAergic interneurons into visual thalamus. I found that this interaction between RGC axons and astrocytes is not dependent on neural activity of RGCs. Using transcriptomic analysis, in situ hybridization, and reporter lines, I observed thalamus-projecting RGCs express SHH and astrocytes in visual thalamus express SHH signaling molecules. My results reveal that SHH signaling between RGC axons and astrocytes is critical for astrocytic fibroblast growth factor 15 (FGF15) expression in developing visual thalamus. Ultimately, FGF15 serves as a potent motogen that is essential for thalamic interneuron migration. These data identify a novel morphogen-dependent and activity-independent mechanism that mediates crosstalk between RGCs and astrocytes to facilitate the recruitment of interneurons into the developing visual thalamus.
Shared Resistance to Aging and ALS in Neuromuscular Junctions of Specific Muscles
Valdez, Gregorio; Tapia, Juan C.; Lichtman, Jeff W.; Fox, Michael A.; Sanes, Joshua R. (PLOS, 2012-04-02)
Normal aging and neurodegenerative diseases both lead to structural and functional alterations in synapses. Comparison of synapses that are generally similar but respond differently to insults could provide the basis for discovering mechanisms that underlie susceptibility or resistance to damage. Here, we analyzed skeletal neuromuscular junctions (NMJs) in 16 mouse muscles to seek such differences. We find that muscles respond in one of three ways to aging. In some, including most limb and trunk muscles, age-related alterations to NMJs are progressive and extensive during the second postnatal year. NMJs in other muscles, such as extraocular muscles, are strikingly resistant to change. A third set of muscles, including several muscles of facial expression and the external anal sphinter, succumb to aging but not until the third postnatal year. We asked whether susceptible and resistant muscles differed in rostrocaudal or proximodistal position, source of innervation, motor unit size, or fiber type composition. Of these factors, muscle innervation by brainstem motor neurons correlated best with resistance to age-related decline. Finally, we compared synaptic alterations in normally aging muscles to those in a mouse model of amyotrophic lateral sclerosis (ALS). Patterns of resistance and susceptibility were strikingly correlated in the two conditions. Moreover, damage to NMJs in aged muscles correlated with altered expression and distribution of CRMP4a and TDP-43, which are both altered in motor neurons affected by ALS. Together, these results reveal novel structural, regional and molecular parallels between aging and ALS.
Specific labeling of synaptic schwann cells reveals unique cellular and molecular features
Castro, Ryan W.; Taetzsch, Thomas; Vaughan, Sydney K.; Godbe, Kerilyn; Chappell, John C.; Settlage, Robert E.; Valdez, Gregorio (2020-06-25)
Perisynaptic Schwann cells (PSCs) are specialized, non-myelinating, synaptic glia of the neuromuscular junction (NMJ), that participate in synapse development, function, maintenance, and repair. The study of PSCs has relied on an anatomy-based approach, as the identities of cell-specific PSC molecular markers have remained elusive. This limited approach has precluded our ability to isolate and genetically manipulate PSCs in a cell specific manner. We have identified neuron-glia antigen 2 (NG2) as a unique molecular marker of S100 beta+ PSCs in skeletal muscle. NG2 is expressed in Schwann cells already associated with the NMJ, indicating that it is a marker of differentiated PSCs. Using a newly generated transgenic mouse in which PSCs are specifically labeled, we show that PSCs have a unique molecular signature that includes genes known to play critical roles in PSCs and synapses. These findings will serve as a springboard for revealing drivers of PSC differentiation and function.
Use of Kinase Inhibitors to Illuminate Signaling Pathways in Breast Cancer
Smith, Nicole R. (Virginia Tech, 2018-02-01)
In the United States, breast cancer is the most commonly diagnosed cancer and is the second most common cause of cancer-related deaths among women. Among the various subtypes of breast cancer, 25-30% of diagnoses present themselves as human epidermal growth factor receptor 2 positive (HER-2+). HER-2 is a protein receptor located on the cell surface that interacts with other proteins and signaling molecules to translate extracellular signals into cellular process such as cell growth and replication. However, in breast cancer, there is a drastic increase in the number of HER-2 proteins on the cell surface, that causes excessive cell growth and proliferation, and ultimately tumor formation. The most frequent treatment of HER-2+ breast cancers includes the use of a single agent inhibitor that directly blocks the HER-2 protein to prevent over-signaling and cell growth. However, after continuous use, breast cancer cells develop drug resistance, as other proteins such as the insulin-like growth factor 1 receptor (IGF-1R) and the protein kinase B (AKT) can also interfere and cause cell growth and replication. In this study, we propose that the use of a multi-agent treatment targeting the HER-2, IGF-1R, and AKT proteins will be more effective than a single-agent treatment of HER-2 alone. Through protein analysis by mass spectrometry, we intend to illuminate the different cellular responses to both treatment types. The results indicate that the single drug treatment targeting Her-2 appears to increase processes related cellular repair, while the multi-drug treatment indicates an increase in processes related to programmed cell death; both treatments appear to block the transmission of protein signaling.

Browsing by Author "Valdez, Gregorio"

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