Faculty Bibliography

OBJECTIVE: To assess the effectiveness of l-cystine dimethyl ester (CDME), an inhibitor of cystine crystal growth, for the treatment of cystine urolithiasis in an Slc3a1 knockout mouse model of cystinuria. MATERIALS AND METHODS: CDME (200 mug per mouse) or water was delivered by gavage daily for 4 weeks. Higher doses by gavage or in the water supply were administered to assess organ toxicity. Urinary amino acids and cystine stones were analyzed to assess drug efficacy using several analytical methods. RESULTS: Treatment with CDME led to a significant decrease in stone size compared with that of the water group (P = .0002), but the number of stones was greater (P = .005). The change in stone size distribution between the 2 groups was evident by micro computed tomography. Overall, cystine excretion in urine was the same between the 2 groups (P = .23), indicating that CDME did not interfere with cystine metabolism. Scanning electron microscopy analysis of cystine stones from the CDME group demonstrated a change in crystal habit, with numerous small crystals. l-cysteine methyl ester was detected by ultra-performance liquid chromatography-mass spectrometer in stones from the CDME group only, indicating that a CDME metabolite was incorporated into the crystal structure. No pathologic changes were observed at the doses tested. CONCLUSION: These data demonstrate that CDME promotes formation of small stones but does not prevent stone formation, consistent with the hypothesis that CDME inhibits cystine crystal growth. Combined with the lack of observed adverse effects, our findings support the use of CDME as a viable treatment for cystine urolithiasis.

Gestational exposure to alcohol can result in long-lasting behavioral deficiencies generally described as fetal alcohol spectrum disorder (FASD). FASD-modeled rodent studies of acute ethanol exposure typically select one developmental window to simulate a specific context equivalent of human embryogenesis, and study consequences of ethanol exposure within that particular developmental epoch. Exposure timing is likely a large determinant in the neurobehavioral consequence of early ethanol exposure, as each brain region is variably susceptible to ethanol cytotoxicity and has unique sensitive periods in their development. We made a parallel comparison of the long-term effects of single-day binge ethanol at either embryonic day 8 (E8) or postnatal day 7 (P7) in male and female mice, and here demonstrate the differential long-term impacts on neuroanatomy, behavior and in vivo electrophysiology of two systems with very different developmental trajectories. The significant long-term differences in odor-evoked activity, local circuit inhibition, and spontaneous coherence between brain regions in the olfacto-hippocampal pathway that were found as a result of developmental ethanol exposure, varied based on insult timing. Long-term effects on cell proliferation and interneuron cell density were also found to vary by insult timing as well as by region. Finally, spatial memory performance and object exploration were affected in P7-exposed mice, but not E8-exposed mice. Our physiology and behavioral results are conceptually coherent with the neuroanatomical data attained from these same mice. Our results recognize both variable and shared effects of ethanol exposure timing on long-term circuit function and their supported behavior.

Abstract The direct conversion of human fibroblasts to neuronal cells, termed human induced neuronal (hiN) cells, has great potential for future clinical advances. However, previous studies have not provided an in-depth analysis of electrophysiological properties of adult fibroblast-derived hiN cultures. We have examined the electrophysiological profile of hiN cells by measuring passive and active membrane properties, as well as spontaneous and evoked neurotransmission. We found that hiN cells exhibited passive membrane properties equivalent to perinatal rodent neurons. In addition, 30% of hiN cells were incapable of action potential (AP) generation and did not exhibit rectifying membrane currents, and none of the cells displayed firing patterns of typical glutamatergic pyramidal neurons. Finally, hiN cells exhibited neither spontaneous nor evoked neurotransmission. Our results suggest that current methods used to produce hiN cells provide preparations in which cells do not achieve the cellular properties of fully mature neurons, rendering these cells inadequate to investigate pathophysiological mechanisms.

Learning and memory and the underlying cellular correlate, long-term synaptic plasticity, involve regulation by posttranslational modifications (PTMs). Here we demonstrate that conjugation with the small ubiquitin-like modifier (SUMO) is a novel PTM required for normal synaptic and cognitive functioning. Acute inhibition of SUMOylation impairs long-term potentiation (LTP) and hippocampal-dependent learning. Since Alzheimer's disease (AD) prominently features both synaptic and PTM dysregulation, we investigated SUMOylation under pathology induced by amyloid-beta (Abeta), a primary neurotoxic molecule implicated in AD. We observed that SUMOylation is dysregulated in both human AD brain tissue and the Tg2576 transgenic AD mouse model. While neuronal activation normally induced upregulation of SUMOylation, this effect was impaired by Abeta42 oligomers. However, supplementing SUMOylation via transduction of its conjugating enzyme, Ubc9, rescued Abeta-induced deficits in LTP and hippocampal-dependent learning and memory. Our data establish SUMO as a novel regulator of LTP and hippocampal-dependent cognition and additionally implicate SUMOylation impairments in AD pathogenesis.

Understanding the dynamic axon-glial cell interaction underlying myelination is hampered by the lack of suitable imaging techniques. Here we demonstrate third harmonic generation microscopy (THGM) for label-free imaging of myelinating Schwann cells in live culture and ex vivo and in vivo tissue. A 3D structure was acquired for a variety of compact and noncompact myelin domains, including juxtaparanodes, Schmidt-Lanterman incisures, and Cajal bands. Other subcellular features of Schwann cells that escape traditional optical microscopies were also visualized. We tested THGM for morphometry of compact myelin. Unlike current methods based on electron microscopy, g-ratio could be determined along an extended length of myelinated fiber in the physiological condition. The precision of THGM-based g-ratio estimation was corroborated in mouse models of hypomyelination. Finally, we demonstrated the feasibility of THGM to monitor morphological changes of myelin during postnatal development and degeneration. The outstanding capabilities of THGM may be useful for elucidation of the mechanism of myelin formation and pathogenesis.

High-frequency ripple oscillations, observed most prominently in the hippocampal CA1 pyramidal layer, are associated with memory consolidation. The cellular and network mechanisms underlying the generation of the rhythm and the recruitment of spikes from pyramidal neurons are still poorly understood. Using intracellular, sharp electrode recordings in freely moving, drug-free mice, we observed consistent large depolarizations in CA1 pyramidal cells during sharp wave ripples, which are associated with ripple frequency fluctuation of the membrane potential ("intracellular ripple"). Despite consistent depolarization, often exceeding pre-ripple spike threshold values, current pulse-induced spikes were strongly suppressed, indicating that spiking was under the control of concurrent shunting inhibition. Ripple events were followed by a prominent afterhyperpolarization and spike suppression. Action potentials during and outside ripples were orthodromic, arguing against ectopic spike generation, which has been postulated by computational models of ripple generation. These findings indicate that dendritic excitation of pyramidal neurons during ripples is countered by shunting of the membrane and postripple silence is mediated by hyperpolarizing inhibition.

Introduction: We assessed (1) the differences in the function of urinary proteins between children with cystinuria and kidney stones (CYS), and healthy controls (HC), and (2) the presence of diagnostic biomarkers for CYS. Material and methods: We compared urinary proteomes of 2 children with CYS and 2 age- and gender-matchedHC, using liquid chromatography-mass spectrometry (LC-MS/MS). Relative protein abundance was estimated using spectral counting. Proteins of interest were selected using the following criteria: 1) >5 spectral counts; 2) >2-fold difference in spectral counts; and 3) <0.05 p-value for the Fisher's Exact Test. Protein function was analyzed using the DAVID online bioinformatics resource, and Cytoscape was used to investigate the key nodes of unique proteins. Results: Of the 623 proteins identified by proteomic analysis, 180 exhibited at least 2-fold difference between CYS and HC. Of those, 94 proteins were up-regulated in CYS, 26 of which were involved in response to wounding, 21 in inflammatory response, 18 in immune response, and 4 in cellular response to oxidative stress. 86 proteins were down-regulated in CYS, 26 of which were involved in cell adhesion. 140 proteins were found only in children with CYS, 33 of which met the selection criteria. Proteinprotein interaction modeling of CYS unique proteins identified actin, vimentin, heat shock 70 kDa protein (HSP70), inter-alpha-trypsin-inhibitor heavy chain (ITIH), and matrix metalloproteinase-9 (MMP-9) as the key nodes, proteins associated with fibrosis pathways. Conclusions: We provide proteomic evidence of oxidative injury, inflammation, wound healing and fibrosis in children with cystinuria and kidney stones. We speculate that oxidative stress and inflammation may cause remodeling via actin and vimentin pathways, leading to fibrosis. Additionally, we identified ITIH and MMP-9 as potential diagnostic biomarkers and novel therapeutic targets in CYS. These unique proteins merit further investigation

In myelinated peripheral axons, Kv1 potassium channels are clustered at the juxtaparanodal region and at an internodal line located along the mesaxon and below the Schmidt-Lanterman incisures. This polarized distribution is controlled by Schwann cells and requires specific cell adhesion molecules (CAMs). The accumulation of Kv1 channels at the juxtaparanodal region depends on the presence of Caspr2 at this site, as well as on the presence of Caspr at the adjacent paranodal junction. However, the localization of these channels along the mesaxonal internodal line still persists in the absence of each one of these CAMs. By generating mice lacking both Caspr and Caspr2 (caspr(-/-)/caspr2(-/-)), we now reveal compensatory functions of the two proteins in the organization of the axolemma. Although Kv1 channels are clustered along the inner mesaxon and in a circumferential ring below the incisures in the single mutants, in sciatic nerves of caspr(-/-)/caspr2(-/-) mice, these channels formed large aggregates that were dispersed along the axolemma, demonstrating that internodal localization of Kv1 channels requires either Caspr or Caspr2. Furthermore, deletion of both Caspr and Caspr2 also resulted in widening of the nodes of Ranvier, suggesting that Caspr2 (which is present at paranodes in the absence of Caspr) can partially compensate for the barrier function of Caspr at this site even without the formation of a distinct paranodal junction. Our results indicate that Caspr and Caspr2 are required for the organization of the axolemma both radially, manifested as the mesaxonal line, and longitudinally, demarcated by the nodal domains.

INTRODUCTION: Familial dysautonomia (FD) is a rare hereditary sensory and autonomic neuropathy (type III). The disease is caused by a point mutation in the IKBKAP gene that affects the splicing of the elongator-1 protein (ELP-1) (also known as IKAP). Patients have dramatic blood pressure instability due to baroreflex failure, chronic kidney disease, and impaired swallowing leading to recurrent aspiration pneumonia, which results in chronic lung disease. Diminished pain and temperature perception result in neuropathic joints and thermal injuries. Impaired proprioception leads to gait ataxia. Optic neuropathy and corneal opacities lead to progressive visual loss. AREAS COVERED: This article reviews current therapeutic strategies for the symptomatic treatment of FD, as well as the potential of new gene-modifying agents. EXPERT OPINION: Therapeutic focus on FD is centered on reducing the catecholamine surges caused by baroreflex failure. Managing neurogenic dysphagia with effective protection of the airway passages and prompt treatment of aspiration pneumonias is necessary to prevent respiratory failure. Sedative medications should be used cautiously due to the risk of respiratory depression. Non-invasive ventilation during sleep effectively manages apneas and prevents hypercapnia. Clinical trials of compounds that increase levels of IKAP (ELP-1) are underway and will determine whether they can reverse or slow disease progression.