Ultrafast Optomechanical Strain in Layered GeS.

Strong coupling between light and mechanical strain forms the foundation for next-generation optical micro- and nano-electromechanical systems. Such optomechanical responses in two-dimensional materials present novel types of functionalities arising from the weak van der Waals bond between atomic layers. Here, by using structure-sensitive megaelectronvolt ultrafast electron diffraction, we report the experimental observation of optically driven ultrafast in-plane strain in the layered group IV monochalcogenide germanium sulfide (GeS). Surprisingly, the photoinduced structural deformation exhibits strain amplitudes of order 0.1% with a 10 ps fast response time and a significant in-plane anisotropy between zigzag and armchair crystallographic directions. Rather than arising due to heating, experimental and theoretical investigations suggest deformation potentials caused by electronic density redistribution and converse piezoelectric effects generated by photoinduced electric fields are the dominant contributors to the observed dynamic anisotropic strains. Our observations define new avenues for ultrafast optomechanical control and strain engineering within functional devices.

The CX3CL1 intracellular domain exhibits neuroprotection via insulin receptor/insulin-like growth factor receptor signaling.

CX3CL1, also known as fractalkine, is best known for its signaling activity through interactions with its cognate receptor CX3CR1. However, its intrinsic function that is independent of interaction with CX3CR1 remains to be fully understood. We demonstrate that the intracellular domain of CX3CL1 (CX3CL1-ICD), generated upon sequential cleavages by α-/ß-secretase and γ-secretase, initiates a back signaling activity, which mediates direct signal transmission to gene expression in the nucleus. To study this, we fused a synthetic peptide derived from CX3CL1-ICD, named Tet34, with a 13-amino acid tetanus sequence at the N terminus to facilitate translocation into neuronal cells. We show that treatment of mouse neuroblastoma Neuro-2A cells with Tet34, but not its scrambled control (Tet34s), induced cell proliferation, as manifested by changes in protein levels of transcription factors and progrowth molecules cyclin D1, PCNA, Sox5, and Cdk2. Further biochemical assays reveal elevation of phosphorylated insulin receptor ß subunit, insulin-like growth factor-1 receptor ß subunit, and insulin receptor substrates as well as activation of proliferation-linked kinase AKT. In addition, transgenic mice overexpressing membrane-anchored C-terminal CX3CL1 also exhibited activation of insulin/insulin-like growth factor-1 receptor signaling. Remarkably, we found that this Tet34 peptide, but not Tet34s, protected against endoplasmic reticulum stress and cellular apoptosis when Neuro-2A cells were challenged with toxic oligomers of ß-amyloid peptide or hydrogen peroxide. Taken together, our results suggest that CX3CL1-ICD may have translational potential for neuroprotection in Alzheimer's disease and for disorders resulting from insulin resistance.

Activated CX3CL1/Smad2 Signals Prevent Neuronal Loss and Alzheimer's Tau Pathology-Mediated Cognitive Dysfunction.

Neurofibrillary tangles likely cause neurodegeneration in Alzheimer's disease (AD). We demonstrate that the CX3CL1 C-terminal domain can upregulate neurogenesis, which may ameliorate neurodegeneration. Here we generated transgenic (Tg-CX3CL1) mice by overexpressing CX3CL1 in neurons. Tg-CX3CL1 mice exhibit enhanced neurogenesis in both subgranular and subventricular zones. This enhanced neurogenesis correlates well with elevated expression of TGF-ß2 and TGF-ß3, and activation of their downstream signaling molecule Smad2. Intriguingly, the enhanced adult neurogenesis was mitigated when Smad2 expression was deleted in neurons, supporting a role for the CX3CL1-TGF-ß2/3-Smad2 pathway in the control of adult neurogenesis. When Tg-CX3CL1 mice were crossed with Alzheimer's PS19 mice, which overexpress a tau P301S mutation and exhibit age-dependent neurofibrillary tangles and neurodegeneration, overexpressed CX3CL1 in both male and female mice was sufficient to rescue the neurodegeneration, increase survival time, and improve cognitive function. Hence, we provide in vivo evidence that CX3CL1 is a strong activator of adult neurogenesis, and that it reduces neuronal loss and improves cognitive function in AD.SIGNIFICANCE STATEMENT This study will be the first to demonstrate that enhanced neurogenesis by overexpressed CX3CL1 is mitigated by disruption of Smad2 signaling and is independent of its interaction with CX3CR1. Overexpression of CX3CL1 lengthens the life span of PS19 tau mice by enhancing adult neurogenesis while having minimal effect on tau pathology. Enhancing neuronal CX3CL1, mainly the C-terminal fragment, is a therapeutic strategy for blocking or reversing neuronal loss in Alzheimer's disease or related neurodegenerative disease patients.

Neurological dysfunctions associated with altered BACE1-dependent Neuregulin-1 signaling.

Inhibition of BACE1 is being pursued as a therapeutic target to treat patients suffering from Alzheimer's disease because BACE1 is the sole ß-secretase that generates ß-amyloid peptide. Knowledge regarding other cellular functions of BACE1 is therefore critical for the safe use of BACE1 inhibitors in human patients. Neuregulin-1 (Nrg1) is a BACE1 substrate and BACE1 cleavage of Nrg1 is critical for signaling functions in myelination, remyelination, synaptic plasticity, normal psychiatric behaviors, and maintenance of muscle spindles. This review summarizes the most recent discoveries associated with BACE1-dependent Nrg1 signaling in these areas. This body of knowledge will help to provide guidance for preventing unwanted Nrg1-based side effects following BACE1 inhibition in humans. To initiate its signaling cascade, membrane anchored Neuregulin (Nrg), mainly type I and III ß1 Nrg1 isoforms and Nrg3, requires ectodomain shedding. BACE1 is one of such indispensable sheddases to release the functional Nrg signaling fragment. The dependence of Nrg on the cleavage by BACE1 is best manifested by disrupting the critical role of Nrg in the control of axonal myelination, schizophrenic behaviors as well as the formation and maintenance of muscle spindles.

Phenotypic characterization of disseminated cells with TSC2 loss of heterozygosity in patients with lymphangioleiomyomatosis.

RATIONALE: Lymphangioleiomyomatosis (LAM), occurring sporadically (S-LAM) or in patients with tuberous sclerosis complex (TSC), results from abnormal proliferation of LAM cells exhibiting mutations or loss of heterozygosity (LOH) of the TSC genes, TSC1 or TSC2. OBJECTIVES: To identify molecular markers useful for isolating LAM cells from body fluids and determine the frequency of TSC1 or TSC2 LOH. METHODS: Candidate cell surface markers were identified using gene microarray analysis of human TSC2⁻(/)⁻ cells. Cells from bronchoalveolar lavage fluid (BALF), urine, chylous effusions, and blood were sorted based on reactivity with antibodies against these proteins (e.g., CD9, CD44v6) and analyzed for LOH using TSC1- and TSC2-related microsatellite markers and single nucleotide polymorphisms in the TSC2 gene. MEASUREMENTS AND MAIN RESULTS: CD44v6(+)CD9(+) cells from BALF, urine, and chyle showed TSC2 LOH in 80%, 69%, and 50% of patient samples, respectively. LAM cells with TSC2 LOH were detected in more than 90% of blood samples. LAM cells from different body fluids of the same patients showed, in most cases, identical LOH patterns, that is, loss of alleles at the same microsatellite loci. In a few patients with S-LAM, LAM cells from different body fluids differed in LOH patterns. No patients with S-LAM with TSC1 LOH were identified, suggesting that TSC2 abnormalities are responsible for the vast majority of S-LAM cases and that TSC1-disease may be subclinical. CONCLUSIONS: Our data support a common genetic origin of LAM cells in most patients with S-LAM, consistent with a metastatic model. In some cases, however, there was evidence for genetic heterogeneity between LAM cells in different sites or within a site.

Mesenchymal-epithelial interactions involving epiregulin in tuberous sclerosis complex hamartomas.

Patients with tuberous sclerosis complex (TSC) develop hamartomas containing biallelic inactivating mutations in either TSC1 or TSC2, resulting in mammalian target of rapamycin (mTOR) activation. Hamartomas overgrow epithelial and mesenchymal cells in TSC skin. The pathogenetic mechanisms for these changes had not been investigated, and the existence or location of cells with biallelic mutations ("two-hit" cells) was unclear. We compared TSC skin hamartomas (angiofibromas and periungual fibromas) with normal-appearing skin of the same patient, and we observed more proliferation and mTOR activation in hamartoma epidermis. Two-hit cells were not detected in the epidermis. Fibroblast-like cells in the dermis, however, exhibited allelic deletion of TSC2, in both touch preparations of fresh tumor samples and cells grown from TSC skin tumors, suggesting that increased epidermal proliferation and mTOR activation were not caused by second-hit mutations in the keratinocytes but by mesenchymal-epithelial interactions. Gene expression arrays, used to identify potential paracrine factors released by mesenchymal cells, revealed more epiregulin mRNA in fibroblast-like angiofibroma and periungual fibroma cells than in fibroblasts from normal-appearing skin of the same patient. Elevation of epiregulin mRNA was confirmed with real-time PCR, and increased amounts of epiregulin protein were demonstrated with immunoprecipitation. Epiregulin stimulated keratinocyte proliferation and phosphorylation of ribosomal protein S6 in vitro. These results suggest that hamartomatous TSC skin tumors are induced by paracrine factors released by two-hit cells in the dermis and that proliferation with mTOR activation of the overlying epidermis is an effect of epiregulin.

Long-Term Effects of Sirolimus on Human Skin TSC2-Null Fibroblast‒Like Cells.

Tuberous sclerosis complex (TSC) is an autosomal-dominant disorder characterized by hamartomatous tumors of the skin, kidneys, brain, and lungs. TSC is caused by mutations in the TSC1 and TSC2 genes, which result in hyperactivation of the mTOR, leading to dysregulated cell growth and autophagy. Rapamycin (sirolimus) shrinks TSC tumors, but the clinical benefits of sirolimus are not sustained after its withdrawal. In this study, we studied the cellular processes critical for tumor formation and growth, including cell proliferation and cell size. TSC2-/- and TSC2+/- cells were isolated from TSC skin tumors and normal-appearing skin, respectively. Cells were incubated with sirolimus for 72 hours. Withdrawal of sirolimus from TSC2-/- cells resulted in a highly proliferative phenotype and caused cells to enter the S phase of the cell cycle, with persistent phosphorylation of mTOR, p70 S6 kinase, ribosomal protein S6, and 4EB-P1; decreased cyclin D kinase inhibitors; and transient hyperactivation of protein kinase B. Sirolimus modulated the estrogen- and autophagy-dependent volume of TSC2-/- cells. These results suggest that sirolimus may decrease the size of TSC tumors by reducing TSC2-/- cell volume, altering the cell cycle, and reprogramming TSC2-null cells.

Antagonism of peripheral inflammation reduces the severity of status epilepticus.

Status epilepticus (SE) is one of the most serious manifestations of epilepsy. Systemic inflammation and damage of blood-brain barrier (BBB) are etiologic cofactors in the pathogenesis of pilocarpine SE while acute osmotic disruption of the BBB is sufficient to elicit seizures. Whether an inflammatory-vascular-BBB mechanism could apply to the lithium-pilocarpine model is unknown. LiCl facilitated seizures induced by low-dose pilocarpine by activation of circulating T-lymphocytes and mononuclear cells. Serum IL-1beta levels increased and BBB damage occurred concurrently to increased theta EEG activity. These events occurred prior to SE induced by cholinergic exposure. SE was elicited by lithium and pilocarpine irrespective of their sequence of administration supporting a common pathogenetic mechanism. Since IL-1beta is an etiologic trigger for BBB breakdown and its serum elevation occurs before onset of SE early after LiCl and pilocarpine injections, we tested the hypothesis that intravenous administration of IL-1 receptor antagonists (IL-1ra) may prevent pilocarpine-induced seizures. Animals pre-treated with IL-1ra exhibited significant reduction of SE onset and of BBB damage. Our data support the concept of targeting systemic inflammation and BBB for the prevention of status epilepticus.

The intracellular domain of CX3CL1 regulates adult neurogenesis and Alzheimer's amyloid pathology.

The membrane-anchored CX3CL1 is best known to exert its signaling function through binding its receptor CX3CR1. This study demonstrates a novel function that CX3CL1 exerts. CX3CL1 is sequentially cleaved by α-, ß-, and γ-secretase, and the released CX3CL1 intracellular domain (CX3CL1-ICD) would translocate into the cell nucleus to alter gene expression due to this back-signaling function. Amyloid deposition and neuronal loss were significantly reduced when membrane-anchored CX3CL1 C-terminal fragment (CX3CL1-ct) was overexpressed in Alzheimer's 5xFAD mouse model. The reversal of neuronal loss in 5xFAD can be attributed to increased neurogenesis by CX3CL1-ICD, as revealed by morphological and unbiased RNA-sequencing analyses. Mechanistically, this CX3CL1 back-signal likely enhances developmental and adult neurogenesis through the TGFß2/3-Smad2/3 pathway and other genes important for neurogenesis. Induction of CX3CL1 back-signaling may not only be a promising novel mechanism to replenish neuronal loss but also for reducing amyloid deposition for Alzheimer's treatment.

Bone marrow-derived keratinocytes are not detected in normal skin and only rarely detected in wounded skin in two different murine models.

OBJECTIVE: Because the ability of bone marrow-derived cells (BMDCs) to repopulate tissues and the possible mechanisms of repopulation remain controversial, we used two distinct murine models to determine whether BMDCs can repopulate epidermal keratinocytes during either steady-state homeostasis or after tissue injury. METHODS: The accessibility of skin keratinocytes makes it an excellent tissue to assess BMDC repopulation. In the two murine models, BMDCs from either male homologous B6, 129S Rosa26 mice that constitutively express ss-galactosidase or male hemizygote C57 BL/6-Tg(ACTbEGFP)1Osb/J mice expressing enhanced green fluorescent protein were transplanted via tail vein injection into control lethally irradiated (9.5 Gy) congenic female recipients and the percentage of keratinocytes derived from the transplanted BMDCs, both with and without wounding, was carefully determined. RESULTS: Analysis of bone marrow, thymus, spleen, and lymph nodes confirmed complete engraftment of donor BMDCs 6 months post-bone marrow transplantation. However, during steady-state homeostasis, bone marrow-derived keratinocytes could not be detected in the epidermis. In a skin wound-healing model, the epidermis contained only rare bone marrow-derived keratinocytes (< 0.0001%) but did contain scattered bone marrow-derived Langerhans cells. CONCLUSIONS: These results suggest that BMDCs do not significantly contribute to steady-state epidermal homeostasis and are not required or responsible for providing keratinocyte stem cells and keratinocyte repopulation following skin injury.

BACE1 Deficiency Causes Abnormal Neuronal Clustering in the Dentate Gyrus.

BACE1 is validated as Alzheimer's ß-secretase and a therapeutic target for Alzheimer's disease. In examining BACE1-null mice, we discovered that BACE1 deficiency develops abnormal clusters of immature neurons, forming doublecortin-positive neuroblasts, in the developing dentate gyrus, mainly in the subpial zone (SPZ). Such clusters were rarely observed in wild-type SPZ and not reported in other mouse models. To understand their origins and fates, we examined how neuroblasts in BACE1-null SPZ mature and migrate during early postnatal development. We show that such neuroblasts are destined to form Prox1-positive granule cells in the dentate granule cell layer, and mainly mature to form excitatory neurons, but not inhibitory neurons. Mechanistically, higher levels of reelin potentially contribute to abnormal neurogenesis and timely migration in BACE1-null SPZ. Altogether, we demonstrate that BACE1 is a critical regulator in forming the dentate granule cell layer through timely maturation and migration of SPZ neuroblasts.

Inhibiting BACE1 to reverse synaptic dysfunctions in Alzheimer's disease.

Over the past two decades, many studies have identified significant contributions of toxic ß-amyloid peptides (Aß) to the etiology of Alzheimer's disease (AD), which is the most common age-dependent neurodegenerative disease. AD is also recognized as a disease of synaptic failure. Aß, generated by sequential proteolytic cleavages of amyloid precursor protein (APP) by BACE1 and γ-secretase, is one of major culprits that cause this failure. In this review, we summarize current findings on how BACE1-cleaved APP products impact learning and memory through proteins localized on glutamatergic, GABAergic, and dopaminergic synapses. Considering the broad effects of Aß on all three types of synapses, BACE1 inhibition emerges as a practical approach for ameliorating Aß-mediated synaptic dysfunctions. Since BACE1 inhibitory drugs are currently in clinical trials, this review also discusses potential complications arising from BACE1 inhibition. We emphasize that the benefits of BACE1 inhibitory drugs will outweigh the concerns.

Tau Protein Mediates APP Intracellular Domain (AICD)-Induced Alzheimer's-Like Pathological Features in Mice.

Amyloid precursor protein (APP) is cleaved by gamma-secretase to simultaneously generate amyloid beta (Aß) and APP Intracellular Domain (AICD) peptides. Aß plays a pivotal role in Alzheimer's disease (AD) pathogenesis but recent studies suggest that amyloid-independent mechanisms also contribute to the disease. We previously showed that AICD transgenic mice (AICD-Tg) exhibit AD-like features such as tau pathology, aberrant neuronal activity, memory deficits and neurodegeneration in an age-dependent manner. Since AD is a tauopathy and tau has been shown to mediate Aß-induced toxicity, we examined the role of tau in AICD-induced pathological features. We report that ablating endogenous tau protects AICD-Tg mice from deficits in adult neurogenesis, seizure severity, short-term memory deficits and neurodegeneration. Deletion of tau restored abnormal phosphorylation of NMDA receptors, which is likely to underlie hyperexcitability and associated excitotoxicity in AICD-Tg mice. Conversely, overexpression of wild-type human tau aggravated receptor phosphorylation, impaired adult neurogenesis, memory deficits and neurodegeneration. Our findings show that tau is essential for mediating the deleterious effects of AICD. Since tau also mediates Aß-induced toxic effects, our findings suggest that tau is a common downstream factor in both amyloid-dependent and-independent pathogenic mechanisms and therefore could be a more effective drug target for therapeutic intervention in AD.

JNK-interacting protein 1 mediates Alzheimer's-like pathological features in AICD-transgenic mice.

Amyloid precursor protein, which generates amyloid beta peptides, is intimately associated with Alzheimer's disease (AD) pathogenesis. We previously showed that transgenic mice overexpressing amyloid precursor protein intracellular domain (AICD), a peptide generated simultaneously with amyloid beta, develop AD-like pathologies, including hyperphosphorylated tau, loss of synapses, and memory impairments. AICD is known to bind c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1), a scaffold protein that associates with and activates JNK. The aim of this study was to examine the role of JIP1 in AICD-induced AD-like pathologies in vivo, since the JNK pathway is aberrantly activated in AD brains and contributes to AD pathologies. We generated AICD-Tg mice lacking the JIP1 gene (AICD; JIP1(-/-)) and found that although AICD; JIP1(-/-) mice exhibit increased AICD, the absence of JIP1 results in decreased levels of hyperphosphorylated tau and activated JNK. AICD; JIP1(-/-) mice are also protected from synaptic loss and show improved performance in behavioral tests. These results suggest that JIP1 mediates AD-like pathologies in AICD-Tg mice and that JNK signaling may contribute to amyloid-independent mechanisms of AD pathogenesis.

Antibody microarrays: analysis of cystic fibrosis.

Cystic fibrosis (CF) is the most common autosomal recessive disease in the USA and Europe, whose life-limiting phenotype is manifest on epithelial cells throughout the body. The principal cause of morbidity and mortality is a massively proinflammatory condition in the lung. The mutation responsible for most cases of CF is [ΔF508]CFTR. However, the penetrance of the disease is quite variable, and adverse events leading to hospitalization cannot be easily predicted. Thus, there is a strong need for prognostic endpoints that might serve to identify impending clinical problems long before they happen. Our approach has been to search for proteomic signatures in easily accessed biological fluids that might identify the molecular basis for adverse events. We describe here a workflow that begins with patient-derived bronchial brush biopsies and progresses to analysis of serum and plasma from patients on antibody microarrays.

Blood-brain barrier damage, but not parenchymal white blood cells, is a hallmark of seizure activity.

It has long been held that chronic seizures cause blood-brain barrier (BBB) damage. Recent studies have also demonstrated that BBB damage triggers seizures. We have used the BBB osmotic disruption procedure (BBBD) to examine the correlation between BBB opening, pattern of white blood cell (WBCs) entry into the brain and seizure occurrence. These findings were compared to results from resected epileptic brain tissue from temporal lobe epilepsy (TLE) patients. We confirmed that a successful BBB osmotic opening (BBBD) leads to the occurrence of acute epileptiform discharges. Electroencephalography (EEG) and time-joint frequency analysis reveal EEG slowing followed by an increase in the 10-20Hz frequency range. Using green fluorescent protein (GFP)-labeled WBCs (GFP-WBCs) suspended in Evans Blue we found that, at time of BBB-induced epileptiform discharges, WBCs populated the perivascular space of a leaky BBB. Similar results were obtained at time of pilocarpine seizure. No frank WBCs extravasation in the brain parenchyma was observed. In TLE brain specimens, CD45-positive leukocytes were detected only in the vascular and perivascular spaces while albumin and IgG extravasates were parenchymal. The pattern was similar to those observed in rats. Our data suggest that neither acute-induced nor chronic seizures correlate with WBC brain parenchymal migration while albumin and IgG brain leakage is a hallmark of acute and chronic seizures.

Association of brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2) with recycling endosomes during transferrin uptake.

ADP-ribosylation factors (ARFs) are critical in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)1 and BIG2 activate ARFs by accelerating replacement of bound GDP with GTP. Additional and differing functions of these approximately 200-kDa proteins are now being recognized, as are their independent intracellular movements. Here, we describe the localization in COS7 cells by immunofluorescence microscopy of BIG2, but not BIG1, with structures that have characteristics of recycling endosomes during transferrin (Tfn) uptake and Tfn receptor (TfnR) recycling. Cell content of BIG2 and Rab11, but not TfnR, BIG1, Rab4, or Exo70, was increased after 60 min of Tfn uptake. BIG2, but not BIG1, appeared in density-gradient fractions containing TfnR, Rab11, and Exo70 after 60 min of Tfn uptake. Treatment of cells with BIG2 small interfering RNA (siRNA), but not BIG1 or control siRNAs, decreased BIG2 protein >90% without affecting BIG1, ARF, or actin content, whereas TfnR was significantly increased as was its accumulation in perinuclear recycling endosomes. Tfn release appeared unaffected by BIG1 siRNA but was significantly slowed from cells treated with BIG2 siRNA alone or plus BIG1 siRNA. We suggest that BIG2 has an important role in Tfn uptake and TfnR recycling, perhaps through its demonstrated interaction with Exo70 and the exocyst complex.