Integrated proteogenomic characterization of glioblastoma evolution.

The evolutionary trajectory of glioblastoma (GBM) is a multifaceted biological process that extends beyond genetic alterations alone. Here, we perform an integrative proteogenomic analysis of 123 longitudinal glioblastoma pairs and identify a highly proliferative cellular state at diagnosis and replacement by activation of neuronal transition and synaptogenic pathways in recurrent tumors. Proteomic and phosphoproteomic analyses reveal that the molecular transition to neuronal state at recurrence is marked by post-translational activation of the wingless-related integration site (WNT)/ planar cell polarity (PCP) signaling pathway and BRAF protein kinase. Consistently, multi-omic analysis of patient-derived xenograft (PDX) models mirror similar patterns of evolutionary trajectory. Inhibition of B-raf proto-oncogene (BRAF) kinase impairs both neuronal transition and migration capability of recurrent tumor cells, phenotypic hallmarks of post-therapy progression. Combinatorial treatment of temozolomide (TMZ) with BRAF inhibitor, vemurafenib, significantly extends the survival of PDX models. This study provides comprehensive insights into the biological mechanisms of glioblastoma evolution and treatment resistance, highlighting promising therapeutic strategies for clinical intervention.

In situ silver nanoparticle development for molecular-specific biological imaging via highly accessible microscopies.

In biological studies and diagnoses, brightfield (BF), fluorescence, and electron microscopy (EM) are used to image biomolecules inside cells. When compared, their relative advantages and disadvantages are obvious. BF microscopy is the most accessible of the three, but its resolution is limited to a few microns. EM provides a nanoscale resolution, but sample preparation is time-consuming. In this study, we present a new imaging technique, which we termed decoration microscopy (DecoM), and quantitative investigations to address the aforementioned issues in EM and BF microscopy. For molecular-specific EM imaging, DecoM labels proteins inside cells using antibodies bearing 1.4 nm gold nanoparticles (AuNPs) and grows silver layers on the AuNPs' surfaces. The cells are then dried without buffer exchange and imaged using scanning electron microscopy (SEM). Structures labeled with silver-grown AuNPs are clearly visible on SEM, even they are covered with lipid membranes. Using stochastic optical reconstruction microscopy, we show that the drying process causes negligible distortion of structures and that less structural deformation could be achieved through simple buffer exchange to hexamethyldisilazane. Using DecoM, we visualize the nanoscale alterations in microtubules by microtubule-severing proteins that cannot be observed with diffraction-limited fluorescence microscopy. We then combine DecoM with expansion microscopy to enable sub-micron resolution BF microscopy imaging. We first show that silver-grown AuNPs strongly absorb white light, and the structures labeled with them are clearly visible on BF microscopy. We then show that the application of AuNPs and silver development must follow expansion to visualize the labeled proteins clearly with sub-micron resolution.

Functions and dysfunctions of oligodendrocytes in neurodegenerative diseases.

Neurodegenerative diseases (NDDs) are characterized by the progressive loss of selectively vulnerable populations of neurons, which is responsible for the clinical symptoms. Although degeneration of neurons is a prominent feature that undoubtedly contributes to and defines NDD pathology, it is now clear that neuronal cell death is by no means mediated solely by cell-autonomous mechanisms. Oligodendrocytes (OLs), the myelinating cells of the central nervous system (CNS), enable rapid transmission of electrical signals and provide metabolic and trophic support to neurons. Recent evidence suggests that OLs and their progenitor population play a role in the onset and progression of NDDs. In this review, we discuss emerging evidence suggesting a role of OL lineage cells in the pathogenesis of age-related NDDs. We start with multiple system atrophy, an NDD with a well-known oligodendroglial pathology, and then discuss Alzheimer's disease (AD) and Parkinson's disease (PD), NDDs which have been thought of as neuronal origins. Understanding the functions and dysfunctions of OLs might lead to the advent of disease-modifying strategies against NDDs.

Comparing axon regeneration in male and female mice after peripheral nerve injury.

Axons in the adult mammalian central nervous system fail to regenerate after injury. By contrast, spontaneous axon regeneration occurs in the peripheral nervous system (PNS) due to a supportive PNS environment and an increase in the intrinsic growth potential induced by injury via cooperative activation of multifaceted biological pathways. This study compared axon regeneration and injury responses in C57BL/6 male and female mice after sciatic nerve crush (SNC) injury. The extent of axon regeneration in vivo was indistinguishable in male and female mice when observed at 3 days after SNC injury, and primary dorsal root ganglion (DRG) neurons from injured, male and female mice extended axons to a similar length. Moreover, the induction of selected regeneration-associated genes (RAGs), such as Atf3, Sprr1a, Gap43, Sox11, Jun, Gadd45a, and Smad1 were comparable in male and female DRGs when assessed by quantitative real-time reverse transcription polymerase chain reaction. Furthermore, the RNA-seq analysis of male and female DRGs revealed that differentially expressed genes (DEGs) in SNC groups compared to sham-operated groups included many common genes associated with neurite outgrowth. However, we also found that a large number of genes in the DEGs were sex dependent, implicating the involvement of distinct gene regulatory network in the two sexes following peripheral nerve injury. In conclusion, we found that male and female mice mounted a comparable axon regeneration response and many RAGs were commonly induced in response to SNC. However, given that many DEGs were sex-dependently expressed, future studies are needed to investigate whether they contribute to peripheral axon regeneration, and if so, to what extent.

Neur1 and Neur2 are required for hippocampus-dependent spatial memory and synaptic plasticity.

Neur1 and Neur2, mouse homologs of the Drosophila neur gene, consist of two neuralized homology repeat domains and a RING domain. Both Neur1 and Neur2 are expressed in the whole adult brain and encode E3 ubiquitin ligases, which play a crucial role in the Notch signaling pathways. A previous study reported that overexpression of Neur1 enhances hippocampus-dependent memory, whereas the role of Neur2 remains largely unknown. Here, we aimed to elucidate the respective roles of Neur1 and Neur2 in hippocampus-dependent memory using three lines of genetically modified mice: Neur1 knock-out, Neur2 knock-out, and Neur1 and Neur2 double knock-out (D-KO). Our results showed that spatial memory was impaired when both Neur1 and Neur2 were deleted, but not in the individual knock-out of either Neur1 or Neur2. In addition, basal synaptic properties estimated by input-output relationships and paired-pulse facilitation did not change, but a form of long-term potentiation that requires protein synthesis was specifically impaired in the D-KO mice. These results collectively suggest that Neur1 and Neur2 are crucially involved in hippocampus-dependent spatial memory and synaptic plasticity.

Transient cAMP elevation during systems consolidation enhances remote contextual fear memory.

Memory is stored in our brains over a temporally graded transition. With time, recently formed memories are transformed into remote memories for permanent storage; multiple brain regions, such as the hippocampus and neocortex, participate in this process. In this study, we aimed to understand the molecular mechanism of systems consolidation of memory and to investigate the brain regions that contribute to this regulation. We first carried out a contextual fear memory test using a transgenic mouse line, which expressed exogenously-derived Aplysia octopamine receptors in the forebrain region, such that, in response to octopamine treatment, cyclic adenosine monophosphate (cAMP) levels could be transiently elevated. From this experiment, we revealed that transient elevation of cAMP levels in the forebrain during systems consolidation led to an enhancement in remote fear memory and increased miniature excitatory synaptic currents in layer II/III of the anterior cingulate cortex (ACC). Furthermore, using an adeno-associated-virus-driven DREADD system, we investigated the specific regions in the forebrain that contribute to the regulation of memory transfer into long-term associations. Our results implied that transient elevation of cAMP levels was induced chemogenetically in the ACC, but not in the hippocampus, and showed a significant enhancement of remote memory. This finding suggests that neuronal activation during systems consolidation through the elevation of cAMP levels in the ACC contributes to remote memory enhancement.

LRRK2 and membrane trafficking: nexus of Parkinson's disease.

Recent evidence from genetics, animal model systems and biochemical studies suggests that defects in membrane trafficking play an important part in the pathophysiology of Parkinson's disease (PD). Mutations in leucine-rich repeat kinase 2 (LRRK2) constitute the most frequent genetic cause of both familial and sporadic PD, and LRRK2 has been suggested as a druggable target for PD. Although the precise physiological function of LRRK2 remains largely unknown, mounting evidence suggests that LRRK2 controls membrane trafficking by interacting with key regulators of the endosomal-lysosomal pathway and synaptic recycling. In this review, we discuss the genetic, biochemical and functional links between LRRK2 and membrane trafficking. Understanding the mechanism by which LRRK2 influences such processes may contribute to the development of disease-modifying therapies for PD. [BMB Reports 2019; 52(9): 533-539].

Structural and Molecular Basis for Katanin-Mediated Severing of Glutamylated Microtubules.

Katanin was the first microtubule (MT)-severing enzyme discovered, but how katanin executes MT severing remains poorly understood. Here, we report X-ray crystal structures of the apo and ATPγS-bound states of the catalytic AAA domain of human katanin p60 at 3.0 and 2.9 Å resolution, respectively. Comparison of the two structures reveals conformational changes induced by ATP binding and how such changes ensure hexamer stability. Moreover, we uncover structural details of pore loops (PLs) and show that Arg283, a residue unique to katanin among MT-severing enzymes, protrudes from PL1 and lines the entry of the catalytic pore. Functional studies suggest that PL1 and Arg283 play essential roles in the recognition and remodeling of the glutamylated, C-terminal tubulin tail and regulation of axon growth. In addition, domain-swapping experiments in katanin and spastin suggest that the non-homologous N-terminal region, which contains the MT-interacting and trafficking domain and a linker, confers specificity to the severing process.

ß-Adrenergic signaling is required for the induction of a labile state during memory reconsolidation.

Memory reconsolidation is the process by which previously consolidated memories reenter a labile state through reactivation of the memory trace and are actively consolidated through de novo protein synthesis. Although extensive studies have shown that ß-adrenergic signaling plays a critical role in the restabilization of reactivated memory, its role in the destabilization of long-term memory is not well-studied. In this study, we found that membrane excitability increased in hippocampal CA1 neurons immediately after the retrieval of contextual fear memory. Interestingly, this increase in membrane excitability diminished after treatment with propranolol (a ß-adrenergic receptor antagonist), an NMDA receptor antagonist, and a PKA inhibitor. In addition, we found that administration of propranolol prior to, but not after, the retrieval of fear memory ameliorated the memory impairment caused by anisomycin, indicating that inhibition of ß-adrenergic signaling blocks the destabilization of contextual fear memory. Taken together, these results indicate that ß-adrenergic signaling via NMDA receptors and PKA signaling pathway induces a labile state of long-term memory through increased neuronal membrane excitability.

Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration.

BACKGROUND: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo. METHODS: In vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35. RESULTS: Our screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo. CONCLUSIONS: Here we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

PKCα-mediated phosphorylation of LSD1 is required for presynaptic plasticity and hippocampal learning and memory.

Lysine-specific demethylase 1 (LSD1) is a histone demethylase that participates in transcriptional repression or activation. Recent studies reported that LSD1 is involved in learning and memory. Although LSD1 phosphorylation by PKCα was implicated in circadian rhythmicity, the importance of LSD1 phosphorylation in learning and memory is unknown. In this study, we examined the roles of LSD1 in synaptic plasticity and memory using Lsd1 SA/SA knock-in (KI) mice, in which a PKCα phosphorylation site is mutated. Interestingly, short-term and long-term contextual fear memory as well as spatial memory were impaired in Lsd1 KI mice. In addition, short-term synaptic plasticity, such as paired pulse ratio and post-tetanic potentiation was impaired, whereas long-term synaptic plasticity, including long-term potentiation and long-term depression, was normal. Moreover, the frequency of miniature excitatory postsynaptic current was significantly increased, suggesting presynaptic dysfunction in Lsd1 KI mice. Consistent with this, RNA-seq analysis using the hippocampus of Lsd1 KI mice showed significant alterations in the expressions of presynaptic function-related genes. Intriguingly, LSD1n-SA mutant showed diminished binding to histone deacetylase 1 (HDAC1) compared to LSD1n-WT in SH-SY5Y cells. These results suggest that LSD1 is involved in the regulation of presynaptic gene expression and subsequently regulates the hippocampus-dependent memory in phosphorylation-dependent manner.

Effects of Microtubule Stabilization by Epothilone B Depend on the Type and Age of Neurons.

Several studies have demonstrated the therapeutic potential of applying microtubule- (MT-) stabilizing agents (MSAs) that cross the blood-brain barrier to promote axon regeneration and prevent axonal dystrophy in rodent models of spinal cord injury and neurodegenerative diseases. Paradoxically, administration of MSAs, which have been widely prescribed to treat malignancies, is well known to cause debilitating peripheral neuropathy and axon degeneration. Despite the growing interest of applying MSAs to treat the injured or degenerating central nervous system (CNS), consequences of MSA exposure to neurons in the central and peripheral nervous system (PNS) have not been thoroughly investigated. Here, we have examined and compared the effects of a brain-penetrant MSA, epothilone B, on cortical and sensory neurons in culture and show that epothilone B exhibits both beneficial and detrimental effects, depending on not only the concentration of drug but also the type and age of a neuron, as seen in clinical settings. Therefore, to exploit MSAs to their full benefit and minimize unwanted side effects, it is important to understand the properties of neuronal MTs and strategies should be devised to deliver minimal effective concentration directly to the site where needed.

The Brain-Enriched MicroRNA miR-9-3p Regulates Synaptic Plasticity and Memory.

UNLABELLED: MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate gene expression in many tissues. Although a number of brain-enriched miRNAs have been identified, only a few specific miRNAs have been revealed as critical regulators of synaptic plasticity, learning, and memory. miR-9-5p/3p are brain-enriched miRNAs known to regulate development and their changes have been implicated in several neurological disorders, yet their role in mature neurons in mice is largely unknown. Here, we report that inhibition of miR-9-3p, but not miR-9-5p, impaired hippocampal long-term potentiation (LTP) without affecting basal synaptic transmission. Moreover, inhibition of miR-9-3p in the hippocampus resulted in learning and memory deficits. Furthermore, miR-9-3p inhibition increased the expression of the LTP-related genes Dmd and SAP97, the expression levels of which are negatively correlated with LTP. These results suggest that miR-9-3p-mediated gene regulation plays important roles in synaptic plasticity and hippocampus-dependent memory. SIGNIFICANCE STATEMENT: Despite the abundant expression of the brain-specific microRNA miR-9-5p/3p in both proliferating and postmitotic neurons, most functional studies have focused on their role in neuronal development. Here, we examined the role of miR-9-5p/3p in adult brain and found that miR-9-3p, but not miR-9-5p, has a critical role in hippocampal synaptic plasticity and memory. Moreover, we identified in vivo binding targets of miR-9-3p that are involved in the regulation of long-term potentiation. Our study provides the very first evidence for the critical role of miR-9-3p in synaptic plasticity and memory in the adult mouse.

Effect of Food Deprivation on a Delayed Nonmatch-to-place T-maze Task.

Food deprivation can affect performance on difficult cognitive task, such as the delayed nonmatch-to-place T-maze task (DNMT). The importance of food deprivation on maintaining high motivation for DNMT task has been emphasized, but not many studies have investigated the optimal conditions for depriving rodents to maximize performance. Establishing appropriate conditions for food deprivation is necessary to maintain DNMT task motivation. We applied different conditions of food deprivation (1-h food restriction vs. 1.5-g food restriction; single caging vs. group caging) and measured body weight and the number of correct choices that 8-week-old C57BL/6J mice made during the DNMT task. The 1.5-g food restriction group maintained 76.0±0.6% of their initial body weight, but the final body weight of the 1-h food restriction condition group was reduced to 62.2±0.8% of their initial body weight. These results propose that 1.5-g food restriction condition is effective condition for maintaining both body weight and motivation to complete the DNMT task.