Biology subjects

Klein, R.

Publications and source records attributed to Klein, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Neuroprotective effects of hepatoma-derived growth factor in models of Huntington's disease

Huntingtons disease (HD) is a movement disorder caused by a mutation in the Huntingtin gene, that leads to severe neurodegeneration and inevitable death of the patients. Molecular mechanisms of HD are still not sufficiently understood, and no cure is currently available. Here, we demonstrate neuroprotective effects of hepatoma-derived growth factor (HDGF) in cellular and mouse models of HD. We show that HDGF expression levels in neuronal cell types inversely correlate with cellular vulnerability to HD. Moreover, lack of endogenous HDGF shortened lifespan and worsened rotarod performance of R6/2 HD model mice. AAV-mediated delivery of HDGF into the brain reduced mutant Huntingtin inclusion body load, but had no significant effect on motor behavior or lifespan. Interestingly, both nuclear and cytoplasmic versions of HDGF were equally efficient in rescuing mutant Huntingtin toxicity in cell culture models of HD. Moreover, extracellular application of a recombinant HDGF protein improved viability of mutant Huntingtin-expressing primary neurons and reduced mutant Huntingtin aggregation in neural progenitor cells differentiated from human patient-derived induced pluripotent stem cells (iPSCs). Our findings provide new insights into the pathomechanisms of HD and suggest neuroprotective potential of HDGF in neurodegeneration.

neuroscience↗

bioRxiv2022

Transcriptomics reveals amygdala neuron regulation by fasting and ghrelin thereby promoting feeding

The central amygdala (CeA) consists of numerous genetically-defined inhibitory neurons that control defensive and appetitive behaviors including feeding. Transcriptomic signatures of cell types and their links to function remain poorly understood. Using single-nucleus RNA-sequencing, we describe nine CeA cell clusters, of which four are mostly associated with appetitive and two with aversive behaviors. To analyze the activation mechanism of appetitive CeA neurons, we characterized Serotonin receptor 2a (Htr2a)-expressing neurons (CeAHtr2a) that comprise three appetitive clusters and were previously shown to promote feeding. In vivo calcium imaging revealed that CeAHtr2a neurons are activated by fasting, the hormone ghrelin, and the presence of food. Moreover, these neurons are required for the orexigenic effects of ghrelin. Appetitive CeA neurons responsive to fasting and ghrelin project to the parabrachial nucleus (PBN) causing inhibition of target PBN neurons. These results illustrate how the transcriptomic diversification of CeA neurons relates to fasting and hormone-regulated feeding behavior. HighlightsTranscriptomics reveals four appetitive, two aversive and three unknown CeA clusters Ghrelin and fasting activate CeAHtr2a neurons comprising three appetitive clusters Ghrelins orexigenic effects require the activity of appetitive CeA neurons CeA[->]PBN inhibition is necessary for ghrelin/fasting induced feeding

neuroscience↗

bioRxiv2022

Environmental DNA from archived leaves reveals widespread temporal turnover and biotic homogenization in forest arthropod communities

A major limitation of current reports on insect declines is the lack of standardized, long-term, and taxonomically broad time series. Here, we demonstrate the utility of environmental DNA from archived leaf material to characterize plant-associated arthropod communities. We base our work on several multi-decadal leaf time series from tree canopies in four land use types, which were sampled as part of a long-term environmental monitoring program across Germany. Using these highly standardized and well-preserved samples, we analyze temporal changes in communities of several thousand arthropod species belonging to 23 orders using metabarcoding and quantitative PCR. Our data do not support widespread declines of -diversity or genetic variation within sites. Instead, we find a gradual community turnover, which results in temporal and spatial biotic homogenization, across all land use types and all arthropod orders. Our results suggest that insect decline is more complex than mere -diversity loss, but can be driven by {beta}-diversity decay across space and time.

ecology↗

bioRxiv2022

Tripartite extended amygdala - basal ganglia CRH circuit drives arousal and avoidance behaviour

An adaptive stress response involves various mediators and circuits orchestrating a complex interplay of physiological, emotional and behavioural adjustments. We identified a population of corticotropin-releasing hormone (CRH) neurons in the lateral part of the interstitial nucleus of the anterior commissure (IPACL) - a subdivision of the extended amygdala, which exclusively innervate the substantia nigra (SN). Specific stimulation of this circuit elicits arousal and avoidance behaviour contingent on CRH receptor 1 (CRHR1) located at axon terminals in the SN, which originate from external globus pallidus (GPe) neurons. The neuronal activity prompting the observed behaviour is shaped by IPACLCRH and GPeCRHR1 neurons coalescing in the SN. These results delineate a novel tripartite CRH circuit functionally connecting extended amygdala and basal ganglia nuclei to drive arousal and avoidance behaviour. One-Sentence SummaryBrain centres involved in emotional and motor control are connected through a stress peptide promoting arousal and avoidance behaviour

neuroscience↗

bioRxiv2021

Neural basis of learning to perceive speech through touch using an acoustic-to-vibrotactile speech sensory substitution

The goal of sensory substitution is to convey the information transduced by one sensory system through a novel sensory modality. One example is vibrotactile (VT) speech, for which acoustic speech is transformed into vibrotactile patterns. Despite an almost century-long history of studying vibrotactile speech, there has been no study of the neural bases of VT speech learning. We here trained hearing adult participants to recognize VT speech syllables. Using fMRI, we showed that both somatosensory (left post-central gyrus) and auditory (right temporal lobe) regions acquire selectivity for VT speech stimuli following training. The right planum temporale in particular was selective for both VT and auditory speech. EEG source-estimated activity revealed temporal dynamics consistent with direct, low-latency engagement of right temporal lobe following activation of the left post-central gyrus. Our results suggest that VT speech learning achieves integration with the auditory speech system by "piggybacking" onto corresponding auditory speech representations. Significance statementIn sensory substitution, the information conveyed by one sensory system is used to replace the function of another. Blind individuals, for example, can learn to use a visual-to-acoustic sensory substitution device to navigate the world. We tested the hypothesis that sensory substitution can more generally occur in typical individuals, by exploiting the existence of multi-sensory convergence areas in the brain. We trained hearing participants to recognize speech syllables presented as vibrotactile stimulation patterns. Using fMRI and EEG, we show that vibrotactile speech learning integrates with the auditory speech system by "piggybacking" onto corresponding auditory speech representations. These results suggest that the human brain can bootstrap new senses by leveraging one sensory system to process information normally processed by another.

neuroscience↗

bioRxiv2021

Adolescent Brain Cognitive Development (ABCD) Community MRI Collection and Utilities

The Adolescent Brain Cognitive Development Study (ABCD), a 10 year longitudinal neuroimaging study of the largest population based and demographically distributed cohort of 9-10 year olds (N=11,877), was designed to overcome reproducibility limitations of prior child mental health studies. Besides the fantastic wealth of research opportunities, the extremely large size of the ABCD data set also creates enormous data storage, processing, and analysis challenges for researchers. To ensure data privacy and safety, researchers are not currently able to share neuroimaging data derivatives through the central repository at the National Data Archive (NDA). However, sharing derived data amongst researchers laterally can powerfully accelerate scientific progress, to ensure the maximum public benefit is derived from the ABCD study. To simultaneously promote collaboration and data safety, we developed the ABCD-BIDS Community Collection (ABCC), which includes both curated processed data and software utilities for further analyses. The ABCC also enables researchers to upload their own custom-processed versions of ABCD data and derivatives for sharing with the research community. This NeuroResource is meant to serve as the companion guide for the ABCC. In section we describe the ABCC. Section II highlights ABCC utilities that help researchers access, share, and analyze ABCD data, while section III provides two exemplar reproducibility analyses using ABCC utilities. We hope that adoption of the ABCCs data-safe, open-science framework will boost access and reproducibility, thus facilitating progress in child and adolescent mental health research.

neuroscience↗

bioRxiv2021

Metamodal Coupling of Vibrotactile and Auditory Speech Processing Systems Through Matched Stimulus Representations

It has been postulated that the brain is organized by "metamodal", sensory-independent cortical modules implementing particular computations, leading to the intriguing hypothesis that brain areas can perform tasks (such as word recognition) not just in "standard" sensory modalities but also in novel sensory modalities. Yet, evidence for this theory, especially in neurotypical subjects, has been variable. We hypothesized that effective metamodal engagement of a brain area requires congruence between the novel and standard sensory modalities not only at the task level (e.g., "word recognition") but critically also a match at the algorithmic level (in Marrs terminology), i.e., at the level of neural representation of the information of interest. To test this hypothesis, we trained participants to recognize vibrotactile versions of auditory words using two encoding schemes. The vocoded approach preserved the dynamics and representational similarities of auditory speech while the token-based approach used an abstract phoneme-based code. Although both groups learned the vibrotactile word recognition task, only in the vocoded group did trained vibrotactile stimuli recruit the auditory speech network and lead to increased coupling between somatosensory and auditory speech areas. In contrast, the token-based encoding appeared to rely on paired-associate learning. Thus, matching neural input representations is a critical factor for assessing and leveraging the metamodal potential of cortical modules.

neuroscience↗

bioRxiv2021

Molecular diet analysis in zebra and quagga mussels (Dreissena spp.) and an assessment of the utility of aquatic filter feeders as biological eDNA filters

Molecular gut content analysis is a popular tool to study food web interactions and was recently also suggested as an alternative source for DNA based biomonitoring. However, the overabundant consumers DNA often outcompetes that of its diet during PCR. Blocking approaches are an efficient means to reduce consumer amplification while retaining broad specificity for dietary taxa. We here designed an assay to monitor the eukaryotic diet of mussels and test their utility as biological eDNA filters to monitor planktonic communities. We designed several rDNA primer sets with a broad taxonomic suitability for eukaryotes, which suppress the amplification of mussels. The primers were tested using mussel DNA extracts and the results were compared to eDNA water samples collected next to the mussel colonies. Taxonomic recovery, as well as patterns of alpha and beta diversity, were compared between mussels and water samples. In addition, we analyzed time series samples of mussel samples from different German rivers. Our primer sets efficiently block the amplification of various mussel genera. The recovered DNA reflects a broad dietary preference across the eukaryotic tree of life and considerable taxonomic overlap with filtered water samples. We also recover various taxa of possible commensals and parasites, associated with the mussels. Our protocol will enable large scale dietary analysis in mussels, facilitate aquatic food web analysis, elucidate the ecological impact of invasive bivalves and the rapid survey of mussel aquacultures for pathogens. Moreover, we show that mussels could serve as an interesting complementary DNA source for biomonitoring.

ecology↗

bioRxiv2020

Novel proteostasis reporter mouse reveals different effects of cytoplasmic and nuclear aggregates on protein quality control in neurons

The cellular protein quality control machinery is important for preventing protein misfolding and aggregation, and decline in protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how proteostasis capacity of neurons changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc), a conformationally unstable protein that requires chaperones for proper folding and sensitively reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked impairment in proteostasis in tauopathy mice, but not in Huntingtons disease mice. Mechanistic investigations in primary neuronal cultures demonstrate that cytoplasmic, but not nuclear, aggregates cause defects of cellular protein quality control. Thus, the Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases.

neuroscience↗

bioRxiv2020

Encoding of environmental cues in central amygdala neurons during foraging

In order to successfully forage in an environment filled with rewards and threats, animals need to rely on familiar structures of their environment that signal food availability. The central amygdala (CeA) is known to mediate a panoply of consummatory and defensive behaviors, yet how specific activity patterns within CeA subpopulations guide optimal choices is incompletely understood. In a paradigm of appetitive conditioning in which mice freely forage for food across a continuum of cues, we find that two major subpopulations of CeA neurons, Somatostatin-positive (CeASst) and protein kinase C{delta}-positive (CeAPKC{delta}) neurons can assign motivational properties to environmental cues and encode memory of goal location. While the proportion of food responsive cells was higher within CeASst than CeAPKC{delta} neurons, only the activities of CeAPKC{delta}, but not CeASst, neurons were required for learning of contextual food cues. Since CeAPKC{delta} neurons are known to promote a range of defensive behaviors, our findings point to a model in which CeA circuit components are not organized in specialized functional units but can process both aversive and rewarding information in a context and experience dependent manner. HIGHLIGHTSTwo populations of central amygdala (CeA) neurons, CeAPKC{delta} and CeASst neurons can assign motivational properties to environmental cues and encode memory of goal location. The proportion of food responsive cells was higher among CeASst, than CeAPKC{delta} neurons. The activities of CeAPKC{delta}, but not CeASst, neurons are required for learning of contextual food cues. CeAPKC{delta} neurons represent a "general encoding" population selecting defensive and appetitive responses depending on context.

neuroscience↗

bioRxiv2019

Amyloid-like aggregates cause lysosomal defects in neurons via gain-of-function toxicity

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like {beta}-sheet proteins ({beta} proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, {beta} proteins interact with and sequester AP-31, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-31 expression ameliorates neurotoxicity caused by {beta} proteins. Altogether, our results highlight the link between protein aggregation and neurotoxicity, pointing to lysosomes as particularly vulnerable organelles.

cell biology↗

bioRxiv2019

Presynaptic inhibition of cutaneous afferents prevents chronic itch

Chronic itch represents an incapacitating burden on patients suffering a wide spectrum of diseases. Despite recent advances in our understanding of the cells and circuits implicated in the processing of itch information, chronic itch often presents itself without apparent cause. Here, we identify a spinal subpopulation of inhibitory neurons defined by the expression of Ptf1a involved in gating mechanosensory information self-generated during movement. These neurons receive tactile and motor input and establish presynaptic inhibitory contacts on mechanosensory afferents. Loss of Ptf1a neurons leads to increased hairy skin sensitivity and chronic itch, at least partially mediated through the classic itch pathway involving gastrin releasing peptide receptor (GRPR) spinal neurons. Conversely, chemogenetic activation of GRPR neurons elicits itch which is suppressed by concomitant activation of Ptf1a neurons. These findings shed new light on the circuit mechanisms implicated in chronic itch and open novel targets for therapy developments. Highlights* Ptf1a specifies adult spinal presynaptic neurons contacting cutaneous afferents * Loss of spinal Ptf1a+ neurons leads to self-generated itch and excessive grooming * Absence of Ptf1a+ neurons increases hairy skin sensitivity which triggers scratching * GRPR+ neurons act downstream of Ptf1a+ neurons in spontaneous itch

neuroscience↗

bioRxiv2019

An integrative ENCODE resource for cancer genomics

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

genomics↗

bioRxiv2019

Pan-filovirus one-step reverse transcription-polymerase chain reaction screening assay

Five species within the genera Ebolavirus and Marburgvirus of the family Filoviridae are known to cause severe hemorrhagic fever with high mortality rates in humans and non-human primates. Recent large outbreaks of Ebola virus disease in West Africa (2014 - 2016) and the Democratic Republic of the Congo (2018 - ongoing) have demonstrated the epidemic potential with devastating public health consequences. Several known and novel filovirus species have been found in bats in recent years. However, the role of each virus species in the disease ecology of human disease is still unclear. In particular, the transmission mechanism from potential animal hosts to humans is not known. Therefore, a simple, flexible, cost-effective screening tool for detecting the presence of any (putative) member of the filovirus family in animal samples is needed. In this study, a one-step conventional pan-filovirus RT-PCR assay was developed. The designed universal consensus primers of this screening test target two highly conserved regions of the nucleoprotein (NP) of all currently known filoviruses. The assay was capable of specific amplification of viral RNA of all six primate-pathogenic (human and non-human) filovirus species and resulted in 317 bp long RT-PCR products. This amplicon length renders the assay suitable for flexible application as conventional reverse transcription polymerase chain reaction (RT-PCR) as well as for future use as rapid real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR).

molecular biology↗

bioRxiv2018

Genome-wide association study of diabetic kidney disease highlights biology involved in renal basement membrane collagen

Diabetic kidney disease (DKD) is a heritable but poorly understood complication of diabetes. To identify genetic variants predisposing to DKD, we performed genome-wide association analyses in 19,406 individuals with type 1 diabetes (T1D) using a spectrum of DKD definitions basedon albuminuria and renal function. We identified 16 genome-wide significant loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM) implicated in heritable nephropathies. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of DKD, including albuminuria and end-stage renal disease. Three other loci are in or near genes with known or suggestive involvement in DKD (BMP7) or renal biology (COLEC11 and DDR1). The 16 DKD-associated loci provide novel insights into the pathogenesis of DKD, identifying potential biological targets for prevention and treatment.

genetics↗

bioRxiv2018

In Silico Identification and In Vitro Validation of Novel KPC-2 β-lactamase Inhibitors

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type {beta}-lactamases are often reported as resistant to available {beta}-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non {beta}-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-{beta}-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the {beta}-lactam antibiotic meropenem by four fold.

biochemistry↗

bioRxiv2018

Cortical circuit alterations precede disease onset in Huntington’s disease mice

AbstractHuntingtons disease (HD) is a devastating hereditary movement disorder, characterized by degeneration of neurons in the striatum and cortex. Studies in human patients and mouse HD models suggest that disturbances of neuronal function in the neocortex play an important role in the disease onset and progression. However, the precise nature and time course of cortical alterations in HD have remained elusive. Here, we use chronic in vivo two-photon calcium imaging to monitor the activity of single neurons in layer 2/3 of the primary motor cortex in awake, behaving R6/2 transgenic HD mice and wildtype littermates. R6/2 mice show age-dependent changes in neuronal activity with a clear increase in activity at the age of 8.5 weeks, preceding the onset of motor and neurological symptoms. Furthermore, quantitative proteomics demonstrate a pronounced downregulation of synaptic proteins in the cortex, and histological analyses in R6/2 mice and HD patient samples reveal reduced inputs from parvalbumin-positive interneurons onto layer 2/3 pyramidal cells. Thus, our study provides a time-resolved description as well as mechanistic details of cortical circuit dysfunction in HD.\n\nSignificance statementFuntional alterations in the cortex are believed to play an important role in the pathogenesis of Huntingtons disease (HD). However, studies monitoring cortical activity in HD models in vivo at a single-cell resultion are still lacking. We have used chronic two-photon imaging to investigate changes in the activity of single neurons in the primary motor cortex of awake presymptomatic HD mice. We show that neuronal activity increases before the mice develop disease symptoms. Our histological analyses in mice and in human HD autopsy cases furthermore demonstrate a loss inhibitory synaptic terminals from parvalbimun-positive interneurons, revealing a potential mechanism of cortical circuit impairment in HD.

neuroscience↗

bioRxiv2018

Validation of Prostate Cancer Risk Variants by CRISPR/Cas9 Mediated Genome Editing

GWAS have identified numerous SNPs associated with prostate cancer risk. One such SNP is rs10993994. It is located in the MSMB promoter, associates with MSMB encoded {beta}-microseminoprotein prostate secretion levels, and is associated with mRNA expression changes in MSMB and the adjacent gene NCOA4. In addition, our previous work showed a second SNP, rs7098889, is in LD with rs10993994 and associated with MSMB expression independent of rs10993994. Here, we generate a series of clones with single alleles removed by double guide RNA (gRNA) mediated CRISPR/Cas9 deletions, through which we demonstrate that each of these SNPs independently and greatly alters MSMB expression in an allele-specific manner. We further show that these SNPs have no substantial effect on the expression of NCOA4. These data demonstrate that a single SNP can have a large effect on gene expression and illustrate the importance of functional validation to deconvolute observed correlations. The method we have developed is generally applicable to test any SNP for which a relevant heterozygous cell line is available.\n\nAuthor summaryIn pursuing the underlying biological mechanism of prostate cancer pathogenesis, scientists utilized the existence of common single nucleotide polymorphisms (SNPs) in human genome as genetic markers to perform large scale genome wide association studies (GWAS) and have so far identified more than a hundred prostate cancer risk variants. Such variants provide an unbiased and systematic new venue to study the disease mechanism, and the next big challenge is to translate these genetic associations to the causal role of altered gene function in oncogenesis. The majority of these variants are waiting to be studied and lots of them may act in oncogenesis through gene expression regulation. To prove the concept, we took rs10993994 and its linked rs7098889 as an example and engineered single cell clones by allelic-specific CRISPR/Cas9 deletion to separate the effect of each allele. We observed that a single nucleotide difference would lead to surprisingly high level of MSMB gene expression change in a gene specific and tissue specific manner. Our study strongly supports the notion that differential level of gene expression caused by risk variants and their associated genetic locus play a major role in oncogenesis and also highlights the importance of studying the function of MSMB encoded {beta}-MSP in prostate cancer pathogenesis.

genetics↗