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Weiss, K.

Publications and source records attributed to Weiss, K..

13 recordsLinked to original sources

Critical period plasticity enables credit assignment

Synaptic plasticity is often guided by instructive inputs to neural circuits, but learning only succeeds when these instructions reach neurons that mediate relevant outputs. This creates the credit assignment problem: how does a neuron receive instructions suited to its own behavioral function? Here we report a developmental mechanism that enables credit assignment by using instructive inputs to organize the downstream architecture through which learning is expressed. In the olivocerebellar learning system, the inferior olive provides instructive inputs that guide plasticity within the cerebellum. During circuit assembly in zebrafish, we find these same inputs regulate long-range cerebellar projections during a highly plastic, two-day critical period. Developmental experience caused specific maturation of projections to targets that were coactivated with olivary inputs. Mathematical theory and computational modeling show how the resulting architecture constrains later learning, such that a single input can sculpt downstream connectivity and then leverage that circuit to assign credit. Simulated learning became paradoxically more robust if we reduced plasticity after a developmental critical period, protecting key architecture from corruption during learning. Thus, instructive inputs can first build the circuits they later teach, coordinating development and learning to enable effective credit assignment.

neuroscience↗

Reciprocal regulation of the H3 histamine receptor in Rett syndrome and MECP2 Duplication 1 syndrome: implications for therapeutic development

Rett syndrome (RTT) and MECP2 Duplication syndrome (MDS) are disorders caused by reciprocal decreases and increases in the expression of the transcriptional regulator, Methyl CpG Binding Protein 2 (MeCP2). We previously performed an mRNA expression profiling study of the temporal cortex region from patients diagnosed with RTT and corresponding age, postmortem interval, and sex-matched controls. These studies identified a significant reduction in the expression of the histamine H3 receptor (HRH3). In the current manuscript, we expanded this H3 receptor profiling to additional RTT patient brain samples representing distinct MECP2 mutations and confirmed significantly reduced levels of H3 receptor expression in the majority of patients compared to controls. Using mouse models of RTT and MDS, we observed antiparallel changes in H3 receptor expression across various brain areas, with Hrh3 expression being reduced in RTT model animals and increased in a mouse model of MDS. We then evaluated both a small molecule agonist of the H3 receptor, (R)--methylhistamine (RAMH), and the H3 receptor inverse agonist, pitolisant (Wakix(R)), in RTT and MDS models, respectively, to determine impacts on phenotypes in these disease models. Our results show that RAMH significantly impacted an anxiety phenotype in mice modeling RTT (MecpNull/+), but pitolisant had no effect on the behaviors examined here in MDS animals (MECP2Tg1).

neuroscience↗

Leonardo: a toolset to correct sample-induced artifacts in light sheet microscopy images

Selective plane illumination microscopy (SPIM, also known as light sheet fluorescence microscopy) is the method of choice for studying morphogenesis and function in biological specimens over extended periods, as it permits gentle and rapid volumetric imaging. In inhomogeneous samples, however, sample-induced artifacts, including light absorption, scattering, and refraction, can impact the image quality, particularly as the focal plane gets deeper into the sample. Here, we present Leonardo, the first toolbox designed to address the major sample-induced artifacts by using two modules: (1) DeStripe removes stripe artifacts in SPIM caused by light absorption while preserving fine sample structures; (2) Fuse reconstructs a single high-quality image from dualsided illumination and/or dual-sided detection, while eliminating blur and optical distortions caused by light scattering and refraction. The efficacy of Leonardo is validated on a wide range of biological samples, from minimally invasive experiments on sensitive specimens (translucent embryonic and optically opaque larval zebrafish) to cleared mouse samples up to two centimeters in size. We provide model code and a Napari-based graphical user interface, enabling the SPIM community to easily apply Leonardo to advance light sheet imaging of inhomogeneous and complex specimens.

bioinformatics↗

Interaction with AK2A links AIFM1 to cellular energy metabolism

Apoptosis inducing factor 1 (AIFM1) is a flavoprotein essential for mitochondrial function and biogenesis. Its interaction with MIA40, the central component of the mitochondrial disulfide relay, accounts for some, but not all effects of AIFM1 loss. Our high-confidence AIFM1 interactome revealed novel interaction partners of AIFM1. For one of these interactors, adenylate kinase 2 (AK2), an essential enzyme maintaining cellular adenine nucleotide pools, AIFM1 binding specifically stabilized the isoform AK2A via interaction with its C-terminus. High resolution cryo-EM and biochemical analyses showed that both, MIA40 and AK2A bind AIFM1s C-terminal {beta}-strand, enhancing NADH oxidoreductase activity by locking an active, dimer conformation and, in the case of MIA40, affecting the cofactor binding site. The AIFM1-AK2A interaction is crucial during respiratory conditions. We further identified ADP/ATP translocases and the ATP synthase as AIFM1 interactors, emphasizing its important regulatory role as a central, organizing platform in energy metabolism.

biochemistry↗

Cyclase-associated protein 1 (CAP1) represses MRTF-SRF-dependent gene expression in the mouse cerebral cortex

Serum response factor (SRF) is a ubiquitously expressed transcription factor essential for brain development and function. SRF activity is controlled by two competing classes of coactivators, myocardin-related transcription factors (MRTF) and ternary complex factors (TCF), which introduce specificity into gene expression programs. To date, only few brain studies investigated upstream regulatory mechanisms, which mainly focused on TCF. Since an inhibitory function of monomeric actin towards MRTF-SRF signaling is well-established, we hypothesized a regulatory role for the key actin regulator ADF/cofilin. Surprisingly, ADF/cofilin was largely dispensable for neuronal MRTF-SRF activity. Instead, reporter assays combined with pharmacological and genetic approaches in isolated mouse neurons identified cyclase-associated protein 1 (CAP1) as an important regulator of this pathway. CAP1 promotes cytosolic MRTF retention and represses neuronal MRTF-SRF signaling via an actin-dependent mechanism that requires two specific protein domains. Further, deep RNA sequencing and mass spectrometry in mutant mice proved CAP1s in vivo relevance for this pathway in the cerebral cortex, and led to the identification of neuronal MRTF-SRF target genes. Together, we identified CAP1 as a novel and crucial repressor of neuronal MRTF-SRF signaling.

neuroscience↗

T-CLEARE: A Pilot Community-Driven Tissue-Clearing Protocol Repository

Selecting and implementing a tissue-clearing protocol is challenging. Established more than 100 years ago, tissue clearing is still a rapidly evolving field of research. There are currently many published protocols to choose from, and each performs better or worse across a range of key evaluation factors (e.g., speed, cost, tissue stability, fluorescence quenching). Additionally, tissue-clearing protocols are often optimized for specific experimental contexts, and applying an existing protocol to a new problem can require a lengthy period of adaptation by trial and error. Although the primary literature and review articles provide a useful starting point for optimization, there is growing recognition that many articles do not provide sufficient detail to replicate or reproduce experimental results. To help address this issue, we have developed a novel, freely available repository of tissue-clearing protocols named T-CLEARE (Tissue CLEAring protocol REpository; https://doryworkspace.org/doryviz). T-CLEARE incorporates community responses to an open survey designed to capture details not commonly found in the scientific literature, including modifications to published protocols required for specific use cases and instances when tissue-clearing protocols did not perform well (negative results). The goal of T-CLEARE is to provide a forum for the community to share evaluations and modifications of tissue-clearing protocols for various tissue types and potentially identify best-in-class methods for a given application.

bioinformatics↗

Light sheet autofluorescence lifetime imaging with a single photon avalanche diode array

Single photon avalanche diode (SPAD) array sensors can increase the imaging speed for fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser scanning to widefield geometries. While a SPAD camera in epi-fluorescence geometry enables widefield FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus features masks weak cell autofluorescence and biases lifetime measurements. Here, we address this problem by integrating the SPAD camera in a light sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling fast label-free FLIM of single-cell NAD(P)H autofluorescence. The feasibility of this NAD(P)H light sheet FLIM system was confirmed with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times, and in vivo NAD(P)H light sheet FLIM was demonstrated with live neutrophil imaging in a zebrafish tail wound, also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light sheet geometries, indicating a 30X to 6X frame rate advantage for the light sheet compared to the laser scanning geometry. This light sheet system provides faster frame rates for 3D NAD(P)H FLIM for live cell imaging applications such as monitoring single cell metabolism and immune cell migration throughout an entire living organism.

bioengineering↗

Spatial transcriptomics reveals antiparasitic targets associated with essential behaviors in the human parasite Brugia malayi

Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.

microbiology↗

Genomic and Metabolic Hallmarks of SDH- and FH-Deficient Renal Cell Carcinomas

PurposeSuccinate dehydrogenase-deficient and fumarate hydratase-deficient renal cell carcinomas (SDHRCC and FHRCC) are rare kidney cancers driven by loss of metabolically proximal enzymes. We sought to define and compare the genomic and metabolomic hallmarks of these entities. Experimental DesignWe analyzed SDHRCC and FHRCC tumors with either immunohistochemical evidence of loss of protein expression or genomically-confirmed biallelic inactivation of SDHA/B/C/D/AF2 or FH. Somatic alterations were identified using clinical pipelines, and allele-specific copy number changes were identified using FACETS. Mass-spectrometry-based metabolomic profiling was performed on available SDHRCC and FHRCC tumors. ResultsForty two patients were analyzed (25 FHRCC, 17 SDHRCC). In the germline analysis, 16/17 SDHRCC harbored a germline alteration in SDHB, whereas only 17/22 FHRCC had pathogenic germline FH variants. SDHRCC had a lower mutation burden (p = 0.02) and copy number alteration burden (p = 0.0002) than FHRCC. All SDHRCC presented with deletion of chromosome 1p (overlapping SDHB), whereas FHRCC demonstrated high but not ubiquitous loss of 1q (FH locus). Both SDHRCC and FHRCC demonstrated significant, idiopathic accumulation of the metabolite guanine. FHRCC tumors had elevated levels of urea cycle metabolites (argininosuccinate, citrulline, and fumarate), whereas SDHRCC had elevation of numerous acylcarnitines. These characteristic metabolic changes enabled the identification of a previously unrecognized SDH-deficient RCC. ConclusionDespite sharing similar genetic etiology, SDHRCC and FHRCC represent distinct molecular entities with unique genetic and metabolic abnormalities. Translational RelevanceMutations to the TCA cycle enzymes Succinate Dehydrogenase (SDH) and Fumarate Hydratase (FH) predispose individuals to unique subtypes of renal cell carcinoma (SDHRCC and FHRCC, respectively). Defining the genetic and metabolic hallmarks of these diseases is critical for advancing new diagnostic and therapeutic approaches for these rare but biologically intriguing entities. Despite a superficially similar genetic etiology, SDHRCC and FHRCC demonstrated significantly fewer secondary mutations to other cancer-associated genes and copy number aberrations than FHRCC, and was distinguished by universal loss-of-heterozygosity of chromosome 1p. Metabolomic analysis identified pathways disrupted in both SDHRCC and FHRCC, including the massive accumulation of free guanine, as well as pathways uniquely disrupted in each of the two entities. These metabolomic findings enabled the identification of a previously unidentified case of unclassified RCC with SDH deficiency, suggesting that metabolomic profiling may aid in phenotypic classification of tumors and uncover novel therapeutic targets.

cancer biology↗

microRNA-138 controls hippocampal interneuron function and short-term memory

The proper development and function of neuronal circuits relies on a tightly regulated balance between excitatory and inhibitory (E/I) synaptic transmission, and disrupting this balance can cause neurodevelopmental disorders, e.g. schizophrenia. microRNA-dependent gene regulation in pyramidal neurons is important for excitatory synaptic function and cognition, but its role in inhibitory interneurons is poorly understood. Here, we identify miR-138-5p as a regulator of short-term memory and inhibitory synaptic transmission in the hippocampus. Sponge-mediated miR-138-5p inactivation specifically in parvalbumin (PV)-expressing interneurons impairs spatial recognition memory and enhances GABAergic synaptic input onto pyramidal neurons. Cellular and behavioural phenotypes associated with miR-138-5p inactivation are paralleled by an upregulation of the schizophrenia-associated Erbb4, which we validated as a direct miR-138-5p target gene. Our findings suggest that miR-138-5p is a critical regulator of PV interneuron function, with implications for cognition and schizophrenia. More generally, they provide evidence that microRNAs orchestrate neural circuit development by fine-tuning both excitatory and inhibitory synaptic transmission.

neuroscience↗

Determining pathogenicity of variants of uncertain significance and identification of a founder variant in the epilepsy-associated gene, SZT2

Biallelic pathogenic variants in SZT2 result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTOR signaling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity of SZT2 missense and in-frame deletions. We report a cohort of twelve individuals with biallelic SZT2 variants and phenotypes consistent with SZT2-related neurodevelopmental disorder. The majority of this cohort contained one or more SZT2 variants of uncertain significance (VUS). We developed a novel individualized platform to functionally characterize SZT2 VUSs. We identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis determined this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Overall, we present a FACS-based rapid assay to distinguish pathogenic variants from VUSs in SZT2, using an approach that is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies, DEPDC5, TSC1/2, MTOR and NPRL2/3.

genetics↗

Small-angle neutron scattering solution structures of NADPH-dependent sulfite reductase

Sulfite reductase (SiR), a dodecameric complex of flavoprotein reductase subunits (SiRFP) and hemoprotein oxidase subunits (SiRHP), reduces sulfur reduction for biomass incorporation. Electron transfer within SiR requires intra- and inter-subunit interactions that are mediated by the relative position of each protein, governed by flexible domain movements. Using small-angle neutron scattering, we report the first solution structures of SiR heterodimers containing a single copy of each subunit. These structures show how the subunits bind and how both subunit binding and oxidation state impact SiRFPs conformation. Neutron contrast matching experiments on selectively deuterated heterodimers allow us to define the contribution of each subunit to the solution scattering. SiRHP binding induces a change in the position of SiRFPs flavodoxin-like domain relative to its ferredoxin-NADP+ reductase domain while compacting SiRHPs N-terminus. Reduction of SiRFP leads to a more open structure relative to its oxidized state, re-positioning SiRFPs N-terminal flavodoxin-like domain towards the SiRHP binding position. These structures show, for the first time, how both SiRHP binding to, and reduction of, SiRFP positions SiRFP for electron transfer between the subunits.

biochemistry↗

PARP inhibition in Ewing sarcoma: impact of germline DNA damage repair defects and activation of immunoregulatory pathways

Ewing sarcoma, an oncofusion-driven primary bone tumor, can occur in the setting of various germline mutations in DNA damage repair pathway genes. We recently reported our discovery of a germline mutation in the DNA damage repair protein BARD1 (BRCA1-associated RING domain-1) in a patient with Ewing sarcoma. BARD1 is recruited to the site of DNA double stranded breaks via the poly(ADP-ribose) polymerase (PARP) protein and plays a critical role in DNA damage response pathways including homologous recombination. PARP inhibitors (PARPi) are effective against Ewing sarcoma cells in vitro, though have demonstrated limited success in clinical trials to date. In order to assess the impact of BARD1 loss on Ewing sarcoma sensitivity to PARP inhibitor therapy, we generated the novel PSaRC318 patient-derived Ewing tumor cell from our patient with a germline BARD1 mutation and then analyzed the response of these cells to PARPi. We demonstrate that PSaRC318 cells are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that loss of BARD1 enhances PARPi sensitivity. In certain malignancies, DNA damage can activate the IRF1 (interferon response factor 1) immunoregulatory pathway, and the activation of this pathway can drive immunosuppression through upregulation of the immune checkpoint protein PD-L1. In order to determine the ability of PARPi to alter Ewing tumor immunoregulation, we evaluated whether PARPi results in upregulation of the IRF1-PDL1 pathway. Indeed, we now demonstrate that PARPi leads to increased PD-L1 expression in Ewing sarcoma. Together, these data thus far suggest that while Ewing tumors harboring germline mutations in DNA damage repair proteins may in respond to PARPi in vitro, in vivo benefit of PARPi may only be demonstrated when counteracting the immunosuppressive effects of DNA damage by concurrently targeting immune checkpoint proteins.

cancer biology↗