Biology subjects

Ittner, L. M.

Publications and source records attributed to Ittner, L. M..

6 recordsLinked to original sources

bioRxiv2026

Engineering a Novel Bacterial Encapsulin for Programmable Surface Functionalization: From Single-Target to Mosaic Nanovaccines

Encapsulins are prokaryotic self-assembling protein nanocages with promise as nanovaccine scaffolds. Their utility as modular platforms require tolerance to surface engineering, high-yield soluble production, formulation stability, and controlled antigen (co-)display. Herein, a previously uncharacterized encapsulin from Alkaliphilus metalliredigens is engineered into a SpyCatcher-decorated nanoscaffold (Am-S) that enables controlled surface display of SpyTagged antigens. Cryo-EM confirms that the native encapsulin forms a T = 1 icosahedral nanocage, and that C-terminal SpyCatcher fusion yields Am-S without compromising nanocage assembly, symmetry, or structural integrity. Notably, Am-S exhibits high-yield soluble production in Escherichia coli, remains monodisperse after freeze-thaw and extended storage, and supports efficient SpyTagged peptide conjugation for single- and multi-antigen display. As a proof-of-concept, Am-S is functionalized with Alzheimers disease-associated amyloid-{beta} and/or hyperphosphorylated tau epitopes to generate single-target nanocages displaying either antigen and dual-target mosaic nanocages co-displaying both. In mice, Am-S antigen display enhances antigen-specific IgG responses relative to free antigens and induces predominantly IgG1-biased humoral immunity. Mosaic nanocages elicit antibodies against both targets, with immune sera selectively recognizing amyloid-{beta}- and phosphorylated tau-associated pathology in ex vivo brain sections from Alzheimers disease mouse models. These findings position Am-S as a manufacturable scaffold for developing multi-targeting nanovaccines against complex diseases.

synthetic biology↗

bioRxiv2026

The long noncoding RNA Dory is required for female but not male spatial learning and memory

Large numbers of long noncoding RNAs (lncRNAs) exhibit region- or cell-specific expression and subcellular locations in the mammalian brain. We analyzed the expression and function of the mouse lncRNA 2700046G09Rik (also called Sgms1os1), which we have named Dory due to sex-specific disruption of spatial memory in rodent gene knockout and RNA knockdown. We show that Dory is predominantly expressed in a punctate pattern in nuclei of excitatory neurons in the hippocampus, and in both neuronal and non-neuronal cells in the cerebellum. We show by high resolution RNA sequencing that Dory brain transcripts are composed of 1-3 exons with multiple isoforms. Deletion of the constitutive first exon of Dory by CRISPR-Cas9 genome editing resulted in the impairment of spatial memory and some aspects of balance in female but not male mice. Dory-null mice presented without overt changes in brain morphology. Knockdown of the rat homolog of Dory in dorsal hippocampus using antisense oligonucleotides confirmed inhibition of spatial memory in females only. RNA sequencing and mass spectrometry revealed differential hippocampal gene and protein expression profiles, notably of prolactin, growth hormone and pro-opiomelanocortin, between male and female Dory knockout mice.

neuroscience↗

bioRxiv2024

Long Read Sequencing reveals transgene concatemerization and backbone integration following AAV-driven electroporation of CRISPR RNP complexes in mouse zygotes

Over the last decade CRISPR gene editing has been successfully used to streamline the generation of animal models for biomedical purposes. However, one limitation to its use is the potential occurrence of on-target mutations that may be detrimental or otherwise unintended. These bystander mutations are often undetected using conventional genotyping methods. The use of Adeno-Associated Viruses (AAVs) to bring donor templates in zygotes is currently being deployed by transgenic cores around the world to generate knock-ins with large transgenes. Thanks to a high level of efficiency and the relative ease to establish this technique, it recently became a method of choice for transgenic laboratories. However, a thorough analysis of the editing outcomes following this method is yet to be developed. To this end, we generated three different types of integration using AAVs in two different murine genes (i.e., Ace2 and Foxg1) and employed Oxford Nanopore Technologies long read sequencing to analyze the outcomes. Using a workflow that includes Cas9 enrichment and adaptive sampling, we showed that unintended on-target mutations (sometimes reported using other workflows) can occur when using AAVs. This work highlights the importance of in-depth validation of the mutant lines generated and informs the uptake of this new method.

bioengineering↗

bioRxiv2021

The SARS-CoV-2 spike (S) and the orthoreovirus p15 cause neuronal and glial fusion

Numerous enveloped viruses use specialized surface molecules called fusogens to enter host cells1. During virus replication, these fusogens decorate the host cells membrane enabling them the ability to fuse with neighboring cells, forming syncytia that the viruses use to propagate while evading the immune system. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infect the brain and may cause serious neurological symptoms through mechanisms which remain poorly understood2-4. Here we show that expression of either the SARS-CoV-2 spike (S) protein or p15 protein from the baboon orthoreovirus is sufficient to induce fusion between interconnected neurons, as well as between neurons and glial cells. This phenomenon is observed across species, from nematodes to mammals, including human embryonic stem cells-derived neurons and brain organoids. We show that fusion events are progressive, can occur between distant neurites, and lead to the formation of multicellular syncytia. Finally, we reveal that in addition to intracellular molecules, fusion events allow diffusion and movement of large organelles such as mitochondria between fused neurons. Our results provide important mechanistic insights into how SARS-CoV-2 and other viruses could affect the nervous system circuitries causing neurological symptoms.

neuroscience↗

bioRxiv2021

Differential mitochondrial protein interaction profile between human translocator protein and its A147T polymorphism variant

The translocator protein (TSPO) has been implicated in mitochondrial transmembrane cholesterol transport, brain inflammation, and other mitochondrial functions. It is upregulated in glial cells during neuroinflammation in Alzheimers disease. High affinity TSPO imaging radioligands are utilized to visualize neuroinflammation. However, this is hampered by the common A147T polymorphism which compromises ligand binding. Furthermore, this polymorphism has been linked to increased risk of neuropsychiatric disorders, and possibly reduces TSPO protein stability. Here, we used immunoprecipitation coupled to mass-spectrometry (IP-MS) to establish a mitochondrial protein binding profile of wild-type (WT) TSPO and the A147T polymorphism variant. Using mitochondria from human glial cells expressing either WT or A147T TSPO, we identified 30 WT TSPO binding partners, yet only 23 for A147T TSPO. Confirming that A147T polymorphism of the TSPO might confer loss of function, we found that one of the identified interactors of WT TSPO, 14-3-3 theta (YWHAQ), a protein involved in regulating mitochondrial membrane proteins, interacts much less with A147T TSPO. Our data presents a network of mitochondrial interactions of TSPO and its A147T polymorphism variant in human glial cells and indicate functional relevance of A147T in mitochondrial protein networks.

molecular biology↗

bioRxiv2020

BioID analysis of the cyclin F interactome reveals that ALS-variant cyclin F alters the homeostasis of paraspeckle-associated proteins

BackgroundPreviously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). CCNF encodes for the protein cyclin F, a substrate recognition component of the E3-ubiquitin ligase, SCFcyclin F. We have previously shown that mutations in CCNF cause disruptions to overall protein homeostasis; causing a build-up of ubiquitylated proteins (1) as well as defects in autophagic machinery (2). MethodsHere, we have used an unbiased proteomic screening workflow using BioID, as well as standard immunoprecipitations to identify novel interaction partners of cyclin F, identifying the interaction between cyclin F and a series of paraspeckle proteins. The homeostasis of these new cyclin F interaction partners, RBM14, NONO and SFPQ were monitored in primary neurons using immunoblotting. In addition, the homeostasis of RBM14 was compared between control and ALS/FTD patient tissue using standard IHC studies. ResultsUsing BioID, we found over 100 putative interaction partners of cyclin F and demonstrated that cyclin F closely associates with a number of essential paraspeckle proteins, which are stress-responsive proteins that have recently been implicated in ALS pathogenesis. We further demonstrate that the turnover of these novel binding partners are defective when cyclin F carries an ALS/FTD-causing mutation. In addition the analysis of RBM14 levels in ALS patient post-mortem tissue revealed that RBM14 levels were significantly reduced in post-mortem ALS patient motor cortex and significantly reduced in the neurons of spinal cord tissue. ConclusionOverall, our data demonstrate that the dysregulation of paraspeckle components may be contributing factors to the molecular pathogenesis of ALS/FTD. HighlightsO_LIPreviously, we identified missense mutations in CCNF that are linked to Amyotrophic lateral sclerosis/Frontotemporal dementia (ALS/FTD) and have shown that a single mutation in cyclin F can cause defects to major protein degradation systems in dividing cells. C_LIO_LICyclin F has very few known interaction partners, many of which have roles in cell cycle progression. Accordingly, we used BioID and mass spectrometry to identify novel binding partners of cyclin F that may reveal insight into the role of cyclin F in neurodegeneration. C_LIO_LIMass spectrometry and bioinformatic studies demonstrate that cyclin F interacts with several RNA binding proteins. This includes the essential paraspeckle proteins, RBM14. Notably, this interaction could be validated by standard immunoprecipitations and immunoblotting. Cyclin F could also be found to interact with a series of essential proteins which form the paraspeckle complex. C_LIO_LIWe further evaluated the effect of cyclin F(S621G) on the homeostasis of these novel interaction partners in primary neurons in response to a known paraspeckle inducer, MG132. Notably, we demonstrate significant defects in the homeostasis of RBM14 and SFPQ, but not NONO, when cyclin F carries an S621G mutation. C_LIO_LIUnlike other paraspeckle proteins, RBM14 levels have not previously been reported in the post-mortem brain and spinal cord of ALS patient post-mortem tissue. Here, we note significant defects in the homeostasis of RBM14 in the post-mortem tissue of ALS patients. C_LI

neuroscience↗