The impact of SWI/SNF and NuRD inactivation on gene expression is tightly coupled with levels of RNA polymerase II occupancy at promoters.

SWI/SNF and NuRD are protein complexes that antagonistically regulate DNA accessibility. However, repression of their activities often leads to unanticipated changes in target gene expression (paradoxical), highlighting our incomplete understanding of their activities. Here we show that SWI/SNF and NuRD are in a tug-of-war to regulate PRC2 occupancy at lowly expressed and bivalent genes in mouse embryonic stem cells (mESCs). In contrast, at promoters of average or highly expressed genes, SWI/SNF and NuRD antagonistically modulate RNA polymerase II (Pol II) release kinetics, arguably owing to accompanying alterations in H3.3 and H2A.Z levels at promoter-flanking nucleosomes, leading to paradoxical changes in gene expression. Owing to this mechanism, the relative activities of the two remodelers potentiate gene promoters toward Pol II-dependent open or PRC2-dependent closed chromatin states. Our results highlight RNA Pol II occupancy as the key parameter in determining the direction of gene expression changes in response to SWI/SNF and NuRD inactivation at gene promoters in mESCs.

Assessment of the excitation-inhibition ratio in the Fmr1 KO2 mouse using neuronal oscillation dynamics.

In vitro and ex vivo studies have shown consistent indications of hyperexcitability in the Fragile X Messenger Ribonucleoprotein 1 (Fmr1) knockout mouse model of autism spectrum disorder. We recently introduced a method to quantify network-level functional excitation-inhibition ratio from the neuronal oscillations. Here, we used this measure to study whether the implicated synaptic excitation-inhibition disturbances translate to disturbances in network physiology in the Fragile X Messenger Ribonucleoprotein 1 (Fmr1) gene knockout model. Vigilance-state scoring was used to extract segments of inactive wakefulness as an equivalent behavioral condition to the human resting-state and, subsequently, we performed high-frequency resolution analysis of the functional excitation-inhibition biomarker, long-range temporal correlations, and spectral power. We corroborated earlier studies showing increased high-frequency power in Fragile X Messenger Ribonucleoprotein 1 (Fmr1) knockout mice. Long-range temporal correlations were higher in the gamma frequency ranges. Contrary to expectations, functional excitation-inhibition was lower in the knockout mice in high frequency ranges, suggesting more inhibition-dominated networks. Exposure to the Gamma-aminobutyric acid (GABA)-agonist clonazepam decreased the functional excitation-inhibition in both genotypes, confirming that increasing inhibitory tone results in a reduction of functional excitation-inhibition. In addition, clonazepam decreased electroencephalogram power and increased long-range temporal correlations in both genotypes. These findings show applicability of these new resting-state electroencephalogram biomarkers to animal for translational studies and allow investigation of the effects of lower-level disturbances in excitation-inhibition balance.

Long-Range Amplitude Coupling Is Optimized for Brain Networks That Function at Criticality.

Brain function depends on segregation and integration of information processing in brain networks often separated by long-range anatomic connections. Neuronal oscillations orchestrate such distributed processing through transient amplitude and phase coupling, yet surprisingly, little is known about local network properties facilitating these functional connections. Here, we test whether criticality, a dynamical state characterized by scale-free oscillations, optimizes the capacity of neuronal networks to couple through amplitude or phase, and transfer information. We coupled in silico networks which exhibit oscillations in the α band (8-16 Hz), and varied excitatory and inhibitory connectivity. We found that phase coupling of oscillations emerges at criticality, and that amplitude coupling, as well as information transfer, are maximal when networks are critical. Importantly, regulating criticality through modulation of synaptic gain showed that critical dynamics, as opposed to a static ratio of excitatory and inhibitory connections, optimize network coupling and information transfer. Our data support the idea that criticality is important for local and global information processing and may help explain why brain disorders characterized by local alterations in criticality also exhibit impaired long-range synchrony, even before degeneration of axonal connections.SIGNIFICANCE STATEMENT To perform adaptively in a changing environment, our brains dynamically coordinate activity across distant areas. Empirical evidence suggests that long-range amplitude and phase coupling of oscillations are systems-level mechanisms enabling transient formation of spatially distributed functional networks on the backbone of a relatively static structural connectome. However, surprisingly little is known about the local network properties that optimize coupling and information transfer. Here, we show that criticality, a dynamical state characterized by scale-free oscillations and a hallmark of neuronal network activity, optimizes the capacity of neuronal networks to couple through amplitude or phase and transfer information.

Endovascular occlusion of a persistent stapedial artery prior to stapedotomy: a novel approach.

BACKGROUND: The stapedial artery is an embryonic artery that transiently supplies the cranial vasculature of the human embryo. Postnatal persistence of the stapedial artery may cause conductive hearing loss and pulsatile tinnitus due to its course through the middle ear. We describe a patient with a persistent stapedial artery (PSA) managed by endovascular coil occlusion prior to stapedotomy. METHODS: A 48-year-old woman presented with left-sided conductive hearing loss and pulsatile tinnitus. Ten years earlier the patient had undergone explorative tympanoplasty, which was aborted due to a large PSA. Digital subtraction angiography was performed to confirm the anatomy and endovascular occlusion of the proximal PSA was achieved by deployment of coils. RESULTS: The pulsatile tinnitus improved immediately after the procedure. The size of the artery subsequently decreased and surgery could be performed with only a minor intraoperative bleeding. Successful stapedotomy resulted in postoperative normalization of her hearing with some minor residual tinnitus. CONCLUSION: Endovascular coil occlusion of a PSA is feasible and safe in patients with favorable anatomy and facilitates middle ear surgery. It decreases the size of the artery and minimizes the risk of intraoperative bleeding in patients with a large PSA. The future role of this novel technique in the management of patients with PSA-related conductive hearing loss and pulsatile tinnitus remains to be determined.

Predictive Value of Pediatric Stroke Diagnoses in Administrative Data: A Systematic Literature Review.

BACKGROUND AND AIM: Administrative healthcare data are frequently used for studying incidence, prevalence, risk factors, and outcome of pediatric stroke. However, the accuracy of these data sources is uncertain. The aim of this study was to systematically analyze published data on the positive predictive value (PPV) and sensitivity of diagnoses used to identify pediatric stroke patients in administrative data. METHODS: This systematic review was performed in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. We searched PubMed and Embase for studies, published in year 2000 or later, describing the PPV or sensitivity of diagnoses used to identify children with stroke in administrative data. The search was performed on June 9, 2022. Studies written in other languages than English, with less than 30 participants, and conference abstracts were excluded. RESULTS: Eight studies were included after full-text review from 2,475 potentially eligible records. These included 3,137 children. All studies reported data from high-income countries. Reported PPVs varied considerably across studies and stroke subtypes: acute ischemic stroke, range 0.27-0.89; cerebral venous thrombosis, range 0.45-0.72; spontaneous subarachnoid hemorrhage, range 0.52-0.83; and spontaneous intracerebral hemorrhage, range 0.62-0.66. One study examined sensitivity of an ICD-9 search compared to a radiology report search and found that the ICD search had poor sensitivity (33%). CONCLUSION: Caution is recommended in the use and interpretation of nonvalidated administrative data for pediatric stroke. Data on the PPV and sensitivity of pediatric stroke diagnoses in administrative data remain limited and are only available from high-income countries.

Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition.

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps or require a highly specialized microfluidics device that is not readily available. We developed a micro-scale chromatin immunoprecipitation and sequencing (µChIP-seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.

Hemorrhage due to a pseudoaneurysm on a dural-pial anastomosis after decompression for Chiari malformation type I: case report.

While intracranial aneurysms rarely develop after neurosurgical procedures, delayed pseudoaneurysm formation after foramen magnum decompression (FMD) has never been reported. A 52-year-old woman presented with an atypical subarachnoid hemorrhage in the posterior fossa 12 years after a FMD for symptomatic Chiari malformation type I was performed. A pseudoaneurysm on a dural-pial anastomosis was identified as the bleeding source and successfully occluded by endovascular means with full clinical recovery of the patient. Injury to the distal posterior inferior cerebellar artery related to surgery and postoperative infection likely caused formation of a dural-pial anastomosis. Additionally, hemodynamic stress or dissection may have contributed to delayed pseudoaneurysm formation and rupture.

Acute posterior multifocal placoid pigment epitheliopathy resembling multiple sclerosis.

A 23-year-old man presented with right eye blurred vision; he was diagnosed with acute posterior multifocal placoid pigment epitheliopathy (APMPPE), and his symptoms resolved with prednisolone. Two months later, he developed a right arm weakness that resolved after 3 weeks. MR scan of brain identified changes suggesting multiple sclerosis, with four hyperintense FLAIR lesions; there was contrast enhancement of two lesions and no diffusion restriction. Cerebrospinal fluid showed mononuclear pleocytosis. We eventually diagnosed these as APMPPE-associated CNS lesions. APMPPE is a rare inflammatory chorioretinopathy that rarely can resemble multiple sclerosis clinically and radiologically.

Catecholamines alter the intrinsic variability of cortical population activity and perception.

The ascending modulatory systems of the brain stem are powerful regulators of global brain state. Disturbances of these systems are implicated in several major neuropsychiatric disorders. Yet, how these systems interact with specific neural computations in the cerebral cortex to shape perception, cognition, and behavior remains poorly understood. Here, we probed into the effect of two such systems, the catecholaminergic (dopaminergic and noradrenergic) and cholinergic systems, on an important aspect of cortical computation: its intrinsic variability. To this end, we combined placebo-controlled pharmacological intervention in humans, recordings of cortical population activity using magnetoencephalography (MEG), and psychophysical measurements of the perception of ambiguous visual input. A low-dose catecholaminergic, but not cholinergic, manipulation altered the rate of spontaneous perceptual fluctuations as well as the temporal structure of "scale-free" population activity of large swaths of the visual and parietal cortices. Computational analyses indicate that both effects were consistent with an increase in excitatory relative to inhibitory activity in the cortical areas underlying visual perceptual inference. We propose that catecholamines regulate the variability of perception and cognition through dynamically changing the cortical excitation-inhibition ratio. The combined readout of fluctuations in perception and cortical activity we established here may prove useful as an efficient and easily accessible marker of altered cortical computation in neuropsychiatric disorders.

Endovascular treatment for cerebral venous sinus thrombosis - a single center study.

BACKGROUND: Cerebral venous sinus thrombosis (CVST) is a rare cerebrovascular disorder. The majority of these patients respond favorably to systemic anticoagulation. However, a subset of patients will deteriorate clinically, despite optimal medical therapy. METHODS: Retrospective single center study of 28 consecutive CVST patients treated with systemic anticoagulation and additional endovascular therapy. RESULTS: Median age was 37.5 years (range 15-76 years), there were 21 (75%) women, and 20 (71%) had thrombosis involving ≥2 venous sinuses. Intracranial hemorrhage (ICH) was present at admission in 18 patients (64%). Endovascular therapy consisted of local thrombolysis in 26 (93%) patients; 9 patients (32%) had additional mechanical thrombectomy, and in 2 (7%) patients thrombectomy alone was performed. Complete recanalization at end of the final intervention was achieved in 15 patients (54%), partial recanalization in 11 patients (39%), whereas there was no recanalization in 2 patients (7%). On follow-up imaging, conducted between 3 and 6 months, recanalization further improved to 76%, 19% and 5%, respectively. A favorable outcome (mRS ≤ 2) was achieved in 63% of patients at 3 months, which improved to 79% at 6 months. Post-procedural ICH or volume expansion of preexisting ICH was seen in 9 patients (32%). In total 5 patients died (18%). CONCLUSIONS: Systemic anticoagulation with the addition of endovascular therapy with local thrombolysis and/or mechanical thrombectomy is a potential strategy to obtain recanalization in patients with CVST who deteriorate clinically despite medical therapy or are comatose. Endovascular therapy may increase the risk of ICH.

Risk of Neurological Disorders in Patients With European Lyme Neuroborreliosis: A Nationwide, Population-Based Cohort Study.

BACKGROUND: Lyme neuroborreliosis (LNB), caused by the tick-borne spirochetes of the Borrelia burgdorferi sensu lato species complex, has been suggested to be associated with a range of neurological disorders. In a nationwide, population-based cohort study, we examined the associations between LNB and dementia, Alzheimer's disease, Parkinson's disease, motor neuron disease, epilepsy, and Guillain-Barré syndrome. METHODS: We used national registers to identify all Danish residents diagnosed during 1986-2016 with LNB (n = 2067), created a gender- and age-matched comparison cohort from the general population (n = 20 670), and calculated risk estimates and hazard ratios. RESULTS: We observed no long-term increased risks of dementia, Alzheimer's disease, Parkinson's disease, motor neuron diseases, or epilepsy. However, within the first year, 8 (0.4%) of the LNB patients developed epilepsy, compared with 20 (0.1%) of the comparison cohort (difference, 0.3%; 95% confidence interval, .02-.6%). In the LNB group, 11 (0.5%) patients were diagnosed with Guillain-Barré syndrome within the first year after LNB diagnosis, compared with 0 (0.0%) in the comparison cohort. After the first year, the risk of Guillain-Barré was not increased. CONCLUSIONS: LNB patients did not have increased long-term risks of dementia, Alzheimer's disease, Parkinson's disease, motor neuron diseases, epilepsy, or Guillain-Barré. Although the absolute risk is low, LNB patients might have an increased short-term risk of epilepsy and Guillain-Barré syndrome.

Scale-Free Amplitude Modulation of Neuronal Oscillations Tracks Comprehension of Accelerated Speech.

Speech comprehension is preserved up to a threefold acceleration, but deteriorates rapidly at higher speeds. Current models posit that perceptual resilience to accelerated speech is limited by the brain's ability to parse speech into syllabic units using δ/θ oscillations. Here, we investigated whether the involvement of neuronal oscillations in processing accelerated speech also relates to their scale-free amplitude modulation as indexed by the strength of long-range temporal correlations (LRTC). We recorded MEG while 24 human subjects (12 females) listened to radio news uttered at different comprehensible rates, at a mostly unintelligible rate and at this same speed interleaved with silence gaps. δ, θ, and low-γ oscillations followed the nonlinear variation of comprehension, with LRTC rising only at the highest speed. In contrast, increasing the rate was associated with a monotonic increase in LRTC in high-γ activity. When intelligibility was restored with the insertion of silence gaps, LRTC in the δ, θ, and low-γ oscillations resumed the low levels observed for intelligible speech. Remarkably, the lower the individual subject scaling exponents of δ/θ oscillations, the greater the comprehension of the fastest speech rate. Moreover, the strength of LRTC of the speech envelope decreased at the maximal rate, suggesting an inverse relationship with the LRTC of brain dynamics when comprehension halts. Our findings show that scale-free amplitude modulation of cortical oscillations and speech signals are tightly coupled to speech uptake capacity.SIGNIFICANCE STATEMENT One may read this statement in 20-30 s, but reading it in less than five leaves us clueless. Our minds limit how much information we grasp in an instant. Understanding the neural constraints on our capacity for sensory uptake is a fundamental question in neuroscience. Here, MEG was used to investigate neuronal activity while subjects listened to radio news played faster and faster until becoming unintelligible. We found that speech comprehension is related to the scale-free dynamics of δ and θ bands, whereas this property in high-γ fluctuations mirrors speech rate. We propose that successful speech processing imposes constraints on the self-organization of synchronous cell assemblies and their scale-free dynamics adjusts to the temporal properties of spoken language.

Palbociclib in combination with simvastatin induce severe rhabdomyolysis: a case report.

BACKGROUND: Palbociclib is a selective well-tolerated antineoplastic drug used in the treatment of advanced HER2-negative, estrogen-receptor positive breast cancer that has shown significant improvement in progression-free survival. We present a patient that developed severe rhabdomyolysis with tetra-affection and loss of gait after initiating the first cycle of Palbociclib concomitantly with Simvastatin 40 mg treatment. CASE PRESENTATION: A 71-year-old woman with metastatic breast cancer developed tetraparesis and near fatal rhabdomyolysis after initiation of first cycle Palbociclib. For 10 years prior to this treatment, the patient had been treated with Simvastatin without myalgia or other neuromuscular complaints prior to the first cycle of Palbociclib. The patient was admitted at the neurology department, where Palbociclib and Simvastatin were discontinued. The patient was aggressively hydrated and treated with intravenous immunoglobulin therapy with slowly remission and finally regaining independent gait function. Evaluation showed a negative myositis antibody work-up. Muscle magnetic resonance imaging showed edema in multiple foci, but skeletal muscle biopsy did not show necrosis. Post discharge genetic analysis showed single heterozygosity for nucleotide polymorphism rs4149056. CONCLUSION: We present a patient who developed severe rhabdomyolysis induced by a combination of Palbociclib and Simvastatin treatment. Rhabdomyolysis was most likely induced by toxic plasma concentrations of Simvastatin due to Palbociclibs inhibition of the CYP3A4 enzyme in combination with a decreased hepatic uptake of Simvastatin due to single nucleotide polymorphism rs4149056. The study underscores that combining Simvastatin and Palbociclib should be done cautiously and genetic testing of the rs4149056 SNP is warranted. If present, Simvastatin should be discontinued or replaced with a lesser myopathic statin in regard to patients risk of cardiovascular events.

Pure Alexia: A Combined First-Person Account and Neuropsychological Investigation.

Pure alexia is an acquired reading disorder where patients' ability to read words and text is severely impaired, while their writing is left unaffected. Patients with pure alexia typically recover some reading ability over time, although most never regain their premorbid reading skills. A few studies have reported some behavioral and imaging correlates of such remission; however, little is known about the patients' experience of their reading impairment. This paper contains a first-person account of pure alexia, describing the first author's (K.H.) experience of his remission from severe reading problems immediately following a posterior cerebral artery stroke to the mild pure alexia characterizing his reading ability today. To provide a context for this account, we also present neuropsychological and reading data obtained from K.H. at several time points during his recovery.

Strong long-range temporal correlations of beta/gamma oscillations are associated with poor sustained visual attention performance.

Neuronal oscillations exhibit complex amplitude fluctuations with autocorrelations that persist over thousands of oscillatory cycles. Such long-range temporal correlations (LRTC) are thought to reflect neuronal systems poised near a critical state, which would render them capable of quick reorganization and responsive to changing processing demands. When we concentrate, however, the influence of internal and external sources of distraction is better reduced, suggesting that neuronal systems involved with sustained attention could benefit from a shift toward the less volatile sub-critical state. To test these ideas, we recorded electroencephalography (EEG) from healthy volunteers during eyes-closed rest and during a sustained attention task requiring a speeded response to images deviating in their presentation duration. We show that for oscillations recorded during rest, high levels of alpha-band LRTC in the sensorimotor region predicted good reaction-time performance in the attention task. During task execution, however, fast reaction times were associated with high-amplitude beta and gamma oscillations with low LRTC. Finally, we show that reduced LRTC during the attention task compared to the rest condition correlates with better performance, while increased LRTC of oscillations from rest to attention is associated with reduced performance. To our knowledge, this is the first empirical evidence that 'resting-state criticality' of neuronal networks predicts swift behavioral responses in a sensorimotor task, and that steady attentive processing of visual stimuli requires brain dynamics with suppressed temporal complexity.

Controlling the Temporal Structure of Brain Oscillations by Focused Attention Meditation.

Our focus of attention naturally fluctuates between different sources of information even when we desire to focus on a single object. Focused attention (FA) meditation is associated with greater control over this process, yet the neuronal mechanisms underlying this ability are not entirely understood. Here, we hypothesize that the capacity of attention to transiently focus and swiftly change relates to the critical dynamics emerging when neuronal systems balance at a point of instability between order and disorder. In FA meditation, however, the ability to stay focused is trained, which may be associated with a more homogeneous brain state. To test this hypothesis, we applied analytical tools from criticality theory to EEG in meditation practitioners and meditation-naïve participants from two independent labs. We show that in practitioners-but not in controls-FA meditation strongly suppressed long-range temporal correlations (LRTC) of neuronal oscillations relative to eyes-closed rest with remarkable consistency across frequency bands and scalp locations. The ability to reduce LRTC during meditation increased after one year of additional training and was associated with the subjective experience of fully engaging one's attentional resources, also known as absorption. Sustained practice also affected normal waking brain dynamics as reflected in increased LRTC during an eyes-closed rest state, indicating that brain dynamics are altered beyond the meditative state. Taken together, our findings suggest that the framework of critical brain dynamics is promising for understanding neuronal mechanisms of meditative states and, specifically, we have identified a clear electrophysiological correlate of the FA meditation state.

Polycomb group protein displacement and gene activation through MSK-dependent H3K27me3S28 phosphorylation.

Epigenetic regulation of chromatin structure is essential for the expression of genes determining cellular specification and function. The Polycomb repressive complex 2 (PRC2) di- and trimethylates histone H3 on lysine 27 (H3K27me2/me3) to establish repression of specific genes in embryonic stem cells and during differentiation. How the Polycomb group (PcG) target genes are regulated by environmental cues and signaling pathways is quite unexplored. Here, we show that the mitogen- and stress-activated kinases (MSK), through a mechanism that involves promoter recruitment, histone H3K27me3S28 phosphorylation, and displacement of PcG proteins, lead to gene activation. We present evidence that the H3K27me3S28 phosphorylation is functioning in response to stress signaling, mitogenic signaling, and retinoic acid (RA)-induced neuronal differentiation. We propose that MSK-mediated H3K27me3S28 phosphorylation serves as a mechanism to activate a subset of PcG target genes determined by the biological stimuli and thereby modulate the gene expression program determining cell fate.

Consistency of EEG source localization and connectivity estimates.

As the EEG inverse problem does not have a unique solution, the sources reconstructed from EEG and their connectivity properties depend on forward and inverse modeling parameters such as the choice of an anatomical template and electrical model, prior assumptions on the sources, and further implementational details. In order to use source connectivity analysis as a reliable research tool, there is a need for stability across a wider range of standard estimation routines. Using resting state EEG recordings of N=65 participants acquired within two studies, we present the first comprehensive assessment of the consistency of EEG source localization and functional/effective connectivity metrics across two anatomical templates (ICBM152 and Colin27), three electrical models (BEM, FEM and spherical harmonics expansions), three inverse methods (WMNE, eLORETA and LCMV), and three software implementations (Brainstorm, Fieldtrip and our own toolbox). Source localizations were found to be more stable across reconstruction pipelines than subsequent estimations of functional connectivity, while effective connectivity estimates where the least consistent. All results were relatively unaffected by the choice of the electrical head model, while the choice of the inverse method and source imaging package induced a considerable variability. In particular, a relatively strong difference was found between LCMV beamformer solutions on one hand and eLORETA/WMNE distributed inverse solutions on the other hand. We also observed a gradual decrease of consistency when results are compared between studies, within individual participants, and between individual participants. In order to provide reliable findings in the face of the observed variability, additional simulations involving interacting brain sources are required. Meanwhile, we encourage verification of the obtained results using more than one source imaging procedure.

Quantitative Assessment of Drug Delivery to Tissues and Association with Phospholipidosis: A Case Study with Two Structurally Related Diamines in Development.

Drug induced phospholipidosis (PLD) may be observed in the preclinical phase of drug development and pose strategic questions. As lysosomes have a central role in pathogenesis of PLD, assessment of lysosomal concentrations is important for understanding the pharmacokinetic basis of PLD manifestation and forecast of potential clinical appearance. Herein we present a systematic approach to provide insight into tissue-specific PLD by evaluation of unbound intracellular and lysosomal (reflecting acidic organelles) concentrations of two structurally related diprotic amines, GRT1 and GRT2. Their intratissue distribution was assessed using brain and lung slice assays. GRT1 induced PLD both in vitro and in vivo. GRT1 showed a high intracellular accumulation that was more pronounced in the lung, but did not cause cerebral PLD due to its effective efflux at the blood-brain barrier. Compared to GRT1, GRT2 revealed higher interstitial fluid concentrations in lung and brain, but more than 30-fold lower lysosomal trapping capacity. No signs of PLD were seen with GRT2. The different profile of GRT2 relative to GRT1 is due to a structural change resulting in a reduced basicity of one amino group. Hence, by distinct chemical modifications, undesired lysosomal trapping can be separated from desired drug delivery into different organs. In summary, assessment of intracellular unbound concentrations was instrumental in delineating the intercompound and intertissue differences in PLD induction in vivo and could be applied for identification of potential lysosomotropic compounds in drug development.