GluK1 kainate receptors are necessary for functional maturation of parvalbumin interneurons regulating amygdala circuit function.

Parvalbumin expressing interneurons (PV INs) are key players in the local inhibitory circuits and their developmental maturation coincides with the onset of adult-type network dynamics in the brain. Glutamatergic signaling regulates emergence of the unique PV IN phenotype, yet the receptor mechanisms involved are not fully understood. Here we show that GluK1 subunit containing kainate receptors (KARs) are necessary for development and maintenance of the neurochemical and functional properties of PV INs in the lateral and basal amygdala (BLA). Ablation of GluK1 expression specifically from PV INs resulted in low parvalbumin expression and loss of characteristic high firing rate throughout development. In addition, we observed reduced spontaneous excitatory synaptic activity at adult GluK1 lacking PV INs. Intriguingly, inactivation of GluK1 expression in adult PV INs was sufficient to abolish their high firing rate and to reduce PV expression levels, suggesting a role for GluK1 in dynamic regulation of PV IN maturation state. The PV IN dysfunction in the absence of GluK1 perturbed the balance between evoked excitatory vs. inhibitory synaptic inputs and long-term potentiation (LTP) in LA principal neurons, and resulted in aberrant development of the resting-state functional connectivity between mPFC and BLA. Behaviorally, the absence of GluK1 from PV INs associated with hyperactivity and increased fear of novelty. These results indicate a critical role for GluK1 KARs in regulation of PV IN function across development and suggest GluK1 as a potential therapeutic target for pathologies involving PV IN malfunction.

Recessive TMOD1 mutation causes childhood cardiomyopathy.

Familial cardiomyopathy in pediatric stages is a poorly understood presentation of heart disease in children that is attributed to pathogenic mutations. Through exome sequencing, we report a homozygous variant in tropomodulin 1 (TMOD1; c.565C>T, p.R189W) in three individuals from two unrelated families with childhood-onset dilated and restrictive cardiomyopathy. To decipher the mechanism of pathogenicity of the R189W mutation in TMOD1, we utilized a wide array of methods, including protein analyses, biochemistry and cultured cardiomyocytes. Structural modeling revealed potential defects in the local folding of TMOD1R189W and its affinity for actin. Cardiomyocytes expressing GFP-TMOD1R189W demonstrated longer thin filaments than GFP-TMOD1wt-expressing cells, resulting in compromised filament length regulation. Furthermore, TMOD1R189W showed weakened activity in capping actin filament pointed ends, providing direct evidence for the variant's effect on actin filament length regulation. Our data indicate that the p.R189W variant in TMOD1 has altered biochemical properties and reveals a unique mechanism for childhood-onset cardiomyopathy.

Genetic etiology of progressive pediatric neurological disorders.

BACKGROUND: The aim of the study was to characterize molecular diagnoses in patients with childhood-onset progressive neurological disorders of suspected genetic etiology. METHODS: We studied 48 probands (age range from newborn to 17 years old) with progressive neurological disorders of unknown etiology from the largest pediatric neurology clinic in Finland. Phenotypes included encephalopathy (54%), neuromuscular disorders (33%), movement disorders (11%), and one patient (2%) with hemiplegic migraine. All patients underwent whole-exome sequencing and disease-causing genes were analyzed. RESULTS: We found 20 (42%) of the patients to have variants in genes previously associated with disease. Of these, 12 were previously reported disease-causing variants, whereas eight patients had a novel variant on a disease-causing gene: ATP7A, CHD2, PURA, PYCR2, SLC1A4, SPAST, TRIT1, and UPF3B. Genetics also enabled us to define atypical clinical presentations of Rett syndrome (MECP2) and Menkes disease (ATP7A). Except for one deletion, all findings were single-nucleotide variants (missense 72%, truncating 22%, splice-site 6%). Nearly half of the variants were de novo. CONCLUSIONS: The most common cause of childhood encephalopathies are de novo variants. Whole-exome sequencing, even singleton, proved to be an efficient tool to gain specific diagnoses and in finding de novo variants in a clinically heterogeneous group of childhood encephalopathies. IMPACT: Whole-exome sequencing is useful in heterogeneous pediatric neurology cohorts. Our article provides further evidence for and novel variants in several genes. De novo variants are an important cause of childhood encephalopathies.

Interneuronal GluK1 kainate receptors control maturation of GABAergic transmission and network synchrony in the hippocampus.

Kainate type glutamate receptors (KARs) are strongly expressed in GABAergic interneurons and have the capability of modulating their functions via ionotropic and G-protein coupled mechanisms. GABAergic interneurons are critical for generation of coordinated network activity in both neonatal and adult brain, yet the role of interneuronal KARs in network synchronization remains unclear. Here, we show that GABAergic neurotransmission and spontaneous network activity is perturbed in the hippocampus of neonatal mice lacking GluK1 KARs selectively in GABAergic neurons. Endogenous activity of interneuronal GluK1 KARs maintains the frequency and duration of spontaneous neonatal network bursts and restrains their propagation through the hippocampal network. In adult male mice, the absence of GluK1 in GABAergic neurons led to stronger hippocampal gamma oscillations and enhanced theta-gamma cross frequency coupling, coinciding with faster spatial relearning in the Barnes maze. In females, loss of interneuronal GluK1 resulted in shorter sharp wave ripple oscillations and slightly impaired abilities in flexible sequencing task. In addition, ablation of interneuronal GluK1 resulted in lower general activity and novel object avoidance, while causing only minor anxiety phenotype. These data indicate a critical role for GluK1 containing KARs in GABAergic interneurons in regulation of physiological network dynamics in the hippocampus at different stages of development.

Micro-computed Tomography-Based Collagen Orientation and Anisotropy Analysis of Rabbit Articular Cartilage.

The collagen network is the highly organized backbone of articular cartilage providing tissue tensile stiffness and restricting proteoglycan bleaching out of the tissue. Osteoarthritis (OA) diminishes proper collagen network adaptation. Our aim was to provide quantitative three-dimensional (3D) information of the cartilage collagen network adaptation in early osteoarthritis using high resolution micro-computed tomography (µCT)-imaging. Osteochondral samples from the femoral condyles were collected from healthy (N = 8, both legs) and experimental OA rabbit model with anterior cruciate ligament transection (N = 14, single leg). Samples were processed for cartilage µCT-imaging and histological evaluation with polarized light microscopy (PLM). Structure tensor analysis was used to analyse the collagen fibre orientation and anisotropy of the µCT-images, and PLM was used as a validation for structural changes. Depth-wise comparison of collagen fibre orientation acquired with µCT-imaging and PLM correlated well, but the values obtained with PLM were systematically greater than those measured with µCT-imaging. Structure tensor analysis allowed for 3D quantification of collagen network anisotropy. Finally, µCT-imaging revealed only minor differences between the control and experimental groups.

Changes in subchondral bone structure and mechanical properties do not substantially affect cartilage mechanical responses - A finite element study.

Subchondral bone structure has been observed to change in osteoarthritis (OA). However, it remains unclear how the early-stage OA changes affect the mechanics (stresses and strains) of the osteochondral unit. In this study, we aim to characterize the effect of subchondral bone structure and mechanical properties on the osteochondral unit mechanics. A 3-D finite element model of the osteochondral unit was constructed based on a rabbit femoral condyle µCT data and subjected to creep loading in indentation. Trabecular bone volume fraction, subchondral bone plate thickness, and equilibrium modulus were varied (including experimentally observed changes in early OA) to characterize the effect of these parameters on the osteochondral unit mechanics. At the end of the creep phase, the maximum principal strain at the bone surface of the cartilage-bone interface was decreased by 50% when the trabecular bone volume fraction was reduced from 48% to 28%. The maximum principal stress at the same location was decreased by 36% when plate thickness was reduced by 100 µm (-31%). In cartilage, small changes in the mechanics were seen near the cartilage-bone interface with a considerably thinner (-31%) plate. The changes in trabecular bone volume fraction, subchondral bone thickness and plate equilibrium modulus did not substantially affect the cartilage mechanics. Our results suggest that experimentally observed changes that occur in the subchondral bone structure in early OA have a minimal effect on cartilage mechanics under creep indentation loading; clear changes in the cartilage mechanics were seen only with an unrealistically soft subchondral bone plate.

Effectiveness of clinical exome sequencing in adult patients with difficult-to-diagnose neurological disorders.

OBJECTIVES: Clinical diagnostics in adults with hereditary neurological diseases is complicated by clinical and genetic heterogeneity, as well as lifestyle effects. Here, we evaluate the effectiveness of exome sequencing and clinical costs in our difficult-to-diagnose adult patient cohort. Additionally, we expand the phenotypic and genetic spectrum of hereditary neurological disorders in Finland. METHODS: We performed clinical exome sequencing (CES) to 100 adult patients from Finland with neurological symptoms of suspected genetic cause. The patients were classified as myopathy (n = 57), peripheral neuropathy (n = 16), ataxia (n = 15), spastic paraplegia (n = 4), Parkinsonism (n = 3), and mixed (n = 5). In addition, we gathered the costs of prior diagnostic work-up to retrospectively assess the cost-effectiveness of CES as a first-line diagnostic tool. RESULTS: The overall diagnostic yield of CES was 27%. Pathogenic variants were found for 14 patients (in genes ANO5, CHCHD10, CLCN1, DES, DOK7, FKBP14, POLG, PYROXD1, SCN4A, TUBB3, and TTN) and likely pathogenic previously undescribed variants for 13 patients (in genes ABCD1, AFG3L2, ATL1, CACNA1A, COL6A1, DYSF, IRF2BPL, KCNA1, MT-ATP6, SAMD9L, SGCB, and TPM2). Age of onset below 40 years increased the probability of finding a genetic cause. Our cost evaluation of prior diagnostic work-up suggested that early CES would be cost-effective in this patient group, in which diagnostic costs increase linearly with prolonged investigations. CONCLUSIONS: Based on our results, CES is a cost-effective, powerful first-line diagnostic tool in establishing the molecular diagnosis in adult neurological patients with variable symptoms. Importantly, CES can markedly shorten the diagnostic odysseys of about one third of patients.

Early changes in osteochondral tissues in a rabbit model of post-traumatic osteoarthritis.

Concurrent osteoarthritic (OA) manifestations in bone and cartilage are poorly known. To shed light on this issue, this study aims to investigate changes in subchondral bone and articular cartilage at two time points after anterior cruciate ligament transection (ACLT) in a rabbit model. 2 (N = 16) and 8 (N = 10) weeks after ACLT, the subchondral bone structure, cartilage thickness, Osteoarthritis Research Society International (OARSI) score, fixed charged density (FCD), and collagen orientation angle were analyzed. OA related changes were evaluated by comparing the ACLT to the contralateral (C-L) and control knees. Already 2 weeks after ACLT, higher trabecular number in the medial femoral condyle and femoral groove, greater OARSI score in the femoral condyles, and thinner trabeculae in the lateral tibial plateau and femoral groove were observed in ACLT compared to C-L knees. Only minor changes of cartilage collagen orientation in the femoral condyles and femoral groove and smaller FCD in the femoral condyles, medial tibial plateau, femoral groove and patella were observed. 8 weeks post-ACLT, the surgical knees had thinner subchondral plate and trabeculae, and smaller trabecular bone volume fraction in most of the knee locations. OARSI score was greater in the femoral condyle and lateral tibial plateau cartilage. FCD loss was progressive only in the femoral condyle, femoral groove, and patellar cartilage, and minor changes of cartilage collagen orientation angle were present in the femoral condyles, femoral groove, and lateral tibial plateau. We conclude that ACLT induces progressive subchondral bone loss, during which proteoglycan loss occurs followed by their partly recovery, as indicated by FCD results.

Genetic background of ataxia in children younger than 5 years in Finland.

OBJECTIVE: To characterize the genetic background of molecularly undefined childhood-onset ataxias in Finland. METHODS: This study examined a cohort of patients from 50 families with onset of an ataxia syndrome before the age of 5 years collected from a single tertiary center, drawing on the advantages offered by next generation sequencing. A genome-wide genotyping array (Illumina Infinium Global Screening Array MD-24 v.2.0) was used to search for copy number variation undetectable by exome sequencing. RESULTS: Exome sequencing led to a molecular diagnosis for 20 probands (40%). In the 23 patients examined with a genome-wide genotyping array, 2 additional diagnoses were made. A considerable proportion of probands with a molecular diagnosis had de novo pathogenic variants (45%). In addition, the study identified a de novo variant in a gene not previously linked to ataxia: MED23. Patients in the cohort had medically actionable findings. CONCLUSIONS: There is a high heterogeneity of causative mutations in this cohort despite the defined age at onset, phenotypical overlap between patients, the founder effect, and genetic isolation in the Finnish population. The findings reflect the heterogeneous genetic background of ataxia seen worldwide and the substantial contribution of de novo variants underlying childhood ataxia.

Machine Learning Classification of Articular Cartilage Integrity Using Near Infrared Spectroscopy.

INTRODUCTION: Assessment of cartilage integrity during arthroscopy is limited by the subjective visual nature of the technique. To address this shortcoming in diagnostic evaluation of articular cartilage, near infrared spectroscopy (NIRS) has been proposed. In this study, we evaluated the capacity of NIRS, combined with machine learning techniques, to classify cartilage integrity. METHODS: Rabbit (n = 14) knee joints with artificial injury, induced via unilateral anterior cruciate ligament transection (ACLT), and the corresponding contra-lateral (CL) joints, including joints from separate non-operated control (CNTRL) animals (n = 8), were used. After sacrifice, NIR spectra (1000-2500 nm) were acquired from different anatomical locations of the joints (n TOTAL  = 313: n CNTRL = 111, n CL = 97, n ACLT = 105). Machine and deep learning methods (support vector machines-SVM, logistic regression-LR, and deep neural networks-DNN) were then used to develop models for classifying the samples based solely on their NIR spectra. RESULTS: The results show that the model based on SVM is optimal of distinguishing between ACLT and CNTRL samples (ROC_AUC = 0.93, kappa = 0.86), LR is capable of distinguishing between CL and CNTRL samples (ROC_AUC = 0.91, kappa = 0.81), while DNN is optimal for discriminating between the different classes (multi-class classification, kappa = 0.48). CONCLUSION: We show that NIR spectroscopy, when combined with machine learning techniques, is capable of holistic assessment of cartilage integrity, with potential for accurately distinguishing between healthy and diseased cartilage.

Multiparametric MR imaging reveals early cartilage degeneration at 2 and 8 weeks after ACL transection in a rabbit model.

In this study, the rabbit model with anterior cruciate ligament transection (ACLT) was used to investigate early degenerative changes in cartilage using multiparametric quantitative magnetic resonance imaging (qMRI). ACLT was surgically induced in the knees of skeletally mature New Zealand White rabbits (n = 14). ACL transected and contralateral knee compartments-medial femur, lateral femur, medial tibia, and lateral tibia-were harvested 2 (n = 8) and 8 weeks (n = 6) postsurgery. Twelve age-matched nonoperated rabbits served as control. qMRI was conducted at 9.4 T and included relaxation times T1 , T2 , continuous-wave T1ρ (CWT1ρ ), adiabatic T1ρ (AdT1ρ ), adiabatic T2ρ (AdT2ρ ), and relaxation along a fictitious field (TRAFF ). For reference, quantitative histology and biomechanical measurements were carried out. Posttraumatic changes were primarily noted in the superficial half of the cartilage. Prolonged T1 , T2 , CWT1ρ , and AdT1ρ were observed in the lateral femur 2 and 8 weeks post-ACLT, compared with the corresponding control and contralateral groups (P < .05). Collagen orientation was significantly altered in the lateral femur at 2 weeks post-ACLT compared with the corresponding control group. In the medial femur, all the studied relaxation time parameters, except TRAFF , were increased 8 weeks post-ACLT, as compared with the corresponding contralateral and control groups (P < .05). Similarly, significant proteoglycan loss was observed in the medial femur at 8 weeks following surgery (P < .05). Multiparametric MRI demonstrated early degenerative changes primarily in the superficial cartilage with T1 , T2 , CWT1ρ , and AdT1ρ sensitive to cartilage changes at 2 weeks after surgery.

Anterior cruciate ligament transection of rabbits alters composition, structure and biomechanics of articular cartilage and chondrocyte deformation 2 weeks post-surgery in a site-specific manner.

Anterior cruciate ligament (ACL) injury often leads to post-traumatic osteoarthritis (OA) and articular cartilage degradation, changing biomechanics of the tissue and chondrocytes, and altering the fixed charged density (FCD) and collagen network. However, changes in these properties are not known at a very early time point after ACL rupture, but recognizing early changes might be crucial for successful intervention. We investigated the effects of ACL transection (ACLT) in rabbits on the site-specific biomechanical properties of articular cartilage and chondrocytes, FCD content and collagen network organization, two weeks post-surgery. Unilateral ACLT was performed in eight rabbits, and femoral condyles, tibial plateaus, femoral grooves and patellae were harvested from experimental and contralateral knee joints. An intact control group was used as a reference. We analyzed chondrocyte morphology under pre- and static loading, cartilage biomechanical properties, FCD content and collagen fibril orientation. ACLT caused FCD loss in the lateral and medial femoral condyle, lateral tibial plateau, femoral groove and patellar cartilage (p < 0.05). Minor changes in the collagen orientation occurred in the femoral groove and lateral and medial femoral condyle cartilage (p < 0.05). Cartilage stiffness was reduced in the lateral and medial femoral condyles, and chondrocyte biomechanics was altered in the lateral femoral condyle and patellar cartilage (p < 0.05). We observed loss of FCD from articular cartilage two weeks after ACLT at several joint locations. These changes may have led to decreased cartilage stiffness and altered cell deformation behavior, especially in the femoral condyles.

Elastic, Viscoelastic and Fibril-Reinforced Poroelastic Material Properties of Healthy and Osteoarthritic Human Tibial Cartilage.

Articular cartilage constituents (collagen, proteoglycans, fluid) are significantly altered during osteoarthritis (OA). A fibril-reinforced poroelastic (FRPE) material model can separate the contribution of each constituent on the mechanical response of cartilage. Yet, these properties and their OA related alterations are not known for human tibial cartilage. To answer this gap in the knowledge, we characterized the FRPE as well as elastic and viscoelastic properties of healthy and osteoarthritic human tibial cartilage. Tibial osteochondral explants (n = 27) harvested from 7 cadavers were mechanically tested in indentation followed by a quantification of elastic, viscoelastic and FRPE properties. Then they were histopathologically OARSI graded for the severity of OA. FRPE modeling revealed that non-fibrillar matrix modulus was higher in the healthy group compared to the early OA (p = 0.003) and advanced OA (p < 0.001) groups. The initial fibril network modulus was also higher in the healthy group compared to the early OA (p = 0.009) and advanced OA (p < 0.001) groups. The permeability correlated with the OARSI grade (p = 0.002, r = 0.56). For the first time, the FRPE properties were characterized for human tibial cartilage. This knowledge is crucial to improve the accuracy of computational knee joint models.

Recessive PYROXD1 mutations cause adult-onset limb-girdle-type muscular dystrophy.

OBJECTIVE: To describe adult-onset limb-girdle-type muscular dystrophy caused by biallelic variants in the PYROXD1 gene, which has been recently linked to early-onset congenital myofibrillar myopathy. METHODS: Whole exome sequencing was performed for adult-onset neuromuscular disease patients with no molecular diagnosis. Patients with PYROXD1 variants underwent clinical characterization, lower limb muscle MRI, muscle biopsy and spirometry. A yeast complementation assay was used to determine the biochemical consequences of the genetic variants. RESULTS: We identified four patients with biallelic PYROXD1 variants. Three patients, who had symptom onset in their 20s or 30s, were homozygous for the previously described p.Asn155Ser. The fourth patient, with symptom onset at age 49, was compound heterozygous for p.Asn155Ser variant and previously unknown p.Tyr354Cys. All patients presented with a LGMD-type phenotype of symmetric muscle weakness and wasting. Symptoms started in proximal muscles of the lower limbs, and progressed slowly to involve also upper limbs in a proximal-predominant fashion. All patients remained ambulant past the age of 60. They had restrictive lung disease but no cardiac impairment. Muscle MRI showed strong involvement of anterolateral thigh muscles. Muscle biopsy displayed chronic myopathic changes. Yeast complementation assay demonstrated the p.Tyr354Cys mutation to impair PYROXD1 oxidoreductase ability. CONCLUSION: PYROXD1 variants can cause an adult-onset slowly progressive LGMD-type phenotype.

Genetic Basis of Severe Childhood-Onset Cardiomyopathies.

BACKGROUND: Childhood cardiomyopathies are progressive and often lethal disorders, forming the most common cause of heart failure in children. Despite severe outcomes, their genetic background is still poorly characterized. OBJECTIVES: The purpose of this study was to characterize the genetics of severe childhood cardiomyopathies in a countrywide cohort. METHODS: The authors collected a countrywide cohort, KidCMP, of 66 severe childhood cardiomyopathies from the sole center in Finland performing cardiac transplantation. For genetic diagnosis, next-generation sequencing and subsequent validation using genetic, cell biology, and computational approaches were used. RESULTS: The KidCMP cohort presents remarkable early-onset and severe disorders: the median age of diagnosis was 0.33 years, and 17 patients underwent cardiac transplantation. The authors identified the pathogenic variants in 39% of patients: 46% de novo, 34% recessive, and 20% dominantly-inherited. The authors report NRAP underlying childhood dilated cardiomyopathy, as well as novel phenotypes for known heart disease genes. Some genetic diagnoses have immediate implications for treatment: CALM1 with life-threatening arrhythmias, and TAZ with good cardiac prognosis. The disease genes converge on metabolic causes (PRKAG2, MRPL44, AARS2, HADHB, DNAJC19, PPA2, TAZ, BAG3), MAPK pathways (HRAS, PTPN11, RAF1, TAB2), development (NEK8 and TBX20), calcium signaling (JPH2, CALM1, CACNA1C), and the sarcomeric contraction cycle (TNNC1, TNNI3, ACTC1, MYH7, NRAP). CONCLUSIONS: Childhood cardiomyopathies are typically caused by rare, family-specific mutations, most commonly de novo, indicating that next-generation sequencing of trios is the approach of choice in their diagnosis. Genetic diagnoses may suggest intervention strategies and predict prognosis, offering valuable tools for prioritization of patients for transplantation versus conservative treatment.

Site-specific glycosaminoglycan content is better maintained in the pericellular matrix than the extracellular matrix in early post-traumatic osteoarthritis.

INTRODUCTION: One of the characteristics of early osteoarthritis (OA) is the loss of fixed charged density (FCD) of glycosaminoglycans in the superficial zone of articular cartilage. However, possible local changes in the FCD content of the pericellular matrix (PCM) are not fully understood. Hence, our aim was to investigate the effect of unilateral anterior cruciate ligament transection (ACLT) in rabbit knees on estimated FCD in the PCM compared to that in the ECM, and relate these results with cell morphology. METHODS: Articular cartilage samples were collected from ACLT, contralateral and intact control knee joints from lateral and medial femoral condyles and tibial plateaus, and from the femoral groove and patella. Histological samples were prepared and stained with Safranin-O to estimate the FCD content around the chondrocytes in the PCM and the ECM with digital densitometry. RESULTS: As a result of ACLT, the greatest decreases in the FCD content of the PCM were observed in the superficial zone of the lateral femoral condyle (p = 0.02), medial tibial plateau (p = 0.002) and patellar (p < 0.001) cartilage. The normalized FCD content of the PCM compared to the surrounding ECM was increased most in the femoral condyles (p < 0.01) and medial tibial plateau (p = 0.02) cartilage. The high normalized FCD content of the PCM in the superficial zone of lateral femoral condyle cartilage was consistent with the round cell morphology in that location. CONCLUSIONS: In conclusion, we suggest that certain sites in the knee joint, particularly the lateral femoral condyle cartilage, experience less FCD loss in the PCM than in the ECM in early post-traumatic OA, which could lead to altered cell shape.