BTK inhibition limits microglia-perpetuated CNS inflammation and promotes myelin repair.

In multiple sclerosis (MS), persisting disability can occur independent of relapse activity or development of new central nervous system (CNS) inflammatory lesions, termed chronic progression. This process occurs early and it is mostly driven by cells within the CNS. One promising strategy to control progression of MS is the inhibition of the enzyme Bruton's tyrosine kinase (BTK), which is centrally involved in the activation of both B cells and myeloid cells, such as macrophages and microglia. The benefit of BTK inhibition by evobrutinib was shown as we observed reduced pro-inflammatory activation of microglia when treating chronic experimental autoimmune encephalomyelitis (EAE) or following the adoptive transfer of activated T cells. Additionally, in a model of toxic demyelination, evobrutinib-mediated BTK inhibition promoted the clearance of myelin debris by microglia, leading to an accelerated remyelination. These findings highlight that BTK inhibition has the potential to counteract underlying chronic progression of MS.

Skeletal muscle fibers produce B-cell stimulatory factors in chronic myositis.

Introduction: We aimed to identify B-cell-mediated immunomechanisms in inclusion body myositis (IBM) and polymyositis (PM) as part of the complex pathophysiology. Materials and methods: Human primary myotube cultures were derived from orthopedic surgery. Diagnostic biopsy specimens from patients with IBM (n=9) and PM (n=9) were analyzed for markers of B cell activation (BAFF and APRIL) and for chemokines that control the recruitment of B cells (CXCL-12 and CXCL-13). Results were compared to biopsy specimens without myopathic changes (n=9) and hereditary muscular dystrophy (n=9). Results: The mRNA expression of BAFF, APRIL, and CXCL-13 was significantly higher in IBM and PM compared to controls. Patients with IBM displayed the highest number of double positive muscle fibers for BAFF and CXCL-12 (48%) compared to PM (25%), muscular dystrophy (3%), and non-myopathic controls (0%). In vitro, exposure of human myotubes to pro-inflammatory cytokines led to a significant upregulation of BAFF and CXCL-12, but APRIL and CXCL-13 remained unchanged. Conclusion: The results substantiate the hypothesis of an involvement of B cell-associated mechanisms in the pathophysiology of IBM and PM. Muscle fibers themselves seem to contribute to the recruitment of B cells and sustain inflammation.

Inflammasome in Skeletal Muscle: NLRP3 Is an Inflammatory Cell Stress Component in Inclusion Body Myositis.

Inclusion body myositis (IBM) is a chronic, mostly treatment-resistant, inflammatory myopathy with a pathology that centers around specific interactions between inflammation and protein accumulation. The study aimed to identify the inflammasome as a key event in the complex network of pathomechanisms. Regulation of the inflammasome was assessed in a well-established pro-inflammatory cell culture model using human myoblasts and primary human myotubes. By quantitative PCR, western blot and immunocytochemistry, inflammasome markers including NLRP3 were assessed in muscle cells exposed to the cytokines IL-1ß and IFN-γ. The data were corroborated by analysis of muscle biopsies from patients with IBM compared to other myositis subtypes. In the cell culture model of IBM, the NLRP3 inflammasome was significantly overexpressed, as evidenced by western blot (p = 0.03) and quantitative PCR (p < 0.01). Target genes that play a role in inflammasome assembly, T-cell migration, and MHC-I expression (p = 0.009) were highly co-upregulated. NLRP3 was significantly overexpressed in muscle biopsies from IBM samples compared to disease controls (p = 0.049), including other inflammatory myopathies. Due to the extraordinary features of the pathogenesis and the pronounced upregulation of NLRP3 in IBM, the inflammasome could serve as a key molecule that drives the inflammatory cascade as well as protein accumulation in the muscle. These data can be useful for future therapeutic developments.

Evidence of Two Novel LAMA2 Variants in a Patient With Muscular Dystrophy: Facing the Challenges of a Certain Diagnosis.

Background: Benefits and challenges resulting from advances in genetic diagnostics are two sides of the same coin. Facilitation of a correct and timely diagnosis is paralleled by challenges in interpretation of variants of unknown significance (VUS). Focusing on an individual VUS-re-classification pipeline, this study offers a diagnostic approach for clinically suspected hereditary muscular dystrophy by combining the expertise of an interdisciplinary team. Methods: In a multi-step approach, a thorough phenotype assessment including clinical examination, laboratory work, muscle MRI and histopathological evaluation of muscle was performed in combination with advanced Next Generation Sequencing (NGS). Different in-silico tools and prediction programs like Alamut, SIFT, Polyphen, MutationTaster and M-Cap as well as 3D- modeling of protein structure and RNA-sequencing were employed to determine clinical significance of the LAMA2 variants. Results: Two previously unknown sequence alterations in LAMA2 were detected, a missense variant was classified initially according to ACMG guidelines as a VUS (class 3) whereas a second splice site variant was deemed as likely pathogenic (class 4). Pathogenicity of the splice site variant was confirmed by mRNA sequencing and nonsense mediated decay (NMD) was detected. Combination of the detected variants could be associated to the LGMDR23-phenotype based on the MRI matching and literature research. Discussion: Two novel variants in LAMA2 associated with LGMDR23-phenotype are described. This study illustrates challenges of the genetic findings due to their VUS classification and elucidates how individualized diagnostic procedure has contributed to the accurate diagnosis in the spectrum of LGMD.

Infratentorial ganglioglioma mimicking a cerebellar metastasis.

We report a case of an infratentorial ganglioglioma in a 56-year-old male, who underwent magnetic resonance imaging (MRI) during the diagnostic workup for a suspected lung cancer. The MRI scan revealed a space-occupying lesion of the left lobulus semilunaris superior cerebelli, which was assumed being a metastasis. The asymptomatic lesion was resected to establish the diagnosis. Histologic and immunohistochemical studies showed a ganglioglioma with World Health Organization grade I characteristics. Although ganglioglioma typically exhibits a supratentorial predilection, it should be included in the differential diagnosis of lesions occurring in the cerebellum.

Treatment and outcomes in anti-HMG-CoA reductase-associated immune-mediated necrotising myopathy. Comparative analysis of a single-centre cohort and published data.

OBJECTIVES: Anti-hydroxy-methyl-glutaryl-coenzyme A reductase (HMGCR) antibody-associated myopathy was recognised as a new form of immune-mediated necrotising myopathy (IMNM) a decade ago. Due to the rarity of the disease, only limited data on clinical manifestations and therapeutic outcomes are available. METHODS: We retrospectively analysed a monocentric cohort of HMGCR-associated IMNM patients treated at the University Medical Centre Göttingen. Clinical, laboratory, and biopsy data, as well as treatment outcomes, were analysed. In addition, a literature search was performed on published HMGCR IMNM cohorts in Medline and Web of Science. RESULTS: We identified nine patients; five were female. The median age was 68 years (47-77). Six were statin-exposed and older than statin-naive patients (71 years [65-77] vs. 51 years [47-67]). All had muscle weakness, seven myalgias. Strength (MRC sum score) was 53/65 (46-61) at baseline and increased to 63/65 (50-65) with therapy. Creatine kinase (CK) levels decreased from a median level of 12837 U/L (range 6346-25011) to 624 U/L (35-1564 U/L). All received glucocorticoids (GC) and at least one immunosuppressive therapy. The literature review identified 26 studies comprising 691 patients. 57.9% were female, 61.3% statin exposed. 95.2% had weakness, 39.1% myalgia. Dysphagia affected 28.8%. 84.9% received GC and a median of 1.5 additional immunosuppressants. Compared to published data, our patients had higher baseline CK values (12837 [6346-25011] vs. 6951 [2539-10500], p<0.001), and we used azathioprine and intravenous immunoglobulins (p<0.001) more frequently but methotrexate and rituximab less frequently (p<0.001). CONCLUSIONS: HMGCR-associated IMNM is a rare subset of myositis. With systemic treatment, patients usually achieve partial or complete remission. Optimal treatment has not been established, but glucocorticoids, azathioprine, and methotrexate are generally effective with or without intravenous immunoglobulins.

IDH2 R172 Mutations Across Poorly Differentiated Sinonasal Tract Malignancies: Forty Molecularly Homogenous and Histologically Variable Cases With Favorable Outcome.

IDH2 R172 mutations occur in sinonasal undifferentiated carcinoma (SNUC), large-cell neuroendocrine carcinoma (LCNEC), sinonasal adenocarcinomas, and olfactory neuroblastoma (ONB). We performed a clinical, pathologic, and genetic/epigenetic analysis of a large IDH2-mutated sinonasal tumor cohort to explore their distinct features. A total 165 sinonasal/skull base tumors included 40 IDH2 mutants studied by light microscopy, immunohistochemistry, and genome-wide DNA methylation, and 125 IDH2 wild-type tumors used for comparison. Methylation profiles were analyzed by unsupervised hierarchical clustering, t-distributed stochastic neighbor embedding dimensionality reduction and assessed for copy number alterations (CNA). Thirty-nine histologically assessable cases included 25 (64.1%) SNUC, 8 (20.5%) LCNEC, 2 (5.1%) poorly differentiated adenocarcinomas, 1 (2.7%) ONB, and 3 (7.7%) IDH2-mutated tumors with ONB features. All cases were high-grade showing necrosis (82.4%), prominent nucleoli (88.9%), and median 21 mitoses/10 HPFs. AE1/AE3 and/or CAM 5.2 were positive in all and insulinoma-associated protein 1 (INSM1) in 80% cases. All IDH2 mutants formed one distinct group by t-distributed stochastic neighbor embedding dimensionality reduction separating from all IDH2 wild-type tumors. There was no correlation between methylation clusters and histopathologic diagnoses. Recurrent CNA included 1q gain (79.3%), 17p loss (75.9%), and 17q gain (58.6%). No CNA differences were observed between SNUC and LCNEC. IDH2 mutants showed better disease-specific survival than SMARCB1-deficient (P=0.027) and IDH2 wild-type carcinomas overall (P=0.042). IDH2-mutated sinonasal tumors are remarkably homogeneous at the molecular level and distinct from IDH2 wild-type sinonasal malignancies. Biology of IDH2-mutated sinonasal tumors might be primarily defined by their unique molecular fingerprint rather than by their respective histopathologic diagnoses.

Differential diagnosis of vacuolar myopathies in the NGS era.

Altered autophagy accompanied by abnormal autophagic (rimmed) vacuoles detectable by light and electron microscopy is a common denominator of many familial and sporadic non-inflammatory muscle diseases. Even in the era of next generation sequencing (NGS), late-onset vacuolar myopathies remain a diagnostic challenge. We identified 32 adult vacuolar myopathy patients from 30 unrelated families, studied their clinical, histopathological and ultrastructural characteristics and performed genetic testing in index patients and relatives using Sanger sequencing and NGS including whole exome sequencing (WES). We established a molecular genetic diagnosis in 17 patients. Pathogenic mutations were found in genes typically linked to vacuolar myopathy (GNE, LDB3/ZASP, MYOT, DES and GAA), but also in genes not regularly associated with severely altered autophagy (FKRP, DYSF, CAV3, COL6A2, GYG1 and TRIM32) and in the digenic facioscapulohumeral muscular dystrophy 2. Characteristic histopathological features including distinct patterns of myofibrillar disarray and evidence of exocytosis proved to be helpful to distinguish causes of vacuolar myopathies. Biopsy validated the pathogenicity of the novel mutations p.(Phe55*) and p.(Arg216*) in GYG1 and of the p.(Leu156Pro) TRIM32 mutation combined with compound heterozygous deletion of exon 2 of TRIM32 and expanded the phenotype of Ala93Thr-caveolinopathy and of limb-girdle muscular dystrophy 2i caused by FKRP mutation. In 15 patients no causal variants were detected by Sanger sequencing and NGS panel analysis. In 12 of these cases, WES was performed, but did not yield any definite mutation or likely candidate gene. In one of these patients with a family history of muscle weakness, the vacuolar myopathy was eventually linked to chloroquine therapy. Our study illustrates the wide phenotypic and genotypic heterogeneity of vacuolar myopathies and validates the role of histopathology in assessing the pathogenicity of novel mutations detected by NGS. In a sizable portion of vacuolar myopathy cases, it remains to be shown whether the cause is hereditary or degenerative.

Primary Angiitis of the Central Nervous System: From Psychiatry to Neurology.

We present a case of a 54-year-old man with primary angiitis of the central nervous system (PACNS) who was initially admitted to a psychiatric clinic with a diagnosis of delirium. We discuss the difficulty in establishing the diagnosis of PACNS and provide the reader with some recommendations on how to promptly and correctly diagnose this disease in order to avoid potentially lethal outcomes.

Cell-autonomous role of GFRα1 in the development of olfactory bulb GABAergic interneurons.

GFRα1, a receptor for glial cell line-derived neurotrophic factor (GDNF), is critical for the development of the main olfactory system. The olfactory bulb (OB) of Gfra1 knockout mice shows significant reductions in the number of olfactory sensory neurons, mitral and tufted cells, as well as all major classes of OB GABAergic interneurons. However, the latter do not express significant levels of GFRα1, leaving the mechanism of action of GFRα1 in OB interneuron development unexplained. Here we report that GFRα1 is highly expressed in the precursor cells that give rise to all major classes of OB interneurons, but is downregulated as these neurons mature. Conditional ablation of GFRα1 in embryonic GABAergic cells recapitulated the cell losses observed in global Gfra1 knockouts at birth. GFRα1 was also required for the sustained generation and allocation of OB interneurons in adulthood. Conditional loss of GFRα1 altered the migratory behaviour of neuroblasts along the rostral migratory stream (RMS) as well as RMS glial tunnel formation. Together, these data indicate that GFRα1 functions cell-autonomously in subpopulations of OB interneuron precursors to regulate their generation and allocation in the mammalian OB.

Thalamo-cortical axons regulate the radial dispersion of neocortical GABAergic interneurons.

Neocortical GABAergic interneuron migration and thalamo-cortical axon (TCA) pathfinding follow similar trajectories and timing, suggesting they may be interdependent. The mechanisms that regulate the radial dispersion of neocortical interneurons are incompletely understood. Here we report that disruption of TCA innervation, or TCA-derived glutamate, affected the laminar distribution of GABAergic interneurons in mouse neocortex, resulting in abnormal accumulation in deep layers of interneurons that failed to switch from tangential to radial orientation. Expression of the KCC2 cotransporter was elevated in interneurons of denervated cortex, and KCC2 deletion restored normal interneuron lamination in the absence of TCAs. Disruption of interneuron NMDA receptors or pharmacological inhibition of calpain also led to increased KCC2 expression and defective radial dispersion of interneurons. Thus, although TCAs are not required to guide the tangential migration of GABAergic interneurons, they provide crucial signals that restrict interneuron KCC2 levels, allowing coordinated neocortical invasion of TCAs and interneurons.

Topographical transcriptome mapping of the mouse medial ganglionic eminence by spatially resolved RNA-seq.

BACKGROUND: Cortical interneurons originating from the medial ganglionic eminence, MGE, are among the most diverse cells within the CNS. Different pools of proliferating progenitor cells are thought to exist in the ventricular zone of the MGE, but whether the underlying subventricular and mantle regions of the MGE are spatially patterned has not yet been addressed. Here, we combined laser-capture microdissection and multiplex RNA-sequencing to map the transcriptome of MGE cells at a spatial resolution of 50 µm. RESULTS: Distinct groups of progenitor cells showing different stages of interneuron maturation are identified and topographically mapped based on their genome-wide transcriptional pattern. Although proliferating potential decreased rather abruptly outside the ventricular zone, a ventro-lateral gradient of increasing migratory capacity was identified, revealing heterogeneous cell populations within this neurogenic structure. CONCLUSIONS: We demonstrate that spatially resolved RNA-seq is ideally suited for high resolution topographical mapping of genome-wide gene expression in heterogeneous anatomical structures such as the mammalian central nervous system.

MET signaling in GABAergic neuronal precursors of the medial ganglionic eminence restricts GDNF activity in cells that express GFRα1 and a new transmembrane receptor partner.

GDNF (glial cell line-derived neurotrophic factor) promotes the differentiation and migration of GABAergic neuronal precursors of the medial ganglionic eminence (MGE). These functions are dependent on the GPI-anchored receptor GFRα1, but independent of its two known transmembrane receptor partners RET and NCAM. Here we show that soluble GFRα1 is also able to promote differentiation and migration of GABAergic MGE neurons. These activities require endogenous production of GDNF. Although GDNF responsiveness is abolished in Gfra1(-/-) neurons, it can be restored upon addition of soluble GFRα1, a result that is only compatible with the existence of a previously unknown transmembrane signaling partner for the GDNF-GFRα1 complex in GABAergic neurons. The roles of two candidate transmembrane receptors previously implicated in GABAergic interneuron development--MET, a receptor for hepatocyte growth factor (HGF), and ErbB4, the neuregulin receptor--were examined. GDNF did not induce the activation of either receptor, nor did inhibition of MET or ErbB4 impair GDNF activity in GABAergic MGE neurons. Unexpectedly, however, inhibition of MET or HGF per se promoted neuronal differentiation and migration and enhanced the activity of GDNF on MGE neurons. These effects were dependent on endogenous GDNF and GFRα1, suggesting that MET signaling negatively regulates GDNF activity in the MGE. In agreement with this, Met mutant MGE neurons showed enhanced responses to GDNF and inhibition of MET or HGF increased Gfra1 mRNA expression in MGE cells. In vivo, expression of MET and GFRα1 overlapped in the MGE, and a loss-of-function mutation in Met increased Gfra1 expression in this region. Together, these observations demonstrate the existence of a novel transmembrane receptor partner for the GDNF-GFRα1 complex and uncover an unexpected interplay between GDNF-GFRα1 and HGF-MET signaling in the early diversification of cortical GABAergic interneuron subtypes.

Fibroblast growth factor 2 regulates astrocyte differentiation in a region-specific manner in the hindbrain.

Fibroblast growth factor (FGF)-2 is known to have important pleiotropic effects in neuronal and glial cells during various physiological and pathological events. To investigate the role of endogenous FGF-2 in the differentiation of astrocytes, we studied the expression of glial fibrillary acidic protein (GFAP) in the hindbrain of the FGF-2 null mouse. GFAP was drastically decreased in a region-specific manner in the hindbrain of the adult and developing FGF-2 null mouse without an associated change in the expression of alternate markers for astrocytes. The deficit was evident in the astrocytes of pontine and medullary gray matter but not in the white matter. The astrocytes of the gray and white matter were seen to express FGF-2 and FGF receptors in a distinct pattern. The methylation of histone H3 at lysine 4 residue (H3K4me2) associated with the STAT (signal transducer and activator of transcription)-binding site of the GFAP promoter was significantly decreased in the gray matter of the FGF-2 null mouse, suggesting a role for FGF-2 in the epigenetic regulation of astrocyte differentiation in vivo. These observations underscore the importance of FGF-2 in astroglial differentiation in the hindbrain and the heterogeneity of astrocytes in their requirement for FGF-2 as a differentiation inducing signal.

Expression and functions of fibroblast growth factor 2 (FGF-2) in hippocampal formation.

Among the 23 members of the fibroblast growth factor (FGF) family, FGF-2 is the most abundant one in the central nervous system. Its impact on neural cells has been profoundly investigated by in vitro and in vivo studies as well as by gene knockout analyses during the past 2 decades. Key functions of FGF-2 in the nervous system include roles in neurogenesis, promotion of axonal growth, differentiation in development, and maintenance and plasticity in adulthood. From a clinical perspective, its prominent role for the maintenance of lesioned neurons (e.g., ischemia and following transection of fiber tracts) is of particular relevance. In the unlesioned brain, FGF-2 is involved in synaptic plasticity and processes attributed to learning and memory. The focus of this review is on the expression of FGF-2 and its receptors in the hippocampal formation and the physiological and pathophysiological roles of FGF-2 in this region during development and adulthood.

Expression of leucine-rich-repeat-kinase 2 (LRRK2) during embryonic development.

The LRRK2 gene was recently found to have multiple mutations that are causative for the most common inherited form of late onset Parkinson's disease. In the adult brain, LRRK2 mRNA is broadly expressed, also in regions other than the nigrostriatal system. In order to establish a basis for assessing more detailed functional implications of LRRK2 in development, we provide here an in-depth analysis of its mRNA expression patterns in neural and extra-neural tissues with a focus on murine embryonic development. LRRK2 mRNA is detectable at E8.5 in non-neural and at E10.5 in neural tissues. From E12.5 to E16.5, LRRK2 mRNA is prominently expressed throughout the neocortex and subsequently highly concentrated in ventricular and subventricular zones and cortical plate. In addition, developing cerebellar granule and Purkinje neurons and spinal cord neurons display robust LRRK2 expression. In non-neural tissues LRRK2 was highly expressed in limb interdigital zones, developing kidney glomeruli, and spermatogenetic cells. Together, our results suggest roles for LRRK2 in controlling proliferation, migration, and differentiation of neural cells as well as in morphogenesis of extra-neural tissues.

Fibroblast growth factor-2 deficiency affects hippocampal spine morphology, but not hippocampal catecholaminergic or cholinergic innervation.

The availability of fibroblast growth factor-2 (FGF-2) -deficient mice has permitted studying the role of endogenous FGF-2. Several studies have reported that neocortical but not hippocampal neurons are lost in FGF-2-deficient mice. Here, we show that neuronal densities within the basolateral amygdala are unaltered in FGF-2-/- mice. Moreover, we provide evidence that FGF-2 mutant mice display no obvious alterations in the catecholaminergic or cholinergic innervation of the hippocampus. With regard to the formation of dendritic spines, our studies reveal that endogenous FGF-2 is not essential for hippocampal spinogenesis; however, FGF-2 affects the length of individual spines. Such alterations in spine morphology may be related to disturbances in mental capacities or alterations in neuronal plasticity. Of interest, in this context, animal models of mental retardation develop no alterations in hippocampal spine densities, but display higher numbers of long dendritic spines. Thus, FGF-2 may also affect learning and memory by altering spine morphology.