Microglial Transforming Growth Factor-ß Signaling in Alzheimer's Disease.

Novel technologies such as single-cell RNA and single-nucleus RNA sequencing have shed new light on the complexity of different microglia populations in physiological and pathological states. The transcriptomic profiling of these populations has led to the subclassification of specific disease-associated microglia and microglia clusters in neurodegenerative diseases. A common profile includes the downregulation of homeostasis and the upregulation of inflammatory markers. Furthermore, there is concordance in few clusters between murine and human samples. Apolipoprotein E, which has long been considered a high-risk factor for late-onset Alzheimer's disease, is strongly regulated in both these murine and human clusters. Transforming growth factor-ß plays an essential role during the development and maturation of microglia. In a pathological state, it attenuates their activation and is involved in numerous cell regulatory processes. Transforming growth factor-ß also has an influence on the deposition of amyloid-beta, as it is involved in the regulation of key proteins and molecules. Taken together, this review highlights the complex interaction of apolipoprotein E, the triggering receptor on myeloid cells 2, and transforming growth factor-ß as part of a regulatory axis in microglia at the onset and over the course of Alzheimer's disease.

The Role of TGFß Signaling in Microglia Maturation and Activation.

The pleiotropic cytokine transforming growth factor-beta 1 (TGFß1) plays pivotal roles in different cell types, including immune cells such as T cells, monocytes/macrophages, and microglia. Microglia are essential during physiological and pathological events. Maturation of postnatal microglia, as well as the regulation of the complex functional repertoire of microglia, needs to be carefully orchestrated. However, an understanding of how mammalian microglia maturation and disease-associated microglia activation is regulated remains fragmentary. Here, we summarize recent observations made by employing transgenic approaches to silence microglial TGFß signaling in mice. These revealed that TGFß1 and TGFß signaling are indispensable for microglia maturation, adult microglia homeostasis, and the control of microglia activation in central nervous system pathologies.

Microglial CD74 Expression Is Regulated by TGFß Signaling.

Microglia play important roles during physiological and pathological situations in the CNS. Several reports have described the expression of Cd74 in disease-associated and aged microglia. Here, we demonstrated that TGFß1 controled the expression of Cd74 in microglia in vitro and in vivo. Using BV2 cells, primary microglia cultures as well as Cx3cr1CreERT2:R26-YFP:Tgfbr2fl/fl in combination with qPCR, flow cytometry, and immunohistochemistry, we were able to provide evidence that TGFß1 inhibited LPS-induced upregulation of Cd74 in microglia. Interestingly, TGFß1 alone was able to mediate downregulation of CD74 in vitro. Moreover, silencing of TGFß signaling in vivo resulted in marked upregulation of CD74, further underlining the importance of microglial TGFß signaling during regulation of microglia activation. Taken together, our data indicated that CD74 is a marker for activated microglia and further demonstrated that microglial TGFß signaling is important for regulation of Cd74 expression during microglia activation.

[Therapy of Hemifacial Spasm with Botulinum Toxin: an Update]. / Botulinumtoxin ­ Therapie des Spasmus hemifacialis: Ein Update.

Injections of botulinum toxin can be viewed by now as the therapy of choice in treating hemifacial spasm (HFS). Each of the three botulinum toxin-A preparations have been approved for this indication in Germany. HFS is a frequent disease characterized by involuntary contractions of the muscles of one half of the face innervated by the facial nerve. The symptoms can be either tonic or clonic, intermittant or permanent. Diagnosis is based purely on clinical observation. A magnetic resonance imagingof the skull is appropriate to demonstrate nerve-vessel contact as most frequent cause and to exclude other pathologies.

[Therapy of Sialorrhea with Botulinum Toxin - An Update]. / Therapie der Sialorrhoe mit Botulinumtoxin ­ ein Update.

The most important salivary glands are the paired parotid and submandibular glands. Adults produce 1 to 1.5 liters of saliva which are then regularly swallowed. When the act of swallowing is disturbed, salivation occurs. More rarely, the cause can be found in increased saliva production, for example, when caused through medication. Sialorrhea impairs the quality of life substantially and is frequently often socially stigmatizing. Therapy includes conservative measures such as functional dysphagia therapy, oral or transdermal application of anticholinergics, as well as, in selected cases, radiation and surgical measures. Over the last 20 years, local injection of botulinum toxin has been successfully applied in the treatment of this condition. With approval of incobotulinumtoxinA toxin for children and adults, this procedure will become the therapy of choice for chronic sialorrhea. The results of the phase III registration trials have demonstrated high efficacy and good safety of the injection treatment in both children and adults.

Microglia Development and Maturation and Its Implications for Induction of Microglia-Like Cells from Human iPSCs.

Microglia are resident immune cells of the central nervous system and play critical roles during the development, homeostasis, and pathologies of the brain. Originated from yolk sac erythromyeloid progenitors, microglia immigrate into the embryonic brain parenchyma to undergo final postnatal differentiation and maturation driven by distinct chemokines, cytokines, and growth factors. Among them, TGFß1 is an important regulator of microglial functions, mediating homeostasis, anti-inflammation, and triggering the expression of microglial homeostatic signature genes. Since microglia studies are mainly based on rodent cells and the isolation of homeostatic microglia from human tissue is challenging, human-induced pluripotent stem cells have been successfully differentiated into microglia-like cells recently. However, employed differentiation protocols strongly vary regarding used cytokines and growth factors, culture conditions, time span, and cell yield. Moreover, the incomplete differentiation of human microglia can hamper the similarity to primary human microglia and dramatically influence the outcome of follow-up studies with these differentiated cells. This review summarizes the current knowledge of the molecular mechanisms driving rodent microglia differentiation in vivo, further compares published differentiation protocols, and highlights the potential of TGFß as an essential maturation factor.

[Therapy of sialorrhea with botulinum toxin]. / Therapie der Sialorrhoe mit Botulinumtoxin.

The most important salivary glands are the paired parotid and submandibular glands. Adults produce 1 to 1.5 liters of saliva which are then regularly swallowed. When the act of swallowing is disturbed, salivation occurs. More rarely, the cause can be found in increased saliva production, for example, when caused through medication. Sialorrhea impairs the quality of life substantially and is frequently often socially stigmatizing. Therapy includes conservative measures such as functional dysphagia therapy, oral or transdermal application of anticholinergics, as well as, in selected cases, radiation and surgical measures. Over the last 20 years local injection of botulinum toxin has been successfully applied in the treatment of this condition. With approval of this therapy by the European agencies, this measure will become the therapy of choice for pronounced therapy-resistant sialorrhea.

Postnatal maturation of microglia is associated with alternative activation and activated TGFß signaling.

Microglia are involved in a widespread set of physiological and pathological processes and further play important roles during neurodevelopmental events. Postnatal maturation of microglia has been associated with the establishment of microglia-specific gene expression patterns. The mechanisms governing microglia maturation are only partially understood but Tgfß1 has been suggested to be one important mediator. In the present study, we demonstrate that early postnatal microglia maturation is associated with alternative microglia activation, increased engulfment of apoptotic cells as well as activated microglial Tgfß signaling. Interestingly, microglial Tgfß signaling preceded the induction of the microglia-specific gene expression indicating the importance of Tgfß1 for postnatal microglia maturation. Moreover, we provide evidence that Tgfß1 is expressed by neurons in postnatal and adult brains defining neuron-microglia communication via Tgfß1 as an important event. Finally, we introduce the recently identified microglia marker Tmem119 as a direct Tgfß1-Smad2 target gene. Taken together, the data presented here further increase the understanding of Tgfß1-mediated effects in microglia and place emphasis on the importance of Tgfß1 for microglia maturation and maintenance.

Aged Mouse Cortical Microglia Display an Activation Profile Suggesting Immunotolerogenic Functions.

Microglia are the resident immune cells of the central nervous system (CNS) and participate in physiological and pathological processes. Their unique developmental nature suggests age-dependent structural and functional impairments that might contribute to neurodegenerative diseases. In the present study, we addressed the age-dependent changes in cortical microglia gene expression patterns and the expression of M1- and M2-like activation markers. Iba1 immunohistochemistry, isolation of cortical microglia followed by fluorescence-activated cell sorting and RNA isolation to analyze transcriptional changes in aged cortical microglia was performed. We provide evidence that aging is associated with decreased numbers of cortical microglia and the establishment of a distinct microglia activation profile including upregulation of Ifi204, Lilrb4, Arhgap, Oas1a, Cd244 and Ildr2. Moreover, flow cytometry revealed that aged cortical microglia express increased levels of Cd206 and Cd36. The data presented in the current study indicate that aged mouse cortical microglia adopt a distinct activation profile, which suggests immunosuppressive and immuno-tolerogenic functions.

Growth/differentiation factor-15 deficiency compromises dopaminergic neuron survival and microglial response in the 6-hydroxydopamine mouse model of Parkinson's disease.

Growth/differentiation factor-15 (Gdf-15) is a member of the TGF-ß superfamily and a pleiotropic, widely distributed cytokine, which has been shown to play roles in various pathologies, including inflammation. Analysis of Gdf-15(-/-) mice has revealed that it serves the postnatal maintenance of spinal cord motor neurons and sensory neurons. In a previous study, exogenous Gdf-15 rescued 6-hydroxydopamine (6-OHDA) lesioned Gdf-15(+/+) nigrostriatal dopaminergic (DAergic) neurons in vitro and in vivo. Whether endogenous Gdf-15 serves the physiological maintenance of nigrostriatal DAergic neurons in health and disease is not known and was addressed in the present study. Stereotactic injection of 6-OHDA into the medial forebrain bundle (MFB) led to a significant decline in the numbers of DAergic neurons in both Gdf-15(+/+) and Gdf-15(-/-) mice over a time-period of 14days. However, this decrease was exacerbated in the Gdf-15(-/-) mice, with only 5.5% surviving neurons as compared to 24% in the Gdf-15(+/+) mice. Furthermore, the microglial response to the 6-OHDA lesion was reduced in Gdf-15(-/-) mice, with significantly lower numbers of total and activated microglia and a differential cytokine expression as compared to the Gdf-15(+/+) mice. Using in vitro models, we could demonstrate the importance of endogenous Gdf-15 in promoting DAergic neuron survival thus highlighting its relevance in a direct neurotrophic supportive role. Taken together, these results indicate the importance of Gdf-15 in promoting survival of DAergic neurons and regulating the inflammatory response post 6-OHDA lesion.

Gdf-15 deficiency does not alter vulnerability of nigrostriatal dopaminergic system in MPTP-intoxicated mice.

Growth/differentiation factor-15 (Gdf-15) is a member of the transforming growth factor-ß (Tgf-ß) superfamily and has been shown to be a potent neurotrophic factor for midbrain dopaminergic (DAergic) neurons both in vitro and in vivo. Gdf-15 has also been shown to be involved in inflammatory processes. The aim of this study was to identify the role of endogenous Gdf-15 in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease (PD) by comparing Gdf-15 (+/+) and Gdf-15 (-/-) mice. At 4 days and 14 days post-MPTP administration, both Gdf-15 (+/+) and Gdf-15 (-/-) mice showed a similar decline in DAergic neuron numbers and in striatal dopamine (DA) levels. This was followed by a comparable restorative phase at 90 days and 120 days, indicating that the absence of Gdf-15 does not affect the susceptibility or the recovery capacity of the nigrostriatal system after MPTP administration. The MPTP-induced microglial and astrocytic response was not significantly altered between the two genotypes. However, pro-inflammatory and anti-inflammatory cytokine profiling revealed the differential expression of markers in Gdf-15 (+/+) and Gdf-15 (-/-) mice after MPTP administration. Thus, the MPTP mouse model fails to uncover a major role of endogenous Gdf-15 in the protection of MPTP-lesioned nigrostriatal DAergic neurons, in contrast to its capacity to protect the 6-hydroxydopamine-intoxicated nigrostriatal system.

Magnetic resonance imaging of intraoral hard and soft tissues using an intraoral coil and FLASH sequences.

OBJECTIVES: To ascertain the feasibility of MRI as a non-ionizing protocol for routine dentomaxillofacial diagnostic imaging. Wireless coils were used for MRI of intraoral hard and soft tissues. METHODS: FLASH MRI was applied in vivo with a mandible voxel size of 250 × 250 × 500 µm3, FOV of 64 × 64 × 28 mm3 and acquisition time of 3:57 min and with a maxilla voxel size of 350 µm3 and FOV of 34 cm3 in 6:40 min. Ex vivo imaging was performed in 4:38 min, with a resolution of 200 µm3 and FOV of 36.5 cm3. Cone beam (CB) CT of the mandible and subjects were acquired. MRI was compared to CBCT and histological sections. Deviations were calculated with intraclass correlation coefficient (ICC) and coefficient of variation (cv). RESULTS: A high congruence between CBCT, MRI and specimens was demonstrated. Hard and soft tissues including dental pulp, periodontium, gingiva, cancellous bone and mandibular canal contents were adequately displayed with MRI. CONCLUSIONS: Imaging of select intraoral tissues was achieved using custom MRI protocols with an easily applicable intraoral coil in a clinically acceptable acquisition time. Comparison with CBCT and histological sections helped demonstrate dimensional accuracy of the MR images. The course of the mandibular canal was accurately displayed with CBCT and MRI. KEY POINTS: • MRI is a clinically available diagnostic tool in dentistry • Intraoral hard and soft tissues can be imaged with a high resolution with MRI • The dimensional accuracy of MRI is comparable to cone beam CT.

Microglia-Mediated Neuroinflammation and Neurotrophic Factor-Induced Protection in the MPTP Mouse Model of Parkinson's Disease-Lessons from Transgenic Mice.

Parkinson's disease (PD) is a neurodegenerative disease characterised by histopathological and biochemical manifestations such as loss of midbrain dopaminergic (DA) neurons and decrease in dopamine levels accompanied by a concomitant neuroinflammatory response in the affected brain regions. Over the past decades, the use of toxin-based animal models has been crucial to elucidate disease pathophysiology, and to develop therapeutic approaches aimed to alleviate its motor symptoms. Analyses of transgenic mice deficient for cytokines, chemokine as well as neurotrophic factors and their respective receptors in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD have broadened the current knowledge of neuroinflammation and neurotrophic support. Here, we provide a comprehensive review that summarises the contribution of microglia-mediated neuroinflammation in MPTP-induced neurodegeneration. Moreover, we highlight the contribution of neurotrophic factors as endogenous and/or exogenous molecules to slow the progression of midbrain dopaminergic (mDA) neurons and further discuss the potential of combined therapeutic approaches employing neuroinflammation modifying agents and neurotrophic factors.

TGFß1 increases microglia-mediated engulfment of apoptotic cells via upregulation of the milk fat globule-EGF factor 8.

Milk fat globule-epidermal growth factor-factor 8 (Mfge8) has been described as an essential molecule during microglia-mediated clearance of apoptotic cells via binding to phosphatidylserine residues and subsequent phagocytosis. Impaired uptake of apoptotic cells by microglia results in prolonged inflammatory responses and damage of healthy cells. Although the mechanisms of Mfge8-mediated engulfment of apoptotic cells are well understood, endogenous or exogenous factors that regulate Mfge8 expression remain elusive. Here, we describe that TGFß1 increases the expression of Mfge8 and enhances the engulfment of apoptotic cells by primary mouse microglia in a Mfge8-dependent manner. Further, apoptotic cells are capable of increasing microglial TGFß expression and release and shift the microglia phenotype toward alternative activation. Moreover, we provide evidence that Mfge8 expression is differentially regulated in microglia after classical and alternative activation and that Mfge8 is not able to exert direct antiinflammatory effects on LPS-treated primary microglia. Together, these results underline the importance of TGFß1 as a regulatory factor for microglia and suggest that increased TGFß1 expression in models of neurodegeneration might be involved in clearance of apoptotic cells via regulation of Mfge8 expression.

TGFß1 inhibits IFNγ-mediated microglia activation and protects mDA neurons from IFNγ-driven neurotoxicity.

Microglia-mediated neuroinflammation has been reported as a common feature of familial and sporadic forms of Parkinson's disease (PD), and a growing body of evidence indicates that onset and progression of PD correlates with the extent of neuroinflammatory responses involving Interferon γ (IFNγ). Transforming growth factor ß1 (TGFß1) has been shown to be a major player in the regulation of microglia activation states and functions and, thus, might be a potential therapeutic agent by shaping microglial activation phenotypes during the course of neurodegenerative diseases such as PD. In this study, we demonstrate that TGFß1 is able to block IFNγ-induced microglia activation by attenuating STAT1 phosphorylation and IFNγRα expression. Moreover, we identified a set of genes involved in microglial IFNγ signaling transduction that were significantly down-regulated upon TGFß1 treatment, resulting in decreased sensitivity of microglia toward IFNγ stimuli. Interestingly, genes mediating negative regulation of IFNγ signaling, such as SOCS2 and SOCS6, were up-regulated after TGFß1 treatment. Finally, we demonstrate that TGFß1 is capable of protecting midbrain dopaminergic (mDA) neurons from IFNγ-driven neurotoxicity in mixed neuron-glia cultures derived from embryonic day 14 (E14) midbrain tissue. Together, these data underline the importance of TGFß1 as a key immunoregulatory factor for microglia by silencing IFNγ-mediated microglia activation and, thereby, rescuing mDA neurons from IFNγ-induced neurotoxicity. Interferon γ (IFNγ) is a potent pro-inflammatory factor that triggers the activation of microglia and the subsequent release of neurotoxic factors. Transforming growth factor ß1 (TGFß1) is able to inhibit the IFNγ-mediated activation of microglia, which is characterized by the release of nitric oxide (NO) and tumor necrosis factor α (TNFα). By decreasing the expression of IFNγ-induced genes as well as the signaling receptor IFNγR1, TGFß1 reduces the responsiveness of microglia towards IFNγ. In mixed neuron-glia cultures, TGFß1 protects midbrain dopaminergic (mDA) neurons from IFNγ-induced neurotoxicity.

Localization of reelin signaling pathway components in murine midbrain and striatum.

We investigated the distribution patterns of the extracellular matrix protein Reelin and of crucial Reelin signaling components in murine midbrain and striatum. The cellular distribution of the Reelin receptors VLDLr and ApoER2, the intracellular downstream mediator Dab1, and the alternative Reelin receptor APP were analyzed at embryonic day 16, at postnatal stage 15 (P15), and in 3-month-old mice. Reelin was expressed intracellularly and extracellularly in midbrain mesencephalic dopaminergic (mDA) neurons of newborns. In the striatum, Calbindin D-28k(+) neurons exhibited Reelin intracellularly at E16 and extracellularly at P15 and 3 months. ApoER2 and VLDLr were expressed in mDA neurons at E16 and P15 and in oligodendrocytes at 3 months, whereas Dab1 and APP immunoreactivity was observed in mDA at all stages analyzed. In the striatum, Calbindin D-28k(+)/GAD67(+) inhibitory neurons expressed VLDLr, ApoER2, and Dab1 at P15, but only Dab1 at E16 and 3 months. APP was always expressed in mouse striatum in which it colocalized with Calbindin D-28k. Our data underline the importance of Reelin signalling during embryonic development and early postnatal maturation of the mesostriatal and mesocorticolimbic system, and suggest that the striatum and not the midbrain is the primary source of Reelin for midbrain neurons. The loss of ApoER2 and VLDLr expression in the mature midbrain and striatum implies that Reelin functions are restricted to migratory events and early postnatal maturation and are dispensable for the maintenance of dopaminergic neurons.

A Custom Panel for Profiling Microglia Gene Expression.

Despite being immune cells of the central nervous system (CNS), microglia contribute to CNS development, maturation, and homeostasis, and microglia dysfunction has been implicated in several neurological disorders. Recent advancements in single-cell studies have uncovered unique microglia-specific gene expression. However, there is a need for a simple yet elegant multiplexed approach to quantifying microglia gene expression. To address this, we have designed a NanoString nCounter technology-based murine microglia-specific custom codeset comprising 178 genes. We analyzed RNA extracted from ex vivo adult mouse microglia, primary mouse microglia, the BV2 microglia cell line, and mouse bone marrow monocytes using our custom panel. Our findings reveal a pattern where homeostatic genes exhibit heightened expression in adult microglia, followed by primary cells, and are absent in BV2 cells, while reactive markers are elevated in primary microglia and BV2 cells. Analysis of publicly available data sets for the genes present in the panel revealed that the panel could reliably reflect the changes in microglia gene expression in response to various factors. These findings highlight that the microglia panel used offers a swift and cost-effective means to assess microglial cells and can be used to study them in varying contexts, ranging from normal homeostasis to disease models.

Endogenous transforming growth factor-beta promotes quiescence of primary microglia in vitro.

Microglia are the immune cells of the central nervous system (CNS) and play important roles under physiological and pathophysiological conditions. Activation of microglia has been reported for a variety of CNS diseases and is believed to be involved in inflammation-mediated neurodegeneration. Loss of TGFß1 results in increased microgliosis and neurodegeneration in mice which indicates that TGFß1 is an important regulator of microglial functions in vivo. Here, we addressed the role of endogenous TGFß signaling for microglia in vitro. We clearly demonstrate active TGFß signaling in primary microglia and further introduce Klf10 as a new TGFß target gene in microglia. Moreover, we provide evidence that microglia express and release TGFß1 that acts in an autocrine manner to activate microglial TGFß/Smad signaling in vitro. Using microarrays, we identified TGFß-regulated genes in microglia that are involved in TGFß1 processing, its extracellular storage as well as activation of latent TGFß. Finally, we demonstrate that pharmacological inhibition of microglial TGFß signaling resulted in upregulation of the proinflammatory markers IL6 and iNOS and downregulation of the alternative activation markers Arg1 and Ym1 in vitro. Together, these data clearly show that endogenous TGFß1 and autocrine TGFß signaling is important for microglial quiescence in vitro and further suggest the upregulation of TGFß1 in neurodegenerative diseases as a mechanism to regulate microglia functions and silence neuroinflammation.

Distribution of microglia in the postnatal murine nigrostriatal system.

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra (SN) and the subsequent loss of striatal target innervation. Neuroinflammatory responses have been described for virtually all PD cases analysed. Microglia are the resident immune cells of the central nervous system and, thus, are the mediators of neuroinflammation. Approximately 12% of all central nervous system cells are microglia but the distribution and density of microglia differ within distinct brain regions. Interestingly, the SN has been shown to contain more microglia than adjacent structures. We have analysed changes in microglia numbers and in microglial morphology in the postnatal murine nigrostriatal system at various stages ranging from postnatal day 0 (P0) up to 24 months of age. We clearly show that the microglia numbers in the SN and in the striatum dramatically increase from P0 to P15 and significantly decrease in both areas in 18-month-old and 24-month-old animals. Moreover, microglia in the nigrostriatal system of aged mice show signs of dystrophy and degeneration, such as cytoplasmic inclusions, deramification of their processes and membrane blebbing. Our results support the hypothesis of microglial dystrophy during aging in the murine nigrostriatal system, accompanied by subsequent impairment of normal microglial functions. Microglial dysfunction during aging might be a potential risk factor for the development and/or progression of PD.

2,4-Dichlorophenoxyacetic Acid Induces Degeneration of mDA Neurons In Vitro.

Background: Parkinson's disease (PD) affects 1-2% of the population over the age of 60 and the majority of PD cases are sporadic, without any family history of the disease. Neuroinflammation driven by microglia has been shown to promote the progression of midbrain dopaminergic (mDA) neuron loss through the release of neurotoxic factors. Interestingly, the risk of developing PD is significantly higher in distinct occupations, such as farming and agriculture, and is linked to the use of pesticides and herbicides. Methods: The neurotoxic features of 2,4-Dichlorophenoxyacetic acid (2,4D) at concentrations of 10 µM and 1 mM were analyzed in two distinct E14 midbrain neuron culture systems and in primary microglia. Results: The application of 1 mM 2,4D resulted in mDA neuron loss in neuron-enriched cultures. Notably, 2,4D-induced neurotoxicity significantly increased in the presence of microglia in neuron-glia cultures, suggesting that microglia-mediated neurotoxicity could be one mechanism for progressive neuron loss in this in vitro setup. However, 2,4D alone was unable to trigger microglia reactivity. Conclusions: Taken together, we demonstrate that 2,4D is neurotoxic for mDA neurons and that the presence of glia cells enhances 2,4D-induced neuron death. These data support the role of 2,4D as a risk factor for the development and progression of PD and further suggest the involvement of microglia during 2,4D-induced mDA neuron loss.