The Interplay Between Brain Vascular Calcification and Microglia

Vascular calcifications are characterized by the ectopic deposition of calcium and phosphate in the vascular lumen or wall. They are a common finding in computed tomography scans or during autopsy and are often directly related to a pathological condition. While the pathogenesis and functional consequences of vascular calcifications have been intensively studied in some peripheral organs, vascular calcification, and its pathogenesis in the central nervous system is poorly characterized and understood. Here, we review the occurrence of vessel calcifications in the brain in the context of aging and various brain diseases. We discuss the pathomechanism of brain vascular calcification in primary familial brain calcification as an example of brain vessel calcification. A particular focus is the response of microglia to the vessel calcification in the brain and their role in the clearance of calcifications

Aquaporin 4 is differentially increased and dislocated in association with tau and amyloid-beta

AIMS Neurovascular-glymphatic dysfunction plays an important role in Alzheimer's disease and has been analysed mainly in relation to amyloid-beta (Aβ) pathology. Here, we aim to investigate the neurovascular alterations and mapping of aquaporin 4 (AQP4) distribution and dislocation associated with tau and Aβ. MATERIALS AND METHODS Perfusion, susceptibility weighted imaging and structural magnetic resonance imaging (MRI) were performed in the pR5 mouse model of 4-repeat tau and the arcAβ mouse model of amyloidosis. Immunofluorescence staining was performed using antibodies against AQP4, vessel, astroglia, microglia, phospho-tau and Aβ in brain tissue slices from pR5, arcAβ and non-transgenic mice. KEY FINDINGS pR5 mice showed regional atrophy, preserved cerebral blood flow, and reduced cerebral vessel density compared to non-transgenic mice, while arcAβ mice showed cerebral microbleeds and reduced cerebral vessel density. AQP4 dislocation and peri-tau enrichment in the hippocampus and increased AQP4 levels in the cortex and hippocampus were detected in pR5 mice compared to non-transgenic mice. In comparison, cortical AQP4 dislocation and cortical/hippocampal peri-plaque increases were observed in arcAβ mice. Increased expression of reactive astrocytes were detected around the tau inclusions in pR5 mice and Aβ plaques in arcAβ mice. SIGNIFICANCE We demonstrated the neurovascular alterations, microgliosis, astrogliosis and increased AQP4 regional expression in pR5 tau and arcAβ mice. We observed a divergent region-specific AQP4 dislocation and association with phospho-tau and Aβ pathologies

Postmitotic Hoxa5 Expression Specifies Pontine Neuron Positional Identity and Input Connectivity of Cortical Afferent Subsets

The mammalian precerebellar pontine nucleus (PN) has a main role in relaying cortical information to the cerebellum. The molecular determinants establishing ordered connectivity patterns between cortical afferents and precerebellar neurons are largely unknown. We show that expression of Hox5 transcription factors is induced in specific subsets of postmitotic PN neurons at migration onset. Hox5 induction is achieved by response to retinoic acid signaling, resulting in Jmjd3-dependent derepression of Polycomb chromatin and 3D conformational changes. Hoxa5 drives neurons to settle posteriorly in the PN, where they are monosynaptically targeted by cortical neuron subsets mainly carrying limb somatosensation. Furthermore, Hoxa5 postmigratory ectopic expression in PN neurons is sufficient to attract cortical somatosensory inputs regardless of position and avoid visual afferents. Transcriptome analysis further suggests that Hoxa5 is involved in circuit formation. Thus, Hoxa5 coordinates postmitotic specification, migration, settling position, and subcircuit assembly of PN neuron subsets in the cortico-cerebellar pathway.Peer reviewe

Inorganic phosphate exporter heterozygosity in mice leads to brain vascular calcification, microangiopathy, and microgliosis

Calcification of the cerebral microvessels in the basal ganglia in the absence of systemic calcium and phosphate imbalance is a hallmark of primary familial brain calcification (PFBC), a rare neurodegenerative disorder. Mutation in genes encoding for sodium-dependent phosphate transporter 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), platelet-derived growth factor B (PDGFB), platelet-derived growth factor receptor beta (PDGFRB), myogenesis regulating glycosidase (MYORG), and junctional adhesion molecule 2 (JAM2) are known to cause PFBC. Loss-of-function mutations in XPR1, the only known inorganic phosphate exporter in metazoans, causing dominantly inherited PFBC was first reported in 2015 but until now no studies in the brain have addressed whether loss of one functional allele leads to pathological alterations in mice, a commonly used organism to model human diseases. Here we show that mice heterozygous for Xpr1 (Xpr1$^{WT/lacZ}$ ) present with reduced inorganic phosphate levels in the cerebrospinal fluid and age- and sex-dependent growth of vascular calcifications in the thalamus. Vascular calcifications are surrounded by vascular basement membrane and are located at arterioles in the smooth muscle layer. Similar to previously characterized PFBC mouse models, vascular calcifications in Xpr1$^{WT/lacZ}$ mice contain bone matrix proteins and are surrounded by reactive astrocytes and microglia. However, microglial activation is not confined to calcified vessels but shows a widespread presence. In addition to vascular calcifications, we observed vessel tortuosity and transmission electron microscopy analysis revealed microangiopathy-endothelial swelling, phenotypic alterations in vascular smooth muscle cells, and thickening of the basement membrane

Pericytes regulate vascular immune homeostasis in the CNS.

Pericytes regulate the development of organ-specific characteristics of the brain vasculature such as the blood-brain barrier (BBB) and astrocytic end-feet. Whether pericytes are involved in the control of leukocyte trafficking in the adult central nervous system (CNS), a process tightly regulated by CNS vasculature, remains elusive. Using adult pericyte-deficient mice (Pdgfb ret/ret ), we show that pericytes limit leukocyte infiltration into the CNS during homeostasis and autoimmune neuroinflammation. The permissiveness of the vasculature toward leukocyte trafficking in Pdgfb ret/ret mice inversely correlates with vessel pericyte coverage. Upon induction of experimental autoimmune encephalomyelitis (EAE), pericyte-deficient mice die of severe atypical EAE, which can be reversed with fingolimod, indicating that the mortality is due to the massive influx of immune cells into the brain. Additionally, administration of anti-VCAM-1 and anti-ICAM-1 antibodies reduces leukocyte infiltration and diminishes the severity of atypical EAE symptoms of Pdgfb ret/ret mice, indicating that the proinflammatory endothelium due to absence of pericytes facilitates exaggerated neuroinflammation. Furthermore, we show that the presence of myelin peptide-specific peripheral T cells in Pdgfb ret/ret ;2D2 tg mice leads to the development of spontaneous neurological symptoms paralleled by the massive influx of leukocytes into the brain. These findings indicate that intrinsic changes within brain vasculature can promote the development of a neuroinflammatory disorder

Inorganic phosphate exporter heterozygosity in mice leads to brain vascular calcification, microangiopathy, and microgliosis

Calcification of the cerebral microvessels in the basal ganglia in the absence of systemic calcium and phosphate imbalance is a hallmark of primary familial brain calcification (PFBC), a rare neurodegenerative disorder. Mutation in genes encoding for sodium-dependent phosphate transporter 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), platelet-derived growth factor B (PDGFB), platelet-derived growth factor receptor beta (PDGFRB), myogenesis regulating glycosidase (MYORG), and junctional adhesion molecule 2 (JAM2) are known to cause PFBC. Loss-of-function mutations in XPR1, the only known inorganic phosphate exporter in metazoans, causing dominantly inherited PFBC was first reported in 2015 but until now no studies in the brain have addressed whether loss of one functional allele leads to pathological alterations in mice, a commonly used organism to model human diseases. Here we show that mice heterozygous for Xpr1 (Xpr1WT/lacZ) present with reduced inorganic phosphate levels in the cerebrospinal fluid and age- and sex-dependent growth of vascular calcifications in the thalamus. Vascular calcifications are surrounded by vascular basement membrane and are located at arterioles in the smooth muscle layer. Similar to previously characterized PFBC mouse models, vascular calcifications in Xpr1WT/lacZ mice contain bone matrix proteins and are surrounded by reactive astrocytes and microglia. However, microglial activation is not confined to calcified vessels but shows a widespread presence. In addition to vascular calcifications, we observed vessel tortuosity and transmission electron microscopy analysis revealed microangiopathy—endothelial swelling, phenotypic alterations in vascular smooth muscle cells, and thickening of the basement membrane

Role of Hox Genes in Sub-circuit Diversification During Cortico-Ponto-Cerebellar Map Formation

Cortical input is relayed to the cerebellum mainly via precerebellar pontine nuclei. The pontine nuclei (PN), which include pontine gray and reticulotegmental nuclei (RTN), are largest of the precerebellar nuclei providing principal input to the cerebellum. PN are hypothesized to serve as an integrator of cortical information before sending these signals to the cerebellum (Schwarz and Their, 1999). The pathway from the cerebral cortex to pontine to the cerebellum is crucial for cerebellar function and learning. It is due to their critical role in the cortico-cerebellar pathway, the pontine nuclei have received much attention. Even though the projection of cortical afferents in the PN is well established (reviewed in Kratochwil et al., 2017), the molecular mechanisms underlying this complex circuitry are poorly understood. During my Ph.D., I studied the role of Hoxa5 transcription factor in the development of pontine neurons and in formation of their input-output projections. Cortical afferents, arising in layer V of the cerebral cortex, are mapped onto the pontine nuclei in a topographic manner (Leergaard and Bjaalie, 2007). It has been proposed that cortical afferents in the PN are organized in an inside-out fashion, matching the birthdate of PN neurons (Altman and Bayer, 1987, 1997). Earliest arriving cortico-pontine fibers grow in the core of PN where the earliest born PN neurons have settled (reviewed in Kratochwil et al., 2017). A rostro-caudal organization of cortico-pontine afferents is also suggested such that afferents arising in the visual cortical area project to the anterior, while afferents arising in the somatosensory areas are mapped to the posterior pontine nuclei (Leergaard and Bjaalie, 2007). A previous study from our lab has shown that the PN neurons born from the lower rhombic lip (lRL) of rhombomere 6 (r6) settle in anterior PN while neurons born from rhombomere 8 settle in posterior PN (Di Meglio et al., 2013). As a result, PN neurons can be sub-divided in clusters based on their Hox expression pattern. Anterior PN neurons express Hox2-3 genes while Hox5 genes are expressed only in posterior PN neurons. This suggests presence of an intrinsic topographic organization in the PN based on the rostro-caudal origin or Hox expression of PN neurons. Hox genes are known to influence the topographic organization as well as input-output connectivity of several nuclei in the hindbrain and spinal cord (Bechara et al., 2016; Karmakar et al., 2017; Philippidou and Dasen, 2013). In my thesis, I therefore focused on investigating the role of Hox5 genes, expressed in posterior PN neurons, in the formation of topographic cortico-pontine circuits. We have identified role of Hoxa5 gene in defining the position of PN neurons and orchestrating somatosensory specific input connectivity of the PN neurons. Using mouse genetics and in-utero electroporation as a tool for embryonic gene manipulation, we show that Hoxa5 overexpression leads to change in position of PN neuron towards posterior PN. This is a result of downregulation of Unc5B, a repulsive cue to Netrin, upon ectopic expression of Hoxa5 in migrating PN neurons. The positioning of PN neurons toward posterior PN enables them to receive somatosensory specific cortical input. By using trans-synaptic rabies virus tracing technique, we could also show that Hoxa5 enables PN neuron to receive or attract somatosensory input and avoid visual input from the cerebral cortex irrespective of the PN neuron position. In this thesis, I have also investigated the molecular basis of PN connectivity with the cerebellum. While cortical inputs are mapped onto PN in a topographic manner, the projections from pontine to the cerebellum are present in a fractured map (Leergaard et al., 2006). How continuous cortical maps are transformed to fractured maps in the cerebellum remains unanswered. We hypothesize that the combinatorial expression of Hox genes in PN neurons underlies their ability to project to different parts of the cerebellum. A PN neuron is more likely to express a combination of several Hox genes as we move from the rostral to the caudal part of the PN. To address this question, we used mouse genetics to identify neurons in anterior PN neuron subsets and found that these neurons primarily project to the paraflocculus, a lobule known for its role in the visual system (reviewed in Kheradmand and Zee, 2011), while Hoxa5 positive posterior PN neurons project to several lobules of cerebellum concerned with processing of somatosensory information (Leergaard et al., 2006). Thus, the output connectivity of PN neurons also matches their input connectivity. However, we still do not understand the role of individual Hox genes in shaping the ponto-cerebellar projections. The findings presented in this thesis will serve as a basis to understand involvement of Hox genes in fracturing of the information between cortex and the cerebellum. As a whole, this thesis highlights the role of Hoxa5 gene in orchestrating the topographic input connectivity of pontine nuclei by defining the position of pontine neurons as well as by providing cues to somatosensory cortical afferents for targeting the PN. It also provides basis to understand the role of Hox genes in ponto-cerebellar connectivity

The Long Journey of Pontine Nuclei Neurons: From Rhombic Lip to Cortico-Ponto-Cerebellar Circuitry

The pontine nuclei (PN) are the largest of the precerebellar nuclei, neuronal assemblies in the hindbrain providing principal input to the cerebellum. The PN are predominantly innervated by the cerebral cortex and project as mossy fibers to the cerebellar hemispheres. Here, we comprehensively review the development of the PN from specification to migration, nucleogenesis and circuit formation. PN neurons originate at the posterior rhombic lip and migrate tangentially crossing several rhombomere derived territories to reach their final position in ventral part of the pons. The developing PN provide a classical example of tangential neuronal migration and a study system for understanding its molecular underpinnings. We anticipate that understanding the mechanisms of PN migration and assembly will also permit a deeper understanding of the molecular and cellular basis of cortico-cerebellar circuit formation and function

The Long Journey of Pontine Nuclei Neurons: From Rhombic Lip to Cortico-Ponto-Cerebellar Circuitry

The pontine nuclei (PN) are the largest of the precerebellar nuclei, neuronal assemblies in the hindbrain providing principal input to the cerebellum. The PN are predominantly innervated by the cerebral cortex and project as mossy fibers to the cerebellar hemispheres. Here, we comprehensively review the development of the PN from specification to migration, nucleogenesis and circuit formation. PN neurons originate at the posterior rhombic lip and migrate tangentially crossing several rhombomere derived territories to reach their final position in ventral part of the pons. The developing PN provide a classical example of tangential neuronal migration and a study system for understanding its molecular underpinnings. We anticipate that understanding the mechanisms of PN migration and assembly will also permit a deeper understanding of the molecular and cellular basis of cortico-cerebellar circuit formation and function.publishe