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Familial hemiplegic migraine type 3 (FHM3) is a severe autosomal dominant migraine disorder caused by mutations in the voltage-gated sodium channel Nav1.1 encoded by SCN1A. We determined the functional consequences of three mutations linked to FHM3 (L263V, Q1489K, and L1649Q) in an effort to identify molecular defects that underlie this inherited migraine disorder. Only L263V and Q1489K generated quantifiable sodium currents when coexpressed in tsA201 cells with the human [beta]1 and [beta]2 accessory subunits. The third mutant, L1649Q, failed to generate measurable whole-cell current because of markedly reduced cell surface expression. Compared to WT-[Na.sub.v]1.1, Q1489K exhibited increased persistent current but also enhanced entry into slow inactivation as well as delayed recovery from fast and slow inactivation, thus resulting in a predominantly loss-of-function phenotype further demonstrated by a greater Ioss of channel availability during repetitive stimulation. In contrast, L263V exhibited gain-of-function features, including delayed entry into, as well as accelerated recovery from, fast inactivation; depolarizing shifts in the steady-state voltage dependence of fast and slow inactivation; increased persistent current; and delayed entry into slow inactivation. Notably, the two mutations (Q1489K and L1649Q) that exhibited partial or complete Ioss of function are linked to typical FHM, whereas the gain-of-function mutation L263V occurred in a family having both FHM and a high incidence of generalized epilepsy. We infer from these data that a complex spectrum of [Na.sub.v]1.1 defects can cause FHM3. Our results also emphasize the complex relationship between migraine and epilepsy and provide further evidence that both disorders may share common molecular mechanisms. epilepsy | SCN1A | Nav1.1 | FHM3

Aldolase C is regarded as the brain-specific form of fructose-1,6-bisphosphate aldolase whereas aldolase A is regarded as muscle-specific. In situ hybridization of mouse central nervous system using isozyme-specific probes revealed that aldolase A and C are expressed in complementary cell types. With the exception of cerebellar Purkinje cells, aldolase A mRNA is found in neurons; aldolase C message is detected in astrocytes, some cells of the pia mater, and Purkinje cells. We isolated aldolase C genomic clones that span the entire protein coding region from 1.5 kb 5[prime] to the transcription start site to 0.5 kb 3[prime] to the end of the last exon. The bacterial gene, lacZ, was inserted in two different locations and the constructs tested in transgenic mice. When the protein coding sequences were replaced with lacZ, three of five transgenic lines expressed [Beta]-galactosidase only in cells of the pia mater; one line also expressed in astrocyte-like cells. When lacZ was inserted into the final exon (and all structural gene sequences were retained) transgene expression was observed in astrocytes in all regions of the central nervous system as well as in pial cells. Thus, with the exception of Purkinje cell expression, the behavior of the full-length transgene mimics the endogenous aldolase C gene. The results with the shorter transgene suggest that additional enhancer elements exist within the intragenic sequences. The absence of Purkinje cell staining suggests that the cis elements required for this expression must be located outside of the sequences used in this study.

Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8) moieties from cullins (thus termed “deNEDDylase”) and a subunit of the cullin-RING E3 ligase-regulating COP9 signalosome complex, attenuates proinflammatory NF-κB signaling. We previously showed that CSN5 is up-regulated in human atherosclerotic arteries. Here, we investigated the role of CSN5 in atherogenesis in vivo by using mice with myeloid-specific Csn5 deletion. Genetic deletion of Csn5 in Apoe−/− mice markedly exacerbated atherosclerotic lesion formation. This was broadly observed in aortic root, arch, and total aorta of male mice, whereas the effect was less pronounced and site-specific in females. Mechanistically, Csn5 KO potentiated NF-κB signaling and proinflammatory cytokine expression in macrophages, whereas HIF-1α levels were reduced. Inversely, inhibition of NEDDylation by MLN4924 blocked proinflammatory gene expression and NF-κB activation while enhancing HIF-1α levels and the expression of M2 marker Arginase 1 in inflammatory-elicited macrophages. MLN4924 further attenuated the expression of chemokines and adhesion molecules in endothelial cells and reduced NF-κB activation and monocyte arrest on activated endothelium in vitro. In vivo, MLN4924 reduced LPS-induced inflammation, favored an antiinflammatory macrophage phenotype, and decreased the progression of early atherosclerotic lesions in mice. On the contrary, MLN4924 treatment increased neutrophil and monocyte counts in blood and had no net effect on the progression of more advanced lesions. Our data show that CSN5 is atheroprotective. We conclude that MLN4924 may be useful in preventing early atherogenesis, whereas selectively promoting CSN5-mediated deNEDDylation may be beneficial in all stages of atherosclerosis. [ABSTRACT FROM AUTHOR]

High temperature requirement protein A1 (HtrA1) is a primarily secreted serine protease involved in a variety of cellular processes including transforming growth factor β (TGF-β) signaling. Loss of its activity causes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an inherited form of cerebral small vessel disease leading to early-onset stroke and premature dementia. Dysregulated TGF-β signaling is considered to promote CARASIL pathogenesis, but the underlying molecular mechanisms are incompletely understood. Here we present evidence from mouse brain tissue and embryonic fibroblasts as well as patient skin fibroblasts for a facilitating role of HtrA1 in TGF-β pathway activation. We identify latent TGF-β binding protein 1 (LTBP-1), an extracellular matrix protein and key regulator of TGF-β bioavailability, as a novel HtrA1 target. Cleavage occurs at physiological protease concentrations, is prevented under HtrA1-deficient conditions as well as by CARASIL mutations and disrupts both LTBP-1 binding to fibronectin and its incorporation into the extracellular matrix. Hence, our data suggest an attenuation of TGF-β signaling caused by a lack of HtrA1-mediated LTBP-1 processing as mechanism underlying CARASIL pathogenesis. [ABSTRACT FROM AUTHOR]

A response from the authors of the article "Cerebral small vessel disease-related protease HtrA1 processes latent TGF-β binding protein 1 and facilitates TGF-β signaling" is presented.

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson's disease (PD) and Alzheimer's disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1 *04 subtypes best accounted for the association, strongest with HLA-DRB1 *04:04 and HLA-DRB1 *04:07, and intermediary with HLA-DRB1 *04:01 and HLA-DRB1 *04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1 *04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1 *04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues.

Large artery atherosclerotic stroke (LAS) shows substantial heritability not explained by previous genome-wide association studies. Here, we explore the role of coding variation in LAS by analyzing variants on the HumanExome BeadChip in a total of 3,127 cases and 9,778 controls from Europe, Australia, and South Asia. We report on a nonsynonymous single-nucleotide variant in serpin family A member 1 ( SERPINA1 ) encoding alpha-1 antitrypsin [AAT; p.V213A; P = 5.99E-9, odds ratio (OR) = 1.22] and confirm histone deacetylase 9 ( HDAC9 ) as a major risk gene for LAS with an association in the 3'-UTR (rs2023938; P = 7.76E-7, OR = 1.28). Using quantitative microscale thermophoresis, we show that M1 (A213) exhibits an almost twofold lower dissociation constant with its primary target human neutrophil elastase (NE) in lipoprotein-containing plasma, but not in lipid-free plasma. Hydrogen/deuterium exchange combined with mass spectrometry further revealed a significant difference in the global flexibility of the two variants. The observed stronger interaction with lipoproteins in plasma and reduced global flexibility of the Val-213 variant most likely improve its local availability and reduce the extent of proteolytic inactivation by other proteases in atherosclerotic plaques. Our results indicate that the interplay between AAT, NE, and lipoprotein particles is modulated by the gate region around position 213 in AAT, far away from the unaltered reactive center loop (357-360). Collectively, our findings point to a functionally relevant balance between lipoproteins, proteases, and AAT in atherosclerosis.