Jacob A. Sloane

Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis

Toll receptors in Drosophila melanogaster function in morphogenesis and host defense. Mammalian orthologues of Toll, the Toll-like receptors (TLRs), have been studied extensively for their essential functions in controlling innate and adaptive immune responses. We report that TLR8 is dynamically expressed during mouse brain development and localizes to neurons and axons. Agonist stimulation of TLR8 in cultured cortical neurons causes inhibition of neurite outgrowth and induces apoptosis in a dissociable manner. Our evidence indicates that such TLR8-mediated neuronal responses do not involve the canonical TLR–NF-κB signaling pathway. These findings reveal novel functions for TLR8 in the mammalian nervous system that are distinct from the classical role of TLRs in immunity.

Hyaluronan blocks oligodendrocyte progenitor maturation and remyelination through TLR2

Failure of remyelination is largely responsible for sustained neurologic symptoms in multiple sclerosis (MS). MS lesions contain hyaluronan deposits that inhibit oligodendrocyte precursor cell (OPC) maturation. However, the mechanism behind this inhibition is unclear. We report here that Toll-like receptor 2 (TLR2) is expressed by oligodendrocytes and is up-regulated in MS lesions. Pathogen-derived TLR2 agonists, but not agonists for other TLRs, inhibit OPC maturation in vitro. Hyaluronan-mediated inhibition of OPC maturation requires TLR2 and MyD88, a TLR2 adaptor molecule. Ablated expression of TLR2 also enhances remyelination in a lysolecithin animal model. Hyaluronidases expressed by OPCs degrade hyaluronan to hyaluronan oligomers, a requirement for hyaluronan/TLR2 signaling. MS lesions contain both TLR2+ oligodendrocytes and low-molecular-weight hyaluronan, consistent with their importance to remyelination in MS. We thus have defined a mechanism controlling remyelination failure in MS where hyaluronan is degraded by hyaluronidases into hyaluronan oligomers that block OPC maturation and remyelination through TLR2-MyD88 signaling.

Increased microglial activation and protein nitration in white matter of the aging monkey

Activated microglia are important pathological features of a variety of neurological diseases, including the normal aging process of the brain. Here, we quantified the level of microglial activation in the aging rhesus monkey using antibodies to HLA-DR and inducible nitric oxide synthase (iNOS). We observed that 3 out of 5 white matter areas but only 1 of 4 cortical gray matter regions examined showed significant increases in two measures of activated microglia with age, indicating that diffuse white matter microglial activation without significant gray matter involvement occurs with age. Substantial levels of iNOS and 3-nitrotyrosine, a marker for peroxynitrite, increased diffusely throughout subcortical white matter with age, suggesting a potential role of nitric oxide in age-related white matter injury. In addition, we found that the density of activated microglia in the subcortical white matter of the cingulate gyrus and the corpus callosum was significantly elevated with cognitive impairment in elderly monkeys. This study suggests that microglial activation increases in white matter with age and that these increases may reflect the role of activated microglia in the general pathogenesis of normal brain aging.

Toll-like receptor 3 is a potent negative regulator of axonal growth in mammals.

Toll is a cell surface receptor with well described roles in the developmental patterning of invertebrates and innate immunity in adult Drosophila. Mammalian toll-like receptors represent a family of Toll orthologs that function in innate immunity by recognizing molecular motifs unique to pathogens or injured tissue. One member in this family of pattern recognition receptors, toll-like receptor 3 (TLR3), recognizes viral double-stranded RNA and host mRNA. We examined the expression and function of TLRs in the nervous system and found that TLR3 is expressed in the mouse central and peripheral nervous systems and is concentrated in the growth cones of neurons. Activation of TLR3 by the synthetic ligand polyinosine:polycytidylic acid (poly I:C) or by mRNA rapidly causes growth cone collapse and irreversibly inhibits neurite extension independent of nuclear factor κB. Mice lacking functional TLR3 were resistant to the neurodegenerative effects of poly I:C. Neonatal mice injected with poly I:C were found to have fewer axons exiting dorsal root ganglia and displayed related sensorimotor deficits. No effect of poly I:C was observed in mice lacking functional TLR3. Together, these findings provide evidence that an innate immune pattern recognition receptor functions autonomously in neurons to regulate axonal growth and advances a novel hypothesis that this class of receptors may contribute to injury and limited CNS regeneration.

Metalloendopeptidase EC 3.4.24.15 is necessary for Alzheimer's amyloid-beta peptide degradation.

We have investigated the functional relationship between metalloendopeptidase EC 3.4.24.15 (MP24.15) and the amyloid precursor protein involved in Alzheimer’s disease (AD) and discovered that the enzyme promotes Aβ degradation. We show here that conditioned medium (CM) of MP24.15 antisense-transfected SKNMC neuroblastoma has significantly higher levels of Aβ. Furthermore, synthetic-Aβ degradation was increased or decreased following incubation with CM of sense or antisense-transfected cells, respectively. Soluble Aβ1–42 was degraded more slowly than soluble Aβ1–40, while aggregated Aβ1–42 showed almost no degradation. Pretreatment of CM with serine proteinase inhibitors 4-(2-aminoethyl)benzenesulfonyl fluoride and diisopropyl fluorophosphate completely inhibited Aβ degradation. Additionally, α1-antichymotrypsin (ACT), a serpin family inhibitor tightly associated with plaques and elevated in AD brain, blocked up to 60% of Aβ degradation. Interestingly, incubation of CM of MP24.15-overexpressing cells with ACT formed an SDS-resistant ACT complex, suggesting an ACT-serine proteinase interaction. Recombinant MP24.15 alone did not degrade Aβ. 14C-Diisopropyl fluorophosphate-radiolabeled CM from MP24.15-overexpressing cells contained increased levels of several active serine proteinases, suggesting that MP24.15 activates one or more Aβ-degrading serine proteases. Thus, ACT may cause Aβ accumulation by inhibiting an Aβ-degrading enzyme or by direct binding to Aβ, rendering it degradation-resistant. Identification of the Aβ-degrading enzyme and MP24.15’s role in its activation is underway. Pharmacological modulation of either enzyme may provide a means of regulating Aβ in the brain.

The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

We have previously shown that myelin abnormalities characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. Although the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. We found significant effects of Klotho on oligodendrocyte functions, including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein analysis indicated that Klothos downstream effects involve Akt and ERK signal pathways. Klotho increased OPC maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knock-out mice and control littermates revealed that knock-out mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knock-out mice. Strikingly, at the ultrastructural level, Klotho knock-out mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. To decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes and promote repair in multiple sclerosis.

Age-dependent myelin degeneration and proteolysis of oligodendrocyte proteins is associated with the activation of calpain-1 in the rhesus monkey.

Myelin provides important insulating properties to axons allowing for propagation of action potentials over large distances at high velocity. Disruption of the myelin sheath could therefore contribute to cognitive impairment, such as that observed during the normal aging process. In the present study, age‐related changes in myelin, myelin proteins and oligodendrocyte proteins were assessed in relationship to calpain‐1 expression and cognition in the rhesus monkey. Isolation of myelin fractions from brain white matter revealed that as the content of the intact myelin fraction decreased with age, there was a corresponding increase in the floating or degraded myelin fraction, suggesting an increased breakdown of intact myelin with age. Of the myelin proteins examined, only the myelin‐associated glycoprotein decreased with age. Levels of the oligodendrocyte‐specific proteins 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) and myelin/oligodendrocyte‐specific protein (MOSP) increased dramatically in white matter homogenates and myelin with age. Age‐related increases in degraded CNPase also were demonstrable in white matter in association with increases in activated calpain‐1. Degraded CNPase was also detectable in myelin fractions, with only the floating fraction containing activated calpain‐1. The increases in the activated enzyme in white matter were much greater than those found in myelin fractions suggesting a source other than the myelin membrane for the marked overexpression of activated calpain‐1 with age. In addition, CNPase was demonstrated to be a substrate for calpain in vitro. In summary, changes in myelin and oligodendrocyte proteins occur with age, and they appear to have a significant relationship to cognitive impairment. The overexpression of CNPase and MOSP suggests new formation of myelin by oligodendrocytes, which may occur in response to myelin degradation and injury caused by proteolytic enzymes such as calpain.

Astrocytic hypertrophy and altered GFAP degradation with age in subcortical white matter of the rhesus monkey

Reactive astrocytosis is a well known phenomenon that occurs in the normal aging process of the brain. While many studies indicate astrocytic hypertrophy and glial fibrillary acidic protein (GFAP) content increase with age in the hippocampal formation of certain animal models, it is unclear whether these findings are generalizable to the primate and to other areas of the brain. In this study, we quantitatively assessed age-related changes in astrocytic cell size and density in a rhesus monkey model of normal aging. By GFAP immunohistochemistry, we observed an increase in GFAP(+) cell size but not density in all subcortical white matter areas of the frontal, temporal, and parietal cortices. No significant increases in astrocyte hypertrophy were observed in any gray matter area examined. In addition, Western blotting experiments showed increases in total and degraded GFAP content with age, suggesting altered degradation and possibly production of GFAP occur with age.

Lack of correlation between plaque burden and cognition in the aged monkey

Abstract To assess whether amyloid plaque accumulation in the monkey brain can account for age-related cognitive impairment that begins at about 20 years of age, we measured plaque content in the brains of 14 rhesus monkeys aged 5–30 years. We used immunohistochemistry employing the monoclonal antibody 6E10, which is specific to amino acids 1–17 of the amyloid β peptide to identify amyloid plaques in serial coronal sections of the forebrain. Amyloid plaques accumulate with age, starting at 25 years of age and escalating after 30 years. Until the age of 30, plaques are only found in a few monkeys and are relatively sparse. Results from our group and others show that plaque content and the proportion of individuals afflicted with amyloid plaques increase with age. Although both cognitive dysfunction and plaque content increase with age, amyloid plaque content does not correlate with the cognitive dysfunction observed in elderly monkeys since even in very old subjects some cognitively impaired animals have few amyloid plaques and others with abundant plaques show only minor cognitive impairments. In summary, amyloid plaques appear to accumulate significantly only in monkeys over 25 years of age but do not appear to be a causal factor in age-related cognitive decline of the normal aging rhesus monkey.

A Clear and Present Danger: Endogenous Ligands of Toll-like Receptors

Neurologic disease promoted by microbial pathogens, sterile injury, or neurodegeneration rapidly induces innate immunity in adjacent healthy tissue, which in turn contributes extensively to neurologic injury. With more recent focus on innate immune processes, it appears that necrotic, but not apoptotic, death mechanisms provoke inflammatory responses likely due to the release or production of endogenous ligands that activate resident immune cells of the central nervous system. These ligands comprise a diverse set of proteins, nucleic acids, and glycosaminoglycans, including heat shock proteins, HMGB1, RNA, DNA, hyaluronan, and heparin sulfate, that stimulate innate immune mechanisms largely through Toll-like receptors (TLRs). The blockade of interactions between endogenous ligands and TLRs may enable neuroprotective therapeutic strategies for a variety of neurologic diseases.