Kurt R. Brunden

ApoE-Directed Therapeutics Rapidly Clear β-Amyloid and Reverse Deficits in AD Mouse Models

Reversing Decline? Apolipoprotein E (apoE) normally helps in the clearance of β-amyloid from the brain, a process that is compromised in Alzheimers disease. Cramer et al. (p. 1503, published online 9 February; see the Perspective by Strittmatter) now show that a drug that increases apoE expression rapidly promoted soluble β-amyloid clearance in a mouse model of Alzheimers disease. The drug also improved cognitive, social, and olfactory performance and rapidly improved neural circuit function. Similar therapeutics may potentially help to ameliorate the symptoms of Alzheimers disease and its prodromal states. Bexarotene counters the effects of neurodegenerative disease in mice. Alzheimer’s disease (AD) is associated with impaired clearance of β-amyloid (Aβ) from the brain, a process normally facilitated by apolipoprotein E (apoE). ApoE expression is transcriptionally induced through the action of the nuclear receptors peroxisome proliferator–activated receptor gamma and liver X receptors in coordination with retinoid X receptors (RXRs). Oral administration of the RXR agonist bexarotene to a mouse model of AD resulted in enhanced clearance of soluble Aβ within hours in an apoE-dependent manner. Aβ plaque area was reduced more than 50% within just 72 hours. Furthermore, bexarotene stimulated the rapid reversal of cognitive, social, and olfactory deficits and improved neural circuit function. Thus, RXR activation stimulates physiological Aβ clearance mechanisms, resulting in the rapid reversal of a broad range of Aβ-induced deficits.

Exogenous α-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells

Cytoplasmic inclusions containing α-synuclein (α-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathological hallmarks of Parkinsons disease (PD). Although α-Syn fibrils can be generated from recombinant α-Syn protein in vitro, the production of fibrillar α-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular α-Syn aggregation can be triggered by the introduction of exogenously produced recombinant α-Syn fibrils into cultured cells engineered to overexpress α-Syn. Unlike unassembled α-Syn, these α-Syn fibrils “seeded” recruitment of endogenous soluble α-Syn protein and their conversion into insoluble, hyperphosphorylated, and ubiquitinated pathological species. Thus, this cell model recapitulates key features of LBs in human PD brains. Also, these findings support the concept that intracellular α-Syn aggregation is normally limited by the number of active nucleation sites present in the cytoplasm and that small quantities of α-Syn fibrils can alter this balance by acting as seeds for aggregation.

Advances in tau-focused drug discovery for Alzheimer's disease and related tauopathies

Neuronal inclusions comprised of the microtubule-associated protein tau are found in numerous neurodegenerative diseases, commonly known as tauopathies. In Alzheimers disease — the most prevalent tauopathy — misfolded tau is probably a key pathological agent. The recent failure of amyloid-β-targeted therapeutics in Phase III clinical trials suggests that it is timely and prudent to consider alternative drug discovery strategies for Alzheimers disease. Here, we focus on strategies directed at reducing misfolded tau and compensating for the loss of normal tau function.

Evidence for glial-mediated inflammation in aged APPSW transgenic mice

Chronic expression of inflammatory cytokines, including interleukin-1beta, tumor necrosis factor alpha, and interleukin-6, by glia may underlie the neurodegenerative events that occur within the brains of patients with Alzheimers disease (AD). The present study determined whether these markers of inflammation could be observed within the brains of Tg(HuAPP695.K670N/M671L)2576 transgenic mice (Tg2576) that have recently been shown to mimic many features of AD. Interleukin-1beta- and tumor necrosis factor alpha-immunopositive microglia were localized with thioflavine-positive (fibrillar) Abeta deposits. Moreover, interleukin-6 immunoreactive astrocytes surrounded fibrillar Abeta deposits. These findings provide evidence that Tg2576 mice exhibit features of the inflammatory pathology seen in AD and suggest that these mice are a useful animal model for studying the role inflammation may play in this disease.

Identification of a Novel Extracellular Cation-sensing G-protein-coupled Receptor

The C family G-protein-coupled receptors contain members that sense amino acid and extracellular cations, of which calcium-sensing receptor (CASR) is the prototypic extracellular calcium-sensing receptor. Some cells, such as osteoblasts in bone, retain responsiveness to extracellular calcium in CASR-deficient mice, consistent with the existence of another calcium-sensing receptor. We examined the calcium-sensing properties of GPRC6A, a newly identified member of this family. Alignment of GPRC6A with CASR revealed conservation of both calcium and calcimimetic binding sites. In addition, calcium, magnesium, strontium, aluminum, gadolinium, and the calcimimetic NPS 568 resulted in a dose-dependent stimulation of GPRC6A overexpressed in human embryonic kidney cells 293 cells. Also, osteocalcin, a calcium-binding protein highly expressed in bone, dose-dependently stimulated GPRC6A activity in the presence of calcium but inhibited the calcium-dependent activation of CASR. Coexpression of β-arrestins 1 and 2, regulators of G-protein signaling RGS2 or RGS4, the RhoA inhibitor C3 toxin, the dominant negative Gαq-(305-359) minigene, and pretreatment with pertussis toxin inhibited activation of GPRC6A by extracellular cations. Reverse transcription-PCR analyses showed that mouse GPRC6A is widely expressed in mouse tissues, including bone, calvaria, and the osteoblastic cell line MC3T3-E1. These data suggest that in addition to sensing amino acids, GPRC6A is a cation-, calcimimetic-, and osteocalcin-sensing receptor and a candidate for mediating extracellular calcium-sensing responses in osteoblasts and possibly other tissues.

The Microtubule-Stabilizing Agent, Epothilone D, Reduces Axonal Dysfunction, Neurotoxicity, Cognitive Deficits, and Alzheimer-Like Pathology in an Interventional Study with Aged Tau Transgenic Mice

Neurodegenerative tauopathies, such as Alzheimers disease (AD), are characterized by insoluble deposits of hyperphosphorylated tau protein within brain neurons. Increased phosphorylation and decreased solubility has been proposed to diminish normal tau stabilization of microtubules (MTs), thereby leading to neuronal dysfunction. Earlier studies have provided evidence that small molecule MT-stabilizing drugs that are used in the treatment of cancer may have utility in the treatment of tauopathies. However, it has not been established whether treatment with a small molecule MT-stabilizing compound will provide benefit in a transgenic model with pre-existing tau pathology, as would be seen in human patients with clinical symptoms. Accordingly, we describe here an interventional study of the brain-penetrant MT-stabilizing agent, epothilone D (EpoD), in aged PS19 mice with existing tau pathology and related behavioral deficits. EpoD treatment reduced axonal dystrophy and increased axonal MT density in the aged PS19 mice, which led to improved fast axonal transport and cognitive performance. Moreover, the EpoD-treated PS19 mice had less forebrain tau pathology and increased hippocampal neuronal integrity, with no dose-limiting side effects. These data reveal that brain-penetrant MT-stabilizing drugs hold promise for the treatment of AD and related tauopathies, and that EpoD could be a candidate for clinical testing.

Epothilone D Improves Microtubule Density, Axonal Integrity and Cognition in a Transgenic Mouse Model of Tauopathy

Neurons in the brains of those with Alzheimers disease (AD) and many frontotemporal dementias (FTDs) contain neurofibrillary tangles comprised of hyperphosphorylated tau protein. Tau normally stabilizes microtubules (MTs), and tau misfolding could lead to a loss of this function with consequent MT destabilization and neuronal dysfunction. Accordingly, a possible therapeutic strategy for AD and related “tauopathies” is treatment with a MT-stabilizing anti-cancer drug such as paclitaxel. However, paclitaxel and related taxanes have poor blood–brain barrier permeability and thus are unsuitable for diseases of the brain. We demonstrate here that the MT-stabilizing agent, epothilone D (EpoD), is brain-penetrant and we subsequently evaluated whether EpoD can compensate for tau loss-of-function in PS19 tau transgenic mice that develop forebrain tau inclusions, axonal degeneration and MT deficits. Treatment of 3-month-old male PS19 mice with low doses of EpoD once weekly for a 3 month period significantly improved CNS MT density and axonal integrity without inducing notable side-effects. Moreover, EpoD treatment reduced cognitive deficits that were observed in the PS19 mice. These results suggest that certain brain-penetrant MT-stabilizing agents might provide a viable therapeutic strategy for the treatment of AD and FTDs.

Amyloid β protein (Aβ) removal by neuroglial cells in culture

Abstract Because the mechanisms of Aβ degradation in normal and Alzheimers disease brain are poorly understood, we have examined whether various cortical cells are capable of processing this peptide. Rat microglia and astrocytes, as well as the human THP-1 monocyte cell line, degraded A β 1−42 added to culture medium. In contrast, neither rat cortical neurons or meningeal fibroblasts effectively catabolized this peptide. When Aβ fibrils were immobilized as plaque-like deposits on culture dishes, both microglia and THP-1 cells removed the peptide. Astrocytes were incapable of processing the Aβ deposits, but these cells released glycosaminoglycase-sensitive molecules that inhibited the subsequent removal of Aβ by microglia. This implied that astrocyte-derived proteoglycans associated with the amyloid peptide and slowed its degradation. The addition of purified proteoglycan to Aβ that was in medium or focally deposited also resulted in significant inhibition of peptide removal by microglia. These data suggest that Aβ can be catabolized by microglia and proteoglycans which co-localize with senile plaques may slow the degradation of Aβ within these pathologic bodies.

GPRC6A Null Mice Exhibit Osteopenia, Feminization and Metabolic Syndrome

Background GPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown. Methods/Principal Findings In this study, we created and characterized the phenotype of GPRC6A −/− mice. We observed complex metabolic abnormalities in GPRC6A −/− mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A −/− mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A −/− mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A−/− mice exhibited increments in urine Ca/Cr and PO4/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A −/− mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone. Conclusions/Significance GPRC6A−/− mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.

Proteoglycan-mediated Inhibition of Aβ Proteolysis A POTENTIAL CAUSE OF SENILE PLAQUE ACCUMULATION

Senile plaques of Alzheimers disease brain contain, in addition to beta amyloid peptide (Aβ), multiple proteoglycans. Systemic amyloidotic deposits also routinely contain proteoglycan, suggesting that these glycoconjugates are generally involved in amyloid plaque formation and/or persistence. We demonstrate that heparan sulfate proteoglycan (HSPG) and chondroitin sulfate proteoglycan (CSPG) inhibit the proteolytic degradation of fibrillar, but not non-fibrillar, Aβ at physiological pH. In accordance with the proteolysis studies, high affinity binding of proteoglycans to fibrillar Aβ(1-40) and Aβ(1-42) is observed from pH 4 to 9, whereas appreciable binding of HSPG or CSPG to non-fibrillar peptide is only seen at pH < 6. This differing pH dependence of binding suggests that a lysine residue is involved in proteoglycan association with fibrillar Aβ, whereas a protonated histidine appears to be needed for binding of the glycoconjugates to non-fibrillar peptide. Scatchard analysis of fibrillar Aβ association with proteoglycans indicates a single affinity interaction, and the binding of both HSPG and CSPG to fibrillar Aβ is completely inhibited by free glycosaminoglycan chains. This implies that these sulfated carbohydrate moieties are primarily responsible for proteoglycan•Aβ interaction. The ability of proteoglycans to bind fibrillar Aβ and inhibit its proteolytic degradation suggests a possible mechanism of senile plaque accumulation and persistence in Alzheimers disease.