Natalia V. Koudinova

Essential role for cholesterol in synaptic plasticity and neuronal degeneration

There is no understanding of the role of cholesterol and phospholipids in the mechanisms of synaptic function and neurodegeneration. Here we report that cholesterol disbalance is critical for synaptic transmission and plasticity as investigated by a study of paired pulse facilitation (PPF) and long‐term potentiation (LTP). Extracellular recording of field‐evoked postsynaptic potentials showed enhanced PPF ratio and an impairment of LTP in CA1 subfield of adult rat ex‐vivo hippocampal slices subjected to cyclodextrin‐ or normal human CSF‐HDL3‐mediated cholesterol efflux. Immunofluorescence with antibodies against neurofilament and tau revealed that cholesterol and phospholipids depletion causes alteration of normal hippocampal neurites and the appearance of PHF‐tau in the mossy fibers. We further find that LTP and amyloid β protein increase [14C]acetate label incorporation into newly synthesized hippocampal membrane lipids. Our results indicate the importance of neuronal cholesterol redistribution and synthesis for synaptic plasticity and neurodegeneration.

Photodynamic therapy with Pd-Bacteriopheophorbide (TOOKAD): successful in vivo treatment of human prostatic small cell carcinoma xenografts.

Small cell carcinoma of the prostate (SCCP), although relatively rare, is the most aggressive variant of prostate cancer, currently with no successful treatment. It was therefore tempting to evaluate the response of this violent malignancy and its bone lesions to Pd‐Bacteriopheophorbide (TOOKAD)‐based photodynamic therapy (PDT), already proven by us to efficiently eradicate other aggressive non‐epithelial solid tumors. TOOKAD is a novel bacteriochlorophyll‐derived, second‐generation photosensitizer recently, developed by us for the treatment of bulky tumors. This photosensitizer is endowed with strong light absorbance (ϵ0 ∼ 105 mol−1 cm−1) in the near infrared region (λ=763nm), allowing deep tissue penetration. The TOOKAD‐PDT protocol targets the tumor vasculature leading to inflammation, hypoxia, necrosis and tumor eradication. The sensitizer clears rapidly from the circulation within a few hours and does not accumulate in tissues, which is compatible with the treatment of localized tumor and isolated metastases. Briefly, male CD1‐nude mice were grafted with the human SCCP (WISH‐PC2) in 3 relevant anatomic locations: subcutaneous (representing tumor mass), intraosseous (representing bone metastases) and orthotopically within the murine prostate microenvironment. The PDT protocol consisted of i.v. administration of TOOKAD (4 mg/kg), followed by immediate illumination (650–800 nm) from a xenon light source or a diode laser emitting at 770 nm. Controls included untreated animals or animals treated with light or TOOKAD alone. Tumor volume, human plasma chromogranin A levels, animal well being and survival were used as end points. In addition, histopathology and immunohistochemistry were used to define the tumor response. Subcutaneous tumors exhibited complete healing within 28–40 days, reaching an overall long‐term cure rate of 69%, followed for 90 days after PDT. Intratibial WISH‐PC2 lesions responded with complete tumor elimination in 50% of the treated mice at 70–90 days after PDT as documented histologically. The response of the orthotopic model was also analyzed histologically with similar results. The study with this model suggests that TOOKAD‐based PDT can reach large tumors and is a feasible, efficient and well‐tolerated approach for minimally invasive treatment of local and disseminated SCCP.

Alzheimer's amyloid beta and lipid metabolism: a missing link?

THE EXCELLENT ARTICLE by Baggio et al. was of particular interest to us because it addressed an important issue pertaining to our special subject of Alzheimer’s research: the ‘‘hypothesis that a continuous reshaping in lipid physiology occurs with age and is a critical factor for survival and successful aging.’’ (1) The authors are correct that ‘‘changes in the serum level of protein, lipids, and lipoproteins that are considered risk factors for atherosclerotic vascular diseases in young people may lose their biological significance and assume a different, unknown role with advanced age.’’ But we are beginning to learn that blood lipids are indeed associated with Alzheimer’s disease (AD) and our group and others are discovering that Alzheimer’s may be very closely linked to human lipid metabolism change. First, it has been shown (2) that the lecithin cholesterol acyl transferase (LCAT) activity is significantly decreased in Alzheimer’s patients. LCAT catalyzes an acyltransferase reaction that forms most of the cholesterol esters and is a key step in reverse cholesterol transport in humans. This makes the enzyme of obvious significance in the development of atherosclerosis and heart disease. Moreover, as was demonstrated a decade ago (3), this is also the case with Down’s syndrome (DS), where there are the same blood changes in LCAT activity. This indicates that changes in this enzyme are related to the etiologies of both AD and DS on the one hand and vascular pathology on the other. In addition, there is a remarkable resistance to systemic atherosclerosis in DS (4), which is not yet shown in AD. Second, it has been proposed (5) that membrane destabilization in Alzheimer’s disease as a natural consequence of primary defective lipid composition (5) is important in the membrane damage that leads to brain cell death. The membrane destabilization was found to occur selectively at the areas of neurodegeneration in AD brain, thus potentially accounting for the localized lipid pathology. Our own research activities are focused on understanding in more detail the normal biology of Alzheimer’s amyloid beta protein (Ab). This small protein has a high tendency to form insoluble amyloid aggregates. Amyloid deposition of cerebrovascular amyloid and senile plaques is a characteristic feature of the Alzheimer’s brain and the latter was reported to occur in the brain of heart disease patients (6, 7). Nevertheless, in 1992 it became clear that Ab is not just a pathological protein. At that time several research groups (8–10) detected it in a ‘normal’, soluble (sAb), nonaggregated state in blood and cerebrospinal fluid (CSF) of both normal people and patients with AD. The question why this protein does not aggregate normally became of special interest to many Alzheimer’s research teams. Published data regarding Ab binding to a number of proteins, and in particular to apolipoproteins in a test tube (11, 12), led us to a hypothesis that these interactions reflect an sAb association with lipoproteins, of which apolipoproteins are protein constituents. Evidence described in our recently published papers (13–15) indicates an association of sAb with special class of normal blood and CSF lipoproteins, high density lipoproteins (HDL), believed to be a protective factor in atherosclerotic vascular disease. Furthermore, sAb is secreted by hepatic cells as a part of lipoprotein complex (16). Association of Alzheimer’s amyloid beta with HDL suggests a mechanism for maintaining it in normal soluble, not aggregated state in blood and body fluids of normal individuals (13–15). Moreover, HDL carry LCAT, the key enzyme of cholesterol removal from the body, activity that in the disease is changed (see above). On the basis of the observations described above, we tested the effect of Ab on cholesterol esterification

The levels of soluble amyloid beta in different high density lipoprotein subfractions distinguish Alzheimer's and normal aging cerebrospinal fluid: implication for brain cholesterol pathology?

Several previous studies reported the association of the soluble form of amyloid beta (sA beta) protein, a major constituent of amyloid deposits in Alzheimers disease (AD), with normal blood, cerebrospinal fluid (CSF) and central nervous system high density lipoproteins (HDLs). The present report aimed to elucidate the pattern of sA beta and apolipoprotein (apo) distribution in AD CSF-HDL subfractions. We studied AD CSF-HDL subfractions by SDS/PAGE and immunoblot analysis after CSF fractionation via density flotation ultracentrifugation. AD CSF was characterized by (i) increased sA beta and apo content of the HDL(1), and (ii) sA beta association with apoE and apoJ in HDL(2), HDL(3) and very high density lipoproteins. The finding supports our proposed hypothesis that upregulation of brain cholesterol dynamics is a fundamental event in the pathophysiology of AD and that sA beta binding to apo and lipid may have important structure-functional consequences.

Bypass of Tumor Drug Resistance by Antivascular Therapy

Multidrug resistance (MDR) presents a major obstacle for the successful chemotherapy of cancer. Its emergence during chemotherapy is attributed to a selective process, which gives a growth advantage to MDR cells within the genetically unstable neoplastic cell population. The pleiotropic nature of clinical MDR poses a great difficulty for the development of treatment strategies that aim at blocking MDR at the tumor cell level. Targeting treatment to the nonmalignant vascular network-the lifeline of the tumor-is a promising alternative for the treatment of drug-resistant tumors. The present study demonstrates that MDR in cancer can be successfully circumvented by photodynamic therapy (PDT) using an antivascular treatment protocol. We show that, although P-glycoprotein-expressing human HT29/MDR colon carcinoma cells in culture are resistant to PDT with Pd-bacteriopheophorbide (TOOKAD), the same treatment induces tumor necrosis with equal efficacy (88% vs 82%) in HT29/MDR-derived xenografts and their wild type counterparts, respectively. These results are ascribed to the rapid antivascular effects of the treatment, supporting the hypothesis that MDR tumors can be successfully eradicated by indirect approaches that bypass their inherent drug resistance. We suggest that with progress in ongoing clinical trials, TOOKAD-PDT may offer a novel option for local treatment of MDR tumors.

Alzheimer's Aβ1–40 Peptide Modulates Lipid Synthesis in Neuronal Cultures and Intact Rat Fetal Brain Under Normoxic and Oxidative Stress Conditions

The effect of amyloid β (Aβ), the major constituent of the Alzheimers (AD) brain on lipid metabolism was investigated in cultured nerve cells and in a fetal rat brain model. Differentiated (NGF) and undifferentiated PC12 cells or primary cerebral cell cultures were incubated with [14C]acetate in the absence or presence of Aβ1–40. Incorporation of label into lipid species was determined after lipid extraction and TLC separation. Phosphatidylcholine (PC) and phosphatidylserine (PS) synthesis was increased by Aβ1–40, in a dose dependent manner, an effect which was more pronounced in differentiated PC12 cells. A significant proportion of radioactivity (5–6%) was released into the medium with a radioactivity distribution similar to that of the cellular lipids. Cholesterol and PC were the highest labeled medium lipids. Increasing Aβ1–40 concentration up to 0.1 μg/ml in cerebral cells but not in PC12 cells, caused a relative increase (1.5 fold) in release of PS, while that of PE decreased. Stimulation of PS release may possibly be associated with apoptotic cell death. Aβ1–40 peptide (5 μg) was administered intraperitonealy into rat fetuses (18 days gestation) along with [14C]acetate (2μCi/fetus). After 24 h, the maternal-fetal blood supply was occluded for 20 min (ischemia) followed by 15 min reperfusion. Fetuses were killed and liver and brain tissue subjected to lipid extraction and radioactivity determination after TLC. Aβ1–40 peptide increased synthesis of different classes of lipids up to 20–40% in brain tissue compared to controls. Labeling of liver lipids was decreased by Aβ1–40 by 20–30%. A general decrease in synthesis of lipids was observed after ischemia/reperfusion. Our data suggest that Aβ1–40 peptide regulates normal lipid biosynthesis but under ischemia it compromises it. The latter finding may confirm the oxidative stress etiology in AD and suggests that Aβ1–40 modulation of lipid metabolism may have Alzheimers pathological relevance, particularly at high peptide concentrations.

Biphasic modulation of protein kinase C and enhanced cell toxicity by amyloid beta peptide and anoxia in neuronal cultures

A major feature of Alzheimers disease is the deposition of the amyloid beta peptide (Aβ) in the brain by mechanisms which remain unclear. One hypothesis suggests that oxidative stress and Aβ aggregation are interrelated processes. Protein kinase C, a major neuronal regulatory protein is activated after oxidative stress and is also altered in the Alzheimers disease brain. Therefore, we examined the effects of Aβ1−40 peptide on the protein kinase C cascade and cell death in primary neuronal cultures following anoxic conditions. Treatment with Aβ1−40 for 48 h caused a significant increase in the content and activity of Ca2+‐dependent and Ca2+‐independent protein kinase C isoforms. By 72 h various protein kinase C isoforms were down‐regulated. Following 90 min anoxia and 6 h normoxia, a decrease in protein kinase C isoforms was noticed, independent of Aβ1−40 treatment. A combination of Aβ1−40 and 30‐min anoxia enhanced cytotoxicity as noticed by a marked loss in the mitochondrial ability to convert 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide and by enhanced 4′,6‐diamidino‐2‐phenylindole nuclear staining. Phosphorylation of two downstream protein kinase C substrates of apparent molecular mass 80 and 43 kDa, tentatively identified as the mirystoyl alanine‐rich C‐kinase substrate (MARCKS), were gradually elevated up to 72 h upon incubation with Aβ1−40. Anoxia followed by 30 min normoxia enhanced MARCKS phosphorylation in the membrane but not in the cytosolic fraction. In the presence of Aβ1−40, phosphorylation of MARCKS was reduced. After 6 h normoxia, MARCKS phosphorylability was diminished possibly because of protein kinase C down‐regulation. The data suggest that a biphasic modulation of protein kinase C and MARCKS by Aβ1−40 combined with anoxic stress may play a role in Alzheimers disease pathology. 1

Amyloid-β, Tau Protein, and Oxidative Changes as a Physiological Compensatory Mechanism to Maintain CNS Plasticity under Alzheimer's Disease and Other Neurodegenerative Conditions

In this review, we propose that the neurodegenerative changes in the neurochemistry of amyloid-beta (Abeta) aggregation, tau phosphorylation, cytoskeleton rearrangement, oxidative stress, and lipid peroxidation in Alzheimers disease (AD), and a number of other neurodegenerative diseases, are secondary pathological features. In fact, we believe that these phenomena represent natural compensatory mechanisms for impaired primary neurodegeneration, membrane dynamic deterioration, and/or associated failures of neurotransmission, synaptic function, and neuroplasticity. Physiologically, Abeta, lipid peroxidation, and tau protein may function to sense changes in activity-dependent membrane properties and therefore biochemically modulate membrane lipid homeostasis for more efficient synaptic action. As such, the previously proposed therapeutic tackling of amyloid, tau, oxidative stress, and other brain disease markers may have no ability to cure AD or other devastating central nervous system pathologies and peripheral nervous system diseases. This unfortunate realization provides a wake-up call to the neuroscience community, demanding open-minded approach.

Cholesterol and Alzheimer’s disease: Is there a link?

To the Editor: We read with great interest the article by Simons et al.1 In 1994, we reported an association of soluble form of amyloid β protein with high-density lipoproteins and functional …

In vivo imaging of the neutron capture therapy agent BSH in mice using 10B MRI

Boron neutron capture therapy (BNCT) is an experimental cancer treatment modality requiring the targeting of 10B‐enriched compounds to the tumor, which is then irradiated by low‐energy neutrons. One of the boron‐containing compounds used for this purpose is the mercaptoborane Na2B12H11SH (BSH). The first in vivo MR images of 10B‐enriched BSH are presented here. BSH, injected into the tail vein of mice with implanted M2R melanoma xenografts, was imaged using 3D gradient echo 10B MRI. 10B NMR spectroscopy, localized mainly to the tumor by virtue of the use of a small surface coil, was applied to measure the T1 (2.9 ± 0.3 ms) and T2 (1.75 ± 0.25 ms) values of the 10B signal. The MRI experiments detected levels of about 20 ppm (μg boron / g tissue) at 6 × 6 × 6 mm spatial resolution in a total scan time of 16 min. Magn Reson Med 46:13–17, 2001.