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Homogenates and particulate fractions of goldfish brain incorporated radioac- tivity from y-(B*P)ATP selectively into acidic phospholipids during brief periods of incuba- tion. Phosphatidate and lysophosphatidate became strongly labelled and activity was also found in phosphatidyl inositol phosphate and in phosphatidyl inositol diphosphate. When tetraphenylborate (a K+-complexing agent) was added, a selective stimulation of incorporation of 3*P into phosphatidate occurred. The addition of perchlorate (also known to bind K+) did not produce a similar stimulation, nor did the addition of K+ block the stimulation by tetraphenylborate. The stimulation of the labelling of phospholipids by tetraphenylborate appeared to be the result of multiple actions. Besides the evidence that it acted by stimulating the phosphoinositide phosphodiesterase of brain, data were obtained suggesting that it stimulated diglyceride kinase and blocked endogenous destruction of ATP as well. The stimulation by tetraphenylborate was blocked by addition of atropine but not of arecoline. THE PHOSPHOINOSITIDES, although ubiquitous in animal tissues, frequently exist in trace amounts demonstrable only by tracer methods. The greater content of phos- phoinositides in excitable tissues (FOLCH-PI, 1949; DAWSON and DITTMER, 1961 ; BROCKERHOFF and BALLOU, 1962) and the evidence from labelling experiments of rapid turnover (DAWSON, 1954; HOKIN and HOKIN, 1958; ANSELL and SPANNER, 1959; LEBARON, KISTLER and HAUSER, 1960; HOLZL and WAGNER, 1964; KFOURY and KERR, 1964; SEIFFERT and AGRANOFF, 1965) have suggested an important physio- logical role for these compounds in excitable tissues. Alterations of the labelling of phosphatidyl inositol (PhI) and phosphatidic acid (PhA) have been described in a variety of neural and secretory tissues in response to the appropriate neurohumor or secretagogue (cf. reviews by: HAWTHORNE, 1960; HOKIN and HOKIN, 1960; Cum- BERT, 1967). In sympathetic ganglia increased labelling has been correlated with synaptic transmission (LARRABEE and LEICHT, 1965; LARRABEE, 1968). Recently DURELL, GARLAND and FRIEDEL (1969) have suggested that such alterations are the result of activation of phosphatidyl inositol phosphodiesterase releasing diglyceride and inositol phosphate, but the detailed mechanism of the effect of acetylcholine remains speculative. The present experiments were undertaken to clarify the nature of these alterations in phospholipid labelling. The goldfish was selected because of the current interest in this laboratory in goldfish behaviour and its metabolic conse- quences. Various aspects of the labelling patterns of phospholipids in the goldflsh brain, the effects of acetylcholine in goldfish brain, and the stimulation by tetra- phenylborate of phospholipid labelling comprise the present report.

Lactate's role in the brain is understood as a contributor to brain energy metabolism, but it may also regulate the cerebral microcirculation. The purpose of this systematic review was to evaluate evidence of lactate as a physiological effector within the normal cerebral microcirculation in reports ranging from in vitro experiments to in vivo studies in animals and humans. Following pre-registration of a review protocol, we systematically searched the PubMed, EMBASE, and Cochrane databases for literature covering themes of ‘lactate’, ‘the brain’, and ‘microcirculation’. Abstracts were screened, and data extracted independently by two individuals. We excluded studies evaluating lactate in disease models. Twenty-eight papers were identified, 18 of which were in vivo animal experiments (65%), four on human studies (14%), and six on in vitro or ex vivo experiments (21%). Approximately half of the papers identified lactate as an augmenter of the hyperemic response to functional activation by a visual stimulus or as an instigator of hyperemia in a dose-dependent manner, without external stimulation. The mechanisms are likely to be coupled to NAD + /NADH redox state influencing the production of nitric oxide. Unfortunately, only 38% of these studies demonstrated any control for bias, which makes reliable generalizations of the conclusions insecure. This systematic review identifies that lactate may act as a dose-dependent regulator of cerebral microcirculation by augmenting the hyperemic response to functional activation below 5 mmol/kg, and by initiating a hyperemic response above 5 mmol/kg. Open Science Badges: This article has received a badge for *Pre-registration* because it made the data publicly available. The data can be accessed at www.radboudumc.nl/getmedia/53625326-d1df-432c-980f-27c7c80d1a90/THollyer_lactate_protocol.aspx. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. (Figure presented.).

The first description of Alzheimer's disease (AD) was made in 1907 by Alois Alzheimer (Allgemeine Zeitschrift fur Psyciatrie und Psychisch-Gerichtliche Medizin 64, 3, 1907), although other contemporary physicians had made similar, and rather more complete, assessments of the neuropathological changes present in the AD brain (Fischer, Monatsschr Psychiat Neurol 22, 17, 1907). Our knowledge of AD has increased dramatically and continues to accelerate. This year is 25 years after the publication of a series of papers that, in various ways, articulated the amyloid cascade hypothesis (ACH) for AD (Beyreuther and Masters, Brain Pathol 1, 241–251, 1991; Hardy and Allsop, Trends Pharmacol Sci 12, 383–388, 1991; Selkoe, Neuron 6, 487–498, 1991; Hardy and Higgins, Science 256, 184–185, 1992). This review will cover some familiar territory, but we shall also place the ACH into a wider context, compare it with other hypotheses for AD, explore the evolution of the hypothesis to encompass new findings, and determine, irrespective of the merits of the hypothesis itself, whether it has been useful for the research field, both in academia and in industry. Finally, we shall review how the ACH has led to a number of therapeutic approaches, all of which have, to date, failed to reach their primary efficacy end-points in clinical trials and reflect upon what the future may hold. We review the amyloid cascade hypothesis (ACH) and compare it with other hypotheses that have been posited to explain the initiation and progression Alzheimer's disease. We document the data that support the ACH, and also reflect upon its deficiencies. We list the recent clinical failures of amyloidocentric drugs and anticipate the results that new therapeutic approaches may deliver. This article is part of the 60th Anniversary special issue.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting millions of people worldwide. Apart from age, the major risk factor identified so far for the sporadic form of AD is possession of the epsilon4 allele of apolipoprotein E (APOE), which is also a risk factor for coronary artery disease (CAD). Other apolipoproteins known to play an important role in CAD such as apolipoprotein B are now gaining attention for their role in AD as well. AD and CAD share other risk factors, such as altered cholesterol levels, particularly high levels of low density lipoproteins together with low levels of high density lipoproteins. Statins--drugs that have been used to lower cholesterol levels in CAD, have been shown to protect against AD, although the protective mechanism(s) involved are still under debate. Enzymatic production of the beta amyloid peptide, the peptide thought to play a major role in AD pathogenesis, is affected by membrane cholesterol levels. In addition, polymorphisms in several proteins and enzymes involved in cholesterol and lipoprotein transport and metabolism have been linked to risk of AD. Taken together, these findings provide strong evidence that changes in cholesterol metabolism are intimately involved in AD pathogenic processes. This paper reviews cholesterol metabolism and transport, as well as those aspects of cholesterol metabolism that have been linked with AD.

Inorganic phosphate (Pi) is an important polyanion needed for ATP synthesis and bone formation. Since it is found at millimolar levels in plasma it is usually incorporated as a constituent of artificial cerebrospinal fluid (ACSF) formulations for maintaining brain slices. In this paper we show that Pi limits the extracellular zinc concentration by inducing metal precipitation. We present data suggesting that amino acids like histidine may counteract the Pi-induced zinc precipitation by the formation of soluble zinc complexes. We propose that the interplay between Pi and amino acids in the extracellular space may influence the availability of metals for cellular uptake.

THE distribution of acid phosphatase in the central nervous system as demonstrated by histochemical methods has been investigated by LANDOW, KABAT and NEWMAN (1942), WISLOCKI and DEMPSEY (1948), and by SHIMIZU (1950) in comparative studies that included mice, rats, guinea pigs, rabbits, cats and chickens. Numerous papers deal with the cytological distribution of acid phosphatase in selected regions of the brain of various species, but little attention has been given to gradations of enzyme activity among those regions. The present paper reports a mapping of acid phosphatase activity in the brain of the rhesus monkey. A quantitative histochemical method was used, based upon spectrophotometric measurements of dye extracts from standard size tissue discs taken from brain sections that had been incubated with a substrate for acid phosphatase. Previous mappings of oxidative enzymes (FRIEDE, 1961), and particularly a mapping of lactate dehydrogenase in the brain of the rhesus monkey (FRIEDE and FLEMING, 1963), facilitate comparison of enzyme patterns. MATERIAL AND METHODS Histochemical method. Preliminary testing and standardization of the quantitative method for acid phosphatase activity were done on cat and monkey brains; a detailed account of methodology will be given elsewhere. For the present experiment three adult, healthy rhesus monkeys were killed under pentothal anaesthesia; the brains were removed immediately and placed in 10 % formalin at 4". After 8 hr fixation, the material was cut into slices, 3-5 mm thick. Twenty-four hours after removal of each brain, 30 p frozen sections were cut, rinsed, and stored in chilled distilled water until all material from the brain had been prepared. All sections were transferred to a 4-litre Erlenmeyer flask containing a small portion of the buffer component of the incubation medium. The substrate and diazo salt were mixed with the remaining portion of the buffer and filtered as quickly as possible; the filtrate was added immediately to the sections and buffer in the Erlenmeyer flask and incubation was started. The incubation medium was a mixture of 1 mg of sodium-a-naphthyl phosphate (Signia Chemical Co. St Louis, Missouri) and 1 mg of the stable diazotate of o-amino azotoluene (Fast Garnet GBC) (Dajac Laboratories, Philadelphia) in 1 ml of 0.2 M-Walpole buffer of pH 5 (PEARSE, 1960). For each monkey brain approximately 2 I. of reaction mixture were used. The sections were incubated in an Eherbach water-bath shaker with constant agitation for 1 hr at 38". The reaction was stopped by transferring all sections into a large quantity of 10% formalin. The stained sections were mounted temporarily on a microslide covered with three layers of parafilm. Discs were cut from selected areas of the sections with stainless steel tubes, 1.1 mm and 2.4 mm in diameter. Each disc was placed in a stoppered test tube containing a drop of distilled water. Care was taken not to transfer particles of parafilm into the tubes since these would cause turbidity and false readings. The azo dye was extracted with 1 ml of chloroform-ethanol (1 :l), v/v and the colour density was determined within 10-30 min at 555 mp in microcells in a Beckman DU spectrophotometer. For the data collected in Table I an average of ten samples were taken from the regions indicated for each monkey, and means and standard deviations were calculated. The dependability of this method was tested in various experiments (unpublished data) the results of which are only briefly summarized: tissue fixed in formalin after incubation was used since it was impractical to cut discs from loose sections During the required 24-hr fixation period, some loss of

Opiate drugs, such as codeine, morphine, and heroin, are powerful analgesics, but also are used as drugs of abuse because of their psychogenic properties. Many studies have shown that opiates impact on cellular proliferation in the adult and developing brain, although anatomical pathologies are lacking in in utero exposed infants and opioid knockout mice. Recent research has defined a context-dependent role for the opioid system in neurogenesis in the adult hippocampus with exercise. Opioids have been shown to interact with proliferating cells of the postnatal subventricular zone of the lateral ventricles. The subventricular zone is also a region of adult neurogenesis, a fact that was not well established at the time this earlier research was conducted. Although a relationship between opioids and fetal neurogenesis has yet to be firmly established, many studies have implicated the opioid system in this process. One common factor that links neurogenesis in adult, postnatal, and fetal structures is the involvement of neuronal progenitor cells of the astrocytic lineage. It is therefore of interest that opioids have been consistently shown to impact upon astrocytic proliferation. It is the intention of this paper to review the literature that has established a role for the opioid system in neurogenesis in vivo in fetal, postnatal, and adult animals and to examine the links of opioids to modulation of astrocytic proliferation.

Normal pressure hydrocephalus (NPH) is the most frequent form of chronic hydrocephalus in adults. NPH remains underdiagnosed although between 5% and 10% of all demented patients may suffer from this disorder. As dementia is an increasing demographic problem, treatable forms such as in NPH have become a central issue in neurology. Despite the traditional perception of hydrocephalus being a disorder of disturbed CSF dynamics, in NPH metabolic impairment seems at least as important. So far, the only valid animal model of NPH is chronic adult kaolin hydrocephalus. In this model, opening of alternative CSF outflow pathways leads to normal or near-normal intracranial pressure and CSF outflow resistance. Yet, various metabolic disturbances cause ongoing ventricular enlargement and characteristic symptoms including cognitive decline and gait ataxia. Delayed hippocampal neuronal death, accumulation of beta-amyloid and disturbed cholinergic neurotransmission may contribute to memory dysfunction. Compromised periventricular blood flow, decreased dopamine levels in the substantia nigra and damaged striatal GABAergic interneurons may reflect basal ganglia symptoms. At least in human hydrocephalus cerebrovascular co-morbidity of the white matter plays an important role as well. It seems that in hydrocephalus from a certain 'point of no return' metabolic impairment becomes decoupled from CSF dynamics and, at least partly, self-sustained. This is probably the reason why despite restored CSF circulation by shunting many patients with chronic hydrocephalus still suffer from severe neurological deficits. The present paper offers a comprehensive review of the experimental and clinical data suggesting metabolic disturbances in chronic hydrocephalus.

Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of alpha-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P alpha-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants alpha-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.

NMDA receptors interact with a variety of intracellular proteins at excitatory synapses. In this paper we show that myosin regulatory light chain (RLC) isolated from mouse brain is a NMDA receptor-interacting protein. Myosin RLC bound directly to the C-termini of both NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) subunits, rendering the interaction of myosin RLC with NMDA receptors distinct from that of calmodulin which is considered a NR1-interacting protein. Myosin RLC co-localized with NR1 in the dendritic spines of isolated hippocampal neurons, and was co-immunoprecipitated from brain extracts in a complex with NR1, NR2A, NR2B, PSD-95, Adaptor protein-2 and myosin II heavy chain. The C0 region of NR1 was necessary and sufficient for binding myosin RLC. Ca2+/calmodulin, but not calmodulin alone, displaced recombinant myosin RLC from the carboxy tail of NR1 indicating that myosin RLC and Ca2+/calmodulin can compete for a common binding site on NR1 in vitro. Myosin RLC is the only known substrate for myosin regulatory light chain kinase, which has recently been shown to modulate NMDA receptor function in isolated hippocampal neurons. Our results suggest that an additional level of NMDA receptor regulation may be mediated via a direct interaction with a light chain of myosin II. Thus, myosin RLC–NMDA receptor interactions may contribute to the contractile and motile forces that are placed upon NMDA receptor subunits during changes associated with synaptic plasticity and neural morphogenesis.

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44–750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.

The enzymatic phosphorylation of glycerol and dihydroxyacetone by ATP to ,sn-glycerol-3- phosphate and dihydroxyacetone phosphate respectively in various subcellular fractions of rat hrain was studied. A sensitive radiochemical assay was used where the labelled phosphorylated products were separated from the radioactive substrates by high voltage paper electrophoresis and the radioacti- vity in these compounds determined, Using this assay the glycerol kinase (EC 2.7.1.30) activity was found to be associated with the mitochondrial fraction of the brain. Under optimum conditions 2.45nmol of glycerol was phosphorylated/min per rng of protein. The K, for glycerol was 70~~ at pH 7. This mitochondrial enzyme, like other glycerol kinases from different sources, also phosphory- lated dihydroxyacetone. Under optimum conditions 1.7 nmol of dihydroxyacetone phosphate was formed/min per mg of mitochondrial protein. The K, for dihydroxyacetone was 0.6 mM. Glycerol kinase activity was also present in the cytoplasm of brain. However, the spccific activity of this enzyme in cytosol is about 15% of the mitochondrial glycerol kinase. Compared to glycerol, dihydroxyacetone was phosphorylated by ATP in cytoplasm at a much higher rate. The pH optimum for this soluble dihydroxyacetone kinase was much lower (pH 6.5) than that of the soluble or mitochon- drial glycerol kinase (pH 100). Using ammonium sulfate, brain cytoplasm was fractionated to yield a fraction in which the dihydroxyacetone kinase was enriched 2-3 fold with no glycerol kinase activity. Under optimum conditions 1.0 nmol of dihydroxyacetone was phosphorylated/min per mg protein. The K, for dihydroxyacetone was 60~~. This cytosol fraction was also found to phosphorylate u-gly- ccraldehyde and L-glyceraldehyde at a rate of 3&400/, to that of the dihydroxyacetone phosphorylation. The properties and the possible metabolic role of these enzymes in brain are discussed. GLYCEROL kinase (ATP: glycerol-3-phosphotransfer- ase EC 2.7.1.30) catalyzes the phosphorylation of gly- cerol by AI'P to sn-glycerol-3-phosphate (G-3-P). This enzyme was discovered by KALCKAR (1939) in kidney. The properties of the enzyme were first de- scribed in detail by BUBLITZ & KENNEDY (1954); WIE- LAND & SUYTER (1957). Using an enzymatic assay, WIELAND & SUYTER (1957) concluded that this kinase activity is present only in liver and kidney but not in other mammalian organs. However, later workers using more sensitive assays showed the presence of this enzyme in a wide variety of tissues (for a review see THORNER & PAULUS, 1973). To datc glycerol kinase has not been described in the brain, although there is indirect evidence to suggest its presence in that organ. A number of workers showed that when radioactive glycerol was injected intracranially into rats it was incorporated into the brain glycerolipids (HOKIN & HOKIN, 1958; LAPETINA ef aL, 1969; BENJA- MINS & MCKHANN, 1973; OBRIEN & GEISON. 1974). This suggests that glycerol is phosphorylated in brain to G-3-P and subsequently acylated and converted to different lipids (KENNEDY, 1962). ~~~ ~ ~~

—Two surgical methods are described in the present paper, allowing for the approximate determination of in vivo levels of ATP, lactate, glucose, pyruvate and glycogen in anatomically uninjured cortex and hypothalamus from unanaesthetized rats. It was not possible to obtain such levels for P-creatine in the 2 mm thick samples used in the present investigation. No fundamental difference was observed between the cortical and the hypo-thalamic levels of these substrates nor in their fluxes. The substrate fluxes during ischemia were correlated with electrical activity in the rat cortex and hypothalamus, recorded by means of telemetrically transmitted electroencephalograms. The electrical activity declined precipitously at 9.6 s after decapitation in the cortex, and after 12.1 s in the hypothalamus. High levels of glycogen, glucose and ATP were present at this moment, while P-creatine had declined sharply.

REPORTS of preceding investigations (FRIEDE, 1959~2, 1961 c; FRIEDE and FLEMING, 1962) have contained detailed charts of the distribution of several enzymes in the brain. The purpose of this paper is to compare enzyme patterns with each other and to describe similarities and, particularly, differences of the patterns that were observed. The regional patterns in the brain of seven enzymes which are concerned with glucose metabolism were studied and compared. Most of the data were obtained from histochemical series from rhesus monkey and human brains. These were compared with tissue homogenate assays. The enzymes studied were cytochrome oxidasef succinic dehydrogenase, DPN-diaphorase, TPN-diaphorase, malic dehydrogenase glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase and lactic dehydrogenase. In order to render this article more complete, some previously published data have been included. MATERIALS AND METHODS Because of difficulty in obtaining adequate human material, the systematic work reported in this study was done on rhesus monkey brains. Most of the Observations were compared with observations on human material as it became available during the course of the study. The following material was used: Monkey: Microscopic histochemicai surveys w2ere made by series for LDH, G-6-P, GPD, DPN-diaphorase, TPN-diaphorase, CYO and SD. Extensive spectrophotometric measurements of LDH were made from histochemical sections. SD, DPN-diaphorase, and G-6-P were assayed in homogenates from various regions and the data were compared with the histochemical staining gradients. Plans to assay all the nuclei of the monkey brain had to be discarded because the small size of the brain did not permit accurate macroscopical sampling of smaller nuclei. Random histochemical material was available for SD, LDH, CYO and G-6-P. The distribution of DPN-diaphorase was available from previous mappings (FRIEDE and FLEMING, 1962). In addition, G-6-P, DPN-diaphorase and SD were assayed in homogenates of seventeen representative regions from five normal human brains obtained 3.5 to 10 hours after death. The assay measurements were used for comparison with the histochemical gradations, as well as for comparison with each other (Table 1, line 6). Man: Also included are some observations on the distribution of MDH in the cat brain stem. Methods : Complete details of methodology are given in a separate publication (FRIEDE, FLEMING and KNOLLER, 1963) in which histochemical methods were tested extensively by comparison with assays in tissue homogenates. At this time, it may suffice to indicate that the use of formalin-fixed material was an advantage for histochemical studies of DPN-diaphorase and LDH. When carried out under carefully controlled conditions, fixation prevented loss of these two enzymes from tissue blocks and sections; histochemical data obtained by spectrophotometric measurement of formazan were in excellent agreement with the data of assays of unfixed tissue homogenates. Attempts to elaborate and standardize fixation methods for the other enzymes studied have failed so far.