· Martha Millan Sanchez · Sarah Moghadam · Priyanka Naik ·
· Kara Martin · Ahmad Salehi ·

Hippocampal structural and functional alterations in Alzheimer’s disease (AD), detected by advanced imaging methods, have been linked to significant abnormalities in multiple internal and external networks in this critical brain region. Uncovering the temporal and anatomical pattern of these network alterations would provide important clues into understanding the pathophysiology of AD and suggest new therapeutic strategies for this multi-system and prevalent disorder. Over the last decade, we have focused on studying brain structures that provide major projections to the hippocampus (HC) and the pattern of de-afferentation of this area in mouse models of AD and a related neurodegenerative disorder, i.e. Down syndrome (DS). Our studies have revealed that major inputs into the hippocampal structure undergo significant age-dependent alterations. Studying locus coeruleus (LC), the sole source of noradrenergic terminals for the HC, it has been shown that these neurons show significant age-dependent degeneration in both mouse models of DS and AD. Furthermore, increasing noradrenergic signaling was able to restore cognitive function by improving synaptic plasticity, and possibly promoting microglia recruitment, and amyloid β (Aβ) clearance in transgenic (tg) mouse models of AD. Here, we re-examine the effects of alterations in major inputs to the hippocampal region and their structural and functional consequences in mouse models of neurodegenerative disorders. We will conclude that improving the function of major hippocampal inputs could lead to a significant improvement in cognitive function in both AD and DS.

Science Translational Medicine (cont.)

· Ahmad Salehi · Mehrdad Faizi · Damien Colas ·
· Janice Valletta · Jessania Laguna · Ryoko Takimoto-Kimura ·
· Alexander Kleschevnikov · Steve Wagner · Paul Aisen ·
· Mehrdad Shamloo · Mehrdad Shamloo ·

• Alzforum
• Down Syndrome Research Center at Stanford
• Health 24
• Le Figaro
• Le Monde
• Lucile Packard Children’s Hospital at Stanford
• MIT Technology Review
• Neurology
• Science
• Science Daily
• Scope
• Stanford Medical Center
• The Scientist
• Times Online

Down syndrome (trisomy 21) is the most common cause of mental retardation in children and leads to marked deficits in contextual learning and memory. In rodents, these tasks require the hippocampus and are mediated by several inputs, particularly those originating in the locus coeruleus. These afferents mainly use norepinephrine as a transmitter. To explore the basis for contextual learning defects in Down syndrome, we examined the Ts65Dn mouse model. These mice, which have three copies of a fragment of mouse chromosome 16, exhibited significant deficits in contextual learning together with dysfunction and degeneration of locus coeruleus neurons. However, the postsynaptic targets of innervation remained responsive to noradrenergic receptor agonists. Indeed, despite advanced locus coeruleus degeneration, we were able to reverse contextual learning failure by using a prodrug for norepinephrine called l-threo-3,4-dihydroxyphenylserine, or xamoterol, a β1-adrenergic receptor partial agonist. Moreover, an increased gene dosage of App, Continued on next page…

Science Translational Medicine (cont.)

in the context of Down syndrome, was necessary for locus coeruleus degeneration. Our findings raise the possibility that restoring norepinephrine-mediated neurotransmission could reverse cognitive dysfunction in Down syndrome.

Note In order to access the full text of this article, please go to sites.google.com/site/asalehipub, where you can click the cover to gain permission to reach the full text. We are sorry for the inconvenience.

Cell Stem Cell (cont.)

· Stanley Tamaki · Yakop Jacobs · Monika Dohse ·
· Alexandra Capela · Jonathan Cooper · Michael Reitsma ·
· Dongping He · Robert Tushinski · Pavel Belichenko ·
· Ahmad Salehi · William Mobley · Fred Gage ·
· Stephen Huhn · Ann Tsukamoto · Irving Weissman ·
· Nobuko Uchida ·

Neurodegeneration of purkinje cells. In a mouse model of neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder, accumulation of lipofuscin granules is followed by progressive neuronal cell death. Purkinje neurons in the cerebellum are severely affected as illustrated by loss of cell bodies and dendritic arbors. Massive lipofuscin deposits remain in the purkinje cell layer in the degenerating brain. The work by Tamaki et al. (pages 310–319) demonstrates that transplantation of human neural stem cells leads to neuroprotection of host cells in the NCL mouse brain. The cover image shows a section of cerebellum from a mouse with disease symptoms. Cover design by Monika Dohse.

· Nadia Belichenko · Pavel Belichenko · Alexander Kleschevnikov ·
· Ahmad Salehi · Roger Reeves · William Mobley ·

Down syndrome (DS) can be modeled in mice segmentally trisomic for mouse chromosome 16. Ts65Dn and Ts1Cje mouse models have been used to study DS neurobiological phenotypes including changes in cognitive ability, induction of long-term potentiation (LTP) in the fascia dentata (FD), the density and size of dendritic spines, and the structure of synapses. To explore the genetic basis for these phenotypes, we examined Ts1Rhr mice that are trisomic for a small subset of the genes triplicated in Ts65Dn and Ts1Cje mice. The 33 trisomic genes in Ts1Rhr represent a “DS critical region” that was once predicted to be sufficient to produce most DS phenotypes. We discovered significant alterations in an open field test, a novel object recognition test and in a T-maze task. As in Ts65Dn and Ts1Cje mice, LTP in FD of Ts1Rhr could be induced only after blocking GABA_A-dependent inhibitory neurotransmission. In addition, widespread enlargement of dendritic spines and decreased density of spines in FD were preserved in Ts1Rhr. Twenty of 48 phenotypes showed significant differences between Ts1Rhr and 2N controls. We conclude that important neurobiological phenotypes characteristic of DS are conserved in Ts1Rhr mice. The data support the view that biologically significant trisomic phenotypes occur because of dosage effects of genes in the Ts1Rhr trisomic segment and that increased dosage is sufficient to produce these changes. The stage is now set for studies to decipher the gene(s) that play a conspicuous role in creating these phenotypes.

· Pavel Belichenko · Alexander Kleschevnikov · E. Masliah ·
· Chengbioa Wu · Ryoko Takimoto-Kimura · Ahmad Salehi ·
· Willam Mobley ·

Down syndrome (DS) is a neurological disorder causing impaired learning and memory. Partial trisomy 16 mice (Ts65Dn) are a genetic model for DS. Previously, we demonstrated widespread alterations of pre- and postsynaptic elements and physiological abnormalities in Ts65Dn mice. The average diameter of presynaptic boutons and spines in the neocortex and hippocampus was enlarged. Failed induction of long-term potentiation (LTP) due to excessive inhibition was observed. In this paper we investigate the morphological substrate for excessive inhibition in Ts65Dn. We used electron microscopy (EM) to characterize synapses, confocal microscopy to analyze colocalization of the general marker for synaptic vesicle protein with specific protein markers for inhibitory and excitatory synapses, and densitometry to characterize the distribution of the receptor and several proteins essential for synaptic clustering of neurotransmitter receptors. EM analysis of synapses in the Ts65Dn vs. 2N showed that synaptic opposition lengths were significantly greater for symmetric synapses (approximately 18%), but not for asymmetric ones. Overall, a significant increase in colocalization coefficients of glutamic acid decarboxylase (GAD)65/p38 immunoreactivity (IR) (approximately 27%) and vesicular GABA transporter (VGAT)/p38 IR (approximately 41%) was found, but not in vesicular glutamate transporter 1 (VGLUT1)/p38 IR. A significant overall decrease of IR in the hippocampus of Ts65Dn mice compared with 2N mice for glutamate receptor 2 (GluR2; approximately 13%) and anti-gamma-aminobutyric acid (GABA_A) Continued on next page…

The Journal of Comparative Neurology (cont.)

receptor β2/3 subunit (approximately 20%) was also found. The study of proteins essential for synaptic clustering of receptors revealed a significant increase in puncta size for neuroligin 2 (approximately 13%) and GABA_A receptor-associated protein (GABARAP; approximately 13%), but not for neuroligin 1 and gephyrin. The results demonstrate a significant alteration of inhibitory synapses in the fascia dentata of Ts65Dn mice.

· Ahmad Salehi · Chengbioa Wu · Ke Zhan ·
· William Mobley ·

The most effective treatments for neurodegenerative disorders, including Alzheimer’s disease, will come from studies of the pathogenesis of age-related cognitive failure and understanding of the underlying mechanisms. Given the marked similarities in pathological and clinical phenotypes between Alzheimer’s disease and Down syndrome, studies of the pathogenesis of one can be expected to complement and support those in the other. Alzheimer’s disease and Down syndrome are characterized by dysfunction and loss of several biochemically and anatomically defined neuronal populations. The pathological involvement of hippocampus, in particular, is an early feature of both disorders, as is the degeneration of neurons whose axons innervate this region. Long, thin and poorly myelinated axons project from a number of subcortical and brain stem nuclei to modulate hippocampally mediated cognitive functions. In studies on mouse models of Down’s syndrome, we uncovered evidence for the involvement of a particular neuronal population heavily innervating the hippocampus. In an extensive series of experiments, we found evidence that failed retrograde transport of nerve growth factor signaling in cholinergic neurons of the basal forebrain is linked to their vulnerability and that these changes are caused by increased gene dose and overexpression of the gene for amyloid precursor protein. These findings raise the possibility that intracellular trafficking defects created by changes in amyloid precursor protein expression or processing make an important contribution to pathogenesis and set the stage for studies to explore the molecular mechanisms of degeneration Continued on next page…

Intracellular Traffic and Neurodegenerative Disorders [Book] (cont.)

of cholinergic neurons and to define new therapeutic targets for these neurons. An important unanswered question is whether or not similar mechanisms operate within other vulnerable populations, innervating hippocampus to cause de-afferentation and dysfunction of this critical brain region.

· Pavel Belichenko · Alexander Kleschevnikov · Ahmad Salehi ·
· Charles Epstein · William Mobley ·

Down syndrome (DS) is caused by trisomy of human chromosome 21. Because Ts65Dn and Ts1Cje mice are segmentally trisomic for a region of mouse chromosome 16, they genetically model DS and are used to study pathogenic mechanisms. Previously, we provided evidence for changes in both the structure and function of pre- and postsynaptic elements in the Ts65Dn mouse. Striking changes were evident in the size of the dendritic spines and in the ability to induce long-term potentiation (LTP) in the fascia dentata (FD). To explore the genetic basis for these changes, we examined Ts1Cje mice, which are trisomic for a completely overlapping but smaller segment of mouse chromosome 16. As in the Ts65Dn mouse, there was a regionally selective decrease in the density of dendritic spines (approximately 12%), an increase in the size of spine heads (approximately 26%), a decrease in the length of spine necks (approximately 26%), and reorganization of inhibitory inputs with a relative decrease in inputs to dendrite shafts and spine heads and a significant increase to the necks of spines (6.4%). Thus, all of the Ts65Dn phenotypes were present, but they were significantly less severe. In contrast, and just as was the case for the Ts65Dn mouse, LTP could not be induced unless the selective gamma-aminobutyric acid (GABA_A) receptor antagonist picrotoxin was applied. Therefore, there was conservation of important synaptic phenotypes in the Ts1Cje mice. The analysis of data from this and earlier studies points to genotype-phenotype linkages in DS whose complexity ranges from relatively simple to quite complex.

· Ahmad Salehi · Mehrdad Faizi · Pavel Belichenko ·
· William Mobley ·

Down Syndrome (DS) caused by trisomy 21 is characterized by a variety of phenotypes and involves multiple organs. Sequencing of human chromosome 21 (HSA21) and subsequently of its orthologues on mouse chromosome 16 have created an unprecedented opportunity to explore the complex relationship between various DS phenotypes and the extra copy of approximately 300 genes on HSA21. Advances in genetics together with the ability to generate genetically well-defined mouse models have been instrumental in understanding the relationships between genotype and phenotype in DS. Indeed, elucidation of these relationships will play an important role in understanding the pathophysiological basis of this disorder and helping to develop therapeutic interventions. A successful example of using such a strategy is our recent studies exploring the relationship between failed nerve growth factor (NGF) transport and amyloid precursor protein (App) overexpression. We found that increased dosage of the gene for App is linked to failed NGF signaling and cholinergic neurodegeneration in a mouse model of DS. Herein, we discuss several mouse models of DS and explore the emergence of exciting new insights into genotype-phenotype relationships, particularly those related to nervous system abnormalities. An important conclusion is that uncovering these relationships is enhanced by working from carefully defined phenotypes to the genes responsible.

· Ahmad Salehi · Alexander Kleshevnikov · William Mobley ·

Neurotrophins play an important role in the survival, differentiation, and maintenance of neurons selectively involved in a number of disorders of the nervous system. Nerve growth factor (NGF) plays a vital role for basal forebrain cholinergic neurons (BFCNs), including the maintenance of the cholinergic phenotype in adults. Recognition of this role has suggested the use of NGF to ameliorate the loss of these neurons in Alzheimer’s disease (AD). While clinical studies directed at supplying NGF to patients continue to be pursued, fundamental questions remain as to the relationship between selective vulnerability of cholinergic neurons and the actions of NGF. In this chapter, we review the structure and function of the basal forebrain cholinergic system, its role in higher cognitive functions, and the importance of NGF actions on these cells. Studies that link changes in NGF signaling to the degeneration of BFCNs are then discussed, as are current approaches to NGF-related treatments. Finally, on the basis of recent findings in mouse models of AD and Down syndrome, we suggest that impaired retrograde axonal transport of NGF plays a significant role in pathogenesis. This insight may guide future studies of pathogenesis and innovative treatment for BFCNs and, perhaps, other neurons affected in AD.

· Ahmad Salehi · Jean-Dominique Delcroix · Pavel Belichenko ·
· Ke Zhan · Chengbioa Wu · Janice Valletta ·
· Ryoko Takimoto-Kimura · Alexander Kleschevnikov · Kumar Sambamurti ·
· Peter Chung · Weiming Xia · Angela Villar ·
· William Campbell · Laura Kulnane · Randy Nixon ·
· Bruce Lamb · Charles Epstein · Gorazd Stokin ·
· Larry Goldstein · William Mobley ·

• Alzforum
• Science
• Stanford Medical Center
• The Scientist
• The Wall Street Journal

Down Syndrome (DS) caused by trisomy 21 is characterized by a variety of phenotypes and involves multiple organs. Sequencing of human chromosome 21 (HSA21) and subsequently of its orthologues on mouse chromosome 16 have created an unprecedented opportunity to explore the complex relationship between various DS phenotypes and the extra copy of 300 genes on HSA21. Advances in genetics together with the ability to generate genetically well-defined mouse models have been instrumental in understanding the relationships between genotype and phenotype in DS. Indeed, elucidation of these relationships will play an important role in understanding the pathophysiological basis of this disorder and helping to develop therapeutic interventions. Continued on next page…

Neuron (cont.)

A successful example of using such a strategy is our recent studies exploring the relationship between failed nerve growth factor (NGF) transport and amyloid precursor protein (App) overexpression. We found that increased dosage of the gene for App is linked to failed NGF signaling and cholinergic neurodegeneration in a mouse model of DS. Herein, we discuss several mouse models of DS and explore the emergence of exciting new insights into genotype-phenotype relationships, particularly those related to nervous system abnormalities. An important conclusion is that uncovering these relationships is enhanced by working from carefully defined phenotypes to the genes responsible.

· Jean-Dominique Delcroix · Janice Valletta · Chengbioa Wu ·
· Charles Howe · Chun-Fai Lai · John Cooper ·
· Pavel Velichenko · Ahmad Salehi · William Mobley ·

Nerve growth factor (NGF) activates TrkA to trigger signaling events that promote the survival, differentiation and maintenance of neurons. The mechanism(s) that controls the retrograde transport of the NGF signal from axon terminals to neuron cell bodies is not known. The ‘signaling endosome’ hypothesis stipulates that NGF, TrkA and signaling proteins are retrogradely transported on endocytic vesicles. Here, we provide evidence for the existence of signaling endosomes. Following NGF treatment, clathrin-coated vesicles (CCVs) contain NGF bound to TrkA together with activated signaling proteins of the Ras/pErk1/2 pathway. NGF signals from isolated CCVs through the Erk1/2 pathway. Early endosomes appear to represent a second type of signaling endosomes. We found that NGF induced a sustained activation of Rap1, a small monomeric GTP-binding protein of the Ras family, and that this activation occurred in early endosomes that contain key elements of Rap1/pErk1/2 pathway. We discuss the possibility that the failure of retrograde NGF signaling in a mouse model of Down syndrome (Ts65Dn) may be due to the failure to retrograde transport signaling endosomes. It is important to define further the significance of signaling endosomes in the biology of both normal and degenerating neurons.

Note NGF and Related Molecules in Health and Disease is part of the series Progress in Brain Research.

· Pavel Belichenko · Alexander Kleschevnikov · Ahmad Salehi ·
· Charles Epstein · William Mobley ·

The Ts65Dn mouse is a genetic model for Down syndrome. Although this mouse shows abnormalities in cognitive function that implicate hippocampus as well as marked deficits in hippocampal long-term potentiation, the structure of the hippocampus has been little studied. We characterized synaptic structure in Ts65Dn and control (2N) mice, studying the hippocampus (fascia dentata, CA1) as well as the motor and somatosensory cortex, entorhinal cortex, and medial septum. Confocal microscopy was used to examine immunostained presynaptic boutons and to detail the structure of dendrites after Lucifer yellow microinjection. Both presynaptic and postsynaptic elements were significantly enlarged in Ts65Dn in all regions examined. The changes were detected at the youngest age examined (postnatal day 21) and in adults. In studies detailing the changes in fascia dentata and motor cortex, the enlargement of spines affected the entire population, resulting in the presence of spines whose volume was greatly increased. Electron microscopy confirmed that boutons and spines were enlarged and demonstrated abnormalities in the internal membranes of both. In addition, spine density was decreased on the dendrites of dentate granule cells, and there was reorganization of inhibitory inputs, with a relative decrease in inputs to dendrite shafts and an increase in inputs to the necks of spines. Taken together, the findings document widespread abnormalities of synaptic structure that recapitulate important features seen in Down syndrome. They establish the Ts65Dn mouse as a model for abnormal synapse structure and function in Down syndrome and point to the importance of studies to elucidate the mechanisms responsible for synapse enlargement.

· Ahmad Salehi · Jean-Dominique Delcroix · Dick Swaab ·

Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) occurs early and contributes significantly to cognitive decline in Alzheimer’s disease (AD). Proper function and morphology of BFCNs depends on the supply of nerve growth factor (NGF) from the cortex and the hippocampus. A large number of experiments have shown that decreased supply of NGF at the level of BFCN cell bodies leads to loss of neuronal markers and shrinkage, mimicking what is observed in AD. The delivery of sufficient amounts of NGF signal to BFCN cell bodies depends on the effective participation of several factors including sufficient synthesis and release of NGF, adequate synthesis and expression of NGF receptors by BFCNs, normal signaling and retrograde transport of NGF-receptor complex, and finally effective induction of gene expression by NGF. In the past few years it has become clear that decreased amounts of NGF at the level of BFCN cell bodies is largely due to failed retrograde transport rather than decreased synthesis, binding or expression of NGF receptors in the BFCN terminals. We will discuss in vivo evidence supporting decreased retrograde transport of NGF in a mouse model with BFCN degeneration, and will attempt to match these findings with our studies in postmortem human AD brain. We will speculate about the possible mechanisms of failed NGF retrograde transport and its relationship to AD pathology.

· Ahmad Salehi · Jean-Dominique Delcroix · William Mobley ·

Advances in understanding the biology of neurotrophic factors and their signaling pathways have provided important insights into the normal growth, differentiation and maintenance of neurons. Stimulated by neuropathological observations and genetic discoveries, studies in cell and animal models of neurodegenerative disorders have begun to clarify pathogenetic mechanisms. We examine the intersection of these research themes and identify several potential mechanisms for linking failed neurotrophic factor signaling to neurodegeneration. Studies of nerve growth factor signaling in a mouse model of Down syndrome encourage the views that neuronal dysfunction and atrophy might be linked to failed neurotrophic support and that additional studies focused on this possibility would enhance our understanding of the mechanisms of neurodegenerative disorders and their treatment.

· John Cooper · Ahmad Salehi · Jean-Dominique Delcroix ·
· Charles Howe · Pavel Belichenko · Jane Chua-Couzens ·
· Joshua Kilbridge · Elaine Carlson · Charles Epstein ·
· William Mobley ·

Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive decline in Alzheimer’s disease and Down’s syndrome. With aging, the partial trisomy 16 (Ts65Dn) mouse model of Down’s syndrome exhibited reductions in BFCN size and number and regressive changes in the hippocampal terminal fields of these neurons with respect to diploid controls. The changes were associated with significantly impaired retrograde transport of nerve growth factor (NGF) from the hippocampus to the basal forebrain. Intracerebroventricular NGF infusion reversed well established abnormalities in BFCN size and number and restored the deficit in cholinergic innervation. The findings are evidence that even BFCNs chronically deprived of endogenous NGF respond to an intervention that compensates for defective retrograde transport. We suggest that age-related cholinergic neurodegeneration may be a treatable disorder of failed retrograde NGF signaling.

· Fred van Leeuwen · David Fischer · Darlene Kamel ·
· Jacqueline Sluijs · Marc Sonnemans · Rob Benne ·
· Dick Swaab · Ahmad Salehi · Elly Hol ·

Dinucleotide deletions (e.g. ΔGA, ΔGU) are created by molecular misreading in or adjacent to GAGAG motifs of neuronal mRNAs. As a result, the reading frame shifts to the +1 frame, and so-called “+1 proteins” are subsequently synthesized. +1 Proteins have a wild-type N-terminus, but an aberrant C-terminus downstream from the site of the dinucleotide deletion. Molecular misreading was discovered in the rat vasopressin gene associated with diabetes insipidus and subsequently in human genes linked to Alzheimer’s disease (AD), e.g. β amyloid precursor protein (βAPP) and ubiquitin-B (UBB). Furthermore, βAPP(+1) and UBB(+1) proteins accumulate in the neuropathological hallmarks (i.e. in the tangles, neuritic plaques, and neuropil threads) of AD. As these +1 proteins were also found in elderly nondemented controls, but not in younger ones (<51 years), molecular misreading in nondividing cells might act as a factor that only becomes manifest at an advanced age. Frameshift mutations (UBB(+1)) and pretangle staining (Alz-50 and MC1) seem to occur independently of each other during early stages of AD. We recently detected +1 proteins, not only in proliferating cells present in non-neuronal tissues such as the liver and epididymis, but also in neuroblastoma cell lines. These observations suggest that molecular misreading is a general source of transcript errors that are involved in cellular derangements in various age-related pathologies.

· Tatjana Ishunina · Ahmad Salehi · Dick Swaab ·

The human supraoptic nucleus (SON) is the main production site of plasma vasopressin. Previously, using the Golgi apparatus and cell size as measures for neuronal metabolic activity, an activation of vasopressinergic neurons was found during ageing in the human SON in women but not in men. We hypothesized that the low-affinity neurotrophin receptor p75 (p75(NTR)) might be involved in the mechanism of activation of vasopressin neurons in postmenopausal women, since this receptor was found to be expressed in the SON neurons of aged individuals, and because p75(NTR) expression was shown to be suppressed by estrogens. Therefore, we investigated whether p75(NTR) immunoreactivity in the SON neurons was age- and sex-dependent. For this purpose, we studied paraffin sections of the SON in 32 postmortem brains of control patients ranging in age from 29 to 94 years with an anti-p75(NTR) antibody and determined the area of p75(NTR) immunoreactivity per neuron using an image analysis system. To study whether the p75(NTR) might also participate in the activation of SON neurons, we related Golgi apparatus size to the area of p75(NTR) immunoreactivity per cell in the same patients. We found that the area of p75(NTR) immunoreactivity per cell correlated indeed significantly with age and with Golgi apparatus size only in women but not in men. Therefore, our results suggest that p75(NTR) is involved in postmenopausal activation of vasopressinergic neurons in the human SON.

· Ahmad Salehi · Marta Ocampo · Joost Verhaagen ·
· Dick Swaab ·

In a previous study we showed that the staining of tyrosine kinase receptors (trks), which are high-affinity neurotrophin receptors (NTRs), is strongly diminished in the nucleus basalis of Meynert (NBM) of Alzheimer’s disease (AD) patients, which may explain the lack of effect of NGF therapy in AD patients so far. Since the literature regarding the expression of low-affinity NTRs was rather controversial, the aim of the present study was to examine (i) possible changes in the staining of low-affinity NTRs, i.e., p75 in the human NBM, an area that is severely affected in AD; and (ii) alterations of these receptors in relation to risk factors for AD, e. g., age, sex, and menopause. Brain material of 31 controls and 30 AD patients was obtained at autopsy, embedded in paraffin, and stained immunocytochemically. Using an image analysis system, we quantified p75 immunoreactivity in both cell bodies and fibers at the level of the NBM. Our results showed a significant diminishment of p75 immunoreactivity in both cell bodies and fibers of NBM neurons in AD. We did not find any relationship between age or sex and the expression of p75 receptor in cell bodies. However, there was a clearly positive relationship between age and fiber staining in AD patients which suggests the occurrence of a p75 transport disorder as an early event in the process of AD. These observations and the earlier reported decreased staining of trk receptors show that degeneration of NBM neurons in AD is associated with a decreased neurotrophin responsiveness of NBM neurons in AD and that therapeutic strategies should be directed toward upregulation of receptors or facilitation of transport before an effect of neurotrophins in AD may be expected.

· Tatjana Ishunina · Ahmad Salehi · Michel Hofman ·
· Dick Swaab ·

In the human hypothalamus, arginine-vasopressin (AVP) is produced for a major part by the neurones of the supraoptic nucleus (SON). Since plasma AVP levels in men were reported to be higher than those of women and we did not find a sex difference in the neurone number, a higher vasopressinergic neurone activity was supposed to be present in the SON of men. Therefore we studied the size of the Golgi-apparatus (GA), which has been demonstrated previously to be a sensitive parameter for protein synthetic ability of neurones, in 15 men and 17 women ranging in age from 29 to 94 years. A polyclonal antibody against immunoaffinity purified MG-160, a sialoglycoprotein of the medial cisternae of the GA was applied on paraffin-embedded sections containing the dorsolateral SON (dl-SON) from which 90-95% of neurones are vasopressinergic. SON areas that contain oxytocin (OT) cells were excluded on the basis of adjacent sections stained with a monoclonal antibody against OT. By means of an image analysis system the size of the GA and the cellular profile area were determined in dl-SON neurones with a nucleolus. Our results showed indeed an age-dependent sex difference in the size of the GA that appeared to be twice as large in young men (≤ 50 years old) than in young women of the same age. The size of the GA increased with age in women but not in men. In addition, the mean cell profile area, another measure for neuronal activity, was significantly larger in young men than in young women and was in old women larger than in young women. In conclusion, these data show the presence of a sex-dependent age-difference Continued on next page…

Journal of Neuroendocrinology (cont.)

in the activity of vasopressinergic neurones in dl-SON which may relate to differences in AVP and sex hormone levels and kidney AVP receptors.

· Ahmad Salehi · Dick Swaab ·

An increasing number of studies have appeared in the literature suggesting that Alzheimer’s disease (AD) is a hypometabolic brain disorder. Decreased metabolism in AD has been revealed by a variety of in vivo and postmortem methods and techniques including positron emission tomography and glucose metabolism. We used the size of the Golgi apparatus (GA) and cell profile area as indicators of neuronal activity in postmortem material. Using an antibody against MG-160, a sialoglycoprotein of the medial cisternae of the GA, we were able to visualize and quantify the GA area. In a series of experiments, we tried to relate neuronal metabolism to different hallmarks of AD, i.e. plaques and tangles, and also to genetic risk factors for AD like age and (apolipoprotein E) ApoE polymorphism. Our results showed that in AD there is indeed a clear reduction in brain metabolism in several severely affected brain regions including the nucleus basalis of Meynert (NBM), the CA1 area of the hippocampus and the hypothalamic tuberomamillary nucleus. However, the reduction in neuronal activity did not seem to be caused by the presence of neuropathological hallmarks of AD, i.e. plaques and tangles. There was, however, a clear relationship between the presence of ApoE ε4 alleles and a decrease in GA size. Our data suggest that decreased neuronal activity and neuropathological hallmarks of AD, such as plaques and tangles, are basically independent phenomena. Moreover, ApoE ε4 may participate in the pathogenesis of AD by decreasing neuronal metabolism. The main implication of these findings is that therapeutic strategies in AD should be focussed on reactivation of neuronal metabolism.

· Ahmad Salehi · Victoria Martinez · Dick Swaab ·

In the nucleus basalis of Meynert (NBM) we studied the presence of early cytoskeletal alterations as shown by the antibody Alz-50 in ApoE-typed patients. Using an image analysis system, the area covered by Alz-50 staining and the percentage of neurons stained by Alz-50 were determined. There were no significant differences in the area covered by Alz-50 or in the proportion of Alz-50-stained neurons in the nucleus basalis of Meynert of Alzheimer’s disease (AD) patients with one or two ApoE ε4 alleles as compared with those without any ApoE ε4 allele. However, there was a significant sex difference in Alz-50 staining: female Alzheimer’s disease patients showed more severe early cytoskeletal alterations than males. We also found a significant relationship between the number of Alz-50-stained neurons and the severity of dementia.

· Ahmad Salehi · Chris Pool · Monique Mulder ·
· Rifka Ravid · Nikolas Gonatas · Dick Swaab ·

Alzheimer’s disease (AD) is neuropathologically characterized by neuritic plaques (NPs) and neurofibrillary tangles and functionally by a decreased metabolic rate of neurons. Our previous studies showed that in brain areas which are extensively affected by plaques and tangles, i.e. the CA1 area of the hippocampus and the hypothalamic tuberomamillary nucleus, neuronal protein synthetic ability is significantly lower in AD patients than in controls. However, the presence of tangles as shown by Bodian staining appeared not to be directly related to decreased protein synthetic ability of neurons. In order to study to what extent the metabolic function of neurons might be affected by the other neuropathological hallmark of AD, i.e. NPs, which are presumed to contain neurotoxic compounds, we studied eight severely demented AD patients matched for the ApoE genotype (ApoE 3/3). Using an image analysis system, the size of the neuronal Golgi apparatus (GA) and of the cell profile area was measured as a parameter for protein synthetic activity in the CA1 area of these patients. NPs were stained by Bodian, and subsequently the distance of each neuron with an immunostained GA to the nearest NP was measured. Our results showed that neither NP density nor the distance between NPs and neurons correlated with the protein synthetic ability of neurons as judged by the size of the GA. Based on these results we suggest that in AD the presence of NPs and decreased neuronal protein synthetic ability are basically two independent phenomena.

· Ahmad Salehi · Elisabeth Dubelaar · Monique Mulder ·
· Dick Swaab ·

As reported before, the metabolic activity of nucleus basalis neurons is reduced significantly in Alzheimer patients. Because the apolipoprotein E (ApoE) ε4 genotype is a major risk factor for Alzheimer’s disease (AD), we determined whether the decrease in metabolic activity in nucleus basalis neurons in AD is ApoE-type dependent. The size of the Golgi apparatus (GA) was determined as a measure of neuronal metabolic activity in 30 controls and 41 AD patients with a known ApoE genotype by using an image analysis system in the nucleus basalis of Meynert. A polyclonal antibody directed against MG-160, a sialoglycoprotein of the GA, was used to visualize this organelle. There was a very strong reduction in the size of the GA in the nucleus basalis of AD patients. Furthermore, a strong and significant extra reduction in the size of the GA was found in the nucleus basalis neurons of AD patients with either one or two ApoE ε4 alleles compared with Alzheimer patients without ApoE ε4 alleles. Our data show that the decreased activity of nucleus basalis neurons in AD is ApoE ε4 dependent and suggest that ApoE ε4 participates in the pathogenesis of AD by decreasing neuronal metabolism.

· Ahmad Salehi · Jan Bakker · Monique Mulder ·
· Dick Swaab ·

Neurofibrillary tangles (NFTs) and neuritic plaques (NPs) are the classic neuropathological hallmarks of Alzheimer disease (AD). It is generally assumed that the pathogenic process of AD could start by local neurotoxicity induced by the β-amyloid core of plaques, followed by the appearance of NFTs and eventually cell death. To determine whether or not local neurotoxicity around NPs is indeed a major pathogenetic mechanism, we used an image analysis system to measure the neuronal density around Bodian-stained NPs in the hippocampal CA1 area of eight AD patients. Neuronal density, as measured within two arbitrary concentric circles around NPs with a radius of 74 and 123.5 μm, respectively, was on average 19% and 16% lower than the density in similar control circles without NPs in the same section. Furthermore, neuronal density around NPs was inversely related to their size. To investigate the impact of such a local reduction in cell density around NPs on the entire CA1 area, we also determined the proportion of the CA1 covered by the NPs and the arbitrary concentric circles around them. This appeared to be 16.3% of the total CA1 area, which means that the negative effect of NPs on the cell density can only explain 2.6% of cell death in the entire CA area. In conclusion, this study suggests that although NPs have a local negative effect on neighboring neurons, their contribution to the strong decrease in CA1 cell numbers is limited.

· Ahmad Salehi · Joost Verhaagen · Dick Swaab ·

Note Ahmad Salehi was also one of the editors of Neuronal degeneration and regeneration:from basic mechanisms to prospects for therapy. The book is part of the series Progress in Brain Research.

· Fred van-Leeuwen · Dominique de-Kleijn · Helma van-den-Hurk ·
· Andrea Neubauer · Marc Sonnemans · Jacqueline Sluijs ·
· Soner Köycü · Ravindra Ramdjielal · Ahmad Salehi ·
· Ahmad Salehi · Frank Grosveld · John Burbach ·
· John Burbach ·

• Alzforum
On the cover

Alzheimer’s is characterized by short-term memory loss and plaques and tangles in the cerebral cortex. Aberrant ubiquitin protein, likely resulting from an RNA mutation, is detectable in plaques (vague spots) and tangles (flame shapes, ~40 µm long) in the hippocampus (a region involved in short-term memory) from an Alzheimer’s patient. This mutation mechanism is likely a factor in nonfamilial Alzheimer’s and other neurodegenerative pathologies.

Abstract

he cerebral cortex of Alzheimer’s and Down syndrome patients is characterized by the presence of protein deposits in neurofibrillary tangles, neuritic plaques, and neuropil threads. These structures were shown to contain forms of beta amyloid precursor protein and ubiquitin-B that are aberrant (+1 proteins) in the carboxyl terminus. The +1 proteins were not found in young control patients, whereas the presence of ubiquitin-B+1 in elderly control patients may indicate early stages of neurodegeneration. Continued on next page…

Science (cont.)

The two species of +1 proteins displayed cellular colocalization, suggesting a common origin, operating at the transcriptional level or by posttranscriptional editing of RNA. This type of transcript mutation is likely an important factor in the widely occurring nonfamilial early- and late-onset forms of Alzheimer’s disease.

· Dick Swaab · Paul Lucassen · Paul Lucassen ·
· Rifka Ravid · Ahmad Salehi ·

· Paul Lucassen · F. Tilders · Ahmad Salehi ·
· Dick Swaab ·

· Ahmad Salehi · Joost Verhaagen · Paul Dijkhuizen ·
· Dick Swaab ·

It has been suggested that degeneration of neurons in Alzheimer’s disease is the result of diminished trophic support. However, so far no evidence has been forwarded that neuronal degeneration in Alzheimer’s disease is causally related to insufficient production of neurotrophins. The present study deals with (i) the expression and co-localization of tyrosine kinase receptors (trks) in the human nucleus basalis of Meynert and (ii) alterations of these receptors in Alzheimer’s disease in the nucleus basalis of Meynert, an area severely affected in Alzheimer’s disease. The expression of trkA, trkB and trkC in the nucleus basalis of Meynert of control and Alzheimer’s disease brains was studied using three polyclonal antibodies specifically recognizing the extracellular domain of trkA, trkB and trkC. Brain material of eight controls and seven Alzheimer’s disease patients was obtained at autopsy, embedded in paraffin and stained immunocytochemically. Using an image analysis system, we determined the proportion of trk neurons expressing the different trk receptors in controls and Alzheimer’s disease patients. In control brains, trkA, trkB and trkC were differentially expressed in numerous nucleus basalis of Meynert neurons. The highest proportion of neurons was found to express trkB (75%), followed by trkC (58%) and trkA (54%). Furthermore, using consecutive sections, a clear co-localization of trk receptors was observed in the same neurons. The highest degree of co-localization was observed between trkA and trkB. In Alzheimer’s disease patients, the number of immunoreactive neurons and the staining intensity of individual neurons was reduced dramatically. Continued on next page…

Neuroscience (cont.)

Reduction in the proportion of neurons expressing trkA was 69%, in trkB 47% and in trkC 49%, which indicated a differential reduction in the amount of trk receptors in Alzheimer’s disease. These observations indicate that nucleus basalis of Meynert neurons can be supported by more than one neurotrophin and that the degeneration of these neurons in Alzheimer’s disease is associated with a decreased expression of trk receptors, suggesting a decreased neurotrophin responsiveness of nucleus basalis of Meynert neurons in Alzheimer’s disease.

· Ahmad Salehi · Rifka Ravid · Nikolas Gonatas ·
· Dick Swaab ·

Numerous studies have established the key role of the Golgi apparatus (GA) in post-translational processing, transport and targeting of proteins destined for secretion, lysosomes and plasma membranes. Moreover, several studies performed in our laboratories have shown that the size of the immunocytochemically detected neuronal GA is a reliable index of neuronal activity in aging, Alzheimer’s disease (AD) and amyotrophic lateral sclerosis. It has been suggested that in AD there is decreased neuronal activity, e.g. in terms of glucose metabolism and protein synthetic capability. To further explore the hypothesis of decreased neuronal activity in AD, in this study the size of the GA was measured in pyramidal neurons of the CAI area of the hippocampus of non-demented controls and AD patients. The size of the GA was measured separately in neurons with and without neurofibrillary tangles (NFT). Moreover, in order to establish a correlation between the density of NFT and the size of the GA, the density of extraneuronal NFT was determined around each neuron and related to the size of its GA. The results, quantified by image analysis, indicate that there is a significant reduction in GA size in the neurons of the CAI area of the hippocampus of AD patients. However, there was no significant relationship between the size of the GA and the presence or absence of intracellular NFT. In addition, there was no correlation between the density of extracellular NFT and GA size of adjacent neurons. These findings are consistent with the conclusion that in AD there is evidence of decreased protein processing and secretion in the affected neurons of the CAI area of the hippocampus. Continued on next page…

Journal of Neuropathology & Experimental Neurology (cont.)

However, we failed to detect a relationship between intracellular or extracellular NFT and neuronal protein synthetic ability. These results justify the hypothesis that in AD a primary lesion is hypoactivity of neurons that is not directly linked with the development of intracellular or extracellular NFT.

· Ahmad Salehi · S. Heyn · Nikolas Gonatas ·
· Dick Swaab ·

The nucleus tuberalis lateralis (NTL) and tuberomamillary nucleus (TM), which are located close together in the tuberal region of the human hypothalamus, are differentially affected by Alzheimer’s disease (AD), In the AD, the NTL shows only early cytoskeletal alterations, i.e. pre-tangle stages, while the TM is characterised by advanced Alzheimer’s changes, e.g. neurofibrillary degeneration, senile plaques and amyloid deposition. Earlier we showed that the early cytoskeletal alterations in the NTL are not accompanied by changes in protein synthetic activity. The present study was carried out in order to measure the protein synthetic activity of the neighbouring area, the TM, which is severely affected by advanced Alzheimer changes. A polyclonal antibody against MG-160, a conserved membrane sialoglycoprotein of the Golgi apparatus, was used to stain this organelle and using an image analysis system, the size of the Golgi apparatus was measured as an index for synthetic and secretory activity in 15 Alzheimer patients and 21 controls. A significant decrease in the size of the Golgi apparatus was found in the TM neurons in AD, although the cell profile area remained unchanged. These data suggest that the protein synthetic and secretory activity of TM neurons is indeed decreased in AD.

· Ahmad Salehi · Ahmad Salehi · Michel Hofman ·
· Nikolas Gonatas · Dick Swaab ·

The nucleus tuberalis lateralis (NTL) is located in the basolateral part of the hypothalamus and is only present as a well-delineated nucleus in human and higher primates. In Alzheimer’s disease (AD), NTL neurons show strong early cytoskeletal alterations, as revealed by the antibody Alz-50, but practically no senile plaques or neurofibrillary tangles. To study whether the activity of NTL neurons decreases when cytoskeletal changes appear, i.e., during aging and in AD, we applied a polyclonal antibody raised against the medial cisternae of the Golgi apparatus (GA). The size of the GA and the cell profile of NTL neurons, two established parameters for neuronal activity, were measured by an image analysis system. No significant change in the size of the profiles of the GA or of the neurons was observed in this nucleus during aging or AD. Earlier studies have shown that there is no decrease in cell number in the NTL in AD. We conclude that in the NTL an early hallmark of AD, i.e., cytoskeletal changes as stained by Alz-50, does not correlate with decreased neuronal activity, as reflected by the size of the GA, nor with a decrease in cell number. In addition, we found that the very early occurring and abundant presence of lipofuscin in NTL neurons does not go together with decreased neuronal activity.

· Paul Lucassen · Ahmad Salehi · Chris Pool ·
· Nikolas Gonatas · Dick Swaab ·

The supraoptic (SON) and paraventricular nuclei (PVN) of the human hypothalamus are production sites of vasopressin (AVP) and oxytocin (OXT). Although the hypothalamus is affected in Alzheimer’s disease (AD), previous work has not only shown that in these two nuclei no neurons are lost, neither during aging nor in AD, but that the number of AVP-expressing neurons and their nucleolar size had even increased with age. These observations indicated that the peptide synthesis of the AVP neurons was activated in the oldest age-groups. Recently published, qualitative observations, using the area of the Golgi Apparatus (GA) as a sensitive parameter for neurosecretory activity, confirmed the activation of SON and PVN neurons with age in human; however, in this report the neurons were not identified according to their neuropeptide content. In the present quantitative study we determined whether the AVP neurons were indeed activated as a result of the aging process in controls and AD patients. We applied a polyclonal antiserum directed against the medial cisternae of the GA on formalin-fixed, paraffin-embedded tissue sections taken from the dorsolateral SON (dl-SON) of 10 controls and 10 AD patients, and performed our measurements in this area that is known to be predominantly occupied (90–95%) by AVP neurons. In addition, the sparse OXT cells present in the area of study, were excluded from the measurements on the basis of alternative sections stained for OXT. In the dl-SON, the area occupied by the GA and the cellular profile area per patient were quantified by means of image analysis. Continued on next page…

Journal of Neuroendocrinology (cont.)

The results show a significant increase in GA area with age in controls and in AD, demonstrating an activation of the AVP neurons in the dl-SON of the human hypothalamus in these two conditions. No changes were observed in the cellular profile areas with age, neither in the controls nor in AD, suggesting that the GA area is a much more sensitive parameter for monitoring activity changes in post-mortem material than neuronal size. It is proposed that this activation of AVP cells with age, which has been suggested to be a compensatory response to the age-related loss of AVP receptors in the kidney, might be the basis of the stability of these neurons in aging and AD.