Axonal Transport of Lysosomes Is Unaffected in Glucocerebrosidase-Inhibited iPSC-Derived Forebrain Neurons.

Lysosomes are acidic organelles that traffic throughout neurons delivering catabolic enzymes to distal regions of the cell and maintaining degradative demands. Loss of function mutations in the gene GBA encoding the lysosomal enzyme glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher's disease (GD) and are the most common genetic risk factor for synucleinopathies like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). GCase degrades the membrane lipid glucosylceramide (GlcCer) and mutations in GBA, or inhibiting its activity, results in the accumulation of GlcCer and disturbs the composition of the lysosomal membrane. The lysosomal membrane serves as the platform to which intracellular trafficking complexes are recruited and activated. Here, we investigated whether lysosomal trafficking in axons was altered by inhibition of GCase with the pharmacological agent Conduritol B Epoxide (CBE). Using live cell imaging in human male induced pluripotent human stem cell (iPSC)-derived forebrain neurons, we demonstrated that lysosomal transport was similar in both control and CBE-treated neurons. Furthermore, we tested whether lysosomal rupture, a process implicated in various neurodegenerative disorders, was affected by inhibition of GCase. Using L-leucyl-L-leucine methyl ester (LLoME) to induce lysosomal membrane damage and immunocytochemical staining for markers of lysosomal rupture, we found no difference in susceptibility to rupture between control and CBE-treated neurons. These results suggest the loss of GCase activity does not contribute to neurodegenerative disease by disrupting either lysosomal transport or rupture.

GSK3ß Impairs KIF1A Transport in a Cellular Model of Alzheimer's Disease but Does Not Regulate Motor Motility at S402.

Impairment of axonal transport is an early pathologic event that precedes neurotoxicity in Alzheimer's disease (AD). Soluble amyloid-ß oligomers (AßOs), a causative agent of AD, activate intracellular signaling cascades that trigger phosphorylation of many target proteins, including tau, resulting in microtubule destabilization and transport impairment. Here, we investigated how KIF1A, a kinesin-3 family motor protein required for the transport of neurotrophic factors, is impaired in mouse hippocampal neurons treated with AßOs. By live cell imaging, we observed that AßOs inhibit transport of KIF1A-GFP similarly in wild-type and tau knock-out neurons, indicating that tau is not required for this effect. Pharmacological inhibition of glycogen synthase kinase 3ß (GSK3ß), a kinase overactivated in AD, prevented the transport defects. By mass spectrometry on KIF1A immunoprecipitated from transgenic AD mouse brain, we detected phosphorylation at S402, which conforms to a highly conserved GSK3ß consensus site. We confirmed that this site is phosphorylated by GSK3ß in vitro Finally, we tested whether a phosphomimic of S402 could modulate KIF1A motility in control and AßO-treated mouse neurons and in a Golgi dispersion assay devoid of endogenous KIF1A. In both systems, transport driven by mutant motors was similar to that of WT motors. In conclusion, GSK3ß impairs KIF1A transport but does not regulate motor motility at S402. Further studies are required to determine the specific phosphorylation sites on KIF1A that regulate its cargo binding and/or motility in physiological and disease states.

The role of selective transport in neuronal protein sorting.

To assess whether selective microtubule-based vesicle transport underlies the polarized distribution of neuronal proteins, we expressed green fluorescent protein- (GFP-) tagged chimeras of representative axonal and dendritic membrane proteins in cultured hippocampal neurons and visualized the transport of carrier vesicles containing these proteins in living cells. Vesicles containing a dendritic protein, transferrin receptor (TfR), were preferentially transported into dendrites and excluded from axons. In contrast, vesicles containing the axonal protein NgCAM (neuron-glia cell adhesion molecule) were transported into both dendrites and axons. These data demonstrate that neurons utilize two distinct mechanisms for the targeting of polarized membrane proteins, one (for dendritic proteins) based on selective transport, the other (for axonal proteins) based on a selectivity "filter" that occurs downstream of transport.

Lymphoproliferative responses to mouse mammary tumor virus in lymphocyte subsets of breast cancer patients.

Altered immunologic reactions were observed in breast cancer patients as compared to those in normal subjects. Lymphoproliferative responses to murine mammary tumor virus (MuMTV) were significantly enhanced in peripheral blood mononuclear cells from patients with metastatic disease. These reactivities occurred with mammary tumor virus purified from either mouse milk or infected feline kidney cells but not with Rauscher murine leukemia virus. For the assessment of the role of peripheral blood mononuclear leukocyte subpopulations in the responsiveness to MuMTV, the cell preparations were fractionated according to their ability to form spontaneous rosettes with sheep red blood cells (E-rosettes). The effectiveness of the separation was ascertained by means of cell surface markers, i.e., presence of surface immunoglobulins or a T-cell marker. Leu-1 antigen, and mitogen-induced blastogenesis. The responsiveness to the MuMTV antigen(s) was associated with the T-cell subset, identified as the E-rosetting. Leu-1-positive, and surface immunoglobulin-negative population. Although some subjects with the normal population gave positive reactions, the results reveal an apparent association between high levels of responsiveness to MuMTV within the T-lymphocyte subset and breast cancer disease.

Maximizing differences in the concanavalin A-induced blastogenic responses of lymphocytes from breast cancer patients and controls by the use of alpha-methyl-D-mannoside.

In an attempt to magnify differences in the immune responses of potentially immunosuppressed cancer patients and normal controls, an assessment was made on the effects of the competitive inhibitor alpha-methyl-D-mannoside on the concanavalin A (Con A)-induced blastogenic responses of lymphocytes from each of these populations. Lymphocytes from breast cancer patients with metastatic disease were significantly deficient in their capability to undergo blast transformation regardless of whether the monosaccharide inhibitor was added to the assay cultures. In contrast, lymphocytes from breast cancer patients who did not display metastatic disease were capable of normal blastogenic responses to Con A. The addition of alpha-methyl-D-mannoside to lymphocyte cultures caused a significantly greater inhibition of the blastogenic responses of these patients' cells as compared to cells of normal controls. Thus the monosaccharide seems to serve as a useful reagent for optimizing differences between lymphocyte blastogenic responses of normal donors and those of immunodepressed donors. The results suggest that lymphocytes from breast cancer patients without clinically evident metastases possess some modification of their cell membrane. One possibility discussed was that the number or distribution of receptors for Con A on the membrane of lymphocytes of these patients is deficient.

Erratum: Intracellular amyloid ß oligomers impair organelle transport and induce dendritic spine loss in primary neurons.

The original version of this article unfortunately contained a mistake in the presentation of Fig. 1 in both the PDF and HTML versions of this manuscript [1]. In the right panel of the corrected Fig. 1d, the images of Mock cells, which were visualized with GFP and stained with Abeta oligomer-specific antibody 11A1, were replaced with those of APPWT cells, and instead the images of APPWT cells were replaced with those of Mock cells. These images had been incorrectly placed in the original Fig. 1. The correct version of Fig. 1 is presented below.

Characterization of Caenorhabditis elegans lectin-binding mutants.

We have identified 45 mutants of Caenorhabditis elegans that show ectopic surface binding of the lectins wheat germ agglutinin (WGA) and soybean agglutinin (SBA). These mutations are all recessive and define six genes: srf-2, srf-3, srf-4, srf-5, srf-8 and srf-9. Mutations in these genes fall into two phenotypic classes: srf-2, -3, -5 mutants are grossly wild-type, except for their lectin-binding phenotype; srf-4, -8, -9 mutants have a suite of defects, including uncoordinated movement, abnormal egg laying, and defective copulatory bursae morphogenesis. Characterization of these pleiotropic mutants at the cellular level reveals defects in the migration of the gonadal distal tip cell and in axon morphology. Unexpectedly, the pleiotropic mutations also interact with mutations in the lin-12 gene, which encodes a putative cell surface receptor involved in the control of cell fate. We propose that the underlying defect in the pleiotropic mutations may be in the general processing or secretion of extracellular proteins.

Hippocampal plasticity involves extensive gene induction and multiple cellular mechanisms.

Long-term plasticity of the central nervous system (CNS) involves induction of a set of genes whose identity is incompletely characterized. To identify candidate plasticity-related genes (CPGs), we conducted an exhaustive screen for genes that undergo induction or downregulation in the hippocampus dentate gyrus (DG) following animal treatment with the potent glutamate analog, kainate. The screen yielded 362 upregulated CPGs and 41 downregulated transcripts (dCPGs). Of these, 66 CPGs and 5 dCPGs are known genes that encode for a variety of signal transduction proteins, transcription factors, and structural proteins. Seven novel CPGs predict the following putative functions: cpg2--a dystrophin-like cytoskeletal protein; cpg4--a heat-shock protein: cpg16--a protein kinase; cpg20--a transcription factor; cpg21--a dual-specificity MAP-kinase phosphatase; and cpg30 and cpg38--two new seven-transmembrane domain receptors. Experiments performed in vitro and with cultured hippocampal cells confirmed the ability of the cpg-21 product to inactivate the MAP-kinase. To test relevance to neural plasticity, 66 CPGs were tested for induction by stimuli producing long-term potentiation (LTP). Approximately one-fourth of the genes examined were upregulated by LTP. These results indicate that an extensive genetic response is induced in mammalian brain after glutamate receptor activation, and imply that a significant proportion of this activity is coinduced by LTP. Based on the identified CPGs, it is conceivable that multiple cellular mechanisms underlie long-term plasticity of the nervous system.

Haplotypes of four novel single nucleotide polymorphisms in the nicotinic acetylcholine receptor beta2-subunit (CHRNB2) gene show no association with smoking initiation or nicotine dependence.

Several types of evidence, including experiments with mice that lack the nicotinic acetylcholine receptor beta2-subunit gene (CHRNB2), have suggested that a beta2-containing nicotinic receptor is necessary for at least some of the reinforcing properties of nicotine. However, sequence variations in CHRNB2 have not been reported, and its role in influencing human smoking behavior and nicotine dependence is not known. We screened most of the introns and exons and found five novel single nucleotide polymorphisms (SNPs). We tested four of these SNPs in three large, carefully selected samples: nonsmokers (n = 317) and regular smokers low levels of nicotine dependence (ND, n = 238), or smokers with high-ND (n = 317). None of the four polymorphisms we tested, nor their estimated haplotypes, were associated with smoking initiation or progression to nicotine dependence.

An association study of DRD5 with smoking initiation and progression to nicotine dependence.

A large body of genetic epidemiological data strongly implicate genetic factors in the etiology of smoking behavior. Polymorphisms of genes in the dopaminergic system are plausible functional candidate genes and a linkage and an association study suggested that the type 5 dopamine receptor gene (DRD5) may be etiologically involved. We investigated the association of four DRD5 polymorphisms with smoking initiation and progression to nicotine dependence in a population-based sample of over 900 subjects. For smoking initiation, there was no significant association with the four DRD5 markers we studied; however, maximum likelihood analyses suggested the presence of a haplotype protective against smoking initiation. For progression to nicotine dependence, there were no strongly significant associations with the four DRD5 markers or for the estimated haplotypes. These data are not consistent with a strong etiological role for DRD5 in the etiology of these complex smoking behaviors.

Use of antipsychotic drugs in nursing homes: current compliance with OBRA regulations.

OBJECTIVE: To examine the degree and patterns of compliance with the Omnibus Budget Reconciliation Act (OBRA) regulations regarding the use of antipsychotic drugs in nursing homes. DESIGN: Retrospective chart review of all resident records. PARTICIPANTS: Eight nursing homes: five community, two county-owned, and one university-affiliated Veterans Administration facility. MEASUREMENTS: A structured assessment instrument to track compliance with each aspect of the OBRA regulations regarding antipsychotic drug use. RESULTS: A total of 1573 nursing home residents' pharmacy records were reviewed between August 1994 and March 1996. Two hundred seventy-nine residents were actively taking antipsychotic medications (prevalence = 17.7%). Mean compliance greater than 70% was found for (1) appropriate diagnostic indication (mean = 70.9%), (2) dosage within recommended limits (mean = 90.1%), and 3) documented appropriate target symptoms (mean = 90.4%). Dosages were more likely to exceed limits in those patients with histories of major mental illness, particularly schizophrenia. CONCLUSIONS: Nursing homes were better able to comply with those guidelines that are most specific. Educational interventions now need to focus on behavioral interventions, monitoring of adverse effects, and efficacy. These data are useful in establishing threshold levels of performance and can be used by nursing homes for continuous quality improvement. OBRA continues to impact neuroleptic drug prescribing practices in nursing homes significantly.

Inappropriate medication prescribing in homebound older adults.

OBJECTIVES: Little is known about the prescribing of medications in the growing population of homebound older adults. We report on the prevalence and pattern of inappropriate medications in a nursing home-eligible, homebound population. DESIGN: A cross-sectional design. SETTING: A managed care plan for individuals meeting nursing home eligibility. PARTICIPANTS: 2193 homebound people older than age 60. MEASUREMENTS: We reviewed the pharmacy profiles of all older homebound enrollees. We identified the average number of medications per patient and the most commonly prescribed classes of drugs. The medication profiles were also analyzed in the context of the 26 drugs/groups listed as inappropriate by the explicit criteria of Beers [Arch Intern Med 1997; 157:1531-1536]. RESULTS: A total of 2193 people aged 60 to 106 (mean 82.8 +/- 8.8) were taking an average of 5.3 +/- 2.9 drugs (range 0-22). Cardiac drugs and benzodiazepines were the medications most commonly prescribed. We found 1152 of the total 11,689 prescriptions (9.9%) to be inappropriate. Eight hundred seventy-one (39.7%) of these 2193 residents had at least one inappropriate prescription, and 230 (10.4%) had two or more. Of particular concern were 285 people prescribed excessive doses of temazepam and zoldipem, 211 people taking first-generation antihistamines, 115 taking doxepin or amitriptyline, 106 taking an ergoloid, 98 taking dipyridamole, and 85 prescribed a long-acting benzodiazepine. CONCLUSIONS: Our study revealed a high prevalence of psychotropic medications and inappropriate drug use among older homebound residents, a group that is at the highest risk for adverse drug reactions. Because this group is not subject to oversight by regulatory agencies, further interventional studies and provider education will be important.

Cancer screening in the elderly population.

This article reviews the current state of knowledge regarding cancer screening in the geriatric population. Care of the elderly requires knowledge of underlying physiologic changes, comorbidities, quality-of-life factors, and life expectancies. There is always the danger that ageism may prevent elderly cancer patients from receiving the proper treatment. On the other hand, overzealous treatment can lead to adverse results if elderly patients are not properly targeted based on current evidence of the benefits and risks of specific screening practices.

Defining patterns of benzodiazepine use in older adults.

Benzodiazepines are disproportionately prescribed to older adults. Elderly adults with comorbid medical and psychiatric conditions, elderly adults taking multiple medications, and elderly women are the most likely adults to continuously use benzodiazepines. These are also the groups of elderly who are likely to experience adverse effects, including falls, accidents, and motor vehicle crashes. Despite recommendations for short-term treatment and the potential risks of long-term use, some patients continue to receive benefit for extended time periods, occasionally years. Future research needs to be directed at improved identification of which patients will benefit from intermittent versus continuous treatment while minimizing risk for adverse side effects. In order to advance the study of the risks and benefits of benzodiazepine use, we have proposed a set of definitions for classification of use. These definitions can be used to develop clinical guidelines based on empirically derived clinical research models.

Community formation and community roles among persons with Alzheimer's disease: a comparative study of experiences in a residential Alzheimer's facility and a traditional nursing home.

Based on an ethnographic study in a residential Alzheimer's facility and a traditional nursing home, this article discusses the process of community formation and the maintenance of community roles among individuals suffering from dementia in institutional settings. These include: therapeutic programming that promotes resident independence and choice; flexible and person-centered staff roles; and a physical environment that facilitates social interaction, autonomy, and participation in the activities of daily living. In contrast, institutional programs that are regimented, that follow a medical rather than a social model of care, and that take place in physical environments that have limited options may discourage resident interaction and social bonding, thus inhibiting community formation. Although Alzheimer's disease and other forms of dementia may create difficulties for the realization of community and community roles among institutionalized people, more significant are the environmental conditions in which such individuals live and the programs designed for their care.

Medical readiness training exercise in El Salvador, Central America, 1996.

Medical Readiness Training Exercises (MEDRETEs) can be a valuable training tool for U.S. Army personnel in remote areas of deployment. We report our experience of the MEDRETE in El Salvador in 1996. Working with foreign physicians of the host country was a positive experience in which we learned local customs and mutual cooperation. Evaluation and treatment of nearly 6,000 patients increased goodwill in the community and provided an opportunity for teamwork for Army Reserve medical units, including increased discussions of public health issues. We also report on the field applicability of the advanced laboratory and pharmacy equipment we included in our training.

Evaluation of Army field sanitation during Blazing Trails '87.

US medical personnel serving in Ecuador analyzed how the field sanitation procedures of troops employed in Blazing Trails '87 had impact on living conditions and overall health of the troops. Such a situation allowed evaluation of USAR troop training and preparedness in field sanitation. Results indicate that field sanitation training needs to be improved at the unit level.

Biochemical Analysis of Caenorhabditis elegans Surface Mutants.

A collection of Caenorhabditis elegans mutants that show ectopic surface lectin binding (Srf mutants) was analyzed to determine the biochemical basis for this phenotype. This analysis involved selective removal or labeling of surface components, specific labeling of surface glycans, and fractionation of total protein with subsequent detection of wheat germ agglutinin (WGA) binding proteins. Wild-type and mutant nematodes showed no differences in their profiles of extractable surface glycoproteins or total WGA-binding proteins, suggesting that the ectopic lectin binding does not result from the novel expression of surface glycans. Instead, these results support a model in which ectopic lectin binding results from an unmasking of glycosylated components present in the insoluble cuticle matrix of wild-type animals. To explain the multiple internal defects found in some surface mutants, we propose that these mutants have a basic defect in protein processing. This defect would interfere with the expression of the postulated masking protein(s), as well as other proteins required for normal development.

Cognitive development and female psychology.

Freud was an astute observer, but his understanding of female psychology was distorted by centration on biological forces, the adult neurosis, libido theory, the Oedipus complex, and male psychology. His views on the role of penis envy, narcissism, and masochism in women, and on the female superego, currently are being updated on the basis of new data emerging out of psychoanalytic investigation and direct observational studies. It has become clear, for example, that core feminine gender identity develops very early and that it is mediated in large part by cognitive development and learning rather than by the impact of the observation of genital differences between the sexes. The little girl's level of cognitive organization does not permit her at first to construct logical, consistent categories of female and male, predicated or coordination of criteria of inclusion and exclusion. She puts girls and women into one group out of intuitive recognition that they "go together" and builds up a view of herself as female on the basis of what she feels as her mother's daughter and what she perceives of the way her parents and other significant object see her. Her mother's feelings about her, about herself, and about females in general influence her greatly, and her father's responses to and expectations of her have an important impact on her self-image and self-esteem. Her representation of herself as female at first does not necessarily take genital differences between the sexes into account. The discovery, some time in the second or third year, that the genitals of boys and girls are different from one another presents the girl with a number of problems, however. Her cognitive organization makes it extremely difficult for her to apprehend her complex, incompletely demarcated, and to a large extent internal, unobservable, and impalpable sexual organs. Her initial conclusion is that she lacks desirable body parts which are possessed by other beings. Because of the narcissistic-exhibitionistic libidinal organization of that period of life, this represents a painful narcissistic injury. The intensity of the girl's castration reaction and the degree to which she will be able to overcome it in the course of further development are determined by multiple internal and external developmental factors. Masochism, narcissistic vulnerability, and penis envy are neither limited to the female sex nor normal outcomes of female development. Where they are prominent features in the psychopathology of female patients, it is insufficient to interpret them as emanating from penis envy as a bedrock, primal cause. Detailed clinical examples are presented to illustrate the thesis that through exploration and explication of the multiple sources of such symptomatology in a female analysand is the most effective way of relieving the symptomatology and releasing the potential for emotional growth.

Identification in healthy and pathological character formation.

The character of the ego is built up via a series of identifications carried out in the course of development. As infants are forced by experience to give up the 'internal illusion' of primal identification with the powerful mother, the sense of helplessness that ensues leads to secondary identification to create an 'external illusion' of oneness with the mother during heightened stress or tension. Adequate experiences, leading to increasing trust and tolerance of frustration and tension, promote ego structuralization, self-object differentiation, and oedipal, triadic object relations. Inadequate experiences lead to pathological identifications that interfere with ego development. Oedipal conflicts produce new problems, which are dealt with in part by complex identifications that contribute extensively to the child's personality structure. Superego crystallization derives largely from these later identifications. In optimal circumstances, the identifications out of which the child's character is built become reworked and modified so that it becomes increasingly unique and independent of its sources in others. Stability is never absolute, however. Under stress, reversion is possible to dependence on powerful, charismatic leaders that offers return to the illusion of identificatory union with a powerful, protective, parental 'other'. This can be exploited by potentially dangerous, destructive leaders.