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.

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.

KIF1A is the primary anterograde motor protein required for the axonal transport of dense-core vesicles in cultured hippocampal neurons.

Dense-core vesicles (DCVs) are responsible for transporting, processing, and secreting neuropeptide cargos that mediate a wide range of biological processes, including neuronal development, survival, and learning and memory. DCVs are synthesized in the cell body and are transported by kinesin motor proteins along microtubules to pre- and postsynaptic release sites. Due to the dependence on kinesin-based transport, we sought to determine if the kinesin-3 family member, KIF1A, transports DCVs in primary cultured hippocampal neurons, as has been described for invertebrate neurons. Two-color, live-cell imaging showed that the DCV markers, chromogranin A-RFP and BDNF-RFP, move together with KIF1A-GFP in both the anterograde and retrograde directions. To demonstrate a functional role for KIF1A in DCV transport, motor protein expression in neurons was reduced using RNA interference (shRNA). Fluorescently tagged DCV markers showed a significant reduction in organelle flux in cells expressing shRNA against KIF1A. The transport of cargo driven by motors other than KIF1A, including mitochondria and the transferrin receptor, was unaffected in KIF1A shRNA expressing cells. Taken together, these data support a primary role for KIF1A in the anterograde transport of DCVs in mammalian neurons, and also provide evidence that KIF1A remains associated with DCVs during retrograde DCV transport.

Expression of kinesin superfamily genes in cultured hippocampal neurons.

The nature of the different kinesin family members that function in a single, specific neuron type has not been systematically investigated. Here, we used quantitative real-time PCR to analyze the developmental expression patterns of kinesin family genes in cultured mouse hippocampal neurons, a highly homogeneous population of nerve cells. For purposes of comparison, we also determined the set of kinesins expressed in embryonic and adult hippocampal tissue. Twenty kinesins are expressed at moderate-to-high levels in mature hippocampal cultures. These include 9 plus-end directed kinesins from the Kinesin-1, -2, and -3 families that are known to mediate organelle transport and 6 other members of the Kinesin-3 and -4 families that are candidate organelle motors. Hippocampal cultures express high levels of a Kinesin-13, which regulates microtubule depolymerization, and moderate-to-high levels of Kinesin-9 and -14 family members, whose functions are not understood. Twelve additional kinesins, including 10 known mitotic kinesins, are expressed at moderate levels in embryonic hippocampus but at very low levels in mature cultures and the adult hippocampus. Collectively, our findings suggest that kinesins subserve diverse functions within a single type of neuron.

Dynactin regulates bidirectional transport of dense-core vesicles in the axon and dendrites of cultured hippocampal neurons.

A critical aspect of nerve cell function is peptidergic secretion involving the packaging, transport, and processing of a large group of peptide hormones and other signaling molecules, e.g. brain-derived neurotrophic factor (BDNF). Dense-core vesicles (DCVs) are the organelles that transport these molecules to release sites in both the axon and dendrites of pyramidal neurons. DCVs exhibit complex transport behavior, where these organelles move bidirectionally, reverse direction, pause intermittently, and vary in velocities and run lengths. A key objective in the field of organelle transport is to define the molecules that mediate transport. This study investigated the role of dynactin, a putative opposite-polarity motor coordinator, in the microtubule-based transport of DCVs in primary cultured hippocampal neurons. First, by live cell imaging, we showed similar microtubule-based transport of BDNF, neuropeptide Y (NPY), and tissue plasminogen activator (tPA), consistent with the co-packaging of these DCV cargoes. However, we found higher DCV velocities in both the axon and dendrites than those of previous neuronal studies likely due to faster image acquisition times. Then, using well-characterized dynactin disruptors we demonstrate the need for dynactin in bidirectional transport where overexpression of both p50/dynamitin and the first coiled-coil domain of p150(Glued) (CC1) reduces the flux of DCVs in both directions in the axon and dendrites. We also observed that only CC1 reduces axonal and dendritic run lengths. These results suggest different functions for p50 and p150 in the dynactin complex in DCV transport. These findings are significant because they demonstrate that dynactin functions as a motor coordinator for the transport of DCVs in primary cultured rat hippocampal neurons.

Motifs that mediate dendritic targeting in hippocampal neurons: a comparison with basolateral targeting signals.

One model for dendritic protein sorting in neurons is based on parallels with basolateral targeting in Madin-Darby Canine Kidney (MDCK) epithelial cells. The goal of this study was to further evaluate this model by analyzing the neuronal targeting of several proteins that contain well-defined basolateral sorting motifs. When we expressed FcRgammaII-B2 and CD44, two basolateral markers whose sorting depends on dihydrophobic motifs, they were unpolarized in hippocampal neurons. We also assessed the localization of the Epidermal Growth Factor Receptor (EGFR), a basolateral protein whose sorting signal contains a proline-rich motif and two dihydrophobic motifs. EGFR was restricted to the dendrites in neurons and relied on the same sorting signal for proper targeting. These results show that the dendritic sorting machinery in neurons does not recognize dihydrophobic-based basolateral sorting signals. In contrast, the sorting signal present in EGFR directs both basolateral and dendritic targeting and defines a novel dendritic targeting motif.

Regulation of peptidergic vesicle mobility by secretagogues.

Neuropeptides are released into the extracellular space from large secretory granules. In order to reach their release sites, these granules are translocated on microtubules and thought to interact with filamentous actin as they approach the cell membrane. We have used a green fluorescent protein-tagged neuropeptide prohormone (prepro-orphanin FQ) to visualize vesicle trafficking dynamics in NS20Y cells and cultures of primary hippocampal neurons. We found that the majority of secretory granules were mobile and accumulated at both the tips of neurites as well as other apparently specialized cellular sites. We also used live-cell imaging to test the notion that peptidergic vesicle mobility was regulated by secretagogues. We show that treatment with forskolin appeared to increase vesicle rates of speed, while depolarization with high K+ had no effect, even though both treatments stimulated neuropeptide secretion. In cultured hippocampal neurons the green fluorescent protein-tagged secretory vesicles were routed to both dendrites and axons, indicating that peptidergic vesicle transport was not polarized. Basal peptidergic vesicle mobility rates in hippocampal neurons were the same as those in NS20Y cells. Taken together, these studies suggest that secretory vesicle mobility is regulated by specific classes of secretagogues and that neuropeptide containing secretory vesicles may be released from dendritic structures.

Sorting and directed transport of membrane proteins during development of hippocampal neurons in culture.

Hippocampal neurons in culture develop morphological polarity in a sequential pattern; axons form before dendrites. Molecular differences, particularly those of membrane proteins, underlie the functional polarity of these domains, yet little is known about the temporal relationship between membrane protein polarization and morphological polarization. We took advantage of viral expression systems to determine when during development the polarization of membrane proteins arises. All markers were unpolarized in neurons before axonogenesis. In neurons with a morphologically distinguishable axon, even on the first day in culture, both axonal and dendritic proteins were polarized. The degree of polarization at these early stages was somewhat less than in mature cells and varied from cell to cell. The cellular mechanism responsible for the polarization of the dendritic marker protein transferrin receptor (TfR) in mature cells centers on directed transport to the dendritic domain. To examine the relationship between cell surface polarization and transport, we assessed the selectivity of transport by live cell imaging. TfR-green fluorescent protein-containing vesicles were already preferentially transported into dendrites at 2 days, the earliest time point we could measure. The selectivity of transport also varied somewhat among cells, and the amount of TfR-green fluorescent protein fluorescence on intracellular structures within the axon correlated with the amount of cell surface expression. This observation implies that selective microtubule-based transport is the primary mechanism that underlies the polarization of TfR on the cell surface. By 5 days in culture, the extent of polarization on the cell surface and the selectivity of transport reached mature levels.

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.

The frequency of blood pressure measurements in children in four EDs.

The study's objective was to assess the frequency of triage blood pressure measurements in pediatric patients and the recognition of an elevated blood pressure. The design was retrospective and included chart review. The setting consisted of four emergency departments associated with one medical school, including one level I academic center, two level II Community departments, and a regional children's hospital. A convenience sample of 437 patients, aged 1 month to 18 years, was selected. The frequency of triage blood pressure measurements was recorded. The number of patients whose blood pressure was higher than the 90th percentile for age and sex as established by the Second Task Force on Blood Pressure Control in Children was also recorded. The frequency of a second blood pressure measurement in patients with an elevated initial blood pressure was recorded. All frequency data were stratified by hospital and by age group. The results showed 294/437 (66%) of children had blood pressures measured at triage. Of these measurements, 153/294 (52%) reflected blood pressures greater than the 90th percentile for age and sex, but only 58/153 (38%) of patients with such blood pressures had a second blood pressure measured. Hospitals varied in their frequency of blood pressure measurement. Adolescents had their blood pressure measured more frequently, 981105 (93%) than two to 12-year-olds, 144/185 (78%) or 1-month to 2-year-olds, 52/147 (35%). Frequency of triage blood pressure measurements in children varied by institution and increased in frequency with age.

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.

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.

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.

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.

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.

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.

Susceptibility genes for nicotine dependence: a genome scan and followup in an independent sample suggest that regions on chromosomes 2, 4, 10, 16, 17 and 18 merit further study.

Cigarette smoking is associated with considerable morbidity, mortality, and public health costs. Genetic factors influence both smoking initiation and nicotine dependence, but none of the genes involved have been identified. A genome scan using 451 markers was conducted to identify chromosomal regions linked to nicotine dependence in a collection of 130 families containing 343 genotyped individuals (308 nicotine-dependent) from Christchurch, New Zealand. By pairwise analysis, the best result was with marker D2S1326 which gave a lod score under heterogeneity (H-LOD) of 2.63 (P=0.0012) and a nonparametric linkage (NPL, Zall) score of 2.65 (P=0.0011). To identify regions that warranted further study, rather than comparing the pairwise scores from the scan to theoretical thresholds, we compared them to an empirical baseline, found here to be H-LOD scores of 0.5 and Zall scores of 1.0. We also found a number of large (31-88 cM) regions where many (8-16) consecutive markers yielded small but positive Zall scores. Selected regions of chromosomes 2, 4, 10, 16, 17 and 18 were investigated further by additional genotyping of the Christchurch sample and an independent sample from Richmond, Virginia (91 families with 264 genotyped individuals, 211 nicotine-dependent). Multipoint nonparametric analysis showed the following maximums for the Christchurch sample: Chr. 2 (Zlr=2.61, P=0.005), Chr. 4 (Zlr=1.36, P=0.09), Chr. 10 (Zlr=2.43, P=0.008), Chr. 16 (Zlr=0.85, P=0.19), Chr. 17 (Zlr=1.64, P=0.05), Chr. 18 (Zlr=1.54, P=0.06). Analysis of the Richmond sample showed the following maximums: Chr. 2 (Zlr=1.00, P=0.15), Chr. 4 (Zlr=0.39, P=0.34), Chr. 10 (Zlr=1.21, P=0.11), Chr. 16 (Zlr=1.11, P=0.13), Chr. 17 (Zlr=1.60, P=0.05), Chr. 18 (Zlr=1.33, P=0.09). It is probable that the small samples used here provided only limited power to detect linkage. It may have been difficult therefore to detect genes of small effect, or those that are influencing risk in only a small proportion of the families. When simply judged against the usual standards of linkage significance, none of the individual regions yielded strong evidence in either sample. Some or all of the most positive results in the genome scan of the Christchurch sample, therefore, could be due to chance. However, the presence in the Christchurch scan of multiple large regions containing many consecutive positive markers, coupled with the relatively positive results in these same regions in the Richmond sample, suggests that some of these regions may contain genes influencing nicotine dependence and therefore deserve further study.