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Subacute combined degeneration (SCD) is caused by demyelination of spinal cord white matter secondary to vitamin B12 (cobalamin) deficiency leading to core symptoms of spastic paresis and vibratory and proprioceptive deficits. Most common causes of B12 deficiency revolve around malabsorption and pernicious anemia; however, nitrous oxide (N2O) can also indirectly cause B12 deficiency by inactivating its biologically active form. We report a case of a patient who took advantage of the unregulated N2O market and presented with signs and symptoms of SCD secondary to N2O abuse. Prior to symptom onset, the patient reported approximately 3,000g of N2O inhalation within five days prior to symptom onset in addition to daily use three weeks prior. Work up revealed laboratory and imaging abnormalities consistent with SCD, although B12 levels were normal intrinsic-factor-blocking (IFB) antibodies were present. Appropriate treatment was undertaken, and the patient was followed up at one week and one month with noticeable clinical improvements. Similarities of this patient to literature include the classic presenting symptoms of SCD and the gradual symptomatic improvement with B12 injections and N2O abstinence. This case is remarkable due to SCD occurrence after recreational N2O abuse, objective quantification of N2O intake over a specified time period to induce SCD, occurrence secondary to N2O inhalation, positive IFB antibodies, and symptomatic presentation with B12 values within normal limits. This report highlights the dangers associated with N2O abuse and moving forward awareness of this case can be referenced to aid in educating members of our communities at risk for substance abuse.

Recently, missense mutations in the gene TARDBP encoding TDP-43 have been linked to familial ALS. The discovery of genes encoding these RNA binding proteins, such as TDP-43 and FUS/TLS, raised the notion that altered RNA metabolism is a major factor underlying the pathogenesis of ALS. To begin to unravel how mutations in TDP-43 cause dysfunction and death of motor neurons, investigators have employed both gain- and loss-of-function studies in rodent model systems. Here, we will summarize major findings from the initial sets of TDP-43 transgenic and knockout rodent models, identify their limitations, and point to future directions toward clarification of disease mechanism(s) and testing of therapeutic strategies that ultimately may lead to novel therapy for this devastating disease. This article is part of a Special Issue entitled RNA-Binding Proteins.

γ-Secretase, a multisubunit transmembrane protease comprised of presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective one, participates in the regulated intramembrane proteolysis of Type I membrane proteins including the amyloid precursor protein (APP). Although Aph-1 is thought to play a structural role in the assembly of γ-secretase complex and several transmembrane domains (TMDs) of Aph-1 have been shown to be critical for its function, the importance of the other domains of Aph-1 remains elusive. We screened a series of Aph-1 mutants and focused on nine mutations distributed in six different TMDs of human APH-1aS, assessing their ability to complement mouse embryonic fibroblasts lacking Aph-1. We showed that mutations in TMD4 (G126) and TMD5 (H171) of Aph-1aS prevented the formation of the Nct/Aph-1 subcomplex. Importantly, although mutations in TMD3 (Q83/E84/R85) and TMD6 (H197) of APH-1aS did not affect Nct/Aph-1 subcomplex formation, both mutations prevented further association/endoproteolysis of PS1. We propose a model that identifies critical TMDs of Aph-1 for associations with Nct and PS for the stepwise assembly of γ-secretase components.

Over the past three decades, significant progress has been made in understanding the neurobiology of Alzheimer's disease. In recent years, the first attempts to implement novel mechanism-based treatments brought rather disappointing results, with low, if any, drug efficacy and significant side effects. A discrepancy between our expectations based on preclinical models and the results of clinical trials calls for a revision of our theoretical views and questions every stage of translation—from how we model the disease to how we run clinical trials. In the following sections, we will use some specific examples of the therapeutics from acetylcholinesterase inhibitors to recent anti-Aβ immunization and γ-secretase inhibition to discuss whether preclinical studies could predict the limitations in efficacy and side effects that we were so disappointed to observe in recent clinical trials. We discuss ways to improve both the predictive validity of mouse models and the translation of knowledge between preclinical and clinical stages of drug development.

β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is an aspartyl protease best known for its role in generating the amyloid-β peptides that are present in plaques of Alzheimer's disease. BACE1 has been an attractive target for drug development. In cultured embryonic neurons, BACE1-cleaved N-terminal APP is further processed to generate a fragment that can trigger axonal degeneration, suggesting a vital role for BACE1 in axonal health. In addition, BACE1 cleaves neuregulin 1 type III, a protein critical for myelination of peripheral axons by Schwann cells during development. Here, we asked whether axonal degeneration or axonal regeneration in adult nerves might be affected by inhibition or elimination of BACE1. We report that BACE1 knock-out and wild-type nerves degenerated at a similar rate after axotomy and to a similar extent in the experimental neuropathies produced by administration of paclitaxel and acrylamide. These data indicate N-APP is not the sole culprit in axonal degeneration in adult nerves. Unexpectedly, however, we observed that BACE1 knock-out mice had markedly enhanced clearance of axonal and myelin debris from degenerated fibers, accelerated axonal regeneration, and earlier reinnervation of neuromuscular junctions, compared with littermate controls. These observations were reproduced in part by pharmacological inhibition of BACE1. These data suggest BACE1 inhibition as a therapeutic approach to accelerate regeneration and recovery after peripheral nerve damage.

Morphological evolution of heteroepitaxial nanoislands toward equilibrium (coarsening) is computed using the detailed continuum model that incorporates the quantum size effect. Results reveal the metastability of the "magic" heights, show the morphological transitions and the surface diffusion routes by which a quantum island reaches its stable height, and provide the coarsening laws for the island density and area, thus clarifying the kinetic morphology pathways in the growth of an ultrathin metal films., Accepted for publication in the Journal of Applied Physics

TAR DNA-binding protein-43 (TDP-43), a DNA/RNA-binding protein involved in RNA transcription and splicing, has been associated with the pathophysiology of neurodegenerative diseases, including ALS. However, the function of TDP-43 in motor neurons remains undefined. Here we use both gain- and loss-of-function approaches to determine roles of TDP-43 in motor neurons. Mice expressing human TDP-43 in neurons exhibited growth retardation and premature death that are characterized by abnormal intranuclear inclusions composed of TDP-43 and fused in sarcoma/translocated in liposarcoma (FUS/TLS), and massive accumulation of mitochondria in TDP-43-negative cytoplasmic inclusions in motor neurons, lack of mitochondria in motor axon terminals, and immature neuromuscular junctions. Whereas an elevated level of TDP-43 disrupts the normal nuclear distribution of survival motor neuron (SMN)-associated Gemini of coiled bodies (GEMs) in motor neurons, its absence prevents the formation of GEMs in the nuclei of these cells. Moreover, transcriptome-wide deep sequencing analysis revealed that a decrease in abundance of neurofilament transcripts contributed to the reduction of caliber of motor axons in TDP-43 mice. In concert, our findings indicate that TDP-43 participates in pathways critical for motor neuron physiology, including those that regulate the normal distributions of SMN-associated GEMs in the nucleus and mitochondria in the cytoplasm.

β-Amyloid (Aβ) pathology is an essential pathogenic component in Alzheimer's disease (AD). However, the significance of Aβ pathology, including Aβ deposits/oligomers and glial reactions, to neurodegeneration is unclear. In particular, despite the Aβ neurotoxicity indicated byin vitrostudies, mouse models with significant Aβ deposition lack robust and progressive loss of forebrain neurons. Such results have fueled the view that Aβ pathology is insufficient for neurodegenerationin vivo. In this study, because monoaminergic (MAergic) neurons show degenerative changes at early stages of AD, we examined whether theAPPswe/PS1ΔE9mouse model recapitulates progressive MAergic neurodegeneration occurring in AD cases. We show that the progression forebrain Aβ deposition in theAPPswe/PS1ΔE9model is associated with progressive losses of the forebrain MAergic afferents. Significantly, axonal degeneration is associated with significant atrophy of cell bodies and eventually leads to robust loss (∼50%) of subcortical MAergic neurons. Degeneration of these neurons occurs without obvious local Aβ or tau pathology at the subcortical sites and precedes the onset of anxiety-associated behavior in the mice. Our results show that a transgenic mouse model of Aβ pathology develops progressive MAergic neurodegeneration occurring in AD cases.

β-Site APP-cleaving enzyme 1 (BACE1) is required for the penultimate cleavage of the amyloid-β precursor protein (APP) leading to the generation of amyloid-β peptides that is central to the pathogenesis of Alzheimer's disease. In addition to its role in endoproteolysis of APP, BACE1 participates in the proteolytic processing of neuregulin 1 (NRG1) and influences the myelination of central and peripheral axons. Although NRG1 has been genetically linked to schizophrenia and NRG1 +/− mice exhibit a number of schizophrenia-like behavioral traits, it is not known whether altered BACE1-dependent NRG1 signaling can cause similar behavioral abnormalities. To test this hypothesis, we analyze the behaviors considered to be rodent analogs of clinical features of schizophrenia in BACE1 −/− mice with impaired processing of NRG1. We demonstrate that BACE1 −/− mice exhibit deficits in prepulse inhibition, novelty-induced hyperactivity, hypersensitivity to a glutamatergic psychostimulant (MK-801), cognitive impairments, and deficits in social recognition. Importantly, some of these manifestations were responsive to treatment with clozapine, an atypical antipsychotic drug. Moreover, although the total amount of ErbB4, a receptor for NRG1 was not changed, binding of ErbB4 with postsynaptic density protein 95 (PSD95) was significantly reduced in the brains of BACE1 −/− mice. Consistent with the role of ErbB4 in spine morphology and synaptic function, BACE1 −/− mice displayed reduced spine density in hippocampal pyramidal neurons. Collectively, our findings suggest that alterations in BACE1-dependent NRG1/ErbB4 signaling may participate in the pathogenesis of schizophrenia and related psychiatric disorders.

Loss-of-function mutations in the DJ-1 gene account for an autosomal recessive form of Parkinson's disease (PD). To investigate the physiological functions of DJ-1 in vivo, we generated DJ-1 knockout (DJ-1(-/-)) mice. Younger (

Purpose: Ischemic stroke patients, who meet criteria to be administered t-PA, should receive a patient-focused effort that advocates for quality, efficacious treatment. It’s essential to continuously evaluate the impact that a model of care delivery has on clinical outcomes. Healthcare reform may dictate utilization of current resources and often allocation of those resources are re-aligned to meet financial targets. The model of care delivery referenced in this study includes primary responders on a code stroke team that have transitioned from a unit based resource to an emergency room resource. Quality outcomes are a priority and overcoming obstacles to prevent delays in meeting the national benchmark of door to needle time in less than 60 minutes requires enhanced partnerships. The purpose of this study was to determine the effects that a model of care delivery for acute stroke response had on door to needle time and identify the impact from utilization of value stream mapping. Methods: Retrospective chart reviews were conducted on stroke patients that received t-PA over a 5 year period. Comparative analysis of all response times were reviewed for trends and opportunities that may have also been impacted by the model of care delivery. Immediate review and feedback on every case served as a catalyst for quality improvement, engaging all providers to implement changes that would improve door to needle times. Response time data was analyzed to determine if there was statistical significance, such that all opportunities for improvement to reduce needle to door time to < 60 minutes could be addressed with action plans. Results: Percentage compliance of door to needle time within 60 minutes increased from 17% to 63% during the study period. Opportunities to further reduce door to needle times reveal potential decrease of key response times by approximately 20 minutes. This is evidenced by continued improvement in the average door to needle time by 15 minutes during the study period. Conclusion: Transition of care delivery models with ownership and team engagement can result in a paradigm shift that indicates effective action plans for quality improvement in door to needle times. Further insight from value mapping serves as a guide for lean processes.

A transmembrane aspartyl protease termed β-site APP cleavage enzyme 1 (BACE1) that cleaves the amyloid-β precursor protein (APP), which is abundant in neurons, is required for the generation of amyloid-β (Aβ) peptides implicated in the pathogenesis of Alzheimer's disease (AD). We now demonstrate that BACE1, enriched in neurons of the CNS, is a major determinant that predisposes the brain to Aβ amyloidogenesis. The physiologically high levels of BACE1 activity coupled with low levels of BACE2 and α-secretase anti-amyloidogenic activities in neurons is a major contributor to the accumulation of Aβ in the CNS, whereas other organs are spared. Significantly, deletion ofBACE1inAPPswe;PS1ΔE9mice prevents both Aβ deposition and age-associated cognitive abnormalities that occur in this model of Aβ amyloidosis. Moreover, Aβ deposits are sensitive toBACE1dosage and can be efficiently cleared from the CNS whenBACE1is silenced. However,BACE1null mice manifest alterations in hippocampal synaptic plasticity as well as in performance on tests of cognition and emotion. Importantly, memory deficits but not emotional alterations inBACE1–/–mice are prevented by coexpressingAPPswe;PS1ΔE9transgenes, indicating that other potential substrates of BACE1 may affect neural circuits related to emotion. Our results establish BACE1 and APP processing pathways as critical for cognitive, emotional, and synaptic functions, and future studies should be alert to potential mechanism-based side effects that may occur with BACE1 inhibitors designed to ameliorate Aβ amyloidosis in AD.

APH-1 (anterior pharynx defective) along with nicastrin and PEN-2 (presenilin enhancer) are essential components of the presenilin (PS)-dependent γ-secretase complex. There exist three murineAph-1alleles termedAph-1a,Aph-1b, andAph-1cthat encode four distinct APH-1 isoforms: APH-1aL and APH-1aS derived from differential splicing ofAph-1a, APH-1b, and APH-1c. To determine the contributions of mammalian APH-1 homologs in formation of functional γ-secretase complexes, we generatedAph-1a-/-mice and derived immortalized fibroblasts from these embryos. Compared with littermate controls, the development ofAph-1a-/-embryos was dramatically retarded by embryonic day 9.5 and exhibited patterning defects that resemble, but are not identical to, those ofNotch1,nicastrin, orPSnull embryos. Moreover, in immortalizedAph-1a-/-fibroblasts, the levels of nicastrin, PS fragments, and PEN-2 were dramatically decreased. Consequently, deletion ofAph-1aresulted in significant reduction in levels of high-molecular-weight γ-secretase complex and secretion of β-amyloid (Aβ). Importantly, complementation analysis revealed that all mammalian APH-1 isoforms were capable of restoring the levels of nicastrin, PS, and PEN-2, as well as Aβ secretion inAph-1a-/-cells. Together, our findings establish that APH-1a is the major mammalian APH-1 homolog present in PS-dependent γ-secretase complexes during embryogenesis and support the view that mammalian APH-1 isoforms define a set of distinct functional γ-secretase complexes.

The present study was designed to examine whether brain inflammation caused by systemic administration of lipopolysaccharides (LPS) alters the expression/processing of amyloid precursor protein (APP) and increases the generation of amyloid beta peptide (Abeta). APPswe transgenic (Tg) mice were treated with either LPS or phosphate-buffered saline (PBS). In LPS-treated APPswe mice, Abeta1-40/42 was 3-fold and APP was 1.8-fold higher than those in PBS-treated mice (P < 0.05) by ELISA, Western blots and immunoprecipitation-mass spectrometry (IP-MS) ProteinChip analysis. Numbers of Abeta- and APP-immunoreactive neurons (Abeta(+) and APP(+) neurons) increased significantly in LPS-treated APPswe mice; APP(+) and Abeta(+) neurons in neocortex were associated with an increased number of F4/80-immunoreactive microglia (F4/80(+) microglia) in their anatomical environment. Our findings demonstrate that experimental neuroinflammation increases APP expression/processing and causes intracellular accumulation of Abeta. It remains to be seen whether such events can cause neuronal dysfunction/degeneration and, with time, lead to extracellular Abeta deposits, as they occur in Alzheimer's disease.

Recent studies indicate that nicastrin (NCT) and presenilins form functional components of a multimeric gamma-secretase complex required for the regulated intramembraneous proteolysis of Notch and beta-amyloid (Abeta) precursor protein (APP). To determine whether nicastrin is required for proteolytic processing of Notch and APP in mammals and the role of nicastrin in presenilin/gamma-secretase complex assembly, we generated nicastrin-deficient (NCT-/-) mice and derived fibroblasts from NCT-/- embryos. Nicastrin-null embryos died by embryonic day 10.5 and exhibited several patterning defects, including abnormal somite segmentation, phenotypes that are reminiscent of embryos lacking Notch1 or both presenilins. Importantly, secretion of Abeta peptides is abolished in NCT-/- fibroblasts, whereas it is reduced by approximately 50% in NCT+/- cells; the failure to generate Abeta peptides in NCT-/- cells is accompanied by destabilization of the presenilin/gamma-secretase complex and accumulation of APP-C-terminal fragments. Moreover, APP trafficking analysis in NCT-/- fibroblasts revealed a significant delay in the rate of APP reinternalization compared with that of control cells. Together, these results establish that nicastrin is an essential component of the multimeric gamma-secretase complex in mammals required for both gamma-secretase activity and APP trafficking and suggest that nicastrin may be a valuable therapeutic target for Alzheimer's disease.

Amyloid deposition appears to be an early and crucial event in Alzheimer's disease (AD). To generate animal models of AD, mice expressing full-length amyloid precursor protein (APP), with mutations linked to FAD, have been created. These animals exhibit abnormalities characteristic of AD, including deposits of beta-amyloid (Abeta), neuritic plaques, and glial responses. In studies of cognition in these animals, there have been several reports of memory disturbances well before the appearance of amyloid deposits. We have developed two distinct lines of transgenic mice (C3-3 and E1-2) that express the "Swedish" variant of APP (APP(SWE)) at levels that are approximately three-fold higher than endogenous mouse APP. Both lines have been backcrossed to C57BL/6J mice for 10 generations. Here, we use longitudinal and cross-sectional studies to evaluate the cognitive performance of our animals, where the concentration of Abeta1-42 in brain increases with aging from low levels (2-10 pmol/g) at 6-14 months of age to relatively high levels (60-100 pmol/g) at 24-26 months, when deposits of Abeta were beginning to form. When 12-month-old mice were tested in tasks that assess reference and working memory, transgenic mice from both lines could not be distinguished from nontransgenic littermates. Further study of 24- to 26-month-old transgenic mice (C3-3 line) found no evidence of memory impairment despite the presence of high levels of human Abeta (60-100 pmol/g). Thus, the expression of APP(SWE) at approximately three-fold over endogenous levels, which is sufficient to induce amyloid deposition at advanced ages, does not significantly erode cognitive performance in aged mice.

Protein adducts of the lipid peroxidation product trans-4-hydroxy-2-nonenal (HNE) are features of oxidative damage in neuronal cell bodies in Alzheimer's disease but are also seen in axons of normal as well as diseased individuals. In this study, focusing on the axons of the mouse sciatic nerve, we found that HNE adducts characterize axons of mice from birth to senility. Immunoblots of axonal proteins showed that HNE adducts are only detected in neurofilament heavy subunit (NFH) and, to a lesser extent, neurofilament medium subunit (NFM), both lysine-rich proteins, consistent with the adducts being limited to lysine residues. In vitro, HNE treatment of permeabilized sciatic nerve showed the same specificity, i.e. NFH and NFM are the only proteins that reacted with HNE, providing they are phosphorylated. Quantitative immunoblot analysis of two strains of mice ages 1-33 months showed that the levels of HNE adducts on NFH are consistent throughout life. Additionally, mice transgenic for human superoxide dismutase-1 with G85R mutation show no difference in HNE adduction to NFH compared with controls. Taken together, these studies indicate that HNE adduction to NFH is physiological, and its constancy from birth to senility as well as its dependence on phosphorylation argues that NFH and NFM modification may play a role in protecting the membrane-rich axon from toxic aldehydes resulting from oxidative damage.

Purpose: To determine whether the stroke alert process results in improved outcomes, as reflected in door to lytic times and other outcome measures. Introduction: The diagnosis and treatment of stroke is time-sensitive and should be inclusive of all seven D’s in the “chain of survival” - Detection, Dispatch, Delivery, Door, Data, Decision and Drug (Adams, Stroke, 2007). Early stroke activation is part of the “Delivery” which incorporates transport and management by Emergency Medical Services (EMS). Clinical suspicion of stroke by EMS resulted in a process of early activation which was labeled “Stroke Alert.” This expedited the code stroke process upon arrival, preparing the hospital based stroke team to provide immediate triage and evaluation. The goal was to improve clinical efficiency and possibly clinical outcomes. Methods: • Implementation of a notification process from EMS to ED - Stroke Alert • Incorporated Stroke Alert to include Stroke Response Team (SRT) nurses January 22, 2011 • Retrospective review of internal stroke database (January 22, 2011 to July 2013) for comparative analysis of Stroke Alerts called versus those where no stroke alert was called • Evaluate clinical outcomes directly related to Stroke Alert process Results: From January 22, 2011 to July 2013: Stroke Alert Called: • 37 t-PA patients and 14 of those, 37.8%, met the 60 minute benchmark • Average Door to Lytic time - 65 minutes Stroke Alert NOT Called: • 35 t-PA patients and 10 of those, 28.6% met the 60 minute benchmark • Average Door to Lytic time - 79 minutes Conclusions: The ability for a SRT to meet the golden hour of stroke benchmark occurs more frequently when a Stroke Alert is called to the SRT nurse. Future plans include review of stroke severity scores, length of stay (LOS), and discharge disposition, to determine the impact a Stroke Alert may have on clinical outcomes.

The mechanisms leading to neurodegeneration in ALS (amyotrophic lateral sclerosis) are not well understood, but cytosolic protein aggregates appear to be common in sporadic and familial ALS as well as transgenic mouse models expressing mutant Cu/Zn superoxide dismutase (SOD1). In this study, we systematically evaluated the presence of these aggregates in three different mouse models (G93A, G85R, and G37R SOD1) and compared these aggregates to those seen in cases of sporadic and familial ALS. Inclusions and loss of motor neurons were observed in spinal cords of all of these three mutant transgenic lines. Since a copper-mediated toxicity hypothesis has been proposed to explain the cytotoxic gain-of-function of mutant SOD1, we sought to determine the involvement of the copper chaperone for SOD1 (CCS) in the formation of protein aggregates. Although all aggregates contained CCS, SOD1 was not uniformly found in the inclusions. Similarly, CCS-positive skein-like inclusions were rarely seen in ALS neurons. These studies do not provide strong evidence for a causal role of CCS in aggregate formation, but they do suggest that protein aggregation is a common event in all animal models of the disease. Selected proteins, such as the glutamate transporter GLT-1, were not typically observed within the inclusions. Most inclusions were positively stained with antibodies recognizing ubiquitin, proteasome, Hsc70 in transgenic lines, and some Hsc70-positive inclusions were detected in sporadic ALS cases. Overall, these observations suggest that inclusions might be sequestered into ubiquitin-proteasome pathway and some chaperone proteins such as Hsc70 may be involved in formation and/or degradation of these inclusions.

Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by the expansion of a glutamine repeat in the N-terminus of the huntingtin protein. To gain insight into the pathogenesis of HD, we generated transgenic mice that express a cDNA encoding an N-terminal fragment (171 amino acids) of huntingtin with 82, 44 or 18 glutamines. Mice expressing relatively low steady-state levels of N171 huntingtin with 82 glutamine repeats (N171-82Q) develop behavioral abnormalities, including loss of coordination, tremors, hypokinesis and abnormal gait, before dying prematurely. In mice exhibiting these abnormalities, diffuse nuclear labeling, intranuclear inclusions and neuritic aggregates, all immunoreactive with an antibody to the N-terminus (amino acids 1-17) of huntingtin (AP194), were found in multiple populations of neurons. None of these behavioral or pathological phenotypes were seen in mice expressing N171-18Q. These findings are consistent with the idea that N-terminal fragments of huntingtin with a repeat expansion are toxic to neurons, and that N-terminal fragments are prone to form both intranuclear inclusions and neuritic aggregates.

The neurodegenerative disorders, a heterogeneous group of chronic progressive diseases, are among the most puzzling and devastating illnesses in medicine. Some of these disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis, the prion diseases, and Parkinson's disease, can occur sporadically and, in some instances, are caused by inheritance of gene mutations. Huntington's disease is acquired in an entirely genetic manner. Transgenic mice that express disease-causing genes recapitulate many features of these diseases. This review provides an overview of transgenic mouse models of familial amyotrophic lateral sclerosis, familial Alzheimer's disease, and Huntington's disease and the emerging insights relevant to the underlying molecular mechanisms of these diseases.

We have examined the trafficking and metabolism of the β-amyloid precursor protein (APP), an APP homolog (APLP1), and TrkB in neurons that lack PS1. We report that PS1-deficient neurons fail to secrete Aβ, and that the rate of appearance of soluble APP derivatives in the conditioned medium is increased. Remarkably, carboxyl-terminal fragments (CTFs) derived from APP and APLP1 accumulate in PS1-deficient neurons. Hence, PS1 plays a role in promoting intramembrane cleavage and/or degradation of membrane-bound CTFs. Moreover, the maturation of TrkB and BDNF-inducible TrkB autophosphorylation is severely compromised in neurons lacking PS1. We conclude that PS1 plays an essential role in modulating trafficking and metabolism of a selected set of membrane and secretory proteins in neurons.

To investigate the role of neurofilaments in motor neuron disease caused by superoxide dismutase (SOD1) mutations, transgenic mice expressing a amyotrophic lateral sclerosis-linked SOD1 mutant (SOD1 G37R ) were mated with transgenic mice expressing human neurofilament heavy (NF-H) subunits. Unexpectedly, expression of human NF-H transgenes increased by up to 65%, the mean lifespan of SOD1 G37R mice. Microscopic examination corroborated the protective effect of NF-H protein against SOD1 toxicity. Although massive neurodegeneration occurred in 1-yr-old mice expressing SOD1 G37R alone, spinal root axons and motor neurons were remarkably spared in doubly SOD1 G37R ;NF-H-transgenic littermates.

Superoxide dismutase 1 (SOD1), a ubiquitously expressed enzyme, detoxifies superoxide radicals and participates in copper homeostasis. Mutations in this enzyme have been linked to a subset of autosomal dominant cases of familial amyotrophic lateral sclerosis (FALS), a disorder characterized by selective degeneration of motor neurons. Transgenic mice expressing FALS mutant human (Hu) SOD1 at high levels develop a motor neuron disease, indicating that mutant Hu SOD1 gains properties that are particularly toxic to motor neurons. In this report, we demonstrate that transgenic mice expressing Hu SOD1 with the G37R FALS mutation, but not mice expressing wild-type enzyme, develop focal increases in immunoreactivity in the proximal axons of spinal motor neurons. This SOD1 immunoreactivity and immunoreactivity to hypophosphorylated neurofilament H epitopes are found adjacent to small vacuoles in axons. Using metabolic radiolabeling methods, we show that mutant G37R HuSOD1 as well as endogenous mouse SOD1 are transported anterograde in slow component b in motor and sensory axons of the sciatic nerve. Together, these findings suggest that anterogradely transported mutant SOD1 may act locally to damage motor axons. r 1998 Academic Press

Mutations in presenilin 1 (PS1) cosegregate with approximately 25% of early onset familial Alzheimer's disease (FAD) pedigrees. A variety of in vitro and in vivo paradigms have established that one mechanism by which PS1 variants cause AD is by elevating the production of highly amyloidogenic Abeta1-42/43 peptides. PS1 is homologous to sel-12, a C. elegans protein that facilitates signaling mediated by the Notch/lin-12 family of receptors. Wild-type human PS1 complements an egg-laying defect in C. elegans lacking sel-12, while FAD-linked PS1 variants exhibit reduced rescue activity. These data suggested that mutant PS1 may cause disease as a result of reduction in PS1 function. To test the function of FAD-linked PS1 in mammals, we examined the ability of the A246E PS1 variant to complement the embryonic lethality and axial skeletal defects in mice lacking PS1. Finally, to examine the influence of reduced PS1 levels on Abeta production, we quantified Abeta1-42/43 peptide levels in PS1 heterozygous null mice (PS1[+/-] mice). We now report that both human wild-type and A246E PS1 efficiently rescue the phenotypes observed in PS1(-/-) embryos, findings consistent with the view that FAD-linked PS1 mutants retain sufficient normal function during mammalian embryonic development. Moreover, the levels of Abeta1-42/43 and Abeta1-40 peptides between PS1(+/-) and control mice are indistinguishable. Collectively, these data lead us to conclude that mutant PS1 causes AD not by loss of normal PS1 function but by influencing amyloid precursor protein (APP) processing in a manner that elevates Abeta1-42/43 production.

Mutations in two related genes, presenilin 1 and presenilin 2 ( PS1 and PS2 ), cosegregate with Alzheimer's disease. PS1 and PS2 are highly homologous polytopic membrane proteins that are subject to endoproteolytic cleavage in vivo. The resulting N- and C-terminal derivatives are the preponderant PS-related species that accumulate in cultured cells and tissue. In earlier studies, we demonstrated that PS1 N- and C-terminal derivatives accumulate to 1:1 stoichiometry and that the absolute levels of fragments are established by a tightly regulated and saturable mechanism. These findings led to the suggestion that the levels of PS1 derivatives might be determined by their association with limiting cellular components. In this study, we use in situ chemical cross-linking and coimmunoprecipitation analyses to document that the N- and C-terminal derivatives of either PS1 or PS2 can be coisolated. Moreover, and in contrast to published reports which documented that PS1 and PS2 form stable heteromeric assemblies with the β-amyloid precursor protein (APP), we have failed to provide evidence for physiological complexes between PS1 and PS2 holoproteins or their derivatives with APP.

Huntington's disease (HD) is caused by CAG triplet repeat expansion inIT15which leads to polyglutamine stretches in the HD protein product, huntingtin. The pathological hallmark of HD is the degeneration of subsets of neurons, primarily those in the striatum and neocortex. Specific morphological markers of affected cells have not been identified in patients with HD, although a unique intranuclear inclusion was recently reported in neurons of transgenic animals expressing a construct encoding the N-terminal part (including the glutamine repeat) of huntingtin (Davieset al., 1997). In order to understand the importance of this finding, we sought for comparable nuclear abnormalities in autopsy material from patients with HD. In all 20 HD cases examined, anti-ubiquitin and N-terminal huntingtin antibodies identified intranuclear inclusions in neurons and the frequency of these lesions correlated with the length of the CAG repeat inIT15. In addition, examination of material from the related HD-like triplet repeat disorder, dentatorubral and pallidoluysian atrophy, also revealed intranuclear neuronal inclusions. These findings suggest that intranuclear inclusions containing protein aggregates may be a common feature of the pathogenesis of glutamine repeat neurodegenerative disorders.

Spinal muscular atrophy (SMA), the second most common fatal, autosomal recessive disease of infants, manifests as generalized muscle weakness. The most severe form (Type I, Werdnig-Hoffmann disease) is associated with quadriplegia, respiratory muscle paralysis and death in infancy. Less severe forms are classified as Type II and Type III, based on age of onset and ultimate motor disability. Some spinal motor neurons show chromatolysis and the number of these cells is decreased. Recently, SMA has been mapped to chromosome 5q11.2-13.3 (Gilliam et al., 1990), a region that contains three candidate genes: Survival Motor Neuron (SMN) (Lefebvre et al., 1995); Neuronal Apoptosis Inhibitory Protein (NAIP) (Roy et al., 1995); and p44, a subunit of transcription factor II H (TFIIH) (Carter et al., 1995; Bürglen et al., 1997). Homozygous deletions or deleterious mutations in SMN are present in all SMA patients, and in some affected individuals, deletions have been identified in one or both of the other genes. These extensive deletions may be associated with a more severe phenotype. We have identified and characterized the mouse homologue of SMN, MoSMN, which is 82% identical to SMN at the amino-acid level. Unlike the duplicated human SMN, MoSMN is present in single copy. Like its human counterpart, MoSMN is ubiquitously expressed, but unlike SMN, MoSMN does not appear to be alternatively spliced. In-situ hybridization analysis of the mouse nervous system revealed that MoSMN mRNA is expressed in spinal cord and throughout the brain, with relatively higher levels of expression in the hippocampus and cerebellum.

Mutations in two related genes, PS1and PS2, account for the majority of early onset cases of familial Alzheimer’s disease. PS1 and PS2 are homologous polytopic membrane proteins that are processed endoproteolytically into two fragments in vivo. In the present report we examine the fate of endogenous PS1 and PS2 after overexpression of human PS1 or PS2 in mouse N2a neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished in cultured cells overexpressing human PS2. To define the minimal sequence requirements for “replacement” we expressed familial Alzheimer’s disease-linked and experimental deletion variants of PS1. These studies revealed that compromised accumulation of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic cleavage. Our results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately by competition for limiting cellular factor(s).

The present study provides an experimental model of the apoptotic death of pyramidal neurons in rat olfactory cortex after total bulbectomy. Terminal transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL), DNA electrophoresis, and neuronal ultrastructure were used to provide evidence of apoptosis; neurons in olfactory cortex were counted by stereology. Maximal TUNEL staining occurred in the piriform cortex between 18 and 26 hr postbulbectomy. Within the survival times used in the present study (up to 48 hr postlesion), cell death was observed exclusively in the piriform cortex; there was no evidence of cell death in any other areas connected with the olfactory bulb. Neurons undergoing apoptosis were pyramidal cells receiving inputs from, but not projecting to, the olfactory bulb. The apical dendrites of these neurons were contacted by large numbers of degenerating axonal terminals. Gel electrophoresis of DNA purified from lesioned olfactory cortex showed a ladder pattern of fragmentation. Inflammatory cells or phagocytes were absent in the environment of degenerating neurons in the early stages of the apoptotic process. The present model suggests that deafferentation injury in sensory systems can cause apoptosis. In addition, olfactory bulbectomy can be used for investigating molecular mechanisms that underlie apoptosis in mature mammalian cortical neurons and for evaluating strategies to prevent the degeneration of cortical neurons.

Missense mutations in the beta-amyloid precursor protein gene (APP) co-segregate with a small subset of autosomal dominant familial Alzheimer's disease (FAD) cases wherein deposition of the 39-43 amino acid beta-amyloid (A beta) peptide and neurodegeneration are principal neuropathological hallmarks. To accurately examine the effect of missense mutations on APP metabolism and A beta production in vivo, we have introduced yeast artificial chromosomes (YACs) containing the entire approximately 400 kbp human APP gene encoding APP harboring either the asparagine for lysine and leucine for methionine FAD substitution at codons 670 and 671 (APP(K670N/M671L)), the isoleucine for valine FAD substitution at codon 717 (APP(V7171)) or a combination of both substitutions into transgenic mice. We demonstrate that, relative to YAC transgenic mice expressing wild-type APP, high levels of A beta peptides are detected in the brains of YAC transgenic mice expressing human APP(K670N/M671L) that is associated with a concomitant diminution in the levels of apha-secretase-generated soluble APP derivatives. Moreover, the levels of longer A beta peptides (species terminating at amino acids 42/43) are elevated in YAC transgenic mice expressing human APP(V7171). These mice should prove valuable for detailed analysis of the in vivo effects of the APP FAD mutations in a variety of tissues and throughout aging and for testing therapeutic agents that specifically alter APP metabolism and A beta production.

Reliable methods are needed to assess the impact of synaptic loss on brain function. In this empirical study we demonstrate a novel and efficient method using immunocytochemistry (ICC) and modern stereological techniques to quantify synapses in neocortex of adult primates (Macaca fascicularis). Systematic-uniform-random sections through forebrain from two 10-year-old monkeys were immunostained for estimation of synaptophysin-immunoreactive (synaptophysin-IR) presynaptic boutons (synapses). Adjacent sections were stained with cresyl violet for estimation of total number of neuronal cell bodies. The unbiased Cavalieri method was used to estimate total forebrain and neocortical volumes to a high level of precision (coefficient of error (CE) < or = 0.10)). Synapse-to-neuron ratios varied from 860 in frontal cortex to 2300 in parietal-temporal cortex. The combination of Cavalieri and optical disector methods provided a direct means of estimating approximately 1.25 trillion (x 10(12)) total synaptophysin-immunopositive boutons and approximately 1.01 billion (x 10(9)) cell bodies in neocortex, with low CEs (0.12). Time required to make precise estimates of total neocortical and forebrain volumes and total numbers of synapses and neurons in neocortex was approximately 2-3 h per case from stained sections. The approach is a direct and efficient technique to quantify total synapse and neuron numbers within a defined brain structure.

We tested the hypothesis that synaptic defects in the hippocampus of individuals with Alzheimer disease (AD) correlate with the severity of cognitive impairment. Three postmortem groups were studied: controls with normal and stable cognition; cognitively intact subjects with senile plaque densities diagnostic for possible AD (p-AD) and neurofibrillary changes characteristic of early AD (Braak stage III); and individuals with definite AD and neurofibrillary changes typical of incipient to severe AD (Braak stage III, V, or VI). Synaptophysin (a presynaptic vesicle protein) levels were quantified by immunoblotting of synaptic membrane fractions isolated from hippocampus, entorhinal cortex, caudate nucleus, and occipital cortex. Average synaptophysin levels were reduced in hippocampus when comparing definite AD to controls (55%, p < 0.0001), p-AD to control (25%, p < 0.005), and definite AD to p-AD (30%, p < 0.05), but levels in entorhinal cortex, occipital cortex, and caudate nucleus were either unchanged or less significantly altered than in hippocampus. By univariate analysis, hippocampal synaptophysin levels correlated with neuropsychological measurements, including Mini-mental state examination scores (r = 0.83, p < 0.0001) and Blessed scores (r = 0.74, P < 0.001), and with senile plaque densities (r = 0.89, p < 0.0001). We conclude that synaptic abnormalities in the hippocampus correlate with the severity of neuropathology and memory deficit in individuals with AD, and that this defect may predate neuropsychological evidence for cognitive impairment early in AD.

Mutations in superoxide dismutase 1 (SOD1; EC 1.15.1.1 ) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) Nature (London) 364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996) Science 271, 515–518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one in vivo aberrant property of this ALS-linked SOD1 mutant.

Approximately 10% of cases of Alzheimer's disease are familial and associated with autosomal dominant inheritance of mutations in genes encoding the amyloid precursor protein1, presenilin 1 (PS1)2 and presenilin 2 (PS2)3,4. Mutations in PS1 are linked to about 25% of cases of early-onset familial Alzheimer's disease5. PS1, which is endoproteolytically processed in vivo6, is a multipass transmembrane protein and is a functional homologue of SEL-12 (ref. 7), a Caenorhabditis elegans protein that facilitates signalling mediated by the Notch/LIN-12 family of receptors8,9. To examine potential roles for PS1 in facilitating Notch-mediated signalling during mammalian embryogenesis, we generated mice with targeted disruptions of PS1 alleles (PS1 –/– mice). PS1 –/–embryos exhibited abnormal patterning of the axial skeleton and spinal ganglia, phenotypes traced to defects in somite segmentation and differentiation. Moreover, expression of mRNA encoding Notch 1 and DII 1 (delta-like gene I)10, a vertebrate Notch ligand, is markedly reduced in the presomitic mesoderm of PS1 –/– embryos compared to controls. Hence, PS1 is required for the spatiotemporal expression of Notch 1 and Dll 1, which are essential for somite segmentation and maintenance of somite borders11–13.

Nerve growth factor (NGF) infusion significantly reduces spatial recent memory deficits in aged rats, an effect that has great relevance to the treatment of memory impairments characteristic of patients with Alzheimer’s disease. The present study was designed to examine whether this NGF-induced improvement in spatial recent memory persists after the discontinuation of NGF treatment, an issue of crucial importance for the potential clinical use of this compound. Spatial recent memory was tested in a Morris water maze delayed nonmatch-to-position task. In addition to memory, sensorimotor skills were also examined. Four- and 22-month-old rats were tested preoperatively, infused intraventricularly with recombinant human NGF or vehicle, and tested both during the 4 week infusion period and during the 4 weeks after discontinuation of the infusion. NGF significantly improved spatial recent memory in 22-month-old rats only, during the 4th week of infusion and for up to 4 weeks after discontinuation of the infusion. Although NGF did not affect overall sensorimotor skills during infusion in either age group, sensorimotor skills were significantly improved both 2 and 4 weeks after discontinuation of infusion in 22-month-old rats. These findings demonstrate that the beneficial effects of NGF on spatial recent memory can persist for up to 1 month after discontinuation of infusion and suggest that NGF can be used intermittently for the treatment of age-associated memory dysfunction and Alzheimer’s disease.

High levels of familial Amyotrophic Lateral Sclerosis (ALS)-linked SOD1 mutants G93A and G37R were previously shown to mediate disease in mice through an acquired toxic property. We report here that even low levels of another mutant, G85R, cause motor neuron disease characterized by an extremely rapid clinical progression, without changes in SOD1 activity. Initial indicators of disease are astrocytic inclusions that stain intensely with SOD1 antibodies and ubiquitin and SOD1-containing aggregates in motor neurons, features common with some cases of SOD1 mutant- mediated ALS. Astrocytic inclusions escalate markedly as disease progresses, concomitant with a decrease in the glial glutamate transporter (GLT-1). Thus, the G85R SOD1 mutant mediates direct damage to astrocytes, which may promote the nearly synchronous degeneration of motor neurons.

Mutations in genes encoding related proteins, termed presenilin 1 (PS1) and presenilin 2 (PS2), are linked to the majority of cases with early-onset familial Alzheimer’s disease (FAD). To clarify potential function(s) of presenilins and relationships of presenilin expression to pathogenesis of AD, we examined the expression of PS1 and PS2 mRNA and PS1 protein in human and mouse. Semi-quantitative PCR of reverse-transcribed RNA (RT-PCR) analysis revealed that PS1 and PS2 mRNA are expressed ubiquitously and at comparable levels in most human and mouse tissues, including adult brain. However, PS1 mRNA is expressed at significantly higher levels in developing brain.In situhybridization studies of mouse embryos revealed widespread expression of PS1 mRNA with a neural expression pattern that, in part, overlaps that reported for mRNA encoding specific Notch homologs.In situhybridization analysis in adult mouse brain revealed that PS1 and PS2 mRNAs are enriched in neurons of the hippocampal formation and entorhinal cortex. Although PS1 and PS2 mRNA are expressed most prominently in neurons, lower but significant levels of PS1 and PS2 transcripts are also detected in white matter glial cells. Moreover, cultured neurons and astrocytes express PS1 and PS2 mRNAs. Using PS1-specific antibodies in immunoblot analysis, we demonstrate that PS1 accumulates as ∼28 kDa N-terminal and ∼18 kDa C-terminal fragments in brain. Immunocytochemical studies of mouse brain reveal that PS1 protein accumulates in a variety of neuronal populations with enrichment in somatodendritic and neuropil compartments.

An expression plasmid (MoPrP.Xho), for use in transgenic mice, was developed from the promoter, 5' intronic, and 3' untranslated sequences of the murine prion protein gene. Analyses of mice harboring the MoPrP.Xho construct with cDNA genes encoding the amyloid precursor protein (APP) and human presenilin 1 demonstrated that this vector provides relatively high levels of transgene-encoded polypeptides in brains and hearts of transgenic mice. The MoPrP.Xho vector should be very useful in strategies designed to overexpress a variety of wild-type and disease related mutant transgenes in the heart and brain.