Acute Presentation of Newly Diagnosed Multiple Sclerosis Associated With Polymerase Chain Reaction-Proven Human Herpesvirus 6 Central Nervous System Infection.

We present the case of a 26-year-old male who was found to have human herpesvirus 6 (HHV-6) in his cerebrospinal fluid (CSF) during acute presentation of multiple sclerosis (MS). Paresthesia of the lower extremities was his only symptom during the initial presentation, and workup for MS was not included during this evaluation. A single dose of IV steroids failed to improve his condition, and symptoms became more severe. Upon secondary evaluation, MRI revealed white-matter disease with plaques at multiple levels of the cervical spine and central nervous system (CNS). Lumbar puncture was obtained, and CSF analysis was positive for HHV-6 DNA. After five days of oral steroid treatment and physical therapy for three weeks, his symptoms continued to worsen. MRI at this time demonstrated an increase in the size of previous plaques and new foci of white matter disease. Repeat CSF analysis was negative for HHV-6. The virus' association with relapse of MS has been investigated by many studies. However, there is a lack of literature investigating its role in causing MS disease. In this case report, we highlight the need for further research aimed at determining if HHV-6 is an environmental trigger for MS disease onset.

Rodent models of TDP-43: recent advances.

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.

Specific domains in anterior pharynx-defective 1 determine its intramembrane interactions with nicastrin and presenilin.

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.

Increased expression of PS1 is sufficient to elevate the level and activity of γ-secretase in vivo.

Increase in the generation and deposition of amyloid-ß (Aß) plays a central role in the development of Alzheimer's Disease (AD). Elevation of the activity of γ-secretase, a key enzyme required for the generation for Aß, can thus be a potential risk factor in AD. However, it is not known whether γ-secretase can be upregulated in vivo. While in vitro studies showed that expression of all four components of γ-secretase (Nicastrin, Presenilin, Pen-2 and Aph-1) are required for upregulation of γ-secretase, it remains to be established as to whether this is true in vivo. To investigate whether overexpressing a single component of the γ-secretase complex is sufficient to elevate its level and activity in the brain, we analyzed transgenic mice expressing either wild type or familial AD (fAD) associated mutant PS1. In contrast to cell culture studies, overexpression of either wild type or mutant PS1 is sufficient to increase levels of Nicastrin and Pen-2, and elevate the level of active γ-secretase complex, enzymatic activity of γ-secretase and the deposition of Aß in brains of mice. Importantly, γ-secretase comprised of mutant PS1 is less active than that of wild type PS1-containing γ-secretase; however, γ-secretase comprised of mutant PS1 cleaves at the Aß42 site of APP-CTFs more efficiently than at the Aß40 site, resulting in greater accumulation of Aß deposits in the brain. Our data suggest that whereas fAD-linked PS1 mutants cause early onset disease, upregulation of PS1/γ-secretase activity may be a risk factor for late onset sporadic AD.

Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent ß-amyloid generation.

Assemblies of ß-amyloid (Aß) peptides are pathological mediators of Alzheimer's Disease (AD) and are produced by the sequential cleavages of amyloid precursor protein (APP) by ß-secretase (BACE1) and γ-secretase. The generation of Aß is coupled to neuronal activity, but the molecular basis is unknown. Here, we report that the immediate early gene Arc is required for activity-dependent generation of Aß. Arc is a postsynaptic protein that recruits endophilin2/3 and dynamin to early/recycling endosomes that traffic AMPA receptors to reduce synaptic strength in both hebbian and non-hebbian forms of plasticity. The Arc-endosome also traffics APP and BACE1, and Arc physically associates with presenilin1 (PS1) to regulate γ-secretase trafficking and confer activity dependence. Genetic deletion of Arc reduces Aß load in a transgenic mouse model of AD. In concert with the finding that patients with AD can express anomalously high levels of Arc, we hypothesize that Arc participates in the pathogenesis of AD.

Reduced BACE1 activity enhances clearance of myelin debris and regeneration of axons in the injured peripheral nervous system.

ß-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is an aspartyl protease best known for its role in generating the amyloid-ß 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.

Passive (amyloid-ß) immunotherapy attenuates monoaminergic axonal degeneration in the AßPPswe/PS1dE9 mice.

The role of amyloid-ß (Aß in the neurodegeneration of Alzheimer's disease remains controversial, to a large extent because of the lack of robust neurodegeneration in mouse models of AD. To address this question, we examined the effects of Aß antibodies in the recently described monoaminergic (MAergic) axonal degeneration in AßPPswe/PS1dE9 mice. To determine if Aß accumulation is directly involved in degeneration of MAergic axons, we examined the effects of passive anti-Aß antibody (7B6) administration on Aß pathology and MAergic degeneration in AßPPswe/PS1dE9 mice. Injections of monoclonal antibody (mAb) 7B6 into mice (6 to 9 months of age) resulted in a modest reduction of Aß load in the brains of AßPPswe/PS1dE9 mice. In addition, 7B6 treated AßPPswe/PS1dE9 mice had significantly higher densities of MAergic axons in both cortex and in hippocampus as compared to untreated mutant mice. For example, 7B6 treated mice showed almost 2-fold greater densities of serotonergic (5-HT) axons in the cortex compared to saline treated mice. Similar findings were observed in the catecholaminergic (TH) axons. Our results demonstrate that lowering of Aß levels via passive Aß immunotherapy ameliorates ongoing degenerative processes, supporting a causal link between Aß and neurodegeneration.

Altered distributions of Gemini of coiled bodies and mitochondria in motor neurons of TDP-43 transgenic mice.

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.

Deletion of TDP-43 down-regulates Tbc1d1, a gene linked to obesity, and alters body fat metabolism.

Tat activating regulatory DNA-binding protein (Tardbp or TDP-43), a highly conserved metazoan DNA/RNA binding protein thought to be involved in RNA transcription and splicing, has been linked to the pathophysiology of amyotrophic lateral sclerosis and frontotemporal lobar degeneration and is essential for early embryonic development. However, neither the physiological role of TDP-43 in the adult nor its downstream targets are well defined. To address these questions, we developed conditional Tardbp-KO mice and embryonic stem (ES) cell models. Here, we show that postnatal deletion of Tardbp in mice caused dramatic loss of body fat followed by rapid death. Moreover, conditional Tardbp-KO ES cells failed to proliferate. Importantly, high-throughput DNA sequencing analysis on the transcriptome of ES cells lacking Tardbp revealed a set of downstream targets of TDP-43. We show that Tbc1d1, a gene known to mediate leanness and linked to obesity, is down-regulated in the absence of TDP-43. Collectively, our results establish that TDP-43 is critical for fat metabolism and ES cell survival.

Modeling an anti-amyloid combination therapy for Alzheimer's disease.

As only symptomatic treatments are now available for Alzheimer's disease (AD), safe and effective mechanism-based therapies remain a great unmet need for patients with this neurodegenerative disease. Although gamma-secretase and BACE1 [beta-site beta-amyloid (Abeta) precursor protein (APP) cleaving enzyme 1] are well-recognized therapeutic targets for AD, untoward side effects associated with strong inhibition or reductions in amounts of these aspartyl proteases have raised concerns regarding their therapeutic potential. Although moderate decreases of either gamma-secretase or BACE1 are not associated with mechanism-based toxicities, they provide only modest benefits in reducing Abeta in the brains of APPswe/PS1DeltaE9 mice. Because the processing of APP to generate Abeta requires both gamma-secretase and BACE1, it is possible that moderate reductions of both enzymes would provide additive and significant protection against Abeta amyloidosis. Here, we test this hypothesis and assess the value of this novel anti-amyloid combination therapy in mutant mice. We demonstrate that genetic reductions of both BACE1 and gamma-secretase additively attenuate the amyloid burden and ameliorate cognitive deficits occurring in aged APPswe/PS1DeltaE9 animals. No evidence of mechanism-based toxicities was associated with such decreases in amounts of both enzymes. Thus, we propose that targeting both gamma-secretase and BACE1 may be an effective and safe treatment strategy for AD.

Neuropathologic studies of the Baltimore Longitudinal Study of Aging (BLSA).

The Baltimore Longitudinal Study of Aging (BLSA) was established in 1958 and is one the oldest prospective studies of aging in the USA and the world. The BLSA is supported by the National Institute of Aging (NIA) and its mission is to learn what happens to people as they get old and how to sort out changes due to aging from those due to disease or other causes. In 1986, an autopsy program combined with comprehensive neurologic and cognitive evaluations was established in collaboration with the Johns Hopkins University Alzheimer's Disease Research Center (ADRC). Since then, 211 subjects have undergone autopsy. Here we review the key clinical neuropathological correlations from this autopsy series. The focus is on the morphological and biochemical changes that occur in normal aging, and the early neuropathological changes of neurodegenerative diseases, especially Alzheimer's disease (AD). We highlight the combined clinical, pathologic, morphometric, and biochemical evidence of asymptomatic AD, a state characterized by normal clinical evaluations in subjects with abundant AD pathology. We conclude that in some individuals, successful cognitive aging results from compensatory mechanisms that occur at the neuronal level (i.e., neuronal hypertrophy and synaptic plasticity) whereas a failure of compensation may culminate in disease.

Amyloid pathology is associated with progressive monoaminergic neurodegeneration in a transgenic mouse model of Alzheimer's disease.

beta-Amyloid (Abeta) pathology is an essential pathogenic component in Alzheimer's disease (AD). However, the significance of Abeta pathology, including Abeta deposits/oligomers and glial reactions, to neurodegeneration is unclear. In particular, despite the Abeta neurotoxicity indicated by in vitro studies, mouse models with significant Abeta deposition lack robust and progressive loss of forebrain neurons. Such results have fueled the view that Abeta pathology is insufficient for neurodegeneration in vivo. In this study, because monoaminergic (MAergic) neurons show degenerative changes at early stages of AD, we examined whether the APPswe/PS1DeltaE9 mouse model recapitulates progressive MAergic neurodegeneration occurring in AD cases. We show that the progression forebrain Abeta deposition in the APPswe/PS1DeltaE9 model 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 (approximately 50%) of subcortical MAergic neurons. Degeneration of these neurons occurs without obvious local Abeta 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 Abeta pathology develops progressive MAergic neurodegeneration occurring in AD cases.

Motor neuron disease occurring in a mutant dynactin mouse model is characterized by defects in vesicular trafficking.

Amyotrophic lateral sclerosis (ALS), a fatal and progressive neurodegenerative disorder characterized by weakness, muscle atrophy, and spasticity, is the most common adult-onset motor neuron disease. Although the majority of ALS cases are sporadic, approximately 5-10% are familial, including those linked to mutations in SOD1 (Cu/Zn superoxide dismutase). Missense mutations in a dynactin gene (DCTN1) encoding the p150(Glued) subunit of dynactin have been linked to both familial and sporadic ALS. To determine the molecular mechanism whereby mutant dynactin p150(Glued) causes selective degeneration of motor neurons, we generated and characterized mice expressing either wild-type or mutant human dynactin p150(Glued). Neuronal expression of mutant, but not wild type, dynactin p150(Glued) causes motor neuron disease in these animals that are characterized by defects in vesicular transport in cell bodies of motor neurons, axonal swelling and axo-terminal degeneration. Importantly, we provide evidence that autophagic cell death is implicated in the pathogenesis of mutant p150(Glued) mice. This novel mouse model will be instrumental for not only clarifying disease mechanisms in ALS, but also for testing therapeutic strategies to ameliorate this devastating disease.

Progressive behavioral deficits in DJ-1-deficient mice are associated with normal nigrostriatal function.

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 (<1 year) DJ-1(-/-) mice were hypoactive and had mild gait abnormalities. Older DJ-1(-/-), however, showed decreased body weight and grip strength and more severe gait irregularities compared to wild-type littermates. The basal level of extracellular dopamine, evoked dopamine release and dopamine receptor D2 sensitivity appeared normal in the striatum of DJ-1(-/-) mice, which was consistent with similar results between DJ-1(-/-) and controls in behavioral paradigms specific for the dopaminergic system. An examination of spinal cord, nerve and muscle tissues failed to identify any pathological changes that were consistent with the noted motor deficits. Taken together, our findings suggest that loss of DJ-1 leads to progressive behavioral changes without significant alterations in nigrostriatal dopaminergic and spinal motor systems.

Moderate reduction of gamma-secretase attenuates amyloid burden and limits mechanism-based liabilities.

Although gamma-secretase is recognized as a therapeutic target for Alzheimer's disease, side effects associated with strong inhibition of this aspartyl protease raised serious concerns regarding this therapeutic strategy. However, it is not known whether moderate inhibition of this enzyme will allow dissociation of beneficial effects in the CNS from mechanism-based toxicities in the periphery. We tested this possibility by using a series of mice with genetic reduction of gamma-secretase (levels ranging from 25 to 64% of control mice). Here, we document that even 30% reduction of gamma-secretase can effectively ameliorate amyloid burden in the CNS. However, global reduction of this enzyme below a threshold level increased the risk of developing squamous cell carcinoma as well as abnormal proliferation of granulocytes in a gamma-secretase dosage-dependent manner. Importantly, we demonstrate that there exists a critical gamma-secretase level that reduces the risk of amyloidosis in the CNS and limits tumorigenesis in epithelia. Our findings suggest that moderate inhibition of gamma-secretase represents an attractive anti-amyloid therapy for Alzheimer's disease.

Epidermal growth factor receptor and notch pathways participate in the tumor suppressor function of gamma-secretase.

Gamma-secretase, a unique aspartyl protease, is required for the regulated intramembrane proteolysis of Notch and APP, pathways that are implicated, respectively, in the pathogenesis of cancer and Alzheimer disease. However, the mechanism whereby reduction of gamma-secretase causes tumors such as squamous cell carcinoma (SCC) remains poorly understood. Here, we demonstrate that gamma-secretase functions in epithelia as a tumor suppressor in an enzyme activity-dependent manner. Notch signaling is down-regulated and epidermal growth factor receptor (EGFR) is activated in SCC caused by genetic reduction of gamma-secretase. Moreover, the level of EGFR is inversely correlated with the level of gamma-secretase in fibroblasts, suggesting that the up-regulation of EGFR stimulates hyperproliferation in epithelia of mice with genetic reduction of gamma-secretase. Supporting this notion is our finding that the proliferative response of fibroblasts lacking gamma-secretase activity is more sensitive when challenged by either EGF or an inhibitor of EGFR as ompared with wild type cells. Interestingly, the up-regulation of EGFR is independent of Notch signaling, suggesting that the EGFR pathway functions in parallel with Notch in the tumorigenesis of SCC. Collectively, our results establish a novel mechanism linking the EGFR pathway to the tumor suppressor role of gamma-secretase and that mice with genetic reduction of gamma-secretase represent an excellent rodent model for clarifying pathogenesis of SCC and for testing therapeutic strategy to ameliorate this type of human cancer.

Amyotrophic lateral sclerosis 2-deficiency leads to neuronal degeneration in amyotrophic lateral sclerosis through altered AMPA receptor trafficking.

Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disease is caused by a selective loss of motor neurons. One form of juvenile onset autosomal recessive ALS (ALS2) has been linked to the loss of function of the ALS2 gene. The pathogenic mechanism of ALS2-deficiency, however, remains unclear. To further understand the function of alsin that is encoded by the full-length ALS2 gene, we screened proteins interacting with alsin. Here, we report that alsin interacted with glutamate receptor interacting protein 1 (GRIP1) both in vitro and in vivo, and colocalized with GRIP1 in neurons. In support of the physiological interaction between alsin and GRIP1, the subcellular distribution of GRIP1 was altered in ALS2(-/-) spinal motor neurons, which correlates with a significant reduction of AMPA-type glutamate receptor subunit 2 (GluR2) at the synaptic/cell surface of ALS2(-/-) neurons. The decrease of calcium-impermeable GluR2-containing AMPA receptors at the cell/synaptic surface rendered ALS2(-/-) neurons more susceptible to glutamate receptor-mediated neurotoxicity. Our findings reveal a novel function of alsin in AMPA receptor trafficking and provide a novel pathogenic link between ALS2-deficiency and motor neuron degeneration, suggesting a protective role of alsin in maintaining the survival of motor neurons.

Parkinson's disease alpha-synuclein transgenic mice develop neuronal mitochondrial degeneration and cell death.

Alpha-synuclein (alpha-Syn) is enriched in nerve terminals. Two mutations in the alpha-Syn gene (Ala53--> Thr and Ala30--> Pro) occur in autosomal dominant familial Parkinson's disease. Mice overexpressing the human A53T mutant alpha-Syn develop a severe movement disorder, paralysis, and synucleinopathy, but the mechanisms are not understood. We examined whether transgenic mice expressing human wild-type or familial Parkinson's disease-linked A53T or A30P mutant alpha-syn develop neuronal degeneration and cell death. Mutant mice were examined at early- to mid-stage disease and at near end-stage disease. Age-matched nontransgenic littermates were controls. In A53T mice, neurons in brainstem and spinal cord exhibited large axonal swellings, somal chromatolytic changes, and nuclear condensation. Spheroid eosinophilic Lewy body-like inclusions were present in the cytoplasm of cortical neurons and spinal motor neurons. These inclusions contained human alpha-syn and nitrated synuclein. Motor neurons were depleted (approximately 75%) in A53T mice but were affected less in A30P mice. Axonal degeneration was present in many regions. Electron microscopy confirmed the cell and axonal degeneration and revealed cytoplasmic inclusions in dendrites and axons. Some inclusions were degenerating mitochondria and were positive for humanalpha-syn. Mitochondrial complex IV and V proteins were at control levels, but complex IV activity was reduced significantly in spinal cord. Subsets of neurons in neocortex, brainstem, and spinal cord ventral horn were positive for terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, cleaved caspase-3, and p53. Mitochondria in neurons had terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive matrices and p53 at the outer membrane. Thus, A53T mutant mice develop intraneuronal inclusions, mitochondrial DNA damage and degeneration, and apoptotic-like death of neocortical, brainstem, and motor neurons.

BACE1, a major determinant of selective vulnerability of the brain to amyloid-beta amyloidogenesis, is essential for cognitive, emotional, and synaptic functions.

A transmembrane aspartyl protease termed beta-site APP cleavage enzyme 1 (BACE1) that cleaves the amyloid-beta precursor protein (APP), which is abundant in neurons, is required for the generation of amyloid-beta (Abeta) 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 Abeta amyloidogenesis. The physiologically high levels of BACE1 activity coupled with low levels of BACE2 and alpha-secretase anti-amyloidogenic activities in neurons is a major contributor to the accumulation of Abeta in the CNS, whereas other organs are spared. Significantly, deletion of BACE1 in APPswe;PS1DeltaE9 mice prevents both Abeta deposition and age-associated cognitive abnormalities that occur in this model of Abeta amyloidosis. Moreover, Abeta deposits are sensitive to BACE1 dosage and can be efficiently cleared from the CNS when BACE1 is silenced. However, BACE1 null 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 in BACE1(-/-) mice are prevented by coexpressing APPswe;PS1DeltaE9 transgenes, 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 Abeta amyloidosis in AD.