Viral Infections, Are They a Trigger and Risk Factor of Alzheimer's Disease?

Alzheimer's Disease (AD), a progressive and debilitating condition, is reported to be the most common type of dementia, with at least 55 million people believed to be currently affected. Many causation hypotheses of AD exist, yet the intriguing link between viral infection and its possible contribution to the known etiology of AD has become an attractive focal point of research for the field and a challenging study task. In this review, we will explore the historical perspective and milestones that led the field to investigate the viral connection to AD. Specifically, several viruses such as Herpes Simplex Virus 1 (HSV-1), Zika virus (ZIKV), and severe cute respiratory syndrome coronavirus 2 (SARS-CoV-2), along with several others mentioned, include the various viruses presently considered within the field. We delve into the strong evidence implicating these viruses in the development of AD such as the lytic replication and axonal transport of HSV-1, the various mechanisms of ZIKV neurotropism through the human protein Musashi-1 (MSI1), and the spread of SARS-CoV-2 through the transfer of the virus through the BBB endothelial cells to glial cells and then to neurons via transsynaptic transfer. We will also explore beyond these mere associations by carefully analyzing the potential mechanisms by which these viruses may contribute to AD pathology. This includes but is not limited to direct neuronal infections, the dysregulation of immune responses, and the impact on protein processing (Aß42 and hyperphosphorylated tau). Controversies and challenges of the virus-AD relationship emerge as we tease out these potential mechanisms. Looking forward, we emphasize future directions, such as distinct questions and proposed experimentations to explore, that the field should take to tackle the remaining unanswered questions and the glaring research gaps that persist. Overall, this review aims to provide a comprehensive survey of the past, present, and future of the potential link between viral infections and their association with AD development while encouraging further discussion.

Changes of tRNA-Derived Fragments by Alzheimer's Disease in Cerebrospinal Fluid and Blood Serum.

BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia, affecting individuals over 65. AD is also a multifactorial disease, with disease mechanisms incompletely characterized, and disease-modifying therapies are marginally effective. Biomarker signatures may shed light on the diagnosis, disease mechanisms, and the development of therapeutic targets. tRNA-derived RNA fragments (tRFs), a family of recently discovered small non-coding RNAs, have been found to be significantly enhanced in human AD hippocampus tissues. However, whether tRFs change in body fluids is unknown. OBJECTIVE: To investigate whether tRFs in body fluids are impacted by AD. METHODS: We first used T4 polynucleotide kinase-RNA-seq, a modified next-generation sequencing technique, to identify detectable tRFs in human cerebrospinal fluid and serum samples. The detectable tRFs were then compared in these fluids from control, AD, and mild cognitive impairment patients using tRF qRT-PCR. The stability of tRFs in serum was also investigated by checking the change in tRFs in response to protein digestion or exosome lysis. RESULTS: Among various tRFs, tRF5-ProAGG seemed to be impacted by AD in both cerebrospinal fluid and serum. AD-impacted serum tRF5-ProAGG showed a correlation with the AD stage. Putative targets of tRF5-ProAGG in the hippocampus were also predicted by a computational algorithm, with some targets being validated experimentally and one of them being in a negative correlation with tRF5-ProAGG even using a small size of samples. CONCLUSIONS: tRF5-ProAGG showed the potential as an AD biomarker and may play a role in disease progression.

Nitric Oxide as a Determinant of Human Longevity and Health Span.

The master molecular regulators and mechanisms determining longevity and health span include nitric oxide (NO) and superoxide anion radicals (SOR). L-arginine, the NO synthase (NOS) substrate, can restore a healthy ratio between the dangerous SOR and the protective NO radical to promote healthy aging. Antioxidant supplementation orchestrates protection against oxidative stress and damage-L-arginine and antioxidants such as vitamin C increase NO production and bioavailability. Uncoupling of NO generation with the appearance of SOR can be induced by asymmetric dimethylarginine (ADMA). L-arginine can displace ADMA from the site of NO formation if sufficient amounts of the amino acid are available. Antioxidants such as ascorbic acids can scavenge SOR and increase the bioavailability of NO. The topics of this review are the complex interactions of antioxidant agents with L-arginine, which determine NO bioactivity and protection against age-related degeneration.

Tryptophan in Nutrition and Health 2.0.

This editorial summarizes the eight articles that have been collected for the Special Issue entitled "Tryptophan in Nutrition and Health 2 [...].

Tryptophan in Nutrition and Health.

Tryptophan is a rate-limiting essential amino acid and a unique building block of peptides and proteins [...].

Indoles as essential mediators in the gut-brain axis. Their role in Alzheimer's disease.

Sporadic late-onset Alzheimer's disease (AD) is the most frequent cause of dementia associated with aging. Due to the progressive aging of the population, AD is becoming a healthcare burden of unprecedented proportions. Twenty years ago, it was reported that some indole molecules produced by the gut microbiota possess essential biological activities, including neuroprotection and antioxidant properties. Since then, research has cemented additional characteristics of these substances, including anti-inflammatory, immunoregulatory, and amyloid anti-aggregation features. Herein, we summarize the evidence supporting an integrated hypothesis that some of these substances can influence the age of onset and progression of AD and are central to the symbiotic relationship between intestinal microbes and the brain. Studies have shown that some of these substances' activities result from interactions with biologically conserved pathways and with genetic risk factors for AD. By targeting multiple pathologic mechanisms simultaneously, certain indoles may be excellent candidates to ameliorate neurodegeneration. We propose that management of the microbiota to induce a higher production of neuroprotective indoles (e.g., indole propionic acid) will promote brain health during aging. This area of research represents a new therapeutic paradigm that could add functional years of life to individuals who would otherwise develop dementia.

Insulin Resistance is Associated with Central Pain in Patients with Fibromyalgia.

BACKGROUND: Insulin resistance (IR) is a pathological condition in which cells fail to respond normally to insulin. IR has been associated with multiple conditions, including chronic pain. Fibromyalgia (FM) is one of the common generalized chronic painful conditions with an incidence rate affecting 3% to 6% of the population. Substantial interest and investigation into FM continue to generate  many hypotheses.The relationship between IR and FM has not been explored. IR is known to cause abnormalities in the cerebral microvasculature, leading to focal hypoperfusion. IR also has been shown to cause cognitive impairment in FM patients, as in parkinsonism. As demonstrated by advanced imaging methods, similar brain perfusion abnormalities occur in the brain of patients with FM as with IR. OBJECTIVES: To determine the potential association between FM and IR. SETTING: Subspecialty pain medicine clinics. STUDY DESIGN: Observational cross-sectional study. METHODS: Laboratory data was extracted through a retrospective review of medical records from patients who had met the American College of Rheumatology (ACR) criteria for FM. The Hemoglobin A1c (HbA1c) values from 33 patients with FM were compared with the means of the glycated HbA1c levels of 2 control populations. In addition, established indices of IR [Quantitative Insulin Sensitivity Check Index (QUICKI) and the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR)] were calculated in a subgroup of patients in whom the analytes necessary for these calculations were available. To assess for confounding factors, the associations between HbA1c, QUICKI, HOMA-IR, fasting insulin levels, and glucose, after controlling for age, were explored by multiple analyses of variance with relation to gender and ethnicity. RESULTS: We found an association between IR and FM that was independent of age, gender, and ethnicity. We found that patients with FM belong to a distinct population that can be segregated from the control groups by their HbA1c levels, a surrogate marker of IR. This was demonstrated by analyzing the data after introducing an age correction into a linear regression model. This strategy showed significant differences between patients with FM and control subjects (P < 0.0001 and P  = 0.0002, for 2 separate control populations, respectively). A subgroup analysis using the QUICKI and HOMA-IR showed that all patients with FM in this subgroup (100%) exhibited laboratory abnormalities pointing to IR. LIMITATIONS: Small observational cross-sectional study. There are also intrinsic limitations that are attributed to cross-sectional studies. CONCLUSION: The association demonstrated in this study warrant further investigation, including the pursuit of randomized, double-blind clinical trials to determine the effect of improving insulin sensitivity in FM related pain scores. Such studies could unveil a potential pathogenetic relationship between FM, central pain, and IR. Based on these initial findings, we present the hypothesis that IR may underlie pathological mechanisms leading to central pain. If confirmed, this may lead to a paradigm shift in the management of central pain.

Methodology for Evidence Synthesis and Development of Comprehensive Evidence-Based Guidelines for Interventional Techniques in Chronic Spinal Pain.

BACKGROUND: The re-engineered definition of clinical guidelines in 2011 from the IOM (Institute of Medicine) states, "clinical practice guidelines are statements that include recommendations intended to optimize patient care that is informed by a systematic review of evidence and an assessment of the benefit and harms of alternative care options." The revised definition distinguishes between the term "clinical practice guideline" and other forms of clinical guidance derived from widely disparate development processes, such as consensus statements, expert advice, and appropriate use criteria. OBJECTIVE: To assess the literature and develop methodology for evidence synthesis and development of comprehensive evidence-based guidelines for interventional techniques in chronic spinal pain. METHODS: A systematic review of the literature including methodology of guideline development encompassing GRADE approach for guidance on evidence synthesis with recommendations. RESULTS: Some of the many factors described in 2011 continue as of 2020 and impede the development of clinical practice guidelines. These impediments include biases due to a variety of conflicts and confluence of interest, inappropriate and poor methodological quality, poor writing and ambiguous presentation, projecting a view that these are not applicable to individual patients or too restrictive with the elimination of clinician autonomy, and overzealous and inappropriate recommendations, either positive, negative, or non-committal. Thus, ideally, a knowledgeable, multidisciplinary panel of experts with true lack of bias and confluence of interest must develop guidelines based on a systematic review of the existing evidence. This manuscript describes evidence synthesis from observational studies, various types of randomized controlled trials (RCTs), and, finally, methodological and reporting quality of systematic reviews. The manuscript also describes various methods utilized in the assessment of the quality of observational studies, diagnostic accuracy studies, RCTs, and systematic reviews. LIMITATIONS: Paucity of publications with appropriate evidence synthesis methodology in reference to interventional techniques. CONCLUSION: This review described comprehensive evidence synthesis derived from systematic reviews, including methodologic quality and bias measurement. The manuscript described various methods utilized in the assessment of the quality of the systematic reviews, RCTs, diagnostic accuracy studies, and observational studies.

HIV-related Neuropathy: Pathophysiology, Treatment and Challenges.

HIV-sensory neuropathy (HIV-SN) is a debilitating complication in HIV patients with or without anti-retroviral treatment (ART). Common symptoms of HIV-SN include pain, decreased sensation, paresthesias, and dysesthesias in a symmetric stocking-glove distribution. While HIV-1 protein such as gp120 is implicated in HIV-SN (e.g. impaired large-diameter fiber), ART itself was recently shown to contribute to HIV-SN in HIV patients and impair thin fiber. Multiple host mechanisms may play roles during the pathogenesis of HIV-SN, including neuron-glia interactions in the spinal dorsal horn (SDH), inflammation, mitochondrial dysfunction and endoplasmic reticulum stress. Concurrent infections, such as tuberculosis, also carry a higher likelihood of HIV-SN as well as environmental or genetic predisposition. Pro-inflammatory cytokines such as IL-1, IL2 receptor-alpha, and tumor necrosis factor (TNF) along with abnormal lactate levels have been identified as potential players within the complex pathophysiology of this condition. In this paper, we review the pathophysiology of HIV neuropathy, focusing on the various treatment options available or under investigation. Although several treatment options are available e.g., the capsaicin patch and spinal cord stimulation, symptomatic control of HIV-SN are often challenging. Alternative approaches such as self-hypnosis, resistance exercise, cannabinoids, and acupuncture have all shown promising results, but need further investigation.

Is insulin resistance the cause of fibromyalgia? A preliminary report.

Fibromyalgia (FM) is one of the most frequent generalized pain disorders with poorly understood neurobiological mechanisms. This condition accounts for an enormous proportion of healthcare costs. Despite extensive research, the etiology of FM is unknown and thus, there is no disease modifying therapy available for this condition. We show that most (if not all) patients with FM belong to a distinct population that can be segregated from a control group by their glycated hemoglobin A1c (HbA1c) levels, a surrogate marker of insulin resistance (IR). This was demonstrated by analyzing the data after introducing an age stratification correction into a linear regression model. This strategy showed highly significant differences between FM patients and control subjects (p < 0.0001 and p = 0.0002, for two separate control populations, respectively). A subgroup of patients meeting criteria for pre-diabetes or diabetes (patients with HbA1c values of 5.7% or greater) who had undergone treatment with metformin showed dramatic improvements of their widespread myofascial pain, as shown by their scores using a pre and post-treatment numerical pain rating scale (NPRS) for evaluation. Although preliminary, these findings suggest a pathogenetic relationship between FM and IR, which may lead to a radical paradigm shift in the management of this disorder.

Responsible, Safe, and Effective Use of Biologics in the Management of Low Back Pain: American Society of Interventional Pain Physicians (ASIPP) Guidelines.

BACKGROUND: Regenerative medicine is a medical subspecialty that seeks to recruit and enhance the body's own inherent healing armamentarium in the treatment of patient pathology. This therapy's intention is to assist in the repair, and to potentially replace or restore damaged tissue through the use of autologous or allogenic biologics. This field is rising like a Phoenix from the ashes of underperforming conventional therapy midst the hopes and high expectations of patients and medical personnel alike. But, because this is a relatively new area of medicine that has yet to substantiate its outcomes, care must be taken in its public presentation and promises as well as in its use. OBJECTIVE: To provide guidance for the responsible, safe, and effective use of biologic therapy in the lumbar spine. To present a template on which to build standardized therapies using biologics. To ground potential administrators of biologics in the knowledge of the current outcome statistics and to stimulate those interested in providing biologic therapy to participate in high quality research that will ultimately promote and further advance this area of medicine. METHODS: The methodology used has included the development of objectives and key questions. A panel of experts from various medical specialties and subspecialties as well as differing regions collaborated in the formation of these guidelines and submitted (if any) their appropriate disclosures of conflicts of interest. Trustworthy standards were employed in the creation of these guidelines. The literature pertaining to regenerative medicine, its effectiveness, and adverse consequences was thoroughly reviewed using a best evidence synthesis of the available literature. The grading for recommendation was provided as described by the Agency for Healthcare Research and Quality (AHRQ). SUMMARY OF EVIDENCE: Lumbar Disc Injections: Based on the available evidence regarding the use of platelet-rich plasma (PRP), including one high-quality randomized controlled trial (RCT), multiple moderate-quality observational studies, a single-arm meta-analysis and evidence from a systematic review, the qualitative evidence has been assessed as Level III (on a scale of Level I through V) using a qualitative modified approach to the grading of evidence based on best-evidence synthesis. Based on the available evidence regarding the use of medicinal signaling/ mesenchymal stem cell (MSCs) with a high-quality RCT, multiple moderate-quality observational studies, a single-arm meta-analysis, and 2 systematic reviews, the qualitative evidence has been assessed as Level III (on a scale of Level I through V) using a qualitative modified approach to the grading of evidence based on best evidence synthesis. Lumbar Epidural Injections Based on one high-quality RCT, multiple relevant moderate-quality observational studies and a single-arm meta-analysis, the qualitative evidence has been assessed as Level IV (on a scale of Level I through V) using a qualitative modified approach to the grading of evidence based on best evidence synthesis. Lumbar Facet Joint Injections Based on one high-quality RCT and 2 moderate-quality observational studies, the qualitative evidence for facet joint injections with PRP has been assessed as Level IV (on a scale of Level I through V) using a qualitative modified approach to the grading of evidence based on best evidence synthesis. Sacroiliac Joint Injection Based on one high-quality RCT, one moderate-quality observational study, and one low-quality case report, the qualitative evidence has been assessed as Level IV (on a scale of Level I through V) using a qualitative modified approach to the grading of evidence based on best evidence synthesis. CONCLUSION: Based on the evidence synthesis summarized above, there is Level III evidence for intradiscal injections of PRP and MSCs, whereas the evidence is considered Level IV for lumbar facet joint, lumbar epidural, and sacroiliac joint injections of PRP, (on a scale of Level I through V) using a qualitative modified approach to the grading of evidence based on best evidence synthesis.Regenerative therapy should be provided to patients following diagnostic evidence of a need for biologic therapy, following a thorough discussion of the patient's needs and expectations, after properly educating the patient on the use and administration of biologics and in full light of the patient's medical history. Regenerative therapy may be provided independently or in conjunction with other modalities of treatment including a structured exercise program, physical therapy, behavioral therapy, and along with the appropriate conventional medical therapy as necessary. Appropriate precautions should be taken into consideration and followed prior to performing biologic therapy. Multiple guidelines from the Food and Drug Administration (FDA), potential limitations in the use of biologic therapy and the appropriate requirements for compliance with the FDA have been detailed in these guidelines. KEY WORDS: Regenerative medicine, platelet-rich plasma, medicinal signaling cells, mesenchymal stem cells, stromal vascular fraction, bone marrow concentrate, chronic low back pain, discogenic pain, facet joint pain, Food and Drug Administration, minimal manipulation, evidence synthesis.

Do Regenerative Medicine Therapies Provide Long-Term Relief in Chronic Low Back Pain: A Systematic Review and Metaanalysis.

BACKGROUND: Several cell-based therapies have been proposed in recent years the management of low back pain, including the injection of medicinal signaling cells or mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP). However, there is only emerging clinical evidence to support their use at this time. OBJECTIVE: To assess the effectiveness of MSCs or PRP injections in the treatment of low back and lower extremity pain. STUDY DESIGN: A systematic review and metaanalysis of the effectiveness of PRP and MSCs injections in managing low back and lower extremity pain. DATA SOURCES: PubMed, Cochrane Library, US National Guideline Clearinghouse, prior systematic reviews, and reference lists. The literature search was performed from 1966 through June 2018. STUDY SELECTION: Randomized trials, observational studies, and case reports of injections of biologics into the disc, epidural space, facet joints, or sacroiliac joints. DATA EXTRACTION: Data extraction and methodological quality assessment were performed utilizing Cochrane review methodologic quality assessment and Interventional Pain Management Techniques - Quality Appraisal of Reliability and Risk of Bias Assessment (IPM-QRB) and Interventional Pain Management Techniques - Quality Appraisal of Reliability and Risk of Bias Assessment for Nonrandomized Studies (IPM-QRBNR). The evidence was summarized utilizing principles of best evidence synthesis on a scale of 1 to 5. DATA SYNTHESIS: Twenty-one injection studies met inclusion criteria. There were 12 lumbar disc injections, 5 epidural, 3 lumbar facet joint, and 3 sacroiliac joint studies RESULTS: Evidence synthesis based on a single-arm metaanalysis, randomized controlled trials (RCTs), and observational studies, disc injections of PRP and MSCs showed Level 3 evidence (on a scale of Level I through V). Evidence for epidural injections based on single-arm metaanalysis, a single randomized controlled trial and other available studies demonstrated Level 4 (on a scale of Level I through V) evidence. Similarly, evidence for lumbar facet joint injections and sacroiliac joint injections without metaanalysis demonstrated Level 4 evidence (on a scale of Level I through V). LIMITATIONS: Lack of high quality RCTs. CONCLUSION: The findings of this systematic review and single-arm metaanalysis shows that MSCs and PRP may be effective in managing discogenic low back pain, radicular pain, facet joint pain, and sacroiliac joint pain, with variable levels of evidence in favor of these techniques. KEY WORDS: Chronic low back pain, regenerative therapy, medicinal signaling or mesenchymal stem cells, platelet-rich plasma, disc injection, lumbar facet joint injections, sacroiliac joint injections.

Amyloid-beta protein clearance and degradation (ABCD) pathways and their role in Alzheimer's disease.

Amyloid-ß proteins (Aß) of 42 (Aß42) and 40 aa (Aß40) accumulate as senile plaques (SP) and cerebrovascular amyloid protein deposits that are defining diagnostic features of Alzheimer's disease (AD). A number of rare mutations linked to familial AD (FAD) on the Aß precursor protein (APP), Presenilin-1 (PS1), Presenilin- 2 (PS2), Adamalysin10, and other genetic risk factors for sporadic AD such as the ε4 allele of Apolipoprotein E (ApoE-ε4) foster the accumulation of Aß and also induce the entire spectrum of pathology associated with the disease. Aß accumulation is therefore a key pathological event and a prime target for the prevention and treatment of AD. APP is sequentially processed by ß-site APP cleaving enzyme (BACE1) and γ-secretase, a multisubunit PS1/PS2-containing integral membrane protease, to generate Aß. Although Aß accumulates in all forms of AD, the only pathways known to be affected in FAD increase Aß production by APP gene duplication or via base substitutions on APP and γ-secretase subunits PS1 and PS2 that either specifically increase the yield of the longer Aß42 or both Aß40 and Aß42. However, the vast majority of AD patients accumulate Aß without these known mutations. This led to proposals that impairment of Aß degradation or clearance may play a key role in AD pathogenesis. Several candidate enzymes, including Insulin-degrading enzyme (IDE), Neprilysin (NEP), Endothelin-converting enzyme (ECE), Angiotensin converting enzyme (ACE), Plasmin, and Matrix metalloproteinases (MMPs) have been identified and some have even been successfully evaluated in animal models. Several studies also have demonstrated the capacity of γ-secretase inhibitors to paradoxically increase the yield of Aß and we have recently established that the mechanism is by skirting Aß degradation. This review outlines major cellular pathways of Aß degradation to provide a basis for future efforts to fully characterize the panel of pathways responsible for Aß turnover.

Evidence for lymphatic Aß clearance in Alzheimer's transgenic mice.

Evidence has shown that lymphatic drainage contributes to removal of debris from the brain but its role in the accumulation of amyloid ß peptides (Aß) has not been demonstrated. We examined the levels of various forms of Aß in the brain, plasma and lymph nodes in a transgenic model of Alzheimer's disease (AD) at different ages. Herein, we report on the novel finding that Aß is present in the cervical and axillary lymph nodes of AD transgenic mice and that Aß levels in lymph nodes increase over time, mirroring the increase of Aß levels observed in the brain. Aß levels in lymph nodes were significantly higher than in plasma. At age 15.5months, there was a significant increase of monomeric soluble Aß40 (p=0.003) and Aß42 (p=0.05) in the lymph nodes over the baseline values measured at 6months of age. In contrast, plasma levels of Aß40 showed no significant changes (p=0.68) and plasma levels Aß42 significantly dropped (p=0.02) at the same age. Aß concentration was low to undetectable in splenic lymphoid tissue and several other control tissues including heart, lung, liver, kidneys and intestine of the same animals, strongly suggesting that Aß peptides in lymph nodes are derived from the brain.

Evidence of a novel mechanism for partial γ-secretase inhibition induced paradoxical increase in secreted amyloid ß protein.

BACE1 (ß-secretase) and α-secretase cleave the Alzheimer's amyloid ß protein (Aß) precursor (APP) to C-terminal fragments of 99 aa (CTFß) and 83 aa (CTFα), respectively, which are further cleaved by γ-secretase to eventually secrete Aß and Aα (a.k.a. P3) that terminate predominantly at residues 40 and 42. A number of γ-secretase inhibitors (GSIs), such as N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), have been developed with the goal of reducing Aß to treat Alzheimer's disease (AD). Although most studies show that DAPT inhibits Aß in a dose-dependent manner several studies have also detected a biphasic effect with an unexpected increase at low doses of DAPT in cell cultures, animal models and clinical trials. In this article, we confirm the increase in Aß40 and Aß42 in SH-SY5Y human neuroblastoma cells treated with low doses of DAPT and identify one of the mechanisms for this paradox. We studied the pathway by first demonstrating that stimulation of Aß, a product of γ-secretase, was accompanied by a parallel increase of its substrate CTFß, thereby demonstrating that the inhibitor was not anomalously stimulating enzyme activity at low levels. Secondly, we have demonstrated that inhibition of an Aß degrading activity, endothelin converting enzyme (ECE), yielded more Aß, but abolished the DAPT-induced stimulation. Finally, we have demonstrated that Aα, which is generated in the secretory pathway before endocytosis, is not subject to the DAPT-mediated stimulation. We therefore conclude that impairment of γ-secretase can paradoxically increase Aß by transiently skirting Aß degradation in the endosome. This study adds to the growing body of literature suggesting that preserving γ-secretase activity, rather than inhibiting it, is important for prevention of neurodegeneration.

Major carboxyl terminal fragments generated by γ-secretase processing of the Alzheimer amyloid precursor are 50 and 51 amino acids long.

OBJECTIVES: To understand the cleavage of the amyloid ß protein (Aß) precursor (APP) by γ-secretase and to determine its changes in a representative familial Alzheimer disease (FAD) mutation. METHODS: Transfected cells expressing wild-type and FAD mutant APP were analyzed for changes in the levels of the major secreted Aß species and of the corresponding intracellular C-terminal APP fragments (APP intracellular domain, AICD) generated by γ-secretase, whereas radio-sequencing was used to precisely identify the resulting cleavage site(s). RESULTS: The AICD fragment(s) generated by γ-secretase cleavage comigrated in gels with a 50-residue synthetic peptide used as control, which is smaller than the 59 and 57 residues predicted from Aß ending at positions 40 (Aß40) and 42 (Aß42), respectively. In agreement with previous findings, an FAD mutant form of presenilin 1 (PS1-M139V) significantly increased the longer Aß42 while showing trends toward reducing Aß40. AICD levels were reduced by the mutation, suggesting that γ-secretase activity may be actually impaired by the mutation. Radiosequence analysis in cells expressing wild-type PS1 detected γ-secretase cleavage sites at the Aß peptide bond L(49)-V(50) to generate a 50-amino acid (aa) AICD fragment (AICD50) and the Aß peptide bond T(48)-L(49), generating an AICD of 51 aa (AICD51). No other cleavage sites were reliably detected. CONCLUSIONS: Based on findings that the FAD mutation that increases Aß42 also reduces AICD, we propose that γ-secretase activity is impaired by FAD mutations and predict that physiologic and environmental agents that inhibit γ-secretase will actually induce AD pathogenesis rather that prevent it. Furthermore, we propose that the cleavage site to generate AICD is naturally ragged and occurs predominantly at two sites 48 and 49 aa from the start of the Aß sequence. Thus, end specific antibodies to these two sites will need to be generated to study the quantitative relationships between these two cleavages in sporadic AD and FAD.

Targets for AD treatment: conflicting messages from γ-secretase inhibitors.

Current evidence suggests that Alzheimer's disease (AD) is a multi-factorial disease that starts with accumulation of multiple proteins. We have previously proposed that inhibition of γ-secretase may impair membrane recycling causing neurodegeneration starting at synapses (Sambamurti K., Suram A., Venugopal C., Prakasam A., Zhou Y., Lahiri D. K. and Greig N. H. A partial failure of membrane protein turnover may cause Alzheimer's disease: a new hypothesis. Curr. Alzheimer Res., 3, 2006, 81). We also proposed familal AD mutations increase Aß42 by inhibiting γ-secretase. Herein, we discuss the failure of Eli Lilly's γ-secretase inhibitor, semagacestat, in clinical trials in the light of our hypothesis, which extends the problem beyond toxicity of Aß aggregates. We elaborate that γ-secretase inhibitors lead to accumulation of amyloid precursor protein C-terminal fragments that can later be processed by γ-secretase to yields bursts of Aß to facilitate aggregation. Although we do not exclude a role for toxic Aß aggregates, inhibition of γ-secretase can affect numerous substrates other than amyloid precursor protein to affect multiple pathways and the combined accumulation of multiple peptides in the membrane may impair its function and turnover. Taken together, protein processing and turnover pathways play an important role in maintaining cellular homeostasis and unless we clearly see consistent disease-related increase in their levels or activity, we need to focus on preserving their function rather than inhibiting them for treatment of AD and similar diseases.

Amphiphilic amide nitrones: a new class of protective agents acting as modifiers of mitochondrial metabolism.

Our group has demonstrated that the amphiphilic character of alpha-phenyl-N-tert-butyl nitrone based agents is a key feature in determining their bioactivity and protection against oxidative toxicity. In this work, we report the synthesis of a new class of amphiphilic amide nitrones. Their hydroxyl radical scavenging activity and radical reducing potency were shown using ABTS competition and ABTS(+) reduction assays, respectively. Cyclic voltammetry was used to investigate their redox behavior, and the effects of the substitution of the PBN on the charge density of the nitronyl atoms, the electron affinity, and the ionization potential were computationally rationalized. The protective effects of amphiphilic amide nitrones in cell cultures exposed to oxidotoxins greatly exceeded those exerted by the parent compound PBN. They decreased electron and proton leakage as well as hydrogen peroxide formation in isolated rat brain mitochondria at nanomolar concentration. They also significantly enhanced mitochondrial membrane potential. Finally, dopamine-induced inhibition of complex I activity was antagonized by pretreatment with these agents. These findings indicate that amphiphilic amide nitrones are much more than just radical scavenging antioxidants but may act as a new class of bioenergetic agents directly on mitochondrial electron and proton transport.