Tapan Kumar Khan

An internally controlled peripheral biomarker for Alzheimer’s disease: Erk1 and Erk2 responses to the inflammatory signal bradykinin

Cognitive impairment has recently been found to correlate with changes in peripheral inflammatory signals such as TNF-α and IL-1β. PKC isozymes regulate levels of TNF-α and IL-6 and the release of other cytokines and also show deficits in Alzheimer’s disease (AD) brains and skin fibroblasts. Here, we investigate MAPK Erk1 and Erk2 phosphorylation in response to the inflammatory agonist bradykinin, which activates PKC pathways. An internally controlled comparison of Erk1 and Erk2 produced an AD index that accurately distinguished fibroblasts of AD from those of normal controls and of non-AD dementias. This accuracy was demonstrated for Coriell Cell Repository (Coriell Institute of Medical Research, Camden, NJ) samples, as well as for samples analyzed on gels with autopsy diagnostic confirmation. AD Erk1 and Erk2 index values were inversely correlated with disease duration, suggesting maximal efficacy for early diagnosis. Finally, the results also demonstrate that, when the AD index agreed with the clinical diagnosis on the presence of AD, there was a high probability of accuracy based on autopsy validation. Thus, this peripheral molecular biomarker, based on differential Erk1 and Erk2 phosphorylation, could have important clinical utility for providing increased certainty in the positive diagnosis of AD, particularly in the early phase of disease progression.

Peripheral Biomarkers of Alzheimer's Disease

Currently available diagnostic tests have moved the field closer to early diagnosis of Alzheimers disease (AD); however, a definitive diagnosis is made only with the development of clinical dementia and the presence of amyloid plaques and neurofibrillary tangles at autopsy. An ideal antemortem AD biomarker should satisfy the following criteria: the ability to diagnose AD with high sensitivity and specificity as confirmed by the gold standard of autopsy validation; the ability to detect early-stage disease and track the progression of AD; and monitor therapeutic efficacy. AD biomarker technologies currently under development include in vivo brain imaging with PET and MRI (i.e., imaging of amyloid plaques, biochemical assays in cerebrospinal fluid (CSF) and peripheral tissues. CSF biomarkers have received increased attention in the past decade. However, it is unclear whether these biomarkers are capable of early diagnosis of AD, prior to Aβ accumulation, or whether they can differentiate between AD and non-AD dementias. In addition, CSF biomarkers may not lend themselves to diagnostic screening of elderly patients, given the invasiveness of lumbar puncture, inter-laboratory variability in techniques and sample handling, and the circadian fluctuation of CSF components. Although commonly viewed as an abnormality of the brain, AD is a systemic disease with associated dysfunction in metabolic, oxidative, inflammatory, and biochemical pathways in peripheral tissues, such as the skin and blood cells. This has led researchers to investigate and develop assays of peripheral AD biomarkers (a few with high sensitivity and specificity) that require minimally invasive skin or blood samples.

Alzheimer’s Disease Cerebrospinal Fluid and Neuroimaging Biomarkers: Diagnostic Accuracy and Relationship to Drug Efficacy

Widely researched Alzheimers disease (AD) biomarkers include in vivo brain imaging with PET and MRI, imaging of amyloid plaques, and biochemical assays of Aβ 1 - 42, total tau, and phosphorylated tau (p-tau-181) in cerebrospinal fluid (CSF). In this review, we critically evaluate these biomarkers and discuss their clinical utility for the differential diagnosis of AD. Current AD biomarker tests are either highly invasive (requiring CSF collection) or expensive and labor-intensive (neuroimaging), making them unsuitable for use in the primary care, clinical office-based setting, or to assess drug efficacy in clinical trials. In addition, CSF and neuroimaging biomarkers continue to face challenges in achieving required sensitivity and specificity and minimizing center-to-center variability (for CSF-Aβ 1 - 42 biomarkers CV = 26.5% ; http://www.alzforum.org/news/conference-coverage/paris-standardization-hurdle-spinal-fluid-imaging-markers). Although potentially useful for selecting patient populations for inclusion in AD clinical trials, the utility of CSF biomarkers and neuroimaging techniques as surrogate endpoints of drug efficacy needs to be validated. Recent trials of β- and γ-secretase inhibitors and Aβ immunization-based therapies in AD showed no significant cognitive improvements, despite changes in CSF and neuroimaging biomarkers. As we learn more about the dysfunctional cellular and molecular signaling processes that occur in AD, and how these processes are manifested in tissues outside of the brain, new peripheral biomarkers may also be validated as non-invasive tests to diagnose preclinical and clinical AD.

Bryostatin Effects on Cognitive Function and PKCɛ in Alzheimer’s Disease Phase IIa and Expanded Access Trials

Bryostatin 1, a potent activator of protein kinase C epsilon (PKCɛ), has been shown to reverse synaptic loss and facilitate synaptic maturation in animal models of Alzheimer’s disease (AD), Fragile X, stroke, and other neurological disorders. In a single-dose (25 μg/m2) randomized double-blind Phase IIa clinical trial, bryostatin levels reached a maximum at 1-2 h after the start of infusion. In close parallel with peak blood levels of bryostatin, an increase of PBMC PKCɛ was measured (p = 0.0185) within 1 h from the onset of infusion. Of 9 patients with a clinical diagnosis of AD, of which 6 received drug and 3 received vehicle within a double-blind protocol, bryostatin increased the Mini-Mental State Examination (MMSE) score by +1.83±0.70 unit at 3 h versus –1.00±1.53 unit for placebo. Bryostatin was well tolerated in these AD patients and no drug-related adverse events were reported. The 25 μg/m2 administered dose was based on prior clinical experience with three Expanded Access advanced AD patients treated with bryostatin, in which return of major functions such as swallowing, vocalization, and word recognition were noted. In one Expanded Access patient trial, elevated PKCɛ levels closely tracked cognitive benefits in the first 24 weeks as measured by MMSE and ADCS-ADL psychometrics. Pre-clinical mouse studies showed effective activation of PKCɛ and increased levels of BDNF and PSD-95. Together, these Phase IIa, Expanded Access, and pre-clinical results provide initial encouragement for bryostatin 1 as a potential treatment for AD.

Spatiotemporal Complexity of Fibroblast Networks Screens for Alzheimer's Disease

Drugs to treat Alzheimers disease (AD) have been unsuccessful in preventing its devastating cognitive deficits and progressive neurodegeneration. The lack of a definitive diagnostic for AD has been a major obstacle to AD drug discovery. Here, we describe a novel, highly accurate peripheral diagnostic for AD patients based on quantitatively measured complexity of skin-sampled fibroblast networks. A significant number of samples were studied under double-blind conditions and had autopsy and/or genetic validation. An additional sample confirmed the diagnostic discrimination on freshly obtained skin samples. A sub-sample of these diagnostic differences were induced by oligomerized amyloid-β1-42. Based on the accuracy of these measures that utilize physical principles such as fractal dimension and lacunarity as well as the significant correlation with disease duration, this biomarker profile appears to identify accurately AD patients for therapeutic intervention.

Fibroblast Aggregation Rate Converges with Validated Peripheral Biomarkers for Alzheimer's Disease

The inaccuracy of the diagnosis for Alzheimers disease (AD) has made its therapeutic intervention difficult, particularly early enough to prevent significant neurodegeneration and cognitive dysfunction. Here, we describe a novel, highly accurate peripheral diagnostic for AD patients based on quantitatively measured aggregation rate of human skin fibroblasts. The elevated aggregation rate with increasing cell density in AD cases is the basis of this new biomarker. The new biomarker was successfully cross-validated with two more mature assays, AD-Index, based on the imbalances of ERK1/2, and Morphology, based on network dynamics, and showed 92% overlap. A significant number of cases tested with this new biomarker were freshly obtained (n = 29), and 82% of the cases are hyper-validated cases, i.e., autopsy and/or genetically confirmed AD or non-Alzheimers disease demented patients (Non-ADD) and non-demented age-matched controls. Furthermore, we show that by using a simple majority rule, i.e., two out of the three assays have the same outcome, we significantly increase the agreement with clinical AD diagnosis (100%). Based on the high accuracy of this strategy, the biomarker profile appears to accurately identify AD patients for therapeutic intervention.

Bryostatin and its synthetic analog, picolog rescue dermal fibroblasts from prolonged stress and contribute to survival and rejuvenation of human skin equivalents†

Skin health is associated with the day‐to‐day activity of fibroblasts. The primary function of fibroblasts is to synthesize structural proteins, such as collagen, extracellular matrix proteins, and other proteins that support the structural integrity of the skin and are associated with younger, firmer, and more elastic skin that is better able to resist and recover from injury. At sub‐nanomolar concentrations (0.03–0.3 nM), bryostatin‐1 and its synthetic analog, picolog (0.1–10 nM) sustained the survival and activation of human dermal fibroblasts cultured under the stressful condition of prolonged serum deprivation. Bryostatin‐1 treatment stabilized human skin equivalents (HSEs), a bioengineered combination of primary human skin cells (keratinocytes and dermal fibroblasts) on an extracellular matrix composed of mainly collagen. Fibroblasts activated by bryostatin‐1 protected the structural integrity of HSEs. Bryostatin‐1 and picolog prolonged activation of Erk in fibroblasts to promote cell survival. Chronic stress promotes the progression of apoptosis. Dermal fibroblasts constitutively express all components of Fas associated apoptosis, including caspase‐8, an initiator enzyme of apoptosis. Prolong bryostatin‐1 treatment reduced apoptosis by decreasing caspase‐8 and protected dermal fibroblasts. Our data suggest that bryostatin‐1 and picolog could be useful in anti‐aging skincare, and could have applications in tissue engineering and regenerative medicine.

Protein kinase C activator bryostatin-1 modulates proteasome function

Proteasome activity in ubiquitin‐proteasome pathway plays a pivotal role in degradation and clearance of aggregated, oxidized, damaged, and misfolded unwanted proteins to control protein homeostasis or proteostasis. Proteasome activity decreases with cellular senescence, aging, and age‐related diseases. Therefore, enhancement of impaired proteasome function by molecular biological and/or pharmacological intervention is an active area of research. Bryostatin‐1, a naturally occurring macrocyclic lactone, activates PKC isozymes (specifically, ‐α and ‐ϵ) at sub‐nanomolar concentrations, but downregulates at higher concentrations. Here, we present bryostatin‐1 increased chymotrypsin‐like proteasome activity of 20S assembly at sub‐nanomolar to nanomolar concentrations (0.3‐30 nM). However, proteasome activity decreased at a micromolar concentration of bryostatin‐1 (AG08044 cultured skin: P < 0.005; differentiated SH‐SY5Y cells: P < 0.02). Modulation of proteasome function by bryostatin‐1 was studied in six dermal fibroblast primary cell lines developed both from freshly taken biopsies from healthy donors (n = 2) and obtained from well‐characterized cell repositories (n = 4; without any diseases). Bryostatin‐1 enhanced proteasome activity in cultured skin fibroblasts obtained from banked and freshly isolated skin fibroblasts from skin biopsies at the sub‐nanomolar concentration (P < 0.015). Modulation of proteasome function by bryostatin‐1 was confirmed in neuron‐like differentiated SH‐SY5Y cells. Direct additions of bryostatin‐1 into cell lysates prepared from neuron‐like differentiated SH‐SY5Y, Jurkat cells, and cultured skin fibroblasts were unable to increase proteasome activity indicating that bryostatin‐1 can only modulate proteasome activity when added to live cell culture systems. Standard PKC inhibitors blocked bryostatin‐1 induced proteasome activity modulation suggesting that enhancement of proteasome activity was mediated by PKC modulation.

An Algorithm for Preclinical Diagnosis of Alzheimer's Disease

Almost all Alzheimers disease (AD) therapeutic trials have failed in recent years. One of the main reasons for failure is due to designing the disease-modifying clinical trials at the advanced stage of the disease when irreversible brain damage has already occurred. Diagnosis of the preclinical stage of AD and therapeutic intervention at this phase, with a perfect target, are key points to slowing the progression of the disease. Various AD biomarkers hold enormous promise for identifying individuals with preclinical AD and predicting the development of AD dementia in the future, but no single AD biomarker has the capability to distinguish the AD preclinical stage. A combination of complimentary AD biomarkers in cerebrospinal fluid (Aβ42, tau, and phosphor-tau), non-invasive neuroimaging, and genetic evidence of AD can detect preclinical AD in the in-vivo ante mortem brain. Neuroimaging studies have examined region-specific cerebral blood flow (CBF) and microstructural changes in the preclinical AD brain. Functional MRI (fMRI), diffusion tensor imaging (DTI) MRI, arterial spin labeling (ASL) MRI, and advanced PET have potential application in preclinical AD diagnosis. A well-validated simple framework for diagnosis of preclinical AD is urgently needed. This article proposes a comprehensive preclinical AD diagnostic algorithm based on neuroimaging, CSF biomarkers, and genetic markers.