Innovative Therapy for Alzheimer’s Disease-With Focus on Biodelivery of NGF

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder associated with abnormal protein modification, inflammation and memory impairment. Aggregated amyloid beta (Aβ) and phosphorylated tau proteins are medical diagnostic features. Loss of memory in AD has been associated with central cholinergic dysfunction in basal forebrain, from where the cholinergic circuitry projects to cerebral cortex and hippocampus. Various reports link AD progression with declining activity of cholinergic neurons in basal forebrain. The neurotrophic molecule, nerve growth factor (NGF), plays a major role in the maintenance of cholinergic neurons integrity and function, both during development and adulthood. Numerous studies have also shown that NGF contributes to the survival and regeneration of neurons during aging and in age-related diseases such as AD. Changes in neurotrophic signaling pathways are involved in the aging process and contribute to cholinergic and cognitive decline as observed in AD. Further, gradual dysregulation of neurotrophic factors like NGF and brain derived neurotrophic factor (BDNF) have been reported during AD development thus intensifying further research in targeting these factors as disease modifying therapies against AD. Today, there is no cure available for AD and the effects of the symptomatic treatment like cholinesterase inhibitors (ChEIs) and memantine are transient and moderate. Although many AD treatment studies are being carried out, there has not been any breakthrough and new therapies are thus highly needed. Long-term effective therapy for alleviating cognitive impairment is a major unmet need. Discussion and summarizing the new advancements of using NGF as a potential therapeutic implication in AD are important. In summary, the intent of this review is describing available experimental and clinical data related to AD therapy, priming to gain additional facts associated with the importance of NGF for AD treatment, and encapsulated cell biodelivery (ECB) as an efficient tool for NGF delivery

Autophagy-lysosomal defect in human CADASIL vascular smooth muscle cells

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial progressive degenerative disorder and is caused by mutations in NOTCH3 gene. Previous study reported that mutant NOTCH3 is more prone to form aggregates than wild-type NOTCH3 and the mutant aggregates are resistant to degradation. We hypothesized that aggregation or accumulation of NOTCH3 could be due to impaired lysosomal-autophagy machinery in VSMC. Here, we investigated the possible cause of accumulation/aggregation of NOTCH3 in CADASIL using cerebral VSMCs derived from control and CADASIL patients carrying NOTCH3(RI33C) mutation. Thioflavin-S-staining confirmed the increased accumulation of aggregated NOTCH3 in VSMCR133C compared to VSMCWT. Increased levels of the lysosomal marker, Lamp2, were detected in VSMCR133C, which also showed co-localization with NOTCH3 using double-immunohistochemistry. Increased level of LC3-II/LC3-I ratio was observed in VSMCR133C suggesting an accumulation of autophagosomes. This was coupled with the decreased co-localization of NOTCH3 with LC3, and Lamp2 and, further, increase of p62/SQSTM1 levels in VSMCR133C compared to the VSMCWT. In addition, Western blot analysis indicated phosphorylation of p-ERK, p-S6RP, and p-P70 S6K. Altogether, these results suggested a dysfunction in the autophagy-lysosomal pathway in VSMCR133C. The present study provides an interesting avenue of the research investigating the molecular mechanism of CADASIL.Peer reviewe

Differences in proliferation rate between CADASIL and control vascular smooth muscle cells are related to increased TGF beta expression

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial fatal progressive degenerative disorder. One of the pathological hallmarks of CADASIL is a dramatic reduction of vascular smooth muscle cells (VSMCs) in cerebral arteries. Using VSMCs from the vasculature of the human umbilical cord, placenta and cerebrum of CADASIL patients, we found that CADASIL VSMCs had a lower proliferation rate compared to control VSMCs. Exposure of control VSMCs and endothelial cells (ECs) to media derived from CADASIL VSMCs lowered the proliferation rate of all cells examined. By quantitative RT-PCR analysis, we observed increased Transforming growth factor-beta (TGF beta) gene expression in CADASIL VSMCs. Adding TGF beta-neutralizing antibody restored the proliferation rate of CADASIL VSMCs. We assessed proliferation differences in the presence or absence of TGF beta-neutralizing antibody in ECs co-cultured with VSMCs. ECs co-cultured with CADASIL VSMCs exhibited a lower proliferation rate than those co-cultured with control VSMCs, and neutralization of TGF beta normalized the proliferation rate of ECs co-cultured with CADASIL VSMCs. We suggest that increased TGF beta expression in CADASIL VSMCs is involved in the reduced VSMC proliferation in CADASIL and may play a role in situ in altered proliferation of neighbouring cells in the vasculature.Peer reviewe

Low Levels of Perforin Expression in CD8+ T Lymphocyte Granules in Lymphoid Tissue during Acute Human Immunodeficiency Virus Type 1 Infection

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) responses are detectable shortly after the acute phase of HIV infection, but they cannot control viral replication and prevent development of chronic immune suppression. This article describes a defect in the coexpression of perforin in granzyme A-positive CD8+ T cells in lymphoid tissue from patients with acute HIV infection and a reduction in the perforin-dependent nuclear translocation of granzyme A. Furthermore, intracellular levels of HIV DNA and RNA found in lymphoid tissue were higher (10-100 times) than those found in blood, and blood samples showed more-coordinated cellular perforin/granzyme A expression. This suggests that mechanisms inhibiting CTL-mediated cytotoxicity are operative in lymphoid tissue early in the course of HIV infectio

Differences in proliferation rate between CADASIL and control vascular smooth muscle cells are related to increased TGFβ expression

Cerebral autosomal‐dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial fatal progressive degenerative disorder. One of the pathological hallmarks of CADASIL is a dramatic reduction of vascular smooth muscle cells (VSMCs) in cerebral arteries. Using VSMCs from the vasculature of the human umbilical cord, placenta and cerebrum of CADASIL patients, we found that CADASIL VSMCs had a lower proliferation rate compared to control VSMCs. Exposure of control VSMCs and endothelial cells (ECs) to media derived from CADASIL VSMCs lowered the proliferation rate of all cells examined. By quantitative RT‐PCR analysis, we observed increased Transforming growth factor‐β (TGFβ) gene expression in CADASIL VSMCs. Adding TGFβ‐neutralizing antibody restored the proliferation rate of CADASIL VSMCs. We assessed proliferation differences in the presence or absence of TGFβ‐neutralizing antibody in ECs co‐cultured with VSMCs. ECs co‐cultured with CADASIL VSMCs exhibited a lower proliferation rate than those co‐cultured with control VSMCs, and neutralization of TGFβ normalized the proliferation rate of ECs co‐cultured with CADASIL VSMCs. We suggest that increased TGFβ expression in CADASIL VSMCs is involved in the reduced VSMC proliferation in CADASIL and may play a role in situ in altered proliferation of neighbouring cells in the vasculature. </p

Insulin-Independent and Dependent Glucose Transporters in Brain Mural Cells in CADASIL

Typical cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the human NOTCH3 gene. Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy is characterized by subcortical ischemic strokes due to severe arteriopathy and fibrotic thickening of small vessels. Blood regulating vascular smooth muscle cells (VSMCs) appear as the key target in CADASIL but the pathogenic mechanisms remain unclear. With the hypothesis that brain glucose metabolism is disrupted in VSMCs in CADASIL, we investigated post-mortem tissues and VSMCs derived from CADASIL patients to explore gene expression and protein immunoreactivity of glucose transporters (GLUTs), particularly GLUT4 and GLUT2 using quantitative RT-PCR and immunohistochemical techniques.In vitrocell model analysis indicated that both GLUT4 and -2 gene expression levels were down-regulated in VSMCs derived from CADASIL patients, compared to controls.In vitrostudies further indicated that the down regulation of GLUT4 coincided with impaired glucose uptake in VSMCs, which could be partially rescued by insulin treatment. Our observations on reduction in GLUTs in VSMCs are consistent with previous findings of decreased cerebral blood flow and glucose uptake in CADASIL patients. That impaired ability of glucose uptake is rescued by insulin is also consistent with previously reported lower proliferation rates of VSMCs derived from CADASIL subjects. Overall, these observations are consistent with the development of severe cerebral arteriopathy in CADASIL, in which VSMCs are replaced by widespread fibrosis

Mitochondrial Alterations in PINK1 Deficient Cells Are Influenced by Calcineurin-Dependent Dephosphorylation of Dynamin-Related Protein 1

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential

Immune dysregulation in HIV-1 infected lymphoid tissue

Lymphoid compartments are major sites for HIV- 1 replication. We evaluated cytokines, chemokines and immunological effector function at the single cell level in lymphoid tissues in chronically HIV-1 infected patients. HIV-1 infected lymphoid tissue was characterized by an extensive proinflammatory activation (IL-1alpha, IL-1beta and IL-12) and vigorous Th 1 type cytokine expression (IL-2 and IFN-gamma) while Th2 cytokines remained low (IL-4 and IL- 10). Treatment with highly active anti-retroviral therapy (HAART) resulted in a significant reduction of proinflammatory as well as of Th 1 type of cytokine expression in lymphoid tissue. However, the pool of HIV- 1 DNA containing cells (1 5%) remained virtually unchanged even after more than one year of HAART while the initial 3-8-fold increase of CD8+ T cells was normalized. We hypothesized that lack of elimination of HIV-1 infected cells was due to impaired cytolytic effector function in activated CD8+ T cells that normally are mediated by either Fas-L/Fas interaction or perforin/granzyme A (grA). CD8+ T cells in HIV- 1 infected lymphoid tissue were found to have upregulated Fas-L and grA expression while perforin expression was not concomitantly induced. This was however not true for CD8+ T cells obtained from acutely EBV infected patients, which instead showed upregulation of both perforin and grA expression. Our data indicated a defective cytolytic activity in CD8+ T cells at local sites of HIV-1 replication within lymphoid tissue. To elucidate the molecular mechanism involved in inhibition of perforin expression in HIV- 1 infection, we initiated short term in vitro cultures using freshly isolated peripheral blood cells from HIV-1 seronegative donors. Exogenous addition of Nef protein was found to mediate downregulation of perforin in CD8+ T cells. Sequence alignments and molecular modeling of different Nef proteins suggested that the so-called disordered loop corresponding to residues 148-178 was a likely contributor to the observed Nefmediated downregulation of perforin expression. Macaques infected with a nef-deleted virus displayed high perforin expression within lymphoid tissues in contrast to macaques infected with the wild-type virus, which suggested a role for Nef as perforin modulator in vivo. Furthermore, cytokine mediated regulation of chemokine receptor expression (CCR5CXCR4) was studied in placenta tissue from transmitting (TT) and non-transmitting (TNT) HIV- 1 infected women. We found upregulation of CCR5 combined with IL-2 expression both at the protein and mRNA level in placenta from TT women. This was associated with an increase in the number of gag-pol mRNA expressing cells. In contrast, the placenta from TNT women was characterised by upregulation of Th2 type (IL-4, IL- 10) cytokine expression. We found an association between Th2 type of cytokine (IL-4) expression and Leukemia Inhibitor Factor (LIF) production in placenta from TNT tissue while LIF expression was low in placenta from TT women. Therefore, we investigated whether LIF secretion may play a role in HIV- 1 inhibition. LIF inhibited HIV- 1 replication in a co-receptor independent manner. This inhibition was dependent upon the expression of LIF-R beta (gp 13 0) on the surface of HIV-1 susceptible cells. The identification of LIF as an inhibitor of HIV-1 replication may lead to the development of a novel anti-retroviral treatment

Microglia Impairs Proliferation and Induces Senescence In-Vitro in NGF Releasing Cells Used in Encapsulated Cell Biodelivery for Alzheimer&rsquo;s Disease Therapy

There is no cure yet available for Alzheimer&rsquo;s disease (AD). We recently optimized encapsulated cell biodelivery (ECB) devices releasing human mature nerve growth factor (hmNGF), termed ECB-NGF, to the basal forebrain of AD patients. The ECB-NGF delivery resulted in increased CSF cholinergic markers, improved glucose metabolism, and positive effects on cognition in AD patients. However, some ECB-NGF implants showed altered hmNGF release post-explantation. To optimize the ECB-NGF platform for future therapeutic purposes, we initiated in-vitro optimization studies by exposing ECB-NGF devices to physiological factors present within the AD brain. We report here that microglia cells can impair hmNGF release from ECB-NGF devices in-vitro, which can be reversed by transferring the devices to fresh culture medium. Further, we exposed the hmNGF secreting human ARPE-19 cell line (NGC0211) to microglia (HMC3) conditioned medium (MCM; untreated or treated with IL-1&beta;/IFN&gamma;/A&beta;40/A&beta;42), and evaluated biochemical stress markers (ROS, GSH, &Delta;&Psi;m, and Alamar Blue assay), cell death indicators (Annexin-V/PI), cell proliferation (CFSE retention and Ki67) and senescence markers (SA-&beta;-gal) in NGC0211 cells. MCMs from activated microglia reduced cell proliferation and induced cell senescence in NGC0211 cells, which otherwise resist biochemical alterations and cell death. These data indicate a critical but reversible impact of activated microglia on NGC0211 cells