Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement.

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.

Tau accumulation activates the unfolded protein response by impairing endoplasmic reticulum-associated degradation.

In Alzheimer's disease (AD), the mechanisms of neuronal loss remain largely unknown. Although tau pathology is closely correlated with neuronal loss, how its accumulation may lead to activation of neurotoxic pathways is unclear. Here we show that tau increased the levels of ubiquitinated proteins in the brain and triggered activation of the unfolded protein response (UPR). This suggested that tau interferes with protein quality control in the endoplasmic reticulum (ER). Consistent with this, ubiquitin was found to associate with the ER in human AD brains and tau transgenic (rTg4510) mouse brains, but this was not always colocalized with tau. The increased levels of ubiquitinated protein were accompanied by increased levels of phosphorylated protein kinase R-like ER kinase (pPERK), a marker that indicates UPR activation. Depleting soluble tau levels in cells and brain could reverse UPR activation. Tau accumulation facilitated its deleterious interaction with ER membrane and associated proteins that are essential for ER-associated degradation (ERAD), including valosin-containing protein (VCP) and Hrd1. Based on this, the effects of tau accumulation on ERAD efficiency were evaluated using the CD3δ reporter, an ERAD substrate. Indeed, CD3δ accumulated in both in vitro and in vivo models of tau overexpression and AD brains. These data suggest that soluble tau impairs ERAD and the result is activation of the UPR. The reversibility of this process, however, suggests that tau-based therapeutics could significantly delay this type of cell death and therefore disease progression.

Imbalance of Hsp70 family variants fosters tau accumulation.

Dysfunctional tau accumulation is a major contributing factor in tauopathies, and the heat-shock protein 70 (Hsp70) seems to play an important role in this accumulation. Several reports suggest that Hsp70 proteins can cause tau degradation to be accelerated or slowed, but how these opposing activities are controlled is unclear. Here we demonstrate that highly homologous variants in the Hsp70 family can have opposing effects on tau clearance kinetics. When overexpressed in a tetracycline (Tet)-based protein chase model, constitutive heat shock cognate 70 (Hsc70) and inducible Hsp72 slowed or accelerated tau clearance, respectively. Tau synergized with Hsc70, but not Hsp72, to promote microtubule assembly at nearly twice the rate of either Hsp70 homologue in reconstituted, ATP-regenerating Xenopus extracts supplemented with rhodamine-labeled tubulin and human recombinant Hsp72 and Hsc70. Nuclear magnetic resonance spectroscopy with human recombinant protein revealed that Hsp72 had greater affinity for tau than Hsc70 (I/I0 ratio difference of 0.3), but Hsc70 was 30 times more abundant than Hsp72 in human and mouse brain tissue. This indicates that the predominant Hsp70 variant in the brain is Hsc70, suggesting that the brain environment primarily supports slower tau clearance. Despite its capacity to clear tau, Hsp72 was not induced in the Alzheimer's disease brain, suggesting a mechanism for age-associated onset of the disease. Through the use of chimeras that blended the domains of Hsp72 and Hsc70, we determined that the reason for these differences between Hsc70 and Hsp72 with regard to tau clearance kinetics lies within their C-terminal domains, which are essential for their interactions with substrates and cochaperones. Hsp72 but not Hsc70 in the presence of tau was able to recruit the cochaperone ubiquitin ligase CHIP, which is known to facilitate the ubiquitination of tau, describing a possible mechanism of how the C-termini of these homologous Hsp70 variants can differentially regulate tau triage. Thus, efforts to promote Hsp72 expression and inhibit Hsc70 could be therapeutically relevant for tauopathies.

Cdc37/Hsp90 protein complex disruption triggers an autophagic clearance cascade for TDP-43 protein.

The RNA-binding protein, trans-active response DNA-binding protein 43 (TDP-43), is normally found in the nucleus, but in amyotrophic lateral sclerosis, frontal temporal dementia, and some cases of Alzheimer disease it is cleaved and mislocalized to the cytosol, leading to accumulation. The mechanisms contributing to this are largely unknown. Here, we show that part of the normal clearance cascade for TDP-43 involves the Cdc37/Hsp90 complex. An Hsp90 inhibitor that disrupts the Cdc37/Hsp90 complex reduced TDP-43 levels to a greater extent than a standard Hsp90 ATPase inhibitor. When Cdc37 was depleted, TDP-43 underwent proteolytic clearance that was dependent on nuclear retrotranslocation and autophagic uptake. Accumulation of the microtubule-associated protein tau prevented the clearance of cleaved TDP-43, but not its production. This caused cleaved TDP-43 to accumulate, a feature observed in the brain of persons with Alzheimer disease. Clearance of cleaved TDP-43 was also prevented by knockdown of the autophagic inducer beclin1. Thus, in cells where TDP-43 clearance is normally needed, a system that employs manipulation of the Hsp90 complex and autophagy exists. But when tau accumulation is occurring, cleaved TDP-43 can no longer be cleared, perhaps explaining the emergence of these co-pathologies.

Glucose-regulated protein 94 triage of mutant myocilin through endoplasmic reticulum-associated degradation subverts a more efficient autophagic clearance mechanism.

BACKGROUND: Mutant myocilin accumulates in the endoplasmic reticulum for unknown reasons. RESULTS: Glucose-regulated protein (Grp) 94 depletion reduces mutant myocilin by engaging autophagy. CONCLUSION: Grp94 triages mutant myocilin through ER-associated degradation, subverting autophagy. SIGNIFICANCE: Treating glaucoma could be possible by inhibiting Grp94 and reducing its novel client, mutant myocilin. Clearance of misfolded proteins in the endoplasmic reticulum (ER) is traditionally handled by ER-associated degradation (ERAD), a process that requires retro-translocation and ubiquitination mediated by a luminal chaperone network. Here we investigated whether the secreted, glaucoma-associated protein myocilin was processed by this pathway. Myocilin is typically transported through the ER/Golgi network, but inherited mutations in myocilin lead to its misfolding and aggregation within trabecular meshwork cells, and ultimately, ER stress-induced cell death. Using targeted knockdown strategies, we determined that glucose-regulated protein 94 (Grp94), the ER equivalent of heat shock protein 90 (Hsp90), specifically recognizes mutant myocilin, triaging it through ERAD. The addition of mutant myocilin to the short list of Grp94 clients strengthens the hypothesis that ß-strand secondary structure drives client association with Grp94. Interestingly, the ERAD pathway is incapable of efficiently handling the removal of mutant myocilin, but when Grp94 is depleted, degradation of mutant myocilin is shunted away from ERAD toward a more robust clearance pathway for aggregation-prone proteins, the autophagy system. Thus ERAD inefficiency for distinct aggregation-prone proteins can be subverted by manipulating ER chaperones, leading to more effective clearance by the autophagic/lysosomal pathway. General Hsp90 inhibitors and a selective Grp94 inhibitor also facilitate clearance of mutant myocilin, suggesting that therapeutic approaches aimed at inhibiting Grp94 could be beneficial for patients suffering from some cases of myocilin glaucoma.

ApoER2 function in the establishment and maintenance of retinal synaptic connectivity.

The cellular and molecular mechanisms responsible for the development of inner retinal circuitry are poorly understood. Reelin and apolipoprotein E (apoE), ligands of apoE receptor 2 (ApoER2), are involved in retinal development and degeneration, respectively. Here we describe the function of ApoER2 in the developing and adult retina. ApoER2 expression was highest during postnatal inner retinal synaptic development and was considerably lower in the mature retina. Both during development and in the adult, ApoER2 was expressed by A-II amacrine cells. ApoER2 knock-out (KO) mice had rod bipolar morphogenic defects, altered A-II amacrine dendritic development, and impaired rod-driven retinal responses. The presence of an intact ApoER2 NPxY motif, necessary for binding Disabled-1 and transducing the Reelin signal, was also necessary for development of the rod bipolar pathway, while the alternatively spliced exon 19 was not. Mice deficient in another Reelin receptor, very low-density lipoprotein receptor (VLDLR), had normal rod bipolar morphology but altered A-II amacrine dendritic development. VLDLR KO mice also had reductions in oscillatory potentials and delayed synaptic response intervals. Interestingly, age-related reductions in rod and cone function were observed in both ApoER2 and VLDLR KOs. These results support a pivotal role for ApoER2 in the establishment and maintenance of normal retinal synaptic connectivity.

The Hsp90 kinase co-chaperone Cdc37 regulates tau stability and phosphorylation dynamics.

The microtubule-associated protein tau, which becomes hyperphosphorylated and pathologically aggregates in a number of these diseases, is extremely sensitive to manipulations of chaperone signaling. For example, Hsp90 inhibitors can reduce the levels of tau in transgenic mouse models of tauopathy. Because of this, we hypothesized that a number of Hsp90 accessory proteins, termed co-chaperones, could also affect tau stability. Perhaps by identifying these co-chaperones, new therapeutics could be designed to specifically target these proteins and facilitate tau clearance. Here, we report that the co-chaperone Cdc37 can regulate aspects of tau pathogenesis. We found that suppression of Cdc37 destabilized tau, leading to its clearance, whereas Cdc37 overexpression preserved tau. Cdc37 was found to co-localize with tau in neuronal cells and to physically interact with tau from human brain. Moreover, Cdc37 levels significantly increased with age. Cdc37 knockdown altered the phosphorylation profile of tau, an effect that was due in part to reduced tau kinase stability, specifically Cdk5 and Akt. Conversely, GSK3ß and Mark2 were unaffected by Cdc37 modulation. Cdc37 overexpression prevented whereas Cdc37 suppression potentiated tau clearance following Hsp90 inhibition. Thus, Cdc37 can regulate tau in two ways: by directly stabilizing it via Hsp90 and by regulating the stability of distinct tau kinases. We propose that changes in the neuronal levels or activity of Cdc37 could dramatically alter the kinome, leading to profound changes in the tau phosphorylation signature, altering its proteotoxicity and stability.

The Hsp90 cochaperone, FKBP51, increases Tau stability and polymerizes microtubules.

Imbalanced protein load within cells is a critical aspect for most diseases of aging. In particular, the accumulation of proteins into neurotoxic aggregates is a common thread for a host of neurodegenerative diseases. Our previous work demonstrated that age-related changes to the cellular chaperone repertoire contributes to abnormal buildup of the microtubule-associated protein tau that accumulates in a group of diseases termed tauopathies, the most common being Alzheimer's disease. Here, we show that the Hsp90 cochaperone, FK506-binding protein 51 (FKBP51), which possesses both an Hsp90-interacting tetratricopeptide domain and a peptidyl-prolyl cis-trans isomerase (PPIase) domain, prevents tau clearance and regulates its phosphorylation status. Regulation of the latter is dependent on the PPIase activity of FKBP51. FKB51 enhances the association of tau with Hsp90, but the FKBP51/tau interaction is not dependent on Hsp90. In vitro FKBP51 stabilizes microtubules with tau in a reaction depending on the PPIase activity of FKBP51. Based on these new findings, we propose that FKBP51 can use the Hsp90 complex to isomerize tau, altering its phosphorylation pattern and stabilizing microtubules.

Phosphorylation dynamics regulate Hsp27-mediated rescue of neuronal plasticity deficits in tau transgenic mice.

Molecular chaperones regulate the aggregation of a number of proteins that pathologically misfold and accumulate in neurodegenerative diseases. Identifying ways to manipulate these proteins in disease models is an area of intense investigation; however, the translation of these results to the mammalian brain has progressed more slowly. In this study, we investigated the ability of one of these chaperones, heat shock protein 27 (Hsp27), to modulate tau dynamics. Recombinant wild-type Hsp27 and a genetically altered version of Hsp27 that is perpetually pseudo-phosphorylated (3×S/D) were generated. Both Hsp27 variants interacted with tau, and atomic force microscopy and dynamic light scattering showed that both variants also prevented tau filament formation. However, extrinsic genetic delivery of these two Hsp27 variants to tau transgenic mice using adeno-associated viral particles showed that wild-type Hsp27 reduced neuronal tau levels, whereas 3×S/D Hsp27 was associated with increased tau levels. Moreover, rapid decay in hippocampal long-term potentiation (LTP) intrinsic to this tau transgenic model was rescued by wild-type Hsp27 overexpression but not by 3×S/D Hsp27. Because the 3×S/D Hsp27 mutant cannot cycle between phosphorylated and dephosphorylated states, we can conclude that Hsp27 must be functionally dynamic to facilitate tau clearance from the brain and rescue LTP; however, when this property is compromised, Hsp27 may actually facilitate accumulation of soluble tau intermediates.

Facilitating Akt clearance via manipulation of Hsp70 activity and levels.

Members of the 70-kDa heat shock family can control and manipulate a host of oncogenic client proteins. This role of Hsp70 in both the folding and degradation of these client proteins makes it a potential drug target for certain forms of cancer. The phenothiazine family of compounds, as well as the flavonoid myricetin, was recently shown to inhibit Hsp70-ATPase activity, whereas members of the dihydropyrimidine family stimulated ATPase function. Akt, a major survival kinase, was found to be under the regulation of Hsp70, and when the ATPase activity of Hsp70 was increased or decreased by these compounds, Akt levels were also increased or decreased. Also, increasing Hsp70 levels concurrent with inhibition of its ATPase function synergistically reduced Akt levels to a greater extent than either manipulation alone, providing new insights about client fate decisions. Akt reductions mediated by Hsp70 inhibitors were prevented when Hsp70 expression was silenced with small interfering RNA. Inhibiting Hsp70 ATPase function produced cytotoxic events only in breast cancer cell lines where Akt dysfunction was previously shown, suggesting therapeutic specificity depending on the Hsp70 client profile. Thus, increasing Hsp70 levels combined with inhibiting its ATPase function may serve to dramatically reduce Akt levels and facilitate cell death in certain types of cancer.

Hsc70 rapidly engages tau after microtubule destabilization.

The microtubule-associated protein Tau plays a crucial role in regulating the dynamic stability of microtubules during neuronal development and synaptic transmission. In a group of neurodegenerative diseases, such as Alzheimer disease and other tauopathies, conformational changes in Tau are associated with the initial stages of disease pathology. Folding of Tau into the MC1 conformation, where the amino acids at residues 7-9 interact with residues 312-342, is one of the earliest pathological alterations of Tau in Alzheimer disease. The mechanism of this conformational change in Tau and the subsequent effect on function and association to microtubules is largely unknown. Recent work by our group and others suggests that members of the Hsp70 family play a significant role in Tau regulation. Our new findings suggest that heat shock cognate (Hsc) 70 facilitates Tau-mediated microtubule polymerization. The association of Hsc70 with Tau was rapidly enhanced following treatment with microtubule-destabilizing agents. The fate of Tau released from the microtubule was found to be dependent on ATPase activity of Hsc70. Microtubule destabilization also rapidly increased the MC1 folded conformation of Tau. An in vitro assay suggests that Hsc70 facilitates formation of MC1 Tau. However, in a hyperphosphorylating environment, the formation of MC1 was abrogated, but Hsc70 binding to Tau was enhanced. Thus, under normal circumstances, MC1 formation may be a protective conformation facilitated by Hsc70. However, in a diseased environment, Hsc70 may preserve Tau in a more unstructured state, perhaps facilitating its pathogenicity.

The diarylheptanoid (+)-aR,11S-myricanol and two flavones from bayberry (Myrica cerifera) destabilize the microtubule-associated protein tau.

Target-based drug discovery for Alzheimer's disease (AD) centered on modulation of the amyloid ß peptide has met with limited success. Therefore, recent efforts have focused on targeting the microtubule-associated protein tau. Tau pathologically accumulates in more than 15 neurodegenerative diseases and is most closely linked with postsymptomatic progression in AD. We endeavored to identify compounds that decrease tau stability rather than prevent its aggregation. An extract from Myrica cerifera (bayberry/southern wax myrtle) potently reduced both endogenous and overexpressed tau protein levels in cells and murine brain slices. The bayberry flavonoids myricetin and myricitrin were confirmed to contribute to this potency, but a diarylheptanoid, myricanol, was the most effective anti-tau component in the extract, with potency approaching the best targeted lead therapies. (+)-aR,11S-Myricanol, isolated from M. cerifera and reported here for the first time as the naturally occurring aglycone, was significantly more potent than commercially available (±)-myricanol. Myricanol may represent a novel scaffold for drug development efforts targeting tau turnover in AD.

Akt and CHIP coregulate tau degradation through coordinated interactions.

A hallmark of the pathology of Alzheimer's disease is the accumulation of the microtubule-associated protein tau into fibrillar aggregates. Recent studies suggest that they accumulate because cytosolic chaperones fail to clear abnormally phosphorylated tau, preserving a pool of toxic tau intermediates within the neuron. We describe a mechanism for tau clearance involving a major cellular kinase, Akt. During stress, Akt is ubiquitinated and degraded by the tau ubiquitin ligase CHIP, and this largely depends on the Hsp90 complex. Akt also prevents CHIP-induced tau ubiquitination and its subsequent degradation, either by regulating the Hsp90/CHIP complex directly or by competing as a client protein with tau for binding. Akt levels tightly regulate the expression of CHIP, such that, as Akt levels are suppressed, CHIP levels also decrease, suggesting a potential stress response feedback mechanism between ligase and kinase activity. We also show that Akt and the microtubule affinity-regulating kinase 2 (PAR1/MARK2), a known tau kinase, interact directly. Akt enhances the activity of PAR1 to promote tau hyperphosphorylation at S262/S356, a tau species that is not recognized by the CHIP/Hsp90 complex. Moreover, Akt1 knockout mice have reduced levels of tau phosphorylated at PAR1/MARK2 consensus sites. Hence, Akt serves as a major regulator of tau biology by manipulating both tau kinases and protein quality control, providing a link to several common pathways that have demonstrated dysfunction in Alzheimer's disease.

Hsp90-interacting Co-chaperones and their Family Proteins in Tau Regulation: Introducing a Novel Role for Cdc37L1.

Tau is a microtubule-associated protein that serves as a promoter of microtubule assembly and stability in neuron cells. In a collective group of neurodegenerative diseases called tauopathies, tau processing is altered as a result of gene mutations and post-translational modifications. In particular, in Alzheimer's disease (AD) or AD-like conditions, tau becomes hyperphosphorylated and forms toxic aggregates inside the cell. The chaperone heat shock protein 90 (Hsp90) plays an important role in the proper folding, degradation, and recycling of tau proteins and tau kinases. Hsp90 has many co-chaperones that aid in tau processing. In particular, a few of these co-chaperones, such as FK506-binding protein (FKBP) 51, protein phosphatase (PP) 5, cell division cycle 37 (Cdc37), and S100A1 have family members that are reported to affect Hsp90-mediated tau processing in either a similar or an opposite manner. Here, we provide a holistic review of these selected co-chaperones and their family proteins and introduce a novel Hsp90-binding Cdc37 relative, Cdc37-like-1 (Cdc37L1 or L1) in tau regulation. Overall, the proteins discussed here highlight the importance of studying family proteins in order to fully understand the mechanism of tau pathogenesis and to establish drug targets for the treatment of tauopathies.

Chemical manipulation of hsp70 ATPase activity regulates tau stability.

Alzheimer's disease and other tauopathies have recently been clustered with a group of nervous system disorders termed protein misfolding diseases. The common element established between these disorders is their requirement for processing by the chaperone complex. It is now clear that the individual components of the chaperone system, such as Hsp70 and Hsp90, exist in an intricate signaling network that exerts pleiotropic effects on a host of substrates. Therefore, we have endeavored to identify new compounds that can specifically regulate individual components of the chaperone family. Here, we hypothesized that chemical manipulation of Hsp70 ATPase activity, a target that has not previously been pursued, could illuminate a new pathway toward chaperone-based therapies. Using a newly developed high-throughput screening system, we identified inhibitors and activators of Hsp70 enzymatic activity. Inhibitors led to rapid proteasome-dependent tau degradation in a cell-based model. Conversely, Hsp70 activators preserved tau levels in the same system. Hsp70 inhibition did not result in general protein degradation, nor did it induce a heat shock response. We also found that inhibiting Hsp70 ATPase activity after increasing its expression levels facilitated tau degradation at lower doses, suggesting that we can combine genetic and pharmacologic manipulation of Hsp70 to control the fate of bound substrates. Disease relevance of this strategy was further established when tau levels were rapidly and substantially reduced in brain tissue from tau transgenic mice. These findings reveal an entirely novel path toward therapeutic intervention of tauopathies by inhibition of the previously untargeted ATPase activity of Hsp70.

Chaperone signalling complexes in Alzheimer's disease.

Molecular chaperones and heat shock proteins (Hsp) have emerged as critical regulators of proteins associated with neurodegenerative disease pathologies. The very nature of the chaperone system, which is to maintain protein quality control, means that most nascent proteins come in contact with chaperone proteins. Thus, amyloid precursor protein (APP), members of the gamma-secretase complex (presenilin 1 [PS1] collectively), the microtubule-associated protein tau (MAPT) as well as a number of neuroinflammatory components are all in contact with chaperones from the moment of their production. Chaperones are often grouped together as one machine presenting abnormal or mutant proteins to the proteasome for degradation, but this is not at all the case. In fact, the chaperone family consists of more than 100 proteins in mammalian cells, and the primary role for most of these proteins is to protect clients following synthesis and during stress; only as a last resort do they facilitate protein degradation. To the best of our current knowledge, the chaperone system in eukaryotic cells revolves around the ATPase activities of Hsp70 and Hsp90, the two primary chaperone scaffolds. Other chaperones and co-chaperones manipulate the ATPase activities of Hsp70 and Hsp90, facilitating either folding of the client or its degradation. In the case of Alzheimer's disease (AD), a number of studies have recently emerged describing the impact that these chaperones have on the proteotoxic effects of tau and amyloid- beta accumulation. Here, we present the current understandings of chaperone biology and examine the literature investigating these proteins in the context of AD.

Therapeutic Potential of the Hsp90/Cdc37 Interaction in Neurodegenerative Diseases.

Alzheimer's, Huntington's, and Parkinson's are devastating neurodegenerative diseases that are prevalent in the aging population. Patient care costs continue to rise each year, because there is currently no cure or disease modifying treatments for these diseases. Numerous efforts have been made to understand the molecular interactions governing the disease development. These efforts have revealed that the phosphorylation of proteins by kinases may play a critical role in the aggregation of disease-associated proteins, which is thought to contribute to neurodegeneration. Interestingly, a molecular chaperone complex consisting of the 90 kDa heat shock protein (Hsp90) and Cell Division Cycle 37 (Cdc37) has been shown to regulate the maturation of many of these kinases as well as regulate some disease-associated proteins directly. Thus, the Hsp90/Cdc37 complex may represent a potential drug target for regulating proteins linked to neurodegenerative diseases, through both direct and indirect interactions. Herein, we discuss the broad understanding of many Hsp90/Cdc37 pathways and how this protein complex may be a useful target to regulate the progression of neurodegenerative disease.

Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA).

Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032.

Identification of Apo B48 and other novel biomarkers in amyotrophic lateral sclerosis patient fibroblasts.

AIM: Amyotrophic lateral sclerosis (ALS) is a debilitating fatal neurodegenerative disorder. 90-95% of ALS cases are sporadic with no clear risk factors associated with the disease. Identification of biomarkers associated with ALS may lead to early detection and make it more amenable to therapeutic intervention. MATERIALS & METHODS: SILAC was used to quantitatively analyze the proteomes of ALS and control human fibroblasts. RESULTS: Out of total of 861 proteins identified, 33 were found to be differentially regulated. ApoB48 and Hsp20 were downregulated, while Fibulin-1 was upregulated. CONCLUSION: We report the differential regulation of these proteins in ALS fibroblasts, and their potential as novel biomarkers and possible drug targets for ALS.