J-domain proteins: From molecular mechanisms to diseases.

J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.

Extracellular chaperone networks and the export of J-domain proteins.

An extracellular network of molecular chaperones protects a diverse array of proteins that reside in or pass through extracellular spaces. Proteins in the extracellular milieu face numerous challenges that can lead to protein misfolding and aggregation. As a checkpoint for proteins that move between cells, extracellular chaperone networks are of growing clinical relevance. J-domain proteins (JDPs) are ubiquitous molecular chaperones that are known for their essential roles in a wide array of fundamental cellular processes through their regulation of heat shock protein 70s. As the largest molecular chaperone family, JDPs have long been recognized for their diverse functions within cells. Some JDPs are elegantly selective for their "client proteins," some do not discriminate among substrates and others act cooperatively on the same target. The realization that JDPs are exported through both classical and unconventional secretory pathways has fueled investigation into the roles that JDPs play in protein quality control and intercellular communication. The proposed functions of exported JDPs are diverse. Studies suggest that export of DnaJB11 enhances extracellular proteostasis, that intercellular movement of DnaJB1 or DnaJB6 enhances the proteostasis capacity in recipient cells, whereas the import of DnaJB8 increases resistance to chemotherapy in recipient cancer cells. In addition, the export of DnaJC5 and concurrent DnaJC5-dependent ejection of dysfunctional and aggregation-prone proteins are implicated in the prevention of neurodegeneration. This review provides a brief overview of the current understanding of the extracellular chaperone networks and outlines the first wave of studies describing the cellular export of JDPs.

Cysteine String Protein Controls Two Routes of Export for Misfolded Huntingtin.

Extracellular vesicles (EVs) are secreted vesicles of diverse size and cargo that are implicated in the cell-to-cell transmission of disease-causing-proteins in several neurodegenerative diseases. Mutant huntingtin, the disease-causing entity in Huntington's disease, has an expanded polyglutamine track at the N terminus that causes the protein to misfold and form toxic intracellular aggregates. In Huntington's disease, mutant huntingtin aggregates are transferred between cells by several routes. We have previously identified a cellular pathway that is responsible for the export of mutant huntingtin via extracellular vesicles. Identifying the EV sub-populations that carry misfolded huntingtin cargo is critical to understanding disease progression. In this work we expressed a form of polyglutamine expanded huntingtin (GFP-tagged 72Qhuntingtinexon1) in cells to assess the EVs involved in cellular export. We demonstrate that the molecular chaperone, cysteine string protein (CSPα; DnaJC5), facilitates export of disease-causing-polyglutamine-expanded huntingtin cargo in 180-240 nm vesicles as well as larger 10-30 µm vesicles.

Neurons Export Extracellular Vesicles Enriched in Cysteine String Protein and Misfolded Protein Cargo.

The fidelity of synaptic transmission depends on the integrity of the protein machinery at the synapse. Unfolded synaptic proteins undergo refolding or degradation in order to maintain synaptic proteostasis and preserve synaptic function, and buildup of unfolded/toxic proteins leads to neuronal dysfunction. Many molecular chaperones contribute to proteostasis, but one in particular, cysteine string protein (CSPα), is critical for proteostasis at the synapse. In this study we report that exported vesicles from neurons contain CSPα. Extracellular vesicles (EV's) have been implicated in a wide range of functions. However, the functional significance of neural EV's remains to be established. Here we demonstrate that co-expression of CSPα with the disease-associated proteins, polyglutamine expanded protein 72Q huntingtinex°n1 or superoxide dismutase-1 (SOD-1G93A) leads to the cellular export of both 72Q huntingtinex°n1 and SOD-1G93A via EV's. In contrast, the inactive CSPαHPD-AAA mutant does not facilitate elimination of misfolded proteins. Furthermore, CSPα-mediated export of 72Q huntingtinex°n1 is reduced by the polyphenol, resveratrol. Our results indicate that by assisting local lysosome/proteasome processes, CSPα-mediated removal of toxic proteins via EVs plays a central role in synaptic proteostasis and CSPα thus represents a potential therapeutic target for neurodegenerative diseases.

Increased Expression of the Large Conductance, Calcium-Activated K+ (BK) Channel in Adult-Onset Neuronal Ceroid Lipofuscinosis.

Cysteine string protein (CSPα) is a presynaptic J protein co-chaperone that opposes neurodegeneration. Mutations in CSPα (i.e., Leu115 to Arg substitution or deletion (Δ) of Leu116) cause adult neuronal ceroid lipofuscinosis (ANCL), a dominantly inherited neurodegenerative disease. We have previously demonstrated that CSPα limits the expression of large conductance, calcium-activated K+ (BK) channels in neurons, which may impact synaptic excitability and neurotransmission. Here we show by western blot analysis that expression of the pore-forming BKα subunit is elevated ~2.5 fold in the post-mortem cortex of a 36-year-old patient with the Leu116∆ CSPα mutation. Moreover, we find that the increase in BKα subunit level is selective for ANCL and not a general feature of neurodegenerative conditions. While reduced levels of CSPα are found in some postmortem cortex specimens from Alzheimer's disease patients, we find no concomitant increase in BKα subunit expression in Alzheimer's specimens. Both CSPα monomer and oligomer expression are reduced in synaptosomes prepared from ANCL cortex compared with control. In a cultured neuronal cell model, CSPα oligomers are short lived. The results of this study indicate that the Leu116∆ mutation leads to elevated BKα subunit levels in human cortex and extend our initial work in rodent models demonstrating the modulation of BKα subunit levels by the same CSPα mutation. While the precise sequence of pathogenic events still remains to be elucidated, our findings suggest that dysregulation of BK channels may contribute to neurodegeneration in ANCL.

J protein mutations and resulting proteostasis collapse.

Despite a century of intensive investigation the effective treatment of protein aggregation diseases remains elusive. Ordinarily, molecular chaperones ensure that proteins maintain their functional conformation. The appearance of misfolded proteins that aggregate implies the collapse of the cellular chaperone quality control network. That said, the cellular chaperone network is extensive and functional information regarding the detailed action of specific chaperones is not yet available. J proteins (DnaJ/Hsp40) are a family of chaperone cofactors that harness Hsc70 (heat shock cognate protein of 70 kDa) for diverse conformational cellular tasks and, as such, represent novel clinically relevant targets for diseases resulting from the disruption of proteostasis. Here we review incisive reports identifying mutations in individual J protein chaperones and the proteostasis collapse that ensues.

CSPα-chaperoning presynaptic proteins.

Synaptic transmission relies on precisely regulated and exceedingly fast protein-protein interactions that involve voltage-gated channels, the exocytosis/endocytosis machinery as well as signaling pathways. Although we have gained an ever more detailed picture of synaptic architecture much remains to be learned about how synapses are maintained. Synaptic chaperones are "folding catalysts" that preserve proteostasis by regulating protein conformation (and therefore protein function) and prevent unwanted protein-protein interactions. Failure to maintain synapses is an early hallmark of several degenerative diseases. Cysteine string protein (CSPα) is a presynaptic vesicle protein and molecular chaperone that has a central role in preventing synaptic loss and neurodegeneration. Over the past few years, a number of different "client proteins" have been implicated as CSPα substrates including voltage-dependent ion channels, signaling proteins and proteins critical to the synaptic vesicle cycle. Here we review the ion channels and synaptic protein complexes under the influence of CSPα and discuss gaps in our current knowledge.

Cysteine string protein limits expression of the large conductance, calcium-activated K⁺ (BK) channel.

Large-conductance, calcium-activated K(+) (BK) channels are widely distributed throughout the nervous system and play an essential role in regulation of action potential duration and firing frequency, along with neurotransmitter release at the presynaptic terminal. We have previously demonstrated that select mutations in cysteine string protein (CSPα), a presynaptic J-protein and co-chaperone, increase BK channel expression. This observation raised the possibility that wild-type CSPα normally functions to limit neuronal BK channel expression. Here we show by Western blot analysis of transfected neuroblastoma cells that when BK channels are present at elevated levels, CSPα acts to reduce expression. Moreover, we demonstrate that the accessory subunits, BKß4 and BKß1 do not alter CSPα-mediated reduction of expressed BKα subunits. Structure-function analysis reveals that the N-terminal J-domain of CSPα is critical for the observed regulation of BK channels levels. Finally, we demonstrate that CSPα limits BK current amplitude, while the loss-of-function homologue CSPαHPD-AAA increases BK current. Our observations indicate that CSPα has a role in regulating synaptic excitability and neurotransmission by limiting expression of BK channels.

The large conductance, calcium-activated K+ (BK) channel is regulated by cysteine string protein.

Large-conductance, calcium-activated-K(+) (BK) channels are widely distributed throughout the nervous system, where they regulate action potential duration and firing frequency, along with presynaptic neurotransmitter release. Our recent efforts to identify chaperones that target neuronal ion channels have revealed cysteine string protein (CSPα) as a key regulator of BK channel expression and current density. CSPα is a vesicle-associated protein and mutations in CSPα cause the hereditary neurodegenerative disorder, adult-onset autosomal dominant neuronal ceroid lipofuscinosis (ANCL). CSPα null mice show 2.5 fold higher BK channel expression compared to wild type mice, which is not seen with other neuronal channels (i.e. Cav2.2, Kv1.1 and Kv1.2). Furthermore, mutations in either CSPα's J domain or cysteine string region markedly increase BK expression and current amplitude. We conclude that CSPα acts to regulate BK channel expression, and consequently CSPα-associated changes in BK activity may contribute to the pathogenesis of neurodegenerative disorders, such as ANCL.

Cell line specific modulation of extracellular aß42 by Hsp40.

Heat shock proteins (Hsps) are a set of molecular chaperones involved in cellular repair. They provide protective mechanisms that allow cells to survive potentially lethal insults, In response to a conditioning stress their expression is increased. Here we examined the connection between Hsps and Aß(42), the amyloid peptide involved in the pathological sequence of Alzheimer's disease (AD). Extracellular Aß(42) associates with neuronal cells and is a major constituent of senile plaques, one of the hallmarks of AD. Although Hsps are generally thought to prevent accumulation of misfolded proteins, there is a lack of mechanistic evidence that heat shock chaperones directly modulate Aß(42) toxicity. In this study we show that neither extracellular Aß(42) nor Aß(42/)PrP(C) trigger the heat shock response in neurons. To address the influence of the neuroprotective heat shock response on cellular Aß(42), Western analysis of Aß(42) was performed following external Aß(42) application. Five hours after a conditioning heat shock, Aß(42) association with CAD cells was increased compared to control neurons. However, at forty-eight hours following heat shock Aß(42) levels were reduced compared to that found for control cells. Moreover, transient transfection of the stress induced Hsp40, decreased CAD levels of Aß(42). In contrast to CAD cells, hippocampal neurons transfected with Hsp40 retained Aß(42) indicating that Hsp40 modulation of Aß(42) proteostasis is cell specific. Mutation of the conserved HPD motif within Hsp40 significantly reduced the Hsp40-mediated Aß(42) increase in hippocampal cultures indicating the importance of this motif in regulating cellular Aß(42). Our data reveal a biochemical link between Hsp40 expression and Aß(42) proteostasis that is cell specific. Therefore, increasing Hsp40 therapeutically with the intention of interfering with the pathogenic cascade leading to neurodegeneration in AD should be pursued with caution.

Mitochondrial chaperone DnaJA3 induces Drp1-dependent mitochondrial fragmentation.

Mitochondrial morphology is dynamic and controlled by coordinated fusion and fission pathways. The role of mitochondrial chaperones in mitochondrial morphological changes and pathology is currently unclear. Here we report that altered levels of DnaJA3 (Tid1/mtHsp40) a mitochondrial member of the DnaJ protein family, and heat shock protein (Hsp) co-chaperone of matrix 70 kDa Hsp70 (mtHsp70/mortalin/HSPA9), induces mitochondrial fragmentation. Suppression of DnaJA3 induced mitochondrial fragmentation in HeLa cells. Elevated levels of DnaJA3 in normal Hs68 fibroblast cells and HeLa, SKN-SH, U87 and U251 cancer cell lines induces mitochondrial fragmentation. Mitochondrial fragmentation induction was not observed in HeLa cells when other DnaJA family members, or mitochondrial DnaJ protein HSC20, were ectopically expressed, indicating that the effects on mitochondrial morphology were specific to DnaJA3. We show that the DnaJ domain (amino acids 88-168) of DnaJA3 is sufficient for the induction of mitochondrial fragmentation. Furthermore, an H121Q point mutation of the DnaJ domain, which abrogates interaction and activation of mtHsp70 ATPase, eliminates fragmentation induced by DnaJA3. This suggests that DnaJA3 interaction with mtHsp70 may be critical in mitochondrial morphological changes. DnaJA3-induced mitochondrial fragmentation was dependent on fission factor dynamin-related protein 1 (Drp1). Ectopic expression of the mitofusins (Mfn1 and Mfn2), however, does not rescue DnaJA3-induced mitochondrial fragmentation. Lastly, elevated levels of DnaJA3 inducing mitochondrial fragmentation were associated with reduction in cell viability. Taken together, elevated DnaJA3 induces Drp1-depedendent mitochondrial fragmentation and decreased cell viability.

Identification of bilateral changes in TID1 expression in the 6-OHDA rat model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra and the aggregation of α-synuclein into Lewy bodies. Existing therapies address motor dysfunction but do not halt progression of the disease. A still unresolved question is the biochemical pathway that modulates the outcome of protein misfolding and aggregation processes in PD. The molecular chaperone network plays an important defensive role against cellular protein misfolding and has been identified as protective in experimental models of protein misfolding diseases like PD. Molecular mechanisms underlying chaperone-neuroprotection are actively under investigation. Current evidence implicates a number of molecular chaperones in PD including Hsp25, Hsp70 and Hsp90, however their precise involvement in the neurodegenerative cascade is unresolved. The J protein family (DnaJ or Hsp40 protein family) has long been known to be important in protein conformational processes.We assessed sensory and motor function of control and PD rats and then evaluated the brain region-specific expression levels of select J proteins by Western analysis. Surprisingly, we observed a widespread 26 kDa breakdown product of the J protein, TID1, (tumorous imaginal discs, mtHsp40 or DnaJ3) in a 6-hydroxydopamine (6-OHDA) rat model of PD in which food handling, gait symmetry and sensory performance were impaired. Greater behavioral deficits were associated with lower TID1 expression. Furthermore, direct application of either 6-OHDA or MPP+ (1-methyl-4-phenylpyridinum) to CAD (CNS-derived catecholinaminergic neuronal cell line) cell cultures, reduced TID1 expression levels.Our results suggest that changes in cellular TID1 are a factor in the pathogenesis of PD by impeding functional and structural compensation and exaggerating neurodegenerative processes. In contrast, no changes were observed in CSPα, Hsp40, Hsp70, Hsc70 and PrP(C) levels and no activation of caspase3 was observed. This study links TID1 to PD and provides a new target for therapeutics that halts the PD progression.

Quercetin targets cysteine string protein (CSPalpha) and impairs synaptic transmission.

BACKGROUND: Cysteine string protein (CSPalpha) is a synaptic vesicle protein that displays unique anti-neurodegenerative properties. CSPalpha is a member of the conserved J protein family, also called the Hsp40 (heat shock protein of 40 kDa) protein family, whose importance in protein folding has been recognized for many years. Deletion of the CSPalpha in mice results in knockout mice that are normal for the first 2-3 weeks of life followed by an unexplained presynaptic neurodegeneration and premature death. How CSPalpha prevents neurodegeneration is currently not known. As a neuroprotective synaptic vesicle protein, CSPalpha represents a promising therapeutic target for the prevention of neurodegenerative disorders. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that the flavonoid quercetin promotes formation of stable CSPalpha-CSPalpha dimers and that quercetin-induced dimerization is dependent on the unique cysteine string region. Furthermore, in primary cultures of Lymnaea neurons, quercetin induction of CSPalpha dimers correlates with an inhibition of synapse formation and synaptic transmission suggesting that quercetin interfers with CSPalpha function. Quercetin's action on CSPalpha is concentration dependent and does not promote dimerization of other synaptic proteins or other J protein family members and reduces the assembly of CSPalpha:Hsc70 units (70kDa heat shock cognate protein). CONCLUSIONS/SIGNIFICANCE: Quercetin is a plant derived flavonoid and popular nutritional supplement proposed to prevent memory loss and altitude sickness among other ailments, although its precise mechanism(s) of action has been unclear. In view of the therapeutic promise of upregulation of CSPalpha and the undesired consequences of CSPalpha dysfunction, our data establish an essential proof of principle that pharmaceutical agents can selectively target the neuroprotective J protein CSPalpha.

CSPalpha: the neuroprotective J protein.

Cysteine string protein (CSPalpha, also called DnaJC5) is unique among J proteins. Similar to other J proteins, CSPalpha interacts with and activates the ATPase of Hsc70s (heat shock proteins of 70 kDa), thereby harnessing the ATPase activity for conformational work on client proteins. In contrast to other J proteins, CSPalpha is anchored to synaptic vesicles, as well as to exocrine, endocrine and neuroendocrine secretory granules, and has been shown to have an essential anti-neurodegenerative role. CSPalpha-null organisms exhibit progressive neurodegeneration, behavioural deficits, and premature death, most likely due to the progressive misfolding of one or more client proteins. Here we highlight recent advances in our understanding of the critical role that CSPalpha plays in governing exocytotic secretory functions.

Reduction of PrP(C) in human cerebrospinal fluid after spinal cord injury.

It has been estimated that cerebrospinal fluid (CSF) contains approximately 80 proteins that significantly increase or decrease in response to various clinical conditions. Here we have evaluated the CSF protein PrP(C) (cellular prion protein) for possible increases or decreases following spinal cord injury. The physiological function of PrP(C) is not yet completely understood; however, recent findings suggest that PrP(C) may have neuroprotective properties. Our results show that CSF PrP(C) is decreased in spinal cord injured patients 12 h following injury and is absent at 7 days. Given that normal PrP(C) has been proposed to be neuroprotective we speculate that the decrease in CSF PrP(C) levels may influence neuronal cell survival following spinal cord injury.

Functional role of J domain of cysteine string protein in Ca2+-dependent secretion from acinar cells.

The heat shock protein 70 family members Hsc70 and Hsp70 are known to play a protective role against the onset of experimental pancreatitis, yet their molecular function in acini is unclear. Cysteine string protein (CSP-alpha) is a zymogen granule (ZG) membrane protein characterized by an NH(2)-terminal "J domain" and a central palmitoylated string of cysteine residues. The J domain functions as a cochaperone by modulating the activity of Hsc70/Hsp70 family members. A role for CSP-alpha in regulating digestive enzyme exocytosis from pancreas was investigated by introducing CSP-alpha truncations into isolated acini following their permeabilization with Perfringolysin O. Incubation of acini with CSP-alpha(1-82), containing the J domain, significantly augmented Ca(2+)-stimulated amylase secretion. Effects of CSP-alpha(1-82) were concentration dependent, with a maximum 80% increase occurring at 200 microg/ml of protein. Although CSP-alpha(1-82) had no effects on basal secretion measured in the presence of < or =10 nM free Ca(2+), it did significantly augment GTP-gammaS-induced secretion under basal Ca(2+) conditions by approximately 25%. Mutation of the J domain to abolish its cochaperone activity failed to augment Ca(2+)-stimulated secretion, implicating the CSP-alpha/Hsc70 cochaperone system as a regulatory component of the secretory pathway. CSP-alpha physically associates with vesicle-associated membrane protein 8 (VAMP 8) on ZGs, and the CSP-alpha-VAMP 8 interaction was dependent on amino acids 83-112 of CSP-alpha. Immunofluorescence analysis of acinar lobules or purified ZGs confirmed the CSP-alpha colocalization with VAMP 8. These data establish a role for CSP-alpha in regulating digestive enzyme secretion and suggest that CSP-alpha and Hsc70 modulate specific soluble N-ethylmaleimide-sensitive attachment receptor interactions necessary for exocytosis.

Hsp40 couples with the CSPalpha chaperone complex upon induction of the heat shock response.

In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPalpha). CSPalpha is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich "string" region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPalpha chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Galpha(s). In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPalpha complex. Association of Hsp40 with CSPalpha decreases CSPalpha-CSPalpha dimerization and enhances the CSPalpha-induced increase in steady state GTP hydrolysis of Galpha(s). This newly identified CSPalpha-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPalpha in neuroprotection.