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.

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.

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.