Molecular chaperones as 21st century solutions for neurodegenerative disorders

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Molecular chaperones as 21st century solutions for neurodegenerative disorders

A look at catecholamine-regulated protein 40 (CRP40)

The aging population in developed countries is experiencing an increase in the prevalence of neurodegenerative disorders that are placing a high burden on healthcare systems. The combined problems of lack of sustainable therapies and the absence of validated biomarkers for early diagnosis and intervention make these debilitating disorders a high priority for research. However, before biomarkers and therapies can be developed, scientists need to establish a clearer and more complete understanding of the molecular mechanisms behind neurodegeneration. Recently, researchers have linked the pathogenesis of many neurodegenerative disorders such as Parkinson’s (PD) and Alzheimer’s disease (AD) to abnormal protein folding, oxidative stress, and mitochondrial dysfunction. A specific class of proteins, called molecular chaperones (also known as heat-shock proteins), have emerged as potential targets for neuroprotection because of their ability to modulate abnormal protein folding and aggregation in disease states. We have discovered in our laboratory a novel molecular chaperone protein called Catecholamineregulated protein 40 (CRP40; 40kDa) that is expressed solely in the central nervous system (CNS) and blood. Our preliminary findings indicate CRP40 is an alternative splice variant of a larger, ubiquitously-expressed protein called mortalin, which has been linked to protein homeostasis, pathophysiology of neurodegenerative disorders, and has been shown to protect neurons from oxidative stress. Studies on CRP40 have revealed the presence of a conserved heat-shock motif, upregulated expression under cell stress, as well as its ability to decrease aggregation of proteins, suggesting heat-shock like functions of this novel protein. Interestingly, evidence has revealed that CRP40 may play a role in neurodegenerative disorders characterized by impairments of the dopamine (DA) system, such as Schizophrenia (SCZ) and PD, through its ability to bind DA and colocalize with components of the DA-synthesis pathway. Our group has also discovered that CRP40 expression is dysregulated in human post-mortem SCZ and Parkinson’s brain samples – a compelling finding that requires further study. In this review, we present evidence that CRP40 may play a role in CNS disorders, particularly disorders related to abnormal protein folding and impairments of the dopaminergic system. Further study of CRP40 and its mechanisms may aid in increasing the understanding of molecular chaperone function in neurodegenerative disorders, which is essential for accelerating future developments of biomarkers and sustainable therapeutics.

Neurodegenerative disorders:How molecular chaperones play a role

Neurodegenerative diseases are increasing in prevalence in many developing countries and this trend is set to continue due to the rapidly aging population. They encompass a heterogeneous group of disorders that can be described by progressive and selective loss of neuronal systems that are anatomically or physiologically related. Despite research advances over the past several decades, critical challenges still remain in understanding the pathology and underlying mechanisms behind these debilitating disorders. Converging lines of evidence have suggested a common “protein-conformational” pathogenic pathway for AD, PD, familial amyotrophic lateral sclerosis (FALS), Huntington’s disease (HD) and related polyglutamine (polyQ) expansion diseases. Collectively, these disorders are characterized by the abnormal accumulation of insoluble neuronal or extracellular protein aggregates in the CNS (Hartl 2011, Muchowski 2005). However, the significance of these phenomena to the process of neurodegeneration is incompletely understood. Several groups have proposed that the formation of protein aggregates may be neuroprotective (Caughey 2003, Sisodia 1998). However, any perturbation of protein homeostasis can impair the ability of proteins to function properly and perform their diverse biological roles that are essential for life.

There is still some debate about whether the aggregates formed by disease proteins are toxic. Evidence has suggested that misfolded proteins can induce excitotoxicity or apoptosis via activation of caspase 3, and also affect neuronal synaptic function and axonal transport (Forman 2004). As well, toxic species in neurodegenerative disorders have been linked to various inflammatory responses, including increased oxidative stress, and mitochondrial dysfunction (Lin 2006). Indeed, there seems to be a common link between misfolded proteins and the resulting aberrations that occur in neurodegenerative disorders.

Protein quality or proteostasis is regulated by an integrated network of proteins known as molecular chaperones. Molecular chaperones perform diverse roles in the cell including assisting with de novo protein folding, refolding of denatured proteins, protein trafficking, and degradation (Hartl 2011, Soti 2005). Numerous classes of molecular chaperones have been described thus far. Many of these proteins are also known as heat-shock proteins (Hsp) or stress proteins because it has been found that various cellular stresses upregulate their expression. These may include heat-shock, glucose deprivation, exposure to heavy metals, protein kinase C (PKC) stimulators, amino acid analogues, calcium increasing agents, ischemia, microbial infections and various hormones (Ellis 2006).

Different families of molecular chaperone proteins are classified according to their molecular weights and include the Hsp40, Hsp60, Hsp70, Hsp90, Hsp100, and small Hsp proteins (Ellis 2006). Heat-shock proteins are expressed in the cytosol, nucleus, mitochondria, and endoplasmic reticulum, and typically have long half-lives of about 48 hours (Ellis 2006). Although the function of these proteins is broad, the Hsp70s, Hsp90s, and the chaperonins (Hsp60s) are specifically involved in de novo protein folding and refolding. These classes of proteins are characterized as multicomponent molecular machines that have the ability to recognize exposed hydrophobic amino-acid side chains of misfolded proteins and guide proper folding through adenosine triphosphate (ATP)- and cofactor- mediated mechanisms (Hartl 2011). Due to their various roles, molecular chaperones are currently being investigated in a variety of disorders.

Indeed, molecular chaperone proteins are emerging as important players in a number of neurodegenerative diseases characterized by aberrations in protein conformation. The purported roles of molecular chaperones in neurodegeneration may include the following: 1) control of potentially toxic structural changes in disease-related polypeptides; 2) regulation of aggregation of proteins by directing towards formation of less toxic species; 3) control of degradation of toxic agents and preservation of cellular degradation systems; 4) involvement in cell signalling and apoptotic events stimulated by abnormal protein aggregates, along with preservation of survival pathways; and 5) protection of mitochondria and neurons against oxidative stress evidenced in neurodegenerative disorders (Muchowski 2005). Further understanding of cellular chaperone networks will help to define their roles in neurodegenerative diseases where protein homeostasis has been impaired, and elucidate ways in which they may be manipulated to serve as possible therapeutic factors in these debilitating disorders.

Mortalin: an essential mitochondrial heat-shock protein

The heat-shock protein 70 (Hsp70; 70kDa) family represents one of the most ubiquitous classes of molecular chaperones. Mortalin, also known as the mitochondrial heat-shock protein (also referred to as mtHsp70 or Grp75), is an essential ubiquitously expressed molecular chaperone with multiple roles in mitochondrial biogenesis, maintenance of mitochondrial protein integrity, and regulation of import of mitochondrial proteins into the matrix (Deocaris 2008). Recently, mortalin has been implicated in neurogenesis and neurodegeneration processes and linked to AD, PD, as well as HD (Deocaris 2008). Also, since mortalin has been found to exert various cytoprotective functions that permit cell survival under stressful conditions, it has been implicated in cancer and aging systems (Kaul 2007).

Evidence for the involvement of mortalin in several human diseases has been accumulating over the past decade. Gabriele and colleagues (2010) studied the possible involvement of mortalin in the pathogenesis of SCZ and have shown that antisense knockdown of mortalin in the medial prefrontal cortex resulted in impairments of DA-mediated behaviours including prepulse inhibition and social interaction deficits (Gabriele 2010). Mortalin has also been implicated in AD through several studies. Osorio and colleagues (2007) showed differential expression of mortalin isoforms in hippocampi of AD patients. In another study using an animal model of AD, the ApoE knockout mouse model, it was shown that mortalin sustained ADassociated oxidative damage, suggesting the involvement of this protein in the pathogenesis of AD (Choi 2004). Mortalin has also been linked to neurodegeneration in PD based on interactions with PD-associated proteins, including the redox-sensing DJ-1 protein that maintains mitochondrial function (Kaul 2007).

Mortalin is expressed from chromosome 5q31.1 (Kaul 1995). A recent study found that PD patients display mutations in the mortalin gene that have been associated with impaired mitochondrial function that is critical in protection from neurodegeneration (Burbulla 2010). Two of these mutations were located in the carboxyl-terminal substrate-binding domain of mortalin, indicating that this functional region of mortalin further characterization. The combined evidence from literature underscores the critical role of mortalin as an important mitochondrial stress protein in a variety of neurodegenerative disorders that needs to be investigated in more detail.

Interestingly, several studies have suggested that mortalin may play a role in brain ischemia, as it is upregulated following brain injury (Deocaris 2008). This protein may serve a protective function in ischemia by limiting the accumulation of reactive oxygen species in neurons (Liu 2005). Further, in vivo overexpression of mortalin in rat brain neurons and astrocytes following induction of ischemia significantly reduced infarct volume, improved neurological function and provided protection from oxidative damage in animals (Xu 2009).

Mortalin has been found to interact with the apoptosis-inducing factor (AIF) (Sun 2006). Normally, when AIF is released from the mitochondria, it may be translocated to the nucleus where it can cause caspase-independent apoptosis. It has been suggested that mortalin sequesters AIF and thus suppresses cell death (Sun 2006). Interestingly, this protective ability of mortalin was not retained in Hsp70 deletion mutants lacking the carboxyl-terminal (Ravagnan 2001). The functionality of the mortalin carboxyl-terminal was further confirmed in studies by Sun and colleagues (2006) who found that Hsp70 mutants lacking the entire amino-terminal domain and Hsp70 ATPasedeficient point mutants retained the ability to protect primary astrocytes against ischemic insults in vitro as well as to inhibit AIF translocation to the nucleus. This indicated that the carboxyl-terminal was indeed sufficient for protection against ischemic brain injury (Sun 2006).

Indeed, the carboxyl-terminal of mortalin seems to serve a specific function in neurodegenerative disorders and oxidative stress. McMaster University researchers have recently discovered a novel protein that is related to the carboxyl-terminal of mortalin, and this protein is under investigation for its possible roles in neurodegeneration.

Catecholamine-regulated protein 40 (CRP40): A novel molecular chaperone

The Catecholamine-Regulated Protein 40 is a novel heat-shock like protein that was discovered and characterized by Drs. Mishra and Gabriele of McMaster University (Hamilton, Canada) (Gabriele 2009, Nair 2001). CRP40 is a splice variant of mortalin, showing significant homology to mortalin’s carboxyl-terminus. Specifically, the sequence between exons 10-17 is identical to mortalin, with a promoter region at intron 9 (Gabriele 2009). Interestingly, this carboxyl-terminus homology means that the above discussed PD-related mortalin mutations would affect CRP40 function as well. Like mortalin, CRP40 belongs to the Hsp70 family, as evidenced by the following characteristics: 1) CRP40 exhibits the characteristic and conserved heat-shock motif; 2) expression of CRP40 is inducible by elevated temperature; and 3) the organellar localization motif, which allows Hsp70s to translocate between organelles, is conserved (Gabriele 2009, Nair 2001). Although the full mechanistic functions of CRP40 have not yet been elucidated, evidence suggests that it may play a role as a molecular chaperone, not unlike mortalin. Further study is required to determine how CRP40 functions in protein folding and proteostasis as related to neurodegenerative conditions.

One major distinction between CRP40 and mortalin lies in their localization. While mortalin is expressed in all cells, CRP40 is found exclusively in the CNS and in specific blood cells (Ross 1995). This pattern of expression makes CRP40 of particular interest to research in neurological disorders, especially neurodegeneration, as CRP40 may serve a more specific function in CNS disorders.

CRP40 may have similar roles to mortalin as a chaperone protein, but CRP40 also displays some functions in neuroprotection. Thus far, studies have shown that upregulation of CRP40 in the presence of excess DA can actually be inhibited by treatment of cells with antioxidants, suggesting that CRP40 is a reactant protein to the oxidative stress caused by the natural oxidation of DA (Nair 2001). Further, heat-shocked cells that were subsequently treated with CRP40 protein showed decreased denaturation and aggregation of proteins in comparison to untreated cells — a finding that highlights CRP40 as a heat-shock protein and molecular chaperone (Gabriele 2009, Nair 2001).

Unlike other members of the Hsp70 family, CRP40 contains the tyrosine and aspartate residues necessary for DA binding (Gabriele 2009, Nair 2001). Staining studies show that CRP40 colocalizes in neuronal cells with DA and Tyrosine Hydroxylase (TH), the ratelimiting molecule in DA synthesis (Gabriele 2009, Goto 2001, Nair 2001). This protein actually displays covalent interactions with the catecholamine neurotransmitters, but does not interrelate with serotonin or other amines (Nair 2001, Ross 1993, Ross 1995). In fact, CRP40 expression is inducible by presence of excess DA, which makes it an interesting target for research related to the neurodegenerative disorders with dopaminergic components, like SCZ and PD (Nair 2001).

Recent reports have revealed the importance of CRP40 to these dopaminergic degenerative conditions. Gabriele et al. (2005) conducted a study using human post-mortem SCZ samples obtained from the Stanley Foundation Neuropathology Consortium. Results showed that CRP40 is dysregulated in the affected brain regions and suggest its involvement in the pathogenesis of SCZ. In fact, there was a significant decrease in the protein expression of CRP40 in patients with SCZ (Gabriele 2005). Similarly, in studies using human postmortem PD brain samples, Mortalin/CRP40 were found to be dysregulated in PD-specific brain regions (Jin 2006, Shi 2008). Specifically, there is a quantitative decrease in Mortalin/CRP40 expression with progression of the disease (Shi 2008). These reports present important evidence that CRP40 may act as a potential biomarker for neurological disorders. Future studies will be conducted to determine whether CRP40 and its dysregulation in neurological disorders may be exploited for early disease-detection and monitoring of disease progression.

Since the discovery of CRP40, new findings and evidence have converged to reveal its involvement in neurodegenerative disease pathology. Specifically, CRP40 seems to have great significance to CNS disorders involving DA. As well, CRP40 may be involved in the crucial process of proteostasis that is impaired in neurodegenerative diseases. The Gabriele group is moving forward with this important research and has engaged in studies of both basic and clinical sciences in order to elucidate potential breakthroughs of CRP40 in the realm of neurodegenerative disorders.

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