Current lines of research

Background

Demographic aging is rapidly increasing the prevalence of aging-associated health problems and setting previously unseen pressures on our healthcare systems. Alzheimer’s disease (AD), the most common form of dementia, is quickly becoming the most expensive disease of our times. It was recently estimated that the current annual worldwide expenditure for dementia care (~450 billion €) already equals 1% of global GDP (World Alzheimer Report, 2012). There are currently 35 million patients living with dementia worldwide (about 25 million with AD), a figure that is expected to double in just 20 years.

Currently, only symptomatic treatment options are available for AD. Although recent AD research has focused on how to reverse or delay the cerebral amyloid pathology, amyloid-based therapies have not translated well into humans. Thus, Alzheimer’s disease, particularly its sporadic late-onset form, needs to be approached from various novel perspectives. Better understanding of the molecular basis that underlies the development of the most common, late-onset form of AD (LOAD or sporadic AD) is essential for reaching the goals of efficient diagnosis and therapy. Specifically, to develop effective, disease-modifying treatments for AD and other dementias, we will need to understand the temporal sequence of the early pathophysiological events and how, on the molecular level, the genetic and environmental risk factors interact to initiate a cascade resulting in dramatic degeneration and loss of neurons (
Figure 1).

Aging appears to render the brain increasingly susceptible to various pathogenetic culprits, often related to environmental and life-style factors. The current view is that the presymptomatic period of AD may be very long, perhaps up to 10-20 years. Amyloid PET imaging is now sufficiently advanced for detecting amyloid plaques in the brain of presymptomatic humans (see the
video). Epidemiological data shows that cardiovascular risk factors in midlife are strongly associated with development of dementia/AD in later life. Cardiovascular factors, including increased serum cholesterol, hypertension and type 2 diabetes, are among the most important risk factors for AD. How these environmental risk factors interact with specific genetic risk factors remains enigmatic.

Alzheimer's disease, particularly its most common late-onset form (LOAD), is a complex disease with a strong hereditary component (
Figure 2). Nearly 700 genes have been associated with the risk of LOAD (www.alzgene.org). Yet, very little is known on how these genes, on a molecular and cellular level, actually affect the risk and the pathogenic chain of events in LOAD. Functional characterization of disease-associated genes remains a key challenge in the post-GWAS (genome-wide association study) era of complex disease research. For many of the most important LOAD risk genes, we can currently only guess what these genes and the proteins encoded by them do in the cells, and how they may be connected to AD pathophysiology.

Cerebral accumulation of β-amyloid peptide (Aβ) appears to be one of the earliest pathogenic events in AD (Querfurth & LaFerla, 2010). Moreover, the microtubule-associated protein Tau is a key molecule in development of neurofibrillary tangle (NFT) pathology but also in mediating the neurotoxicity of Aβ. Mitochondrial dysfunction, oxidative damage, insufficient growth factor signaling, excitotoxicity and neuroinflammation all contribute to disease progression. Individual susceptibilities in these domains determined by genetic factors eventually dictate the risk and age of onset for AD. Thus, on the cellular level, the onset and course of AD-like neurodegeneration is determined by a combination of primary pathogenic events and contributing factors, most of which can be measured individually in cell and animal models of disease. However, most conventional assays rely on analysis of cell or tissue extracts and are limited in providing information on the dynamic nature of these events.

Our overall aim is to understand those molecular and cellular events that play key roles in the early stages of neurodegeneration. Moreover, we aim to understand physiological functions of key pathophysiological proteins involved in neurodegenerative diseases. This basic research approach is expected to provide novel mechanisms, lead molecules and drug targets for the development of therapeutics for these highly significant yet unmet medical needs.

Protein-protein interactions at the core of neurodegeneration

The functionality of all cells depends critically on protein-protein interactions, particularly on the formation of multi-protein complexes. It is widely recognized that aberrant protein-protein interactions are associated with numerous pathological disorders - far beyond simple aggregation of amyloidogenic proteins. Better understanding of normal and pathological regulation and interaction dynamics of pathophysiologically important proteins in AD, amyloid precursor protein (APP) and Tau, is needed to fully understand how AD pathogenesis begins and how it could be prevented or slowed down. Quantitative detection of protein interaction dynamics offers a powerful but currently largely underused opportunity for modeling and understanding the fundamental neurodegenerative processes in live cells. In our laboratory, we have developed a technology platform for live-cell analysis of pathways involved in AD pathophysiology.

The traditional methods for studying protein-protein interactions rely on steady-state analysis of protein complexes that have been extracted from their native cellular environment. This is a significant shortcoming for functional studies. We use a technology called Protein-fragment Complementation Assays (PCA), a novel group of methods that allows studying dynamics of PPIs in live cells, to understand basic molecular mechanisms involved in pathophysiology of neurodegenerative diseases. Currently, our focus is on normal cellular regulation of β-amyloid precursor protein (APP) and microtubule-associated protein Tau, molecules that are involved in amyloid plaque and neurofibrillary pathologies in AD, respectively. Our technology platform allows various types of approaches, including mechanistic and functional genomics studies as well as screening of novel small-molecule modulators of PPIs.

Read more about our research on protein-protein interactions in neurodegenerative diseases.

Lipoproteins, their receptors and neuronal cell death mechanisms

Cholesterol has been linked to AD by a large number of epidemiological, genetic, animal and cell-based studies (Di Paolo & Kim, 2011). Brain cholesterol homeostasis is quite independent from the systemic cholesterol regulation, and due to their large membrane content and constant structural change, neurons are highly dependent on supply of cholesterol by glial cells, particularly astrocytes. Interestingly, proteolytic processing of amyloid precursor protein (APP) is strongly connected to neuronal cholesterol homeostasis. Henri's previous work showed that even small changes in subcellular cholesterol distribution strongly affect Aβ production in cells.

The interest in cholesterol's role in AD stems from the genetic finding that the epsilon 4 allele of ApoE gene is the strongest genetic risk factor for late-onset AD (Strittmatter et al, 1993). ApoE gene encodes the main brain lipoprotein and carrier of cholesterol from the astrocytes to neurons. ApoE has multiple functions beyond its lipoprotein role and appears to be an important contributor to AD pathogenesis on many levels, and inheritance of the ApoE4 isoform (carried by ~14% of population) significantly increases the risk of late-onset AD. There are at least 4 known mechanisms through which ApoE4 can increase the risk AD. (1) Neuronal health is dependent on proper ApoE-mediated cholesterol transport from astrocytes to neurons, and ApoE4 has weaker lipid-binding capacity than the other isoforms. (2) Aβ clearance from the brain is regulated by ApoE, and ApoE4 has weaker Aβ-binding capacity than the other isoforms. (3) Cerebovascular integrity is specifically impaired by the ApoE4 isoform. (4) ApoE4 produces truncated, toxic gain-of-function forms in neurons that seem to interfere with neuronal energy homeostasis.

Moreover, the lipoprotein receptors (e.g. ApoER2, VLDL receptor, LRP1) and their downstream signaling pathways may play interesting contributing roles in cell death and survival signaling and AD pathogenesis. We are interested on the roles and interplay of APP and lipoprotein receptors in normal neuronal function and pathogenesis of AD. We have recently identified novel mechanisms how PCSK9 and its target ApoER2 are involved in regulation neuronal cell death and survival (Kysenius et al, 2012). Recently, we have also studied the role of APP in the regulation of cholesterol biosynthesis (Wang et al, 2013). Also, we are interested in developing novel live-cell tools for measuring cholesterol-induced changes in APP trafficking in cells.

Microfluidics offers exciting new opportunities to study axonal biology

Engineered microstructures offer a diverse toolbox for cellular and molecular biologists to direct the placement of cells and small organisms, and to recreate biological functions in vitro: cells can be positioned and connected in a designed fashion, and connectivity and community effects of cells studied. Because of the highly polar morphology and finely compartmentalized functions of neurons, microfabricated cell culture systems and related on-chip technologies have become an important enabling platform for studying development, function and degeneration of the nervous system at the molecular and cellular level (Brunello et al, 2013).

A microfluidic chamber (MFC) method for isolation of axons in CNS neuron cultures allows treatment of axonal compartment independently of somatodendritic compartment. As dendrites and cell somas do not extend further than ~300-400 μm into the microgrooves, the neuronal processes growing through the microchannels (>450 μm) and connecting the two reservoirs are practically only axons. This allow effective segregation of axons from cell somas and cell bodies and elegant ways to treat and study these neuronal cell compartments separately in culture conditions. While the MFC provides excellent imaging, the electrophysiological recording or perfusion possibilities are somewhat limited. We are currently developing novel designs of microfluidic devices to allow more versatile neurobiological applications.

Read more about our neurofluidics research.

Previous work

References

Brunello CA, Jokinen V, Sakha P, Hideyuki T, Nomura F, Kaneko T, Lauri SE, Franssila S, Rivera C, Yasuda K and Huttunen HJ. Microtechnologies to fuel neurobiological research with nanometer precision.
J. Nanobiotechnol., 11:11, 2013. [abstract]

Di Paolo G, Kim TW. Linking lipids to Alzheimer's disease: cholesterol and beyond.
Nat. Rev. Neurosci. 12: 284-296, 2011. [abstract]

Kysenius K, Muggalla P, Mätlik K, Arumäe U and Huttunen HJ. Proprotein convertase PCSK9 regulates neuronal apoptosis by adjusting ApoER2 levels and signaling.
Cell. Mol. Life Sci. 69: 1903-1916, 2012. [abstract]

Querfurth HW, LaFerla FM. Alzheimer's disease.
N. Engl. J. Med. 362: 329-344, 2010. [abstract]

Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen GS, Roses AD. Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease.
Proc Natl Acad Sci U S A 90: 1977-1981, 1993. [abstract]

Wang W, Mutka A-L, Prosenc Zmrzljak U, Rozman D, Tanila H, Gylling H, Remes AM, Huttunen HJ and Ikonen E. Amyloid precursor protein α- and β-cleaved ectodomains exert opposing control of cholesterol homeostasis via SREBP2.
FASEB J. Epub Nov 18, 2013. [abstract]

World Alzheimer Report 2012. Alzheimer's Disease International (
www.alz.co.uk). PDF.