Abstract
Therapeutic options remain very limited for many neurodegenerative diseases. The transactive response DNA binding protein, TDP-43, has emerged as an important player in a diversity of neurodegenerative diseases. TDP-43 protein has important roles in RNA processing, such as transcription, alternative splicing, microRNA (miRNA) processing, mRNA stability and co-regulation of translation along with other factors. However, in many neurological diseases, TDP-43's normal cellular functions are lost and, whereas the protein is normally enriched in the nucleus, in disease states, aberrant forms of this critical protein form inclusion bodies and protein macroaggregates in the cytoplasm, rough ER, micronuclei, and mitochondria. In the past several years, TDP-43 has been defined as a hallmark feature in the etiology of neurodegenerative disorders, including both familial and sporadic ALS, even in the absence of a mutation in the TARDBP gene and it has been gaining wide-spread recognition as an important proteinopathy target. This chapter will focus on the therapeutic modulation of TDP-43 as a rational therapeutic approach to treating patients with cognitive and neurodegenerative disorders.
Keywords
amyotrophic lateral sclerosis, ALS, TDP-43, TARDBP, randomized clinical trial, cytoplasmic aggregates, mislocalization, N-terminal domain (NTD) and nuclear localization signal (NLS), non-homologous end-joining (NHEJ), DNA double-stranded breaks (DSB), neurite outgrowth, primary lateral sclerosis, progressive muscular atrophy and frontotemporal dementia (FTD), nonsense-mediated mRNA decay
Keypoints Box:
- N-terminal domain (NTD) - The beginning of an amino acid sequence of a protein which contains an amine group (-NH2)
- Nuclear localization signal (NLS) - a peptide sequence tag in a protein which directs the protein containing this sequence to be imported into the cell nucleus
- Non-homologous end-joining (NHEJ) - a double strand DNA break repair pathway which does not require a homologous template to repair the DNA damage
- DNA double-stranded breaks (DSB) -
- Neurite outgrowth - a crucial process in that takes place during embryonic development and neurogenesis whereby neuron, during differentiation, grow dendrite and axon projections from their cell bodies; outgrowth of dendrites and axons is guided by cues such as nerve growth factor signals
- Nonsense-mediated mRNA decay
Introduction
Overview of protein aggregates that occur in neurodegenerative disorders and rationale for targeting of TDP-43
There are a number of known pathological proteins that cluster or form aggregates in the brain and which are implicated, either a cause or an effect, of various neurodegenerative conditions. Besides TDP-43, additional well-know proteinopathy conformational, or misfolding disorders include Rho Guanine Nucleotide Exchange Factor (RGNEF), Alzheimer's Disease beta amyloid, which form plaques and hyperphosphorylated tau, which form neurofibrillary tangles, and α-synuclein in Lewy bodies in Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). It is somewhat uncertain as to whether aggregates may represent compensatory responses to cell stresses to forestall or halt death of neuronal, glia or microglial cells, rather than reflecting causal mechanisms in disease \cite{Espay2019}.
The normal biological role of the transactive response DNA binding protein, TDP-43
The transactive response DNA binding protein, TARDBP gene, encodes the TAR DNA-binding protein of 43 kDa (TDP-43), a ubiquitous nuclear protein that binds to both DNA and RNA. TDP-43's primarily function involves regulating pre-mRNA splicing \cite{Jiang_2017}. However, in addition to mRNA alternative splicing, TDP-43 is a critical protein with pleiotropic RNA-processing activities, including transcriptional repression, regulation of non-coding RNA, miRNA biogenesis, RNA stability, RNA trafficking, and auto-regulation of its own protein production \cite{Mejzini_2019}. TDP-43 is expressed early during embryonic development, and is also ubiquitously expressed in a diversity of tissues. In addition to its role in a wide range of RNA regulatory processes, TDP-43 has important functions in neurite outgrowth, projections that emanate from the neuron cell bodies, such as axons and dendrites . In addition, TDP-43 has a role in maintenance of genomic integrity and is a component of the DNA damage response pathway known as non-homologous end-joining (NHEJ). NHEJ is activated in response to DNA double-stranded breaks (DSB)\cite{Mitra_2019}.
Structure of TDP-43
Structurally, TDP-43 is a 414 amino acid protein consisting of four domains: two RNA recognition motifs (RRM1 and RRM2), a C-terminal domain (CTD) and N-terminal domain (NTD). The dynamic structure of TDP-43 NTD has been determined to be solenoid in shape with a physical separation between the C and N terminal domains. The C-terminal domain is implicated in the formation of cytoplasmic aggregates, and TDP-43's ability to form its correct shape. This is likely important for the normal physiological function of the protein rather than its disease-associated accumulation \cite{Mitra_2018}\cite{Afroz_2017}. TDP-43 is a highly conserved nuclear phase-separating protein, with regions of low complexity and intrinsically disordered domains. Phase separation occurs when a homogenous solution of molecules forms a membraneless phase-separated compartment. In the C-terminal region of the protein, the intrinsically disordered domain and a prion-like motif are located, which are regions implicated in the aggregate proteinopathy \cite{Yang_2010}. TDP-43 has a nuclear localization signal (NLS). Both the NTD and NLS are important regions for the nuclear localization of the protein as deletion of these regions results in its mislocalization from of the protein from the nucleus to the cytoplasm. It is there that it forms cytoplasmic inclusions. Importantly, using algorithms to predict the ability of peptide sequences to self-aggregate, domains of TDP-43 were analyzed and the highest proclivity to aggregate was found in the RRM2 and NTD peptide sequences, which should prove very important to screen for peptide inhibitors of TDP-43 based upon the interaction with these domains \cite{Kumar2019}.
The role of TDP-43 in ALS and other neurodegenerative diseases
TDP-43 has emerged as a critical player in neurodegenerative disease. Some notable examples of neurodegenerative diseases for which TDP-43 has been implicated include amyotrophic lateral sclerosis (ALS), a rapidly progressive and fatal group of motor neuron diseases that leads to degeneration and death of both upper- and lower motor neurons (UMN and LMN, respectively), resulting in loss of brain communication with muscles, which causes an inability to move voluntarily and, eventually, leads to the loss of ability to breath, accounting for it's high fatality rate of between 2-5 years after diagnosis; progressive muscular atrophy (PMA), which may be regarded as form of ALS, which affects primarily LMN and, in some patients, also UMN; primary lateral sclerosis (PLS), a slowly progressive motor neuron disease that affects upper neurons, and is more readily managed than ALS and may not necessarily decrease lifespan; frontotemporal dementia (FTD), the second most common form of early-onset dementia, following Alzheimer's disease, which alters personality, behavior, language and mental functions, and has a life expectancy of 6-8 years with the first onset of symptoms; Alzheimer's Disease (AD); Perry Syndrome ; Parkinson's Disease; Lewy body dementia; Huntington’s disease, and chronic traumatic encephalopathy .
TDP-43 and ALS group of neurodegenerative diseases
ALS is the motor neuron disease that occurs most often in late middle life. Classic ALS leads to the progressive death of motor neurons in the cerebral motor cortex and spinal cord, and the gradual loss of respiratory ability, muscle paralysis, and atrophy. Treatment consists of supportive care for respiratory difficulties, symptom relief and nutritional needs. There are currently only two FDA-approved medications for ALS, Riluzole, an anti-glutamatergic compound and Edaravone, an antioxidant, which only provide modest benefit in some patients. ALS often culminates in death within 2-5 years of clinical presentation, although there exist variants that have a slower progression and better prognosis. Approximately 5-10 % of patients with ALS have a familial history (fALS), while the majority of cases, 90-95%, emerge sporadically (sALS), in patients without a family history as a result of indeterminate genetic and environmental etiological causes. Uncovering the genetic variants associated with sALS may be hampered by the methodological approaches employed. For instance, most genetic studies have relied upon short-read and whole exome sequencing platforms, approached more conducive to detecting diseases attributed to one or more single nucleotide polymorphisms. This missing larger structural variants such as deletions, inversions or tandem repeats, which appear likely to play a fundamental role in ALS pathology. In ALS, TDP-43 forms aberrant aggregates in both neurons and glia \cite{Fernandopulle_2019}. Within the CTD is a glycine rich region that is involved in protein-protein interactions and binding to heterogeneous nuclear ribonucleoproteins (hnRNPs). It is in this region that the majority of ALS mutations occur (ref). The TDP-43 NTD was found to form a homodimer in solution and as a homodimer the formation of cytoplasmic aggregates is prevented and the mRNA splicing functions of TDP-43 in the cell nucleus is enhanced \cite{Jiang_2017}. During cell stress TDP-43 relocates to cytoplasmic stress granules. Although TDP-43 is normally present in the nucleus, during pathology it is cleaved and this cleaved form of TDP-43 enters the cell cytoplasm. Cell stress granules form in the cytoplasm and are composed of mRNA and RNA binding proteins following a stress to the cell, such as in the presence of toxins or high temperature. In normal physiology, cell stress granules are dynamic structures that can be assembled and dissembled when the cell stress trigger subsides. In ALS stress granules are persistent in motor neurons and this culminates in the degeneration of the motor neurons.
Mutations and epigenetic variations of TDP-43 and other ALS-linked genes in familial and sporadic neurodegenerative diseases
There are at least 60 known mutations in the TDP-43 gene
which have been identified in ALS (ref). Certain mutations in this gene are
responsible for the development of several other neurodegenerative
disorders. These include primary lateral sclerosis, progressive muscular
atrophy and frontotemporal dementia (FTD). Yet, TDP-43 mutations only account
for an estimated 5-10 % of familial ALS (fALS) patients \cite{Prasad_2019}, while the remainder of fALS mutations occur in other ALS-linked
genes. The most well-characterized of ALS-linked genes are the superoxide dismutase
(SOD1), chromosome 9 open reading frame 72 (C9ORF72) , and
fused in sarcoma (FUS) genes. For fALS-associated genes, all are inherited in a autosomal dominant fashion.
TDP-43 gain-of-function: sequestering of TDP-43 in cytoplasm and formation of aggregates
In ALS there is a perturbation in TDP-43 trafficking between the nucleus and cytoplasm. The TDP-43 protein is predominantly localized to the nucleus under normal endogenous conditions. However, sequestration of the protein in the cytoplasm, results in loss of endogenous TDP-43 function and to the accumulation of insoluble cytoplasmic aggregates. Evidence suggests that the N-terminal domain (NTD) of TDP-43 proteins, a region which contains its nuclear localization signal, homodimerizes with its protein partners and appears important in its function of targeting splicing of RNA. In fact, mutation of the nuclear localization or nuclear export signals results in cytoplasmic or nuclear aggregate formation \cite{Winton_2008}, whereas exogenous accumulation of cytoplasmic TDP-43 has been demonstrated to be specifically cytotoxic in primary rat cortical neurons. Therefore, homeostatic auto-regulation of TDP-43 is critical for its normal function. Normally, TDP-43 binds to the 3’ UTR of its own pre-mRNA, which leads to its undergoing of nonsense-mediated mRNA decay \cite{Ayala_2010}, thereby decreasing the nuclear to cytoplasmic shuttling of the transcript as well as its corresponding translation in the functional protein \cite{Koyama_2016}. Loss of homeostasic nucleo-cytoplasmic localization resulting in either nuclear or cytoplasmic TDP-43 aggregates appears critical is the pathology of all variants of ALS. Pathogenic TDP-43 inclusion bodies, becomes cleaved at the C-terminus, ubiquitinated and hyperphyphorylated, and targeted for autophagosomal removal . (Chang et al., 2016)
TDP-43 loss-of-function: loss of normal DNA and RNA processing functions
TDP-43 is an important protein, which is normally enriched in the nucleus, where it binds to both DNA and RNA. Normally it regulates transcription, mRNA splicing and RNA stability. The attenuation of these critical cell processes is implicated in a variety of neurological disorders, and it has been argued that the loss of normally functioning TDP-43 is of primary importance, and that toxic formation of aggregates is a secondary pathological feature \cite{Vanden2014}.
TDP-43 represents a promising therapeutic target
TDP-43 remains a
promising therapeutic target even when a patient does not have a
mutation evident in the TARDBP gene. For example, the vast majority (~95%) of ALS patients (sporatic and familial), exhibit TDP-43 neuronal
inclusions in their cortical and spinal cord neurons although a mere 5-10% have TDP-43 mutations. TDP-43 was found
to be a major constituent of ubiquitin-positive inclusions in ALS
patients \cite{Arai_2006}, leading to the recognition of this
protein aggregate as a hallmark of ALS \cite{Wolozin_2019}.